Adverse pregnancy outcomes are associated with Plasmodium vivax malaria in a prospective cohort of women from the Brazilian Amazon.

BACKGROUND: Malaria in Brazil represents one of the highest percentages of Latin America cases, where approximately 84% of infections are attributed to Plasmodium (P.) vivax. Despite the high incidence, many aspects of gestational malaria resulting from P. vivax infections remain poorly studied. As such, we aimed to evaluate the consequences of P. vivax infections during gestation on the health of mothers and their neonates in an endemic area of the Amazon. METHODS AND
FINDINGS: We have conducted an observational cohort study in Brazilian Amazon between January 2013 and April 2015. 600 pregnant women were enrolled and followed until delivery. After applying exclusion criteria, 329 mother-child pairs were included in the analysis. Clinical data regarding maternal infection, newborn's anthropometric measures, placental histopathological characteristics, and angiogenic and inflammatory factors were evaluated. The presence of plasma IgG against the P. vivax (Pv) MSP119 protein was used as marker of exposure and possible associations with pregnancy outcomes were analyzed. Multivariate logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy are associated with adverse gestational outcomes such as premature birth (adjusted odds ratio [aOR] 8.12, 95% confidence interval [95%CI] 2.69-24.54, p < 0.0001) and reduced head circumference (aOR 3.58, 95%CI 1.29-9.97, p = 0.01). Histopathology analysis showed marked differences between placentas from P. vivax-infected and non-infected pregnant women, especially regarding placental monocytes infiltrate. Placental levels of vasomodulatory factors such as angiopoietin-2 (ANG-2) and complement proteins such as C5a were also altered at delivery. Plasma levels of anti-PvMSP119 IgG in infected pregnant women were shown to be a reliable exposure marker; yet, with no association with improved pregnancy outcomes.
CONCLUSIONS: This study indicates that P. vivax malaria during the first trimester of pregnancy represents a higher likelihood of subsequent poor pregnancy outcomes associated with marked placental histologic modification and angiogenic/inflammatory imbalance. Additionally, our findings support the idea that antibodies against PvMSP119 are not protective against poor pregnancy outcomes induced by P. vivax infections.

Introduction

Pregnant women are highly susceptible to malaria and its deleterious effects. Recently, the World Health Organization Malaria Report pointed the often-neglected malaria cases in pregnancy (MiP) that occur mainly in Africa, where around 11 million pregnant women were estimated to have developed the disease during 2018 [1]. Africa is characterized by a high and stable transmission of Plasmodium (P.) falciparum. Despite being frequently under the spotlight of malaria control campaigns, infections during pregnancy are still a burden associated with maternal anemia, abortion, premature delivery, and low birth weight (LBW) [2-7]. Few studies have focused on the burden of MiP in low transmission regions like Latin America, where P. vivax is the most prevalent species [1]. Its infections are often considered benign, a paradigm that is frequently supported by inconsistent reports that tend to focus on P. falciparum malaria episodes during pregnancy; this makes the evaluation of the real impact of P. vivax monoinfections somehow challenging [8-11].

In P. falciparum MiP, poor pregnancy outcomes are mainly linked to severe placental inflammation triggered by the sequestration of infected erythrocytes (which characterize placental malaria, PM) [12]. This process results in dramatic histological changes of the placental structure [11,13], which ultimately dysregulate local homeostasis and contribute to poor pregnancy outcomes [2]. This phenomenon is not yet clearly elucidated in P. vivax MiP mainly due to scarce and controversial evidence concerning: (1) placental adhesion [14,15]; (2) severe placental histopathology [9-11]; and (3) subsequent poor gestational outcomes [16-18]. The harmful effects of P. vivax infections are most likely a consequence of systemic inflammation and production of cytokines [19], such as TNF-α and IFN-γ, which are linked to disease severity, or IL-10 that is associated with better disease prognosis [20]. Accordingly, P. vivax-induced inflammation during pregnancy may be implicated in placental structural abnormalities and leukocytes infiltration, as well as in angiogenic imbalance, which altogether can directly or indirectly impact fetal development [11,13,21]. Nevertheless, to our knowledge, studies addressing these parameters together are still missing.

Attenuation of clinical symptoms frequently occurs after naturally acquiring immunity due to frequent exposures to Plasmodium spp. In P. falciparum MiP, loss of protection is mainly attributed to lack of immunity against the P. falciparum erythrocyte membrane protein 1 (PfEMP-1) variant VAR2CSA that binds to chondroitin sulfate A (CSA) in the placenta. In subsequent pregnancies, the development of PfEMP1var2csa specific antibodies is associated with protection from MiP [22-26]; yet, in P. vivax MiP, the establishment of a clear association between pre-existing acquired immunity and improved pregnancy outcomes is yet to be achieved.

Herein, we report the deleterious relationship between P. vivax monoinfections, poor pregnancy outcomes, placental histopathology, cytokine and angiogenic imbalance, and humoral immune response in pregnant women from the Brazilian Amazon. Our results suggest that P. vivax infections during the first gestational trimester are associated with poor pregnancy outcomes. The presence of IgG against P. vivax merozoite surface protein 1 (P. vivax MSP119) is not associated with protection against the deleterious effects of P. vivax MiP.

Methods

Ethics statement

This study was approved by the Ethics Committee from the University of São Paulo (Plataforma Brasil, CAAE: 03930812.8.0000.5467 and 90474318.4.0000.5467), according to resolution n° 466/12 of the Brazilian National Health Committee and in compliance with the Declaration of Helsinki. All subjects or their legal guardians (if minors) gave written informed consent. This study followed a specified analysis plan ( and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement guidelines (.

Setting and study design

We have conducted an observational cohort study in five municipalities (Cruzeiro do Sul, Mâncio Lima, Rodrigues Alves, Marechal Thaumaturgo, and Porto Walter) in the Juruá Valley, located in the Brazilian Amazon southwest region (Acre State, Brazil). This region is considered highly endemic for malaria, with an annual parasite incidence (API) above 100 cases per 1,000 inhabitants. Juruá Valley is also characterized by a significant prevalence of P. vivax infections, responsible for 70–80% of total malaria cases [27,28]. A complete region characterization with detailed procedures and methods used for data and sample collection has been described elsewhere [11,13,21]. Briefly, 600 pregnant women were recruited during their first antenatal care (ANC) visit. These women were followed until delivery, between January 2013 and April 2015. Data was collected on socioeconomic, clinical, and obstetric variables at the time of recruitment, together with peripheral blood samples. In parallel, thick and thin peripheral blood smears were performed to screen for malaria parasites by microscopy, which were later confirmed by PCR. Each pregnant woman was followed by a trained nurse, which involved at least two domiciliary visits, at the second and third trimester, to monitor their clinical state and collect a peripheral blood sample, in addition to the usual prenatal care visit to the health care services. An additional blood sample was collected in each malaria episode during pregnancy. At the time of delivery, clinical data were collected from mother and newborn and performed a placental biopsy and blood sampling. Only women with P. vivax monoinfections and their non-infected counterparts were considered for this study (. We evaluated mother-child pairs at delivery in the Women and Children’s Hospital of Juruá Valley located in Cruzeiro do Sul.

Description of study participants and definitions.

(A) Flow chart detailing exclusion criteria applied prior data analysis; (B) Definitions of pregnancy partitions used in the report. Mixed infection–P. vivax and P. falciparum infections occurring at the same or different times during pregnancy; w—weeks; d—days.

Malaria screening and treatment

Malaria during pregnancy was first diagnosed from thin and thick blood smears by microscopy analysis, which was performed by the endemic surveillance team of Juruá Valley. Upon diagnosis, all women infected with P. vivax during pregnancy were treated with chloroquine (25mg/kg of body weight) in divided doses over a 3-day period. Successful treatment was confirmed subsequently. No hypnozontocidal treatment with primaquine was performed, following the Brazilian Ministry of Health (MoH) guidelines regarding medical prescription in pregnant women [29]. Of note, the Intermittent Preventive Treatment in pregnancy (IPTp) is not employed in Brazil. All samples collected throughout pregnancy (recruitment, malaria episodes, and domiciliary visits) and at delivery were further screened by molecular diagnosis using the real-time PCR technique (PET-PCR) as described elsewhere [13,30].

Exclusion criteria

Pregnant women were excluded from further analysis if they self-informed any alcohol consumption, smoking, and/or if presenting any other diagnosed infectious diseases (e.g. Toxoplasmosis, Rubella, Cytomegalovirus and Herpes simplex (TORCH), HIV, Hepatitis B and C virus, Syphilis), and/or other comorbidities (e.g. hypertension, pre-eclampsia/eclampsia, diabetes mellitus, and newborn with congenital malformation). Likewise, pregnant women infected with other Plasmodium species (P. falciparum or mixed infections) were excluded after confirmation by PET-PCR (.

Laboratory procedures

Peripheral and placental blood (from the maternal side) was collected aseptically in heparinized tubes and separated into plasma and whole blood cells after centrifugation. All samples were stored at -80°C. Total DNA was obtained from whole blood cells using a commercially available extraction kit (QIAmp DNA Mini Kit, Qiagen), following the manufacturer’s instructions. Fragments of placental tissue were collected and stored in 10% neutral buffered formalin at 4°C, until processing. Placental tissue was sectioned and embedded in paraffin using standard procedures; afterward, it was stained with Hematoxylin-Eosin (H&E) or Giemsa for histological examination. All histological evaluation methods were optimized by our group and are described elsewhere [11,13]. Placental images were captured using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc camera. Placental parameters were evaluated and analyzed using Image J software (http://imagej.nih.gov/ij). All measurements were performed by two investigators blinded to group and outcome. Cases that proved to be contradictory between observers were re-evaluated until consensus was reached.

Placental weight and newborn’s anthropometric measurements

Placental weight was measured (grams (g)) immediately after expulsion/extraction using a digital semi-analytical scale (Shimadzu), with a capacity of 3.2 kilograms (kg) and a precision of 0.01 g. Umbilical cord, membranes, and fetal blood contained within the large chorionic vessels and intervillous space, were included in weight measurement. Newborns’ anthropometric measures were obtained after birth (maximum within a 24-hour period) by trained obstetrician nurses. Weight was measured (g) using digital pediatric scales (Toledo), with a maximum capacity of 15 kg and a precision of 5 g. Length, head and chest circumferences were measured using a non-stretching flexible measuring tape and expressed as centimeters (cm). Rohrer’s ponderal index was calculated as the newborn’s weight (g) divided by the cube of the length (cm).

Gestational age estimation and other definitions

Gestational age (GA) was assessed during the first trimester of pregnancy using crown-rump length measured by trans-abdominal ultrasound. In the absence of this examination within this period, the GA was estimated by the woman’s last menstrual period (LMP) together with the earliest ultrasound performed. The LMP method is recommended by the Brazilian MoH for GA calculation. Low birth weight (LBW) was defined as birth weight below 2500 g. Preterm birth was defined as birth before the 37th week of gestation, abortion as birth occurring before the 22nd week of gestation, and stillbirth as fetal death occurring between the 22nd week of gestation and time of delivery (. Term LBW was defined as term birth (after 37th week of gestation) with weight below 2500 g. Small for gestational age (SGA), reduced head circumference, and reduced body length were defined as: birth weight, head circumference and length below the 10th percentile sex-specific for GA according to the INTERGROWTH-21st standards [31]. Maternal anemia was defined as hemoglobin levels lower than 11 g/dL. Placental malaria was diagnosed according to a previously established classification [12].

Angiogenic factors and leptin measurement

Angiogenic factors (angiopoietins 1 and 2 (ANG-1 and ANG-2), soluble TEK receptor tyrosine kinase (sTIE-2), vascular endothelial growth factor A (VEGF-A), receptors fms-like tyrosine kinase-1 (sFlt-1) and soluble vascular endothelial growth factor receptor 2 (sVEGFR-2)), and the leptin hormone were measured in placental plasma (1:20 dilution for all factors) using the DuoSet ELISA development kits (R&D), according to manufacturer’s guidelines.

Cytokines/anaphylatoxins measurement by cytometric bead array (CBA)

Interleukin (IL-) 1β, IL-6, IL-8, IL10, IL-12p70, and TNF-α were detected and quantified in placental plasma by CBA Human Inflammatory kit (BD Biosciences), according to the manufacturer’s protocol. Complement (C3a, C4a, and C5a) activation was evaluated in placental plasma through CBA Human Anaphylatoxin kit (BD Biosciences). Samples were analyzed in a two-laser BD FACSCalibur flow cytometer with CellQuest version 5.2 software (BD Biosciences) and concentrations computed using FCAP array software version 3.0.1 (BD Biosciences).

Measurement of total IgG antibodies against Plasmodium vivax merozoite surface protein 1 (PvMSP119)

For the study of total IgG antibodies against PvMSP119, samples collected at the first time-point (recruitment) were used; yet, we excluded infected pregnant women who were recruited after having already one or more malaria episodes during that pregnancy. Enzyme-linked immunosorbent assay (ELISA) was performed using high-binding 96-well microplates (Costar, Cambridge, MA) coated with Glutathione S-transferase (GST)-PvMSP119 (31.25 ng/well), produced as described elsewhere [32,33]. In parallel, GST was used as a control for non-specific binding. The optimal concentrations were determined by plasma serial titration of positive and negative controls. Plates were incubated at 4°C overnight and washed four times with phosphate buffered saline with 0.05% Tween 20 (PBS-T). PBS-T containing skimmed milk was used for blocking (4%) and diluting plasma and antibodies (1%). Each plasma sample (50 μL/well) was diluted 1:200. After a 1-hour incubation at room temperature, antibody-antigen complexes were detected with HRP-conjugated mouse anti-human IgG (IG266) (Novus, Cat# NBP2-34648H) at a 1:3,000 dilution. Enzyme-substrate reaction was revealed with tetramethylbenzidine (TMB)/H2O2 at room temperature (dark conditions). After 10 minutes, the reaction was interrupted with 2N H2SO4. Absorbance values were measured at 450/595 nm using a CLARIOstar Plus plate reader (BMG Labtech). Corrected absorbance values were obtained by subtracting absorbance readings with GST ran on the same microplate. Absorbance data used in statistical analyses correspond to corrected values. Reactivity indices (RIs) were calculated as the ratio between each test sample’s corrected absorbance values and a cut-off value for antigen, corresponding to the average corrected absorbance for samples from 10 malaria-naïve blood donors plus 3 standard deviations. Positive samples were considered as having RIs greater than 1.

Statistical analysis

Data were analyzed using Stata/SE 14.2 (Stata, College Station, TX, USA) and GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). Continuous variables were summarized using mean and standard deviation (SD) values as well as median values and interquartile ranges (IQRs). Categorical variables were summarized using frequencies and percentages. Differences between groups were evaluated using the nonparametric Kruskal-Wallis test followed by the Dunn’s post hoc multiple comparison test and the Mann-Whitney test as appropriate. Differences between categorical data and proportions were analyzed using Pearson′s chi-squared test or Fisher’s exact test depending on the type of variables. All p values were 2-sided and interpreted at a significance level of 0.05. To compare the association between malaria and other variables (clinical characteristics, newborns’ anthropometric measurements, placental parameters, angiogenic and inflammatory factors, and anti-PvMSP119 IgG), we used linear regression, adjusting for maternal age, number of gestations, residence, education level, and occupation. To assess associations between malaria and adverse pregnancy outcomes, the presence of anti-PvMSP119 IgG and outcomes in the context of MiP, adjusted odds ratios (aOR) with 95% confidence intervals (CI) were estimated using multiple logistic regression, which included infection by malaria (no / yes), maternal age (≥ 18 years old / ≤ 17 years old), number of gestations (two or more / one), residence (urban / rural), education level (≥ secondary / ≤ primary), occupation (others / housewife) and presence of anti-PvMSP119 IgG (no / yes) as explanatory variables and adverse pregnancy outcomes (yes / no) as response variables. The first category for each explanatory variable was considered as the reference.

Results

Study population and baseline characteristics

From 600 pregnant women enrolled in the major study, 329 (54.8%) were eligible for further analysis after applying the exclusion criteria and malaria parasite species diagnosis by PET-PCR (. Of these, 170 (51.7%) were classified as non-infected and 159 (48.3%) as P. vivax (Pv)-infected during gestation (. The overall socio-demographic profile of the stratified participants is depicted in . Group stratification was performed by the trimester in which the first infection occurred, considering that time of infection is known to be associated with MiP poor pregnancy outcomes [34]. illustrates a standard gestational window with the hypothetical poor outcomes in unsuccessful pregnancies. shows some heterogeneity between the groups since pregnant women in the infected groups tend to be younger, with reduced educational level and rural residence. Occupation also varies significantly between the groups, but no difference was found regarding the number of previous pregnancies. The aforementioned characteristics were used as explanatory variables in the following multiple logistic and linear regressions analysis due to reported heterogeneity and their possible impact as confounding effects.

P. vivax infections during the first trimester are associated with MiP poor pregnancy outcomes

Malaria in pregnancy is known to impact proper fetal development [2]. We assessed pregnancy outcomes and newborns’ anthropometric measurements to ascertain if P. vivax infections at a given gestational trimester are associated with impaired fetal development and poor outcomes. Multiple logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy increase the odds of premature birth (aOR 8.12, 95%CI 2.69–24.54, p < 0.0001), LBW (aOR 4.34, 95%CI 1.49–12.62, p = 0.007), as well as LBW in those born at term (aOR 4.75, 95%CI 1.06–21.35, p = 0.04). First trimester infections were also associated with reduced head circumference (aOR 3.58, 95%CI 1.29–9.97, p = 0.01) and newborn’s length (aOR 4.48, 95%CI 1.66–12.06, p = 0.003) (. No differences were observed for the overall birth weight and length (. Though, in line with our findings, we observed statistically significant differences in head and chest circumference of children born from women infected with P. vivax during the first trimester and those born from non-infected women (median [IQR], head–Pv-1st tri 34.0 cm [32.0–34.0] vs. NI 34.0 cm [33.0–35.0], p = 0.007; chest–Pv-1st tri 33.0 cm [32.0–34.0] vs NI 34.0 cm [33.0–35.0], p = 0.006) (. Our results suggest that P. vivax infections during the first gestational trimester promote preterm birth and impair fetal development, possibly due to clinical manifestations associated with MiP.

Association between adverse pregnancy outcomes and P. vivax infections according to the trimester of first infection.

Forest plot of odds ratios of adverse events in newborns from women infected during pregnancy compared with newborns from non-infected women is shown. n/N—number of events by total number of individuals in each group; aOdds Ratio—adjusted Odds Ratio; CI—confidence interval; tri—gestational trimester. Odds ratios were adjusted (aOR) for maternal age, gravidity status, residence, education level and occupation. Preterm birth–birth < 37th week of gestation; Low birth weight–birth weight < 2500 g; Term low birth weight–birth weight < 2500 g from 37th week of gestation; Small for gestational age, Reduced head circumference and Reduced body length–birth weight, head circumference and length (respectively) < 10th centile for sex-specific gestational age. P-values were estimated through multiple logistic regression methods.

Anthropometric measurements of newborns according to maternal infection and trimester of first infection.

(A) Newborn weight; (B) Head circumference; (C) Chest circumference; and, (D) Length. NI–non-infected (N = 160–169); Pv–P. vivax-infected pregnant women (N = 130–150); Pv-1st tri–P. vivax infection 1st trimester (N = 38–46); Pv-2nd tri–P. vivax infection 2nd trimester (N = 43–54); Pv-3rd tri–P. vivax infection 3rd trimester (N = 49–50). Weight is given in grams (g). Head and chest circumferences, and body length are informed in centimeters (cm). Data are represented as Tukey boxplots, with the bottom and top of each box representing the first and third quartiles; the line inside the box is the median, and the whiskers represent the lowest and the highest data within 1.5 IQR of the first and upper quartiles; and the circles, outliers. Differences between groups were determined by multiple liner regression (adjusted for maternal age, gravidity status, residence, education level, and occupation).

Clinical and infection characteristics of pregnant women with P. vivax MiP

As for the aforementioned outcomes, pregnant women infected with P. vivax had some clinical characteristics that are worth mentioning (. Women infected during the first trimester had reduced gestational age at delivery when compared with non-infected counterparts (median [IQR], Pv-1st tri 39.0 weeks [37.0–40.0] vs. NI 40.0 weeks [39.0–40.0], p = 0.006). These results are in line with our observations that suggest an association between first trimester infections and pregnancy outcomes such as LBW and preterm delivery (. Reduced maternal weight gain was also observed in this group (median [IQR], Pv-1st tri 10.0 Kg [7.0–12.0] vs. NI 13.5 Kg [10.0–16.8], p = 0.004). Anemia is frequently found in pregnant women infected with P. vivax, which has been suggested to be one of the leading causes of impaired fetal development during P. vivax MiP [16,35]. However, a statistically significant reduction in hematocrit was only observed for the overall Pv-infected group (median [IQR], Pv 34.8% [32.4–37.5] vs. NI 36.6% [34.5–38.6], p = 0.008) and increased clinical anemia in those infected during the third trimester (Pv-3rd tri 28.3% vs. NI 14.1%, p = 0.02). Additionally, women in the Pv-infected group reported more malaria episodes before current pregnancy than their non-infected counterparts regardless the infection trimester (. Pregnant women infected during the first trimester had higher chances of having three or more infections than infected women from other groups. Parasitemia tend to be similar for the first infection across gestational trimesters; yet, it seems to decrease on average from the second infection onward (. 8.1% of women infected in the first trimester had past PM, while 4.6 and 4.3% of women infected during the second and third trimesters had active acute PM, respectively. This was established by the presence of hemozoin in milder or moderate amounts. However, it was absent in more than 90% of the examined placentas, regardless of the infection’s gestational trimester. Our observations support the idea that systemic effects rather than PM are linked to the outcomes observed during P. vivax MiP in our cohort.

P. vivax infections across gestational trimesters lead to diverse placental histologic alterations and angiogenic and cytokine imbalance

Reduced PM prevalence is frequently observed in P. vivax infections because, contrary to P. falciparum infections, P. vivax sequestration in the placenta is controversial [14,15] and studies that report this event frequently have reduced sample sizes. Nevertheless, placental histologic changes had been observed [9-11]. To determine whether Pv-infections across the gestational trimesters result in considerable histologic and cytological alterations, we have evaluated several histologic parameters in more than 100 placentas from Pv-infected women (. We observed that placentas from Pv-infected women had a significant increase in syncytial nuclear aggregates (SNA), fibrin deposition, monocytes and leukocytes infiltrate (median [IQR], SNA–Pv 15.0 [11.0–21.0] vs. NI 13.0 [10.0–17.0], p = 0.001; fibrin–Pv 2.5 [1.9–2.8] vs. NI 1.9 [1.9–2.8], p = 0.001; monocytes–Pv 8.0 [6.0–13.0] vs NI 4.0 [2.0–6.0], p < 0.0001; leucocytes–Pv 21.5 [14.0–29.0] vs. NI 15.0 [9.0–21.0], p = 0.004) (. Differences between placentas from non-infected women and those from women infected in the first trimester were observed regarding SNA and monocyte infiltrate (median [IQR], SNA–Pv-1st tri 19.0 [11.0–22.0] vs. NI 13.0 [10.0–17.0], p = 0.02; monocytes–Pv-1st tri 7.0 [5.0–9.5] vs NI 4.0 [2.0–6.0], p = 0.01) (. Interestingly, infections closer to term tend to yield higher levels of fibrin deposition and monocytes infiltrate at delivery (. These findings are representative of placental anomalies that can be extended to angiogenic and inflammatory mediators in placental plasma (. First and third trimester infections led to reduced levels of angiopoietin-2 (ANG-2) and increased levels of soluble TEK receptor tyrosine kinase (sTIE-2), respectively (median [IQR], ANG-2 –Pv-1st tri 4.4 ng/mL [1.0–6.6] vs. NI 6.8 ng/mL [3.5–12.4], p = 0.03; sTIE-2 –Pv-3rd tri 18.5 ng/mL [11.7–24.7] vs. NI 13.9 ng/mL [9.1–19.0], p = 0.008). Overall, factors such as sFlt-1, sVEGFR-2, and the hormone leptin were also altered in placentas from Pv-infected women (. Regarding inflammatory mediators, only IL-12 and C5a showed more consistent and significant alterations, as they were reduced at delivery due to first and third trimester infections (median [IQR], IL-12 –Pv-1st tri 3.3 pg/mL [2.6–3.8] vs. NI 3.6 pg/mL [3.0–4.2], p = 0.009; C5a –Pv-1st tri 807.1 pg/mL [534.9–913.9] vs. NI 1117.2 pg/mL [814.8–1475.5], p = 0.03; Pv-3rd tri 673.0 pg/mL [445.9–871.3] vs. NI 1117.2 pg/mL [814.8–1475.5], p = 0.006) (. Altogether, our observations suggest that poor pregnancy outcomes associated with first trimester infections may be linked to placental inflammation, structural abnormalities and angiogenic imbalance during P. vivax MiP.

Evaluation of placental histological parameters according to maternal infection and trimester of first infection.

(A) Syncytial nuclear aggregates; (B) Fibrin deposition score; (C) Monocytes infiltrate (CD68+); (D) Leukocytes infiltrate (CD45+). Histologic parameters were evaluated by microscopy through H&E (syncytial nuclear aggregates and fibrin deposition) or immunohistochemistry (monocytes and leukocytes). NI–non-infected (N = 153–155); Pv–P. vivax-infected pregnant women (N = 118–128); Pv-1st tri–P. vivax infection 1st trimester (N = 33–37); Pv-2nd tri–P. vivax infection 2nd trimester (N = 42–44); Pv-3rd tri–P. vivax infection 3rd trimester (N = 43–47). Data are represented as Tukey boxplots, with the bottom and top of each box representing the first and third quartiles; the line inside the box is the median, and the whiskers represent the lowest and the highest data within 1.5 IQR of the first and upper quartiles; and the circles, outliers. Differences between groups were determined by multiple liner regression (adjusted for maternal age, gravidity status, residence, education level, and occupation).

Antibodies against P. vivax MSP119 are not associated with protection from poor pregnancy outcomes

The impact of gravidity on P. vivax MiP outcomes is still inconclusive [16,35,36]. Possibly the absence of pregnancy-specific antigens in P. vivax and its restricted distribution to areas of low malaria transmission may impair immunity acquisition across pregnancies [8,37]. As such, other antigens might be targeted by the immune system to confer some degree of protection from P. vivax MiP. Therefore, we measured antibodies against the C-terminal fragment of the P. vivax merozoite surface protein 1 (PvMSP119). We used it as a surrogate marker of previous exposures ( and sought possible associations between antibody titers and improved pregnancy outcomes. Infected pregnant women who were recruited with more than one malaria episode during their current pregnancy were removed from the analysis, to target antibodies acquired upon any malaria episode occurred before pregnancy. We observed that Pv-infected women previously exposed to the parasite (Pv-PvMSP1Ab+) had higher titers of IgG when compared with non-infected pregnant women with prior exposure (NI-PvMSP1Ab+) (median [IQR], Pv-PvMSP1Ab+ 65.1 [14.1–91.6] vs. NI-PvMSP1Ab+ 4.0 [2.1–9.8], p < 0.0001) (). However, no associations were observed between IgG titers and outcomes, suggesting that the presence of these antibodies do not protect pregnant women from the adverse outcomes of P. vivax MiP (. Accordingly, no statistically significant differences were observed for birth weight, head and chest circumference, and body length, between P. vivax-infected women who had PvMSP119-specific antibodies and those who did not have them (.

Anthropometric measurements of newborn, according to previous exposure to P. vivax (PvMSP1).

(A) Antibody profile among non-infected and P. vivax-infected pregnant women; (B) Forest plot of odds ratios of adverse pregnancy outcomes in infected women with anti-PvMSP1 IgG compared with infected women without anti-PvMSP1 IgG; (C) Newborn weight; (D) Head circumference; (E) Chest circumference; and, (F) Length. NI-PvMSP1Ab-—non-infected non-exposed (N = 106–112); NI- PvMSP1Ab+—non-infected exposed (N = 54–58); Pv- PvMSP1Ab-—P. vivax-infected non-exposed (N = 19); Pv- PvMSP1Ab+—P. vivax-infected exposed (N = 80–95). PvMSP1- Plasmodium vivax merozoite surface protein 1; Ab—Antibody; LBW—low birth weight; SGA—small for gestational age; HC—head circumference. RI—Reactivity indices were calculated as the ratio between each test sample’s corrected absorbance values and a cut-off value for antigen, corresponding to the average corrected absorbance of samples from 10 malaria-naïve blood donors plus 3 standard deviations. Weight in grams (g). Head and chest circumferences, and body length in centimeters (cm). Data are represented as Tukey boxplots, with the bottom and top of each box representing the first and third quartiles; the line inside the box is the median, and the whiskers represent the lowest and the highest data within 1.5 IQR of the first and upper quartiles; and the circles, outliers. Odds ratios were adjusted (aOR) for maternal age, gravidity status, residence, education level and occupation using multiple logistic regression. Differences among the groups were determined by multiple linear regression (adjusted for maternal age, gravidity status, residence, education level and occupation).

Overall, our results suggest that P. vivax infections during the first gestational trimester are associated with poor pregnancy outcomes most likely due to placental histologic and physiologic abnormalities, which are unlikely to be prevented by the presence of IgG against PvMSP119, acquired during previous P. vivax-malaria episodes.

Discussion

MiP is considered a public health problem worldwide, as it is a significant risk factor for abortion, stillbirth, LBW, and maternal anemia [2-6]. Here, we describe an important association between P. vivax MiP and adverse gestational events, placental histopathological changes, and a reduction of the neonate’s anthropometric profile. However, a protective association between IgG against PvMSP119 and improved pregnancy outcomes was not observed.

P. vivax is widely distributed worldwide, particularly in Asia, Pacific regions, and Latin America. Though, longitudinal studies on gestational P. vivax-malaria are still limited, especially regarding placental histopathological analysis [8-11]. Although P. vivax MiP is poorly associated with severe malaria, this species’ infections are far from benign and may have severe consequences for maternal and fetal health [16,17,35,36,38-40]. The gestational outcomes associated with MiP can vary not only with the species but also with variables such as transmission intensity and stability, number of previous pregnancies, and trimester of infection [7,37]. In our study, no association was found between gravidity and improved pregnancy outcomes (), suggesting that gestational-dependent mechanisms that govern immunity against P. falciparum during pregnancy may not be relevant in infections caused by P. vivax. In low transmission settings, where P. vivax is more prevalent, gravidity-dependent immunity acquisition is uncertain and inconsistently reported [8,16,35,39]. This could be related to the inexistence of known pregnancy-related P. vivax proteins targeted by the immune system [14,15].

The burden of first trimester infections, was scarcely addressed by researchers until recent years [7] despite knowing that perturbation in early pregnancy imposes severe fetal development consequences [41,42]. In our study, P. vivax first trimester infections were found associated with LBW and reduced newborn’s length. The proportion of pregnancies with LBW observed by us was consistent with other studies conducted in Latin America [38,43] and other P. vivax transmission areas [7]. These findings are relevant as LBW is strongly associated with neonatal morbidity and mortality, growth inhibition and cognitive impairment in children, and chronic diseases in adulthood [44]. Similar to what we reported recently on P. falciparum MiP, P. vivax infections also impact fetal cephalic perimeter development [13]. However, a previous report using the same study population reported that one antenatal P. vivax malaria episode in another trimester than the first trimester was enough to impair fetal development, which was aggravated by recurrent parasite exposure during pregnancy [36].

A distinctive characteristic of P. vivax is the occurrence of multiple malaria episodes due to reinfections or relapses, with the latter being potentiated by the absence of primaquine treatment in pregnant women due to its teratogenic nature [45]. Taken that and the higher prevalence of peripheral parasitemia observed during the second gestational trimester, also described in other studies [46,47], we hypothesize that inflammation modulation during pregnancy might favor parasite multiplication and relapses.

The mechanisms by which P. vivax infections lead to MiP outcomes are still unclear; yet, systemic events such as maternal anemia and generalized inflammation have been discussed as relevant to P. vivax MiP pathology [16,35,40]. Our data evidence a significant hematocrit reduction in pregnant women with P. vivax; though, no severe maternal anemia was observed. Only a significant frequency of anemia at delivery was detected among women with third trimester infections. Although maternal anemia is a well-known risk factor for intrauterine growth retardation (IUGR) and LBW in P. vivax-infected women [2,16-18,35,36,40], this is unlikely to be influencing pregnancy outcomes in our cohort.

Contrary to P. falciparum infections, where pregnancy complications and adverse outcomes are often associated with PM [4,48-50], in P. vivax infections, PM is unlikely to be the reason why infected women experience complications during pregnancy. Previous studies indicate that PM caused by P. vivax is modest and not associated with placental sequestration of infected erythrocytes [9-11,40], despite evidence of binding to CSA and placental tissue sections [14,15]. Our observations corroborate previous studies, as only 8.6% of the analyzed placentas were classified as having PM. Nonetheless, most placentas from P. vivax-infected pregnant women present histopathological changes. Placental lesions and histopathological changes are often difficult to observe in P. vivax-infected pregnant women, probably due to the low sample size reported in the studies [9-11,40]. By performing histologic analysis of a considerable number of placentas, we have shown that P. vivax infections during the first trimester promote the increase of SNA and monocyte infiltrate at delivery. Monocyte infiltrate and inflammation have been frequently associated with reduced birth weight and preterm birth in pregnant women infected with P. falciparum [51-54]. Therefore, we hypothesize that this may also occur in P. vivax infections and that placental inflammation independent of PM may be linked to P. vivax MiP adverse outcomes, opposing previous observations suggesting that P. vivax monoinfections do not promote placental inflammation and that adverse outcomes have an alternative etiologic nature [55].

Dysregulation of angiogenic and inflammatory factors are often associated with placental insufficiency and impaired fetal development in P. falciparum MiP [2]. The imbalance of angiopoietins has been associated with PM and LBW in pregnant women infected with P. falciparum [56] and observed in P. vivax-infected patients with thrombocytopenia [57]. In fact, in P. vivax infections, we observed diminished placental levels of ANG-2, sFlt-1, and leptin and increased sTIE-2 and sVEGFR-2 levels at delivery. It is unclear whether placental vasomodulation is being promoted or inhibited in our cohort, as diminished sFlt-1 and increased sVEGFR-2 support angiogenesis and vasodilation [58], while decreased ANG-2 and increased sTIE-2 suggest an anti-angiogenic environment [59]. On the other hand, in our study, the placental levels of classic inflammatory cytokines at delivery were not altered, reflecting a picture from past events. It is plausible that cytokines at delivery are already downregulated, which is in line with previous observations that showed inflammatory cytokines associated with episodes of P. vivax malaria during pregnancy that are lost at delivery [60]. Yet, a decrease in the plasma levels of IL-12 and C5a at delivery, particularly upon infections that occur during the first and third trimesters, was observed by us. IL-12 has been previously associated with P. vivax infections during pregnancy [60], and C5a is considered a hypothetical key driver of PM pathogenesis, being associated with placental insufficiency and SGA babies, and correlated negatively and positively with ANG-1 and ANG-2, respectively [61,62]. Moreover, our study shows that both placental C5a and ANG-2 are diminished at delivery when women are infected for the first time during the first trimester. These observations oppose a previous hypothesis assuming that P. vivax infections would promote the same augmented levels of C5a as those observed during P. falciparum MiP [61]. Altogether, our data suggest that inflammatory responses acting against P. vivax infections in the first trimester may contribute to chronic placental damage and insufficiency. This might occur through angiogenic and vascular remodeling mechanisms, which, when altered, promote abnormal placental/fetal development and poor pregnancy outcomes, despite early pharmacological intervention with antimalarials.

Currently, the main strategy to control/treat Plasmodium spp. infections relies on antimalarials and vaccines, channeling the research resources to develop these approaches. Regarding P. falciparum MiP, vaccines such as PAMVAC have been tested to induce immunity acquisition to VAR2CSA. However, there are no known orthologs in P. vivax that the immune system can directly target to protect women during pregnancy. Alternatively, the combat of disease symptoms in pregnant women can be done through the natural generation of antibodies against blood-stage parasite antigens. Antibodies against P. vivax MSP119 were shown to be associated with protection against symptomatic malaria in non-pregnant individuals [63,64] and those against P. vivax VIR, and Duffy-binding protein (PvDBP) were shown protective of birth weight reduction in pregnant women infected with P. vivax [65,66], suggesting that targeting non-pregnancy related antigens may also improve gestational outcomes. Nonetheless, in our study, we did not observe a statistically significant association between antibodies against P. vivax MSP119 and improved pregnancy outcomes, which is in line with previous observations showing that P. vivax MSP119 antibodies are associated with infection but do not correlate with protection from maternal anemia or reduced birth weight [66].

P. vivax MiP is still poorly understood, representing major challenges for pregnant women and their concepts. Here, we have shown that P. vivax MiP impairs newborn’s development and leads to placental tissue damage. We conclude that P. vivax infections during the first trimester are associated with poor pregnancy outcomes and that the presence of antibodies against P. vivax MSP119 does not protect pregnant women from adverse gestational outcomes. Abnormal pregnancies in this context might occur due to placental inflammation and dysregulation of local homeostasis, regardless the absence of parasite sequestration and prompt treatment administration given to pregnant women in the endemic areas of the Brazilian Amazon.

(DOCX)

Click here for additional data file.

Characteristics of Anthropometric Measurements of Newborns, according to the gestational trimester in which the first infection occurred.

(DOCX)

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Placental Parameters of Non-infected and P. vivax-infected women, according to the gestational trimester in which the first infection occurred.

(DOCX)

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Angiogenic factors in placental plasma from Non-infected and P. vivax-infected women, according to the gestational trimester in which the first infection occurred.

(DOCX)

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Inflammatory factors in placental plasma from Non-infected and P. vivax-infected women, according to the gestational trimester in which the first infection occurred.

(DOCX)

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Anthropometric characteristics of the newborns, according to maternal previous exposure to P. vivax (PvMSP1).

(DOCX)

Click here for additional data file.

Association between adverse pregnancy outcomes and P. vivax infections according to gravidity.

(DOCX)

Click here for additional data file.

Analysis plan.

(DOCX)

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Study database.

(XLSX)

Click here for additional data file.

11 Dec 2020

Dear Dr. Marinho,

Thank you very much for submitting your manuscript "Adverse pregnancy outcomes are associated with P. vivax malaria in a prospective cohort of women from the Brazilian Amazon" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The authors have studied the impact of P. vivax infection on pregnancy outcome in a large prospective cohort of Brazilian women. Maternal history of infection during the pregnancy, placental histopathology, levels of angiogenic factors and cytokines, and levels of PvMSP1-19 antibodies are related to various measures of pregnancy outcome. The authors conclude that P. vivax infection, particularly during the first trimester, negatively affects pregnancy outcome.

The authors use the terms malaria in pregnancy and placental malaria more or less randomly. In my opinion, placental malaria should be reserved for infections, where a placental disease focus is known or highly likely. Whereas this is generally the case for P. falciparum infection among women living in areas with stable parasite transmission, it remains highly controversial whether selective placental accumulation of P. vivax-infected erythrocytes occurs. As the authors do not provide such evidence, I strongly recommend that the authors use the term malaria in pregnancy (or pregnancy-associated malaria) throughout. This recommendation is supported by the fact that the authors did not find a statistically significant relation between their outcome variables and parity. Such a relation would be strongly expected in the case of bona fide placental malaria.

The study appears well designed and it is nicely presented. My overriding concern is whether the uninfected and infected women can be compared directly, as they are throughout the manuscript. The reason for this concern is Table 1, where it is stated that 44/159 infected women lived in a rural area, whereas only 8/170 of the non-infected did. This difference is very highly significant – but not commented on in the manuscript. It is therefore an open – and critical – question whether it is justified to attribute the differences between the infected and uninfected women (only) to P. vivax infection. While it appears reasonable to suspect that a strongly pro-inflammatory infection such as P. vivax malaria would negatively affect pregnancy, it is also very highly likely that rural residence for many reasons other than P. vivax exposure predisposes for a pro-inflammatory status. The authors should comment on this, and definitely control for this likely confounding factor in their analyses wherever possible (i.e., a multivariate approach). Additional factors reported in Table 1 that might bias the data in the same direction (albeit less pronounced manner) are age, occupation, and educational level.

Reviewer #2: Objectives of this study are well stated. Many important details of the methods, especially of sampling from the participants, are omitted. I expect that previous publications provide this information, but readers of this article should not have to seek out those papers in order to understand the study.

It would be helpful to know (briefly) how the women in this study were recruited – before or during pregnancy, at home or clinic or hospital, and how followed – monthly, active or passive follow-up? What exactly defines a woman ‘with P vivax?’ – detected infection at any time in the pregnancy, or at parturition, or in the placenta, or any of these? In general we need to be told when samples were taken – some can obviously be taken only at or around parturition, but others could be taken at various times during pregnancy: e.g. at what point in the study were blood samples taken from women studied for total IgG antibodies against Plasmodium vivax Merozoite Surface Protein 1 (PvMSP1-19)? (Was evidence being sought for P vivax infection before pregnancy had started, or at some stage in the current pregnancy before enrolment?)

Were tests for Pv taken at first recruitment irrespective of symptoms (e.g. fever); were women asked to attend for any fever; were blood films taken routinely at scheduled visits to the clinic, or only if there were symptoms? Did the ‘endemic surveillance team’ actively seek women with fever, or women in pregnancy, to enrol into the study?

Page 11. When assessed by microscopy, were placental samples ‘blinded’ as to P vivax infection in the mother?

Reviewer #3: - Details on the timing of recruitment and follow up in the study are not adequately described. At what gestational age were women recruited into the study? How many malaria episodes occurred during pregnancy?

- Given that the women were not followed during pregnancy unless they were symptomatic, how can the authors rule out that they did not experience a P. falciparum infection at some point in pregnancy that was asymptomatic?

- Table 1 should include statistical analyses to compare across the different groups.

- How were previous P. vivax infections in the current or prior pregnancies confirmed? (Line 312)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Univariate analyses are used throughout. A multivariate approach is strongly recommended (see preceding section).

Reviewer #2: Many data are presented in detail in this paper, both in text and Figures, so that the important findings are difficult to identify. It would be useful to decide a hierarchy of importance of findings and make sure that these are conveyed prominently.

P14 line 290: “All p values were 2-sided, and interpreted at a significance level of 0.05.” Mention should be made that there are very large numbers of statistical tests in the Tables and figures – will any adjustment for multiple testing be made to the interpretations of ‘significance’?

In Fig 4 please clarify why denominators in column 2 don’t add up to the same total on each line.

Reviewer #3: - In Fig 8, why would newborns have longer length in the uninfected group depending on their PvMSP1 Ab status?

- It seems possible that the PvMSP1 antibodies at enrolment could be due to a recent infection in that pregnancy prior to enrolment, rather than an exposure prior to pregnancy. Consideration of the timing of infection and timing of enrolment are important to interpret these serology data.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Until the data have been re-analysed with inclusion of confounding factors, I cannot confidently evaluate the appropriateness of the authors’ interpretations of their data, or the conclusions they draw.

Reviewer #2: There is frequent reference (or implication) in this paper to a finding being the cause of some later event or difference. For example: p32 lines 557-558: “we have observed an important impact of P. vivax infection on fetal growth…”. In fact association has been observed, but you have not demonstrated a causal link (implied by ‘impact’). The above, for example, would be better written as ‘we have observed a significant association between P vivax infection and impaired fetal growth…’. Several implications or assumptions of causality recur throughout the paper and would be better reworded appropriately (including in the last sentence of Discussion).

Reviewer #3: - The statement on lines 73-75 is not entirely accurate. The study by Bardaji et al (2017), for example, explored some of these interactions between Pv infection in pregnancy and birth outcomes.

- The statement on lines 83-85 should be revised since these associations were observed in specific groups, not generally between infected vs non-infected women, except for preterm birth as shown in Fig 2. The statement should also reflect the data in Figures 3 and 4.

- Line 86. Placental malaria is defined by the presence of parasites or hemozoin in the placenta, not by these histological changes in the placenta. This sentence should be revised accordingly. Only 8 women in the study had placental malaria and the results should be interpreted with some caution.

- I don’t agree with the statement on line 351. The difference is only significant for infections in the second trimester. The results seem to be skewed by the high error bars.

- The strong conclusions from the serology data for PvMSP1-19 are premature. The authors state that they used this marker as a surrogate of past exposure to P. vivax and yet they conclude that these antibodies are associated with protection. Was there a correlation between PvMSP1 Abs and placental outcomes or clinical outcomes? The discussion of pre-existing immunity should be removed or revised substantially.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: One woman in her first pregnancy is a primigravida, more than one are primigravidae. Please correct ‘primigravida’ (and ‘multigravida’) throughout the paper, including in Tables and Figures, where they should be plural nouns. p.21 line 348: 'Pv-infected women experienced more adverse pregnancy outcomes'…Several references are made to ‘more’ or ‘higher’ – when referring to differences between groups; in these situations please add ‘on average’ or ‘mean’ or ‘median’ before the comparative word, and make sure you indicate with whom or against what the comparison is being made. For example P32 line 571 “we observed that primigravida who presented infection in the second trimester had higher parasitemia.” Higher than whose? Than you would expect? Or than multigravidae with 2nd trimester infections? Or than primigravidae with infections in the first trimester? Etc… Please look out for comparatives without a comparator, and clarify accordingly.

Throughout the paper, especially in the Discussion, redundant words are commonly used to start sentences. Try omitting these and see if it makes any difference to the clarity of the text (I don’t think it does): examples:

‘Additionally’, ‘Finally’ (best avoided because you can never say the final word on any subject), ‘Not surprisingly’, ‘Further’, ‘In addition’, ‘Remarkably’, ‘Furthermore’, ‘Moreover’, ‘Controversially’, ‘Thus’.

Reviewer #3: Minor comments

- Line 159. Please state EIR or some other measure of malaria transmission in the study area.

- Specify source of PvMSP1-19 protein used for ELISAs and concentration coated on the plate.

- Table 3. Include the units for the parasitemia

- Minor grammatical errors, as follows:

- Line 61, delete ‘occurring’

- Line 122, rephrase ‘it is detected’

- Line 150, change the term ‘benignity paradigm’

- Line 183, should be ‘first’

- Line 240 should be ‘10th percentile’

- Line 308 should be ‘pregnant women’

- Line 316, change ‘exert’ to ‘may be detrimental’

- Line 377, remove ‘the’

- Line 379, ‘in’ should be ‘on’

- Line 385, Change the word ‘patent’, perhaps to ‘apparent’ or another term

- Line 387, change ‘multigravidity status’ to ‘in multigravida’

- Line 430, should be ‘alterations in several’

- Line 457 should be ‘cytokine’

- Line 460, remove period before bracket

- Line 461, change ‘of’ to ‘in’

- Line 484, please change the term ‘tenuous’ – the meaning here is not clear

- Line 489, change ‘heighten’ to ‘greater’

- Line 509, change to samples collected at enrollment

- Line 540 is repetitive with line 535

- Line 560, ‘of’ should be ‘on’

- Line 561, add a comma after infections

- Line 579, remove ‘the existence of’, line 580 ‘supports’

- Line 602, change ‘though’ to ‘however’

- Line 627 should be ‘protection’

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: The importance of this study is diminished by the profusion of material - more brevity in the introduction and discussion, and a clarification of findings of importance, would make it more accessible to readers. At present the paper would do better for a thesis-Chapter than a journal publication.

Reviewer #3: The authors present the results of a longitudinal study of P. vivax malaria in a population of pregnant women in Brazil. This study focuses exclusively on women with P. vivax infection identified through passive case detection and at delivery compared to non-infected pregnant women. Extensive data are provided on the clinical outcomes, placental histology, newborn characteristics and analyses of various biomarkers. The study reveals that P. vivax infection in pregnancy, particularly during the first trimester, can be detrimental to the health of the newborn, prompting re-consideration of the severity of infections with this species in pregnancy. While the study provides very important data on P. vivax MiP that will contribute significantly to the field, there are a number of limitations and these should be discussed.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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24 Mar 2021

Submitted filename: REPLY_REVIEWERS_COMMENTS_FINAL.docx

Click here for additional data file.

15 Apr 2021

Dear Dr. Marinho,

We are pleased to inform you that your manuscript 'Adverse pregnancy outcomes are associated with P. vivax malaria in a prospective cohort of women from the Brazilian Amazon' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Hans-Peter Fuehrer

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Editor: Write out Plasmodium in title

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Adequate methodology with no remaining concerns.

Reviewer #3: The revisions are acceptable.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results adequately presented and analysed.

Reviewer #3: The revisions are acceptable.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Appropriate conclusions justified by the results obtained.

Reviewer #3: The revisions are acceptable.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: None.

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: All my concerns regarding the earlier version of this manuscript have been alleviated. I congratulate the authors with a nice study that constitutes a valuable addition to the litterature.

Reviewer #3: The authors have substantially revised the manuscript based on their new statistical analyses. All concerns that I raised have been addressed in this new version.

**********

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Reviewer #1: Yes: Lars Hviid

Reviewer #3: No

22 Apr 2021

Dear Dr. Marinho,

We are delighted to inform you that your manuscript, "Adverse pregnancy outcomes are associated with P. vivax malaria in a prospective cohort of women from the Brazilian Amazon," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Table 1

Demographic characteristics of the mothers participating in the study, according to infection and gestational trimester of first infection.

Characteristics	Non-Infected (N = 170)	P. vivax (N = 159)	p-valuea	P. vivax - 1st tri (N = 52)	p-valueb	P. vivax - 2nd tri (N = 54)	p-valuec	P. vivax - 3rd tri (N = 53)	p-valued
Age, years, median (IQR)	23.0 (19.0–28.0)	21.0 (17.0–26.0)	0.002	22.5 (18.0–25.0)	0.12	19.0 (17.0–26.0)	0.002	21.0 (17.0–26.0)	0.01
Gravidity, median (IQR)	2.0 (1.0–3.0)	2.0 (1.0–3.0)	0.38	2.0 (1.0–3.0)	0.13	1.0 (1.0–3.0)	0.31	2.0 (1.0–3.0)	0.11
Education level, n (%)			< 0.0001		0.001		0.004		< 0.0001
No education	0	3 (1.9)		0		0		3 (5.7)
Primary	7 (4.1)	17 (10.7)		4 (7.7)		4 (7.4)		9 (17.0)
≥ Secondary	163 (95.9)	139 (87.4)		48 (92.3)		50 (92.6)		41 (77.3)
Occupation, n (%) e			0.001		0.18		0.03		< 0.0001
Farmer	3 (1.8)	9 (5.7)		4 (7.9)		1 (1.8)		4 (7.5)
Housewife	77 (45.3)	86 (54.1)		22 (43.1)		30 (55.6)		34 (64.2)
Student	36 (21.2)	41 (25.8)		12 (23.5)		16 (29.6)		13 (24.5)
Other occupation	54 (31.7)	22 (13.8)		13 (25.5)		7 (13.0)		2 (3.8)
Rural residence, n (%)	8 (4.7)	44 (27.7)	< 0.0001	12 (23.1)	< 0.0001	16 (29.6)	< 0.0001	16 (30.2)	< 0.0001

Abbreviations: N, total number of individuals; tri, trimester; NA, not applicable. Results are presented as median and interquartile range (IQR) or total number of events (n) and percentage (%). Statistical tests were applied according to the type of variable (Mann-Whitney, Chi-square or Kruskal-Wallis with Dunn’s corrections).

a Differences between Non-Infected and P. vivax group.

b Differences between Non-Infected and P. vivax infection in the 1st trimester.

c Differences between Non-Infected and P. vivax infection in the 2nd trimester.

d Differences between Non-Infected and P. vivax infection in the 3rd trimester.

e Occupation was recorded in 158 P. vivax-infected pregnant.

Table 2

Clinical characteristics of the mothers participating in the study, according to infection and gestational trimester of first infection.

Characteristics	Non-Infected (N = 170)	P. vivax (N = 159)	p-valuea	P. vivax - 1st tri (N = 52)	p-valueb	P. vivax - 2nd tri (N = 54)	p-valuec	P. vivax - 3rd tri (N = 53)	p-valued
Clinical outcomes
Gestational age at delivery, weeks, median (IQR) e	40.0 (39.0–40.0)	39.0 (38.0–40.0)	0.09	39.0 (37.0–40.0)	0.006	39.0 (39.0–40.0)	0.25	40.0 (38.0–40.0)	0.33
Maternal weight gain, Kg, median (IQR) f	13.5 (10.0–16.8)	10.8 (8.0–14.0)	< 0.0001	10.0 (7.0–12.0)	0.004	11.0 (7.5–15.0)	0.10	10.8 (8.0–15.0)	0.26
Hematocrit during pregnancy, % g	36.6 (34.5–38.6)	34.8 (32.4–37.5)	0.008	34.9 (32.7–37.8)	0.37	34.5 (32.4–36.9)	0.11	34.9 (32.4–37.5)	0.24
Hematocrit at delivery, % h	36.2 (33.6–39.1)	35.4 (32.4–37.6)	0.70	36.1 (33.0–39.1)	0.93	35.3 (32.8–37.3)	1.00	34.2 (30.7–37.4)	0.33
Hemoglobin during pregnancy, g/dL, median (IQR) i	12.2 (11.6–12.8)	11.7 (10.8–12.3)	0.16	12.0 (10.9–12.4)	0.67	11.5 (10.7–12.1)	0.54	11.6 (10.8–12.6)	0.96
Hemoglobin at delivery, g/dL, median (IQR) j	11.8 (11.2–12.8)	11.6 (10.7–12.5)	0.64	12.1 (10.8–12.6)	0.97	11.6 (11.0–12.3)	1.00	11.3 (10.1–12.3)	0.32
Anemia, n (%) k	24 (14.1)	32 (20.1)	0.15	10 (19.2)	0.38	7 (13.0)	1.00	15 (28.3)	0.02
Antenatal care visits, median (IQR) l	8.0 (6.0–10.0)	6.0 (5.0–8.0)	< 0.0001	6.0 (4.0–9.0)	0.07	6.0 (5.0–8.0)	0.004	6.0 (5.0–8.0)	0.003
Placental weight, g, median (IQR) m	577.4 (508.3–658.9)	541.8 (482.7–613.3)	0.12	521.4 (455.0–605.3)	0.06	554.6 (500.9–641.5)	0.89	541.4 (497.8–614.5)	1.00
Gestational outcomes, n (%)n
Abortion	1 (0.6)	6 (3.8)	0.06	6 (11.5)	0.001	0	1.00	NA	-
Stillbirth	2 (1.2)	1 (0.6)	1.00	NA	-	0	1.00	1 (1.9)	0.56
Preterm birth	6 (3.5)	17 (10.7)	0.02	10 (19.2)	0.001	3 (5.6)	0.45	4 (7.6)	0.25
Low birth weight	9 (5.3)	14 (9.3)	0.20	8 (17.4)	0.01	4 (7.4)	0.52	2 (4.0)	1.00
Term low birth weight	4 (2.4)	8 (5.0)	0.25	4 (7.7)	0.09	3 (5.6)	0.36	1 (1.9)	1.00
Small for gestational age	16 (9.6)	15 (10.1)	1.00	6 (13.3)	0.42	7 (13.0)	0.45	2 (4.0)	0.26
Malaria history, n (%) o
Previous episodes before current gestation	92 (54.1)	139 (87.4)	< 0.0001	47 (90.4)	< 0.0001	47 (87.0)	< 0.0001	45 (86.5)	< 0.0001
Previous episodes in other gestations	7 (7.8)	25 (28.4)	0.0004	7 (21.2)	0.05	7 (26.9)	0.01	11 (36.7)	0.0004

Abbreviations: N, total number of individuals; tri, trimester; Kg, kilograms; g, grams; NA, not applicable. Results are presented as median and interquartile range (IQR) or total number of events (n) and percentage (%). Statistical tests were applied according to the type of variable (Mann-Whitney, Chi-square or Kruskal-Wallis with Dunn’s corrections or Multiple linear regression, adjusted for maternal age, gravidity, residence, education, and occupation).

a Differences between Non-Infected and P. vivax group.

b Differences between Non-Infected and P. vivax infection in the 1st trimester.

c Differences between Non-Infected and P. vivax infection in the 2nd trimester.

d Differences between Non-Infected and P. vivax infection in the 3rd trimester.

e Gestational age was recorded in 168 non-infected and 157 P. vivax infected pregnant women.

f Maternal weight gain was recorded in 163 non-infected and 126 P. vivax infected pregnant women. It was determined as the final weight minus the initial weight during pregnancy.

g Hematocrit during pregnancy was recorded in 152 non-infected and 136 P. vivax infected pregnant women.

h Hematocrit at delivery was recorded in 116 non-infected and 101 P. vivax infected pregnant women.

i Hemoglobin during pregnancy was recorded in 157 non-infected and 136 P. vivax infected pregnant women.

j Hemoglobin at delivery was recorded in 116 non-infected and 100 P. vivax infected pregnant women.

k Defined as hemoglobin <11 g/dL.

l Antenatal care visits were recorded in 164 non-infected and 139 P. vivax infected pregnant women.

m Placental weight was recorded in 155 non-infected and 128 P. vivax infected pregnant women.

n Definition of gestational outcomes: Abortion–birth < 22nd week of gestation; Stillbirth–fetal death between the 22nd week of gestation and delivery; Preterm birth–birth < 37th week of gestation; Low birth weight–birth weight < 2500 g; Term low birth weight–birth weight < 2500 g from 37th week of gestation; Small for gestational age–birth weight for sex-specific gestational age < 10th centile.

o Malaria before current gestation (in all life) and Malaria in previous pregnancy (in other previous pregnancies) were recorded in 158 P. vivax infected pregnant women.

Table 3

Characteristics of the infection during pregnancy.

Characteristics	P. vivax (N = 159)	P. vivax - 1st tri (N = 52)	P. vivax - 2nd tri (N = 54)	P. vivax - 3rd tri (N = 53)
Gestational age at 1st infection, weeks, median (IQR)	21 (11.0–30.0)	9.0 (7.0–11.0)	20.5 (16.0–24.0)	34.0 (30.0–37.0)
Number of infections, n (%)
One infection	70 (44.0)	15 (28.8)	19 (35.2)	36 (67.9)
Two infections	41 (25.8)	14 (26.9)	14 (25.9)	13 (24.5)
Three or more infections	48 (30.2)	23 (44.3)	21 (38.9)	4 (7.6)
Parasitemia at 1st infection, median (IQR) a	997.6 (152.5–3943.0)	976.1 (226.8–4546.0)	1019.0 (78.9–3943.0)	977.6 (157.4–3409.1)
Parasitemia 2nd or more infection, median (IQR)	1052.6 (111.2–4253.0)	846.8 (83.6–3951.0)	1132.9 (153.8–3887.0)	794.1 (113.0–6647.0)
Placental malaria, n (%) b
No	117 (91.4)	34 (91.9)	40 (90.9)	43 (91.5)
Active Acute	4 (3.1)	0	2 (4.6)	2 (4.3)
Active Chronic	0	0	0	0
Past	7 (5.5)	3 (8.1)	2 (4.5)	2 (4.2)
Hemozoin, n (%) c
No	119 (93.7)	34 (94.4)	42 (95.5)	43 (91.5)
Mild	6 (4.7)	0	2 (4.5)	4 (8.5)
Moderate	2 (1.6)	2 (5.6)	0	0
Severe	0	0	0	0

Abbreviations: N, total number of individuals; tri, trimester. Results are presented as median and interquartile range (IQR) or total number of events (n) and percentage (%).

a Parasitemia at first infection was recorded in 110 P. vivax infected pregnant women. Values presented in 103 DNA copies obtained by PET-PCR quantification, after nucleotide extraction from 200 μL of blood cell concentrate.

b Placental malaria was assessed in 128 P. vivax infected pregnant women.

c Hemozoin was assessed in 127 P. vivax infected pregnant women. Mild: focal presence in small amounts; moderate: small spots or larger deposits in many locations; severe: large amount presented widely in the tissue sections.