Despite that over 90 million pregnancies are at risk of Plasmodium vivax infection annually, little is known about the epidemiology and impact of the infection in pregnancy.
Methodology and principal findings
We undertook a health facility-based prospective observational study in pregnant women from Guatemala (GT), Colombia (CO), Brazil (BR), India (IN) and Papua New Guinea PNG). Malaria and anemia were determined during pregnancy and fetal outcomes assessed at delivery. A total of 9388 women were enrolled at antennal care (ANC), of whom 53% (4957) were followed until delivery. Prevalence of P. vivax monoinfection in maternal blood at delivery was 0.4% (20/4461) by microscopy [GT 0.1%, CO 0.5%, BR 0.1%, IN 0.2%, PNG 1.2%] and 7% (104/1488) by PCR. P. falciparum monoinfection was found in 0.5% (22/4463) of women by microscopy [GT 0%, CO 0.5%, BR 0%, IN 0%, PNG 2%]. P. vivax infection was observed in 0.4% (14/3725) of placentas examined by microscopy and in 3.7% (19/508) by PCR. P. vivax in newborn blood was detected in 0.02% (1/4302) of samples examined by microscopy [in cord blood; 0.05% (2/4040) by microscopy, and 2.6% (13/497) by PCR]. Clinical P. vivax infection was associated with increased risk of maternal anemia (Odds Ratio-OR, 5.48, [95% CI 1.83–16.41]; p = 0.009), while submicroscopic vivax infection was not associated with increased risk of moderate-severe anemia (Hb<8g/dL) (OR, 1.16, [95% CI 0.52–2.59]; p = 0.717), or low birth weight (<2500g) (OR, 0.52, [95% CI, 0.23–1.16]; p = 0.110).
In this multicenter study, the prevalence of P. vivax infection in pregnancy by microscopy was overall low across all endemic study sites; however, molecular methods revealed a significant number of submicroscopic infections. Clinical vivax infection in pregnancy was associated with maternal anemia, which may be deleterious for infant’s health. These results may help to guide maternal health programs in settings where vivax malaria is endemic; they also highlight the need of addressing a vulnerable population such as pregnant women while embracing malaria elimination in endemic countries.
More than 90 million pregnancies are exposed to P. vivax infection every year. While it is well known that pregnant women have an increased risk of P. falciparum infection and disease, much less is known on the epidemiology and the impact of P. vivax in pregnancy. A health-facility based observational study was conducted in pregnant women living in five vivax endemic countries aimed to determine the burden of the infection in pregnancy and its impact on the mother and the newborn health. We found that the prevalence of P. vivax malaria in pregnant women attending the routine antenatal clinic visits was overall low across all sites, however submicroscopic infections were unexpectedly high in some areas. Pregnant women with clinical malaria experienced an increased risk of anemia, which may have a deleterious impact on infant health. These findings may be useful for guiding maternal health programs in vivax endemic settings, as well as for malaria elimination activities.
Citation: Bardají A, Martínez-Espinosa FE, Arévalo-Herrera M, Padilla N, Kochar S, Ome-Kaius M, et al. (2017) Burden and impact of Plasmodium vivax in pregnancy: A multi-centre prospective observational study. PLoS Negl Trop Dis 11(6): e0005606. https://doi.org/10.1371/journal.pntd.0005606
Editor: Photini Sinnis, Johns Hopkins Bloomberg School of Public Health, UNITED STATES
Received: November 24, 2016; Accepted: April 27, 2017; Published: June 12, 2017
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: This information will only be available after acceptance.
Funding: The PregVax collaborative project was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number PREGVAX FP7-HEALTH-201588. It also received co-funding from the Malaria in Pregnancy Consortium (MiPc) through a grant from the Bill & Melinda Gates Foundation [Grant No. 46099], and from the Ministry of Science and Innovation of Spain. Ivo Mueller was supported by a National Health and Medical Research Council (NHMRC) Senior Research Fellowship, Myriam Arevalo-Herrera was supported by the Centro Latino Americano de Investigación en Malaria (CLAIM) sponsored by NIAID/ICEMR (U19AI089702), and Azucena Bardají was supported by the Government of Spain, Ramon y Cajal Fellowship [Grant No. RYC-2013-14512]. The study was coordinated by Fundació Clínic per a la Recerca Biomèdica (FCRB) – ISGlobal/Barcelona Centre for International Health Research (CRESIB) in Spain, which provided project and data management and logistic support, and co-coordinated by the Centers for Disease Control and Prevention (CDC) in Atlanta, USA. ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
More than a third of the world′s population is at risk of Plasmodium vivax infection, 90% of whom live in the Asia and the Pacific regions, while the rest live in the American (6%) and African (3%) regions. [1–3] In comparison with P. falciparum, P. vivax infection has received little attention mostly because it has been considered a relatively benign disease. [3, 4] However, in recent years due to the increasing evidence of the severity of P. vivax infection and the recognition of its economic impact in endemic countries, a greater interest has been devoted to this neglected and complex species. [3, 5] This renewed interest is also enhanced by the recognition that with intensified control efforts P. vivax has become the predominant malaria species and the major challenge to malaria elimination outside Africa.
It is well recognized that pregnant women have an increased risk of P. falciparum infection and disease, and its effects on maternal and infant health have been well documented in sub-Saharan Africa.  Less is known about the burden of P. vivax in pregnancy and its impact on maternal and infant health. In 2007, it was estimated that the number of pregnancies at risk of P. vivax was at least 92 million globally.  Some studies on the effect of P. vivax infection in pregnancy have suggested that the infection is associated with maternal anemia, miscarriage, congenital malaria and preterm delivery, as well as with severe disease. [8–17] These studies were mostly done in Asia, and with some exceptions,[12, 18–20] were based on limited numbers or case reports, thus providing an incomplete picture of the overall burden and impact of P. vivax in pregnancy. In particular, there were limited data from Latin America.
A comprehensive description of the burden and impact of P. vivax infection in pregnancy across different malaria transmission settings is needed to guide control strategies. We present here the results of a prospective observational study in pregnant women exposed to vivax malaria that aimed to determine the burden of P. vivax malaria and its impact on pregnancy outcomes. The study followed a multicenter approach including different epidemiological settings broadly representing of P. vivax endemic areas; one Asian country, one country from the Pacific region and three countries from the Americas. The objectives of the study were to estimate the prevalence of P. vivax infection at different time points overall and for each of the sites, and the incidence of P. vivax malaria during pregnancy. It also aimed to assess the clinical impact of P. vivax infection during pregnancy on maternal health and fetal outcomes.
The study protocol was reviewed and approved by the national and/or local ethics review boards in each of the study sites, the Institutional Review Board (IRB) of the Centers for Disease Control and Prevention (CDC), and the Hospital Clinic of Barcelona Ethics Review Committee. The study was conducted in accordance with the Good Clinical Practice Guidelines, the Declaration of Helsinki, and local rules and regulations of each partner country.
Study design and study areas
This was a health facility-based prospective observational study of pregnant women attending routine antenatal care (ANC) clinics undertaken between June 2008 and October 2011 in five P. vivax endemic countries—Colombia (CO), Guatemala (GT), Brazil (BR), India (IN) and Papua New Guinea (PNG)–of different malaria endemicity characteristics (Table 1). The historic levels of malaria transmission varied across study sites from hypoendemic in Guatemala, India and Colombia, mesoendemic in Brazil, to hyperendemic in PNG. P. vivax was the predominant species in all sites except for PNG where P. vivax co-existed with P. falciparum (50%) and other species (P. malariae and P. ovale, 5%).
Unselected pregnant women of any age, gestational age, and parity, attending the ANC clinic at each study site, independently of parasitological or disease status, were invited to participate, and after signing a written informed consent were recruited into the study. There were four study visits: recruitment visit, coinciding with an ANC visit; two subsequent scheduled ANC visits one month apart; and at delivery. Project personnel were trained on study procedures that were standardized across all study sites. At each study visit and regardless of the presence of symptoms suggestive of malaria, a capillary blood sample was collected to prepare two thick and thin blood smears and two filter papers (Whatmann 3MM) to determine Plasmodium parasitemia. From the same samples the hemoglobin (Hb) concentration was measured at enrolment, at scheduled ANC study visits, at delivery and at any other time the woman was suspected to have malaria. In addition, demographic, obstetric and clinical information were recorded on standardized questionnaires. Pregnant women were encouraged to deliver at the study health facility. In case of home delivery, they were advised to come to the study health facility within the first week of giving birth.
A passive surveillance system to identify study women presenting with clinical malaria was set up at each study health facility. If the women reported any signs and/or symptoms suggestive of clinical malaria, a capillary blood sample was collected to prepare two thick and thin blood smears and two filter papers to determine Plasmodium parasitemia, and for determination of Hb concentration.
At delivery, the pregnancy outcome and the clinical information on the mother and the neonate were collected. Placental blood from all women delivering at the health facility was collected onto filter paper for PCR molecular analysis, and two impression smears were stained with Giemsa and read following standard procedures for parasitemia determination.  For the preparation of the impression smears a 2.5x2.5 cm3 sample from the placenta, that should include the full thickness of the tissue from the maternal to the fetal side, was cut and put in contact with the slide after being dried with a piece of filter paper. In a randomly selected subsample of women (10%) a placental biopsy was collected, following a similar procedure (2.5x2.5 cm3 sample) to that for the preparation of the impression smear. The biopsy was kept at 4°C in 50 mL of 10% neutral buffer formalin, processed for histological examination, and stained with haematoxylin and eosin as previously described.  The histological examination of placental biopsies for malaria infection was performed by local pathologists trained for the purposes of the study (S1 File). Cord blood samples were collected after birth for parasitemia determination. As soon as the umbilical cord was clamped, cut, and separated from the newborn study trained staff collected 5mL cord blood sample taken from the cord with a syringe and needle. Cord blood was used for preparation of two thick and thin blood smears to determine Plasmodium parasitemia, and two filter papers to perform PCR molecular analysis. Newborn samples were collected by heel prick after medical assessment was complete and within the first 12 hours of life also for parasitemia determination.
In GT, BR and PNG gestational age was assessed by the Ballard’s method for deliveries that occurred at the health facility within the first 72 hours after birth . In CO and IN gestational age was assessed by ultrasound assessment performed at enrolment. All newborns were weighed on a digital scale, accurate to the nearest gram, within the first 2 hours of life and examined for any clinical abnormalities. For deliveries occurred at home, the study personnel obtained information on the pregnancy outcome through home visits. All women and newborns with malaria infection or anemia were treated according to the national guidelines in each country. Strategies for malaria control in pregnancy differed across study sites. While these relied on active detection of infection with microscopy at each ANC visit in some countries, in other countries passive detection of cases, or weekly prophylaxis with chloroquine (CQ) until delivery were carried out (Table 1).
At enrolment a subsample of 1500 women (300 per study site) were randomly selected for the determination of the prevalence of P. vivax and P. falciparum infection by PCR methods. Likewise, a subsample of 1500 women (300 per study site) was randomly selected at delivery for the same purpose. The 1500 women from whom samples were obtained at delivery were different from those selected at enrolment. A total of 500 placental samples (100 per site) and 500 cord blood samples (100 per site) were also analysed following same methodology. A simple random sampling method was used for the selection of women in each country, and for each time point and compartment. The sample size for prevalence by PCR was agreed among study investigators according to preliminary results of the first 100 selected samples in each site that showed a prevalence of P. vivax monoinfection of 7.5% by PCR, and on the availability of resources for molecular analyses in the study. PCR assays were not performed on newborn blood samples.
To estimate the impact of submicroscopic P. vivax and P. falciparum infections on maternal anemia and low birth weight (LBW), a nested case-control study was conducted. All countries contributed to this pooled analysis. A definition for moderate-severe anemia as Hb less than 8g/dL was agreed for the purpose of the analysis. All moderate-severe anemia cases (n = 342), and LBW cases (<2500g) (n = 327) existing across countries for which a blood smear and a filter paper were available, were included in the case control analysis. A total of 414 controls to anemia cases, and a total of 410 controls to LBW cases, were randomly selected. Logistic regression models used to evaluate the impact of P.vivax submicroscopic infections on anemia and LBW were adjusted by site and P. falciparum infections. Similarly, for the evaluation of the impact of P. falciparum submicrocopic infections, models were adjusted by site and P. vivax infections.
Giemsa-stained thick and thin blood slides were read onsite in all countries following WHO standard quality-controlled procedures to establish parasite presence and density of Plasmodium asexual stages.  Two independent expert malaria microscopists read all slides and results were registered in two separate forms. Discrepant results (positive vs. negative) were resolved by a third reading done by a different microscopist. A blood slide was declared as negative only when no parasites were found after reading 200 fields. Results were expressed in parasites/μL after counting the number of parasites per 500 white blood cells or reaching 500 parasites; counting was normalized using estimated leukocyte counts of 8000/μL. External validation of a blood slides subsample (100 slides per country) was done at the Hospital Clinic and at the Hospital Sant Joan de Deu, in Barcelona, Spain. Hb was measured by Coulter Counter (except in PNG where it was done by Hemocue, HemoCue, Ltd, Angelhom, Sweden; accuracy of 0.1 g/dL) using 50–100 μL collected in a 0.5mL EDTA tube (microtainer).
Molecular detection of Plasmodium species in samples from CO, GT, BR and PNG was performed by Real Time PCR at the Istituto Superiore di Sanità (ISS) in Rome, Italy. Samples from India were analysed, due to local regulatory requirements, at the International Center for Genetic Engineering and Biotechnology (ICGEB), in New Delhi using the same protocol as that at ISS but adapted for the sake of instrument sensitivity (3rd step at amplification was 72°C for 25 sec instead of 72°C for 5 sec). DNA was extracted from whole blood-spot filter paper from maternal peripheral blood collected at ANC, at delivery and during passive case detection, and from placental and cord blood using Purelink Genomic DNA Kit (Invitrogen). P. vivax and P. falciparum infections were detected with a LightCycler 480 system (Roche). Species-specific primers and Taqman probes were selected from the sequence of the small 18S rRNA subunit as previously described by Veron et al 2009.  Briefly, pre-incubation was at 95°C for 10 min; amplification at 95°C for 10 sec, 50°C for 20 sec and 72°C for 5 sec for 50 cycles. All reactions were in duplicate in a final volume of 20 μL. The sensitivity of the PCR assay performed at the ISS for detection of P. falciparum infections was between 10–100 times higher in comparison to the microscopy. An external validation of the PCR methods used by the ISS and the ICGEB, for detection of P. vivax and P. falciparum species, was performed by the Malaria in Pregnancy Consortium (MiPc) (http://www.mip-consortium.org) in a subset of 20 samples. Internal validation between ISS and ICGEB was also done.
Data management and statistical analysis
A standardised system for data entry, data management, and statistical analysis was established. All clinical and laboratory data were collected using standardised questionnaires. The data collection and management was performed using the OpenClinica open source software, version 2.0. Copyright OpenClinica LLC and collaborators, Waltham, MA, USA, www.OpenClinica.com. All data were double entered by two independent data clerks at each of the sites. There was a specific URL link to access the data entry software. In PNG data were doubled-entered into form-specific databases (FoxPro 9·0, Microsoft, USA). Validation and cleaning were done using the same software, and statistical analysis was performed using Stata 13 (Stata Corporation, College Station, TX, USA). Differences between proportions were compared using the Pearson′s chi-squared test or Fisher's exact test depending on type of variables. For continuous variables, Student’s T-tests were used to compare the groups. Incidence rates were calculated as the number of new episodes/person-year at risk using the Poisson distribution in the exposed and unexposed groups, with primigravid women as the comparator group. The impact of P. vivax infection on maternal and newborn health was determined through a multicenter-pooled analysis. The case control study for submicroscopic infections with and anemia was analyzed using logistic regression models. We adjusted all regression models for possible operational confounding variables such as country and previous malaria episodes. In the analyses of P. falciparum infections, they were included in the models as being free of P. vivax (see definitions section). Likewise, in the analyses of P. vivax infections, they were included as being free of P. falciparum. Multivariate analyses were performed by a forward-stepwise procedure, using p<0.05 and p>0.10 from the likelihood ratio test, as enter and remove criteria respectively. Results from the estimated models were expressed as OR and 95% CI. Missing values were coded as such and excluded from analysis.
P. vivax microscopic monoinfection was defined as the presence of asexual P. vivax parasites of any density and absence of other Plasmodium species on the blood smear. P. vivax clinical malaria episode was defined as the latter plus any signs or symptoms suggestive of malaria (axillary temperature ≥37.5°C or history of fever in the last 24 hours, headache, arthromyalgias, and/or pallor). P. vivax submicroscopic infection was defined as a PCR that was positive for P. vivax and negative for P. falciparum, with a concomitant blood film negative by microscopy. P. falciparum microscopic monoinfection was defined as the presence of asexual P. falciparum of any density and absence of other Plasmodium species on the blood smear. P. falciparum clinical malaria episode was defined as the latter plus any signs or symptoms suggestive of malaria. P. falciparum submicroscopic infection was defined as a PCR that was positive for P. falciparum and negative for P. vivax, with a concomitant blood film negative by microscopy. The duration of any single malaria episode was estimated as 28 days.
Congenital malaria was defined as presence of asexual Plasmodium parasites of any species in the cord blood or in the newborn′s peripheral blood at delivery, regardless of clinical symptoms or signs in the neonate. Placental infection was classified according to a previously established definition.  Briefly, acute infection was defined as the presence of parasites, with absent or minimal pigment deposition within fibrin or cells within fibrin, chronic infection as the presence of parasites and a significant amount of pigment deposition, and past infection as the presence of pigment with absence of parasites. Prematurity was defined as gestational age < 37 weeks. LBW was defined as birth weight <2500g.
Baseline characteristics of study women
A total of 9388 pregnant women were recruited across all sites (2043 in CO; 2009 in GT; 1657 in BR; 1982 in IN; 1697 in PNG), and 4957 (53%) of them were followed up until delivery (Fig 1). Table 2 the baseline characteristics of the study participants by site (S1 Table). Mean age of study women was 23.6 years [standard deviation (SD) = 5.6], mean gestational age 23 weeks (SD = 8); nearly 40% (3759/9468) of the women were primigravidae. At enrolment, overall mean Hb level was 10.5 g/dL (SD 1.8), and 57% (5201/9105) of women were anemic (Hb<11g/dL). Around 70% of women (6471) were enrolled at their first ANC visit versus at later ANC visit.
* Number recruited at antenatal care per country: 2009 in Guatemala, 2043 in Colombia, 1657 in Brazil, 1982 in India, and 1697 in PNG.
Prevalence of P. vivax mono-infection
The overall prevalence of P. vivax mono-infection detected by microscopy was 0.8% (73/9299) at enrolment and 0.4% (20/4461) at delivery. Of those women who were parasitemic for P. vivax 57.6% (42/73) were symptomatic at enrolment and 20% (4/20) at delivery. The prevalence of P. vivax mono-infection by PCR assessed in a subsample of women was 8.5% (124/1455) at enrolment and 6.9% (104/1488) at delivery, and of those 9.7% (12/124) and 4.8% (5/104) reported symptoms, respectively (Fig 2).
Prevalence of P. falciparum mono-infection
The overall prevalence of P. falciparum mono-infection detected by microscopy was 1.3% (124/9305) at enrolment and 0.5% (22/4463) at delivery. Of those women who were parasitemic for P. falciparum 16.1% (20/124) were symptomatic at enrolment and 27.3% (6/22) at delivery. No P. falciparum infection was detected by microscopy in GT and BR neither at enrolment or delivery, nor in India at delivery. The prevalence of P. falciparum mono-infection by PCR assessed in a subsample of women was 6.9% (80/1157) at enrolment and 2% (24/1191) at delivery, and of those 10% (8/80) and 12.5% (3/24) reported symptoms, respectively (Fig 3).
* Not available data. Technical problems in the performance of the molecular analyses (PCR) for P. falciparum occurred at the reference laboratory in India. No biological specimens remained available after that sufficient to repeat the analyses in an alternative laboratory.
There were no mixed infections (P. vivax plus P. falciparum) detected by microscopy during pregnancy or at delivery in CO, GT, BR and IN. In PNG mixed infections were observed in 9/818 (1.1%) of placental impression smears only. The prevalence of mixed infections detected by PCR was 2.2% (26/1165), 0.3% (4/1191), 0.3% (1/379) at enrolment, delivery, and in cord blood, respectively. No mixed infections were detected in the placental blood.
Incidence of malaria
Data on incidence of malaria is shown in Table 5. The incidence rate of P. vivax infection detected by microscopy was not associated with parity (Incidence Rate Ratio-IRR for multigravidae-women with 4 or more pregnancies-, 1.14 [95% CI 0.70–1.87]; p = 0.619). For P. falciparum the IRR for multigravidae was 0.39, [95% CI 0.25–0.62]; p<0.001). A total of 898 passive case detection visits (528 in PNG, 183 in CO, 75 in BR, 62 in GT and 50 in IN) were registered among study women who reported symptoms or had signs suggestive of malaria infection. Of those, 823 women had a blood smear collected, and in 141 (17%) of them malaria infection was confirmed by microscopy; 68 cases were due to P. vivax, 72 cases were due to P. falciparum, and one case was due to P. malariae. In 85% (117/137) malaria episodes women were found to be anemic (Hb<11g/dL) and 14% (19/137) severely anemic (Hb<7g/dL). Only 8 women with clinical malaria (2 with P. vivax and 6 with P. falciparum infection, and all from CO) required hospital admission (data from admitted women in PNG not available). On average, 2% (86/4227) of study women reported at delivery a history of having had a malaria episode during the current pregnancy, and 2.3% (90/3830) having taken antimalarial drugs during the same pregnancy. See S2 Table for incidence data disaggregated by PCD or ADI, overall and by country.
Plasmodium parasite densities
Overall, parasite densities in pregnant women throughout the study were low in most cases. The geometric mean density (GMD) for P. vivax infections on maternal peripheral blood at any time during the study was 1275 parasites/μL (95% CI 906.5–1643.5); being 385 parasites/μL (95% CI 226.9–543.1) and 2396 parasites/μL (95% CI 1579.5–3212.5) the GMDs for P. vivax asymptomatic and symptomatic infections, respectively. The GMD for P. falciparum infections on maternal peripheral blood was 1678 parasites/μL (95% CI 1268.6–2087.4); being 918 parasites/μL (95% CI 681.7–1154.3) and 4087 parasites/μL (95% CI 2362.1–5811.8) the GMDs for P. falciparum asymptomatic and symptomatic infections, respectively.
Parasite densities on placental blood ranged for P. vivax from 0.6 parasites/μL in the positive case detected in Brazil to 25.5 parasites/μL arithmetic mean (SD 31.8) in PNG. For P. falciparum densities in the placenta ranged from 21.4 parasites/μL arithmetic mean (SD 25.5) in CO up to 6255 parasites/μL arithmetic mean (SD 7845) in placentas from PNG.
Overall, 10.2% (505/4943) of babies born to women enrolled in the study had LBW (range 2.8% in CO– 16.5% in PNG), and the prevalence of pre-term birth was 12.6% (455/3590) across countries (Table 6). At delivery, on average 53.4% (2285/4278) of study women were anemic (range 17.8% in BR– 88.3% in IN), and 4.5% of them (193/4278) were severely anemic. Most of the documented deliveries (96%) took place at a health facility (hospital/health center) attended by skilled staff. During the study follow up there were three maternal deaths and their clinical diagnoses at the time of death were recorded; one in CO due to P. falciparum malaria, one in PNG due to post-partum hemorrhage with no evidence of malaria at any time during pregnancy, and one in IN associated with P. vivax malaria and anemia.
Impact of P. vivax and P. falciparum malaria on maternal and newborn outcomes
At enrolment women with P. vivax or P. falciparum clinical malaria were more than 5-times as likely to be anemic compared to non-infected women (adjusted OR, 5.48, [95% CI 1.82–16.41]; p = 0.009), and adjusted OR, 5.57, [95% CI 1.22–25.34]; p = 0.012, respectively) (Table 7). In contrast, those women with clinical malaria at delivery, whether infected with P. vivax (p = 0.754) or P. falciparum (p = 0.122), did not show an increased risk of anemia at the same time point compared to non-infected or infected-asymptomatic women. No association was found between P. vivax or P. falciparum clinical malaria at enrolment (p = 0.100, and p = 0.760, respectively) or at delivery (p = 0.120, and p = 0.060, respectively) and an increased risk of LBW.
Women with P. vivax malaria at delivery detected by microscopy did not have an increased risk of anemia (adjusted OR, 0.62, [95% CI 0.22–1.81]; p = 0.384) or LBW (adjusted OR, 0.90, [95% CI 0.20–4.10]; p = 0.895), compared to uninfected women. However, those women with P. falciparum malaria showed an increased risk for both anemia (adjusted OR, 4.07, [95% CI 1.93–8.58]; p = 0.0002) and LBW (adjusted OR, 4.28, [95% CI 1.75–10.44]; p = 0.0014). In the nested case-control sub-analysis, women with P. vivax submicroscopic infection at delivery did not show an increased risk of moderate-severe anemia (adjusted OR, 1.16, [95% CI 0.52–2.59]; p = 0.717) or LBW (adjusted OR, 0.52, [95% CI 0.23–1.16]; p = 0.110) compared to uninfected women (negative by microscopy and by PCR). Likewise, P. falciparum submicroscopic infections at delivery were not associated with moderate-severe anemia (adjusted OR, 2.93, [95% CI 0.24–36.23]; p = 0.402) or LBW (adjusted OR, 1.97, [95% CI 0.46–8.46]; p = 0.359).
Malaria infection in the placenta (whether detected by impression smear or PCR) was not associated with an increased risk of LBW (adjusted OR, 0.67, [95% CI 0.07–6.18]; p = 0.725 for P. vivax, and adjusted OR, 8.61, [95% CI 0.79–94.09]; p = 0.077, for P. falciparum) or anemia (adjusted OR, 2.04, [95% CI 0.70–5.92]; p = 0.189 for P. vivax, and adjusted OR, 0.77, [95% CI 0.16–3.67]; p = 0.746, for P. falciparum). In contrast, a statistically significant association was found for placental infections detected by histology with LBW (adjusted OR, 2.36, [95% CI 1.48–3.76]; p = <0.001) and anemia (adjusted OR, 1.66, [95% CI 1.09–2.51]; p = 0.017). Histology did not allow distinguishing between species, but of the infected placentas according to histology a higher proportion were positive for P. falciparum (11.4%) compared to P. vivax (4.8%) by impression smear or PCR.
This is, to our knowledge, the first multicenter prospective observational study on the burden of P. vivax in pregnant women from different malaria endemic areas. Overall, the proportion of pregnant women attending the ANC who were infected with vivax malaria detected by microscopy was low across the different endemic settings included in the study. However, P. vivax infection in pregnancy was associated with negative consequences for maternal health in those that were symptomatic, which may have a negative impact also for the health of the neonate. Importantly, most of the P. vivax infections in pregnancy were undetected with microscopy, the routinely used screening method across study sites.
The majority of the information on P. vivax in pregnancy comes from studies carried out in Asia.  There is much less information from Latin America, where P. vivax is responsible for 65 to 100% of malaria cases; a community-based study conducted in the Brazilian Amazon in 2002 showed that 1.9% of the pregnant women were infected with P. vivax, and a study from Colombia conducted between 2005 and 2011 reported that 11.6% of pregnant women were infected at delivery with P. vivax.  However, in the current study, the frequency of P. vivax infection detected by microscopy was below 2% in all countries and at all time points. Recent efforts in the deployment of malaria preventive measures, as it was the case of Guatemala in this study, together with the introduction of new more effective antimalarial drugs for case management, may have played a role in the decrease in the prevalence of infections observed in this study below microcopy-detection levels, but still allowing a considerable proportion of the population harboring sub-microscopic parasitemia levels.
Mixed infections were rare, except in PNG, which is different from the situation in Africa where most non-falciparum infections in pregnancy are mixed infections.  Histology was the method showing the highest sensitivity for detection of malaria infection in the placenta compared to microscopy and PCR, and the presence of malaria pigment more frequent than of parasites in the infected placentas.
In most study countries, optical microscopy is used for case detection of malaria infection, and for routine screening of malaria at ANC visits (where the latter is recommended). However, the fact that PCR methods detected significantly more infections than microscopy suggest that many infections were undetected and that microscopy is not a sensitive method for routine screening of malaria, particularly in P. vivax endemic areas due to the generally low parasite densities. 
The available data on the impact of P. vivax in pregnancy are scarce, and they have rarely been analysed disaggregated from the effects of P. falciparum infection. Thus, understanding the main effects of P vivax monoinfection in pregnancy was a main objective of this study. It was observed that during pregnancy pregnant women with clinical P. vivax monoinfection had a 5-fold higher risk of having anemia (Hb<11g/dL) compared to non-infected women. In malaria endemic areas, there is evidence on the impact of maternal anemia on maternal mortality and morbidity, as well as on neonatal and infant health and survival. [6, 8] In contrast, asymptomatic P. vivax infection was not associated with an increased risk of maternal anemia of any degree. The evidence from this study also indicated that it is rare for P. vivax, and for P. falciparum to establish an infection in the newborn.
The two maternal deaths reported in Colombia and India were associated with P. falciparum and P. vivax infections, respectively, underpinning that malaria in pregnancy can be a contributor to maternal mortality in these low transmission settings. These results claim the maternal and neonatal health programs in P. vivax endemic areas to recognize that vivax malaria is associated with negative consequences for the health of pregnant women and consequently for that of the neonate. Clinical and epidemiologic surveillance systems need to be integrated into maternal and reproductive health programs to detect malaria cases in pregnant women for prompt and effective case management.
The results of this study can also contribute to evaluate whether current strategies for malaria control during pregnancy adopted in areas where P. vivax is predominant are adequate, and if they are aligned with most recent evidence. In this study, performance of passive surveillance was better in detecting Plasmodium infections (17% positivity by blood smear among women with malaria symptoms) compared to active detection at routine visits (2.1% and 0.94% of women infected at enrolment and at delivery visits, respectively). As malaria transmission diminishes, the approach to the detection of infected pregnant women may change from active detection at each ANC visit to passive detection to capture the infections (febrile, symptomatic cases) that are mostly associated with poor pregnancy outcomes. As malaria burden decreases the sustainability of an active approach might be questionable, as shown in some countries in the Americas, nor cost-effective. Surveillance is of particular relevance for those countries that are in the pathway towards malaria elimination and will need to shift from measuring reductions in morbidity and mortality, to detecting infections and measuring transmission.
P. vivax is becoming the dominant species in low transmission areas.  Accurate data on P. vivax burden depending on the level of malaria endemicity is crucial to designing effective malaria control and elimination policies. However, P. vivax poses unique epidemiological challenges. In contrast to P. falciparum, P. vivax can arise from dormant hypnozoites leading to relapses weeks or months after, that can be indistinguishable from a primary infection.  The prevention of these relapses is considered to be the most important strategy for interrupting P. vivax transmission.  In pregnant women the use of primaquine, a member of the 8-aminoquinoline family and the only drug currently licensed for radical cure of P. vivax malaria, is contraindicated for safety reasons, putting them at a higher risk of clinical relapses. The role that pregnant women may play as asymptomatic parasite reservoirs needs to be further investigated, as this might represent a limitation for effective malaria control and elimination.
There are limitations to this study. The study was able to capture data and collect samples from pregnant women at the time of delivery for more than half of study women, but this still represented a low follow-up rate. This limitation is not uncommon in health facility based studies from settings where most women deliver at home, as it was the case for most of study sites. Comparison of baseline characteristics between complete and lost to follow-up women showed that differences were related primarily to characteristics of the site or site-related, Although statistical analyses were adjusted by site, these dissimilarities need to be carefully considered as this may imply that the findings might not be generalized to the women who do not deliver at the health facility.
The evaluation of gestational age ultrasound assessment was not available at those facilities, and the Ballard′s method was used in some of the study countries. This might represent a limitation, as the latter is not as reliable method as ultrasound is. PCR assays for detection of malaria infection were performed at a different laboratory in India due to regulatory issues that did not allow analyzing samples at the study central laboratory at the ISS in Rome. Although the same standardized protocols were used in both laboratories, and that internal and external validations were performed in a subset of samples, this might have introduced some potential bias by pooling of data obtained from different laboratories. In addition, much of the data shown in this study is reported as aggregate to provide an estimate of the wider geographic situation. However, the interpretation of these data has to take into consideration the differences in some of the estimates across sites. Also, some women (2.4%) reported having received antimalarial treatment during pregnancy. Though analyses of the impact of microscopic and sub-microscopic infection were adjusted by previous malaria episodes and study site, it needs to be taken into consideration as this may have impacted the results. Lastly, the low proportion of microscopically detected infections and that PCR determination of malaria infection could not be performed in all samples did represent a limitation for the pooled analysis on the impact of microscopic and submicroscopic infections. The lack of association of submicroscopic and placental infections (except those detected by histology) with anemia and LBW needs to be viewed with caution as it could be related to a sample size issue, and thus not being enough placental infections to detect a clinically important difference.
This multicenter study in representative areas endemic for vivax malaria shows that the prevalence of P. vivax malaria was overall low across study sites. However, the prevalence of submicroscopic infections was significant in some areas, which may have implications for detection of malaria infections with the currently used diagnostic tools. Clinical episodes due to P. vivax infection were associated with maternal anemia, which may be also detrimental for infant’s health. These findings can be relevant for guiding maternal health programs in settings where vivax malaria is endemic, as well as for monitoring, evaluation and surveillance activities in countries that are in the pathway towards malaria elimination.
S1 File. Further details of the methods used in the histological evaluation of placentas.
S1 Table. Comparison of baseline characteristics between lost to follow-up women and women with complete follow-up.
S2 Table. Incidence of malaria detected through passive case detection (PCD) and through active detection of infection (ADI), overall and by species and country.
We are grateful to the women who participated in the study across all sites. We also thank all local study teams and health staff of the Fray Bartolomé de las Casas (FBC) Municipality Hospital (Guatemala), the Fundaçâo de Medicina Tropical Dr. Héctor Vieira Dourado (FMT-HVD) Outpatient Clinic and the Gebes Medeiros Health Post in Manaus (Brazil), the Hospital de San José of Tierralta (Colombia), the Sardar Patel Medical College associated Hospital in Bikaner (India), and the Alexishafen Health Centre, the Yagaum Health Center and the Modilon General Hospital in Madang (PNG). We thank John Jairo Aponte and Caterina Guinovart from ISGlobal-CRESIB for providing technical support in data management and statistical plan, and preparation of protocol. We would also like to thank Julie Gutman from CDC-Malaria Branch for assistance with managing in coordination issues between the MiPc, CDC and ISGlobal-CRESIB. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
Full list of members/authors of the PregVax Study Group: Flor E. Martínez-Espinosa (FEME), Camila Bôtto-Menezes (CBM) and Luiz Carlos de Lima Ferreira (LF) for the Gerência de Malária, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), Manaus, Brazil. Myriam Arévalo-Herrera (MAH) for the Centro Internacional de Vacunas (CIV) / Facultad de Salud, Universidad del Valle, Cali, Colombia. Pedro Rovetto (PR) for the Department of Pathology, Universidad del Valle, Cali, Colombia. Norma Padilla (NP), María Eugenia Castellanos (MEC) and Renata Mendizábal de Cabrera (RM) for the Centro de Estudios en Salud, Universidad del Valle de Guatemala (CES-UVG), Guatemala, Guatemala. Werner Raúl de Leon Pérez (WL) for the Department of Pathology, Hospital de San Juan de Dios, Guatemala, Guatemala. Swati Kochar (SK), Sanjay K. Kochar (SKK), Dev Raaj Arya (DRA) and Dhanpat K. Kochar (DKK) for the Sardar Patel Medical College (SPMC), Bikaner, India. Maria Ome-Kaius (MOK), Regina A. Wangnapi (RW), Inoni Betuela (IB), Sarah Hanieh (SH), Heather Huaupe (HH), Peter Siba (PS), Leanne J. Robinson (LR), Alexandra J. Umbers (AU), Paula Samol (PS) and Holger Unger (HU) for the PNG Institute of Medical Research (PNG IMR), Madang, Papua New Guinea. Ivo Mueller (IM) for The Walter and Eliza Hall Institute (WEHI), Parkville, Australia. Stephen Rogerson (SR) for the University of Melbourne, Melbourne, Australia. Chetan Chitnis (CC) and Dhiraj Hans (DH) for the International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. Michela Menegon (MM) and Carlo Severini (CS) for the Istituto Superiore di Sanità (ISS), Rome, Italy. Azucena Bardají (AB), Hernando del Portillo (HDP), Carlota Dobaño (CD), Alfredo Mayor (AM), Jaume Ordi (JO), Mireia Piqueras (MP), Sergi Sanz (SS) and Clara Menéndez (CM) for the ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic—Universitat de Barcelona, Barcelona, Spain. Mats Wahlgren (MW) for the Karolinska Institute (KI), Stockholm, Sweden. Laurence Slutsker (LS) and Meghna Desai (MD) for the Centers for Disease Control and Prevention (CDC), Center for Global Health, Division of Parasitic Diseases and Malaria Atlanta, GA, USA.
- Conceptualization: CM MD LS HdP JO CD AM AB.
- Data curation: CBM MEC NP MAH MOK IM SK SKK MP AB SS.
- Formal analysis: AB SS MP.
- Funding acquisition: CM HdP CD JO MD LS AB.
- Investigation: FEME CBM NP MEC MAH SK AB SKK DKK MOK IB SR IM MW MD MM CS CC DH LF PR RM WL DRA RW SH HU LR AU PS HH.
- Methodology: AB CM HdP JO CD AM MD LS MW.
- Project administration: AB MP CM FEME NP MAH SK MOK IM CD AM HdP JO MW.
- Resources: CBM MEC MAH MOK IB RW SH HH LJR AJU PS HH SR SK CD AM MP AB SS IM.
- Software: SS AB.
- Supervision: AB CM MD FEME MAH NP IM MOK PS SR DKK SKK SK.
- Validation: CM AB MP AM SS.
- Visualization: AB.
- Writing – original draft: AB CM.
- Writing – review & editing: AB FEME MAH NP SK MOK CBM MEC DKK SKK IB IM SR CC DH MM CS HdP CD AM JO MP SS MW LS MD CM.
- 1. Guerra CA, Snow RW, Hay SI: Mapping the global extent of malaria in 2005. Trends Parasitol 2006, 22(8):353–358. pmid:16798089
- 2. Hay SI, Guerra CA, Tatem AJ, Noor AM, Snow RW: The global distribution and population at risk of malaria: past, present, and future. Lancet Infect Dis 2004, 4(6):327–336. pmid:15172341
- 3. Price RN, Tjitra E, Guerra CA, Yeung S, White NJ, Anstey NM: Vivax malaria: neglected and not benign. Am J Trop Med Hyg 2007, 77(6 Suppl):79–87. pmid:18165478
- 4. Baird JK: Neglect of Plasmodium vivax malaria. Trends Parasitol 2007, 23(11):533–539. pmid:17933585
- 5. Mueller I, Galinski MR, Baird JK, Carlton JM, Kochar DK, Alonso PL, del Portillo HA: Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite. Lancet Infect Dis 2009, 9(9):555–566. pmid:19695492
- 6. Desai M, ter Kuile FO, Nosten F, McGready R, Asamoa K, Brabin B, Newman RD: Epidemiology and burden of malaria in pregnancy. Lancet Infect Dis 2007, 7(2):93–104. pmid:17251080
- 7. Dellicour S, Tatem AJ, Guerra CA, Snow RW, ter Kuile FO: Quantifying the number of pregnancies at risk of malaria in 2007: a demographic study. PLoS Med 2010, 7(1):e1000221. pmid:20126256
- 8. Rodriguez-Morales AJ, Sanchez E, Vargas M, Piccolo C, Colina R, Arria M, Franco-Paredes C: Pregnancy outcomes associated with Plasmodium vivax malaria in northeastern Venezuela. Am J Trop Med Hyg 2006, 74(5):755–757. pmid:16687675
- 9. Kochar DK, Saxena V, Singh N, Kochar SK, Kumar SV, Das A: Plasmodium vivax malaria. Emerg Infect Dis 2005, 11(1):132–134. pmid:15705338
- 10. Nosten F, McGready R, Simpson JA, Thwai KL, Balkan S, Cho T, Hkirijaroen L, Looareesuwan S, White NJ: Effects of Plasmodium vivax malaria in pregnancy. Lancet 1999, 354(9178):546–549. pmid:10470698
- 11. Martinez-Espinosa FE, Daniel-Ribeiro CT, Alecrim WD: Malaria during pregnancy in a reference centre from the Brazilian Amazon: unexpected increase in the frequency of Plasmodium falciparum infections. Mem Inst Oswaldo Cruz 2004, 99(1):19–21. pmid:15057341
- 12. Singh N, Shukla MM, Sharma VP: Epidemiology of malaria in pregnancy in central India. Bull World Health Organ 1999, 77(7):567–572. pmid:10444880
- 13. Sholapurkar SL, Gupta AN, Mahajan RC: Clinical course of malaria in pregnancy—a prospective controlled study from India. Trans R Soc Trop Med Hyg 1988, 82(3):376–379. pmid:3068848
- 14. Carvalho BO, Matsuda JS, Luz SL, Martinez-Espinosa FE, Leite JA, Franzin F, Orlandi PP, Gregoracci GB, Lacerda MV, Nogueira PA et al: Gestational malaria associated to Plasmodium vivax and Plasmodium falciparum placental mixed-infection followed by foetal loss: a case report from an unstable transmission area in Brazil. Malar J 2011, 10:178. pmid:21708032
- 15. Castellanos ME, Bardaji A, Menegon M, Mayor A, Desai M, Severini C, Menendez C, Padilla N: Plasmodium vivax congenital malaria in an area of very low endemicity in Guatemala: implications for clinical and epidemiological surveillance in a malaria elimination context. Malar J 2012, 11:411. pmid:23217209
- 16. Singh N, Singh MP, Wylie BJ, Hussain M, Kojo YA, Shekhar C, Sabin L, Desai M, Udhayakumar V, Hamer DH: Malaria prevalence among pregnant women in two districts with differing endemicity in Chhattisgarh, India. Malar J 2012, 11:274. pmid:22882903
- 17. Singh N, Saxena A, Shrivastava R: Placental Plasmodium vivax infection and congenital malaria in central India. Ann Trop Med Parasitol 2003, 97(8):875–878. pmid:14754501
- 18. McGready R, Lee SJ, Wiladphaingern J, Ashley EA, Rijken MJ, Boel M, Simpson JA, Paw MK, Pimanpanarak M, Mu O et al: Adverse effects of falciparum and vivax malaria and the safety of antimalarial treatment in early pregnancy: a population-based study. Lancet Infect Dis 2012, 12(5):388–396. pmid:22169409
- 19. Rijken MJ, McGready R, Boel ME, Poespoprodjo R, Singh N, Syafruddin D, Rogerson S, Nosten F: Malaria in pregnancy in the Asia-Pacific region. Lancet Infect Dis 2012, 12(1):75–88. pmid:22192132
- 20. Poespoprodjo JR, Fobia W, Kenangalem E, Lampah DA, Warikar N, Seal A, McGready R, Sugiarto P, Tjitra E, Anstey NM et al: Adverse pregnancy outcomes in an area where multidrug-resistant plasmodium vivax and Plasmodium falciparum infections are endemic. Clin Infect Dis 2008, 46(9):1374–1381. pmid:18419439
- 21. Rogerson SJ, Mkundika P, Kanjala MK: Diagnosis of Plasmodium falciparum malaria at delivery: comparison of blood film preparation methods and of blood films with histology. J Clin Microbiol 2003, 41(4):1370–1374. pmid:12682116
- 22. Ismail MR, Ordi J, Menendez C, Ventura PJ, Aponte JJ, Kahigwa E, Hirt R, Cardesa A, Alonso PL: Placental pathology in malaria: a histological, immunohistochemical, and quantitative study. Hum Pathol 2000, 31(1):85–93. pmid:10665918
- 23. Ballard JL, Novak KK, Driver M. A simplified score for assessment of fetal maturation of newly born infants. J Pediatr 1979; 95:769–74. pmid:490248
- 24. Shute PG, Maryon M: AN IMPROVED TECHNIQUE FOR STAINING MALARIA PARASITES WITH GIEMSA STAIN. Arch Roum Pathol Exp Microbiol 1963, 22:887–894. pmid:14166976
- 25. Veron V, Simon S, Carme B: Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples. Exp Parasitol 2009, 121(4):346–351. pmid:19124021
- 26. WHO/HTM/GMP: WHO, World Malaria Report 2014. Geneva. In.; 2014.
- 27. Martinez-Espinosa FE: Malaria e gravidez na regiâo Amazônica: prevalencia de infecçâo em mulheres de idade do Municipio de Coari, 2001 [tese doutorado]. 2003.
- 28. Arango EM, Samuel R, Agudelo OM, Carmona-Fonseca J, Maestre A, Yanow SK: Molecular detection of malaria at delivery reveals a high frequency of submicroscopic infections and associated placental damage in pregnant women from northwest Colombia. Am J Trop Med Hyg 2013, 89(1):178–183. pmid:23716408
- 29. Saute F, Menendez C, Mayor A, Aponte J, Gomez-Olive X, Dgedge M, Alonso P: Malaria in pregnancy in rural Mozambique: the role of parity, submicroscopic and multiple Plasmodium falciparum infections. Trop Med Int Health 2002, 7(1):19–28. pmid:11851951
- 30. Parekh FK, Hernandez JN, Krogstad DJ, Casapia WM, Branch OH: Prevalence and risk of Plasmodium falciparum and P. vivax malaria among pregnant women living in the hypoendemic communities of the Peruvian Amazon. Am J Trop Med Hyg 2007, 77(3):451–457. pmid:17827359
- 31. Menendez C, Romagosa C, Ismail MR, Carrilho C, Saute F, Osman N, Machungo F, Bardaji A, Quinto L, Mayor A et al: An autopsy study of maternal mortality in Mozambique: the contribution of infectious diseases. PLoS Med 2008, 5(2):e44. pmid:18288887
- 32. White NJ: Determinants of relapse periodicity in Plasmodium vivax malaria. Malar J 2011, 10:297. pmid:21989376
- 33. Douglas NM, Nosten F, Ashley EA, Phaiphun L, van Vugt M, Singhasivanon P, White NJ, Price RN: Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and effect of antimalarial kinetics. Clin Infect Dis 2011, 52(5):612–620. pmid:21292666
- 34. WHO: Making Pregnancy Safer: The Critical Role of the Skilled Attendant, A joint statement by WHO, ICM and FIGO. Geneva: World Health Organization. [http://whqlibdoc.who.int/publications/2004/9241591692.pdf]. In.; 2004.