Malaria transmission in Senegal is highly stratified, from low in the dry north to moderately high in the moist south. In northern Senegal, along the Senegal River Valley and in the Ferlo semi-desert region, annual incidence is less than five cases per 1000 inhabitants. Many nomadic pastoralists have permanent dwellings in the Ferlo Desert and Senegal River Valley, but spend dry season in the south with their herds, returning north when the rains start, leading to a concern that this population could contribute to ongoing transmission in the north.A modified snowball sampling survey was conducted at six sites in northern Senegal to determine the malaria prevention and treatment seeking practices and parasite prevalence among nomadic pastoralists in the Senegal River Valley and the Ferlo Desert. Nomadic pastoralists aged 6 months and older were surveyed during September and October 2014, and data regarding demographics, access to care and preventive measures were collected. Parasite infection was detected using rapid diagnostic tests (RDTs), microscopy (thin and thick smears) and polymerase chain reaction (PCR). Molecular barcodes were determined by high resolution melting (HRM).Of 1800 participants, 61% were male. Sixty-four percent had at least one bed net in the household, and 53% reported using a net the night before. Only 29% had received a net from a mass distribution campaign. Of the 8% (142) who reported having had fever in the last month, 55% sought care, 20% of whom received a diagnostic test, one-third of which (n = 5) were reported to be positive. Parasite prevalence was 0.44% by thick smear and 0.50% by PCR. None of the molecular barcodes identified among the nomadic pastoralists had been previously identified in Senegal.While access to and utilization of malaria control interventions among nomadic pastoralists was lower than the general population, parasite prevalence was lower than expected and sheds doubt on the perception that they are a source of ongoing transmission in the north. The National Malaria Control Program is making efforts to improve access to malaria prevention and case management for nomadic populations.The online version of this article (doi:10.1186/s12936-017-2055-x) contains supplementary material, which is available to authorized users. Senegal is a malaria-endemic country in the Sahel zone of West Africa. Malaria control interventions have been scaled up dramatically in the last decade, resulting in a substantial decrease in parasite prevalence, and possibly contributing to a significant drop in all cause under five mortality . MalariaWhile reasons for such travel include business, school, and visiting family and/or friends, a group of travelers of concern to the National Malaria Control Program (NMCP) were nomadic pastoralists. Senegal, as with many countries in the Sahel, hosts a large number of nomadic pastoralists, primarily of the Fulani ethnic group, who move with their flocks to find pasture. In Senegal, these pastoralists may have more permanent dwellings in the north, but spend dry seasons in the comparatively moist south, returning north during rainy season. A large number of pastoralists move with their livestock from southern high malaria transmission zones toward the Ferlo and the Senegal River Valley every rainy season . AnecdotThe Senegal National Malaria Control Program has set an ambitious target to reach pre-elimination status by 2018 . InterveDue to their mobility, nomadic populations are very challenging to study. They are likely to be missed during census activities, and if included, are not associated with a defined geographic location where they may be reliably found. While large family groups may travel together, the population composition of any given locality where they may congregate fluctuates as family groups arrive and leave. In Senegal, when they arrive in the north during the rains, they may stay with family members who are part of the non-nomadic population. Hence, development of a probability sampling frame is extremely difficult, if not impossible. Respondent driven sampling (RDS) was developed to study hidden populations , and whi2 with a population of 511,432 inhabitants [This study was carried out from September to October 2014 in the Ferlo Desert and the Senegal River Valley. The Ferlo Desert is a vast Sahelian plain just south of the Senegal River in northeastern Senegal of over 75,000 kmabitants , 16. Theabitants . In the Nomadic pastoralists aged 6 months and older, who were returning or had just returned to the north from the south during rainy season were recruited . Study participants reported no acute illness, had axillary temperature < 37.5 °C, and provided written informed consent before being enrolled in the study. For children, informed consent was provided by their parents or legal guardian. Potential participants were excluded if they lived permanently in the district, were not members of the nomadic community, had an acute febrile illness, or had previously taken part in the study. Given the nature of the sampling design, with participants being recruited and coming to the interviewers to participate, it was important not to become seen as care seeking opportunity, so as not to falsely inflate the parasite prevalence among an already stigmatized population. Therefore, potential participants with acute febrile illness were excluded from the study, and offered malaria testing, with treatment if the result was positive.A snowball sampling survey, using a modified respondent driven sampling methodology was conducted, as described above. Chief nurses at the health posts that served each community and were familiar with them selected leaders in the nomadic community to serve as initial seeds that would recruit other participants. Each seed was asked to recruit three other individuals, including one participant under the age of 15 years, so as to include children in the sample. While this strategy made the sample more representative of the target population, it made it impossible to use classic RDS methodology and analysis, which depends on each participant having an independent social network from which they can recruit. We did not discourage recruitment of household members. Interviews were held at a designated central location chosen to facilitate participation. Each subsequently recruited participant received the same instructions, with recruitment continuing until the sample size of 1800 was reached (300 in each of the six sites). To calculate sample size, a target proportion of 50% for key variables such as care seeking and net use, with a confidence level of 95% and a confidence interval of , and a design effect of 3 were used, giving 300 participants per site, a total of 1800 participants.After informed consent, a questionnaire was administered for collecting socio-demographic data , knowledge of malaria prevention and treatment, ownership and use of insecticide treated nets, care seeking practices for illness within the last month, recent travel history, and plans for future travel   and two drops for the thin and thick smear. For molecular analysis, three to four drops of blood were spotted on Whatman 903 protein saver card  filter paper and dried at room temperature for further extraction of Plasmodium genus was performed using the following primers rPLUf 5′-TTA AAA TTG TTG CAG TTA AAA CG-3′ and rPLUr 5′-CCT GTT GTT GCC TTA AAC TTC-3′. The second round PCR was performed with primers specific to four Plasmodium species:DNA was extracted from whole blood with the QIAamp DNA blood mini kit . The ssrRNA gene was amplified as previously described , 19 withPCR reactions were performed in a final volume of 20 μl: GoTaq (Promega Green Master Mix 2X) 6 μl, forward primer 10 pmol/μl (1 μl), reverse primer 10 pmol/μl (1 μl), DNA 2 μl, and 10 μl of distilled water. The PCR program was as follows: initial denaturation at 94 °C for 4 min, then 35 cycles at 94 °C for 30 s, 55 °C for 1 min  or 58 °C for 1 min , 72 °C for 1 min, final extension at 72 °C for 4 min, and 4 °C continuous at the end.Samples were analyzed to determine if they were monogenomic and genetically distinct using a 24 single nucleotide polymorphism (SNP) molecular barcode, as described elsewhere , 21.Excel  was used for data entry, and statistical analysis was performed using STATA version IC 12  and Epi-Info 7 . Given that this was a non-probability sample , proportions were calculated for categorical data, and means for continuous data, but confidence intervals were not presented.The project was discussed with community members, health post chief nurses, and district medical officers prior to the study, and community consent was obtained from community leaders. Written informed consent was obtained from all participants or guardians prior to their enrollment. The study protocol received a non-research determination from CDC Atlanta  and was approved by the ethical review committee of the Ministry of Health, Senegal . Participation was strictly voluntary, and patients who were ineligible due to fever were offered diagnosis and treatment free of charge, as indicated by national policy.In the six study districts in the Senegal River Valley and the Ferlo, 1854 participants were screened and 1800 included (300 participants per district). Fifty-four were excluded from the study: 48 were ineligible (45 febrile and three permanent residents) and six refused to participate. Fever was the only cause of ineligibility in five of the six sites; in Ranerou, three permanent residents presented for screening. The six cases of refusal were attributed to fear among children (Table The mean age of participants was 25.1 years (range 1–85 years); 61% were male. Children under 15 years constituted approximately one-third (32%) of participants, in accordance with the recruitment instructions, 13% were aged 15–20 years, and 56% were older than 20 years. The majority (88%) belonged to the Pulaar ethnic group, also known as Peul, Fula, or Fulani, a primarily pastoralist ethnic group found across the Sahel. The Serer ethnic group represented 12%, and was found exclusively in the Linguere district. The majority was illiterate; only 4% could read and understand French, and 2% could read and understand their language in Arabic script.Among nomadic pastoralists, 73% had heard of malaria. While 44% said they had received health messages (from any source) in the last 3 months, 29% had received health messages specifically related to malaria. However, of those who responded (n = 1563), 87% knew the importance of sleeping under a bednet; 38% knew that pregnant women should take sulfadoxine-pyrimethamine (SP) during pregnancy to prevent malaria; and 45% knew about the importance of seeking care for malaria in a timely manner. The most common source of messages was radio, which was a means of receiving health messages for 93% of those who reported receiving them.Though 64% reported having at least one bednet in their household, only 29% reported having received a net from a mass distribution campaign. Overall, 38% reported having received a net at a health facility, with 52% saying they paid for it and 48% saying they received it for free. Of the 33% who had purchased a net somewhere other than a health facility, 81% purchased it at a weekly market. Of those who responded to a question regarding the most convenient place to obtain a bednet (n = 1676), 45% said a health facility, followed by 25% at a weekly market, while 14% wanted the opportunity to purchase a net from home. Of all participants, 53% reported having used a bednet the night before the survey, 26% reported using them every night, and 61% used one some nights. The primary reason for not using a bednet every night (40%) was lack of access to one, followed by lack of mosquitoes (39%).Among the 1792 who responded, 8% (n = 142) reported having suffered a febrile illness in the previous month. Of these, 27% reported taking no action, while 55% sought care either at a formal health facility or from a community health worker . While 23% had self-medicated at home , only 6% sought the services of a traditional practitioner . Of those that did not seek care and answered the question as to why they did not (n = 56), 36% said it was too far, 25% lacked sufficient funds, 16% said it was unnecessary, and 16% did not have time. Of all those who sought care, 20% reported that a malaria diagnostic test had been done. Of those who knew a diagnostic test had been performed (n = 17), five reported that the result was positive, three of whom reported being treated with an ACT , and two of whom were unsure what they received.While 404 (23%) reported having suffered an illness of any kind in the last month, 329 responded as to what they did for that illness: 42% went to a formal health facility, 7% went to a community level provider, 7% went to a pharmacy or informal drug vendor, 26% sought no treatment, and 13% took medicines they had at home. The primary means of transportation for care seeking was a horse or donkey cart (75%), followed by walking (15%), and while the trip took an hour or less for 48%, it took two or more hours for 21%. When asked about challenges in seeking care, 67% stated that it was too far. While there were few complaints as to the treatment received once there, 95% stated that they would favor training a member of their group to perform case management using RDT and ACT.Most participants were recently arrived, with 15% having arrived in the last 2 weeks, 55% having arrived within the last 4 weeks, 76% within the last 8 weeks, and 96% within the last 12 weeks. Planned duration in the district was short, with 61% planning to stay 3 months or less, and 93% planning to stay 4 months or less. Only 38% said they followed the same migration patterns every year. While participants were asked from where they had come and to where they were going, the inability to the connect the location names given with known place names made analyzing this information impossible.Plasmodium falciparum was the only malaria species detected. All that were positive by thick smear were also positive by RDT and PCR, and all that were positive by PCR were also positive by RDT. There were two RDT positives that were negative by PCR and thick smear. Malaria positive individuals (by PCR) were exclusively found in the districts of Kanel (n = 3), Ranerou (n = 3), and Pété (n = 3)  Table . One hadOf those who were ineligible to participate due to febrile illness, 7/45 (16%) were positive by RDT: one in Pété (1/7), four in Kanel (4/12), and two in Ranerou (2/2), the same districts in which there were asymptomatic positives. In the Linguere, Dagana, and Podor sites, none of the 24 febrile potential participants had positive RDTs. If these patients are included in the overall total, parasite prevalence by RDT would be 0.98%; blood slide and PCR results are not available as these individuals did not undergo informed consent, but were offered case management for fever.P. falciparum isolates gave analyzable barcodes , which showed an incomplete partial similarity (22/24) to an isolate from Thies in 2012 , and prompt and effective case management was low. Contrary to expectations, the prevalence of malaria was very low. None of the barcodes had been previously identified among a library of over 1500, giving no information as to parasite origin.Senegal conducts a continuous Demographic and Health Survey (cDHS), with annual results and a period of data collection covering January to October, largely during low transmission season. For the year of this survey (2014), the cDHS reported that 74% of households owned at least one ITN, 46% slept under a bednet the previous night (40% under an ITN), and treatment was sought for 54% of children under 5 years who had fever in the previous 2 weeks . We founThe cDHS 2014 found parasite prevalence among children under 5 years in northern Senegal was 0.1%, compared to 5.9% in southern Senegal, the region in which nomadic pastoralists spend the dry season . Given tWhile in this sample, participants with malaria infection were only found among Pulaar (Fulani), lower susceptibility to malaria among Fulani than among sympatric ethnic groups was noted first in 1995 in Burkina Faso  and subsThe study team noted that the nomadic pastoralists among whom the study was conducted were exceptionally welcoming, understanding of the importance of malaria control, and open to collaboration; the primary difficulties in conducting the study were the mobility of the population and very difficult access by vehicle, particularly after rains. This group is often stigmatized and socially marginalized, an attitude which may hamper attempts at outreach and malaria control . ConstruThere are number of possible limitations in a study of this nature. While we tried to compensate for some of the innate limitations of snowball sampling by limiting the number of participants one person could recruit, the sample is not a probability sample and may not be representative. Some information collected routinely by household surveys (bednet treatment status) was not possible to obtain. We did not collect information on household composition, and were unable to determine if two respondents were members of the same household. We were also not able to use the information given to map travel routes, and thus were unable to learn about risk for origin and destination points. We were also unable to directly compare parasite prevalence with that of sympatric populations, either in the north or the south. The study was not designed to investigate symptomatic cases of malaria at the health posts; this may be of interest as a follow up study. We did not recruit and enroll febrile patients, not wanting the study to be seen as an opportunity to seek care, and thus leading to enrollment of a large number of febrile participants. Due to this decision, we did not collect filter paper on and were not able to perform molecular analysis for the seven individuals who were ineligible due to acute febrile illness and who had a positive RDT. While none of the barcodes matched others collected in higher transmission zones of Senegal, these areas have not been comprehensively mapped, and it may be possible, as further molecular studies are completed, to understand the genomic background more comprehensively.While malaria prevalence among this population appears to be low, this may also indicate low levels of exposure. Serologic studies have not been done on this population to determine antibody responses and levels, and while susceptibility to uncomplicated malaria may be reduced among the Fulani, relative risk of severe malaria has not been studied, and malaria epidemics have been documented in other nomadic pastoralist groups . EffortsThis study examined malaria prevention, care and treatment seeking behavior, and parasitemia among asymptomatic nomadic pastoralists in northern Senegal. Low access to health messaging and insecticide treated nets, and challenges with care seeking underlines the need to design strategies to reach this group as Senegal moves toward elimination. The low prevalence of malaria infection among this group suggests that they may be at risk of severe disease. Further investigation is needed to clarify to what extent they may contribute to ongoing transmission in northern Senegal, and to understand the burden of symptomatic malaria among this population.


Chitinase-3-like 1 (CHI3L1) is a glycoprotein elevated in paediatric severe malaria, and an emerging urinary biomarker of acute kidney injury (AKI). Based on the hypothesis that elevated CHI3L1 levels in malaria are associated with disease severity, the relationship between plasma CHI3L1 levels, AKI and mortality was investigated in Ugandan children enrolled in a clinical trial evaluating inhaled nitric oxide (iNO) as an adjunctive therapy for severe malaria.Plasma CHI3L1 levels were measured daily for 4 days in children admitted to hospital with severe malaria and at day 14 follow up. AKI was defined using the Kidney Disease: Improving Global Outcomes consensus criteria. This is a secondary analysis of a randomized double-blind placebo-controlled trial of iNO versus placebo as an adjunctive therapy for severe malaria. Inclusion criteria were: age 1–10 years, and selected criteria for severe malaria. Exclusion criteria included suspected bacterial meningitis, known chronic illness including renal disease, haemoglobinopathy, or severe malnutrition. iNO was administered by non-rebreather mask for up to 72 h at 80 ppm.CHI3L1 was elevated in patients with AKI and remained higher over hospitalization (p < 0.0001). Admission CHI3L1 levels were elevated in children who died. By multivariable analysis logCHI3L1 levels were associated with increased risk of in-hospital death  and all-cause 6 month mortality  following correction for iNO and AKI. Treatment with iNO was associated with delayed CHI3L1 recovery with a daily decline of 34% in the placebo group versus 29% in the iNO group (p = 0.012). CHI3L1 levels correlated with markers of inflammation , endothelial activation  and intravascular haemolysis .CHI3L1 is a novel biomarker of malaria-associated AKI and an independent risk factor for mortality that is associated with well-established pathways of severe malaria pathogenesis including inflammation, endothelial activation, and haemolysis.Trial registration Clinicaltrials.gov, NCT01255215. Registered December 7th 2010The online version of this article (10.1186/s12936-018-2225-5) contains supplementary material, which is available to authorized users. Acute kidney injury (AKI) is an important complication in severe malaria that is associated with increased mortality in both paediatric –6 and adl-arginine in adults with severe malaria improved endothelial recovery [Decreased bioavailable nitric oxide is a common feature in both children and adults with severe malaria, and clinical trials to increase nitric oxide (NO) have been conducted –14. Althrecovery , and iNOrecovery .Despite early reports that inhaled NO (iNO) was associated with increased splanchnic and renal blood flow , a meta-Plasmodium falciparum-infected erythrocytes in vitro induced CHI3L1 transcription and secretion of CHI3L1 protein [Chi3l1 did not affect inflammatory responses or outcome [Chitinase-3-like 1 protein (CHI3L1) is a 39 kDa secreted glycoprotein produced by a variety of cell types in response to inflammation, including activated macrophages, neutrophils, and fibroblasts. CHI3L1 is highly expressed in healthy kidney tissue  and is f protein . While C outcome .In this secondary analysis of an iNO intervention trial, CHI3L1 was investigated as a biomarker of morbidity and mortality in paediatric severe malaria, the longitudinal kinetics of CHI3L1 were explored in children hospitalized with severe malaria, and the impact of iNO therapy on CHI3L1 normalization was evaluated.P. falciparum by RDT , selected severe malaria criteria , and plasma sample available for CHI3L1 testing. Exclusion criteria included: known chronic illness, severe malnutrition, known haemoglobinopathy, prior treatment with quinine in the emergency department, suspicion of acute bacterial meningitis. Study gas was delivered continuously by non-rebreather mask for up to 72 h, as previously described [The study was conducted between 2011 and 2013 at the Jinja Regional Referral Hospital in Jinja, Uganda. All children were treated with intravenous artesunate followed by oral artemisinin-based combination therapy. Inclusion criteria were age 1–10 years, escribed .2 and using the Schwartz equation to back calculate creatinine based on the child’s height. The children with AKI were further classified by stage: stage 1 , stage 2 , and stage 3  [Creatinine and BUN were assessed at the point of care using i-STAT CHEM8+ or Crea cartridges . Creatinine measured by i-STAT is calibrated traceable to the isotype dilution mass spectrometry reference measurement and is free of interference from haemoglobin, bilirubin, and glucose . Estimat1.73 m2) . The inc1.73 m2) .2 EDTA anticoagulated plasma samples were collected daily from children during hospitalization (day 1–4), and at follow up (day 14) and stored at − 80 °C until testing. Cystatin C and CHI3L1 were measured by ELISA  with the investigator blinded to treatment group and outcome [K outcome . LDH act outcome , endothe outcome , and inf outcome , 28. Bio outcome , 28.Data were analysed using Stata/SE v14.0, GraphPadPrism v7.03, and R . For accR  and lme410(CHI3L1) levels at admission as the independent variable  remained independently associated with an increase in Cystatin C following correction for creatinine , but not BUN. The relationship between log10(CHI3L1) and BUN was not significant when adjusting for either creatinine or Cystatin C (p > 0.05), and the relationship between log10(CHI3L1) and creatinine was not significant when adjusting for Cystatin C or BUN (p > 0.05).Levels of CHI3L1 were available for 159 children at admission  was associated with a 4.10-fold increased risk of in-hospital death  following adjustment for treatment arm and AKI status using a log binomial model. Further, a one unit increase in log10(CHI3L1) was associated with a 3.21-fold increased risk of death by 6 months  following adjustment for treatment arm and AKI status.CHI3L1 levels were compared at admission with subsequent in-hospital mortality and all-cause 6 month mortality. CHI3L1 levels in the highest quartile were significantly associated with death (Table 10(CHI3L1) was associated with a 2.13-fold increased risk of death , and a 3.11-fold increased risk of death  if all children < 5 years of age LTFU survived. This analysis was then expanded to all children in the cohort (including children > 5 years of age that were not followed). Assuming all children LTFU or > 5 years of age died, log10(CHI3L1) was associated with a 1.99-fold increased risk of death by 6 months . Assuming all children LTFU or > 5 years of age survived, log10(CHI3L1) was associated with a 3.10-fold increased risk of death by 6 months .Survival data were not available at 6 months follow up for 16 children lost to follow up (LTFU) and 19 children > 5 years of age and not followed to 6 months (according to the study protocol). To evaluate whether the association between CHI3L1 and 6 month mortality was robust to various assumptions a sensitivity analysis was performed first assuming all children < 5 years of age LTFU died , markers of inflammation ; and markers of haemolysis .LME models were used to explore the relationship between the longitudinal time course of CHI3L1 in patients in the placebo and treatment arms of the iNO trial. Assuming that differences at baseline between trial arms were due to chance alone , we observed that CHI3L1 was elevated at baseline and decreased over the first 3 days of hospitalization at a different rate in children receiving iNO compared to placebo. The baseline (day 1) CHI3L1 concentration  decreased by 34% per day (95% CI 31–38) in the placebo group and 29% per day (95% CI 25–33) in the iNO group (p = 0.007)  macrophage activation, and inhibiting apoptosis –38. In tThere are limited, and conflicting, data related to the relationship between CHI3L1 and NO. CHI3L1 has been positively correlated with NO levels in tissue culture supernatant from herniated lumbar discs , and exhAcute kidney injury is a common complication in severe malaria but its pathogenesis is unclear. Peripheral parasitaemia is not associated with worsening kidney function in children or adults with severe malaria , 10, butWhile the mechanisms leading to severe malaria-associated AKI are not well understood, it is likely a heterogeneous syndrome characterized by reduced renal blood flow due to dehydration, hypotension, and impaired microcirculatory function as a result of parasite sequestration and reduced bioavailable nitric oxide. A subset of children may be at risk of developing direct tubular damage as a result of prolonged ischaemia, endothelial activation, inflammation, and cell-free haemoglobin and haem-mediated injury. In this cohort, an increase in CHI3L1 was associated with significant increases in creatinine, Cystatin C, and BUN by linear regression, and the relationship between CHI3L1 and Cystatin C remained significant following adjustment for creatinine. CHI3L1 levels also correlated with markers of immune activation , endothelial activation , and haemolysis , pathways of injury that are well described in paediatric severe malaria , 46–48. In this study elevated CHI3L1 levels at admission were a risk factor for in-hospital and all-cause 6 month mortality independent of kidney function and treatment group. These results are consistent with reports from adults where CHI3L1 is an independent predictor of all-cause mortality in type II diabetes , 50, heaThis study has several strengths including a randomized trial design with detailed clinical follow up and daily assessment of renal function and plasma CHI3L1 levels. Further, the majority of children were followed up to 6 months allowing us to evaluate the association between CHI3L1 and post-discharge mortality in children with severe malaria. Limitations include a lack of data on renal recovery and long-term renal function in the children. Further, urine was not collected to evaluate plasma versus urine levels of CHI3L1.In this study, CHI3L1 was validated as an independent biomarker of morbidity and mortality in children with severe malaria that is associated with the presence and severity of AKI. This provides further evidence that AKI is an important complication in children with severe malaria associated with endothelial activation and inflammation. Additional studies to evaluate the long-term implications of AKI on kidney function in surviving children are urgently needed.Additional file 1. Dataset for malaria, CHI3L1, acute kidney injury, and mortality.


Plasmodium species in the Brazilian Amazon.Brazil has seen a great decline in malaria and the country is moving towards elimination. However, for eventual elimination, the control program needs efficient tools in order to monitor malaria exposure and transmission. In this study, we aimed to evaluate whether seroprevalence to the circumsporozoite protein (CSP) is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium vivax, P. falciparum, and P. malariae  were detected using the enzyme-linked immunosorbent assay technique. Human leukocyte antigen (HLA)-DRB1 and DQB1 genes were typed using Luminex® xMAP® technology.Cross-sectional surveys were conducted in a rural area of Porto Velho, Rondônia state. Parasite infection was detected by microscopy and polymerase chain reaction. Antibodies to the sporozoite CSP repeats of P. vivax CSP peptide (62%) was higher than P. falciparum (49%) and P. malariae (46%) CSP peptide. Most of the studied individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although the majority of the population was not infected at the time of the survey, 74.3% of parasite-negative individuals had antibodies to at least one of the CSPs. Importantly, among individuals carrying the haplotypes DRB1*04~DQB1*03, there was a significantly higher frequency of PfCS responders, and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders. In contrast, HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P. vivax and P. falciparum CSP repeats, and the haplotype DRB1*01~DQB1*05 was also associated with non-responders, including non-responders to P. malariae.The prevalence of immunoglobulin G against P. vivax, P. falciparum, and P. malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II molecules play an important role in antibody response and require further study with a larger sample size. It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.Our results show that in low transmission settings, naturally acquired antibody responses against the CSP repeats of The online version of this article (10.1186/s40249-018-0428-1) contains supplementary material, which is available to authorized users. Please see Additional file Malaria remains an acute public health problem, particularly in Sub-Saharan Africa, despite an impressive reduction in the number of cases globally. According to the latest estimates from the World Health Organization, there were 212 million new cases of malaria and 429 000 deaths in 2015 worldwide [Plasmodium vivax representing 88.3%, P. falciparum 11.7%, and only a few cases of P. malariae being reported  [P. falciparum malaria are good, a progressive increase in P. vivax cases remains a challenge [P. vivax infections, the presence of asymptomatic carriers and the possibility of relapse impose additional challenges for surveillance. In addition, there is no current method for diagnosing hypnozoite carriers and P. vivax infections that recur after drug treatment can be attributed to relapse arising from the dormant liver stages (hypnozoites) or recrudescence originating from asexual blood-stage parasites that survived drug treatment, or a reinfection resulting from a new mosquito inoculation [In Brazil, major successful control efforts have contributed to the number of cases declining from 615 246 in 2000 to 143 145 in 2014, a reduction of 76.7% [The challenge to sustain and ensure malaria elimination is to detect asymptomatic infections in order to block transmission. In endemic areas, the risk of infection has been routinely estimated by parasite prevalence using microscopy and rapid diagnostic tests (RDTs) or estimated using the entomological inoculation rate (EIR) , 10. MicP. falciparum and P. vivax transmission intensity and exposure has gained recognition in the monitoring of changes in transmission, and in identifying hotspots of transmission and high-risk groups [Prior to the molecular diagnosis era, serology was used to identify subjects with evidence of current or recent infection, with this method having the potential to detect not only ongoing blood stage infections but also symptomatic or asymptomatic infections –15. In rk groups –19.P. falciparum, are a predictor of parasite exposure over time and are a good indicator of malaria transmission intensity [Numerous studies conducted in a variety of epidemiological settings have revealed that the levels and seroprevalence of sporozoite antibodies, especially those against the repetitive epitope of circumsporozoite protein (CSP) of ntensity –24. Howentensity , 26. Morntensity –29.P. vivax (VK210), HLA-DR3, and HLA-DR5, with poor responses against N-terminal region [P. falciparum multiple antigen peptide vaccine, the formulation elicited high levels of parasite-specific antibodies in volunteers expressing DQB1*0603, DRB1*0401, or DRB1*1101 class II molecules [In naturally exposed individuals in the Brazilian Amazon, the expression of HLA-DR7 has been associated with poor responses against the CSP repetitive region of l region –30. In aolecules . HLA claPlasmodium species: P. falciparum, P. vivax, and P. malariae. Most of the studies on the antibody responses to CSPs of P. vivax and P. falciparum point out an association with malaria exposure [In Brazil, a number of cross-sectional studies have aimed to characterize antibody response to CSPs of all three exposure , 32.P. falciparum, P. vivax, and P. malariae is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of these species in a rural area of Porto Velho, Rondônia state. This endemic area has experienced a decline in P. falciparum cases, a progressive increase in P. vivax cases, and has had a few reported cases of P. malariae. The influence of HLA-DRB1 and HLA-DQB1 allelic products on the antibody responses was also taken into consideration.In this study, we aimed to test whether seroprevalence to CSPs of ∘54′13′′W 8∘45′43′′S), municipality of Rondônia state, Brazilian Amazon.This study comprised of cross-sectional surveys being conducted in a rural community of Porto Velho .Plasmodium spp.) and species . The amplification protocols have been described previously [All individuals answered a questionnaire and were asked if they had any symptoms, such as fever, headaches, chills, myalgia, and/or nausea. After the epidemiological survey was conducted, blood was drawn from each participant by venipuncture and malaria infection was diagnosed by microscopic examination of Giemsa-stained blood smears and PCR using primers specific for genus  per 200 leukocytes in thick blood films, multiplying this by the number of individual leukocytes, and dividing this by 200. All smear-positive individuals were treated with antimalarial drugs, as according to the protocol of the Brazilian Ministry of Health.Subjects were considered positive for P. vivax, all three variant sequences were used: P. vivax VK210 DGQPAGDRAAGQPAG-(DRADGQPAG)2, P. vivax VK247 (ANGAGNQPG)3-ANGAGN, and P. vivax-like (APGANQEGGAA)3. For P. falciparum and P. malariae, sequences (NANP)8 and (GNAA)2-GNDA-(GNAA)4 were used, respectively.Synthetic peptides representing the repeated immune-dominant epitope of the CSP were used as an antigen for the detection of immunoglobulin G (IgG) antibodies. For , Carlsbad, CA, USA) were coated with 5μg/ml of the synthetic peptides representing CSP repeats of P. vivax (PvCS), P. falciparum (PfCS), and P. malariae (PmCS), and incubated overnight at 4 °C. Individual plasma samples were assayed at a dilution of 1:100 for one hour at 37 °C. Bound antibodies were detected by IgG-peroxidase conjugated goat anti-human IgG , followed by O-phenylenediamine dihydrochloride  and hydrogen peroxide. The optical densities (ODs) were recorded on an ELISA reader (BioTek ELx800™) at 490 nm. On each plate, reactive standard sera and non-reactive standard sera from individuals living in non-endemic areas who have never been exposed to malaria infection were placed. Results were expressed as reactivity index (RI), which was calculated by dividing the mean OD values of tested samples by the mean plus three standard deviations (SDs) of negative controls tested simultaneously. Samples with an RI greater than 1 were considered positive for the CSP peptide tested. In the case of P. vivax, positive samples presenting RI > 1 for any of the three CSP repeats representing P. vivax CSP genotypes  were considered.The enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to the CSP was conducted, as previously described . BrieflyGenomic DNA was isolated from whole blood with ethylenediaminetetraacetic acid (EDTA) using QIAamp® DNA Blood Midi Kit . The concentration and quality of all DNA were analysed using NanoDrop® ND-1000 . HLA-DRB1 and HLA-DQB1 genes were typed using a Luminex® Multi-analyte profiling system . This system uses LABType® SSO One Lambda typing kit  and is based on the PCR sequence-specific oligonucleotide probes. The highly polymorphic exons 2 of the HLA-DRB1 and HLA-DQB1 genes were amplified using the kit-specific primer pairs. The 5′ ends of the upstream primers were labeled with biotin. Hybridized amplicons were labeled with streptavidin, R-phycoerythrin, and quantified on the Luminex® 100™ flow analyzer . The hybridized patterns were classified using HLA Visual 2.0 software  according to the manufacturer’s instructions.χ2) test. Associations between antibody levels and epidemiological data were assessed with Spearman’s rank correlation. Overall associations of antibody responses with the alleles from each HLA-DRB1* and HLA-DQB1* loci were evaluated by comparing the allele frequencies between seronegative (non-responders) and seropositive (responders) subjects using a Fisher’s exact test 2 × 2 contingency table. Statistical significance was set at P < 0.05.Analysis were done using Epi Info™ 2002 , GraphPad Prism version 7.0 , and R version 3.4.0  . Differen = 285). Only 72 individuals were recruited from the outpatient clinic, but they lived in the same area as the other participants and had similar exposure to malaria infections.Details about the study population are summarized in Table P. falciparum, and one had a mixed infection). It is worth noting that all individuals testing positive by microscopy were also positive by PCR, except five individuals who were found positive only by PCR.Parasite prevalence determined by microscopy and/or PCR was 27% , headaches (76%), chills (54%), myalgia (57%), and nausea (43%).P < 0.01) and PmCS . Most of the individuals had antibodies to at least one of the three peptides (72%), 34% had antibodies to all three peptides and 28% were non-responders. Although P. malariae was not detected by microscopy or PCR in the study population, the prevalence of antibodies to PmCS (46%) was similar to PfCS (49%)  or number of past malaria infections . We did not find any association between antibody responses and age .The prevalence of naturally acquired antibodies to PvCS, PfCS, and PmCS was measured. Of the study participants, 72% presented IgG against at least one of the three peptides. The prevalence of IgG against PvCS (62%) was higher than PfCS  was not infected at the time of the survey (n = 257), 74.3% of the parasite-negative individuals had antibodies to at least one of the CSPs. Among the parasite-negative individuals, the prevalence of antibodies to PvCS was significantly higher when compared to PfCS (P < 0.01) and PmCS (P < 0.0001), and 25.7% were non-responders .Individuals positive for malaria were assigned as having malaria in the last ≤1 month. Figure P. vivax, P. falciparum, and P. malariae in 280 individuals. The frequency of each allele in responders and non-responders to all three CSPs are summarized in Table Considering that HLA class II molecules play an important role in antibody response and that 28% of individuals did not respond to all three CSP peptides, we evaluated the influence of HLA-DRB1 and HLA-DQB1 alleles on the naturally acquired IgG responses to the CSPs of Common allele groups ranged from 13 at the HLA-DRB1 locus and five at the HLA-DQB1 locus. The predominant HLA-DRB1 allelic groups were HLA-DRB1*04 (16%) and HLA-DRB1*13 (14%), and the most frequent HLA-DQB1 were HLA-DQB1*03 (38%) and HLA-DQB1*06 (21%). Although 72% of the population were positive to at least one of the peptides, the frequency of HLA-DRB1*01 was significantly higher in non-responders to PvCS and PfCS and HLA-DQB1*05 in non-responders to PvCS and PfCS. In contrast, we observed a higher frequency of HLA-DQB1*02 genotype in responders to PfCS peptide. Antibodies to PmCS showed no association with particular HLA-DRB1 and HLA-DQB1 genotypes.Associations attributed to individual DRB1 and DQB1 allele groups were partially reflected by the DRB1~DQB1 haplotypes. These allele groups contributed to 10 common DRB1~DQB1 haplotypes.Frequencies of responders and non-responders to each peptide according to the haplotypes are described in Fig. P. falciparum has been decreasing (under 11%), P. vivax has become the predominant species (88%), and only a few cases of P. malariae have been reported in Rondônia state. This epidemiological situation gave us the opportunity to investigate whether seroprevalence to the CSPs of P. falciparum, P. vivax, and P. malariae is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of these species in a rural area of Porto Velho, Rondônia.Malaria elimination is on the agenda of the control program in Brazil, and effective monitoring of disease transmission is crucial. From 2007 to 2017, P. vivax and P. falciparum were higher than those estimated based on blood analysis and/or PCR, and revealed the presence of anti-CSP antibody to P. malariae. This is not surprising as a high prevalence of anti-CSP antibody to P. malariae has been reported in five states of the Amazon, including Rondônia [P. malariae infection by PCR, this species seems to be more common than suggested by official data based on microscopy. Studies using nested PCR have found significantly more cases of P. malariae infection in patients from Rondônia (10%) and Mato Grosso (11.9%) [P. malariae has the ability to persist in a single host for decades as a chronic, low-density, asymptomatic infection, and there is a risk of recurrence decades after initial exposure, even when the infected individuals have left the endemic region [P. malariae reported cases in Rondonia have been rare, the high prevalence of antibodies to the CSP repeats of P. malariae in the absence of detected cases during the surveys could be due to the presence of antibodies cross-reacting with P. vivax, P. falciparum or with the simian P. brasilianum antigens [P. malariae and P. brasilianum share identical CS repeat sequences and do not segregate into distinct types in all genomic markers used so far [Our results show that antibody responses against the CSP repeats of Rondônia . Althoug (11.9%) , 5, 32. c region –42. Evenantigens . Indeed,d so far .P. falciparum, P. vivax and P. malariae [P. vivax, the presence of blood stage infection does not necessarily indicate a new infection as it may be due to a relapse. It is not known if the presence of hypnozoites/relapses could influence serological profile, especially those against sporozoites.Overall, production of anti-CSP antibodies was independent of a blood-stage infection. Current or recent blood infections appear to have little impact on seropositivity and do not seem to boost the anti-CSP response, as prevalence and levels of anti-CSP antibodies were not significantly different between parasite-positive and parasite-negative individuals. This is consistent with other studies that showed the absence of anti-CS antibody responses in individuals with confirmed exposure to malariae , 45–48. Plasmodium species in the area.Numerous studies that evaluated anti-CSP antibody responses in endemic populations showed that an anti-sporozoite humoral immune response was independent of a blood stage infection, but correlates with exposure and increases with age –51. We wAs infection and exposure refer to infective bites by a mosquito regardless whether it results in a blood-stage infection, it is difficult to obtain data that reflect individual natural exposure to infective bites. On the other hand, several studies showed that anti-CSP antibody responses change with seasonal or geographical transmission intensity. In low transmission settings, anti-CSP antibodies formed at a slower rate and decayed at a faster rate compared to anti-blood-stage antibodies . In contAlthough we were unable to establish a correlation between time since the last malaria infection and specific anti-CSP responses, the prevalence of antibodies to PvCS and PfCS was higher in individuals for whom the time elapsed since the last malaria infection was more than one year ago.P. falciparum and P. vivax infection. Non-responsiveness to CSP in a proportion of individuals living in endemic areas is a well-documented finding and several studies have reported an association between specific HLA alleles and immune response to malaria antigen [P. malariae. Interestingly, according to the Brazilian National Registry of Bone Marrow Voluntary Donors, the allelic frequency of DRB1*01 in the Amazon region is 19.73% and in Rondônia, it is 19.79% [P. vivax MSP-9 antigen in the Brazilian Amazon, and HLA-DQB1*05 along with HLA-DRB1*13 has been associated with a reduced susceptibility to severe malaria in Gambian children [One major limitation of our study is that serological measures were performed from a single cross-sectional survey at the malaria transmission season and changes in prevalences and the persistence of antibodies to the CSP after the end of the transmission was not evaluated. Interestingly, in our study, a proportion of naturally exposed individuals remained unresponsive to all CSP repeats 28%), even after a history of repeated malaria exposures and current %, even a antigen . In the s 19.79% . In addichildren , 54.In contrast, we observed a higher frequency of DRB1*07~DQB1*02 and DRB1*04~DQB1*03 haplotypes in responders to PfCS, and DRB1*16~DQB1*03 haplotype for both PfCS and PvCS. These antibody responses did not seem to be related to exposure as no difference was observed between time of residence, number of previous malaria infection, and time since the last malaria infection, and the presence of these alleles.P. vivax, P. falciparum, and P. vivax measured in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure, especially in an area with a high prevalence of P. vivax. Furthermore, HLA class II alleles  were associated with antibody response for CSP of P. vivax and P. falciparum, and it will be of interest to consider a HLA analysis when using serosurveillance among genetically diverse populations. In the context of malaria elimination, factors affecting the acquisition and maintenance of antimalarial antibodies are important for the development of serosurveillance tools.Our results show that in a low transmission setting, naturally acquired antibody responses against the CSP repeats of Additional file 1:Multilingual abstracts in the six official working languages of the United Nations. (PDF 666 kb)


Plasmodium falciparum malaria are two major public health problems in the state of Odisha, India. The prevalence of sickle cell gene in the western part of Odisha is 13.1%,1P. falciparum contributes 23% of cases and 15% of malaria-related deaths in India. Various African studies have shown that, even though SCA protects from P. falciparum infection, the risk of severe illness and death due to malaria is higher.P.falciparum malaria in patients with SCA, it was recently found that fetal hemoglobin (HbF), a physiological hemoglobin usually found higher in patients with SCA had a negative epistatic interaction with HbS during protection against malaria.P. falciparum malaria in cases with normal hemoglobin genotypes has been widely studied and found to be protective against severe disease manifestation. So it is necessary to investigate this association in the regions with high prevalence of sickle cell gene and high endemicity of P. falciparum malaria. This study aims to find out the effect of HbF level on the clinical manifestation of severe P.falciparum malaria in patients with SCA.Sickle cell anemia (SCA) and P. falciparum malaria admitted in the Department of Medicine of this institute were included in this study. The mean age of patients was 25.4±8.8 years  with 58.7% (27/46) being males. The hemoglobin variants including HbF was estimated by Cation-Exchange high performance liquid chromatography (CE-HPLC) using Variant II −β-thalassemia short program . The mean % HbF level was found to be 18. 2 ± 4.9 %; ranging from 6.0 to 29.0 %.This prospective study was undertaken at the Sickle Cell Clinic and Molecular Biology Laboratory, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, Odisha, India. Forty-six adult patients with SCA along with severe r, −0.356; p, 0.015), which indicates that patients with higher % HbF level had lower total hemoglobin level.The severity of malaria was defined by WHO guideline in 2010.p, 0.01). The % HbF differences in the patients with severe malarial anemia, cerebral malaria, and fatality, has been illustrated in In another comparison, we found that there was a trend in increasing % HbF level in patients with severe malarial anemia compared to patients without it. A similar tendency was observed in patients with cerebral malaria. The average % HbF level also significantly increased averagely in patients who died compared to patients who survived .Like HbS, alpha thalassemia has also been found to have a protective role against P. falciparum malaria in patients with SCA, derived that HbF has a negative role in protection against severe disease manifestation. It has been found that HbF provides protection from P. falciparum malaria by diminishing the growth of the parasites inside the RBCs.P. falciparum infection. This might be due to the peripheral selection and not for its increased synthesis of HbF in patients with SCA.From the three factors above, which are associated with severity and mortality due to Though, HbF in SCA is found to be supportive in reducing episodes of vaso-occlusive crises and requirements of blood transfusion,


Plasmodium falciparum transmission relies exclusively on sexual-stage parasites, several malaria control strategies aim to disrupt this step of the life cycle. Thus, a better understanding of which individuals constitute the primary gametocyte reservoir within an endemic population, and the temporal dynamics of gametocyte carriage, especially in seasonal transmission settings, will not only support the effective implementation of current transmission control programmes, but also inform the design of more targeted strategies.Since P. falciparum gametocyte carriage in a village in Mali with intense seasonal malaria transmission. A cohort of 500 individuals aged 1–65 years was recruited for this study. Gametocyte prevalence was measured monthly using Pfs25-specific RT-PCR, and analysed for the effects of host age and gender, seasonality, and multiclonality of P. falciparum infection over 1 year.A 1-year prospective cohort study was initiated in June 2013 with the goal of assessing the longitudinal dynamics of P. falciparum infections (51–89%) in this population were accompanied by gametocytaemia throughout the 1-year period. Gametocyte prevalence among P. falciparum-positive individuals (proportion of gametocyte positive infections) was associated with age (p = 0.003) but not with seasonality (wet vs. dry) or gender. The proportion of gametocyte positive infections were similarly high in children aged 1–17 years (74–82% on median among 5 age groups), while older individuals had relatively lower proportion, and those aged > 35 years (median of 43%) had significantly lower than those aged 1–17 years (p < 0.05). Plasmodium falciparum-positive individuals with gametocytaemia were found to have significantly higher P. falciparum multiclonality than those without gametocytaemia .Most P. falciparum-positive individuals. Furthermore, the presence of multiple P. falciparum genotypes in an infection, a common feature of P. falciparum infections in high transmission areas, is associated with gametocyte prevalence.Taken together, these results suggest that a substantial proportion of Pf-positive individuals carries gametocytes throughout the year, and that age is a significant determinant of gametocyte prevalence among these The online version of this article (10.1186/s12936-017-2123-2) contains supplementary material, which is available to authorized users. Plasmodium falciparum, the most lethal malaria parasite causing 99% of malaria-related deaths in 2015, is the predominant malaria parasite in Africa. Human-to-mosquito transmission of P. falciparum relies exclusively on the gametocyte stage of parasite development, and this step in the transmission cycle has been targeted by several malaria control strategies. A better understanding of which individuals constitute the primary gametocyte reservoir within an endemic population, and the temporal dynamics of gametocyte carriage, especially in seasonal transmission settings, is needed to support the effective implementation of existing strategies, and to further guide the design of more effective transmission control programmes.Although malaria incidence and mortality rates have steadily declined from 2000 to 2015  in a seasonal transmission area, a 1-year prospective cohort study was conducted in Kenieroba, Mali, from June 2013 to May 2014 [To gain a better understanding of the year-round dynamics of Ethical clearance for this study was provided by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases and the Ethics Committee of the Faculty of Medicine, Pharmacy, and Odontostomatology, University of Bamako. The study is registered with Clinicaltrials.gov (Identifier No. NCT01829737). Written informed consent was obtained from all participants aged ≥ 18 years, or the parents or guardians of individuals aged < 18 years.In June 2013, a prospective cohort study was initiated in Kenieroba, Mali, to assess the population-level dynamics of asexual and sexual parasite carriage over 1 year. Malaria transmission in this area is seasonal, with most cases occurring during the June-December rainy season , 15. The® Cell Reagent (Qiagen) for nucleic acid stabilization. Filter paper samples were stored under dry conditions at room temperature, and used for total parasite detection [® samples were stored at − 80 °C until nucleic acid extraction and molecular analyses were performed. For participants who reported malaria symptoms  at any time during the study, thin and thick blood smears were prepared, Giemsa-stained, and read by experienced microscopists for parasitological diagnosis. Confirmed cases were referred to the Kenieroba Health Centre for standard-of-care anti-malarial treatment after samples collection. It should be noted that microscopic detection of parasites was only performed on samples collected from such symptomatic individuals for parasitological diagnosis of malaria cases, and not for general detection of P. falciparum infection in asymptomatic individuals in this study.Finger-prick blood samples were collected as dried blood spots (DBS) on Whatman 3MM filter paper and also as whole blood in RNAprotectetection . RNAprotP. falciparum infection by species-specific nested-PCR as previously described [® Cell Reagent, P. falciparum gametocyte prevalence was measured by RT-PCR targeting expression of the female gametocyte specific Pfs25 gene. Total RNA extraction was performed using the RNeasy Plus 96 Kit (Qiagen), with an additional on-column DNase I treatment to eliminate any remaining contaminating genomic DNA (gDNA). RNA was eluted in 100 µL of RNase-free H20 and stored at − 80 °C until molecular assays were performed. Prior to the RT-PCR assay for gametocyte detection, all extracted RNA samples were tested for gDNA contamination with a 40-cycle Pfs25-targeted PCR, which was analysed with the LabChip GX and the HT-DNA 5 K LabChip capillary electrophoresis setup in accordance with the manufacturer’s protocol . After confirming absence of gDNA, RNA samples were used as templates in a 1-step Pfs25-targeted RT-PCR assay for gametocyte detection. Primers and probe used in this assay were previously described by Wampfler et al. [DBS samples were screened for escribed . Using wr et al. . The RT-r et al. : the lowr et al. . TherefoPlasmodium falciparum multiclonality was reported for each sample in two ways, as described elsewhere [P. falciparum genotypes in each infection calculated by the COIL program [Multiclonality was assessed among Pf-positive samples at four time-points , which were selected to reflect periods of varying transmission intensities during the year. Multiclonality was measured using a SNP DNA barcode assay as previously described , 17. Plalsewhere . In brie program ., termed “population gametocyte prevalence” in this manuscript, and (2) the number of gametocyte-positive samples out of P. falciparum-positive (by nPCR) blood samples . The latter measure was used for all statistical analysis. The proportion of gametocyte positive infections between wet (June to December) and dry (January to May) season time-points were compared by Mann–Whitney test. To analyse the effect of age on proportion of gametocyte positive infections, the cohort was categorized into seven age groups such that each group had a similar number of individuals (60–91 in each group). Proportion of gametocyte positive infections in each age group was then determined at each of the 12 time-points (one data point per month). Age-effect (12 proportion data for each age group) was analysed by One-way ANOVA test followed by Tukey’s multiple comparison test. Similarly, the proportion of gametocyte positive infections in each gender was calculated for each of the 12 time-points, and the gender-effect (n = 12 for each gender) was evaluated using Mann–Whitney test. Multiclonality was compared between gametocyte-positive and -negative samples using Mann–Whitney test for PmP, and Chi square test for COI.Cross-sectional Pf gametocyte prevalence was calculated at each time-point in two ways; (1) the number of gametocyte-positive blood samples out of the total number of blood samples collected at the respective time-pointPlasmodium infections were caused by P. falciparum  [P. falciparum parasites  were evaluated in this study, irrespective of potential, but rare infections by other Plasmodium spp.During the 1-year study period, a total of 469 clinical malaria cases were recorded among 232 individuals; viz. 46.4% (232 out of 500) of all study participants experienced at least one clinical malaria episode during the year . The highe year) ; therefoP. falciparum prevalence (by nested PCR) was previously determined at 2-week intervals within this cohort over the 1-year study period [P. falciparum-positive samples . Cross-sectional P. falciparum gametocyte prevalence in the entire cohort  showed a seasonal profile, ranging from a minimum of 8.3% in May 2014 to a maximum of 30.1% in October 2013, similar to the seasonal effect seen in population total P. falciparum prevalence  at one-month intervals over 1 year. The proportion of gametocyte positive infections ranged from 51.3%  to 88.9% , with no significant effect of seasonality on proportion . A previous assessment of the impact of several host factors, including age, gender, and red blood cell polymorphisms  identified significant age and gender effects on persistent total P. falciparum positivity, while all other factors tested showed no significant association [P. falciparum-positive individuals were identified as gametocyte-positive throughout the year, when cross-sectional population gametocyte prevalence was analysed, similar age and gender effects were observed on gametocyte positivity as was observed on total P. falciparum-positivity  in a multiple linear regression analysis after adjusting for age and gender  samples were identified as gametocyte positive. Hence, the chance of gametocyte positivity in symptomatic individuals was significantly higher than that in asymptomatic individuals .To assess age effect, the cohort was categorized into seven age groups and the proportion was measured for each age group at each of the 12 months during the year Fig. a. ProporP. falciparum positivity over 1 year and multiclonality at multiple time-points throughout the year [P. falciparum infections and gametocyte positivity was investigated. Both PmP and COI were compared between gametocyte-positive and gametocyte-negative infections at 2 wet season and 2 dry season time-points. There was a general trend of higher PmP and COI in gametocyte-positive compared to gametocyte-negative infections throughout the year, although these differences were not statistically significant at individual time-points except in November . There was no effect of seasonality or gender on proportion of gametocyte positive infections. When age-effect was assessed, the likelihood of carrying gametocytes was similarly high in groups of children aged ≤ 17 years. However, adults aged > 35 years had a significantly lower proportion of gametocyte infections than most groups of children aged ≤ 17 years, while adults aged 18–35 years showed an intermediate proportion. Interestingly, this study also found that P. falciparum infections with gametocytaemia had relatively higher multiclonality than those without gametocytaemia.In this study, a highly sensitive molecular method was used to measure changes in gametocyte positivity over a 1-year period in an area with intense, seasonal malaria transmission in Mali. Subsequently, the effects of age and gender on the proportion of gametocyte positive infections, as well as the relationship between gametocyte positivity and multiclonality of P. falciparum carriage in an area where malaria transmission is intense and seasonal [P. falciparum infections at peak transmission and that age and gender were significant determinants of Pf positivity throughout the year. Furthermore, persistent parasite carriage was significantly associated with reduced risk of developing clinical malaria over the 1-year study period [P. falciparum-positive individuals in this population were also gametocyte-positive. Therefore, it was not surprising that the cross-sectional population gametocyte prevalence showed a generally similar profile over the 1-year study period as population total P. falciparum prevalence [The current report is an expansion of a year-long cohort study conducted to investigate the longitudinal dynamics of seasonal . This iny period . In the evalence . This obevalence , 18–20, evalence , 21.P. falciparum infections did not differ significantly between the wet and dry seasons . Overalion Fig.  is in lietection , 28. TheP. falciparum positive individuals was observed in children aged 1–17 years. Children have commonly been associated with higher risk of gametocyte carriage than adults [Age was found to be a significant determinant of proportion of gametocyte positive infections in this area. However, similarly high gametocyte proportion among n adults . In somen adults , 28. In n adults , the chaP. falciparum infection. Multiclonality has effects on various aspects of P. falciparum infection, from outcome of infection [P. falciparum infections have been reported to last longer and have increased likelihood to produce gametocytes relative to single-clone infections [The occurrence of multiple distinct parasite clones is a common feature of nfection , 30 to tnfection , 32. Witfections . In thisP. falciparum-positive individuals (detected by nested PCR) at the time of sample collection. However, Pfs25 transcript abundance can exceed DNA copy number, therefore, this study could miss some gametocyte positive cases (with lower gametocyte density) in “P. falciparum-negative” samples. Lastly, direct skin feed and/or direct membrane feeding assay was not performed in this study. Therefore, gametocyte positivity may not necessarily directly link with infectivity in the field.There are a few limitations to this study. First, the volumes of blood samples collected in this study were not standardized, making it impossible to measure total parasite and gametocyte densities. With this limitation in mind, it should be noted that several associations found in this study may be explained by asexual parasite or gametocyte densities. For example, children might be more likely to carry infections at higher asexual parasite densities, and consequently higher gametocyte positivity and also higher COI. Second, the study had limited power to evaluate the relationship between gametocyte carriage and RBC polymorphisms, such as ABO/Rh types, haemoglobin phenotype, and G6PD deficiency and α-thalassaemia genotypes. Third, the 1-year study design places a limit on the study to assess these interactions beyond a single transmission year. It may be necessary to re-evaluate the effects of various risk factors of gametocyte prevalence as transmission dynamics change over time. Fourth, gametocyte positivity was analysed only for P. falciparum infections, and that individuals aged ≤ 17 years who have P. falciparum infections all have high likelihoods of also carrying gametocytes. These findings will help guide the design of transmission studies and intervention strategies that target the gametocyte reservoir in this and perhaps other areas with similar human population structure and P. falciparum transmission dynamics. Furthermore, high multiclonality is associated with high gametocyte positivity, suggesting that intra-species competition in P. falciparum infection favours gametocyte development in this population.Results from this study indicate that in this area of intense, seasonal malaria transmission, there is a high prevalence of gametocyte carriage among Additional file 1. Age and gender effects on cross-sectional population gametocyte prevalence among the entire cohort during the wet and dry seasons. Gametocyte positivity was measured by RT-PCR once a month for 1 year. Data from November (A) and April (B) are presented as representative time-points for wet and dry seasons, respectively. Black bars and gray bars represent the proportion of gametocyte-positives and –negatives, respectively, within each category. Similar age and gender patterns were consistently observed at all other time-points throughout the year.Additional file 2. Effect of RBC polymorphisms on gametocyte positive rate.Additional file 3. Comparison of multiclonality between gametocyte-negative and -positive in Pf-positive individuals at four time-points. (A) Polymorphic proportion  and (B) complexity of infection  among Pf-positive individuals at June (n = 119), November (n = 110), February (n = 51), and April (n = 42) time-points. For PmP, red boxes represent gametocyte-negative (Nega) and blue boxes represent gametocyte-positive (Posi) individuals. PmP and COI were compared between gametocyte carriers and non-carriers using Mann–Whitney and Chi square tests, respectively. Only the November time-point showed a significant difference in PmP (p = 0.029) or COI (p = 0.025) based on gametocyte status.


Plasmodium falciparum antigens, as well as their relationship with parasite infection and clinical outcomes, were assessed in mothers and their children. Antibody responses among 207 Mozambican pregnant women at delivery against MSP119, EBA175, AMA1, DBLα and parasite lysate , as well as the surface of infected erythrocytes, were assessed by enzyme-linked immunosorbent assay and flow cytometry. The relationship between antibody levels, maternal infection and clinical outcomes was assessed by multivariate regression analysis.Difficulties to disentangle the protective versus exposure role of anti-malarial antibodies hamper the identification of clinically-relevant immune targets. Here, factors affecting maternal IgG and IgMs against Placental infection was associated with an increase in maternal levels of IgGs and IgMs against a broad range of parasite antigens. The multivariate analysis including IgGs and IgMs showed that the newborn weight increased with increasing IgG levels against a parasite lysate, whereas the opposite association was found with IgMs. IgGs are markers of protection against poor pregnancy outcomes and IgMs of parasite exposure.P. falciparum when assessing immune responses effective against malaria in pregnancy.Adjusting the analysis for the simultaneous effect of IgMs and IgGs can contribute to account for heterogeneous exposure to The online version of this article (10.1186/s12936-018-2331-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum infection during pregnancy, characterized by the accumulation of parasites in the intervillous spaces of the placenta, is a major disease that affects birth outcome in sub-Saharan Africa [n Africa , causingn Africa . In contn Africa . In condn Africa , suggestn Africa .P. falciparum infected erythrocytes that binds chondroitin sulfate A (CSA) [P. falciparum in pregnancy [Immunoglobulin G (IgG) antibodies against placental infected erythrocytes (IE) and VAR2CSA , the maj A (CSA)  to enabl A (CSA) , increas A (CSA) . These a A (CSA) , 9–11, l A (CSA) –15 and m A (CSA) , 16. How A (CSA) –22, but regnancy –28.P. falciparum [P. falciparum in the absence of information before delivery. Specifically, the objective was to assess the relationship of IgG and IgM against P. falciparum antigens in Mozambican pregnant women at delivery with parasite infection and adverse clinical impacts in the mothers and newborns. Pregnancy-specific antigens , general (non-pregnancy specific) antigens  and a parasite lysate  were included. IgG but also IgM were assessed given their contribution for protection against infection [Recent studies show how the power to identify immune correlates of protection in children –31 and plciparum , 22, 27 lciparum . Howeverlciparum . The aimnfection , both innfection .P. falciparum is perennial and of moderate intensity with a warm rainy season from November to April, and a cool dry season during the rest of the year.The study was conducted at the Centro de Investigação em Saúde de Manhiça (CISM), located in the Manhiça District, Maputo Province, southern Mozambique. The area has been described in detail elsewhere . TransmiHIV-negative pregnant women resident in the Manhiça study area were recruited during the third trimester of pregnancy at the antenatal clinic of the Maragra Health Post (MHP), in the south of the study area, from September 2005 to March 2007 . After dP. falciparum detection by real-time quantitative PCR (RTqPCR) in duplicates [At delivery, maternal peripheral and cord blood samples collected into EDTA vacutainers were centrifuged and plasma stored at − 20 °C for future antibody determinations. Thin and thick smears of peripheral and cord blood were Giemsa-stained and examined for malarial parasites according to quality-control procedures . Haemoglplicates . Tissue plicates . When thChildren were followed up until age 24 months, with weekly active case detection from birth to age 10.5 months, and monthly home visits from 10.5 to 24 months of age. Children were examined and their parasitaemia and haematocrit determined if they presented fever (axillary temperature ≥ 37.5 °C) or their guardians referred history of fever in the preceding 24 h. Additionally, passive case detection was carried out at the MHP and Manhiça District Hospital (MDH) through the morbidity surveillance system to monitor attendances to the outpatient clinics and admissions to hospital.Informed consent was sought to enroll pregnant women and their newborn children in the study upon birth. The protocol was approved by the National Mozambican Ethics Review Committee and the Hospital Clínic of Barcelona Ethics Review Committee. The trial was conducted according to the ICH Good Clinical Practice guidelines and reviewed by a Local Safety Monitor and a Data and Safety Monitoring Board. The trial was registered in ClinicalTrials.gov .P. falciparum R29-rosetting + line) [Escherichia coli, refolded if the protein was insoluble and purified (> 95%) following previously described methods [Recombinant merozoite surface protein 1  , erythro + line)  were pro methods . Enzyme- methods , 41. Rea methods  and OD vWhole-parasite lysate was prepared by three freezing/thawing cycles of asynchronous in vitro cultures of 3D7, R29 and E8B laboratory strains at a 5% level of parasitaemia and 1% haematocrit, as previously described . Non-infPlac1 and IEPlac2), five pediatric isolates (IECh1–Ch4 and IESEV) from Manhiça [Cryopreserved O+ erythrocytes infected by two placental isolates previously confirmed to express VAR2CSA  , those with one previous pregnancy  and those with more than two previous pregnancies . Age was categorized as ≤ 20, 21–24 and ≥ 25 years. Maternal anaemia was considered if the haemoglobin level was < 11 g/dL and low birth weight if the newborn weight was < 2500 g. Distance to the river was calculated with Hawth’s tools in ArcGIS software (ESRI) as the mean distance of each neighbourhood centroid to the Incomati river, and neighbourhoods were classified as those adjacent to the river (≤ 2.5 km) or those at a higher distance (> 2.5 km) . Season P. falciparum antigens, the association of antibody levels (log transformed ODs or MFIs) with P. falciparum infection , placental inflammation, parity, neighbourhood, season and use of insecticide treated nets (ITN) or indoor residual spraying (IRS) were assessed by linear regression models. For those variables with negative values (IgGs against infected erythrocytes), prior to applying the log transform a constant value was added to the data to obtain a minimum value less than 1. All the analyses were done univariate and multivariate, adjusting for all the other independent variables. To determine the relationship of antibody responses with clinical outcomes, parasite infection and clinical outcomes in mothers and their children, the association of antibody levels (independent variable) with maternal haematocrit and weight of the newborn (dependent variable) was assessed, including a multivariate analysis with all the antibody responses as independent variables. Negative binomial regression models were used to evaluate the effect of doubling the levels of antibodies on the incidence of multiple malaria episodes in children up to 12 months of age [P. falciparum infection in the mother. To assess if parity modified the associations, an interaction term was included in the regression models, and ratios and 95% confidence intervals (95% CI) for each antibody responses were estimated after stratifying the regression models. Analyses were performed using Stata 11  and significance was defined at p < 0.05.Continuous variables were compared by ANOVA, and categorical variables by Chi squared test or Fisher’s exact test. To identify factors that affect maternal IgG and IgM against s of age . Analyses of age  and P. fThe analysis of antibody responses included 207 women Table , 42 20% and age SEV), as well as with maternal IgMs against MSP1, EBA175, AMA1 and parasite lysate  than in negative cords .Maternal IgM against MSP1, EBA175, AMA1 and DBLα were lower in women with placental inflammation compared to women without inflammation Table . IgMAMA1ch3 were 1.32-fold higher in neighbourhoods close to the river (< 2.5 km) compared to those at more than < 2.5 km . There were no significant differences according to season .The use of ITNs was associated with reduced levels of maternal IgGs against DBLα  with twofold increase in antibody levels, respectively; Additional file ch1) and cord IgGs . The multivariate analysis including all antibody levels showed that newborn weight increased with levels of IgGlysate whereas decreased with levels of IgMlysate  were at higher risk of placental infection as detected by histology . This in–20 year P. falciparum antigens and not only against CS2 may have a role in controlling parasite infections and reducing the adverse consequences of malaria in pregnancy.In this maternal cohort, placental infection is associated with an increase of IgG, as already shown in studies conducted in the same study area , 28, butNewborn weight increased with increasing IgG levels against a parasite lysate, whereas the opposite association was found with IgM, suggesting that IgG are markers of protection against poor pregnancy outcomes and IgM of parasite exposure. This study confirms the need for developing methods to control for heterogeneous exposure to the parasite when aiming to identify those immune responses that have a potential role in protection against infection and disease. Further studies are needed to assess novel approaches to disentangle exposure from protection in immunological studies assessing the role of antibodies against the adverse consequences of malaria infection in situations of heterogeneous malaria exposure.Additional file 1: Table S1. Association of antibody responses with maternal infection, use of insecticide treated nets and indoor residual spraying. Table S2. Association between antibody levels and weight of newborn, maternal hemoglobin and incidence of malaria in children. Table S3. Association of antibody levels with maternal infection, pregnancy outcomes and incidence of malaria in children.


Plasmodium vivax infection annually, little is known about the epidemiology and impact of the infection in pregnancy.Despite that over 90 million pregnancies are at risk of P. vivax monoinfection in maternal blood at delivery was 0.4% (20/4461) by microscopy  and 7% (104/1488) by PCR. P. falciparum monoinfection was found in 0.5% (22/4463) of women by microscopy . 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 . Clinical P. vivax infection was associated with increased risk of maternal anemia , while submicroscopic vivax infection was not associated with increased risk of moderate-severe anemia (Hb<8g/dL) , or low birth weight (<2500g) .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 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.In this multicenter study, the prevalence of  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.More than 90 million pregnancies are exposed to  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. ; p<0.001). A total of 898 passive case detection visits  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  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 Data on incidence of malaria is shown in 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.Overall, parasite densities in pregnant women throughout the study were low in most cases. The geometric mean density (GMD) 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.Parasite densities on placental blood ranged for 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.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 . At deliP. vivax or P. falciparum clinical malaria were more than 5-times as likely to be anemic compared to non-infected women , and adjusted OR, 5.57, [95% CI 1.22–25.34]; p = 0.012, respectively) (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  or at delivery  and an increased risk of LBW.At enrolment women with ctively) . In contP. vivax malaria at delivery detected by microscopy did not have an increased risk of anemia  or LBW , compared to uninfected women. However, those women with P. falciparum malaria showed an increased risk for both anemia  and LBW . 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  or LBW  compared to uninfected women (negative by microscopy and by PCR). Likewise, P. falciparum submicroscopic infections at delivery were not associated with moderate-severe anemia  or LBW .Women with P. vivax, and adjusted OR, 8.61, [95% CI 0.79–94.09]; p = 0.077, for P. falciparum) or anemia . In contrast, a statistically significant association was found for placental infections detected by histology with LBW  and anemia . 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.Malaria infection in the placenta (whether detected by impression smear or PCR) was not associated with an increased risk of LBW  compared to non-infected women. In malaria endemic areas, there is evidence on the impact of maternal anemia on maternal mortality and morbidity,[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 available data on the impact of orbidity, as well orbidity,, 8 In coP. 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 two maternal deaths reported in Colombia and India were associated with 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  compared to active detection at routine visits . 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  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.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 becoming the dominant species in low transmission areas. [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. [P. vivax transmission. [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.n areas.  Accuratefection.  The prevmission.  In pregnThere 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 waThe 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.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.This multicenter study in representative areas endemic for vivax malaria shows that the prevalence of S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.