The use of molecular techniques has put in the spotlight the existence of a large mass of malaria sub-microscopic infections among apparently healthy populations. These sub-microscopic infections are considered an important pool for maintained malaria transmission.Plasmodium vivax gametocytes in circulation, gametocyte density and the parasite infectivity to Anopheles mosquitoes, a study was designed to compare three groups of volunteers either experimentally infected with P. vivax sporozoites  or naturally infected patients . In order to determine gametocyte stage, a quantitative reverse transcriptase PCR (RT-qPCR) assay targeting two sexual stage-specific molecular markers was used. Parasite infectivity was assessed by membrane feeding assays (MFA).In order to assess the appearance of P. vivax gametocytes could be detected starting at day 7 without giving rise to infected mosquitoes during 13 days of follow-up. Asymptomatic carriers, with presumably long-lasting infections, presented the highest proportion of mature gametocytes and were as infective as acute patients.In early infections P. vivax asymptomatic carriers in malaria transmission should be considered when new policies are envisioned to redirect malaria control strategies towards targeting asymptomatic infections as a tool for malaria elimination.This study shows the potential role of The online version of this article (doi:10.1186/s12936-016-1104-1) contains supplementary material, which is available to authorized users. Plasmodium falciparum and Plasmodium vivax. While P. falciparum is highly prevalent in the African continent, P. vivax is more prevalent in Central Asia, Southeast Asia and the American continent [P. vivax infection is characterized by induction of periodical asymptomatic infection or clinical episodes due to spontaneous activation of hypnozoites, dormant parasite liver forms that do not occur in P. falciparum infections [P. vivax has a preference for young erythrocytes whereas P. falciparum usually invades circulating erythrocytes of any age. Both species develop and multiply asexually inside erythrocytes where a small proportion of the parasite population switches from the asexually replicating to the sexual stage in a process initiated by a differentiation step [P. falciparum is known to differentiate into gametocytes in a relatively late phase of parasite blood development, P. vivax appears to produce mature gametocytes significantly earlier, due to the shorter developmental cycle in comparison with P. falciparum, which is believed to result in greater transmissibility [Despite several features being similar among different species of malaria parasites, there are important biological differences between the two most relevant to human infections, ontinent . P. vivafections . In blooion step . While Psibility , 5.P. vivax in vitro cultures and the lack of suitable animal models, the study of P. vivax gametocyte maturation has been limited to the use of parasite samples from naturally infected patients [P. vivax gametocyte production and infectivity in early, acute and asymptomatic infections.Although gametocytes may be present at early stages of an infection, they require a maturation process where factors such as gametocyte density could influence infectivity to mosquitoes . Moreovepatients –10. Furtpatients , 12. A bpatients . The curSamples used in this study were obtained from studies approved by the Institutional Review Board (IRB) of the Malaria Vaccine and Drug Development Centre (MVDC) under the codes CIV-01-042009, CIV 08-102010 and CIV 009. Samples from volunteers were collected anonymously and not linked to the identity of the donor. Written informed consent (IC) was obtained from each volunteer at enrolment. All volunteers were adults aged 18–55 years (Table P. vivax). Tierralta (Department of Cordoba) is located at 5 m asl in the northern region of the country. In 2014 the town reported 840 malaria cases (94.8 % P. vivax).Field activities were conducted in two endemic regions of Colombia, Buenaventura and Tierralta. Buenaventura  is the main port of Colombia on the Pacific Coast, located at 7 m asl, with a population of ~400,000 inhabitants and 307 malaria cases reported in 2014  in Buenaventura were included in a group denominated ‘acute patients’; all had non-complicated P. vivax malaria symptoms and were diagnosed by thick blood smear (TBS) and later confirmed by quantitative PCR (qPCR).A total of 46 individuals from early, acute and asymptomatic conditions were studied between January and December 2013  was performed every 2 days starting at day 7 post-infection until the parasites were detected by microscopy (days 11–13). Kinetics of parasitaemia was measured by qPCR at the same time points as MFA [P. vivax, chloroquine 25 mg/kg provided in three doses and primaquine 0.50 mg/kg daily for 14 days) [The time line of infectivity and gametocyte maturation in early infections was assessed using samples collected from naïve and semi-immune volunteers participating in a hallenge . Sixteens as MFA . These v14 days) .Volunteers (n = 14) participating in a cross-sectional epidemiological survey carried out in June 2012 in La Delfina and Zacarias  with low malaria transmission were selected and enrolled. After a population census, inhabitants of randomly selected houses were asked to supply blood samples to perform TBS and qPCR. At day 7 after PCR parasite detection, a blood sample was collected to perform MFA.Symptomatic volunteers (n = 16) were recruited in malaria diagnosis outpatient clinics of endemic settings in Buenaventura  and Monteria (Cordoba) between January and December 2013. A sample was collected at the time of consultation for MFA. Parasitaemia was estimated by qPCR.Whole blood (400 µL) was collected by venipuncture in tubes containing EDTA anticoagulant, and used as follows: 50 ml for TBS preparation, and a 200 µL aliquot which was added to 400 µl of Tempus RNA stabilization buffer  and preserved for molecular analysis at −20 °C. The remaining sample was immediately transported to the laboratory and placed at −20 °C.Anopheles albimanus mosquito colony was used for MFA as described before [g for 3 min at 37 °C, packed red blood cells (RBCs) were washed with warm serum-free RPMI 1640 medium  and reconstituted to 50 % haematocrit using equal volumes of pooled AB+ non-immune human serum. For each assay, 200 female mosquitoes (3–4 days after emergence) were sugar and water starved overnight and fed with the reconstituted P. vivax field isolate.Infected blood samples from acute and asymptomatic infected volunteers were sent at 37 °C from endemic areas to the nearest mosquito colony in Buenaventura or Monteria (<4 h in transit). Samples from experimentally infected volunteers was collected and tested in MFA at central laboratories in Cali within 4 h of collection. A laboratory-reared d before . BrieflyAn. albimanus mosquitoes were placed in feeding boxes and were subject to overnight starvation prior to feeding. Fed mosquitoes were transported in cages to secure infection rooms and kept under strict security and laboratory conditions (constant 80 % humidity and 26 °C temperature). Infected volunteers were subjected to mosquitoes directly feeding on one arm and after 10 min of exposure, mosquitoes were transported and kept under safe and tightly controlled laboratory conditions [For early infections, batches of 30 adult (3–4 days old) nditions .On day 7 post feeding, mosquitoes were dissected and examined microscopically at 40× for the presence of oocysts in the midgut after being stained with 2 % mercurochrome. Mosquito infections prevalence was expressed as infection percentage. Mosquito intensity was determined as the arithmetic mean of oocysts count per infected mosquito.P. vivax infections in mosquito midgut was performed by qPCR targeting the 18S ribosomal RNA gene. Primers and amplification conditions were described previously [High throughput methods for detecting infection in whole mosquitoes have shown that 18s PCR provides a reliable approximation of mosquito infection rates on day 7 post infection . Moleculeviously . In summPvs16 and Pvs25, which are P. falciparum orthologues known to define immature gametocytes and stage V female gametocytes, respectively [Plasmodium or human homologous sequences using PlasmoDB and NCBI Blast in order to eliminate the chances of non-specific amplification.In order to determine gametocyte stage, a quantitative reverse transcriptase PCR (RT-qPCR) was used to determine two sexual stage-specific molecular markers; g for 30 min at 4 °C and the supernatant removed. After drying for 2 min, the pellet was suspended in 350 μL of lysis buffer and 350 μL of 70 % ethanol, and the whole volume transferred to a spin cartridge. Columns were centrifuged and washed with buffers one and two, RNA was eluted from the cartridge columns using Ultra-Pure water. RNA sample was added to 2 μL of DNase I, 2 μL of 10X DNase I buffer, and inactivated with 4 μL of DNase inactivation reagent. A second round of DNase digestion was performed to ensure genomic DNA removal. cDNA was obtained using a Super Script III Kit , according to the manufacturer’s instructions.RNA was extracted from the 600 µl (200 µl whole blood +400 Tempus RNA stabilization buffer) aliquots conserved in Tempus RNA stabilization Buffer  using the PureLink RNA Mini Kit  following the manufacture conditions. Briefly, 200 µL of 1X PBS were added to the samples, mixed thoroughly using vortex, centrifuged at 12,00018S rRNA gene of P. vivax in an independent reaction without reverse transcriptase. Sensitivity of this assay was determined using tenfold serial dilutions of cDNA. The melt curves were checked to control primer dimers. cDNA from malaria-negative individuals was used as negative amplification controls.Gene expression was performed in triplicate by RT-qPCR  using SYBR Green and oligonucleotide primers as described . For oocyst prevalence Fisher’s exact test was used. A p value <0.05 was considered statistically significant.The data were analysed using SPSS software version 19, IBM and Graph Pad Prism 6, GraphPad PrismP. vivax sporozoites (early infections) were confirmed by qPCR to harbour P. vivax blood infection starting on day 7 after exposure to infected mosquitoes. PCR results confirmed blood infection in the two early infection sub-groups: semi-immune volunteers n = 9 (1–84 parasites/µL) and naïve volunteers n = 7 (57–105 parasites/µL) , 5096  and 13.93 (1–43), respectively, .Expression of gametocyte-stage specific genes was used to determine the course of gametocyte development and maturation. Gametocyte markers’ efficiency curves are shown in Additional file An. albimanus was assessed by both mosquito DFA and MFA performed simultaneously followed by evaluation of oocyst counts using both microscopy and qPCR, 7 days after experimental mosquito infection. Feeding assays were performed every 2 days from day 7 until day 13 when parasite patency was confirmed by TBS and volunteers received curative anti-malarial treatment. None of the samples obtained from individuals in early stages of infection (n = 16) showed infectivity to mosquitoes when assessed by either microscopy or PCR for the presence of oocysts.The infectivity of gametocytes from early infected volunteers to Pvs16 (mean 123 copies/µL) and Pvs25 (mean 3473 copies/µL). Eight out of 16 symptomatic patients successfully infected mosquitoes with an oocyst average prevalence of 57 % as determined by MFA.Naturally infected symptomatic patients (n = 16) showed the presence of gametocytes (400–2820/µL) by TBS, and were additionally positive for the presence of gametocyte-specific transcripts corresponding to the following genes: P. vivax was 12.4 and 4.5 % in La Delfina and Zacarias, respectively. Individuals who did not present microscopically detectable parasites and showed no symptoms on the day of diagnosis were followed up for 7 days to confirm the asymptomatic condition. A sub-set of 14 asymptomatic volunteers was enrolled to assess the parasite transmission potential by MFA. Remarkably, 56 % of the asymptomatic carriers were infective to An. albimanus mosquitoes. Mosquitoes fed with blood from asymptomatic volunteers developed a low infection prevalence of 2.5 and 5 % from La Delfina and Zacarias, respectively, with oocyst loads ranging between one and ten per midgut (Table Pvs16 transcripts (mean 2395 copies/µL) and Pvs25 transcripts (mean 46.30 copies/µL).The prevalence of asymptomatic sub-microscopic Pvs25/Pvs16 in comparison with non-infective samples; however no statistically significant differences were observed  was determined using the 18s gene expression as reference. Expression of immature gametocyte marker Pvs16 was significantly higher in asymptomatic volunteers (172.3) compared to relative expression from acute  and early infection samples . This trend was similar for the mature stage V gametocyte marker Pvs25, which showed a maximum relative expression in asymptomatic volunteers. Acute and early groups showed a low proportion of sexual stage parasites (mean ratio 0.58 for immature gametocytes and 0.25 for mature gametocytes)  and maintaining a low proportion of mature gametocytes, similar to the model of gametocyte sequestration in the bone marrow in lciparum . On the Anopheles mosquitoes with blood from sub-microscopic asymptomatic carriers underscores the importance of targeting all parasite carriers as they should be considered potential malaria transmitters. In P. falciparum it has been experimentally shown that stage V gametocytes observed in both sub-microscopic and microscopic gametocytaemia contributed similarly to malaria transmission [P. falciparum-infected children were more important as the reservoir than adults and sub-microscopic infections [P. vivax and An. dirus mosquitoes resulted in 13 % of infective samples with low oocyst counts [P. falciparum showed that low levels of gametocytes (<200/µL) are infective to An. gambiae; however, at higher densities a plateau region is reached [P. vivax gametocytes are infective to mosquitoes in more than half of the cases.Successful infections of smission . In regifections . In regifections . A previt counts . In this reached . In thisThe low proportion of mature gametocytes in acute and early infections in comparison with asymptomatic carriers appears to indicate that in early infections, the asexual parasite development and replication prevails in comparison to the sexual parasite development. However, the environmental changes that define the parasite cell fate remain largely unknown. The comparison between early, acute and asymptomatic infections could be useful for understanding the switch from asexual to sexual replication with potential applications towards development of novel drugs targeting malaria transmission.P. vivax strains reported early successful feeds at days 7 and 10 of patency [P. vivax strain, by multiple factors related to the human host [Whereas it was possible precisely ascertain that gametocytes in experimentally induced infections corresponded to 13-day infections, this was not the case with naturally infected acute individuals. Early studies on neurosyphilitic patients using different  patency , 27. Togman host , and posman host , 29.Pvs25 expression earlier than those from semi-immune individuals who had experienced multiple previous malaria episodes and were known to have specific anti-Plasmodium vivax antibodies before experimental infection [An interesting observation here is that human immune response appears to influence gametocyte appearance. As shown in Fig. nfection . A bettenfection , 30–32. P. vivax gametocyte maturation process and presents evidence supporting the role of asymptomatic sub-microscopic infections in malaria transmission. These results should be considered when new policies are envisioned to redirect malaria control strategies towards targeting asymptomatic infections as a tool for malaria elimination. Further longitudinal studies on asymptomatic volunteers, aimed at assessing roles of the host’s immune response and signalling pathways associated with the asymptomatic condition, are ongoing.This study provides preliminary evidence on the


Plasmodium vivax is the most frequent parasite. The objective of this analysis was to estimate the proportion of relapses in the burden of vivax malaria using the statistical rule stating that any case of vivax malaria occurring less than 90 days following a first episode is a relapse.Malaria is a public health problem in French Guiana. P. vivax malaria, and a total of 1,226 episodes of vivax malaria among which 559 were relapses (45.5%). For 194 patients having had falciparum malaria followed by vivax malaria it was estimated that 19% of the vivax episodes occurred less than 90 days following the falciparum episode and thus were possibly relapses due to the activation of latent hypnozoites. Despite the number of vivax cases and the number of relapses, there were only 28 recorded primaquine prescriptions .A total of 622 subjects were followed for 2,9 years with 336 first single episodes of P. vivax cases, many of which in children, are caused by latent hypnozoites, only a minority of them benefit from primaquine radical cure. The obstacles to this are discussed and suggestions are made to reduce the burden of vivax malaria in Camopi and other remote health centres in French Guiana.The present study points out that despite the fact that nearly half of the  Recently, data was presented suggesting that, in French Guiana, an interval under 90 days between two P. vivax infections was most likely a relapse, whereas over 90 days it was presumed to be a re-infection[Malaria remains a public health problem in French Guiana, a French overseas territory in South America-3. Ninete forest. Much ofe forest. Before nfection. In Frenst 2011. The children come to the health centre approximately once a month, generally for treatment or for prevention (i.e. vaccination). Every six months, a researcher verifies that all newborn children are included in the cohort, that all children are present in the village, and that all the malaria data is collected. Data for children for whom follow-up by the health centre had been interrupted was right censored at the interruption date. Given the isolation of Camopi and the fact that malaria diagnosis and health care is delivered free of charge, it was assumed that all malaria cases were recorded by the health centre.The study took place in Camopi, an Amerindian village in the Amazon forest along the Oyapock River, which delineates the border with Brazil. A retrospective open cohort study was carried out with data from all children born between January 1, 2001 and December 31There has been a recent malaria decline which was concomitant with a number of events: (i) the distribution of rapid diagnostic tests, starting in 2007, clearing the vegetation around homes starting in 2007(2); (ii) the “Harpie operation” involving over 1,000 soldiers and military police, which have, since March 2008, dismantled illegal mining sites, sent back arrested miners to their country, and destroyed on-site mining equipment, with possible effects on malaria transmission; (iii) insecticide-treated bed nets started being distributed in 2010; and (iv) the creation of the regional health agency which led to an intensification of trans-border cooperation.Plasmodium species identification were made in the Camopi health centre. Blood smears were first analysed in Camopi by a trained nurse and then cross-checked by the Parasitology Unit of Cayenne General Hospital, the reference centre for parasitological diagnosis in French Guiana. Malaria was defined as temperature > 38°C at the time of consultation or fever within the past 48 hours associated with Plasmodium asexual forms on a thin blood smear (detection threshold: 50 Plasmodium/μl). The treatment prescribed was also collected in the medical records.All clinical malaria episodes, their date of occurrence, P. vivax received an unsupervised three-day treatment of chloroquine . In Camopi, treatment with a combination of primaquine (14 days) and chloroquine (3 days) was supposedly initiated in 2005, following a first relapse. This prescription of primaquine implies respecting French prescription rules: systematic screening for G6PD deficiency (contra-indication if case of G6PD deficiency) this is performed in Cayenne and thus leads to delays in obtaining results; in addition prescription requires a nominative temporary use authorization from the state drug authority, which requires paperwork by the physician and delays between the transmission of the paperwork and the authorization. This procedure has been simplified in 2012 to reduce prescription delays. For primaquine prescription, the data from the medical records was cross-checked by looking at the list of patients having benefitted from a temporary authorization for the use of primaquine.During the study period, all confirmed cases of n where x is the fraction of patients experiencing n relapses[The number of vivax cases per year was retrieved with a differentiation between relapses according to the 90-day criteria . Incidence rates were calculated by age group, and by year using multiple failure models. In order to obtain the relapse rate, a graph was produced based on the fact that the relationship between the numbers of patients who experience one or more, two or more, three or more etc relapses is exponential summarized by the formula xAll parents were given an explanation and written consent was obtained for the study. The protocol was approved by the CCTIRS (Comité Consultatif pour le Traitement des Informations de Recherche en Santé), and the database was declared to the Commission Nationale Informatique et Libertés, the French regulatory authority.P. vivax malaria, and a total of 1,226 episodes of vivax malaria among which 559 were relapses (45.5%). For 194 patients having had falciparum malaria followed by vivax malaria it was estimated that 19% of the vivax episodes occurred less than 90 days following the falciparum episode and, thus, were possibly relapses due to the activation of latent hypnozoites.There were 622 subjects followed for a total of 2,9 years with 336 first single episodes of Figure vivax single infections and 559 relapses according to the 90-day criteria in 622 subjects in Camopi there were 28 recorded primaquine prescriptions . In 2011-2012, in French Guiana, there were approximately 2,000 malaria cases of which 75% were vivax malaria. During the same period there were 259 prescriptions of primaquine, thus 30% of the number of reported vivax cases.For a total of 1,226 P. falciparum infection could have activated latent hypnozoites[Although all vivax primary infections do not lead to subsequent relapses, calculations showed that around half would lead to subsequent relapses. Similarly, 19% of patients having had falciparum malaria had a vivax malaria episode within 90 days, suggesting the nozoites. In thisThis sobering observation should stimulate to propose solutions to fix the problem. First, the G6PD problem: Nearly all pregnant women from remote areas deliver in one of the three hospitals in French Guiana, including women from Camopi. Thus, neonates coming from malaria endemic areas could be easily screened for G6PD deficiency before hospital discharge, and their result written in their health book. Another alternative would be point of care testing, with the possibility of using Beutler’s fluorescent spot test on site, or to validate rapid G6PD tests on the field,9. FinalA large portion of the vivax malaria problem in these Amerindian children is linked to the activation of latent hypnozoites. Despite research aiming to differentiate primary infections from relapses,10, despThe authors declare that they have no competing interests.MN wrote the first draft & analyzed the data. AS BC DL provided data. MN AS CB MD DL PB MV BC participated in the data interpretation and final manuscript. All authors read and approved the final manuscript.


Plasmodium vivax is the most prevalent malaria species in the American region. Brazil accounts for the higher number of the malaria cases reported in pregnant women in the Americas. This study aims to describe the characteristics of pregnant women with malaria in an endemic area of the Brazilian Amazon and the risk factors associated with prematurity and low birth weight (LBW).P. vivax infection. Among women with only P. vivax malaria, the risk of preterm birth and low birth weight decreased in multigravidae . The risk of preterm birth decreased with higher maternal age  and among those women who reported higher antenatal care (ANC) attendance .Between December 2005 and March 2008, 503 pregnant women with malaria that attended a tertiary health centre were enrolled and followed up until delivery and reported a total of 1016 malaria episodes. More than half of study women (54%) were between 20–29 years old, and almost a third were adolescents. The prevalence of anaemia at enrolment was 59%. Most women (286/503) reported more than one malaria episode and most malaria episodes  were due to P. vivax is the prevailing species among pregnant women with malaria in the region and shows that vivax clinical malaria may represent harmful consequences for the health of the mother and their offsprings particularly on specific groups such as adolescents, primigravidae and those women with lower ANC attendance.This study shows that  Plasmodium vivax. According to the World Health Organization, the American region was responsible in the same year for nearly 20% of the total P. vivax malaria cases reported in the world [In 2011 the region of the Americas reported nearly half million malaria cases, 77% of them were due to P. vivax endemic areas and of them that 2.9 million pregnancies (3.1%) occurred in the American region [In 2007 it was estimated that 92.9 million pregnancies occurred in gnancies .1% occurn region .P. falciparum infection and disease, and the impact of P. falciparum infection on pregnancy outcomes, such as maternal anaemia, low birth weight (LBW) and prematurity are well documented, at least in sub-Saharan Africa [P. vivax infection can be associated with low birth and anaemia, as it has been described in Thailand, India and Papua (Indonesia) [P. vivax infection during pregnancy is associated with maternal anaemia, low birth weight, preterm delivery and miscarriage [It is well known that pregnant women have a higher risk for n Africa , 6. Therdonesia) –12. In tcarriage –18.The current study aims to characterize malaria among pregnant women from an endemic area of the Brazilian Amazon, and to assess in this population group the risk factors associated with poor pregnancy outcomes.The study protocol was approved by the Research Ethics Committee of the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD) (study registration No. 0836-05/2005) and the same committee approved an amendment to the protocol in 2010 (study registration No. 2013). We obtained informed written consent from all participants and from the guardians on behalf of the pregnant women less than 18 years old.The city of Manaus  is located in the Brazilian Amazon region, capital of the Amazonas state, northern Brazil. Manaus is one of the top six cities contributing to the Gross National Product, which together represent approximately 25% of the Brazilian Gross National Product. The population of the city of Manaus was of 1,802.525 inhabitants according to the 2010 census. It is the most populous city in the northern region and the seventh in Brazil. The majority of the population (99.5%) lives in urban areas . ApproxiP. vivax malaria in pregnant women consists of oral chloroquine  followed by weekly prophylaxis with chloroquine . During the study period weekly prophylaxis recommendation changed from getting started after the second P. vivax malaria episode during pregnancy to start after the first one. The treatment is completed with primaquine six months after delivery [The Brazilian National Malaria Control Program relies on early diagnosis and treatment of malaria cases in general population . Since 2delivery . The chldelivery . There idelivery .Plasmodium species confirmed by microscopy, were invited to participate in the study. The FMT-HVD is a tertiary health care centre that runs a passive case detection system (outpatient clinic) for pregnant woman among other curative services. After written informed consent was given, blood samples were collected for parasitaemia determination and anaemia screening. According to Brazilian national guidelines, all confirmed malaria cases received treatment, and clinical follow up was done on days (D) 3, 7, 14, 21, 28 and 35 after treatment initiation. After D35, follow up visits were scheduled every four weeks until delivery. In the event of a new malaria episode the woman initiated the same sequence of follow up visits again. All pregnant women with anaemia received ferrous sulphate and folic acid according to the Brazilian national guidelines.From December 2005 to March 2008, pregnant women of any gestational age attending to the outpatient clinic of the FMT-HVD, and diagnosed with malaria due to any Information on the number of antenatal visits, date of birth, birth weight, gender of the newborn, and the occurrence of any congenital malformation was retrieved through the Live Birth Information System (SINASC) . Birth wPlasmodium species in peripheral blood of any density by microscopy in pregnant women with symptoms. For the purpose of the analysis, the duration of any single malaria episode was estimated as 28 days, in order to distinguish between the same episode and a new one. Low birth weight was defined as a birth weight less than 2,500 g. Low birth weight was classified, in turn, as very low birth weight , and as extreme low birth weight . For the analysis of birth weight in relation to gestational age, Brazilian standardized growth curves [th percentile, appropriate for gestational age when it was between the 10th and the 90th percentiles, and small for gestational age when it was below the 10th percentile [A malaria case was defined as the presence of asexual forms of any h curves . Sex speh curves . NewbornP. vivax during pregnancy and 503 women with any Plasmodium species during pregnancy. The comparison between the proportions in the group with and without prematurity and LBW, respectively, was validated by the chi-square statistical test. To calculate the odds ratio (OR) logistic regression analysis was used. To evaluate the association between maternal factors, and prematurity and LBW multivariate analysis was performed using backward stepwise logistic regression, removing variables with a p value of 0.05. All episodes occurred during pregnancy were included and were further categorised in single episode, two episodes or at least 3 episodes during pregnancy.All clinical and laboratory data from patients were collected through standardized questionnaires and recorded in Epi Info software . Analysis was performed using STATA software . The analysis was stratified in two groups: 356 women with only Between December 2005 and March 2008, a total of 583 pregnant women were enrolled in the study at the FMT-HVD, of which 503 were finally included in the analysis .See Plasmodium vivax infection frequency in the first malaria episode during the pregnancy was 80.5% (395/491), followed by Plasmodium falciparum , and mixed P. falciparum and P. vivax infection . Most women  reported two or more malaria episodes during the same pregnancy. The 503 study women reported a total of 1016 malaria episodes (ranging from one to eight episodes per woman). Most episodes were due to P. vivax , followed by P. falciparum , and mixed P. falciparum and P. vivax infection . Most women  had only malaria due to P. vivax during pregnancy. The frequency distribution of P. vivax in the first malaria episode during pregnancy among pregnant women increased over the study period from 76.8% (199/259) in the first half of the study to 84.5% (196/232) in the second half of the study. The frequency of P. falciparum decreased from 20.9% (54/259) in the first half of the study to 15.1% (35/232) in the second half over the same period.P. vivax malaria during pregnancy, the risk of premature delivery decreased with higher maternal age at enrolment , higher parity  and in those women with higher attendance to ANC . Maternal anaemia at enrolment was significantly associated with an increased risk of premature delivery  in the univariate analysis but this was not confirmed in the adjusted analysis.Plasmodium species   see .P. vivax malaria during pregnancy, the risk of LBW decreased with higher parity .Plasmodium species, the risk of LBW was highest among primigravidae and in those women with anaemia at the time of enrolment  attended to ANC at least four times during pregnancy, according to World Health Organization recommendations, which is consistent with the antenatal care coverage data reported in the area. Nevertheless more than a quarter of them had less than the recommended number of visits, and this was exactly the group who showed the greater risk of suffering the negative effects of malaria during pregnancy. The reasons behind poor utilization of antenatal care services in this area are still unknown. The health seeking behaviour, barriers to access and use of health services, or other determinants associated with economic, environmental, or cultural factors could explain poor compliance.Plasmodium infection. This screening was performed monthly until delivery allowing the detection of any subsequent infection, regardless the presence of symptoms. Secondly, the study women followed weekly chemoprophylaxis with chloroquine until delivery. The positive effect of this intervention on poor pregnancy outcomes, such as LBW and maternal anaemia, and prevention of relapses, has been demonstrated in several studies, yet none of the studies included pregnant women from Latin America [The total of malaria episodes included in this study (n = 1016) represented 67.9% out of the 1496 malaria episodes in pregnant women reported in the Manaus area during the study period [ America , 34. It P. vivax malaria, the frequency of relapses in control groups (no primaquine) was 20%, but this review did not include data from pregnant women or from Latin America [P. vivax malaria since radical treatment of P. vivax infections with primaquine is contraindicated during pregnancy.Interestingly, this study reveals that more than one third of the study women (36%) had more than one malaria episode during the same pregnancy despite the weekly chemoprophylaxis with chloroquine. The contraindication during pregnancy of radical cure with primaquine due to safety issues, putting women at a higher risk of subsequent episodes, as well as the chloroquine resistance reported in the area may explain this finding. To date, no study has shown if pregnant women have an increased risk of relapses compared to non-pregnant populations. In a Cochrane meta-analysis aimed to compare alternative primaquine regimens for preventing relapses in people with  America . AlthougP. vivax malaria can be a considerable cause of maternal morbidity, which is consistent with previous studies from this and other P. vivax endemic areas [The prevalence of maternal anaemia found in this study 59%) was considerably higher compared with the prevalence of anaemia found among women attending routine ANC from two studies conducted in the area in women with similar characteristics attending the routine antenatal care, being at 26% in 2008  and at 3% was conic areas , 16.P. vivax gradually prevailed over P. falciparum, are aligned with the change in the pattern observed in general population in this region [P. vivax malaria have not changed in recent years in Brazil [P. falciparum malaria in 2006.The change in the distribution of species observed over the study period, showing that s region . This aln Brazil . Thus, tPlasmodium sp. infection, each episode of malaria exposes the mother to the malaria paroxysm, which may be associated with increased uterine activity and thus triggering a premature labour [P. vivax paroxysm have shown all the cellular and noncellular mediators involved [P. falciparum placental malaria, and to be embryotoxic, cause necrosis of the implanted embryo, and increase the risk of uterine contraction resulting in foetal expulsion [The prevalence of LBW seen was similar to that observed in the general population (8.1%), which is similar in terms of demographic and social characteristics to that in our study . LBW wase labour . The stuinvolved . One of xpulsion .th and 90th percentiles), being only few of them (9%) below the 10th percentile (IUGR). This is in smaller than IUGR rates reported in other Brazilian studies (13%) [P. vivax predominates, prematurity appears to be among the main detrimental outcomes of malaria in pregnancy, differently to what occurs in P. falciparum areas where transmission is stable and LBW is the main outcome associated with malaria infection in the pregnant woman followed by pre-term birth.In this study, as shown in es (13%) . Hence, There are studies that indicate that male and female foetuses behave differently upon the same adverse maternal environment, resulting in different foetal and neonatal morbidity and mortality outcomes . StudiesP. vivax malaria on pregnancy outcomes, as a comparison group of non-infected women were not enrolled.A limitation of this study was that birth weight was obtained from the SINASC system, however, among a subsample of newborns, there was a high correlation between this weight and that collected from the newborn card by study staff. Gestation age was established by ultrasound in 61.2% (295/482) and it is known that last menstrual period is subject to reporting bias. Other poor pregnancy outcomes in this study population such as miscarriage and stillbirth are reported elsewhere . Also, iP. vivax is the prevailing species among malaria cases in pregnant population in the region and shows that P. vivax malaria in pregnancy may represent detrimental consequences for the health of the mother and their offsprings particularly for specific groups such as primigravidae, adolescents, and those with lower attendance to antenatal services with P. vivax clinical malaria during pregnancy. These groups deserve special attention in the design and deployment of preventive and curative strategies for malaria control during pregnancy.In conclusion, this study shows that


Plasmodium falciparum malaria cause substantial morbidity in Sub-Saharan Africa, especially as co-infecting pathogens. We examined the relationship between presence of P. falciparum DNA in plasma samples and clinical malaria as well as the impact of atazanavir, an HIV-1 protease inhibitor (PI), on P. falciparum PCR positivity.HIV-1 and P. falciparum 18s rRNA gene to detect the presence of malaria DNA in 215 of the 221 participants enrolled in Blantyre and Lilongwe, Malawi. We also studied the closest sample preceding the first malaria diagnosis from 102 persons with clinical malaria and randomly selected follow up samples from 88 persons without clinical malaria.ACTG study A5175 compared two NNRTI-based regimens and one PI-based anti-retroviral (ARV) regimen in antiretroviral therapy naïve participants. We performed nested PCR on plasma samples for the PCR positivity was observed in 18 (8%) baseline samples and was not significantly associated with age, sex, screening CD4+ T-cell count, baseline HIV-1 RNA level or co-trimoxazole use within the first 8 weeks. Neither baseline PCR positivity (p = 0.45) nor PCR positivity after initiation of antiretroviral therapy (p = 1.0) were significantly associated with subsequent clinical malaria. Randomization to the PI versus NNRTI ARV regimens was not significantly associated with either PCR positivity (p = 0.5) or clinical malaria (p = 0.609). Clinical malaria was associated with a history of tuberculosis (p = 0.006) and a lower BMI (p = 0.004).P. falciparum DNA was detected in 8% of participants at baseline, but was not significantly associated with subsequent development of clinical malaria. HIV PI therapy did not decrease the prevalence of PCR positivity or incidence of clinical disease. Plasmodium falciparum malaria are two of Sub-Saharan Africa’s major contributors to morbidity and mortality. Although the interaction between the two diseases was initially dismissed [HIV-1 and ismissed , more coismissed –7.P. falciparum infection never fully develops as individuals remain susceptible to both clinical disease and asymptomatic infections [Although the majority of deaths from malaria are in children under 5 years of age, pregnant women and immunocompromised individuals (including those with HIV-1 infection) are also at increased risk for poor outcomes . Adaptivfections . This asfections ,11, othefections . Very fefections ,14.P. falciparum in tissue culture [in vivo has not yet been fully delineated. In one recently completed adult clinical trial comparing nevirapine-based (NNRTI) treatment to ritonavir-boosted lopinavir-based (PI) treatment, lopinavir therapy was not associated with lower rates of malarial antigen detection or clinical disease [HIV protease inhibitors (PIs) are in wide use for treatment of HIV infection in the US and Europe, but in resource limited settings, PIs are primarily reserved for second-line therapy. Although PIs inhibit replication of  culture , their i disease ,17. Howe disease . In thisIn the present study, we used a highly sensitive nested polymerase chain reaction (nPCR) to determine whether subpatent parasitemia was a predictor of clinical malaria and if treatment with atazanavir was associated with lower rates of subpatent parasitemia or clinical disease in study participants recruited to a clinical trial of antiretroviral therapy that was conducted in Malawi.IRB approval and informed consent were obtained from all participants in the parent study (see reference below). The UCSD Human Research Protections Program approved the use of the already collected patient samples in the ACTG study for this study.3 counts of < 300 to lamivudine/zidovudine plus efavirenz, emtricitabine/tenofovir-DF plus efavirenz, or didanosine plus emtricitabine plus atazanavir (not ritonavir boosted) [P. falciparum parasitemia on Malawian study enrollees. Cases of malaria were prospectively defined as ‘confirmed’ when study participants presented for clinical care with a compatible clinical syndrome  and were found to have Plasmodium sp. parasites on a peripheral blood smear and as ‘probable’ when study participants presented with a compatible clinical syndrome and specific treatment for P. falciparum was initiated or recommended without laboratory confirmation by a positive blood smear.PEARLS  randomized treatment-naïve individuals with CD4+ T-cells/mmboosted) . PEARLS P. falciparum parasitemia is illustrated schematically in P. falciparum DNA . Baseline samples were defined as those obtained prior to the initiation of antiretroviral therapy (N = 113) or within 8 weeks following entry into the clinical trial (N = 102). Two participants were removed from the analysis because they had active malaria at entry into the ARV study. Among the 18 study participants with positive baseline samples, another sample obtained 4–12 weeks later was requested to test for persistence of subpatent parasitemia. For the 122 participants who had a clinical malaria diagnosis during the study, PCR testing for subpatent parasitemia was also performed on the closest available specimen prior to the first diagnosis of clinical malaria. These samples were drawn an average of 31 days (range 1–60 days) prior to the malaria diagnosis. For those who never had a clinical malaria diagnosis , a 2-stage restricted sampling design was used to control for 2 potential confounding factors: time of year  and exposure time to ARVs. Time since ARV initiation was divided into four intervals: <12 weeks, 12–24 weeks, 24 - < 96 weeks, and 96+ weeks. “Early” co-trimoxazole use refers to administration in the first 8 weeks of study.Plasma samples were obtained prior to study entry and at regular intervals during follow-up based on the sampling schedule dictated by the clinical trial. The sampling strategy for the evaluation of P. falciparum detects a 205bp portion of the 18s rRNA gene. A nested PCR reaction was performed on 40μL of supernatant from the extraction in a 50μL PCR reaction (2.5 U Taq polymerase (Invitrogen), 3mM MgCl2, 500nM dNTPs (Fermentas), and 50 pmol of each primer (IDT). In the first round of the nPCR, the gene was amplified using primers fPLU: 5’-TTA AAA TTG TTG CAG TTA AAA CG-3’, and rPLU: 5’-CCT GTT GTT GCC TTA AAC TTC-3’. Denaturation was performed at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, and elongation at 72°C for 2 minutes, with a final extension at 72°C for 5 minutes. 2μL of the outer PCR product was used as the template for the inner PCR reaction  using primers fFAL: 5’-TTAAACTGGTTTGGGAAAACCAAATATATT-3’ and rFAL: 5’-ACACAATGAACTCAATCATGACTACCCGTC-3’. For the inner PCR, denaturation was performed at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, and elongation at 72°C for 1 minute, with a final extension at 72°C for 5 minutes. The PCR products from the inner PCR were run on a 1% agarose gel  and stained with ethidium bromide. The PCR result was qualitative and could detect parasite DNA in the plasma down to 1.8 parasite genomes/5μL of extracted DNA. The sensitivity threshold was defined by performing serial dilutions starting from a known parasitemia, followed by DNA extraction and then PCR.200μL of plasma were used to extract DNA using Qiagen Mini Blood Kit (Qiagen) as per manufacturer’s instructions. The nested PCR (nPCR) for Categorical variables were compared by Fisher’s exact test and continuous variables were compared with Wilcoxon Rank-Sum Test. Univariable logistic regression examined the association between subpatent parasitemia at baseline (or during follow-up) and clinical malaria diagnosis during study follow-up. To control for confounders, effect modifiers, and other prognostic factors, multi-variable logistic regression was used including covariates identified as associated with either development of clinical malaria during follow-up, or with subpatent status. Adding subpatent status tested for independent association provided by a plasma DNA finding at baseline. Since all of the participants placed on atazanavir were switched to NNRTI therapy after a median of 102 weeks in the trial and some participants were crossed over from NNRTIs to PIs or vice versa due to HIV outcome failures over the course of the study, the effect of PI therapy on subpatent parasitemia and clinical malaria outcomes were investigated using an as-treated analysis. Because we did not see any effect of drug therapy on subpatent parasitemia or clinical malaria outcomes, an intent-to-treat analysis was used for the remainder of our analysis.Of the 219 participants analyzed, 147 (67%) were female and 72 (33%) were male and the mean age was 33 (range 18–65). PCR positivity was observed in 18  of 215 baseline samples. There was a trend toward a greater likelihood of PCR positivity in those with a lower BMI (p = 0.114) and lower hemoglobin levels (p = 0.066). There was no significant association between PCR positivity and early cotrimoxazole use, age, gender, CD4+ T-cell count or plasma HIV-1 RNA levels. None of the 37 participants with a prior tuberculosis diagnosis were found to have PCR positivity (p = 0.027) .Of the 219 participants, 56% (122) developed probable (28/122) or confirmed (94/122) clinical malaria. Neither PCR positivity at baseline (p = 0.45) nor during follow-up visits (p = 1.0) was associated with the subsequent development of confirmed or probable malaria over the course of the study. 50% of those with PCR positivity at baseline had a bout of clinical malaria within the first 24 weeks while 24% of those without PCR positivity at baseline had clinical malaria in the same time period . The sensitivity of PCR positivity for predicting clinical malaria in this interval was only 16% (95% CI: 8–23).PCR positivity was detected in only 7 (6.9%) of the closest plasma samples collected prior to the onset of the first bout of clinical malaria in 102 individuals during the trial. Among the 88 controls that were selected because they did not experience clinical malaria during the study, PCR positivity was detected at the same frequency (6.8%) , furtherPCR positivity persisted after the initiation of antiretroviral therapy. If a baseline sample was PCR positive, we continued testing follow up samples from the same individual until they were no longer positive. Of the 18 positive samples at baseline, a follow-up sample was available for 17; 76% (13/17) of these samples remained positive (follow-up samples obtained 4–8 weeks after baseline). Among the 13 remaining positive at the second determination, only 30% (4/13) remained positive when a third sample collected 1–8 weeks later was tested. The patterns of persistent PCR positivity were not associated with NNRTI use (p = 1.0) or early co-trimoxazole use (p = 1.0). Persistent positivity was not associated with a diagnosis of clinical malaria over the course of the trial .A total of 122 of the 219 participants (56%) had at least one probable or confirmed malaria diagnosis during the course of the three-year study, for a total of 238 clinical events. Clinical malaria (confirmed or probable) was observed more frequently in those with history of tuberculosis  and with a lower body mass index . Those using cotrimoxazole in the first 8 weeks were more likely to develop clinical malaria  but this association did not persist once adjusted for baseline BMI and TB history. Other baseline characteristics including CD4+ T-cell count, plasma HIV-1 RNA level and hemoglobin levels were not significantly associated with development of clinical malaria during the study . ConfirmP. falciparum co-infection influences the morbidity and mortality of HIV disease and vice versa. As additional data have accumulated it appears that some of the contradictory findings fueling this debate have been driven by the dynamic and complex interplay among these two pathogens and the human immune system and are heavily influenced by the underlying prevalence of each of the two pathogens in a given population [3, it is not surprising that about half of the participants had documented clinical events and some of them had more than 1 event over the course of the trial. Our primary goal was to determine whether the detection of P. falciparum DNA in plasma samples obtained from those with relatively advanced HIV-associated immunodeficiency was a predictor of clinical malaria or, alternatively, that PCR positivity in the absence of clinical disease identifies partially immune individuals who have the capacity to immunologically control the organism.Over the past decade, there has been substantial discussion and debate about the extent to which pulation ,21. We spulation . The incpulation . Given t3[P. falciparum DNA in the plasma at study entry and development of clinical malaria were more common in those with a lower BMI. PCR positivity at study entry was also more common in those with lower hemoglobin levels and in those with no prior history of TB. In contrast, development of probable or confirmed clinical malaria was positively associated with a history of TB. Although these seemingly contradictory findings are driven by relatively small numbers of patients, one could speculate that those without a prior history of tuberculosis were more immunologically competent and, thus, able to maintain P. falciparum in a subpatent state while those with a prior history of tuberculosis were more likely to develop clinical disease when they were exposed to the parasite.In our study we found that PCR positivity was not significantly associated with subsequent development of clinical malaria. Neither lower pre-entry CD4+ T-cell counts nor higher plasma HIV-1 RNA levels at study entry were significantly associated with either PCR positivity or clinical malaria. While other studies have documented an association between lower CD4+ T-cell counts and increased episodes of malaria, these studies included patients with a relatively wide range of CD4+ T-cell counts and ours was restricted to candidates for antiretroviral therapy and composed only of those with CD4+ T-cell counts below 300/mm3. We did et al, demonstrated that several PIs, including atazanavir, reach concentrations in the plasma that exceed the EC50 for anti-malaria activity in vitro [in vitro, supporting the possibility that PIs reach levels in the range required for anti-malarial activity [in vitro (2.5–11.6 μM) [in vitro. In a recently reported study of 745 HIV-infected African women, ritonavir boosted lopinavir did not decrease the incidence of clinical malaria or subpatent parasitemia  [In addition, we did not observe an association between HIV protease inhibitor therapy and PCR positivity or clinical malaria in our small study. Andrews, in vitro . Sera fractivity ,26. Atazactivity  overlapp11.6 μM) , though en test) ,17. In aen test) . This reP. falciparum DNA in this population of HIV-1 infected adults residing in a hyperendemic area for malaria was not significantly associated with subsequent clinical disease. Our study has a number of important limitations. First, the study was retrospective using available samples from a previously completed antiretroviral drug study. Although many cases of malaria were reported among the Malawi participants, plasma samples were not routinely collected at the time of the malaria episode. The samples available were collected at study entry and at regular intervals during the study for the purpose of assessing the efficacy of antiretroviral therapy. Although we did not see an association between parasitemia and the amount of time elapsed to subsequent diagnosis of clinical malaria, the samples were collected, on average, 31 days prior to the malaria diagnosis. The low prevalence of baseline and follow-up PCR positivity and clinical malaria cases limited the statistical power to detect the effect of the PI-based therapy. Lastly, we had access to frozen plasma samples, not blood spots. Although others have previously reported that parasite DNA can be found in the plasma of patients with clinical malaria [P. falciparum infection has not been previously reported. The quantitative and qualitative relationships between levels of detection of P. falciparum DNA in blood (or dried blood spots) compared to plasma have not been extensively studied. However, in one study using quantitative PCR, parasite DNA was detected in blood in 3.5-fold more samples than in plasma [P. falciparum infection on HIV-1 and vice versa have been conducted using clinical evidence of malaria as a marker for P. falciparum. Larger scale longitudinal studies of PCR positivity and of clinical disease in regions of varying malaria endemnicity, in additional HIV-infected populations, are needed to more conclusively examine the clinical implications of PCR positivity and of the impact of PCR positivity on the natural history of HIV-1 infection. Finally such studies, when combined with more focused studies of malaria-specific immunity, could provide important insights into the immunological correlates of protective immunity.In conclusion, detection of  malaria ,29, the n plasma . Althoug


P. vivax (over 85%) at ANC and DU. Peripheral parasitemia was significantly associated with fever within past week, rural origin of subjects, and first/second pregnancies in multivariate analysis, with the highest risk factor associated with fever followed by rural residence. Strikingly in cohort, anaemia was prevalent in 86% at ANC as compared to 72% at DU, whereas severe anaemia was 13.6% and 7.8% at ANC and DU. Even more anaemia prevalence was observed in MIP group (88% and 89% at ANC and DU), whereas severe anaemia was 23% and 21%, respectively. In view of observed impact of anaemia, parasitemia and asymptomatic infection of P. vivax during pregnancy and delivery suggest prompt diagnosis regardless of symptoms and comprehensive drug regime should be offered to pregnant women in association with existing measures in clinical spectrum of MIP, delivery, and its outcome.The escalating burden, pathogenesis, and clinical sequel of malaria during pregnancy have combinatorial adverse impact on both mother and foetus that further perplexed the situation of diagnosis, treatment, and prevention. This prompted us to evaluate the status of population at risk of MIP in Hazaribag, Jharkhand, India. Cross-sectional study was conducted over a year at Sadar Hospital, Hazaribag. Malaria was screened using blood smear and/or RDT. Anaemia was defined as haemoglobin concentration. Pretested questionnaires were used to gather sociodemographic, clinical, and obstetrical data. The prevalence of MIP was 5.4% and 4.3% at ANC and DU, and 13.2% malaria was in women without pregnancy. Interestingly, majority were asymptomatically infected with Plasmodium falciparum and Plasmodium vivax, is responsible for 515 million clinical cases [ Plasmodium vivax, the most widespread parasite causing human malaria, is responsible for estimated 130–435 million infections annually and is the major cause of malaria in most of Asia and Latin America [ P. vivax infection is commonly considered to be much more benign than Plasmodium falciparum infection, historical evidence suggests significant mortality associated with P. vivax malaria in the preantimalarial era [ P. vivax malaria has been increasingly recognized over the past few years [ P. vivax whereas some buffering, bordering, and adjoining regions have lower prevalence of P. falciparum and mixed infection. The other human infecting Plasmodium parasites, like P. ovale, P. malariae, and P. knowlesi, are the rarest in Indian isolates and these parasites neither were observed during our investigation nor have been reported previously from Jharkhand.Malaria in tropical regions, which is caused by the protozoan parasitesal cases . Plasmod Plasmodium infections than their nonpregnant peers. The adverse outcomes of these infections are primarily felt by primigravidae [The emergence and spread of drug resistance to commonly used chemotherapeutics are major factors contributing to this increasing burden and most of the mortality and morbidity are borne by children and pregnant women. Pregnant women and their infants are susceptible to common and preventable infectious diseases including malaria but are woefully left unscreened and untreated. According to an estimate, approximately 125 million pregnant women worldwide are exposed to the risks of malaria in pregnancy (MIP) each year, resulting in 200,000 infant deaths . Every ygravidae , 11, altgravidae .In spite of severe and fatal consequences of malaria during pregnancy for the mother, foetus, and newborn child, the harmful effects can be substantially prevented and reduced  either bP. falciparum malaria during pregnancy is a well-known cause of maternal and fetal morbidity and mortality. Although P. vivax infection has received less attention than P. falciparum infection, it is clearly an important contributor to both maternal anaemia and low birth weights [ P. vivax malaria is endemic [ P. vivax infection during pregnancy has been recognized for many years [ P. vivax infection were more commonly anaemic and delivered lower birth weight neonates, compared with uninfected women, but the effects were less pronounced than those associated with P. falciparum infection [ P. vivax infection was most common during the first pregnancy, and the prevalence of such infection peaked early during the second trimester. weights –23 where endemic . Althougny years , the impnfection , 22. In  P. falciparum and P. vivax are associated with adverse pregnancy outcomes, these studies primarily focused on symptomatic pregnant women infected with vivax [ vivax associated malaria during pregnancy, particularly from Jharkhand, an understudied and tribal dominant region with perennial malaria transmission zone where malaria is rampant and causing sizable annual malaria deaths, second to Orissa in India as per the latest observations published by Dhingra et al. [Limited and past MIP studies in India have demonstrated the important contribution of malaria to maternal and neonatal morbidity and mortality , 23, 24.th vivax , 25. Rela et al.  and Hussa et al. , which r vivax infection during pregnancy in India, it prompted us to investigate with an objective to better define the estimate of MIP, the prevalence of asymptomatic malaria, and the relative contribution of P. falciparum and P. vivax during pregnancy and at delivery. To the best of our knowledge, such profile, epidemiological association, and clinical correlation have not been investigated before on isolates of malaria in pregnancy from Hazaribag, Jharkhand, among malaria endemic regions of India. Most significantly, our investigation will be the first report attempting to evaluate the interplay among anaemia, pregnancy, and asymptomatic malaria, stratified according to clinical groups in adult population residing in a perennial transmission zone with a codominance of P. vivax and P. falciparum prevalent region. Thestudy was conducted at Hazaribag in the state of Jharkhand in east India, with the ultimate goal of enhancing the development of evidence-based policies to reduce the burden of disease due to MIP in this region of India.Thus, in view of the limited information on asymptomatic and P. falciparum, P. vivax, and mix infection accounting for 44%, 44%, and 7% of the cases, respectively [ P. falciparum, P. vivax, and mix infection, respectively [This study consisted of cross-sectional surveys conducted in three units, that is, antenatal care units (ANCs), delivery units (DUs), or the inpatient antepartum ward of Sadar Hospital in Hazaribag districts of Jharkhand, India . Jharkhaectively . The proectively . The preHazaribag  is selected to represent a rural-cum semiurban district with low but perennial transmission of malaria. Hazaribag had a yearly average SPR of 7.3% for symptomatic individuals over the last three years, with P. falciparum, P. vivax, and mix infection accounting for 14%, 73%, and 13% of the cases, respectively [http://dx.doi.org/10.1155/2015/740512). Most interestingly, with the monthly climatic temperature when compared with monthly malaria episode, we observed significant correlation between ambient temperature and subsequent rise and fall in malaria episode as shown in Supplementary Figure-1B. The recent (2010–2012) data on malaria epidemiology has been analyzed during investigation in this project and we observed the increasing trend of malaria episodes as shown in Supplementary Figure-2A-C, despite consistent interventions and preventive measures implemented by various national and international bodies.ectively . The majThus, the selected study district is meant to provide a representation of typical conditions that would be found in malaria endemic districts of Jharkhand.The District Level Household and Facility Survey conducted between December 2007 and April 2008 revealed that 56% of women had at least one antenatal clinic (ANC) visit and 18% overall had institutional deliveries including 59% in urban areas but only 13% in rural settings . Sadar HThe study had two components with recruitment targeted to all the women presenting to antenatal care unit (ANC) and delivery units (DUs). For the ANC component, pregnant women aged ≥17 years who reported to the study site for routine care were screened and enrolled; those were willing and consented to participate in our study. For the DU component, women aged ≥18 years who presented for delivery and were willing to provide written informed consent were enrolled. Inclusion in the study protocol was based on the considerations like residency and availability status in the study region, no history of hereditary diseases and/or no known severe disease at the time of conceiving and/or at first ANC attendance, voluntary and consented participation in our study, and no immediate illness due to other infectious diseases or malaria in preconception and/or during present pregnancy at the time of first attendance at ANC. Exclusion from study was based on either refusal to give signed consent or unwilling for sampling, clinically suspected or identified cases of HIV and hepatitis B infection, and stringently those who are apparently and at first sight so weak due to unknown reason compounded by pregnancy that may not sustain sampling stress and may lead to undesired complications. P. vivax, P. falciparum, and mix infection, respectively, whereas at DU, we found 32, 2, and 3 cases of P. vivax, P. falciparum, and mix infection, respectively, at Sadar Hospital, Hazaribag , reproductive history including gravidity, history of fever and antimalarial drug use, and use of antimalarial prevention measures. A complete physical examination including the determination of gestational age was assessed by palpation of uterine fundus height combined with information on last menstrual period; measurement of auxiliary temperature with digital thermometer and other vital signs was also performed. Peripheral venous blood (3–5 mL) was collected from all the attendees for malaria blood film preparation, rapid diagnostic test (RDT), and haemoglobin determination apart from other biochemical and molecular investigations. Women with positive RDT results or who were anaemic were referred immediately to the hospital physician for treatment. The hospital staffs were informed of additional parasitaemic individuals identified through blood smears so that they could be appropriately treated.Pregnant women enrolled at the DUs were interviewed, with data collection focused on sociodemographic and anthropometric characteristics, obstetric complications, history of fever and antimalarial use during pregnancy and the use of antimalarial prevention measures, birth outcome, and mode of delivery. Peripheral venous blood (3–5 mL) was collected after delivery for malaria blood film preparation and/or rapid diagnostic test (RDT) and haemoglobin determination apart from other biochemical and molecular investigations. Women with positive RDT or blood smear results were referred for treatment. Apart from malaria prevalence study in DU, we have also collected clinical and demographic data and samples based on the mode of delivery, that is, normal, caesarean, and stillbirth delivery, and further on the mode of birth/delivery outcome, that is, preterm, postterm, and term delivery; details were presented in Supplementary Table-1. To assess the gestational age, we mainly adopted the simplest method, that is, symphysis-pubis fundal height (SFH) measurement , most widely used method over the globe especially in resource poor settings like ours. Assessments were performed by trained nurses followed by gynaecologist. However, in case of any undesired measurement or dought over positioning of foetus, they were confirmed by ultrasound to record the gestational ages.μL of blood [ Plasmodium species. All slides were cross-checked using stringent diagnostic criteria to diagnose Plasmodium infection with our trained technical staff. The commercial (RDT kit) First Response Malaria pLDH/HR2 combo test kits  were also used as per the manufacturer's guideline as a screening tool for diagnosing malaria in pregnant women. We have used the PCR technique also to diagnose malaria but in selective samples not in all the samples due to budgetary constraint. The selective samples were all the MIP positive samples at ANC and DU verified by PCR, those subjects who were disputed on microscopy and RDT also verified by PCR, and clinically most suspected cases with strong sign and symptoms but microscopically negative samples were also verified by PCR.Thick and thin smears prepared from peripheral blood of ANC and DU subjects were Giemsa-stained and examined under high power. The parasite density was evaluated by counting the number of asexual forms of parasites for every 200 leukocytes, assuming a leukocytes count of 8000 leukocytes/of blood . The thiHaemoglobin (Hb) levels were recorded at the first ANC and DU visit. Determining the concentrations of haemoglobin (Hb) was performed in peripheral blood samples using a portable HemoCue haemoglobinometer  as stated by the manufacturer. The concentration of Hb was recorded on the study questionnaire and double-checked by the laboratory technician. Women were classified as anaemic (Hb < 11 g/dL) and then categorized as being moderately to severely anaemic, with haemoglobin <8 g/dL and <7 g/dL, respectively, as the primary outcome, and being mild to nonanaemic (Hb ≥ 9 g/dL) according to , 34.2. A documented fever was defined as an auxiliary temperature ≥37.5°C.Severe malaria was defined as a malaria attack associated with any of the following: cerebral malaria, severe anaemia, renal failure, pulmonary oedema, hypoglycaemia, shock, spontaneous bleeding, or repeated convulsions . MaternaAll human blood samples used in this study were collected after obtaining written consent from the study participants under protocols activities approved by the Institutional Ethics Committee (IEC) of the Vinoba Bhave University, Hazaribag, Jharkhand, and human ethical guidelines as reflected in the guidelines of the Medical Ethics Committee, Ministry of Health, Government of India. Present study does not involve any minor/children. Thus, signed and written approval was given by adult subject herself. All study participants were included only after informed consent. The study protocol and consent proposal are approved from IEC, VBU, having memo number VBU/R/888/2012, dated 05-06-2012.t-test was used for evaluating significance for normally distributed data and when data were not normally distributed; nonparametric tests (Mann-Whitney U) test were used to analyze the data. Categorical data are presented as frequency counts (percent) and compared using the Chi-square or Fisher's exact statistic as appropriate. Continuous data are presented as means (± standard error) and compared using the t-test or analysis of variance as appropriate. The age of the recruited subject was between 18 and 37 years, whereas mean age was 26.7 years. We have presented participants' ages in ranges based on their responses (Supplementary Table-1). Risk factors for either P. falciparum or P. vivax parasitemia were evaluated by univariate analysis and then adjusted for significant predictors in multivariate analysis. Simple and multiple logistic regressions were used to analyze potential risk factors associated. Precisely, to investigate the association between the various independent variables  and malaria parasitemia, we began by performing simple logistic regressions with each independent variable. Next, we applied multiple backward logistic regression models and all covariables present in univariate were kept in model, independent of their significance, in univariate analysis due to their possible relevance in the final results; thus, we could analyze their possible influence when considered together with the other variables. Similar strategies were followed for factors associated with haemoglobin and anaemia during pregnancy and malaria in pregnancy; risks were assessed using haemoglobin or anaemia as dependent variables and all other factors as independent variables. The differences were considered statistically significant when the p value obtained was <0.05.All clinical, demographic, and anthropometric information were carefully checked for correctness and inconsistencies were resolved before analysis. Data were entered in MS-Excel and analyses were performed using SPSS version 16  and Graphpad Prism version 5.0 . For comparisons of means between two groups of subjects, Student's Most pregnant women attending ANC were in the 18 to 38 years of age range and had some level of formal education (Supplementary Table-1). The vast majority of participants were Hindi speaking (97.6%) and nonsmoking (98.7%). Most owned their own home (75.4%) and were engaged in household work (76.7%) with a small proportion involved in farming (12.3%). They had attended a median of one ANC visit (range 0–9) during their current pregnancy and almost one-third of the attendees were primigravidae (33.3%). Slightly more than half of participants presented to the ANC in the latter half of pregnancy whereas 44.6% presented prior to 20 weeks. Less than half of the participants reported taking iron/folate supplements (46.3%) while 33.2% were taking multivitamins. In terms of malaria prevention activities, most pregnant women reported having untreated bed nets in their homes and using them recently, but very few had ITNs (Supplementary Table-2). Similarly, only 9 of the women were taking prophylaxis for malaria and most of them  were unable to identify the drug they were taking and the rest (two), who were able to identify the drug, were taking chloroquine.μL . P. falciparum was identified in 4.4% of parasitaemic individuals while P. vivax was found in 86.8% and 8.8% of infections were mixed. Peripheral parasitemia was over four times more likely among women living in rural areas when compared with those from urban or semiurban subjects  and among primigravidae and secundigravidae relative to multigravidae . Parasitaemia was more commonly encountered in pregnant women who had a history of fever within the week prior to enrollment or were febrile at the time of the study visit . The majority of positive malaria tests occurred from July to January with the greatest number in between August and October, corresponding to the monsoon season. Further multivariate analysis was performed in order to identify the association between specific demographic, socioeconomic, and malaria prevention activities and the risk of parasitemia. Among pregnant women attending ANCs, first/second pregnancies, fever in the past week, and residence in rural areas were significantly associated with peripheral parasitemia as shown in A positive diagnostic test for malaria was obtained in 5.4% (68/1271) of the total cohort . Blood s P. falciparum was identified in 5.4% (2/37), P. vivax in 86.5% (32/37), and mixed infection in 8.1% (3/37). The mean density of parasitemia in the women with positive blood smears was 16,395 asexual forms/μL . The peripheral parasitemia density was significantly higher in primigravid women than in those who had one or more prior pregnancies . Pregnant women with peripheral parasitemia were more likely to have either a self-reported fever or fever measured at enrollment than those who were aparasitaemic . A sizable proportion of women presenting to the rural origin were parasitaemic as compared to semiurban and urban origin and this difference was significant  (p = 0.0001). Asymptomatic malaria infections were present in 70% of women with peripheral parasitemia (26/37) as compared to 30% symptomatic infection (11/37). Pregnant women with peripheral parasitemia were more likely to have either a self-reported fever or fever measured at enrollment than those who were aparasitaemic .Like the ANC cohort, most pregnant women attending DUs were aged 20–36 years and had some level of formal education (Supplementary Table-1). All were nonsmokers (100%) and nearly all spoke Hindi (97.2%). Most owned their own home (73.9%) and were involved in household work (84.3%); a minority engaged in farming (14.6%). Study participants had attended a median of three ANC visits (range 0–9) and about slightly less than two-thirds were primigravidae and secundigravidae (Supplementary Table-1). The majority of pregnant women reported having untreated bed nets in their homes and using them recently but ITN ownership was uncommon (Supplementary Table-2). Only three women were taking chemoprophylaxis for malaria and none knew the name of the medication that they were taking. Only 4.3% of the women enrolled at the DUs had peripheral parasitemia (either a positive blood smear or RDT). 0.0001) . Primigrp = 0.004). More women with peripheral parasitemia had severe anaemia (5.7%) than those without parasitemia (2.6%) and the difference was significant (p = 0.02).As observed in the ANC participants, most episodes of parasitemia occurred in July to September during the monsoon season. For DU participants with peripheral parasitemia, 83.7% had anaemia as compared to 47.6% of those who did not have parasitemia , women from the scheduled tribes (85%), and women in the two lowest wealth quintiles (over 70%). The prevalence of anaemia among adults in Jharkhand is higher than in almost all other states in India . Anaemia was prevalent among ANC participants whereas severe anaemia was reasonably observed in the investigated cases (Supplementary Table-1). More than two-thirds of the DU participants were anaemic whereas 7.8% had severe anaemia . Of thesN) for all the signs and symptoms were also presented in  vivax strain as described earlier  of the positive cases of malaria in pregnancy subjects at ANC were asymptomatic with peripheral parasitemia compared to 29.4% symptomatic MIP cases, whereas 75.7% were asymptomatic cases with peripheral parasitemia compared to 24.3% symptomatic infection during malaria in pregnancy at DU. Based on the data collected on sign and symptoms from the pregnant women attendees at ANC and DU subjects, we performed positive predictive value (PPV)  and mult earlier  both at N = 1271 at ANC and N = 870 at DU) as well as in malaria in pregnancy at ANC (N = 68) and DU (N = 37) subjects as presented in Multivariate logistic regression showed that malaria infection, ferritin, iron, haemoglobin, and formal education were significantly associated with a higher risk of anaemia in overall cohort  of home, which is usually conducted by government agencies, was reported more in rural areas as compared to urban and semiurban zone of Hazaribag, though its seasonal usage of IRS in those areas regarded as perennial transmission may be suggestive of vector resistance and subsequent higher prevalence of disease. Our observations warrant the potential need to enhance the IRS and distribution of ITNs in and around the investigated district. P. vivax is associated with a high burden of anaemia and remarkable severe anaemia during pregnancy and malaria in pregnancy in endemic population of Hazaribag.We report that P. vivax has shown 2-fold higher risk of moderate anaemia than uninfected subject [Overall, there was significant burden of anaemia among women in Jharkhand and particularly during pregnancy . Our obs subject , 47.Thus, regardless of transmission level and the level of prepregnancy immunity against malaria, maternal anaemia remains the most frequent adverse consequences of malaria during pregnancy . The sym P. vivax infection. This prevalence is similar to that reported by Brutus et al. [ Plasmodium vivax infection on haemoglobin concentration varies from negligible to dramatic [ vivax infection is reasonably well documented, the clinical, developmental, and socioeconomic consequences are largely unknown. Population-based estimates of mortality in severely anaemic individuals with vivax malaria have not been established but recent studies from Latin America, New Guinea, and the Indian subcontinent have identified deaths in patients with severe vivax anaemia [High prevalence of anaemia was observed and strongly correlated with asymptomatics et al.  and Dougs et al. , 51. Recs et al. –54. The dramatic , 55–57.  anaemia , 59. HowThe very low rate of ownership of insecticide treated bed nets (ITNs) and awareness suggests that this component of the enhanced malaria control programme (EMCP) has not effectively reached this vulnerable population although it was encouraging to find that many households had bed nets and that they were used on a regular basis. However, our investigation suggests that approaches for ITN distribution and enhancing community awareness about the importance of their use need to be addressed as similarly observed and proposed by earlier investigation in adjacent region by Hamer et al. .http://www.nvbdcp.gov.in/Doc/Diagnosis-Treatment-Malaria-2013.pdf). Since the intensity of transmission and the prevalence of malaria in pregnant women in Jharkhand are comparatively lesser than in many areas in sub-Saharan Africa, notably, sulfadoxine pyrimethamine was commonly used in Africa as intermittent preventive treatment of pregnant women (IPTp) [ P. vivax infection with pregnancy synergistically contributes to maternal anaemia in a low and perennial malaria transmission setting.Despite the change in drug policy in 2008 in the studied state (Jharkhand), the availability and implementation of combination therapy, that is, artesunate plus sulfadoxine pyrimethamine, are a major concern. It has been well documented that chloroquine resistance has been rising in India –63; thisn (IPTp) , which mOne major limitation of this study is that we were unable to access the placental malaria due to limitation of our study design. Although the study was restricted to women delivering in the hospital, a sizable number of (more than 60%) women give birth outside Sadar Hospital, Hazaribag. vivax malaria during pregnancy and delivering at Hazaribag district Sadar Hospital. In spite of restricted and facility based study, we preferentially covered marginalized, tribes, and remote population of the investigated rural-cum semiurban district, Hazaribag. The majority of the districts and particularly malaria endemic districts in Jharkhand have similar geographical, socioeconomic, demographic, literacy, and basic amenities including health facility and awareness. Thus, our observation may be utilized for baseline information for further comprehensive and multicentric study design, in strengthening MIP associated preventive measures and screening methods within the state of Jharkhand.Further, a longitudinal study instead of cross-sectional one would have provided better estimate of MIP in this region and probably our study design may have given underestimate as compared to actual risk population. This has also been apprehended and suggested by Hamer et al. . Despite P. vivax is set to become the dominant Plasmodium species [ vivax malaria and vivax-associated anaemia have received very little attention. Salient findings of this study are as follows:There is high prevalence of anaemia during pregnancy and in delivering women in the malaria endemic population of Hazaribag, Jharkhand. Plasmodium vivax infection during pregnancy and in delivering women.Prevalence of anaemia is significantly associated with P. vivax infection at both ANC and DU.The most significant observation was the high prevalence of asymptomaticTaken together, these observations are quite indicative and emphasize the need to actively diagnose and treat malaria infection during ANC visit in the areas of perennial transmission. Additionally, in view of the sizable population at risk in this malaria endemic region of India, we are suggestive of few priority practice amendments and reorientation of policies for MIP prevention strategies:There is an urgent need to enhance the ITN availability, use, and awareness both in population and health worker.Distribution of ITNs at first ANC visit will be lucrative alternative for preventive strategy.There should be priority consideration of early case detection and management of asymptomatic pregnant women through restructuring the need of active and passive surveillance strategy in endemic as well as in nonendemic zone.In view of the asymptomatic prevalence of coinfection, we need to further strengthen and emphasize the robust screening strategies, curative attention, and safe treatment facilities at the community level health centres.Further, integrated investigation is desperately needed to understand the magnitude and prevalence of asymptomatic malaria infection linking as an important infected reservoir to continue malaria transmission. Precisely, our finding highlights the public health importance of integrated genus-wide malaria control strategies using diagnostic tests including RDTs and ensuring the availability of safe and effective drugs for the treatment of pregnant women in areas of Plasmodium coendemicity.As the global control and elimination of malaria progress, species ; yet, thExtensive malarial epidemiology related to study sites mentioned in methodology section, detailed demographic information about the subjects investigated during this study as part of method section of manuscript text and some additional and supportive findings mentioned in the results section, which further consolidate and substantiate our observation of asymptomatic prevalence of malaria anaemia in the investigated region has been given in Supplementary material attached to this article.