The objective of this study was to investigate malaria induced changes in fetal growth during the 3An observational study of 876 pregnant women (398 primi- and secundigravidae and 478 multigravidae) was conducted in Tanzania. Fetal growth was monitored with ultrasound and screening for malaria was performed regularly. Birth weight and fetal weight were converted to z-scores, and fetal growth evaluated as fetal weight gain from the 26th week of pregnancy.P = 0.008, multiple linear regression). Fifty-eight percent (28/48) of the primi- and secundigravidae had malaria in the first half of pregnancy, but an effect on fetal growth was observed in the 3rd trimester with an OR of 4.89 for the fetal growth rate belonging to the lowest 25% in the population . At an individual level, among the primi- and secundigravidae, 27% experienced alterations of fetal growth immediately after exposure but only for a short interval, 27% only late in pregnancy, 16.2% persistently from exposure until the end of pregnancy, and 29.7% had no alterations of fetal growth.Malaria infection only affected birth weight and fetal growth among primi- and secundigravid women. Forty-eight of the 398 primi- and secundigravid women had malaria during pregnancy causing a reduction in the newborns z-score of −0.50 (95% CI: −0.86, −0.13, rd trimester, despite infections occurring much earlier in pregnancy, and different mechanisms might operate leading to different patterns of growth alterations. This study highlights the need for protection against malaria throughout pregnancy and the recognition that observed changes in fetal growth might be a consequence of an infection much earlier in pregnancy.The effect of malaria infections was observed during the 3 Plasmodium falciparum is associated with maternal anemia P. falciparum invades the erythrocytes, and the effect on BW has been attributed to maternal anemia Pregnancy associated malaria (PAM) caused by rd trimester of pregnancy after a malaria infection.To ensure that preventive strategies are targeting the most vulnerable time-points in pregnancy a better understanding of fetal growth alterations after PAM is warranted th of April 2008, reference number NIMR7HQ/R.8a/Vol. IX/688). All procedures were conducted in accordance with the Declaration of Helsinki and Good Clinical and Laboratory Practices. All participants gave informed written consent.Ethical approval was granted by the Tanzania Medical Research Coordinating Committee (MRCC) . Women with symptoms or sign of malaria had an immediate blood smear investigation in addition to the RDT. Infections requiring hospital admission were treated with Quinine . RDT positive women, who had been RDT positive within two weeks before the ANV, also had an immediate blood smear examination. Parasite HRP-2 antigen can circulate in the blood stream after clearance of the malaria parasites, and only women who were consistently blood smear positive were treated At all visits venous blood was collected and at delivery both venous and placental blood. Malaria was diagnosed using rapid diagnostic test (RDT)  st dose in the 2nd trimester after a GA of 20 weeks and 2nd dose in the 3rd trimester, at least four weeks apart. If included early in pregnancy the 1st dose was given at an extra clinic visit at a GA of 20. If having received a 1st dose of IPTp before inclusion, but earlier than recommended by WHO  with sulfadoxine-pyrimethamine (1500 mg/75 mg)  was given as directly observed treatment; 1Trans-abdominal ultrasound examination was performed at inclusion, ANV2, ANV3, and ANV4 . At inclusion GA was estimated using crown-rump length (crown-rump length<75 mm) nd trimester/); ANV3-ANV4 (early 3rd trimester); ANV4-Delivery (late 3rd trimester); ANV3-Delivery (entire 3rd trimester)) using the fetal weight (kg) in the beginning of each growth interval as the reference To account for dependency of growth velocity on initial weight, fetal growth was evaluated as the relative weight gain in g/week*kg in four fixed growth intervals of at least 14 days To exclude variability due to GA and sex, EFW and BW were converted to z-scores using sex-specific standard weight charts developed from healthy pregnancies in the same cohort as a reference th centile for Δz was used as a cut-off to define insufficient weight gain. The effect of malaria was categorized in four groups: 1) Normal growth as Δz ≥25th centile in all growth intervals following a malaria infection, 2) Immediate effect as Δz <25th centile in the growth interval when the infection occurred or in the first growth interval after a malaria infection, followed by growth intervals where Δz ≥25th centile, 3) Late effect as a Δz<25th centile in a growth interval not immediately following the infection, and 4) Persistent effect as a Δz <25th centile immediately after a malaria infection and until the end of pregnancy.Changes in z-score over time were investigated to further evaluate alterations in fetal growth. Only offspring with an eligible BW and data from at least two growth intervals were evaluated. Primarily, the growth intervals ANV2-ANV3, ANV3-ANV4, ANV4-Delivery were used for evaluation. Alternative growth intervals  were used if a woman had incomplete attendance. Changes in z-scores (Δz) were calculated for the malaria negative group and used as a reference. Theoretically, a normally growing fetus will have a Δz close to zero, whereas a Δz>0 represents excess fetal weight gain and a Δz<0 insufficient weight gain. Evaluation of changes in z-scores can be affected by the fetal pulsatile growth pattern For analysis, women were considered to be malaria negative if they never had malaria based on RDT and microscopy results from the time they were enrolled. Women, who contracted malaria (RDT and/or microscopy positive), were considered to belong to the malaria group from when they were diagnosed until delivery. In growth intervals preceding the time of detection of the malaria infection they were excluded from the analysis. Hence, women contracting malaria between ANV2 and ANV3 were not included in analyses of the growth interval ANV2-ANV3, but considered malaria positive in all other growth intervals.2 test, Fisher’s exact test, Mann Whitney ranksum, and Student t-test, and all with two-sided P-values. The effect of malaria on BW (as z-score) was investigated using multiple linear regression and on relative weight gain (dichotomized as lowest 25% and highest 75%) using multiple logistic regression. Crude and adjusted coefficients/odds ratios were calculated. Factors with a P<0.20 in univariate analysis were entered into the multivariate models. Using a step-wise backward elimination approach final models were obtained including variables with a P<0.10. A P<0.05 was considered significant. Final models only included women without missing values.All data were documented and validated using Microsoft Office Access 2003. Statistical analyses were performed in Stata 10 (Stata Corporation) using, when appropriate, ChiIn total, 1171 pregnant women were screened and 995 women met the inclusion criteria. Of these 21 women had miscarriage, 11 withdrew consent, 34 were lost to follow-up, 5 moved out of the district, and 924 women completed follow-up. Of these, 48 were excluded due to (some with multiple conditions) preeclampsia (28), twin pregnancy (16), and/or severe congenital malformation (6), leaving 876 women-newborn pairs for analysis. The included women were more often multigravidae, had a known HIV status and lower educational level compared to the excluded women  (Characteristics of the 876 mother-newborn pairs are shown in  2 test) . Except  2 test)  and S3. The majority of ANV2, 3 and 4 was conducted at a GA of 26 (698/843 (82.3%) estimated fetal weights (EFW)), 30 (727/837 (86.9%) EFW) and 36 (688/792 (86.9%) EFW) weeks, respectively. Women showing early or late for their bookings had fetal weight estimated within 1.5 weeks of these GA. The timing of the ANV was comparable for the malaria positive and malaria negative women , whereas malaria infection did not affect the BW among multigravidae. Furthermore, the newborns Z-score on BW was statistically significant lower among the malaria positive compared to the malaria negative primi- and secundigravidae  vs. −0.06 , P = 0.003, Students t-test). Malaria infection did not affect the GA at delivery  vs. 3100 g , delivery .rd trimester of pregnancy in the malaria positive group compared to the malaria negative group. The relative weight gain had unequal variance and we therefore dichotomized the women into those with a relative fetal weight gain belonging to the lowest 25% and those with a weight gain belonging to the highest 75%, to allow adjusting for confounding factors using multiple logistic regression. In the 3rd trimester, in the univariate analyses, there were a statistical significantly higher proportion of fetuses belonging to the lowest 25% among the malaria positive compared to the malaria negative group . Malaria infection was also associated with a relative fetal weight gain belonging to the lowest 25% in all three growth intervals in the 3rd trimester after adjusting for confounding factors (ANV3-ANV4 adj. odds ratio (OR): 2.72, 95% CI: 1.18–6.23, P = 0.019; ANV4-Delivery adj. OR: 2.48, 95% CI: 1.04–5.91, P = 0.040; ANV3-Delivery adj. OR: 4.89, 95% CI: 2.03–11.79, P<0.001)  . In univve group . Fetal gve group . The chave group  and S6. P<0.001) . Only coTo further elucidate how and when fetal growth in the last half of pregnancy was affected by malaria we evaluated the change in z-score over time. To illustrate the importance of gravidity when assessing the consequences of malaria on fetal growth multigravidae were also included in these analyses. Fifty-two of the 72 women (72.2%) with malaria had an eligible BW and data available for at least two growth intervals. Among the primi- and secundigravid women, the majority (26/37) experienced alterations in fetal growth after a malaria infection. An equal distribution between an immediate, a late and a persistent effect on fetal growth was observed. Among the majority of the multigravid women (10/15) the fetal growth was not affected. Furthermore, a persistent and a late alteration of fetal growth seemed to result in the lowest BW . In FiguAmong primi- and secundigravidae, there was a trend towards decreased head circumference at birth . Previous findings indicate that the timing of the infection matters st and 2nd trimester the placental development is particularly sensitive to pathology, and if the trophoblast invasion of the maternal spiral arteries are disrupted, current and later placental function can be impaired et alrd trimester after infections occurring before a GA of 20 weeks. This indicates that the effect of PAM might be observed with considerable delay, which is in line with the pattern observed in our study. In the 3rd trimester when nutrient requirement of the fetus is at its highest level, insufficient placental development and function could have severe consequences Most infections in our cohort were detected at inclusion . In future studies, inclusion as soon as possible after conceiving should be sought. Furthermore, maternal HIV infection might also affect fetal growth We investigated malaria induced changes in fetal growth using both a reference population and each individual fetus as his/her own control. This approach enabled us to capture changes that may not cause LBW, but do hinder the newborn in obtaining its inherent growth potential. By utilizing ultrasound to estimate GA and a local standard weight chart for reference we circumvented some of the difficulties encountered in previous malaria studies on assessment of intrauterine growth To validate our ultrasound data we investigated the correlation between fetal weight gain in the different growth intervals and BW, independent of malaria infection, and found a significant correlation (data not shown). Alterations in fetal growth has been evaluated using changes in z-scores One limitation of the study was that the ultrasonographs were not blinded to the women’s current or previous malaria status. Furthermore, we did not observe consistent statistically significant changes in head and abdominal circumference. This might be due the possibly decrease in accuracy when estimating fetal size using single biometric parameters P. falciparum malaria exposure can be observed in the 3rd trimester. The effects on fetal growth often occurred after a considerable delay in women who were diagnosed with malaria at the first antenatal visit and who subsequently strictly followed the antenatal program and the recommended measures to prevent malaria infections. The study was carried out in an area where malaria transmission has declined considerably and the results highlight the importance of developing interventions which can protect women early in pregnancy.In conclusion, we provide evidence that alteration of fetal growth after Table S1Comparison of included and excluded mother-newborn pairs.(DOCX)Click here for additional data file.Table S2Comparison of characteristics for malaria positive and malaria negative primi- and secundigravid mothers and their fetuses/newborns.(DOCX)Click here for additional data file.Table S3Comparison of characteristics for malaria positive and malaria negative multigravid mothers and their fetuses/newborns.(DOCX)Click here for additional data file.Table S4Characteristics of all malaria positive women.(PDF)Click here for additional data file.Table S5rd trimester relative fetal weight gain ((g/week)*kg) dichotomized as belonging to the lowest 25% or the highest 75% for primi- and secundigravidae.Factors associated with 3(DOCX)Click here for additional data file.Table S6Factors associated Z-score at delivery among primi- and secundigravidae. Z-score was adjusted for sex of newborn and gestational age at delivery.(DOCX)Click here for additional data file.


Plasmodium falciparum infections and malaria clinical cases are reported during the dry season. This study aims to assess the relationship between IgG antibody (Ab) responses to gSG6-P1 mosquito salivary peptide and the prevalence of P. falciparum infection in children during the dry season in the Senegal River Valley. The positive association of the Ab response to gSG6-P1, as biomarker of human exposure to Anopheles vector bite, and P. falciparum infectious status  will allow considering this biomarker as a potential indicator of P. falciparum infection risk during the dry season.The Northern part of Senegal is characterized by a low and seasonal transmission of malaria. However, some P. falciparum infection. Collected sera were used to evaluate IgG response to gSG6-P1 by ELISA. Association between parasitological and clinical data (infected-asymptomatic or infected-symptomatic) and the anti-gSG6-P1 IgG levels were evaluated during this period.Microscopic examination of thick blood smears was performed in 371 and 310 children at the start (January) and at the end (June) of the dry season, respectively, in order to assess the prevalence of P. falciparum infection was very low to moderate according to the studied period and was higher in January (23.5%) compared to June (3.5%). Specific IgG response was also different between uninfected children and asymptomatic carriers of the parasite. Children with P. falciparum infection in the dry season showed higher IgG Ab levels to gSG6-P1 than uninfected children.The prevalence of P. falciparum exposure observed during dry season and, by consequences, could be used for the survey of potential pre-elimination situation.The results strengthen the hypothesis that malaria transmission is maintained during the dry season in an area of low and seasonal transmission. The measurement of IgG responses to gSG6-P1 salivary peptide could be a pertinent indicator of human malaria reservoir or infection risk in this particular epidemiological context. This promising immunological marker could be useful for the evaluation of the risk of  Anopheles gambiae complex (An. gambiae s.s. and Anopheles arabiensis) and for Anopheles funestus, which breed in fresh water and in swampy habitats with much vegetation, respectively. In this area, Plasmodium falciparum is virtually the only species of Plasmodium. The incidence of clinical cases and mortality peaks between September and November, and then rapidly declines. According to statistical data of the National Malaria Control Programme (NMCP), this region of Senegal presents the lowest national malaria attack rates, with less than five malaria declared cases per 1,000 inhabitants in 2009[Malaria transmission in the Northern part of Senegal is low and occurs mainly between August and October due to a single rainy season from July to October. This siP. falciparum human carriers were found during the dry season, despite a very low level of parasite transmission[P. falciparum carriers in human populations and the evaluation of their risks of exposure to malaria vectors are needed to carry out an effective malaria control in this particular area, considered suitable for malaria pre-elimination by the NMCP.In several epidemiologic studies, a proportion of smission-5. The ssmission. In Nortsmission. The ideAnopheles mosquito injects saliva into the vertebrate host. The study of salivary proteins and their effects on the immune response of vertebrate host represents an innovative research tool to characterize the mechanisms of pathogens’ transmission. Therefore, the study of human-vector relationship appeared promising in the perspective of an effective control of vector-borne diseases. Numerous studies have shown that Anopheles salivary proteins are antigenic. In particular, it has been shown that children living in malaria-endemic areas developed IgG response to whole saliva of An. gambiae which was associated with the intensity of human exposure to vector bites[P. falciparum or Plasmodium vivax infection showed higher IgG response to Anopheles dirus[Anopheles darlingi saliva[An. gambiae saliva may also be useful for assessing exposure in travellers, and thus could be a biomarker of vector exposure in individuals weakly and transiently exposed to Anopheles bites[Anopheles salivary proteins represents an immunological marker of human exposure to vector bites and thus a potential indicator of the risks of Plasmodium transmission, as observed for various vector-borne diseases[An. gambiae (gSG6) protein has been identified and validated as a biomarker candidate of exposure to Afro-tropical malaria vectors[Anopheles and it was demonstrated that specific IgG response was especially a relevant biomarker in a context of low and seasonal exposure to malaria vectors[During its blood meal, the or bites. Moreoveor bites. It was les dirus and Anopi saliva, than unes bites. Altogetdiseases,13. With vectors,15. Inde vectors-18.P. falciparum infection during the dry season in the Senegal River valley and ii) to evaluate the association between IgG responses to gSG6-P1 salivary peptide and different clinical statuses  of P. falciparum infection in children. The final objective was to assess the potential use of the gSG6-P1 as a biomarker of P. falciparum infection risk during dry season.This present study aimed i) to appraise the prevalence of The study was conducted in Northern Senegal, in the Senegal River Valley. This site is a typical sahelian area with less than 400 mm of rainfall per year. Malaria transmission is described as low, seasonal and occurs mainly during the rainy season between August and October with a rate of infective bites/person/night <1.A longitudinal follow-up was performed from October 2008 to January 2010 in children between one and nine years of age, in Agniam, Niandane, Pendao, Guede and Fanaye, as previously described,20. In tThe present study was approved by the National Ethics Committee of the Ministry of Health of Senegal . Oral and written informed consents were obtained from the parents or the legal guardians of the children.Anopheles bites was extremely low in this area during the dry season and remained similar between villages[The design of the entomological, parasitological and immunological monitoring  of each village  have been previously reported. This prvillages. TherefoP. falciparum prevalence and gametocyte carriage. The slides were transferred to a nearby laboratory, stained with Giemsa and analysed under a microscope by experienced technicians. In parallel, sera collected by finger prick into BD microtainer® tubes  were centrifuged and 300 μL of serum was collected and stored at −20°C until use for immunological tests by ELISA. Axillary temperature of each participant was also measured.681 thick blood smears were collected during the two mentioned visits (January and June 2009) to estimate P. falciparum malaria diagnosis and the clinical status of the disease, children were stratified in three different groups: uninfected , infected-asymptomatic and infected-symptomatic . Children presenting a PMBS  without any clinical sign, fever (axillary temperature ≥37.5°C) and/or a history of fever reported during the corresponding month, were considered infected-asymptomatic; while in the presence of fever and/or history of fever, they were classified as infected-symptomatic.According to the An. gambiae s.l.  have been described as the predominant species and the only vectors of P. falciparum[An. funestus, Anopheles wellcomei, Anopheles pharoensis and Anopheles ziemanni were also collected in low densities. At each visit, entomological sampling was performed using four adult volunteers per village (two indoors and two outdoors). Human Biting Rate (HBR) was estimated by the number of bites per person per night (BHN). It was calculated by dividing the number of An. gambiae caught by the total person-night for the period[Entomological data collected by human landing catches and Pyrethrum spray catches during the follow-up were previously reported and alciparum. Other se period.Anopheles specificity and its immunogenicity, as previously described[The gSG6-P1 peptide was designed using bioinformatics to maximize its escribed,15. It w2O2) and absorbance (OD) was measured at 405 nm.ELISAs were carried out on sera to quantify IgG response to the gSG6-P1 peptide as previously described. BrieflyAnopheles exposed individuals  in order to quantify the non-specific background Ab level and to calculate the specific immune response threshold: TR = mean (∆ODneg) + 3SD = 0.180. An exposed individual was then classified as an immune responder (IR) if its ΔOD > 0.180.Individual results were expressed as the ΔOD value: ΔOD = ODxODn, where ODx represents the mean of individual optical density (OD) value in both wells with gSG6-P1 antigen and ODn the individual OD value for each serum without gSG6-P1 antigen. Specific anti-gSG6-P1 IgG response were also assayed in non-2 test was used to compare P. falciparum prevalence between January and June, and also between IgG immune responders to gSG6-P1 and non-responders. Mann–Whitney U test was used for the comparison of IgG levels between uninfected and infected-asymptomatic or infected-symptomatic children. The same test was used to compare differences between uninfected and infected children. Differences of IgG response levels between more than three groups were calculated using the non parametric Kruskal-Wallis test. The association between P. falciparum infection and immune responders to gSG6-P1 peptide was calculated using the relative risk (RR), and 95% confidence intervals (CI) were calculated by the Miettinen method. All differences were considered significant at p < 0.05.Data were analysed with Graph Pad Prism® . ChiP. falciparum infection in children during dry season. The prevalence of P. falciparum infection was higher in January 2009 (23.5%) than in June 2009 (3.5%) . No difference in P. falciparum infection was observed between children under 5 years of age and the older ones [As indicated in Table EIR = 0).P. falciparum infection  and uninfected. Comparison showed that the median of specific IgG Ab level was significantly higher in P. falciparum-infected children compared to uninfected ones during the dry season  during the dry season . Indeed, specific IgG responses were significantly higher in both infected-asymptomatic and infected-symptomatic children compared to uninfected ones . But, no statistical difference was observed in IgG Ab responses between infected-asymptomatic and infected-symptomatic children, . Similar results were obtained when data were analysed separately in January and in June 2009  and non-immune responders (∆OD ≤ 0.180) , in January  than in June (3.5%). This finding confirmed previous studies in a neighbouring area[P. falciparum infection rate decreases throughout the dry season (December to June)[In the first part of the study,  setting. P. falcing area,22 in whto June).Anopheles could be explained by a lack of sensitivity of entomological sampling methods used, in a special context where a very small number of An. gambiae s.l. is collected. Indeed, An. gambiae s.s and An. arabiensis were already proven to be highly anthropophilic and predominant malaria vectors in the Senegal River Valley[Anopheles infection rate of 1.37% was observed one year later during the dry season[Anopheles[The paradox observed in this area, between low or no transmission (EIR = 0) and high or moderate malaria parasite prevalence, has previously been described in a neighbouring area and elser Valley. Anophely season. This suAnopheles,26,27, aAn. gambiae gSG6-P1 salivary peptide could be a pertinent biomarker of P. falciparum asymptomatic infection. Such a distinction of asymptomatic P. falciparum infected individuals compared to uninfected children appears considerable for malaria control in low endemicity areas, and especially during the dry season. Indeed, most of infected children are asymptomatic carriers of the parasite and long-term asymptomatic carriage of P. falciparum throughout the dry season is commonly observed[P. falciparum to uninfected Anopheles vectors which remain after the rainy season, contributing to the maintenance of malaria transmission in the dry season and its re-starting after the first rains[P. falciparum asymptomatic infection could be used as a valuable tool in a malaria pre-elimination context.In this regard, the present study shows that IgG response to gSG6-P1 was higher in infected-asymptomatic children than in uninfected ones. Statistical significance between infected-asymptomatic and uninfected children was stronger (p < 0.01) when data were cumulated (January and June 2009); probably due to the huge amount of overlap between groups during these two visits. However, IgG response to gSG6-P1 peptide remained significantly higher in infected-asymptomatic children than in uninfected ones when data were analysed separately in January p = 0.04) and in June (p = 0.02). So, IgG response to observed,4,28. In and in Jst rains,29. TherP. falciparum infection. This result is in accordance with previous results in Senegal showing that children with subsequently clinical P. falciparum malaria presented higher anti-saliva IgG response than did non-infected children[et al.[An. darlingi saliva Ab response could indicate human exposure to P. vivax and biomarker of P. vivax in Brazil. Recently, a study in Burkina-Faso showed a positive correlation between malaria incidence and IgG response against the recombinant protein of An. gambiae (gSG6)[Anopheles bites in numerous contexts, could also be a useful indicator of malaria infection and/or morbidity risks in this particular context. It is quite conceivable that salivary peptides could complete in the near future, routine thick blood smears performed in the framework of active case detection of malaria infection or in the evaluation of vector control strategies. Indeed, it was shown that it was possible to assess by ELISA, IgG response to gSG6-P1 on eluates of blood drops collected on blotting paper[Anopheles bites as an immunological indicator of disease risk remains to be largely studied. For example, there is a lack of a conventional threshold of immune response to salivary proteins/peptide at which an individual is at risk of malaria infection. The intensity of exposure or transmission varies according to the area and season. Despite these limitations, data of the current study indicate the potentiality to use biomarkers of human exposure to vectors as a reliable and useful tool to detect malaria human reservoirs and help predicting malaria risk, and this in a context of very low exposure to vectors, such as malaria pre-elimination settings. This point appeared to be clearly relevant in regard to the observed results in June (end of dry season with very low exposure to Anopheles bites) where the risk of P. falciparum infection was seven times higher in immune responders than in non-responders.In addition, the present study showed that high IgG levels to gSG6-P1 could be predictive indicators of children. In addin[et al. have shoe (gSG6). In Kenye (gSG6). Altogetng paper,32,33. Tng paper. FurtherP. falciparum infection prevalence during the dry season, since it was only determined by microscopy examination. However, this gold standard technique for malaria screening has limited sensitivity for detection of very low parasite densities. Indeed, it has been shown that, in areas with low or very low intensity of malaria transmission, sub-microscopic infections were commonly seen[P. falciparum infection has been revealed with microscopic detection, suggesting that malaria transmission occurs during the dry season in this area.A limitation of this study could be linked to an under-evaluation of nly seen,36. It wnly seen-39. NeveP. falciparum infections were observed in the Northern Senegal River Valley. Parasitological data have been correlated with anti-gSG6-P1 IgG responses, indicating that this Anopheles salivary-antigen specific response could be a pertinent epidemiological biomarker of P. falciparum exposure during the dry season. Furthermore, the analysis revealed that high levels of gSG6-P1 specific IgG response could be used as an indicator of infection risk and even to discriminate uninfected and infected-asymptomatic carriers of the parasite during the dry season. This biomarker could be very useful for national malaria control programs to identify asymptomatic carriers of P. falciparum in low and seasonal transmission areas and, by consequences, in a context of malaria pre-elimination.During the low-transmission season (dry season), The authors declare that they have no competing interests.ABS participated in field surveys, carried out the immunological assessments, analysed data and drafted the manuscript. JBS, LG, SS, MP, AMS, EH, NF participated in study coordination, field surveys, data collection and microscopic examination. SBC participated to data analysis. GR and FR participated in study design, preparation and writing of the manuscript. All authors contribute to revision the manuscript and read and approved the final version.Prevalence ofP. falciparum infection during the dry season, stratified by age group. The table summarizes P. falciparum infection between children under 5 years of age and the older ones during the dry season.Click here for fileIgG response levels to gSG6-P1 peptide according to malaria status. Box plots show gSG6-P1 specific IgG response levels (∆OD) according to three P. falciparum infection statuses. Boxes display the median ∆OD value, 25th and 75th percentiles. The whiskers show the 5th/95th percentiles and the dots indicate the outliers. Differences between two or three groups were tested using Mann Whitney test and Kruskal Wallis test, respectively.Click here for file


Plasmodium vivax malaria, which undergoes gametocytogenesis earlier during infection. Pvs25 and Pvs28 are transmission-blocking vaccine candidates for P. vivax malaria. Assessment of genetic diversity of the vaccine candidates will provide necessary information for predicting the performance of vaccines, which will guide us during the development of malaria vaccines.Transmission-blocking vaccines (TBVs) have been considered an important strategy for disrupting the malaria transmission cycle, especially for pvs25 and pvs28 from 30 P. vivax isolates from Yunnan Province, identifying five amino acid haplotypes of Pvs25 and seven amino acid haplotypes of Pvs28. Among a total of four mutant residues, the predominant haplotype of Pvs25 only had the I130T substitution. For Pvs28, a total of eight amino acid substitutions were identified. The predominant haplotype of Pvs28 had two substitution at positions 52 (M52L) and 140 (T140S) with 5-6 GSGGE/D tandem repeats at the end of fourth EGF-like domain. Most amino acid substitutions were common with previous reports from South Asian isolates. Although the nucleotide diversity of pvs28 (π = 0.0034 ± 0.0012) was significantly higher than pvs25 (π = 0.0013 ± 0.0009), it was still conserved when compared with the blood stage vaccine candidates.We sequenced the coding regions of pvs25 and pvs28, suggesting antigenic diversity may not be a particular problem for Sal I based TBVs in most P. vivax-endemic areas of China.Genetic analysis revealed limited genetic diversity of  Plasmodium vivax, the most widespread species of human malaria parasites, is responsible for the majority of malaria cases outside of Africa, and the most prevalent form of relapsing malaria. Although not as lethal as P. falciparum malaria, P. vivax has caused substantial morbidity for human populations residing in Asia and South America [P. vivax malaria has displayed tremendous resilience to control efforts, which is in part due to two biological characteristics of this parasite. One is the formation of hypnozoites in the liver, which is responsible for relapses of the disease. The other is that P. vivax undergoes gametocytogenesis before manifestation of the disease symptoms, making transmission possible before treatment. America ,2. ThrouP. vivax is the predominant Plasmodium species in China, and in recent years, P. vivax cases accounted for more than 90% of all malaria cases [vivax malaria transmission is a major challenge. Yunnan is one of the two provinces that have year-round local transmission of P. vivax and P. falciparum. It is located in southwest China and borders Myanmar to the west and Laos and Vietnam to the south [P. vivax malaria and 44,465 cases of P. falciparum malaria have been reported between 1991 and 2006 in Yunnan [P. vivax malaria and 5,821 cases of P. falciparum malaria have been reported between 2006 and 2009 in Yunnan [ia cases . As The ia cases , interruhe south . A totaln Yunnan . A totaln Yunnan ,7-9. Mosn Yunnan ,10. DespPlasmodium zygote and ookinete surface [Transmission-blocking vaccines (TBVs), which target the sexual stages of malaria parasites to prevent their further development within mosquitoes, are considered an important strategy for disrupting malaria transmission ,12. To d surface , and the surface . Among t surface , yeasts  surface ,16 and b surface  exhibits surface  and oocy surface -17. The  surface  and huma surface ,20. A ph surface . It has  surface .Plasmodium infections [pvs25 and pvs28 genes has been surveyed in several Asian countries, including South Korea [Polymorphisms represent a major impediment in vaccine design. An effective vaccine must either be based on conserved regions or incorporate multiple allelic forms of the antigen. As antigenic polymorphisms limit the immunogenicity and immunoreactivity of vaccines, it is necessary to understand antigenic variations in order to generate effective vaccines against natural fections . Pvs25 afections . To dateth Korea , Indonesth Korea , Iran [2th Korea , India [th Korea , Bangladth Korea . In thisP. vivax and P. falciparum. Since 2001, there has been a strong seasonal pattern of malaria incidence characterized by a peak of infection occurring from June to July and another peak from October to November [Anopheles minimus and Anopheles dirus [Yunnan Province is one of the most highly endemic regions in China with year-round transmission of November . CountieNovember ,27. The es dirus .P. vivax malaria using Giemsa stained thin smear examination by malaria clinic staff. After informed consent/assent was obtained from either adults or parents or legal guardians of children, ~100 μl of finger-prick blood were collected from each patient on filter papers. The patients were Yunnan local residents, 13 to 50 years old, living in Baoshan , Ruili  and Longchuan   according to the manufacturer's protocol. DNA was eluted in 60 μl of H2O. pvs25 (GenBank: AF083502.1) and pvs28 (GenBank: AF083503.2) of the Salvador I  strain were used as reference sequences. The full-length open reading frames (ORFs) of pvs25 and pvs28 were amplified using the following primer pairs: pvs25F (5'-CACTTAGCCAAAATGAACTC-3') and pvs25 R (5'-AAAGGACAAGCAGGATGATA-3') for pvs25; pvs28F (5'-CTACCACAGCTTGCTGTTCC-3') and pvs28R (5'-TGACATCATGAAGAAGGCG-3') for pvs28. The PCR reaction contained 2 μl of 10 × KOD-Plus buffer, 2 μl of 2 mM dNTPs, 0.8 μl of 25 mM MgSO4, 1.0 μl of 10 μM of each primer, 0.2 units of KOD-Plus DNA polymerase , and 0.5 μl genomic DNA template in a final volume of 20 μl. Amplification conditions were as follows: initial denaturing at 94°C for 2 min, 35 cycles of 94°C for 15 sec, 56°C (for pvs28) or 54°C (for pvs25) for 15 sec, and 68°C for 1 min, and a final extension at 68°C for 5 min. PCR products were cleaned up using Exonuclease I and Shrip alkaline phosphatase treatment according to the manufacturer's instructions .Three circles, 2.5 mm in diameter, were punched out from each dried blood spot and Pvs25 and pvs28 ORFs were sequenced using the ABI Prism® BigDye™ Terminator cycle sequencing kit  on an ABI PRISM® 310 Genetic Analyzer . Sequencing primers are: pvs25F (5'-GAAACCCTAGGCAAAGCATG-3') and pvs25R (5'- GGGACTTTGCCA ATAGCACA-3') for pvs25, and pvs28F (5'-AACTGTGGAGACTACGCTG-3') and pvs28R (5'-ATATTACAAGAGCACATGGTG-3') for pvs28.pvs25 and pvs28 were aligned with the reference sequences using the CLUSTAL W program [π) and its standard error (SE) were calculated with the Jukes and Cantor method using MEGA 4.0. The number of haplotypes (h), haplotypes diversity (Hd), segregating sites (S) were computed using the options available in DnaSP 5.0 [The ORFs of  program  with mannaSP 5.0 .Pvs25 and pvs28 genes were successfully amplified in 30 P. vivax samples obtained from Yunnan. The sequence of pvs25 (645 bp) and pvs28 (712 bp) were determined by direct sequencing of the PCR products. The 30 pvs25 sequences contained four polymorphic nucleotide sites , which resulted in four amino acid substitutions . These substitutions resulted in a total of five amino acid haplotypes, hitherto referred to as Pvs25-I, II, III, IV and V  and eight nonsynonymous substitutions , which resulted in eight amino acid substitutions . A total of seven amino acid haplotypes were identified: Pvs28-I, II, III, IV, V, VI and VII (Table The 30 ABG29072.1), North Korea (GenBank: AAV33640.1) [AB091730.1) [ABS70906.1-ABS70935.1) [BAA94348.1-BAA94350.1) [BAC66001.1) [ACJ54129.1, ACJ54128.1) [BAC66003.1) [AAV33639.1) [BAA94346.1) [BAA94347.1) [ADJ18839.1) [Compared with previous reports of worldwide isolates, Pvs25-I haplotype showed 100% identity with isolates from Vietnam (GenBank: 33640.1) , Thailan91730.1)  and Mexi70935.1) . The Pvs66001.1) . The Pvs54128.1) , Thailan66003.1) , Indones33639.1) , India (18839.1) .ACJ54136.1) [BAA94352.1, BAA94353.1, BAA94371.1, BAA94362, BAA94363) [BAC66011.1) [BAA94370.1, BAA94369.1) [ADJ18881.1) [The Pvs28-I haplotype in Yunnan isolates was also common in isolates from Iran (GenBank: 54136.1)  and BangAA94363) . The Pvs66011.1)  and Bang94369.1) . Parasit18881.1)  and ChinThe Pvs25 and Pvs28 amino acid substitutions identified from worldwide isolates including those from China are summarized in Tables Among a total of 24 amino acid substitutions in Pvs28 from worldwide isolates, M52L and T140S were the most common substitutions from different geographic regions ,24-26,31pvs25. There were a total of 12 polymorphic sites that yielded eight haplotypes in pvs28. Nucleotide diversity of pvs28 (π = 0.0034 ± 0.0012) was significantly higher than that of pvs25 (π = 0.0013 ± 0.0009) (p < 0.05)  [In this study, we assessed the level of genetic diversity of d India) ,25. Becapvs25 (π = 0.0013) had a much lower level of diversity than P. vivax blood stage proteins DBP (π = 0.0086-0.0184) [P. falciparum sexual stage proteins pfs25 (π = 0.0035) [pvs25 suggested that genetic polymorphism of pvs25 was limited in Yunnan isolates. Although the nucleotide diversity of pvs28 was significantly higher (π = 0.0034) than that of pvs25, it was still conserved when compared with the blood stage vaccine candidates. Although direct sequencing of the PCR products in our study might underestimate the genetic diversity of these two genes, the effect should be minor since our study areas are malaria hypoendemic and mixed infections should not be prevalent. The limited diversity of sexual stage antigens such as P25 and P28 is perhaps attributed to the expression of these proteins in mosquito stages, which should avoid immune selection in the humans.One of the major obstacles to the development of an effective malaria vaccine is the genetic polymorphism of many of the genes in natural parasite populations that otherwise would be promising vaccine candidates. A number of studies have shown that TBVs candidates have limited polymorphism compared to antigens expressed in asexual stage parasites ,35. Our -0.0184)  and MSP1-0.0184) . In addi 0.0035) , the lowP. vivax recognized corresponding molecules expressed by field-isolated parasites in Thailand. Our data showed limited genetic diversity of pvs25 and pvs28 in the Yunnan isolates. The major amino acid haplotype of Pvs25 from this region was also shared with that from Thailand isolates, it provids a good prospect on implication of TBVs in China malaria areas.It is very important to evaluate the effect of genetic polymorphism of Pvs25 and Pvs28 on the efficacy of Sal-I based TBVs in different malaria epidemiological areas. The transmission-blocking assays conducted in Thailand clearly demonstrated that antisera to recombinant Pvs25 and Pvs28 based on the Sal-I strain of P. vivax parasites from Yunnan. Further studies on a larger sample scale and comparison analysis with Central China isolates will certainly improve our understanding of genetic polymorphisms of TBVs candidates in the malaria endemic areas of China.Altogether, our study offers a first glimpse of the genetic diversity of two TBVs candidate antigens in Yunnan Province, China. Yet, this study only offered limited information of TBVs are one of the important strategies for controlling malaria transmission. A key point in vaccine optimization is to understand the extent of genetic diversity of candidate antigens. The TBVs candidates Pvs25 and Pvs28 from Yunnan isolates showed limited genetic diversity. Further studies encompassing larger malaria endemic areas are needed to provide a thorough evaluation of situation in China.The authors declare that they have no competing interests.YC conceived of the study and helped to draft the manuscript. YY collected the blood spots specimens. HF carried out the studies, statistical analysis and drafted the manuscript. LZ, XZ, GW, YP, YL, YL participated in the molecular genetic studies and sequence alignment. LC helped with statistical analysis and critically revised the manuscript. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.


We investigated the prevalence of malaria and associated risk factors in children living in urban Ghana. Malaria prevalence was associated with low hemoglobin concentration, low socioeconomic status, and higher age. Our findings indicate that African urban poor are seriously affected by malaria and that irrigated agriculture may increase this risk. Anopheles mosquito breeding decreases with increasing proximity to the center of urban areas   Figure . CommuniIn the selected communities, we conducted a cross-sectional house-to-house survey to assess malaria parasitemia and hemoglobin (Hb) concentration in children 6 to 60 months of age. A team consisting of technicians and trained enumerators went house to house to collect data. Houses were selected arbitrarily and queried regarding the presence of children <5 years of age. For each community, the whole area was covered to account for spatial heterogeneity. If most compounds or houses had children <5 years of age, houses were omitted to obtain the target sample size of 150 children from the community.Informed consent was obtained from each child's caregiver. Thick and thin blood films were collected and read according to standard World Health Organization protocols. Hb levels were assessed by using a blood hemoglobin photometer . Children with parasitemias or Hb levels <8.0 g/dL were provided free treatment at a local clinic. The epidemiologic data were related to proximity to sites of urban agriculture, socioeconomic status based on household assets following a World Bank template , and posPlasmodium-positive and -negative slides, and P. falciparum, 2 were P. malariae, and 1 was a mixed infection with P. falciparum and P. malariae. The average Hb level was 10.82 g/dL (SD 1.47), and 78 (4.5%) of 1,738 children had moderate-to-severe anemia (Hb <8.0 g/dL). Overall malaria parasite prevalence was 14.9%  and was higher in communities around urban agricultural sites than in control communities . In a univariate analysis , Hb concentration ; moderate-to-severe anemia ; having netting in front of  windows, doors, or both ; socioeconomic status ; and age  showed significant associations with presence of malaria parasites. Reported bed net use by a household was 33% (range 6%–53% in different communities) but was not significantly associated with presence of malaria parasites in the blood.A total of 1,757 children from 938 households in 12 different communities were enrolled in the study. A generalized linear mixed model (GLMM) approach, using an SAS macro  that allowed a logistic link function, was used to investigate the association between putative predictor variables and malaria parasite prevalence. Covariates with p<0.1 in the univariate analysis were entered in the multivariate model. Household was nested within community, and both variables were treated as random effects. Age was divided into the following groups: 6–12, 13–24, 25–36, 37–48, and 49–60 months. Hb was entered as a continuous variable. Malaria parasitemia was significantly associated with Hb , age group , and socioeconomic status . The effect of urban agriculture was marginally below significance (p = 0.0647), possibly because of reduced statistical power. Having netting in front of windows or doors was no longer significant (p = 0.3638), presumably because presence of nets was associated with a higher socioeconomic status (p<0.001).In urban agricultural communities, GLMM analysis with parasitemia as the outcome was conducted with age group, distance to an urban agricultural site, socioeconomic status, and house effects. The Hb level was omitted because it was likely to be the result of malaria infection and its inclusion could obscure the effect of distance. Two of the districts, Mle (p = 0.021) and Kbu (p = 0.014), showed decreases in prevalence with distance from an urban agricultural site; the odds of infection were reduced ≈50% every 100 m from the site. However, these results need to be interpreted with caution because it is difficult to detect a putative decrease in prevalence with distance against the noise introduced by small, unidentified, often transitory, breeding sites. Their presence may explain why 2 sites, Rom (p = 0.043) and Dzor (p = 0.039), showed a significant increase in prevalence with distance, while 2 others, Air and Koto, showed a significant effect when distance in 100-m intervals was cross-tabulated with prevalence . Since unidentified breeding sites may also introduce unknown data structuring that cannot be incorporated into a GLMM, the probabilities obtained may be lower than are appropriate.Anopheles gambiae S form breeding in irrigation water at urban agricultural sites and resting at higher densities in houses in urban agricultural communities.The parasitemia levels obtained in this study are worrisome because high-density urban African populations are not often considered particularly vulnerable to malaria infection. In other West African urban areas, malaria prevalence rates from 2% to 16% have been reported with large variation between communities ,13. ReceThese findings are based on a point prevalence survey in the dry season. Although we continue to obtain data during the wet season, analysis of data indicates that the urban poor in Africa may be at higher risk for malaria than expected and that malaria can no longer be regarded only as a rural phenomenon. This finding is of great concern because in Africa the current urban population growth rate of 3.5% is >3 times the rural population growth rate, and by 2015 a total of 25 countries in sub-Saharan Africa will have urban populations larger than the rural populations . Althoug


Intermittent preventive treatment during pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is widely recommended in sub-Saharan Africa to reduce the risk of malaria and improve birth outcomes. However, there are reports that the efficacy of IPTp with SP is waning, especially in parts of Africa where antimalarial resistance to this drug has become widespread.We conducted a cross-sectional study of 565 HIV-uninfected women giving birth at Tororo District Hospital in southeastern Uganda. The primary objective of the study was to measure associations between use of SP during pregnancy from antenatal records and the risk of adverse outcomes including placental malaria, low birth weight, maternal parasitemia and maternal anemia. The proportion of women who reported taking 0, 1, 2, and 3 doses of SP during pregnancy was 5.7%, 35.8%, 56.6% and 2.0% respectively. Overall, the prevalence of placental malaria was 17.5%, 28.1%, and 66.2% by placental smear, PCR, and histopathology, respectively. In multivariate analyses controlling for potential confounders, ≥2 doses of SP was associated with non-significant trends towards lower odds of placental malaria by placental smear , placental malaria by PCR , placental malaria by histopathology , low birth weight , maternal parasitemia  and maternal anemia . Using a composite outcome, ≥2doses of SP was associated with a significantly lower odds of placental malaria, low birth weight, maternal parasitemia, or maternal anemia .In this area of Uganda with intense malaria transmission, the prevalence of placental malaria by histopathology was high even among women who reported taking at least 2 doses of SP during pregnancy. The reported use of ≥2 doses of SP was not associated with protection against individual birth and maternal outcome measures but did protect against a composite measure of any adverse outcome. Plasmodium falciparum at the time of delivery Malaria in pregnancy remains a widespread problem in sub-Saharan Africa, where approximately one in four pregnant women has evidence of infection with dhfr) and dihydropteroate synthase (dhps) genes. The dhfr/dhps quintuple mutant has been strongly associated with SP treatment failure and has recently reached levels >90% in parts of East Africa Despite the widespread adoption of IPTp with SP as policy in Africa, there is concern for the continued efficacy of this intervention due to the spread of resistance, especially in East Africa. Sulfadoxine-pyrimethamine belongs to the antifolate class of antimalarial drugs and resistance is mediated by the ordered accumulation of point mutations in the dihydrofolate reductase  SP use documented from antenatal card if attended antenatal care, 2) HIV status known and negative, 3) absence of reported antimalarial therapy other than SP in the previous 1 month, and 4) provision of informed consent. A standardized questionnaire was administered to all enrolled women including review of their government issued antenatal card. Information collected as part of the questionnaire included demographics, previous pregnancies, bednet use, education level, ownership of household items, and the number and timing of doses of SP  and other medications. Delivery outcomes were assessed and birth weight obtained using a digital scale . Data on gestational age was not collected because information on last menstrual period was often missing and if present thought to be inaccurate. Biological samples collected included maternal finger prick for blood smear and hemoglobin measurement and placental blood and tissue biopsy.This study was conducted in Tororo, an area of southeastern Uganda with high malaria transmission intensity and an entomological inoculation rate recently estimated to be 125 infectious bites per person per year in 2011–12 . Study participants were women giving birth at Tororo District Hospital (TDH), a government hospital that provides antenatal services and free HIV testing to all pregnant women. Using a cross-sectional study design, all pregnant women with singleton births delivering at TDH who were known to be HIV-uninfected were screened for enrollment if they delivered between Monday 8∶00 am through Friday 4∶00 pm from February 28Hemoglobin measurements from maternal blood were made using a portable spectrophotometer . Maternal and placental thick blood smears were stained with 2% Giemsa for 30 minutes and examined for malaria parasites by standard microscopy. Parasite density was estimated by counting the number of asexual parasites per 200 white blood cells and calculating parasites per µL, assuming a white blood cell count of 8,000 cells per µL. A smear was judged to be negative if no parasites were seen after review of 500 high-powered fields. Final microscopy results were based on a rigorous quality control system with re-reading all blood smears by a second microscopist and resolution of any discrepancies by a third microscopist. PCR for the detection of malaria parasites were performed on placental blood stored on filter paper using nested PCR as previously described Placental biopsies of approximately 1–2 cm×1–2 cm from the maternal side were collected using scissors and placed in 10% neutral buffered formalin. After 24 hours, biopsies were trimmed with a razor blade to 1×1 cm size and formalin replaced with fresh neutral buffered formalin. Following 1–3 months of storage, placental tissue was embedded in paraffin wax, microtome sectioned and stained with 2% Giemsa, and hematoxylin and eosin. Histopathological slides were examined using standard and polarized light microscopy for hemozoin pigment in intervillous fibrin, malaria parasites, and macrophages with hemozoin pigment using standardized criteria as previously described Data were double entered in Access , and analyses performed using STATA . The primary exposure variable of interest was IPTp use with SP as indicated on the participant’s antenatal card. Given the distribution of the number of SP doses reported taken, SP usage was dichotomized into <2 doses vs. ≥2 doses of IPTp. Comparisons of characteristics between the two SP usage groups were made using the chi-squared or t-test. Three definitions of placenta malaria were used: 1) any parasitemia by placental blood smear, 2) detection of parasites by PCR, and 3) any evidence of placental malaria by histopathology. Maternal peripheral parasitemia was defined as a positive blood smear and maternal anemia defined as a hemoglobin level <11 gm/dL. Low birth weight was defined as <2500 gm. Univariate and multivariate analyses of associations between SP usage and outcomes of interest were performed using logistic regression. Covariates adjusted for in multivariate analyses included maternal age, gravidity, bednet use, level of education, transmission season, and a wealth index generated using principal component analysis as previously described A total of 581 women were screened and 565 enrolled . ReasonsOverall the risk of placental malaria as defined by placental blood smear, placental PCR, and histopathology were 17.5%, 28.1%, and 66.2%, respectively. Among 466 placental blood samples that were negative by blood smear, 62 (13.3%) were positive by PCR suggesting the presence of sub-patent parasitemia. Histopathology was the most sensitive measure of placental malaria with 235 of 404 (58.2%) of samples negative for both placental blood smear and PCR having histopathological evidence of placental malaria, the majority (76.2%) of which only had evidence of malarial pigment indicative of past infection. Associations between SP usage and various measures of placental malaria are presented in Associations between the duration since the last reported dose of SP taken and the risk of a positive placental blood smear and placental malaria by histopathology are presented in Considering birth outcomes, 545 infants survived, 13 were stillborn, and 7 died shortly after birth prior to discharge. Overall the risk of LBW was 9.9%. Compared to <2 doses of SP, ≥2 doses of SP was associated with a modest trend towards a lower odds of LBW , but this did not reach statistical significance after controlling for potential confounders . When evIn this cross-sectional study of HIV-uninfected women giving birth at a district hospital in Uganda, 94.3% of women had documentation of receiving at least one dose of SP during pregnancy and 58.6% received at least 2 doses of SP. At the time of this study, national recommendations were that women take at least 2 doses of SP during pregnancy. Despite the high coverage of IPTp with SP in this population, the risk of placental malaria was 17.5% by placental blood smear and 66.2% by histopathology. Given the small numbers of women who did not report taking SP or who reported taking >2 doses during pregnancy, comparisons were only made between those with <2 doses of SP versus those with ≥2 doses of SP. Receiving ≥2 doses of SP was not significantly associated with protection against placental malaria, LBW, maternal parasitemia, or maternal anemia, however, there was significant protection when using a composite including any of these adverse outcomes.IPTp with SP replaced weekly chloroquine prophylaxis for the prevention of malaria in pregnancy in the mid 1990’s and is currently recommended as policy for most countries in sub-Saharan Africa. A systematic review published in 2007 of 4 trials comparing IPTp with 2-dose SP to case management or placebo reported that IPTp with SP was associated with a reduced risk of placental malaria, LBW, and maternal anemia in vivo studies of SP for the treatment of symptomatic malaria have been used in the past, however, SP monotherapy is no longer recommended for the treatment of malaria in Africa. The WHO now recommends evaluation of the in vivo efficacy of SP in populations with asymptomatic parasitemia as a method of monitoring the effectiveness of IPTp with SP dhfr and dhps target genes and the prevalence of the dhfr/dhps quintuple mutant has been correlated with in vivo SP resistance dhfr/dhps quintuple mutant increased from 7% in 1996–2000 to 88% in 2008–09 dhfr/dhps quintuple mutant has surpassed 90% dhps codon 581 associated with high level SP resistance has now been reported in areas of Tanzania and Kenya Monitoring the efficacy of IPTp with SP is challenging as it is generally not considered ethical to perform randomized clinical trials with a placebo arm in high transmission areas. Surrogate data from This study has several limitations. First, we used a cross-sectional study design rather than a placebo-controlled randomized trial since SP for IPTp is the standard of care and withholding it was not felt to be an option. Although we attempted to control for potential confounding factors, the possibility of bias due to unmeasured factors cannot be ruled out, limiting our ability to make causal inferences. Second, SP use was based on documentation from antenatal cards and not measured using a prospective study design. However, reports from antenatal cards are likely accurate given that SP administration is directly observed in the antenatal clinic. Third, over 92% of women in this study reported taking 1–2 doses of SP during pregnancy, therefore we lacked the statistical power to compare outcomes between groups that reported taking no SP or more than 2 doses of SP. Fourth, we were unable to obtain accurate data on gestational age or molecular markers of SP resistance markers, limiting the scope of our analyses. Finally, we cannot rule out the possibility of a type II error, i.e. the possibility that SP provided protection against individual outcomes we were unable to detect due to a lack of statistical power.In summary, growing evidence from this study and others raise questions about the continued efficacy of IPTp with 2-dose SP for the prevention of placental malaria in areas of East Africa with a high prevalence of SP resistance. Although these data do not provide evidence that IPTp with SP should be abandoned, there is certainly a need to evaluate strategies for chemoprevention in pregnancy that will reduce this risk of placental malaria. Several studies have investigated whether increasing the number of recommended doses of SP would improve efficacy. In a randomized controlled trial from an area with low SP resistance in Mali, 3-dose SP was associated with a lower risk of placental malaria, LBW and preterm birth compared with 2-dose SP


Previous data have suggested that regulatory T cells (Tregs) balance protective immune responses with immune mediated pathology in malaria. This study aimed to determine to test the hypothesis that Treg proportions or absolute levels are associated with parasitaemia and malaria symptoms.low T cells. Three patient groups were assessed: patients with symptomatic Plasmodium falciparum malaria (S), subjects with asymptomatic P. falciparum parasitaemia (AS) and uninfected control individuals (C).Treg cells were quantified by flow cytometry as CD4+ CD25+, Foxp3+, CD127S, AS and C groups had similar absolute numbers and percentage of Tregs . Levels of parasitaemia were not associated with Treg percentage (p = 0.47).P. falciparum parasitaemia in the Peruvian Amazon; but further study to explore these mechanisms is needed.Neither relative nor absolute regulatory T cell numbers were found to be associated with malaria-related symptomatology in this study. Immune mechanisms other than Tregs are likely to be responsible for the state of asymptomatic  Plasmodium falciparum-infected individuals show a wide spectrum of clinical manifestations that range from a state of asymptomatic infection to severe life-threatening forms, such as hyperparasitaemia, hypoglycaemia, cerebral malaria, respiratory distress, and vital organ dysfunction    was found [r = 0.02, P. falciparum hypoendemic region of the Peruvian Amazon, absolute or relative numbers of Tregs relates to malaria symptomatology. These results stand in contrast to previous studies in mouse and from humans in other malaria regions [This study showed that Symptomatic (S), Asymptomatic (AS) and Control (C) groups had similar absolute numbers and percentage of Tregs in peripheral blood, 3.9%, 3.5% and 3.5% respectively, and a relationship between Treg percentage and parasitaemia was not found Figures  and 2. T regions ,9,17-20,P. vivax infections were also found to have increased proportions of CD4+ CD25+ Foxp3+ Treg cells in Thailand [et al.[+ cell counts. In contrast to our observations, this previous study showed that the CD25+ Foxp3+ Treg cell counts were inversely proportional to level of parasitaemia [Some studies have compared numbers of Tregs with severe or uncomplicated malaria, and found also increased number of Tregs during convalescence negatively associated with the magnitude of Th1 memory response in children , and theThailand  and BrazThailand . A diffesitaemia .P. falciparum antigens [These results showed that the absolute numbers and proportion of Tregs were similar among symptomatic and asymptomatic groups despite a higher parasitaemia in S group vs. AS group, confirming that the relationship between number of parasites and Tregs activity is still not clear. Therefore, at least in the context of the hypoendemic setting, total numbers of Tregs are not implicated in the symptomatology or control of parasite proliferation. These results agree with previous reports that showed a more efficient Th1 and Th2 response toward antigens . A studyantigens . These rAlthough the data presented here did not include analysis of cytokines associated with Tregs, antigen-specific Tregs or possibly a lower expression of molecules involved in Treg cell mediated modulation of immune responses might contribute to the control of symptoms related to symptomatic vs. asymptomatic parasitaemia.Little is known about characteristics of the malaria parasites themselves associated with the state of asymptomatic infections. A recent study of asymptomatic infected children found an increased tumor necrosis factor receptor type II (TNFRII) expression on Tregs confirming the activation of these cells even in subclinical parasitaemia .The lack of relationship of number or proportion of Tregs to symptomatology in the present study is consistent with previous work where the duration of clinical symptoms and Treg cell number were measured; these studies did not find a significant correlation between these two parameters .The discrepancy among all of these studies, including the current results, may related to differences in study population, study design, Treg phenotypes, age of individuals, cumulative exposure to malaria, clinical outcome of the disease, working conditions, clinical outcome, among others variables.P. falciparum malaria requiring frequent infections over a period of five years and sustained frequent infections in order to maintain an ability to resist symptoms during malaria infection. With these data the function of Tregs might be different in these regions because of different transmission context, intensity and other unknown factors. Another important consideration is that patients with symptomatic parasitaemia might only have had incidental parasitemia along with a separate concomitant infection responsible for causing the fever; we did not assess this possibility in this study.There are several important limitations of this study. First, pro- and anti-inflammatory cytokines associated with the regulation by Tregs were not measured. The concept that transmission intensity influences the efficiency of acquiring malaria-specific immunological memory has been reported widely. If the majority of infections occur within a sub-population who frequently travel or live in higher transmission regions (i.e. Africa), then these highly exposed individuals might receive the quantity of infections conventionally considered necessary develop protective immunity against symptoms during infection. Conversely, if infections are acquired locally, then there would be low transmission in the whole population and one would expect most of the infections to be symptomatic. This expectation is based upon literature regarding the development of naturally acquired immunity to vs. asymptomatic P. falciparum infected individuals from a malaria hypoendemic region, typified by the transmission patterns founds in the Peruvian Amazon region. Deeper phenotypic and functional characterization of Tregs in malaria in this and comparable regions may yield new insights into the immunological control of malaria symptomatology in the face of asymptomatic parasitaemia.The present work is the first to report about number and proportion of Tregs in symptomatic The authors declare that they have no competing interests.KT designed the study, carried out the immunological analysis and wrote the paper. EV and JB performed some experiments and contribute to the paper writing. JCH carried out the statistical analysis. JV and DG supervised the work and study design, wrote and reviewed the final versions of the manuscript. All authors read and approved the final manuscript.


Plasmodium vivax was used to identify blood donors infected with malaria parasites.When selecting blood donors in transfusion centres, one important problem is to identify, during screening, individuals with infectious diseases that can be transmitted by blood, such as malaria, especially when the parasite densities are very low. This problem is particularly severe in endemic areas, such as the Brazilian Amazon. In the present study, molecular diagnostic  of P. vivax. The experiment was designed for hybridization of the cytochrome c oxidase genes of the mitochondrial genome (GenBank GI63022502). The serological data were obtained using enzyme-linked immunosorbent assay - ELISA  and VDRL (Syphilis) from the Blood Bank System of the Haematology and Haemotherapy Centre of Pará.Samples from 595 blood donors were collected in seven haemotherapy centres in northern Brazil located in areas at risk for malaria transmission, and the analyses were performed by real-time PCR with TaqMan probes on 7500 Real-Time PCR Systems, to genotype the mitochondrial DNA region specific to P. vivax at the time of blood donation. This percentage was higher than the altered serological results (reactive or inconclusive) of the prevalence of anti-HIV (0.67%), anti-hepatitis C virus (0.34%), anti-hepatitis B surface antigen (0.67%), anti-human T-lymphotropic virus I/II (1.18%), anti-Chagas disease (0.17%) and syphilis (VDRL) (0.50%), but not higher than anti-hepatitis B core antigen antibodies (4.37%). This result indicates the need to use more sensitive methods of diagnosing malaria in blood banks.The assay identified eight individuals in the sample (1.34%) infected with P. vivax in a high proportion of clinically healthy donors, highlighting the potential risk for transfusion-transmitted malaria. Additionally, this molecular diagnostic tool can be adopted as a new laboratory screening method in haemotherapy centres, especially in malaria-endemic areas.The real-time PCR with TaqMan probes enabled the identification of  Plasmodium have been described, including studies carried out in South America[Malaria is recognized as a serious public health problem, occurring in tropical and regions such as Africa, Asia and Central and South America. In 2010, there were an estimated 216 million new cases of malaria, with 655,000 deaths[ America.Plasmodium vivax, Plasmodium falciparum and Plasmodium malariae. Plasmodium vivax is the most prevalent species, causing approximately 83.7% of reported cases in 2009[In Brazil, malaria is endemic in the Amazon region, with 98% of the cases in the year 2009. Malaria transmission is unstable and usually focal, and the period of highest transmission occurs after the rainy season. The vast geographical extent and the climatic conditions of the Amazon region favour transmission of the species  in 2009.Anopheles mosquito but also by congenital transmission and, rarely, by blood transfusion and the sharing of needles and syringes[Malaria is transmitted by the bite of the female syringes,6. The hsyringes. Howeversyringes.The risk of transfusion-transmitted malaria (TTM) has been associated with the difficulty in identifying infected potential donors, most with low numbers of parasites circulating in the blood (incomplete immunity), as well as the ability of this parasite to remain viable in stored blood bags, even after the storage process,10. ThusAdditionally, in cases of TTM, depending on the number of parasites in the inoculum, the symptoms of malaria may begin days or weeks after transfusion, presentCurrently, in countries where malaria is endemic, such as Brazil, TTM control is based on clinical interviews of potential donors to identify residents and travellers from high-risk endemic areas, individuals with infection and a history of malaria and those with a history of fever that precedes blood donation. Additionally, in Brazil, laboratorial diagnosis in blood banks, in endemic areas with active transmission and in non-endemic areas (whose candidates for blood donation are from cities located in endemic areas) is recommended for the detection of parasites,15.Plasmodium detection in endemic areas but cannot identify parasites at low densities. This technique detects quantities between 5 and 20 parasites/μL of blood, and the results depend on the experience of the microscopist[P. falciparum antigen[The scientific community has been concerned with the low sensitivity and specificity of the different methods used for laboratory investigation of malaria parasites. In practice, the gold-standard technique, optical microscopy in thick blood smears, is used widely for oscopist,15-18. Ioscopist,19, enzy antigen,17. ELIS antigen, but thiPlasmodium by PCR, such as the nested PCR[Methods based on molecular biology have been used to detect different types of sted PCR,23. Thissted PCR,18,24-27sted PCR,18. The sted PCR,26,27.The detection of malaria infection by real-time PCR is considered, at the moment, the best molecular biology technique available and shows that real-time PCR has high sensitivity and specificity to detect malaria parasites in the blood,24,27. TP. vivax through the amplification of mitochondrial DNA (mtDNA), the use of this sequence for malaria diagnosis was first described in 2009[The present study employed real-time PCR with TaqMan probes to detect  in 2009, and in A total of 595 blood samples from the blood donor archives of seven haemotherapy centres in the state of Pará, Brazil, were investigated during April to July 2010. The samples came from municipalities that had a variable annual parasite incidence (API)  in 2009. Each API was classified as low in relation to the endemic level of malaria based on the Brazilian Ministry of Health data registered in 2009. Donors Blood samples were collected in EDTA anti-coagulant. From each individual 3 mL of venous blood was obtained from the archives of blood donor samples of the Haematology and Haemotherapy Centre of Pará, Brazil, corresponding to the period from April to July 2010. All samples were stored at −20°C until DNA extraction.DNA was extracted by the phenol/chloroform method, with moP. vivax genomic DNA was based on primer extension and subsequent hybridization with specific probes. DNA segments were amplified by the method of presence and absence by real-time PCR with TaqMan® probes, 7500 Real-Time PCR Systems and 7500 System SDS software . For this purpose, the probe was labelled (6-FAM) differently from the DNA region of interest. The primers and probes were designed with the software Custom TaqMan Genomic Assays File Builder, Version 3.0, and a kit was developed.In general, the strategy for determining the presence or absence of Plasmodium vivax mtDNA was genotyped according to the manufacturer's instructions (Life Technologies). The reactions were prepared in duplicate at a final volume of 15 μL. The reactions were normalized by the use of an endogenous internal positive control (Life Technologies). As experimental calibrators of the reaction, samples from individuals with proven infection and not infected with P. vivax were used. The experiment was designed for hybridization of the cytochrome c oxidase genes of the mitochondrial genome. This sequence was based on the P. vivax genome (Cox I - Access Number GI63022502) available at GenBank. The primers used were previously published[ublished, with moP. vivax genetic material in the haemotherapy centres investigated in the present study. A Student-T and chi-square were used as statistical test to compare the samples’ attributes.A ratio test was used to measure the percentage of donors who had P. vivax DNA in blood samples derived from blood donor archives of the Haematology and Haemotherapy Centre of Pará, Brazil. A total of 595 donor samples from seven haemotherapy centres were evaluated, all in municipalities considered endemic for malaria in the state of Pará according to the API classification. In donor samples investigated by real-time PCR, eight cases were identified positive for the presence of P. vivax DNA (Table P. vivax occurred during the months of May (two/eight cases), April (five/eight cases) and June (two/eight cases). The positive samples originated from two haemotherapy centres: three samples from donors at the Regional Blood Bank of Santarém and five samples from the Blood Bank of Abaetetuba. All samples positive for P. vivax belonged to municipalities with low API.The present study evaluated the applicability of real-time PCR to the detection of A Table . The colP. vivax included three samples from women (three/166 women) and five from men (five/429 men). All positive samples were within the age range of 18 to 26 years, and their average age was 23.12 , but no difference relating to gender (p=0.317) of donors , and rapid diagnostic test. Thus, these techniques show even lower sensitivity than optical microscopy in thick blood smears,17,19.Three previous publications based on molecular biology techniques,23,28 dec oxidase specific to P. vivax and P. falciparum. The main advantage of this method compared to the others is due to the diagnostic target (mtDNA), which is more numerous than nuclear DNA. The number of copies/cell of the mtDNA genome can range from 20 to 100[The third method uses the0 to 100,33.In the present study, the method previously developed has beenAlthough malaria affects all ages, the present study showed a higher concentration of positive donors between the ages of 18 and 26 , where all positive belong this range. This result confirms the greater risk of and exposure to malaria infection in adults. This pattern may be the result of professional activities that foster individual exposure to the mosquito vector.P. vivax with altered serology (reactive and inconclusive cases) for different blood-borne diseases, the positivity for malaria was more common than the positivity for other pathogens. Because the presence of any serological alteration, by itself, is reason for discarding the donor’s blood, the presence of P. vivax mtDNA also increases the risk of infection and necessitates the disposal of the donated blood. Therefore, testing for Plasmodium or its vestiges should be recommended.By comparing, within the same analysed samples, the real-time PCR results for P. vivax by classical molecular diagnosis (standard PCR) in haemotherapy centres has been described[P. vivax and P. falciparum, with an average positivity rate of 2.3% among blood donors from four blood banks of the Amazon. The present study found a P. vivax-positive rate of 1.34% in two haemotherapy centres, the Blood Bank of Abaetetuba and the Regional Blood Bank of Santarém, both located in the state of Pará, in municipalities described to have low risk for malaria transmission with API 0.2 and 3.5, respectively. Interestingly, seven positive cases of P. vivax were detected in donors living in the cities where the blood banks are located , and only one case was from a donor residing in a rural area near the Blood Bank of Abaetetuba. However, considering the continuous flow between urban and rural areas (mainly due to economic activities) and the proximity of the blood centres to the forest, the actual infection sites may not have been in urban areas.The detection of escribed. In thatP. vivax mtDNA is a feasible, accurate, sensitive marker for detecting low-level malaria parasitaemia. All these data highlight the need to adopt new identification techniques with better sensitivity and specificity for malaria research, such as real-time PCR.Therefore, the presence of the P. vivax by real-time PCR was able to identify carriers of this parasite among a relatively high proportion of clinically healthy blood donors. The results from this study indicate that real-time PCR-based laboratory screening for the presence of P. vivax is a method that is necessary, appropriate and inexpensive, with superior sensitivity and specificity compared to previously described methods, and can be adopted as part of the laboratory screening in haemotherapy centres, especially in malaria-endemic areas.The method to detect mtDNA of The authors declare that they have no competing interests.SBS participated in the sample collection, performed RT-PCR, and drafted the manuscript. MR did the DNA extractions from the blood samples and performed RT-PCR method. SS contributed to data interpretation. MGC and ARS designed RT-PCR assay, data interpretation, and wrote the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum infection, and anaemia is a well-known outcome. The prevalence and haematological impact of P. falciparum parasitaemia were determined among HIV-infected individuals in Nigeria. Parasite detection was carried out using microscopy and Polymerase Chain Reaction (PCR). Haemoglobin concentration was determined using an automated machine while CD4+ T-cells count was analyzed using flow cytometer. Thirty-seven (18.5%) out of the 200 HIV individuals enrolled had malaria parasites detected in their blood. All the positive cases were detected by PCR while only 20 (10%) were detected by thick blood microscopy. The mean haemoglobin concentration and packed cell volume (PCV) of HIV individuals with malaria parasitaemia were lower compared to those without malaria parasitaemia but the difference was not statistically significant. Also no significant difference was observed in malaria positivity in respect tosex and mean CD4+ cell count. The study highlights the effects of P. falciparum parasitaemia on the haematologic and immune components of HIV individuals.Malaria and HIV are the two most important health challenges of our time. Haematologic abnormalities are features in  The two greatest health challenges facing Africa today are human immunodeficiency virus (HIV) infection and malaria , 2. WorlPlasmodium parasitaemia differs in instances of asymptomatic and clinical malaria, and the degree of parasitaemia may influence the pathological and biochemical presentations of individuals presenting with either of these conditions [There have been some controversies on the incidence of malaria among HIV individuals. While some studies reported high incidence of malaria in HIV individuals in malaria endemic areas  others hnditions , 10. Repnditions , 12.P. falciparum, has been shown to be useful in the detection or measurement of very low parasitaemia [P. falciparum, which is useful in determining genetic variability of parasite isolates [Different PCR-based assays have been used in diagnosis and as reference or standard for the assessment of the sensitivity and specificity of microscopy and commonly deployed rapid diagnostic tests. PCR assays though used mainly for research purposes may indeed be of clinical value in some selected situations –15. PCR sitaemia , 17. Theisolates .falciparum malaria in HIV patients using both microscopy and stevor PCR. The study also looked at the effect of age, sex, CD4+ T-cell count, and haemoglobin level on prevalence falciparum parasiteamia.This study therefore determines the prevalence of asymptomatic Two hundred HIV-seropositive confirmed individuals who came for normal routine medical checkup at the institute of human virology of Nigeria (IHVN), Ladoke Akintola University of Technology (LAUTECH) Teaching Hospital Osogbo, Osun State Nigeria, were recruited into the study. Their HIV status was confirmed using Determine HIV1/2 kit  followed by Unigold or stat pak concurrently according to serial algorithm of the Federal Government of Nigeria . Those w5 mLs of blood was aseptically drawn by venipuncture into ethylene diaminetetra-acetic acid anticoagulant bottles. The blood was used for the assessment of thin and thick films for malaria parasitemia, estimation of haematological parameters, and CD4+ T-cell count estimation. Two drops of blood were also spotted on filter paper, air dried, placed in individual sealed plastic bags, and kept at room temperature for DNA extraction and PCR analysis. The haemoglobin concentration of the collected blood samples was determined using an autoanalyser-Sysmex KX-21 . Blood was collected into heparinized capillary tube up to two-third of its length for packed cell volume (PCV) estimation. Anaemia was defined according to the WHO criteria . For malμL of distilled water was used to elute the DNA and stored at −20°C until further analysis.DNA was extracted from the dried blood spots on filter paper using the Ultra Clean Blood Spin Kit  following the manufacturer's protocol. 100 μL containing 5.0 μL ×10 reaction buffer, 200 μM of dNTPs, 1.25 units of Taq DNA polymerase, 0.4 pM of each primer , and 5.0 μL of DNA extract. The PCR programme included denaturation at 93°C for 3 minutes followed by 25 cycles of 30 seconds at 93°C, 50 seconds at 50°C, and 30 seconds at 72°C and a final extension period of 3 minutes at 72°C. Two microlitres of the first-round PCR product were used in the second round amplification which was performed in a reaction mixture of 50 μL containing 5.0 μL ×10 reaction buffer, 200 μM of dNTPs, 1.25 units of Taq DNA polymerase, and 0.4 pM of each primer (P17 and P24). Conditions for the nested reaction included denaturation at 93°C for 3 seconds followed by 25 cycles of 30 seconds at 93°C, 50 seconds at 55°C, and 30 seconds at 72°C and a final extension period of 3 minutes at 72°C. DNA extracted from laboratory adapted P. falciparum (FCR-3 strain) was used as positive control and water as negative control. PCR products were subjected to electrophoresis on 2% agarose gels and visualized using Syngene gel documentation system  after staining with ethidium bromide. The primer sequences for the stevor PCR were as previously described [Stevor gene PCR protocol was carried out as previously described . Brieflyχ2) was also used to test the association between categorical variables. All tests were carried out using the 5% level of significance.The data were analyzed using SPSS version 16.0 statistical software. Analysis of variance (ANOVA) and regression analysis were used to investigate the relationship between the variables. Chi-square , 20 (10%) were positive for malaria by microscopy while 37 (18.5%) were positive by stevor gene PCR. The mean age of the studied individuals was 36.24 (SD ± 10.10) years. Of the overall infected individuals, 27 (72.97%) were females while 10 (27.03%) were males. The mean haemoglobin concentration and packed cell volume (PCV) of the HIV-positive individuals were 10.85 g/dL (SD ± 1.89) and 32.53% (SD ± 4.41), respectively, while the mean CD4+ count was 391.31 (SD ± 237.14). The breakdown of the general characteristics of the study population is shown in P = 0.12). Similarly the mean haemoglobin level of malaria positive (10.86 g/dL SD ± 1.87) HIV individuals was lower in comparison to their malaria negative (10.80 g/dL SD ± 1.71) individuals and the difference was not statistically significant (P = 0.87). Also for the other parameters (sex and PCV) no significant difference was observed  individuals will experience increased incidence of malaria infection [μL) and decreases in the proportion of patients with a CD4 T-cell count less than 200/μL (28.7% to 13.2%) after successful malaria treatment in HIV-infected individuals [In our study, highest prevalence of malaria parasitaemia was observed among HIV-positive group that had the higher CD4+ T-cells count. Contrary to our observation, many studies have reported that malaria coinfection with HIV decreases CD4+ T cells counts , 28. Altnfection , 33. HIVnfection , 32. A pividuals .P. falciparum parasitaemia without any direct contribution to anaemia. The need for constant screening and treatment of HIV individuals living in malaria endemic area for malaria parasitaemia is therefore suggested. Improved diagnostic technique is also suggested in other to effectively detect these parasites.In conclusion, our study observed that 18.5% of the HIV individuals have


P < 0.05). Plasmodium falciparum occurred in all, and none had mixed infection. The average birth weights of the babies delivered of nonmalarious pregnant women were higher than those delivered by malarious pregnant women, though not significant (P > 0.05). Malaria parasitaemia occurred more frequently in primigravidae than multigravidae.The study was designed to determine the true prevalence of congenital, cord, and placental malaria in General Hospital Minna, North Central Nigeria. Peripheral blood smears of near-term pregnant women, as well as the placental, cord, and peripheral blood smears of their newborn babies, were examined for malaria parasites, using the Giemsa staining technique. Out of 152 pregnant women screened, 21 (13.82%) of them were infected with malaria parasites. Of the 152 new born babies, 4 (2.63%) showed positive peripheral parasitaemia. Placental parasitaemia was 7/152 (4.61%), while cord blood parasitaemia was 9/152 (5.92%). There were strong associations between peripheral and cord malaria parasitaemia and congenital malaria ( Congenital malaria was first described in 1876 .Congenital malaria has been documented for many years, but it was previously thought to be uncommon especially in indigenous populations; more recent studies, however, suggest that incidence has increased, and values between 0.30 to 33.00% have been observed from both endemic and nonendemic areas [Malaria and pregnancy are generally believed to be mutually aggravating conditions. The pathological changes due to malaria and the physiological changes associated with pregnancy have a synergistic effect on the course of each other .Pregnancy exacerbates malaria through a nonspecific hormone-dependant depression of the immune system; protective antiplasmodial activity is suppressed at pregnancy .The following hypotheses to explain the altered immunity associated with pregnancy were offered.P. falciparum to multiply; pregnant women display a bias towards type-2 cytokines and are therefore susceptible to diseases requiring type-1 responses for protection like TB, malaria, and P. falciparum has the unique ability of cytoadhesion; chondroitin sulfate A and hyaluronic acid are the adhesion molecules for parasite attachment to placental cells [Reduced lymphoproliferative response sustained by elevated levels of serum cortisol, loss of cell-mediated immunity in the mother, the presence of placenta, a new organ in the primigravidae, allows the parasite to bypass the existing host immunity, or allows placenta-specific phenotypes of al cells .In pregnancies complicated by malaria, both fetal growth retardation and preterm birth contribute to low birth weight . Malaria2 with a population of 1.2 million. Minna has a tropical climate with mean annual temperature, relative humidity, and rainfall of 30.20°, 61.00%, and 1334.00 cm, respectively. The climate presents two distinct seasons: a rainy season (April–October) and dry season (November–March). Minna is an endemic area for malaria.The study was carried out at General Hospital Minna, North Central Nigeria. Minna, the capital of Niger State, Nigeria, is located within longitude 6°33′E and latitude 9°37′N, covering a land area of 88 kmThe study was conducted from October 2010 to February 2011. The subjects were near-term (close to delivery) pregnant women who were delivered of their babies at the hospital. Sample sizes were determined from the number of pregnant women that attended antenatal care during the period of study. The nonpregnant women served as control to help compare malaria prevalence in pregnancy only. The pregnant women did not take any chemoprophylaxis and did not use mosquito treated nets for protection. The HIV status of the subjects were not determined.All work was performed according to the guidelines for human experimentations in clinical research stated by the Federal Ministry of Health of Nigeria. This study was approved by the ethical committee of General Hospital Minna, Nigeria. All women gave oral informed consent.Blood samples were obtained from the peripheral blood of 152 near-term pregnant women using sterile syringes. After delivery, blood was collected from the placentae by placental biopsies. The placentae were incised between the maternal and foetal surface and a small quantity of blood pipette out from the intervillous with a sterile syringe. Cord blood was collected immediately after separation of placenta, and peripheral blood of neonates was collected by heel pricking using a sterile blood lancet on clean glass slides. Labelled ethylene-diamine tetraacetic acid (EDTA) bottles were used in collecting blood samples for parasitological examinations. Blood collections were made possible with the assistance of midwives on duty during child delivery. Blood samples were also collected from 100 nonpregnant women. The ages, types of birth, and weights of newborn babies were recorded.P. falciparum per microlitre; a result was considered negative after a reading of 1000 leucocytes in the microscope (×1000). Parasitaemia was graded as low (1–999/μL), moderate (1000–9999/μL), and high (>10000/μL). Transplacental passage of Plasmodium falciparum was confirmed by detection of malaria parasite within 7 days of birth.Thick and thin blood films were stained with 10% Giemsa and read for malaria parasites by two trained microscopists following standard quality control procedure. Parasitaemia was expressed as the number of asexual forms of All data were analysed using SPSS version 10.1 for windows. Descriptive statistics were computed for all relevant data. Chi square analysis was used to compare proportions within and among groups, for statistical significance.P > 0.05) using the chi-square. The inclusion of nonpregnant women helped to compare the prevalence of malaria. Also, there was no significant difference in infection rates between the primigravidae (15.79%) and the multigravidae (11.84%), (P > 0.05). Of the 152 newborn babies screened, 4 (2.63%) had positive peripheral parasitaemia. Examination of the 152 placentae gave a prevalence of 4.63% (7) for malaria parasite, out of which 4 placentae were for the 4 babies with positive peripheral parasitaemia and known showed any clinical symptom. The only malaria parasite encountered in this study was the Plasmodium falciparum.The prevalence of malaria infection in blood smears of near-term pregnant mothers, their newborns, cords, placentae, and nonpregnant women was shown in P < 0.05) higher than the 411 p/uL recorded for nonpregnant women. Average parasite density for newborns was 128 p/uL, slightly lower than that for the cords which was 196 p/uL; however, both were significantly lower than parasite density recorded in the placentae (mean = 689 p/μL). Parasite density was significantly (P < 0.05) higher in neonates from primigravid (1281/uL) mothers than multigravids (134/uL) , 5 of which came from multigravidae (6.58%) and 4 from primigravidae (5.26%). The average parasite density was 1,415 p/uL for pregnant women which was significantly ((134/uL) . Parasit(134/uL) .P > 0.05). Also, newborns from primigravid mothers weighed averagely less than those from multigravid mothers; again the difference was not significant .On the average, the birth weights of babies from parasitaemia mothers were lower than those of babies from nonparasitaemia mothers, but not significantly so. This disagrees with the widely held view that babies with parasitized placentae often have low birth weight. It also contradicts the findings of Adebami et al. , who staCongenital malaria was previously thought to be rare, especially, in areas of malaria endemicity. Though this study appears not to support this view, an increasing prevalence of congenital malaria among newborns in other areas of malaria endemicity has been observed.The authors declare that there is no conflict of interests.