Plasmodium falciparum infection prevalence in children 0.5–14 years old was 9.3% (95%CI:5.9–14.5) and 22.8% (95%CI:17.3–29.4) in the two surveys. Risk factors for infection after the short rains included households not being sprayed ; low community net ownership ; and low community SES . Risk factors after the long rains included household poverty  and community poverty ; household IRS or high community ITN ownership were not protective. Despite high IRS coverage and equitable LLIN distribution, poverty was an important risk factor for malaria suggesting it could be beneficial to target additional malaria control activities to poor households and communities. High malaria prevalence in some clusters and the limited protection given by pyrethroid IRS and LLINs suggest that it may be necessary to enhance established vector control activities and consider additional interventions.Malaria prevalence remains high in many African countries despite massive scaling-up of insecticide treated nets (ITN) and indoor residual spraying (IRS). This paper evaluates the protective effect of pyrethroid IRS and ITNs in relation to risk factors for malaria based on a study conducted in North-West Tanzania, where IRS has been conducted since 2007 and universal coverage of ITNs has been carried out recently. In 2011 community-based cross-sectional surveys were conducted in the two main malaria transmission periods that occur after the short and long rainy seasons. These included 5,152 and 4,325 children aged 0.5–14 years, respectively. Data on IRS and ITN coverage, household demographics and socio-economic status were collected using an adapted version of the Malaria Indicator Survey. Children were screened for malaria by rapid diagnostic test. In the second survey, haemoglobin density was measured and filter paper blood spots were collected to determine age-specific sero-prevalence in each community surveyed.  An estimated 17% global reduction in malaria incidence has been achieved between 2000 and 2010, however despite this progress 174 million episodes of malaria were estimated to have occurred in Africa alone in 2010 Thirty-one (72%) endemic countries in Africa were reported to use both IRS and ITN in at least some areas in 2010 In Tanzania the Malaria Indicator Survey of 2007–2008 found a malaria prevalence in children under five was 18.1% Establishing determinants of infection and evaluating the effectiveness of vector control interventions can identify possible ways to improve malaria control. This study uses observational data from two large cross sectional surveys conducted in 2011 to investigate the protective effect of LLINs and IRS in relation to risk factors for malaria infection in Muleba district, North-West Tanzania.The study area has been described elsewhere Plasmodium falciparum parasite rate The National Malaria Indicator Survey 2007–2008 reported a malaria prevalence of 41% in children under five in Kagera region etrex legend H®, Garmin International Inc. USA). Each cluster was composed of a buffer zone at least 1 km wide surrounding a core sampling area approximately 1 km in radius where the surveys were conducted; the core areas were comprised of between one and five hamlets with a minimum of 200 households.The trial area was divided into 50 clusters following the mapping and enumeration of every household with hand held Global Positioning System devices  have been conducted in Muleba district with the pyrethroid lambdacyhalothrin  between 2007 and 2011. Spraying was carried out by Research Triangle Institute (RTI) with funding from PMI. At the time of the surveys for this study, the villages had been sprayed four or five times depending on year of enrolment, most recently in January-February 2011. The other intervention is high coverage of LLINs. Tanzania has a national pregnant women voucher scheme (TNVS) for the routine distribution of LLINs. LLIN coverage has been increased further by two mass distribution campaigns; the first targeting children under five in 2009/2010 nd February-25th March 2011) was conducted after the short rainy season and survey two (27th June –3rd August 2011) after the long rainy season. The first survey was approximately one month after IRS and immediately before the UCC and the second was approximately five months after IRS and three months after the UCC. These surveys form the baseline assessment for an ongoing cluster randomised control trial (CRT) to compare the effect of the combined use of IRS and LLINs versus LLINs alone on malaria transmission .Two household cross-sectional surveys were undertaken to collect data in the two annual malaria transmission seasons. Survey one  or Kihaya  after written informed consent had been obtained. Data on IRS coverage, bed net ownership and usage, demographics of household members, and other household characteristics including factors related to socio economic status (SES) were gathered using an adapted version of the standard Malaria Indicator Survey P. falciparum, Orchid Biomedical Systems, Goa, India and Survey 2: CareStart™ Malaria DiaSys, Wokingham, UK). In survey two, haemoglobin densities were measured  and filter paper blood spots were collected for the detection of antibodies to P. falciparum blood stage antigen. Cases with malaria infection detected by RDTs were treated with artemisinin-based combination therapy (ACTs). For serological analysis, in survey two, all pregnant women, plus one adult randomly selected from every third selected household were clinically examined.A maximum of two children per household were selected at random and clinically examined the following day at a central location in the village. Axillary temperature and thick and thin blood smears were taken, and rapid diagnostic tests (RDT) for malaria parasites were conducted  and membrane surface protein (MSP-119) from blood spots from all study children and a sample of individuals aged over 14 years old 19) were calculated using the mixture model function A standard ELISA was used to detect antibodies to Data were collected by interviewers using Pendragon™ Forms  on Personal Digital Assistants (PDA), and then transferred into a Microsoft Access database . All statistical analysis was done in STATA 12 .For the purpose of this analysis ‘ITNs’ was used to refer to LLINs, pre-treated nets that were less than 1 year old and nets that had been re-treated within the last year.Plasmodium falciparum malaria infection was defined as P. falciparum alone or as a mixed species infections as detected by RDT. Plasmodium falciparum infection prevalence was calculated for the study area overall and by cluster. Unless otherwise stated analysis was completed and the results are given for children aged 6 months to 14 years. SES quintiles were created using Principle Component Analysis of the following household characteristics: number of rooms, household crowding, level of schooling of the household head, type of house  and ownership of livestock, farmland, bicycles, mobile phones and radios P. falciparum infection status.For all children and separately those aged less than five years study site and cluster level anaemia prevalence estimates  were calculated for: moderate to severe (<8 g/dL) and any anaemia (cut off varied with age) 19). Age standardised sero-prevalence was calculated for each cluster using direct standardisation against the total population studied.Evaluations of current control programmes generally use observational data as trials cannot be feasibly conducted. One problem with this approach to evaluation is that the uptake of vector control measures is sometimes highest in the areas with the highest risk of malaria. Serology has been used historically P. falciparum infection in children 6 months to 14 years old were analysed for each survey using logistic regression allowing for clustering using the survey commands in STATA 12. Sero-prevalence data was only available for 44 out of the 50 clusters thus only these were included in the risk factor analysis. Household altitude, housing density at the cluster level and mean cluster altitude were not included in the final models as they were not confounders for the risk factors of interest and they are on the causal pathway for cluster sero-prevalence.Risk factors for individual Individual level factors: Age, gender, slept under an ITN the previous night•Household level factors:•Crowding, number residents, SES quintiles (survey 2 only),Household ITN ownership (at least one ITN per two residents)Household ITN usage IRS in 2011•Cluster level factors:Age-adjusted sero-prevalence (from the survey 2 as a proxy for transmission intensity)Community SES (from survey two)IRS coverage (from survey one approximately one month after spraying)Proportion of cluster residents that used an ITN the previous nightProportion of households with adequate ITNs (at least one ITN per two residents)P. falciparum infection with a p-value of <0.2 were fitted to a multi variable logistic regression model using the step-wise technique and retained if the adjusted p-value was <0.05. It was pre-specified that interactions between malaria transmission intensity and the following factors would be investigated: household IRS; ITN use the previous night; and any factors relating to IRS or ITN use that were independently associated with P. falciparum infection.All factors associated with The trial was approved by the ethics review committees of the Kilimanjaro Christian Medical College, the National Institute for Medical Research Tanzania and the London School of Hygiene and Tropical Medicine. Written informed consent was obtained from all participants or the parents/guardians if under 15 years old.In survey one 5,000 households were selected, of which 64.6% were eligible with children aged 0.5–14 years and consented to participate in the study. Twelve households (0.2%) refused to participate. There were 9,233 children aged between 6 months and 14 years, of these 5,802 were selected for inclusion and of whom 88.6% participated . The perPlasmodium falciparum infection prevalence determined by RDTs in children aged 6 months to 14 years old was 9.3% (95% CI 5.9–14.5) in survey one and 22.8% (95% CI 17.3–29.4) in survey two. Figure 2 shows that P. falciparum infection prevalence was heterogeneous between clusters. Cluster estimates ranged from 0 to 55.7% in survey one and 1.3 to 61.5% in survey two. The maps  show that the highest transmission was in the south-west of the district. Plasmodium falciparum infection prevalence increased with age, reaching a peak in six year olds at 11.6% in survey one and 28.9% in survey two. In survey two 9.6%  of pregnant women and 10.3%  of non-pregnant adults (aged over 14 years) had P. falciparum infection.P. falciparum infection – in children was 4.7%  and 4.3%  in the first and second survey respectively. Of children with P. falciparum infection, 50.7% (95% CI = 33.4–67.9%) had fever in survey one and 18.7% (95% CI = 12.6–26.9%) in survey two. The odds of fever were 62.1 (95% CI 25.9–148.9) times higher in those that were P. falciparum positive compared to P. falciparum negatives in survey one and 18.9 (95% CI 9.7–36.7) times higher in survey two. Amongst children with P. falciparum, the percentage of fever cases that could be attributed to P. falciparum infection (attributable fraction) were 96.8% and 93.5% in survey one and two respectively. Of all fever cases in the community 73.7% and 76.7% could be attributed to P. falciparum infection in survey one and two P. falciparum infection does not vary with age . Of the children that had fever, 76.2% (95% CI 58.3–88.0%) and 75.2% (95% CI = 63.5–84.1%) were P. falciparum positive in survey one and two respectively.The prevalence of clinical malaria – defined as fever (axillary temperature ≥37.5?C) and P. falciparum infection .Anaemia prevalence data was only collected in survey two. In children under 5 years 55%  had some level of anaemia and 6.2% (95% CI = 4.5–8.5) had moderate to severe anaemia (<8 g/dL). Moderate-severe cluster estimates ranged from 0 to 23.8%. The odds of moderate-severe anaemia were eight times higher if the child had 19 or GLURP) in all residents was 37.3% (95% CI = 35.9–38.7%). Cluster estimates (adjusted for age) ranged from 3.1% to 79.7% . There was a positive association between cluster sero-prevalence and cluster P. falciparum infection . In children the odds of P. falciparum infection were 12.2 times higher for sero-positives. Sero-prevalence was 72.8% and 18% in children with and without P. falciparum infection (RDT) respectively.Total sero-prevalence , and malaria transmission intensity  . Infection was considerably lower in sprayed than unsprayed houses . There was weak evidence  that the size of this effect was stronger in areas with low malaria transmission intensity . The odds of P. falciparum infection were lower in clusters with more than 15% of households with one or more ITN per two residents . Children in clusters in the highest SES tertile have a much lower odds of P. falciparum infection than those that live in the lowest tertile villages . No other factors that were investigated were independently associated with P. falciparum infection including sleeping under an ITN the previous night. Household SES was not assessed in survey one.In survey one, vey two) . CompareP. falciparum infection was independently associated with age, household SES, community SES and high malaria transmission intensity. The odds of infection were similar in children aged 1 to 14 years of age . The odds of P. falciparum infection in the least poor villages was approximately halved compared to the poorest villages . The odds of P. falciparum infection were 1.5 times higher for each 10% rise in cluster sero-prevalence. No other factors that were investigated were independently associated with P. falciparum infection in survey two including: sleeping under an ITN the previous night, cluster ITN coverage nor whether the house was sprayed.In survey two, s of age . The oddApproximately one month after the spraying (first survey) reported coverage was 95.3% Table 1 Detailed information on net usage and ownership has been reported elsewhere P. falciparum infection in children less than 15 years old were: house not being sprayed; historically high transmission in the cluster; low community wealth; low community coverage of ITN ownership and; child being aged 5–9 years old. During the second malaria season (after the UCC) the main risk factors associated with P. falciparum infection in children were: historically high transmission in the cluster; low household wealth; low community wealth and; age of child (<1 year olds at the lowest risk).The results show that in Muleba during the first annual malaria transmission season the main risk factors for P. falciparum infection and spray coverage after adjusting for other factors; coverage was high in all clusters and thus unlikely to be a concern. In the second malaria season when malaria prevalence was higher, household IRS status was not associated with P. falciparum infection. Lambdacyhalothrin has a residual effect of 3 to 6 months The protective effect of household IRS in the first malaria season was within the range reported by other studies P. falciparum infection and reported individual use of an ITN the previous night. This finding is inconsistent with the vast amount of evidence from randomised control trials that showed the use of ITNs greatly reduced malaria morbidity and mortality P. falciparum infection, while verified usage was associated with a lower odds of P. falciparum infection. Thirdly, there was no additional benefit to using nets when high coverage of IRS has been achieved as reported in Burundi No association was seen between P. falciparum infection, clusters with higher community ITN coverage had lower malaria prevalence in the first survey. This suggests that ownership may be a better indicator for the protection given by ITNs than individual net use if self-reported ITN use is unreliable While reported individual net use was not associated with a decrease in During this analysis the combined use of IRS and ITNs was not compared to the use of one alone. There were few individuals that used a net alone and most net use (95%) was combined with IRS due to the high IRS coverage. Therefore we investigated the effect of each intervention adjusted for the other in multi-variable regression.The apparent absence of personal protection provided by ITNs in this area with high IRS coverage suggests the need for trials comparing the combined use of IRS and ITNs compared to one alone. Current evidence as to whether there is an additional benefit from using ITNs in an area with high IRS is inconclusive P. falciparum infection, highlighting the importance of both household and community level poverty. Although malaria is considered a disease of poverty P. falciparum infection and the distance from the household to the nearest health facility after adjusting for other factors. It may be that it is not the actual distance but the accessibility due to poverty that is important, especially as health care and ACTs are not provided free of charge in Tanzania P. falciparum infection after adjusting for age and background malaria transmission; wall and floor material were not associated. Temu and collegues also found an association between grass roofing and malaria infection In both surveys there was clear evidence of individual infection prevalence being associated with low socio-economic characteristics of the community. In survey two, in which household SES was available, it was shown that living in a poor household and a poor cluster were both independently associated with having increased odds of Infection prevalence was higher after the long rainy season  than after the short rainy season (survey one = 9.3%). However the estimates do not show the complete seasonal changes and may not capture the peak in cases. The 8.5% prevalence reported in children under five in Kagera region in the 2012 malaria indicator survey P. falciparum infection was about 75% in both surveys. This suggests that malaria is responsible for a large proportion of the fever cases and thus disease burden in the area. The prevalence of symptomatic cases  was very similar between the two surveys as a higher percentage of the infected individuals had fever in the first malaria season. This could be explained if the second survey was conducted after the peak in symptomatic cases, but cannot be verified as the surveys were cross-sectional.The percentage of fever that can be attributed to The level of any anaemia (55%) in our study was much lower than the estimates reported in the 2004–5 Demographic Health Survey in Kagera region (71%) IRS acceptance appears to be high even after six years of spraying as illustrated by very high reported coverage (95%) and that 95% of all households reported that they would accept IRS in the future. Most people appear to understand the purpose of IRS and this aids compliance. ITN ownership and usage has increased between the two surveys as reported elsewhere Sero-prevalence was used in this study to adjust for background malaria exposure and thus transmission levels when assessing the impact of the interventions. This is important in observational studies since uptake of the interventions may increase with risk of disease and the impact of IRS may differ between places of differing transmission intensities. The substantial heterogeneity in transmission in this study underscores the need for this adjustment. Sero-prevalence data has been used previously to adjust for malaria transmission intensity when evaluating the effectiveness of vector control interventions on Bioko Island In conclusion, in this area where IRS coverage was high and LLIN distribution was equitable


Pyruvate kinase (PK) deficiency, causing hemolytic anemia, has been associated to malaria protection and its prevalence in sub-Saharan Africa is not known so far. This work shows the results of a study undertaken to determine PK deficiency occurrence in some sub-Saharan African countries, as well as finding a prevalent PK variant underlying this deficiency.Blood samples of individuals from four malaria endemic countries  were analyzed in order to determine PK deficiency occurrence and detect any possible high frequent PK variant mutation. The association between this mutation and malaria was ascertained through association studies involving sample groups from individuals showing different malaria infection and outcome status.The percentage of individuals showing a reduced PK activity in Maputo was 4.1% and the missense mutation G829A (Glu277Lys) in the PKLR gene  was identified in a high frequency. Heterozygous carrier frequency was between 6.7% and 2.6%. A significant association was not detected between either PK reduced activity or allele 829A frequency and malaria infection and outcome, although the variant was more frequent among individuals with uncomplicated malaria.This was the first study on the occurrence of PK deficiency in several areas of Africa. A common PKLR mutation G829A (Glu277Lys) was identified. A global geographical co-distribution between malaria and high frequency of PK deficiency seems to occur suggesting that malaria may be a selective force raising the frequency of this 277Lys variant. Infectious diseases have been one of the major causes of mortality during most of human evolution. For many diseases, mortality and hence reproductive success are influenced by certain individual genotype. Consequently, some aspects of modern patterns of human genetic diversity should have been determined by diseases dating from prehistoric times Plasmodium falciparum in vitro cultures using human PK-deficient blood Pyruvate kinase (PK) deficiency, caused by mutations in the pyruvate kinase, liver and RBC (PKLR) gene (chromosome 1q21) is one of the most recently described erythrocyte abnormalities associated to malaria. Evidences of its protective effect were obtained both in murine models PK catalyzes the conversion of phosphoenolpyruvate (PEP) into pyruvate with the synthesis of ATP in the last step of glycolysis. PEP and pyruvate are involved in a great deal of energetic and biosynthetic pathways and the regulation of PK activity has proven to be of great importance for the entire cellular metabolism High frequencies of PK deficiency have not yet been recorded in malaria endemic areas but a systematic analysis has never been performed. Considering the previous knowledge of co-distribution between malaria endemicity and protective polymorphisms, we questioned if a PK variant could be exceptionally prevalent in malaria endemic areas. Therefore, the aims of the present study were: i) to determine PK deficiency occurrence in sub-Saharan African countries, ii) to assess frequency of PK variants underlying this deficiency, iii) to investigate possible associations between PK deficiency and malaria infection.This study is based on the molecular analysis of six sets of blood samples collected in four sub-Saharan African areas – Mozambique, Angola, Equatorial Guinea and Sao Tome and Principe see  - and inIn this study, 296 unrelated whole blood samples from individuals who attended to the Central Hospital of Maputo (Mozambique) between September and December 2008 were analyzed: 144 from children (6 months to 14 years-old) who presented to the Emergency Services of the Pediatric Department with some kind of complaint, and 152 from healthy blood donor adults (16 to 65 years-old) who presented to the Blood Bank. In order to increase the sample size of the set with a malaria outcome characterization, an additional group of 151 DNA samples extracted from blood samples collected from 3 months to 15 years-old children in Mozambique Plasmodium infection status were considered at collection time. In the Blood Bank, the blood samples were randomly collected from blood donors. In the admission, a solubility test for rapid detection of hemoglobin S  and IHMT . A detailed work plan, questionnaires and informed consent forms were submitted to the Ethical Committees of the participant institutions in the study, which approved the survey. Each individual and parent/tutor of the children was informed of the nature and aims of the study and was told that participation was voluntary; written informed consent was obtained from each person (or parent/tutor). Blood sample collection followed strict requirements set by the Ethical Committees: blood samples from children who attended to the Pediatric Department were the remaining volume of the samples previously collected for the medical diagnosis; in the Blood Bank, during the blood donation, a small volume was put aside in a tube. In this way, no extra blood collection was needed and the patient, blood donor and the routine health services were not significantly disturbed. All ethical aspects related with the other sets of samples collected in previous studies, are described in the respective reports Plasmodium species specific primers In the Central Hospital of Maputo, the rapid test OptiMAL-IT  was used for malaria diagnosis in all the patients with suspicion of malaria infection, and a blood smear was prepared for microscopic visualization to confirm diagnosis; later, all samples were amplified by Polymerase Chain Reaction (PCR), using Plasmodium, fever , hemoglobin level of Hb≤5 g/dL and/or any of these symptoms: coma, prostration or convulsions; (ii) Uncomplicated Malaria (UM): positive PCR for any Plasmodium species, fever and hemoglobin level of Hb>5 g/dL; and (iii) Asymptomatic Infection (AI): positive PCR for any Plasmodium species in the absence of fever  or other symptoms of clinical illness; (iv) No infection (NI): negative PCR and absence of fever or other symptoms of clinical illness.Malaria outcome was defined as follows: (i) Severe Malaria (SM): positive PCR for any species of et al.Based on malaria infection and symptoms data, the 144 samples from the Pediatric Department of Central Hospital of Maputo collected in 2008 were organized in the following malaria outcome groups: SM (18 samples); UM (27 samples) and NI (99 samples). The 152 samples from the Blood Bank were organized in the following groups: AI (4 samples) and NI (148 samples). Outcome groups were also defined using the same criteria for the set of isolates from Angola  and for the set of isolates previously collected in Mozambique , both described in Machado PK activity was measured in lyzed erythrocytes from all the 296 fresh blood samples  collected in Mozambique in 2008, with an enzymatic assay adapted from Beutler et al.Samples with a PK activity value less than or equal to 75% of the normal control sample activity were analyzed by the Single Strand Conformational Polymorphism (SSCP) method (described in Manco STF (to test for differentiation between populations), expected and observed values of heterozygosity and to perform Hardy–Weinberg equilibrium tests. Prediction of the possible impact of the amino acid substitution on the structure and function of the human PK protein was performed with the Polyphen software The association between alleles and malaria outcome groups was assessed by Pearson's chi-square tests and Fisher's exact test, this latter considered when there were a few cases in each comparison group (less than five), using the Simple Interactive Statistical Analysis software (SISA) P. falciparum. Nineteen of the infected individuals were also anemic. Four (2.6%) of the 152 samples from the adult blood donors in Blood Bank showed an asymptomatic infection with P. falciparum (see Ninety-eight from the 144 samples collected in the Pediatric Department (68%) in Mozambique in 2008 were from children with a hemoglobin concentration <9 g/dL (considered anemic) and 41 samples (28.5%) were infected with arum see From the 296 samples set, 12 (4.1%) presented PK activity values between 39% and 75% of the normal control activity (established in an average of 3.2 U/g Hb) see : 8 from A migration pattern alteration was observed in the amplicon of exon 7 of 5 out of 12 samples with low activity (41.7%) by SSCP see : 4 from 2 = 14.38 (P<0.00), OR = 5.58 (95% CI: 2.07–15.03). Of the 6 samples with the lowest PK activity values , 5 had the mutation. All the 6 other samples with an activity between 47% and 75% of the normal activity were wild type.No association was found between the 829A allele and anemia (2.7–9 g/dL Hb). Conversely, a strong association was found between the allele 829A and PK deficient activity: χAs visualized in the 3D PK structure simulation see , this 27The mutation G829A was found in the other three African countries, always in heterozygosis: in 11 samples from Angola (6.7%), 1 sample from Equatorial Guinea (2.6%) and 2 samples from Sao Tome and Principe (3.0%). Allele 829A frequencies were 3.4%, 1.3% and 1.5%, respectively. In the Mozambican group from 2005, the frequency of individuals heterozygous for 829A was 5.3%, giving an allele frequency of 2.6%. The mutation was not found in the control group from Portugal. Considering all the Mozambican 447 samples, a frequency of carrier individuals of 5.8% and 829A allele frequency of 3.0% were estimated.STF were non-significant between all pairs of African populations  .The observed genotype frequencies  were according to Hardy-Weinberg expectations for all populations . Estimates of Six-hundred and eleven DNA samples belonging to individuals characterized for their infection and malaria disease outcome status were analyzed: 459 samples from children  from Angola and Mozambique and 152 samples from adults (4 AI and 148 NI), from Mozambique. No significant differentiation between samples from Angola and Mozambique were observed, so all samples together were considered for this analysis.2 = 0.02, P = 1.00; OR = 1.07, 95% CI: 0.41–2.80). No significant association was found either between 829A allele and infection  or between PK deficient activity (low enzyme activity) and infection (P = 0.30), though 11 from the 12 samples with PK reduced activity were non-infected.Allele 829A frequencies were as follows see : in chilThis is the first study aimed at determining PK deficiency occurrence as well as at studying a potential widespread PKLR mutation in the African continent.In the first instance, PK deficiency was studied in samples from Maputo, Mozambique, measuring PK activity in anemic individuals, as this is described as a symptom of the disease. However, anemia was neither associated to PK reduced activity nor 829A allele. The overall prevalence rate of PK reduced activity was 4.1% in the study population (5.3% from blood donors and 2.8% from children). Although children samples were, most of them, clinical cases with a considerable anemic status, a higher PK deficiency prevalence was not found in these samples and no association was detected between PK low activity and anemia. In this regard, a study carried in 2002 revealed that 74% of the children under five and 50% of the women in reproductive age from Mozambique was anemic STF values were not significantly different between them. These differences may be explained by sample size bias  or design bias . In addition, genetic substructure among geographic regions cannot be excluded as a hypothesis for this disparity. Differences in malaria selective pressure are not a probable cause, since it has probably been similar in all these regions in the past.The missense mutation G829A (Glu277Lys) was identified in 41.7% of Mozambican PK deficient isolates with a strong association with reduced activity phenotype. This mutation was then searched in additional Mozambican samples and other sub-Saharan regions and the 829A allele was detected in all of them at allele frequencies between 1.3%  and 3.4% (in Angola). The allele 829A was not present in the Portuguese samples. Although two African groups could be established according to these frequencies , Prevalence of PK deficiency seems to vary greatly among ethnic groups and geographic regions, as well as the mutations in the PKLR gene. Some authors have estimated a prevalence of 1∶20 000 in the general white population The PK deficiency recorded in Mozambique (4.1%) and 829GA heterozygous prevalence (2.6–6.7%) determined from unrelated individuals from sub-Saharan populations is, to our knowledge, the highest estimated worldwide so far. We initially hypothesized that this would be the result of a strong malaria pressure, but a significant association between both PK low activity and 829A and malaria infection and outcome was not found. However, only 12 samples were available for testing a possible effect of low enzyme activity on severity of malaria and 20 samples for testing a possible effect of 829A allele meaning that larger numbers are required to formally conclude. Moreover, since this was a cross-sectional study, infection and malaria outcome groups were established according to a malaria phenotype in a specific time point (the collection day), that may not accurately reflect the true individual phenotype. Nevertheless, there was higher mutation prevalence in the uncomplicated malaria group supporting that further analysis is essential to complete the present study.P. falciparum and Plasmodium vivax are widely distributed and the estimated number of annual malaria episodes in this country is 1.5 million The Glu277Lys mutation here identified has been previously reported in the PKLR mutation database The simulation of this Glu277Lys substitution on the human PK protein suggested that this mutation is likely to be non-functional. This residue is extremely well conserved and the result complies with the prediction from SIFT from a previous work To conclude, a geographical co-distribution between malaria and PK-deficiency seems to occur: the Middle East and sub-Saharan Africa are the regions with the highest PK deficiency prevalence described so far, as determined in the present study. These are regions with a strong malaria pressure, suggesting that malaria may be an agent of contribute to the selection of PK deficiency variants in these regions. Conversely, the prevalence of PK deficiency is extremely low in the general white populations. Moreover, some of the genes that confer resistance to malaria are among the most variable genes in the human genome Additional studies with a larger sampling effort including longitudinal malaria clinical history characterization and a search of the variant 277Lys in other malaria endemic regions will be conducted to clarify the results in this survey.Table S1List of primers and annealing temperatures (a.t.) used in the amplification of PKLR promoter (Prom) and coding regions by PCR.(DOCX)Click here for additional data file.


Plasmodium falciparum malaria. The sequestration of parasitized red blood cells (PRBCs) to brain microvascular endothelium has been shown to contribute to the pathophysiology of cerebral malaria. Recent studies reported increased levels of von Willebrand factor (VWF) and reduced activity of VWF-cleaving protease, ADAMTS13 , in patients with cerebral malaria.Cerebral malaria is one of the most severe manifestations of ADAMTS13 gene with cerebral malaria was examined in 708 Thai patients with P. falciparum malaria.Association of six single nucleotide polymorphisms (SNPs) of the P-value = 0.0057; OR = 0.27; 95% CI = 0.096-0.76). Significant association was also detected between 115 cerebral malaria and 593 non-cerebral malaria  patients .Among six SNPs, the derived allele of a SNP located in intron 28, rs4962153-A, was significantly associated with protection against cerebral malaria when 115 cerebral malaria patients were compared with 367 mild malaria patients (Fisher's exact Excessive adhesion of PRBCs to the platelet-decorated ultra-large VWF (ULVWF) appears to enhance the sequestration of PRBCs to cerebral microvascular endothelium. The genetic association observed in the present study implies that the regulation of platelet-decorated ULVWF strings by ADAMTS13 may play a role in the development of cerebral malaria. Plasmodium falciparum malaria infection, is an important cause of death. The excessive sequestration of parasitized red blood cells (PRBCs) to brain microvascular endothelium has been suggested to specifically contribute to the pathophysiology of cerebral malaria [Malaria is a major cause of morbidity and mortality in tropical countries. Approximately 250 million clinical cases of malaria occur, resulting in almost 1 million deaths each year. Cerebral malaria, one of the most serious complications of falciparum malaria [It has been demonstrated that levels of von Willebrand factor (VWF) are significantly increased in malaria patients compared with non-malarial patients with fever or control subjects . Further malaria -5 and de malaria . The pla malaria ,7. TogetADAMTS13 gene (OMIM *604134), spanning approximately 45 kb, is located on chromosome 9q34 [ADAMTS13 mutations causing congenital thrombotic thrombocytopaenic purpura (TTP), a rare life-threatening disease characterized by thrombocytopenia, microangiopathic haemolytic anaemia, renal failure, and neurological deficits including coma, have been identified [falciparum malaria. Although ADAMTS13 is a candidate gene for cerebral malaria, no genetic association study of ADAMTS13 in malaria patients has been conducted. This study reports a significant association of common ADAMTS13 polymorphism with protection against cerebral malaria in Thai patients with falciparum malaria.The human ne OMIM *04134, spome 9q34 . To datene OMIM *04134, spentified . These sP. falciparum living in north-west of Thailand, near the border with Myanmar, were recruited in this study. Malarial infection by P. falciparum was confirmed by a positive blood smear for the presence of asexual form of P. falciparum. Clinical manifestations of malaria were classified according to the definitions and associated criteria by the World Health Organization. The mild malaria group consisted of 367 patients with mild symptoms characterized by fever without other causes of infection and by lack of the manifestations of severe malaria as described below. The non-cerebral severe malaria group comprised 226 patients with severe malaria which was defined as one with one of the following signs of severity or evidence of vital organ dysfunction: high parasitaemia , hypoglycaemia (glucose level < 22 nmol/L), severe anaemia (haematocrit < 20% or haemoglobin level < 7 g/dL), a serum creatinine level > 3.0 mg/dL. The cerebral malaria group consisted of 115 patients characterized by an unrousable coma and exclusion of other causes of coma. The patients were ≥13 years old, and the average ages of the patients with mild malaria, non-cerebral severe malaria and cerebral malaria were 25.2, 20.5 and 28.6 years, respectively. All patients underwent treatment at the Hospital for Tropical Disease, Faculty of Tropical Medicine, Mahidol University . This study was approved by the institutional review board of Faculty of Tropical Medicine, Mahidol University, and the Research Ethics Committee of the Graduate School of Comprehensive Human Sciences, University of Tsukuba. Written informed consent was obtained from every patient.A total of 708 Thai patients infected with Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp blood kit according to the manufacturer's instruction .ADAMTS13 gene by using the Tagger algorithm implemented in the Haploview software version 4.2 with the default settings  parameter, r2, threshold of ≥ 0.8). Accordingly, five SNPs were selected as tag SNPs  and 45 Han Chinese in Beijing, China (CHB) ,10, fiveADAMTS13 SNPs  located in exon12 of ADAMTS13 was analysed in 128 Thai malaria patients  by using the polymerase chain reaction (PCR)-direct sequencing. The PCR was performed using the following primers: 5'-ACCCAGCTGGAGTTCATGTC-3' and 5'-CCTATGACTCTGCCCTGTCC-3', and the PCR products were subjected to direct sequencing with ABI prism 3100 Genetic Analyzer (Applied Biosystems).The TaqMan SNP Genotyping Assay was used to genotype six P-value ≤ 0.05 was considered statistically significant. To address the problem of multiple testings, permutation P-values were calculated for single SNPs from 100,000 permutations using the Haploview software, version 4.2. Haplotype frequency and pairwise LD parameter, r2, were estimated using the Haploview software. The haplotype association test based on the permutation procedure was conducted using the Haploview software. In the haplotype association test, to find a haplotype showing stronger association with cerebral malaria than SNP, permutation P-values were calculated for single SNPs and haplotypes from 100,000 permutations.Deviation from Hardy-Weinberg equilibrium was tested in each malaria group by chi-square test. The allele frequency was compared between two malaria groups by Fisher's exact test. The odds ratio (OR) and 95% confidence interval (CI) were calculated by Woolf's method. In this study the direction of association was determined based on the derived allele regardless of the allele frequency. The association of rs4962153 with platelet count obtained from 449 malaria patients before treatment (on Day 0) was assessed by analysis of covariance (ANCOVA) adjusted for age (years), gender , and symptom , where dummy variables were used for symptom.ADAMTS13 gene were analysed in this study . There was no significant difference in allele frequency of rs4962153-A between non-cerebral severe and cerebral malaria groups , although a slight tendency towards protection against cerebral malaria was observed in this comparison as well. When mild and severe malaria groups were combined as a non-cerebral malaria group, the frequency of rs4962153-A was significantly higher in non-cerebral malaria than in cerebral malaria , while the permutation P-value was not significant.Six SNPs, two in exon and four in intron, of the dy Table . All thedy Table . AssociaThe SNP rs11575933 (P475S) has been reported to influence the proteolytic activity of ADAMTS13 [r2 values among six ADAMTS13 SNPs were not high in this study .Since platelet-decorated ULVWF strings are cleaved and regulated by ADAMTS13 ,7, rs496ADAMTS13 was significantly associated with protection against cerebral malaria in 708 Thai malaria patients. The genetic association observed in the present study supports the above-mentioned role of ADAMTS13 in the development of cerebral malaria, although the functional significance of rs4962153-A remains to be studied. A better understanding of the regulation of platelet-decorated ULVWF strings by ADAMTS13 would provide new insights into the development of the treatment of cerebral malaria. For instance, to eliminate ULVWF and replace ADAMTS13, plasma exchange might potentially be considered a new treatment.The reduced activity of ADAMTS13 in malaria patients may allow overproduction of ULVWF multimers. The following adhesion of PBRCs to platelet-decorated ULVWF strings would result in excessive sequestration of PBRCs to brain microvascular endothelium. This has been proposed as one of important mechanisms of adhesion of PBRCs to endothelium in cerebral malaria . The preADAMTS13 is capable of being increased and decreased by rs4962153-A depending on the situation, the detected association of rs4962153-A may be a false positive in either study. To examine the present results, the effect of rs4962153 on the transcription and/or plasma levels of ADAMTS13 in malaria patients and healthy controls should be investigated in the future.A previous study has shown that not only plasma ADAMTS13 activity but also plasma ADAMTS13 concentration were reduced in cerebral or severe malaria patients compared to healthy controls . Since tADAMTS13 gene for Thai malaria patients was not conducted, the possibility that the association between rs4962153 and cerebral malaria detected in the present study has been caused by LD from the other polymorphisms cannot be excluded. The analysis of LD based on the HapMap database revealed that, in the Asian HapMap populations (JPT + CHB), rs4962153 was in strong LD with rs739468, rs3124765, and rs3094379 with r2 of 1, 1, and 0.86, respectively. These three SNPs are on the chromosome 9 open reading frame 7 (C9orf7) gene, located adjacent to ADAMTS13. Flower protein, the ortholog of C9orf7 in Drosophila, has been suggested to function as a Ca2+ channel to control Ca2+ influx in insect salivary gland [C9orf7 gene remains to be studied. However, it appears unlikely that polymorphism of C9orf7 is primarily associated with cerebral malaria, since Ca2+ channel is not currently implicated in the pathogenesis of cerebral malaria. Since this is the first study reporting the significant association of ADAMTS13 polymorphism with cerebral malaria, further independent studies are required to examine whether the present finding can be replicated.Since variation screening of the ry gland . The funADAMTS13, rs4962153, was found to be significantly associated with cerebral malaria in 708 Thai patients with P. falciparum malaria. The present findings support the hypothesis that the regulation of platelet-decorated ULVWF strings by ADAMTS13 may play a role in the development of cerebral malaria.PRBCs adhere to the platelet-decorated ULVWF, leading to enhancement of the sequestration of PRBCs to cerebral microvascular endothelium. The platelet-decorated ULVWF strings are cleaved and regulated by ADAMTS13. It has been reported that plasma ADAMTS13 function is significantly reduced in cerebral malaria patients compared to healthy controls. In this study, a SNP of The authors declare that they have no competing interests.SK participated in the design of this study, carried out the genotyping, conducted statistical analyses, and wrote the manuscript. IN helped to genotype the samples. IN, JP, DN, PN and HH collected blood samples and extracted DNA from the samples. JP participated in the design of the study and coordination. NT was involved in the interpretation of the data and preparation of the manuscript. JO conceived of the study, participated in its design, helped to perform statistical analyses, and helped to write the manuscript. All authors read and approved the final manuscript.


Malaria is a major cause of paediatric morbidity and mortality. As no clinical features clearly differentiate malaria from other febrile illnesses, and malaria diagnosis is challenged by often lacking laboratory equipment and expertise, overdiagnosis and overtreatment is common.Plasmodium mitochondrial DNA was performed on DNA extracted from whole blood and species-specific PCR was done on positive samples.Children admitted with fever at the general paediatric wards at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania from January to June 2009 were recruited consecutively and prospectively. Demographic and clinical features were registered. Routine thick blood smear microscopy at MNH was compared to results of subsequent thin blood smear microscopy, and rapid diagnostics tests (RDTs). Genus-specific PCR of Among 304 included children, 62.6% had received anti-malarials during the last four weeks prior to admission and 65.1% during the hospital stay. Routine thick blood smears, research blood smears, PCR and RDT detected malaria in 13.2%, 6.6%, 25.0% and 13.5%, respectively. Positive routine microscopy was confirmed in only 43% (17/40), 45% (18/40) and 53% (21/40), by research microscopy, RDTs and PCR, respectively. Eighteen percent (56/304) had positive PCR but negative research microscopy. Reported low parasitaemia on routine microscopy was associated with negative research blood slide and PCR. RDT-positive cases were associated with signs of severe malaria. Palmar pallor, low haemoglobin and low platelet count were significantly associated with positive PCR, research microscopy and RDT.The true morbidity attributable to malaria in the study population remains uncertain due to the discrepancies in results among the diagnostic methods. The current routine microscopy appears to result in overdiagnosis of malaria and, consequently, overuse of anti-malarials. Conversely, children with a false positive malaria diagnosis may die because they do not receive treatment for the true cause of their illness. RDTs appear to have the potential to improve routine diagnostics, but the clinical implication of the many RDT-negative, PCR-positive samples needs to be elucidated. Malaria is a major cause of paediatric morbidity and mortality in many developing countries, particularly in sub-Saharan Africa,2.In many areas where malaria is endemic it is highly overdiagnosed. FebrileOverworked health-workers in understaffed institutions may contribute to the overdiagnosis and overtreatment of malaria,7. FurthBlood smear microscopy, the long-time gold standard for malaria diagnostics, is increasingly supplemented or replaced by rapid diagnostic tests (RDTs). Lack ofPolymerase chain reaction (PCR) is a reliable technique for identifying malaria cases. In someWith a diminishing number of cases and deaths attributable to malaria, the World Health Organization (WHO) recommends anti-malarial treatment to be based on laboratory results, when available, also in children. The curPlasmodium falciparum, may limit the applicability of the results to the general Tanzanian population. Both clinical and laboratory findings may be biased by the fact that many of the children had received prior treatment with antibiotics and anti-malarials and that two-thirds were referred from other hospitals or clinics. Since the study did not investigate systematically other causes of febrile illness, it is uncertain how many of the identified malaria cases had concomitant serious infections caused by bacteria or other agents.The study indicates that the current routine diagnostic method, while efficient at detecting high malaria parasitaemia, leads to gross overdiagnosis of malaria and, consequently, overuse of anti-malarials. Overdiagnosis of malaria may hamper the identification and treatment of the real cause of the febrile illness and lead to death from bacterial sepsis and other infections. The diagnostic challenges understandably make clinicians opt to safeguard using both anti-malarials and antibacterial drugs, however, this has potential harmful consequences in terms of increasing emerging antibiotic and anti-malarial drug resistance. The current study supports the notion that diagnostic algorithms employing RDTs may contribute to improving malaria diagnosis and management.The authors declare that they have no competing interests.GEAS and BB were involved in all stages of this study. MRF participated in the coordination of the fieldwork. NL was involved in the design of the study. CH was involved in the laboratory work and development and optimization of the PCR-method. All authors contributed to the data interpretation and writing of the manuscript. All authors have read and approved the final manuscript.Univariate analysis of predictors of positive malaria results by PCR, blood smear and RDT.Click here for fileSensitivity, specificity, false positive rate and false negative rate for the various diagnostic methods.Click here for fileLogistic regression of predictors of positive PCR or positive blood slide or positive.Click here for file


Plasmodium falciparum infection during pregnancy is characterized by malaria-related anaemia, placental malaria and does not always result in clinical symptoms. This situation is associated with poor pregnancy outcomes. The aim of this study was to determine the extent of asymptomatic P. falciparum infection, its relation with anaemia as well as the most cost-effective technique for its diagnosis in healthy pregnant women living in Kinshasa, Democratic Republic of the Congo.In areas of high malaria transmission, Plasmodium falciparum infection was diagnosed using rapid diagnostic test (RDT), microscopy and polymerase chain reaction (PCR). Haemoglobin concentration was also determined.In a cross-sectional study design, information on socio-demographic characteristics and cost data were collected in healthy pregnant women attending antenatal care consultations. P. falciparum infection using microscopy, RDTs and PCR, were respectively 21.6%, 27.4% and 29.5%. Taking PCR as a reference, RDTs had a sensitivity of 81.6% and a specificity of 94.9% to diagnose asymptomatic P. falciparum infection. The corresponding values for microscopy were 67.3% and 97.4%. The prevalence of anaemia was 61.1% and asymptomatic malaria increased five times the odds (p < 0.001) of having anaemia. RDTs were more cost-effective compared to microscopy. Incremental cost-effectiveness ratio was US$ 63.47 per microscopy adequately diagnosed case.In total, 332 pregnant women were enrolled. RDT and microscopy data were available for all the blood samples and 166 samples were analysed by PCR. The prevalence of asymptomatic These alarming results emphasize the need to actively diagnose and treat asymptomatic malaria infection during all antenatal care visits. Moreover, in DRC, malaria and anaemia control efforts should be strengthened by promoting the use of insecticide-treated nets, intermittent preventive treatment with sulphadoxine-pyrimethamine and iron and folic acid supplements. P. falciparum transmission, where a high proportion of the population is semi-immune, most pregnant women with malaria are asymptomatic[Malaria infection in pregnant women is a major problem in sub-Saharan Africa (SSA) with significant risks for the mother and their offspring. Malaria in pregnancy is characterized by a secondary anaemia and the presence of parasites in the placenta known as placental malaria, leading to low birth weight, source of perinatal morbidity and mortality. In areas of high ptomatic-4.The sequestration of infected erythrocytes in the intervillous spaces induces a local inflammation and a massive infiltration of immune cells  and is often observed and described as ‘inflammatory placental malaria’,5. This P. falciparum infection during pregnancy is, therefore, essential to prevent poor pregnancy outcomes, but presents a great challenge. The absence of clinical signs decreases the chances of a clinical diagnosis and a management guided by a decisional algorithm. Moreover, microscopic diagnosis is often challenged by fluctuating and very low parasite densities, due to placental parasite sequestration, the most common form of asymptomatic malaria during pregnancy[P. falciparum histidine-rich protein 2 (HRP2) are more sensitive than microscopy and appear to be reliable predictors of adverse outcomes of malaria in pregnancy[P. falciparum infection during pregnancy in malaria-endemic areas.Diagnosis of asymptomatic regnancy. Previouregnancy. Howeverregnancy. While RP. falciparum infection and its contribution to anaemia among pregnant women. The latest studies were conducted in 1988 and 2003 and reported a prevalence of malaria infection among women attending antenatal clinics (ANCs) to be respectively 22% and 49%[In the Democratic Republic of the Congo (DRC), previous studies have evaluated the prevalence of malaria and placental infection, the pra and 49%. These pP. falciparum infection during pregnancy and its relation to anaemia. In addition, their cost and cost-effectiveness to diagnose asymptomatic P. falciparum infection during pregnancy, in semi-rural setting in Kinshasa, were assessed.Therefore, this study determined through microscopy, RDTs and PCR the prevalence of asymptomatic This study was conducted at the Centre Hospitalier de Kingasani II (CHK), commonly known as Maternité des Soeurs. With twenty to thirty deliveries daily, it is the most frequented Centre in Kinshasa and countrywide. Located in the heart of the semi-rural areas of the south-eastern suburbs of Kinshasa, the maternity provides invaluable health care at an accessible rate.P. falciparum infection was determined in apparently healthy pregnant women, going to the CHK for their first antenatal care (ANC) visit. Data and blood samples were collected prior to any routine administration of intermittent preventive treatment (IPT). Participants were recruited from July 27th, 2012 to August 27th, 2012. Women not providing written informed consent or presenting fever, muscle aches or other symptoms suggestive for malaria were excluded. Women who received anti-malarial treatment within the past two weeks were also excluded from the study. A structured questionnaire was used to obtain information on age, parity, and gestational age, level of education, previous or current use of anti-malarial drugs and the use of bed nets.In a cross-sectional survey, the prevalence of asymptomatic Blood samples were collected from a finger prick for laboratory analysis which included: thick blood smears for microscopy, RDT performance, determination of haemoglobin concentration and molecular analysis. For molecular analysis, blood was collected on a filter paper (Whatmann 3 MM), dried thoroughly, put in individual zip lock plastic bags containing desiccant and stored at room temperature (< 25°C) until completion of the study and then transported to the Tropical Diseases Research Centre (TDRC) in Zambia for molecular analyses.P. falciparum infection. Giemsa-stained thick blood smears (TBS) were used for microscopy. Blood slides were examined using light microscopy at 1,000 × magnification. Hundred microscopic fields were examined in the thick smear before concluding that a blood slide was negative. All slides were read twice by experienced microscopists. If the discrepancy was greater than 15%, a third reader was used to confirm diagnosis. The parasite density per microlitre of blood was computed using the following formula: /Number of leucocytes. Besides microscopy, rapid diagnostic tests (RDTs) were also performed. In this study the SD Bioline Malaria Ag Pf® detecting HRP2 was used.Microscopy, RDTs and PCR were used to detect As microscopy in pregnancy has a lower sensitivity due to placental sequestering of parasites and RDTs have internal validity problems, species specific PCR was conducted on all microscopy negative samples but RDT positive samples. In addition, 166 randomly chosen samples were analysed using nested Plasmodium species diagnostic PCR assay. Molecular analysis was performed at TDRC Ndola, Zambia.The blood spot samples of 5 mm diameter size were soaked in 0.5% saponin in phosphate-buffered saline (PBS), incubated for 10 minutes at room temperature in a 1.5-ml tube, and centrifuged at 14000 rpm. The supernatant was discarded and blood spots were washed in 1 ml of PBS. One hundred Fifty microlitres of a 2% Chelex-100 resin work solution  and 50 μl of water (pH 9.5) were added to the sample in a1.5-ml tube and incubated at 100°C for 10 minutes. After centrifugation at 10,000 g for 1 min, the supernatant was collected and stored at -20°C, prior to the PCR assay.Plasmodium falciparum-specific fragment amplification using FAL1 (5' TTAAACTGGTTTGGGAAAACCAAATATATT3') and FAL2 (5’ ACACAATGAACTCAA TCATGACTACCCGTC 3') specific primers[Nested PCR assay was performed as a two-step procedure. Firstly, amplification of Plasmodium genus specific fragment was carried out as follow: PCR mixture containing buffer, dNTPs, MgCl2, primers, Taq polymerases and sterile water. All PCR reactions were carried out in a total volume of 20 μl. One μl of the purified template DNA was used for the first reaction, in which the fragment spanned by rPLU5 (5' CCTGTT GTTGCCTTAAACTTC 3') and rPLU6 (5' TTAAAATTGTT GCAGTTAAAACG 3') was amplified. Secondly, a 1 μl aliquot from the product of the first PCR reaction was subsequently used as a template for  primers. PositivP. falciparum.PCR products were detected by running 20 μl of DNA product on a 3% agarose gel, small fragments (Eurogentec®), which was subsequently stained with a 0.5 μg/ml ethidium bromide solution and visualized under ultraviolet transillumination. The specific size of the PCR product (second amplification) was 205 bp for Determining the concentration of haemoglobin was performed with a portable HemoControl® device. Anaemia was defined by a haemoglobin concentration <11 g/dL. Anaemia was classified as severe anaemia Hb <7 g/dl, moderate anaemia Hb: 7–9.9 g/dl and mild anaemia Hb: 10-10.9 g/dl..The population of pregnant women in the health zone of Kingasani was estimated at 7,187. With anData was entered and stored in Epi info™7. Descriptive statistics were employed for the analysis of socio-demographic data. Frequencies were used to assess the prevalence of asymptomatic malaria and anaemia in pregnant women. The χ2 test was used to investigate associations between categorical variables. Odds ratios (ORs), 95% confidence intervals (CIs) were calculated and p < 0.05 values were considered to be statistically significant. Multivariate logistic regression models were constructed to identify factors associated with asymptomatic malaria or anaemia during pregnancy. Based on a priori knowledge and a P value less than 0.05 considered as significant, the following variables: Age, sex, parity, marital status, ownership of bed net, presence of lattice on windows, malaria infection and geophagia were included in the model. Backward regression technique was used to construct the model. Statistical analyses were performed using SPSS statistical program, version 17 .The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of microscopy and RDT  were determined with PCR as gold standard, for the 166 samples (50%) of which PCR results were available.Financial data were obtained by conducting interviews with Hospital managers and laboratory staff in Kingasani health Zone. Costs were collected in Congolese Francs (CDF) and converted to US dollars . The cost for one thick blood smear includes: a glass slide, cotton, lancet, Giemsa and other stains, immersion oil, alcohol and gloves. The personnel cost for the service provided corresponded to the time spent from drawing blood samples, preparing the thick and thin smears, staining, reading and reporting the results. At CHK II as in all public maternities in Kinshasa, RDT kits are donated and the personnel service is free of charge. In the present study, for RDT diagnostic strategy, the effective contact time with those seeking care, which accounted for the personnel cost, and the cost of RDT kit were the main input parameters to figure out the amount of money a pregnant women would pay. The effective contact time was comprised of: drawing blood samples from patients, applying samples onto the test, test reading and reporting of results. The time was recorded on a “time sheet” by laboratory personnel for every RDT and microscopy service provided. Laboratory staff work eight hours from Monday to Friday and 5 hours on Saturday, thus 68 hours  weekly. Assuming a month of four weeks; laboratory staff work 272 hours  monthly. Staff salary data were provided by the staff manager.This study was approved by the Ethical Committee board of the University of Kinshasa. Test results of the thick blood smear were revealed to all participants three days after sample collection. All women were given mebendazole, iron and folic acid on a routine basis and were treated with SP regardless of the laboratory test results.vs. 23.6 weeks (SD ± 5.8) in multigravida (p < 0.01)  = 22-33), and 26% were primigravidae, the median gravidity was 3 (IQR = 1-5). Eighty one percent of the subjects were married and 66.2% had not completed secondary school. Thirty-seven percent (37%) of the pregnant women had started their antenatal care in the third trimester. The mean gestational age in primigravidae at the time of the first prenatal visit was 21.5 weeks (SD ± 4.2) P. falciparum infection using microscopy, RDTs and PCR, were respectively 21.6% (95% CI:17.4-26.6%), 27.4% (95% CI:22.5-32.6%) and 29.5% (95% CI:22.7-37.1%). Plasmodium malariae was diagnosed in five women by microscopy, but was not confirmed by PCR. The median parasite density was 126/μl (IQR:105-162). In bivariate analyses, age, gravidity, marital status and the use of bed nets demonstrated a significant association with asymptomatic malaria. The regression model shows that being younger than 20 years old was an independent risk factor associated with asymptomatic P. falciparum infection , while being married  and spending the night under a bed net  was associated with low prevalence of asymptomatic P. falciparum infection . Of these anaemic pregnant women, 70 21.1%), 132 (39.7%), 1 (0.3%), had respectively, mild, moderate and severe anaemia. In multivariate analyses, asymptomatic malaria increased five times the likelihood of having anaemia   possessed a bed net. Of these, 69.8% stated that their nets were treated with insecticide and 81.8% reported having slept under the bed net the night before the interview  were positive by PCR (95% CI: 40.8- 84.6)  and 94.9% (95% CI:89.2-98.1) for microscopy and RDTs, respectively. RDTs demonstrated a positive predictive value (PPV) of 87.0% (95% CI:73.7-95.1%) and a negative predictive value (NPV) of 92.5% (95% CI:86.2-96.5%). The corresponding values for microscopy were 91.7% (95% CI:77.5-98.2%) and 87.7% (95% CI:80.8-92.8%). Microscopy combined to RDT demonstrated a sensitivity of 81.6% (95% CI:68.0-91.2), a specificity of 94.9% (95% CI:89.2-98.1), a PPV of 87.0% (95% CI:73.7-95.1%) and NPV) of 92.5% (95% CI:86.2-96.5%). The differences were not significant for any comparison  and 81.6% (95% CI:68.0-91.2) to diagnose asymptomatic On the basis of the 166 samples for which PCR results were available, malaria RDT was the most effective with the number of cases correctly diagnosed being 40 (90.9%) compared to 33(88.6%) for microscopy Table .In the most frequented maternity in Kinshasa, located in the heart of the semi-rural areas Kinshasa, the prevalence of asymptomatic malaria at the first antenatal visit was 27% and 21%, using respectively RDT and microscopy. PCR showed that almost one third and one fifth were missed by microscopy and RDT respectively. Microscopy results (21%) were similar to the 22% value reported in 1988.P. falciparum infection may have been somewhat lower, because the study was carried out during the dry season, period subjected to a mild/low malaria transmission. Therefore, another survey during the rainy season may be informative.These values are paradoxically lower than the 42% found in Lubumbashi, an area of low malaria transmission. AsymptoP. falciparum-infected but asymptomatic individuals tend to have low and submicroscopic parasite densities[Parasite density was also found to be very low in all infected pregnant women. ensities,27. Moreensities.P. falciparum infection compared to married women. In addition, owning a mosquito net was associated with being married, as previously reported by Pettifor et al. in 2008[P. falciparum infection. This indicates that malaria endemicity may be lower than expected impairing the development of immunity against pregnancy-related malaria during successive pregnancies. Similar observations were made in other low malaria-endemic areas[Other studies also found a protective effect of bed nets against malaria in pregnancy. Other s in 2008. This inic areas-33, streic areas. It is, ic areas.P. falciparum infection in early pregnancy has been associated with adverse outcomes such as low birth weight LBW[et al. in 2008[Pregnant women were coming for their first antenatal visit at the time of enrolment, thus prior to any administration of IPTp with SP. However ight LBW. The useight LBW, Pettifo in 2008 and the  in 2008. However in 2008.P. falciparum infection. This prevalence is similar to those reported in Kisangani in 2000 (65%) and Lubumbashi (in average 65%) in 2003[High prevalence of anaemia was observed and strongly correlated with asymptomatic  in 2003,21. Howe in 2003. A limit in 2003, was notP. falciparum infection. In addition, all the RDT positive but microscopy negative samples were analysed by PCR and it showed that 65% were in fact submicroscopic infections. The PCR negative samples could be either explained by the persistence of HRP-2 circulation in the blood more than two weeks even after successful clearance of infected erythrocytes in the bloodstream or by the sequestration of the parasites in the placenta while HRP-2 circulate[In the present study, no significant difference was found between microscopy and RDT using PCR as the gold standard. This observation was also made elsewhere,40. Combirculate. PCR shoThis study demonstrated that RDT technique was more cost-effective than microscopy. This finding is supported by others studies carried out in other settings,43. In mP. falciparum infection. These results are quite alarming and emphasize the need to actively diagnose and treat asymptomatic malaria infection using appropriate techniques during ANC visits in areas of high malaria transmission, regardless of IPTp. On the other hand, though the use of ITNs in DRC is slightly increasing, the promotion of ITN use should be further encouraged. The present study suggests that RDTs may replace blood smears screening of asymptomatic P. falciparum infection in pregnancy in some malaria-endemic settings by virtue of their ease-to-use, ability to detect sub-microscopic infection and cost-effectiveness compared with microscopy.This study reports a high prevalence of anaemia among pregnant women in the health zone of Kingasani, Kinshasa and anaemia is strongly correlated to asymptomatic AOR: Adjusted Odds-ratio; CI: Confidence Interval; DRC: Democratic Republic of the Congo; IQR: Inter Quartile Range; OR: Odds-ratio; PCR: Polymerase Chain Reaction; PNLP: Programme National de Lutte contre le Paludisme; PRDT: Rapid Diagnostic Test; SSA: Sub-Saharan Africa; TBS: Thick Blood Smear.The authors declare that they have no competing interest.JMR participated in the conception and design of the study protocol, conducted the study, draft the manuscript and participated in molecular analysis. AI and L participated in the design of the study protocol and conducted the study. RIL, PL, JPV reviewed the manuscript and provided critical inputs. All authors read and approved the final manuscript.


No effect was observed on non-malarial febrile episodes.The overall incidence of malaria was 3.0/child-year ; no differences were found in malaria rates between genotypes . However, this association strongly depended on age: among children aged 6-17 months, those with α+-thalassaemia and malaria depends on age. Our data suggest that protection by α+-thalassaemia is conferred by more efficient acquisition of malaria-specific immunity.In this population, the association between α Reports on its effect on uncomplicated malaria are inconsistent. A study in Vanuatu showed that, among children aged < 5 years, homozygous (but not heterozygous) α+-thalassaemia was associated with an increased incidence of uncomplicated malaria due to both Plasmodium vivax and Plasmodium falciparum ; paradoxically, among children aged 5-9 years, there was no evidence of such an association [+-thalassaemia against uncomplicated malaria due to P. falciparum.Case-control studies -4 and twociation ,9 or proociation  by α+-th+-thalassaemia was also strongly protective against hospitalization for disease episodes caused by infections other than malaria [In a prospective case-control study in Papua New Guinean children, α malaria .+-thalassaemia on rates of fevers due to malaria and fever due to other causes, and evaluated to what extent these effects depended on age.In this study among preschool children, it was aimed to assess effects of αP. falciparum) is intense and perennial, with entomological inoculation rates of 35-400 infectious bites per person-year [The study was conducted in February 2008-March 2009 in Handeni District, north-eastern Tanzania. The population is a mixture of many different tribes, residing in scattered hamlets, and mainly comprises poor farmer families engaged in subsistence farming. Transmission of malaria , a census was conducted and all resident children aged < 60 months were registered. Parents and guardians were invited to bring their children for screening. A medical examination was performed by a clinical officer, anthropometric indices were calculated as the average of two recordings taken at consecutive days, and venous blood samples were collected. An aliquot of blood was centrifuged immediately after collection, and a 90 μL red cell pellet including the buffy coat was mixed with 90 μL phosphate-buffered saline and 180 μL of a DNA stabilizing buffer  and stored at 4°C for subsequent genotyping. Plasma was stored in liquid nitrogen. A second aliquot of whole blood was examined by haematology analyzer  the same day.Plasmodium infection and age class . Children with Plasmodium infection at baseline were treated with artemether-lumefantrine .Children were eligible for randomization when aged 6-59 months, and with height-for-age z-score < -1.5 SD. Children with weight-for-age z-score < -3 SD, haemoglobin concentration < 70 g/L, signs of severe or chronic disease, those unlikely to comply with interventions, or whose parents/guardians refused consent were excluded. Eligible children were randomly allocated to receive daily supplements with zinc alone, zinc in combination with other micronutrients, micronutrients alone, or placebo. Block randomization was used within six strata defined by A clinical officer was on 24-h duty at the research clinic. For any child reporting with fever (axillary temperature ≥ 37.5°C) or a history of fever according to the guardian, a finger-prick blood sample was collected to detect the presence of malaria parasites. For all children with a positive dipstick we measured whole blood concentrations of haemoglobin and C-reactive protein. Children were treated free of charge, and referred to the district hospital when indicated.P. falciparum and other Plasmodium species) was detected by rapid test . This test has a sensitivity of 96% for blood samples with > 50 parasites/μL as determined by microscopy [In samples collected at baseline and from sick children, the presence of parasite-specific lactate dehydrogenase ; the -αreaction . This tyn Africa , with prn Africa ,10,14; oP. falciparum-specific histidine-rich protein-2 (HRP2) in samples collected during the first malaria episode were measured using a commercial enzyme-linked immunosorbent assay kit . This protein is released into the plasma at schizont rupture, and its plasma concentration may more accurately represent total body parasite biomass, because it also reflects the presence of sequestered parasites that remain undetected with conventional microscopy [Whole-blood concentrations of haemoglobin and C-reactive protein were measured using point-of-care tests . Plasma concentrations of C-reactive protein and ferritin for survey samples were measured in The Netherlands  on a Beckman Coulter Unicel DxC880i system according to the manufacturer's instructions. Plasma concentrations of croscopy .Analyses were performed using SPSS , CIA (v2.1.2)  and STATPlasmodium-infected participants were treated at baseline to clear parasitaemia before the start of surveillance. Episodes with parasitaemia thresholds of 5,000 and 10,000 parasites/μL were considered as secondary outcomes. Children were presumed to be protected against new malaria infections for 14 days after treatment with artemether-lumefantrine. Recurrent symptoms during this period were assumed to be part of the initial episode and were not counted as separate episodes. Additional outcomes were hospital admissions or death due to infection-related causes (combined end-point), non-malarial febrile episodes , and the severity of malaria episodes as indicated by parasite density, haemoglobin concentrations, concentrations of whole blood C-reactive protein and plasma HRP2.The primary outcome, an episode of malaria, was pre-defined as a guardian-reported history of fever accompanied by either an axillary temperature ≥ 37.5°C or inflammation (whole blood C-reactive protein concentration ≥ 8 g/L), plus a positive result for the pLDH rapid dipstick test. Elevated C-reactive protein concentrations are probably indicative of recent malaria episodes in currently afebrile individuals with parasitaemia ,19, and Plasmodium infection, distance between homestead and clinic, and height-for-age z-score and micronutrient intervention) influenced the estimates. Because it was anticipated that the effect of α+-thalassemia would depend on age, a stratified analysis was conducted within each of the three age classes used for randomization, and interaction was assessed using a Cox regression model. It was also explored whether there was evidence of an interaction between α+-thalassaemia and the intervention by including interaction terms in the Cox regression model. Incidence and incidence ratios were calculated based on time to first episodes, and Cox regression models were used to obtain adjusted hazard ratios.In the primary analysis, group rates were compared using Cox regression with robust estimates of the standard error to account for multiple episodes within children. It was explored to what extent adjustment for baseline factors that were prognostic for malaria  were assessed by ANOVA and multiple linear regression.Associations between αOf 1,029 children screened, 612 children were enrolled , virtually all due to P. falciparum (261), with similar prevalence values among genotypes. Homozygotes had lower haemoglobin concentrations and height-for-age z-scores, seemed slightly younger, appeared to have a higher prevalence of iron deficiency, and used mosquito nets less frequently than those with a normal genotype. They also less frequently received zinc supplements, with or without multi-nutrients  had a normal genotype, 41% (251) were heterozygous, 9% (55) were homozygous; genotyping could not be performed for 2 children. Of 2,462 episodes of reported fever, 1,618 had a positive dipstick test result; 1,572 malaria episodes were recorded in 526 child-years of observation. The incidence of malaria was 3.0/child-year; 507 children (83%) experienced at least one malaria episode, recurrent episodes occurred in 395 (63%) of children. The remaining 890 febrile episodes were classified as not being due to malaria; of these, 27 (3%) were associated with a positive result for a malaria dipstick test, but in these cases, fever could not be confirmed and plasma C-reactive protein concentration was < 8 mg/L. The incidence of non-malarial fever cases was 1.69/child-year. Hospital referral for infection-related causes occurred in 68 cases, of which 30 were malaria . Malaria incidence decreased with age class ; the corresponding incidence estimates for episodes with haemoglobin concentration < 80 g/L were 0.82, 0.33 and 0.09 episodes/child-year, respectively).Plasmodium infection at baseline, distance to the research clinic, stunting and experimental intervention. Stratification by age-class, however, showed that heterozygotes had increased malaria rates when aged < 18 months , whereas they were protected against malaria when aged ≥ 18 months .Rates of malaria were similar among genotypes Table , whetherThe rate of episodes with haemoglobin concentrations < 80 g/L was highest among homozygotes and lowest among children with a normal genotype, regardless of age.+-thalassaemia seemed associated with increased parasite densities  from values observed in children with a normal genotype (-3.0 g/L).Lastly, the rate of non-malaria febrile episodes was similar between genotypes Table ; stratif+-thalassaemia was associated with increased rates of malaria in children aged < 18 months, but with protection against malaria in older children. There was no evidence that α+-thalassaemia was associated with the severity of malaria episodes as measured by haemoglobin concentrations and other indicators.In this cohort, αPlasmodium-infected children were treated at baseline, the specificity of our case definition may have gradually decreased with time, as more children became asymptomatically infected. Similar results were obtained, however, when restricting the analysis to cases with parasite densities > 10,000/μL or > 100,000/μL. Because these case definitions are more specific for detecting true malaria cases, it is unlikely that a low specificity affected the validity of the conclusions drawn in this report.Although all +-thalassaemia in the youngest children. It remains unclear however, what mechanism could underlie the increased incidence in the youngest children. It has been put forward that Plasmodium parasites preferentially invade reticulocytes [Plasmodium parasites [+-thalassaemia [Because effect estimates were adjusted for factors associated with malaria risk (including distance to the research facility and intervention), it is unlikely that a difference in external factors  biased the estimates and would be responsible for the increased risk associated with αulocytes ,27. Thusarasites . Howeverassaemia .Others hypothesized that increased parasite-induced surface expression of neo-antigens on thalassaemic erythrocytes results in enhanced binding of IgG antibody and more rapid clearance of parasitized erythrocytes in the spleen . ClearanP. falciparum in both heterozygotes and homozygotes for α+-thalassaemia early in life may result in protection against severe malaria, and (at older age) uncomplicated malaria due to the same species. In Vanuatu, the incidence of malaria due to P. falciparum and P. vivax was increased in children aged < 5 years with homozygous α+-thalassaemia relative to children with normal genotype [+-thalassaemia was found to be associated with protection against malaria in children aged 6 months to 20 years [Nevertheless, the findings reported here contribute to existing epidemiological evidence that predisposition to malaria due to genotype , but the20 years . This pr+-thalassaemia becomes evident shall vary with transmission intensity. This may at least in part explain differences between studies; under conditions of intense transmission, such as in the present study, the difference attained would become evident earlier in life than in conditions of less intense transmission such as encountered in Vanuatu [Because the intensity of malaria exposure determines how fast protective immunity is obtained, the age at which a difference in protective immunity between children with and without α Vanuatu .+-thalassaemia is associated with an increased frequency of malaria in children aged 6-18 months may seem to contradict reports from hospital-based studies that α+-thalassaemia protects against severe malarial anaemia [+-thalassaemia than in their peers with normal genotype.The finding that α anaemia ,6, which anaemia . An incr anaemia . In thal+-thalassaemia were found to be protected against the decline in haemoglobin concentration associated with mild and asymptomatic infections [+-thalassaemia has been providing a survival advantage in the past.When analysing the decline in haemoglobin concentration between baseline and first malaria episode, we did not find evidence of such protection. It should be noted, however, that the average decline in haemoglobin concentration during malaria episodes was relatively small, and that very few 19) episodes occurred whereby haemoglobin concentrations dropped below 60 g/L. This is probably due to good access to treatment in our study, and haematological gains due to the nutrient interventions. By contrast, in a pilot survey in the same area in 2006, children with α9 episodeA likely explanation for the increased rates of episodes with haemoglobin concentrations < 80 g/L observed in heterozygotes and homozygotes Table  is that +-thalassaemia on malaria rates were age-dependent: it was associated with increased rates in children aged < 18 months as opposed to decreased rates in older children. There was no evidence that α+-thalassaemia was associated with the severity of malaria episodes.In pre-school Tanzanian children living in an area of intense transmission, and with height-for-age z-score < -1.5 SD, effects of αThe authors declare that they have no competing interests.JV was responsible for data collection and analysis, administration and drafted the manuscript; HV assisted in statistical analyses. EJSJ, EVM and AB assisted in data collection; RJK and AYD were responsible for laboratory analyses in The Netherlands. HV and HFJS were responsible for supervision. All authors participated in data interpretation and critical revision of the report for intellectual content; and provided final approval of the submitted version.