Intravenous artesunate has replaced quinine as the first-line therapy for severe imported malaria, given its anti-malarial superiority shown in clinical trials conducted in endemic countries. Evidence for red blood cell (RBC) exchange in patients with severe malaria treated with artesunate is lacking. This retrospective cohort study describes the experience at Hospital Clinic of Barcelona with the use of artesunate for severe malaria and the joint use of RBC exchange in selected cases.Patients treated for severe malaria at Hospital Clinic of Barcelona between August 2013 and January 2015 were included in this retrospective study. Severe malaria was defined according to WHO criteria. Data were extracted from electronic hospital records. A log-linear mixed model approach was used to estimate parasite clearance times.Plasmodium falciparum (90.5 %). Sixteen patients (42 %) had severe malaria cases and were treated with intravenous artesunate. Four patients underwent RBC exchange within a period of 15 h after the first dose of artesunate (range 9–21 h). The procedure lasted a median of 2 h (IQR 1.8–2 h), using a median of 12 (IQR 11–14) units of packed RBCs to replace a median of 3794 ml (IQR 2977–4343). The technique was well-tolerated without haemodynamic complications. There were no deaths. The regression model showed an estimated time to 95 % decay of 21.6 h (95 % CI 17.3–28.8). When assessing effect modification by RBC exchange, there was no difference in the parasite elimination rate (p = 0.286).Within the study period, 42 patients were diagnosed of malaria at this centre, of which 38 had In this study RBC exchange failed to show benefits in terms of parasite clearance probably due to the small number of patients analysed. The evidence for exchange transfusion remains limited. The management of severe imported malaria remains a challenge of consensus, since the elaboration of clinical guidelines always has to deal with the lack of evidence in non-endemic countries. The design of clinical trials randomizing cases of severe imported malaria is unrealistic nowadays, due to the high number of subjects required, the heterogeneous group of affected travellers in terms of parasitaemia and clinical presentation/complications., and ethical problems given the evidence from endemic regions , 2. The Different strategies have been proposed as adjunctive therapies, mainly helping pharmacologic treatment to reduce parasitaemia. Whole blood exchange transfusion has been suggested and used with this purpose. Its theoretical benefits are based on the removal of: (i) infected circulating red blood cells (RBCs) (those sequestered in microvasculature are not affected); (ii) rigid non-infected RBCs, with a rheological effect; (iii) plasma toxins, minimizing systemic inflammatory response and sequestration. It has demonstrated a rapidly reduction in parasite biomass , but hasThis study reports our experience with the use of artesunate for treating severe malaria and the use of RBC exchange in selected cases treated at Hospital Clinic of Barcelona in Spain between August 2013 and January 2015.This retrospective cohort study includes all patients with severe malaria treated with intravenous artesunate at Hospital Clínic, in Barcelona, Spain, between August 2013 and January 2015. Hospital Clínic is a national reference centre for Tropical Imported Diseases. In 2013, artesunate became available in Hospital Clínic for the treatment of patients with severe malaria. Patients with severe malaria are initially managed in the intensive care unit and subsequently, when they have improved, transferred to a conventional ward.Plasmodium falciparum and fulfilled one or more WHO criteria  during 3 days.RBC exchange was performed at the decision of the treating physician in conjunction with Haemotherapy specialists. In general, it was considered as an adjunctive treatment in patients who had high baseline parasitaemia  and shock, respiratory failure or cerebral malaria. RBC exchange was also performed if patients were severely ill and parasitaemia did not decrease at least 25 % compared to baseline after 8–12 h of artesunate. RBC exchanges were performed in a COBE Spectra blood separator  using the RBCX software. Sex, height, weight, patient’s haematocrit, average haematocrit in the replacement red blood cells units, desired end haematocrit for the patient, fluid balance and the fraction of infected RBC remaining at the end were introduced at the start of the procedure. Once the initial data is entered, the replacement volume of RBC to be exchanged proposed by the COBE Spectra software was followed. ACD-A was used as anticoagulant at a infusion rate of 1.2 mL/min/L total blood volume with prophylactic calcium and magnesium infusion administration . Fresh lAll patients included into the study were identified prospectively in the clinical routine practice. A retrospective review of their electronic records was performed and an anonymized case report form was filled out. This included the following data: demographics and travel history; WHO clinical, analytical and radiological severity criteria; dates and times on symptoms initiation, hospital attendance, blood films performed and treatment commencement; the number of artesunate doses and other antimicrobials previously administered; complications and mortality. Data on RBC exchange was provided by the Department of Hemotherapy and Hemostasis, which keeps a registry of the apheresis performed in the hospital.The data collected were entered into Epi Info 7  and then transferred to Stata 13.1  for the analysis. Categorical variables were described by counts and percentages, whereas median and interquartile ranges (IQR) were used to summarize continuous variables. Comparisons between groups were performed with Fisher exact test and Mann–Whitney test for categorical and continuous variables, respectively.Parasite clearance times (PCT) were estimated since the administration of the first dose of artesunate. Initially, the parasite loads were calculated from the parasite counts expressed in percentages and the number of RBC per microlitre in each blood film. The variation of parasite loads was analysed using a log-linear mixed-effects regression model incorporating a Gaussian random intercept. This resulted in an estimation of the dynamics of parasitaemia, assuming a single exponential model, allowing calculating the decrease of log parasitaemia per unit of time (hour) or parasite elimination rate. The interaction between RBC exchange and time was included in the model to evaluate the possible modification of the effect of artesunate in clearing parasitaemia.The average reduction rate per hour was obtained based on the coefficient of the regression model , as well as its 95 % confidence interval. The model estimates obtained were used to calculate the time to 50 % (PCT50 %), 75 % (PCT75 %), 90 % (PCT90 %), 95 % (PCT95 %) and 99 % (PCT99 %) clearance of parasitaemia. 95 % confidence intervals (95 % CI) and p values were obtained for all estimates in the model.The Ethics Committee of Hospital Clínic approved this study. Data collection forms were completely anonymous. As it was a retrospective study, the Ethics Committee approved waiver of individual consent from patients whose routine data was used.P. falciparum (90.5 %), and 16 of those (42.11 %) were identified as severe malaria cases. All 16 were treated with IVA, and 4 additionally with RBC exchange. Their baseline characteristics are summarized and compared in Table From August 2013 to January 2015, 42 patients presented with malaria, 38 by The median age was 40 years old (IQR 33–50), with a high proportion of male patients . Most patients were travellers who had arrived from sub-Saharan Africa , followed by immigrants “visiting friends and relatives”—temporary visitors- (VFR) . None of the travellers completed a full course of chemoprophylaxis. Two patients were residents in endemic countries travelling to Spain .Half of the cohort presented three or more severity criteria, the most frequent being hyperparasitaemia (93.75 %), prostration (50 %), renal impairment (37.50 %) and jaundice (37.50 %). The median time to seek for health advice was 5 days (range 2.50–11.70) since the onset of symptoms. The median initial parasitaemia was 12.5 % (range 3.95–32.50 %). Four patients (25 %) presented with an initial parasitaemia above 30 %.Patients finally treated with RBC exchange had a higher median initial parasitaemia compared to those who were treated with IVA alone (46.50 vs 7.30 %) and presented with more severity criteria Table .Four patients (25 %) received RBC exchange as an adjunctive treatment to IVA. This technique was initiated a median of 15.45 h after the first dose of IVA (IQR 9.05–20.58). The median duration of the technique was 2 h (IQR 1.80–2), using a median of 12 units of packed RBCs (IQR 11–14) to replace a median of 3794 ml (IQR 2977–4343).3, respectively. The median difference in blood calcium and potassium before and after the procedure was 0.15 mg/dl and −0.2 mmol/l, respectively. All patients survived and none of them had long-term renal or neurological sequelae. Two cases of delayed haemolysis could be detected: they completely recovered and did not show more complications.The procedure was well tolerated without haemodynamic or hydroelectrolytical complications. The median difference in haematocrit and platelet count before and after RBC exchange was 2 % and −13,500/mmThe values of parasitaemia at each timepoint for each patient were analysed to estimate the PCT Fig. . Table 3Based on the estimates of this model, the mean time to 50 % (PCT50), 90 % (PCT90), 95 % (PCT95) and 99 % (PCT99) parasite clearance were calculated Table . PCT werIn the present study, it is shown in the largest case series of severe imported malaria that use RBC exchange in a cohort treated with artesunate that IVA reduces quickly parasitaemia and that RBC exchange does not seem to add any benefit in terms of parasite clearance times.The results of this retrospective follow-up study confirm the speed of action of IVA when used for imported severe falciparum malaria, reducing parasite clearance time in a similar way to other cohorts –7. This Regarding the use of RBC exchange as an adjunctive therapy, it was found that it does not significantly contribute to parasite clearance . HoweverIn this study, a mixed effects model for log parasitaemia with fixed and random effects for initial parasitaemia, and fixed effects for exchange transfusion and time was used Table . The autThe development of safer apheresis therapies that can be used for severe malaria, such as RBC exchange, may have an impact on the medical management of the disease and there are many aspects of its use as adjunctive therapy susceptible of further investigation. One of the main questions about using RBC exchange for severe malaria together with IVA is which would be the accurate moment to initiate the apheresis. There is no debate that anti-malarial drug is the first step for this medical emergency. Its use is envisioned due to its good safety profile, confirmed by the data in this cohort, and its plausible biological benefits. One of the hypothetical concerns when using RBC exchange immediately after AVI is that the drug could be partially removed within the RBC exchange since artesunate has shown to have high binding to RBC .Therefore, the use of RBC exchange should target to optimize IVA effect, not to replace it, not even diminish it. Since artesunate half-life is less than 15 min and 30–60 min for its metabolite dihydroartemisinin , the recThis case series illustrates the experience with the use of AVI for treating severe malaria, and the use of RBC exchange as adjunctive therapy. No serious adverse effects of RBC exchange transfusion were encountered in this small series. The study provides valuable data about technical procedure of RBC exchange and about monitoring microbiological response to artesunate. Although there remains limited evidence to support RBC exchange transfusion in severe malaria, it may be considered in special circumstances, such as the suspicion of artemisinin resistant hyperparasitemic falciparum malaria, the unavailability of IVA or in settings where parasite clearance may be prolonged, such as splenectomy.A randomized clinical trial would provide some evidence about the prompt indication and initiation of RBC exchange when using artesunate, but it is currently unaffordable. Further multicentric, cohort studies should be performed to gradually increase the level of evidence to determine which selected patients with imported severe malaria may benefit from this adjunctive treatment and at which moment after artesunate administration will not reduce the drug’s benefit.


Plasmodium falciparum parasite and the factors influencing severity of disease are still not fully understood. Human single nucleotide polymorphisms SNPs associated with Knops blood group system; carried by complement receptor 1 may be associated with the pathology of P. falciparum malaria, and susceptibility to disease.The complex interactions between the human host and the a/b, McCoya/b, Swain-Langley1/2 and KCAM+/- in Ghanaian patients with malaria and determine possible associations between these polymorphisms and the severity of the disease. Study participants were patients (n = 267) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as uncomplicated malaria (n = 89), severe anaemia (n = 57) and cerebral malaria (n = 121) and controls who did not have a detectable Plasmodium infection or were symptomless carriers of the parasite (n = 275). The frequencies were determined using a post-PCR ligation detection reaction-fluorescent microsphere assay, developed to detect the SNPs defining the antigens. Chi-square/Fisher’s exact test and logistic regression models were used to analyse the data.The objective of this study was to determine the genotype and haplotype frequencies of the SNPs defining the Knops blood group antigens; Kna, McCb, Sl2 and KCAM- were found in the Ghanaian population. Apart from small significant differences between the groups at the Sl locus, no significant allelic or genotypic differences were found between the controls and the disease groups or between the disease groups. The polymorphisms define eight different haplotypes H1(2.4%), H2(9.4%), H3(59.8%), H4(0%), H5(25.2%), H6(0.33%), H7(2.8%) and H8(0%). Investigating these haplotypes, no significant differences between any of the groups were found.As expected, high frequencies of the alleles Knb, Sl2, Knb and KCAM- or combined haplotypes do not seem to confer any protective advantage against malaria infection or resulting disease severity. Based on these findings, in a very well-characterized population, malaria does not seem to be the selective force on these alleles.The results confirm earlier findings of high frequencies of certain CR1 alleles in Africa; and shed more light on earlier conflicting findings; the alleles McC In spite of the significant decrease in malaria-associated morbidity and mortality in recent years, approximately 216 million cases of malaria and an estimated 655.000 deaths were recorded in 2010 according to the World Malaria Report, 2011 [Plasmodium falciparum and the factors influencing severity of disease are still not fully understood. Although never demonstrated in vivo, it is believed that the formation of rosettes; the spontaneous binding of P. falciparum-infected erythrocytes to uninfected erythrocytes [P. falciparum malaria [P. falciparum erythrocyte membrane protein 1 (PfEMP1) [The complex interactions between the human host and hrocytes , are ass malaria  through  malaria -6. Compl malaria . The two malaria . The pot(PfEMP1) .P. falciparum malaria [Several polymorphic traits of CR1 have been reported; molecular weight polymorphisms, defined by deletion and duplication of sections of complement control proteins resulting in variations in the number of C3b binding sites . In addi malaria .a/Knb), rs41274768 [a/McCb), rs17047660 [+/KCAM-), rs6691117 [+/Yka-), rs3737002 [Several antigens have been described in the Knops blood group system on CR1; Knops antigens a and b  or A (Knb) at nucleotide position 4708, causing the amino acid (aa) change V1561M [a) or G (McCb) at nucleotide position 4795 (K1590E change) [+) or G (KCAM-) at position 4870 (I1615V change) [The Kn antigens are determined by a G (Kne V1561M . The McC change) . Swain-L change) ,21. The  change) .et al. found that both the Sl2 and McCb allele frequencies were very rare in European-Americans  compared to African-Americans  and 39% (McCb)), whereas the frequencies in a Malian population (n = 139) were 70% (Sl2) and 49% (McCb), and suggested that Sl2 might offer a protective advantage against severe malaria [et al. identified the molecular basis for the McC and Sl alleles and determined the frequencies in West Africa ) and another Malian population (n = 99). The allele frequencies were 79% (Sl2) and 31% (McCb) in pooled West African samples and 76% (Sl2) and 30% (McCb) in those from Mali [b alleles were associated with protection against any form of severe malaria [b allele were associated with decreased susceptibility to cerebral malaria [+ of 20% and Knb of <0.01% in African populations is taken from The Blood Group Antigen Facts Book [The association between polymorphisms in the Knops blood group system and malaria has been investigated in several studies with focus on McC and Sl alleles. In 2000, Moulds  malaria . In 2001rom Mali ,25 and irom Mali , but fin malaria ,25. In t malaria . The KCActs Book .et al. in Brazil [b and KCAM-, was found to be more frequent in individuals infected with P. falciparum (14%) compared to the controls (2%) [a/Knb genotype and the Knb allele were more frequent in patients with P. falciparum malaria compared to controls  and the study indicated that the Knb allele and possibly the H8 haplotype are associated with susceptibility to P. falciparum infection, however, the sample size was very low; with only 25–35 individuals per group [A new SNP in the Knops blood group system was recently identified and 12 haplotypes were described, defined by this new SNP at position 4646A > G (Asn1540Ser) in combination with SNPs determining Kn, McC, Sl, Sl4, Sl5 and KCAM . These hn Brazil . The H8 ols (2%) . Furtherer group .The purpose of the present study was to examine the possible association between the CR1 polymorphisms, Kn, McC, Sl and KCAM alleles, eight derived haplotypes and severity of malaria using samples from children between 0 and 15 years of age admitted to Korle-Bu Teaching Hospital in Accra, Ghana to confirm/disconfirm earlier studies.P. falciparum parasitaemia of more than 10,000/μL and an axillary temperature of more than 37.5°C. Patients who fulfilled the criteria for uncomplicated malaria , severe malarial anaemia  or cerebral malaria , as described in detail elsewhere, were enrolled [P. falciparum in a thick blood smear from a nearby community, Dodowa. Both patients and controls were included after signed consent from patients or guardians after receiving standardized information in local language. In both Accra and Dodowa, the population is a mixture of several ethnic groups, possibly, with a slightly more uniform population in Dodowa. However, the dominating ethnic group in both Accra and Dodowa is Ga-Adangme. The study population is well described with very thorough patient characterization and has been studied extensively, among other things, with regards to interleukin 10, tumour necrosis factor and Mannose-binding lectin genotypes [The study population was children between 0 and 15 years of age admitted to the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997. The general inclusion criteria were asexual enrolled ,29. Patienotypes ,29.The sVenous blood samples were collected in test tubes containing EDTA at admission. Plasma was separated within two hours after collection, and pellets were frozen at -20°C. Genomic DNA was extracted as described previously . Whole gCR1 exon 29 based on NCBI Reference Sequence: NG_007481.1 which contains all four SNPs. Primers and conditions are provided in Table 2O, 2 μl AHPol HS 7.5 mM , and 2.5 μl 3 μM primermix. The reaction was performed in a 96-well PCR plate  in a VWRi Duo Cycler . Selected samples were sequenced to verify the genotyping of the Kna/b and KCAM+/- alleles. Controls for the McC and Sl SNPs were kindly provided by Dr JoAnn Moulds.PCR primers were designed to amplify a region of et al.[2O pr. sample. The LDR primermix consisted of three primers pr SNP; two allele-specific primers and one common. The common primer was 5’-phosphorylated and 3’-biotinylated and unique 24 bp tag sequences were attached to each allele-specific primer at the 5’ end by the manufacturer . All the LDR primers were designed based on NCBI Reference Sequence: NG_007481.1 and the tags were chosen as complementary to anti-tag sequences attached to unique fluorescent Luminex MagPlex-TAG™ microspheres . A biotin-labelled product will only be formed by the allele-specific and common primer due to the 3’ mismatched base. Tag sequences, LDR primers and conditions are shown in Table A genotyping assay was developed, based on the similar methods for the determination of cytochrome P450 2B6 alleles by Mehlotra et al.; a post-Deviations from the Hardy-Weinberg equilibrium were tested using the Court Lab Calculator . Cut-offet al.[Calculations were performed using SAS ver. 9.2, . Haplotypes were determined based on the definitions described by Covas et al.. Chi-squSamples were collected from children from 0 to 15 years of age. No significant differences were observed in the mean age or the proportion of males between the control and disease groups except for the severe anaemia group where the mean age was lower than controls (P = 0.93 ) and the proportion of males was higher than controls P = 0.02) , due to a higher frequency of Sl1/lower frequency of Sl1/Sl2 than expected. Analysing Hardy-Weinberg in the different groups at the Sl locus, showed that the CC group , the SA group  and the CM group  all deviated from Hardy Weinberg, whereas the UM group  was the only one in Hardy-Weinberg equilibrium. The deviations in three of the groups were very small and seemed to reflect a small anomaly, so the Sl locus was not excluded from further analysis. The distribution in the UM, SA and CM groups was compared to the CC group and to each other. At the Sl locus, the UM group differed significantly from the CC and SA groups and showed a tendency to differ from the CM group with a higher frequency of the Sl2 allele  . The McCb allele was more common with a total frequency of 8.7% and 35.5% in the McCb/b and the McCa/b genotype, respectively. The Sl2 and KCAM- alleles were very common with high frequencies of both the homozygotes  and the heterozygotes . Logistic regression, adjusted for age and sex, tested for an association between the genotypes and parasitaemia or disease outcome . No significant associations were found between any genotype at the four loci and level of parasitaemia (p > 0.1) and between any genotype and disease outcome (p > 0.3).The genotype frequencies at the four loci in the different groups are shown in Table b allele . At the Sl loci Ghana differed from both Mali  and Kenya  by a higher frequency of the Sl2 allele and slightly differed from The Gambia . Only few data for the KCAM and Kn loci are known. At the KCAM loci, Ghana showed a higher frequency of the KCAM- allele compared to an earlier publication . No differences were found at the Kn loci.Furthermore, the allele frequencies for McC and Sl were compared against earlier findings from studies in various African countries Table . The GhaThe SNPs of the four loci were constructed into haplotypes. The frequencies and definitions of the eight haplotypes are shown in Table When comparing the distributions of the haplotypes in the four groups, no significant differences were found between any of the groups (p > 0.3). A study in three Brazilian groups: European Brazilian, Asian Brazilians and African Brazilians defined the CR1 haplotypes . All thrBased on the described pathogenetic and epidemiological evidence, it was hypothesized that there would be an association between polymorphisms in the Knops blood group system and malaria severity. In this present study, no evidence was found for such an association.b (27% vs 1%) and Sl2 (83% vs 1%) alleles compared to Caucasian populations [- frequency compared to Caucasians (91% vs 2%), while frequencies of the Kna were similar in Africans and Caucasians concurring with an earlier publication [The McC and Sl-polymorphisms have previously been studied in African populations including Mali ,20, a poulations . Kn and lication . The UM et al.[From the various alleles at the four loci, eight different haplotypes can be constructed based on definitions described in Covas et al.. Six of et al.. H2 and et al. showed that low CR1 copy numbers on the erythrocytes could affect the ability to remove circulating immune complexes, one of the most important functions of CR1 [Thus, it seems that the polymorphisms are not associated with any of the studied complications of falciparum malaria. Earlier studies have shown conflicting data when investigating the possible association between the McC and Sl polymorphisms and malaria ,24. Seves of CR1 , in chils of CR1 .et al. tested different recombinant constructs of CR1 and showed that the selective pressure on the Sl2 and McCb alleles does not appear to be P. falciparum malaria since no differences in complement-regulatory functionality, rosetting, or binding to PfEMP1 were found between the alleles [et al. showed that the alleles McCb and Sl2 are associated with resistance to Mycobacterium tuberculosis infection in a Malian population [Conversely, malaria may not be the driving force behind the selective pressure of CR1 polymorphisms. Recently, Tetteh-Quarcoo  alleles . Based o alleles , and thipulation , which sb and Sl2, and Knb and KCAM- do not seem to confer any protective advantage against malaria infection or resulting disease severity. Furthermore, combining the loci into haplotypes showed no significant association with disease severity either. Based on these findings, in a very well-characterized population, malaria does not seem to be the selective force on these alleles.The results from this study confirm earlier findings of high frequencies of certain CR1 alleles in Africa and shed more light on the conflicting earlier findings: McCPlasmodium falciparum erythrocyte membrane protein one; PCR: Polymerase chain reaction; SA: Severe anaemia; Sl: Swain-Langley; SNP: Single nucleotide polymorphism; TMAC: Tetramethyl-ammonium chloride; UM: Uncomplicated malaria.CC: Controls; CM: Cerebral malaria; CR1: Complement receptor one; FMA: Fluorescent microsphere assay; Kn: Knops antigen; LDR: Ligase detection reaction; LHR-D: Long homologous repeat D; McC: McCoy; MFI: Median fluorescent intensity; PfEMP1: The authors declare that they have no competing interests.HHH acquired and analysed the data, carried out the laboratory work and wrote the manuscript. MA, JAK and ICB contributed to study design and interpretation of data, supervised DNA work and revised the manuscript for important intellectual content. QBG, OPR, FNN and TGT contributed to concept development and study design, edited and revised the manuscript critically for content. All authors read and approved the final manuscript.


Plasmodium. However, most malaria deaths are caused by Plasmodium falciparum [Malaria is one of the most dangerous tropical diseases worldwide, resulting in approximately 1.5-2.7 million deaths per year [Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is one of the key regulators of the malaria parasite life cycle in both sexual and asexual blood-stages. Inhibition of PfPKG stops differentiation and transmission of the parasites, indicating that this kinase is a promising drug target for malaria [The  malaria -5. Howev malaria . TherefoE. coli. For the cGMP binding studies we used fluorescence polarization (FP) assay and for the activation studies we used microfluidic mobility-shift assay (MSA).To investigate the role of the CNB-D in PfPKG activation, we generated a deletion mutant without the N-terminal 400 amino acid residues (Δ400), which is missing a potential auto-inhibitory sequence. We expressed the deletion construct (PfPKG 401-853) containing the catalytic domain and the PfCNB-D in A about 240 nM) in comparison to the full length PfPKG (KA about 70 nM [The deletion construct showed similar affinity for cGMP (40 nM) like the isolated PfCNB-D. In activation, the Δ400 mutant showed an approximately 3 fold increased activation constant for cGMP (Kut 70 nM ). UnexpeA for cGMP), suggesting that the cGMP binding at the CNB-D mainly triggers the activation of this mutant.In addition, disrupting cGMP binding through mutating the conserved arginine 492 to lysine (R492K) abolished the cGMP-dependent activation of the deletion mutant  has a low basal activity and can be activated efficiently by cGMP. This suggests that controlling PfPKG activity may not depend on the interaction between the inhibitory sequence and the catalytic domain. Upon cGMP binding, conformational changes in the regulatory (R) domain, especially in the area that bridges the regulatory and catalytic domains, induce kinase activation.


P. falciparum. This study aimed at investigating the influence of gametocyte carriage on; the prevalence of fever and splenomegaly, blood pressure, heart rate and haematological indices in children <15 years, in the Mount Cameroon area.Cardiovascular parameters can be impaired by repeated infections with A cross-sectional study was carried out, from February to July 2013. A child with axillary body temperature ≥37.5 °C was considered febrile and splenomegaly was investigated by palpation. Systolic and diastolic blood pressures as well as heart rate were assessed by non-invasive methods. Malaria parasites were detected and density assessed from Giemsa-stained thin and thick blood films. An auto haematology analyser was used to obtain complete blood count values such as haemoglobin (Hb), haematocrit (Hct), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and mean corpuscular haemoglobin (MCH). Univariate analyses were used to examine influence of gametocyte carriage on fever and splenomegaly while, multiple linear regression models were used to evaluate influence of independent variables on the dependent variables.P = 0.03, P = 0.002) prevalence of fever and splenomegaly  respectively than their aparasitaemic (AP) and asexual parasite positive (ASP Pos) equivalents . The presence of asexual malaria parasitaemia significantly influenced the MCV (P = 0.03), MCH (P = 0.03) and heart beats /min (0.03) while gametocytaemia significantly influenced the Hb (P < 0.001), Hct (P < 0.001), RBC (P < 0.001) and systolic blood pressure (P < 0.05).Of a total of 454 children examined, malaria parasitaemia, fever, splenomegaly and gametocyte carriage were detected in 36.6, 21.6, 14.3 and 7.3 % of them respectively. Children who were asexual parasite and gametocyte positive (ASP + Gam Pos) had significantly highest (Gametocyte carriage significantly influenced the prevalence of fever, splenomegaly and some cardiovascular indices. In effect, children concurrently having asexual parasitaemia and gametocytes had significantly lower, Hct, Hb levels, RBC and platelet counts and systolic blood pressure. Plasmodium. Globally, an estimated 3.3 billion people in 97 countries and territories are at risk of being infected with malaria parasite and developing disease [Malaria is a febrile illness caused by protozoans of the genus  disease . Majorit disease .P. falciparum hold a prominent place in the history of malaria [Malaria transmission from an infected human host to a female anopheline mosquito is mediated through highly specialized sexual-stage parasites, i.e., gametocytes. The gametocytes of  malaria . Several malaria . However malaria .P. falciparum transmission areas [Splenomegaly has been reported as one of the common pathologies among children with multiple parasitic aetiologies . Splenomon areas . A palpaon areas , 8. Indion areas , 9. Thouon areas , repeateon areas . Impairmon areas –14 reprePlasmodium induces haematological alterations involving major cell lines such as RBCs, leucocytes and thrombocytes which may lead to complications in malaria pathology characterised by splenomegaly, anaemia, thrombocytopenia and disseminated intravascular coagulation [et al. [As an intraerythrocytic parasite of the fluid component of the cardiovascular system, gulation , 15. Thi [et al. , 16 who  [et al.  have bee [et al. , 19–21. et al. [The study was carried out in Muea, a semi-rural setting in the Mount Cameroon area. The coordinates of the study area ranged from altitude 540 m, latitude 04°10.464’N, longitude 009° 18.168’E to 556 m, 04°10.015’N and 009°18.009’E. The study area has been described in detail by Sumbele n = Z2pq/d2 [n = the sample size required, z = 1.96: confidence level test statistic at the desired level of significance, p = 85.4 %: proportion of malaria prevalence, q = 1-p: proportion of malaria negative children and d = acceptable error willing to be committed. The minimum sample size was estimated as n = 192. This was adjusted to an optimum of at least 450 samples taking into consideration the recently reported decline in malaria parasite prevalence [A cross-sectional study was carried out from February to July 2013, which included the rainy season (March - October) reported as the peak malaria transmission period in the Mount Cameroon area . After o Z2pq/d2  where n Clinical evaluation was carried out by trained medical personnel. The clinical evaluation consisted of: measurement of body temperature, examination of the spleen, measurement of blood pressure and heart rate. Axillary body temperature was measured using a digital thermometer. A child with a body temperature ≥37.5 °C was considered febrile. The tip of the spleen was felt by pressing the abdomen under the left coastal border and splenomegaly was graded according to the classification of Hackett on a scale of 1–5 , 26. ChiFour (4) mL of venous blood was collected from each participant into sterile disposable syringes. A small portion was used for the preparation of thick and thin blood films. The remainder was dispensed into labelled ethelenediaminetetraacetate (EDTA) tubes, and transported in a cool box (temp 8–10 °C) to the Malaria Research Laboratory, University of Buea for further analysis.Plasmodium species were observed on the blood film. Parasitaemia was classified as low (<1000 parasites/μL of blood), moderate (1000–4999 parasites/μL of blood) and high (≥5000 parasites/μL of blood). Slides were read by two independent parasitologists and in the case of any disparity they were read again by a third parasitologist. Blood samples in the EDTA tubes were transported to the St. Albert the Great Reference Medical Diagnostic Centre, for blood count analysis. The complete blood count analysis was obtained using an auto haematology analyser (TC Hemaxa 1000) following the manufacturer’s instructions. Parameters such as Hb concentration, Hct, RBC, WBC, granulocyte, lymphocyte and platelet counts, MCV, MCHC and MCH were obtained. Anaemia was defined as Hb <11 g/dL and was further classified into severe (Hb ≤ 6 g/dL), moderate (Hb = 6.1–8 g/dL) and mild (8.1–10.9 g/dL) anaemia [9/L. Thrombocytopaenia was defined as platelet count <150,000/μL.The thick and thin blood films prepared on glass slide at the time of blood sampling were stained with 10 % Giemsa and examined following standard protocols . Parasit anaemia . Leucopeχ2). The non parametric test, Kruskal-Wallis test, was used to compare the mean cardiovascular indices in the different age groups  and malaria parasitaemia status . The mean temperature of children with different parasitaemic status was compared using analysis of variance (ANOVA). Gametocyte counts were log transformed before analysis. Pearson’s correlation coefficient was used to examine the relation between temperature and heart rate. Cardiovascular indices with a P-value ≤0.2 in the univariate analyses were entered into a multiple linear regression (MLR) model with each haematological variable, systolic and diastolic blood pressure as the dependent variable. The MLR (enter) models were run after controlling for age to examine the influence of the following independent variables; sex, temperature, asexual parasitaemia, gametocyte carriage and spleen size. All 454 participants were included in the model. Significant levels were measured at 95 % confidence interval (CI) with significant differences set at P < 0.05.Data was entered into spread sheets using Microsoft Excel, validated and analysed with the Statistical Package for Social Sciences (SPSS) version 19 . Data were summarized into means and standard deviations (SD), and percentages were used in the evaluation of the descriptive statistics. Proportions were compared using the Chi-square test . Gametocytaemia, splenomegaly, fever, leucopaenia and thrombocytopaenia were detected in 7.3, 14.3, 21.6, 24.6 and 18 % of the children respectively, as shown in Table P = 0.55) in children who were ASP Pos  and those with ASP + Gam . Although not statistically significant (P = 0.26), children with ASP only had a higher geometric mean parasite density/μL of blood (859) when compared with those with ASP + Gam (639).Low, moderate and high malaria parasitaemia were common in 56.5 % (CI = 48.8–63.9 %), 29.8 % (CI = 23.3–37.3 %) and 13.7 % (CI = 9.2–19.8 %) of the children respectively. The prevalence of low, moderate and high malaria parasitaemia were comparable (P = 0.56) in males (51) and females (61). Although children of the ≤ 5 years age group had the highest GMGD/μL of blood (69) when compared with the 10–14 years (30) and the 6–10 years (51) age groups, the differences were however not statistically significant (P = 0.12).Out of the 454 children examined, 33  were gametocyte positive with a geometric mean gametocyte density (GMGD) of 56/μL of blood (range = 15–306 gametocytes/μL). The GMGD/μL was comparable  was observed between temperature and heart rate. The prevalence of fever was comparable (P = 0.70) in children of the age group ≤ 5 , 6–10  and > 10 years  irrespective of the malaria parasitaemia status. However, the prevalence of fever was significantly highest (P = 0.03) in children who were ASP + Gam Pos (39.4 %) than their equivalents as revealed in Table The mean temperature in the study population was 37.0 ± 0.64 P < 0.001) in children with ASP + Gam (54.5 %) when compared with those AP (21.4 %) and ASP Pos (38.2 %).The prevalence of anaemia in the study population was 28.9 % (131), while mild, moderate and severe anaemia was prevalent in 24.2 % (110), 2.6 % (12) and 2.0 % (9) of the children respectively. Anaemia prevalence was significantly highest (P = 0.68) in the different age groups . Similarly, AES was comparable (P = 0.21) in AP (2.6 ± 1.4), ASP Pos (3.3 ± 1.5) and ASP + Gam Pos (2.6 ± 1.4). Overall, the prevalence of mild enlargement was 7.9 % (36), while 6.4 % (29) of the children had very enlarged spleens. Mild enlargement and very enlarged spleen were significantly highest (P = 0.001) in children with ASP + Gam (21.2 and 12.1 % respectively) than their counterparts. Likewise children with ASP + Gam had significantly highest (P = 0.001) prevalence of splenomegaly  as shown in Fig. Out of the 454 children examined, 65 14.3 %) had enlarged spleens with an AES of 2.8 ± 1.5. The AES was comparable  prevalence of splenomegaly (33.3 and 40 % respectively) in comparison with the aparasitaemic and those with asexual parasites only as shown in Fig. Children with ASP + Gam in the < 5 years and the 6–10 years age groups had significantly highest (P = 0.003).A comparison of prevalence of anaemia revealed a higher prevalence of anaemia in children with splenomegaly than in those with normal spleen Fig. . HoweverP < 0.001), Hb levels (P < 0.001), RBC counts (P = 0.029), platelet counts (P = 0.02) and systolic blood pressure (P = 0.049) when compared with their AP and ASP Pos counterparts. Although not statistically significant, the mean WBC and lymphocyte counts and diastolic blood pressure were lower in children who were ASP + Gam Pos than those AP and those ASP Pos only. The mean MCV, MCH, MCHC and MPV were comparable in all three groups of children.As shown in Table P = 0.03). The size of the spleen significantly influenced the Hb (P = 0.01), Hct (P = 0.01) and RBC (P = 0.001). Temperature had a significant influence on Hb (P = 0.01), Hct (P = 0.03), lymphocyte counts (P = 0.02), MCH (P = 0.01) and heart beats/min (P < 0.001). The presence of asexual parasitaemia significantly influenced the MCV (P = 0.03), MCH (P = 0.03) and heart beats/min (0.03) while gametocytaemia significantly influenced the Hb (P < 0.001), Hct (P < 0.001), RBC (P < 0.001) and systolic blood pressure (P < 0.05) as shown in Table After controlling for the effect of age on the independent variables, sex had a significant influence on MCV  is significantly lower than that reported by Sumbele et al.  in the s [et al.  during t [et al. , the preer cases . Never tet al. [et al. [Parasite prevalence and level of exposure are closely related and clinically immune infections have been reported to have lower gametocytemia as a result of having anti-parasite immunity, conferring protection against high-density parasitaemia . The lacet al.  in child [et al. . The hig [et al.  is used et al. [P. falciparum malaria by releasing pro- and anti-inflammatory cytokines such as TNF-α, interferon-γ and other cytokines, and activating other inflammatory cells. However, excessive secretion of the pro-inflammatory cytokines contributes to disease severity [The lowered WBC and lymphocyte counts noticed in children harbouring gametocytes and asexual parasite is unusual as the aparasitaemic and those harbouring asexual parasite only had similar mean WBC and lymphocyte counts. However, the observed malaria associated decrease in WBC and lymphocyte counts is in line with the decrease in lymphocyte count in untreated malaria reported by Sumbele et al. . Furtherseverity , 36.et al. [et al. [Findings from the multiple linear regression analysis revealed that the presence of gametocytes significantly influenced the Hb, Hct, RBC and systolic pressure. In like manner, a comparison of haematological variables revealed these variables to be significantly lowest in children harbouring both gametocytes and asexual parasites. The reduction in these red cell indices in gametocyte positive children is not unusual as Sumbele et al.  and Nach [et al.  reported [et al. .P. falciparum infection is known to cause low platelet levels and disseminated intravascular coagulation with bleeding [One of the additive significant influences of gametocyte carriage is the lowered systolic blood pressure observed in children who had both asexual parasites and gametocytes when compared with those aparasitaemic and those harbouring only asexual parasites. This may be associated to the fever observed in this group of children. Fever and excessive sweating, accompanied with lack of rehydration, lead to continuous dehydration. During hyperthermia, one of the mechanisms involved in regulating the temperature by the body is vasodilation. This is caused by inhibition of the sympathetic centres in the hypothalamus . More albleeding , resultiet al. [Observation from the study revealed the presence of mild and very enlarged spleens in the study population. Splenic enlargement has been highlighted as a consequence of malaria parasitaemia  and multet al.  reportedFindings from the study demonstrated a significant influence of the spleen size on red cell indices such as the Hb level, Hct, and RBC while, temperature in addition to the Hb and Hct significantly influenced the MCH. The effects may be attributed to the role of the spleen in clearing infected , 47 and P. falciparum malaria as the disease gets toward complication. The observed increase of the heart rate could help balance the provision of oxygen to the tissues.Although not significant, the heart rate was higher in gametocyte positive children than those aparasitaemic and those having asexual parasites only. As well, the temperature of the child was significantly associated with the heart rate. The significant association of temperature with the heart rate is not unexpected as an increase of only 1 °C raises the heart rate by about 10 beats per minute. This explains the faster heart beats observed in cases with fever . The higIn addition to the gametocytaemia, increased spleen size, the trophozoite count also had significant influence on haematological indices such as MCV and MCH and cardiovascular indices like the heart beats/min. It is worth noting that despite all variations in the hemodynamic parameters, majority of the values remained within normal ranges. However, our findings can serve as a relevant warning signal, highlighting the hidden danger of the impact of gametocyte carriage, asymptomatic malaria parasitaemia and uncomplicated malaria on the cardiovascular system. The fact that some children did not even display signs of malaria infection is a strong proof that regular investigations are invaluable in malaria endemic areas. The more infections are persistent in an area of transmission, the more risky the population will be, irrespective of whether the patient has asymptomatic or uncomplicated malaria.The results showed that in a malaria endemic setting such as the Mount Cameroon area, gametocyte carriage significantly influenced the prevalence of fever, splenomegaly and some cardiovascular indices. In effect, children concurrently having asexual parasitaemia and gametocytes had significantly lower, Hct, Hb levels, RBC and platelet counts and systolic blood pressure when compared with gametocyte negative children. Further investigations with ultrasound cardiography will surely bring out more information on the cardiac profile of these children.


Introduction. Thrombocytopenia is frequently noticed with P. falciparum malaria but is less reported and studied with P. vivax. Materials and Methods. The study was conducted in the Department of Medicine, SBKS MI & RC, Pipariya. We included patients who were diagnosed with vivax malaria. The data regarding their clinical and hematological profile was collected and analysed. Result. A total of 66 patients were included. 42 (63%) had platelet count <100000/mm3. Mean platelet count was 1,18,650, range being 8000/mm3–6,10,000/mm3. Amongst those with thrombocytopenia, 16 (38.09%) had anemia, 14 (33.33%) had serum creatinine >1.2 gm/dL, 15 (35.71%) had jaundice (s. bilirubin > 1.2), 2 (4.76%) had altered sensorium, 6 (14.28%) had ARDS, 2 needed ventilator support, and 1 expired. Amongst those with normal platelet count, 5 (20.83%) had anemia and 1 had jaundice whereas none had elevated s. creatinine, altered sensorium, or lung involvement. Conclusion. Thrombocytopenia is now being seen more commonly with vivax malaria. Patients with platelet count <1 lac/cumm have more severe disease. Malaria is a disease of global importance. The World Health Organization (WHO) has reported a worldwide annual incidence of 247 million cases and malarial mortality of one million per year . Plasmodium falciparum  is known to cause thrombocytopenia and severe malaria but severe cases in Plasmodium vivax (P. vivax) malaria patients have been reported in the last decade. With implementation of molecular diagnosis, it became evident that P. vivax monoinfection could also be involved in multiple organ dysfunction and severe life-threatening disease as seen in P. falciparum infection [β1 [ P. vivax infected RBCs adhering to lung endothelial cells and to the placental tissue ex vivo indicates that, in vivax malaria, mechanisms similar to those associated with falciparum malaria severity may be involved [Infection withnfection , 3. Manynfection ; bridgesnfection  and stimion [β1 . Recent involved . P. vivax monoinfection. They reported all severe manifestations in vivax malaria in children including severe anemia, thrombocytopenia, cerebral malaria, acute respiratory distress syndrome (ARDS), hepatic dysfunction, renal dysfunction, and abnormal bleeding. They reported thrombocytopenia in 61.5% children and bleeding symptoms in 10.8% cases [ P. vivax malaria, with 25.6% requiring platelet transfusions [All the complications seen in falciparum malaria are now reported with vivax also –10. A st8% cases . A studysfusions . P. vivax malaria are available, most of them were done on pediatric population or included children also. There is lack of studies assessing thrombocytopenia and severity in P. vivax exclusively in adults. Hence, we carried out this study to find out the status of thrombocytopenia in P. vivax in adult population and find if it has any association with severity.Thus, although many studies regarding thrombocytopenia inIt was a prospective cohort study. Prior permission for the study was taken from institutional ethical committee. All patients diagnosed with malaria on peripheral smear were enrolled for the study. The patients with falciparum infection either alone or in combination with vivax or who did not give written informed consent were excluded. The patients with underlying disease which may cause similar complications like dengue and sepsis were also excluded.All patients were subject to the routine laboratory investigations like hematological profile (complete blood count with platelet count), urine routine microscopy, renal function test, serum electrolytes, liver function test, blood sugar levels, and chest X-ray. Viral markers were done in patients with icterus to rule out viral hepatitis. Ultrasonography was also done to study the size and echo texture of the liver and gall bladder, intrahepatic or extrahepatic bile duct dilatation, and signs of portal hypertension. Other special investigations like CT scan of head and arterial blood gas analysis (ABG) were done when indicated. Presenting symptoms and signs were noted along with the information on complications developed.Thrombocytopenia was defined as platelet count <1 lac/cumm. Anemia was defined as hemoglobin <10 gm/dL. Respiratory distress was defined as oxygen saturation less than 94% at room air or deep breathing (acidotic breathing) or a rapid respiratory rate (>32/min) . The patP. falciparum monoinfection or mixed P. vivax and P. falciparum infection or coinfection with dengue or associated sepsis). 100 patients had P. vivax monoinfection as diagnosed on peripheral blood smear. Hence, total 100 patients with P. vivax monoinfection were included in the study. The age of included patients ranged from 18 to 50 years. 42 (42%) had platelet count <1 lac/cumm. Average platelet count amongst them was 1,18,651 ± 1,01,937/cumm and mean hemoglobin was 10.69 ± 2.35 mg%.A total of 120 patients diagnosed on peripheral smear with malaria were enrolled for the study. 20 were excluded for various reasons (The patients were divided into two groups based on platelet count (<1 lac/cumm and >1 lac/cumm) for further analysis. Baseline characters were similar amongst the two groups .Gastrointestinal symptoms , shortness of breath, and CNS symptoms  were more common in <1 lac/cumm group whereas upper respiratory tract symptoms  were more common in >1 lac/cumm group . IcterusOn lab investigation  there waMalaria is a disease that affects almost all blood components. The essential pathological feature of severe falciparum malaria is sequestration of erythrocytes in the deep vascular beds of vital organs leading to cerebral malaria, renal failure, hepatic dysfunction, or ARDS. Hemolysis, reduced cell deformity of parasitized and nonparasitized erythrocytes, increased splenic clearance, reduced platelet production and survival, and increased splenic uptake of platelets cause severe anemia and thrombocytopenia leading to bleeding diathesis. P. falciparum. Since the beginning of the 1970s, reports of similar degree of thrombocytopenia in P. vivax and P. falciparum infections started coming in [ P. vivax malaria were published in the late 1990s, probably due to the surge in the availability of automated machines. Around the same time, reports of severe, complicated malaria with P. vivax infection also started being published; but regardless of being described as a complication by WHO, thrombocytopenia is not considered to be a severity criterion by itself [ per se.Initially thrombocytopenia was thought to be a feature ofoming in . Most ofy itself  due to t P. vivax malaria but they had distinct selection criteria of the enrolled patients or included only pediatric patients [μL) [ P. vivax infection; some have reported multiple complications, but none of these studied adult population exclusively [ P. vivax monoinfection [Many studies have shown a wide range of thrombocytopenia and severity inpatients , 14. No nts [μL) , 16. Onllusively –20. Repolusively , while mlusively , 16. Anonfection . The sig P. vivax [ Plasmodium vivax malaria [Although some studies have reported association of thrombocytopenia with severe malaria , 24, mosP. vivax . Tanwar P. vivax . Kochar  malaria . P. vivax malaria in adults. Most have included both adult and pediatric populations and have not excluded the other causes of thrombocytopenia. Also, there is no study which has assessed the association of thrombocytopenia with severity in P. vivax malaria in adults. Our study included only adult population. We excluded P. falciparum, dengue positive as well as sepsis patients as these are the major causes of thrombocytopenia in our region. We also assessed the association of thrombocytopenia with severity in adult P. vivax patients. We observed both sequestration-related and nonsequestration-related complications  in our study population. These were significantly more common in patients with platelet count <1 lakh/cumm. ARDS and need of blood or platelet transfusion and inotropes as well as mechanical ventilation were also seen in platelet count <1 lac/cumm group. This group also recorded mortality in one case. Thus, this study highlights the fact that all complications of P. falciparum can be seen with P. vivax and that the parameter of platelet count <1 lac/cumm may be used as a marker of severity and increased chances of complications. It identifies the set of patients who will need aggressive management.Thus, there has been a lack of studies on thrombocytopenia in P. vivax malaria is changing. Similar incidence of thrombocytopenia is now seen in vivax and falciparum malaria patients. All complications seen in falciparum positive cases are being seen in vivax positive cases also. Thrombocytopenia may not be a cause of mortality by itself, but it can be a marker of increased severity and need of aggressive management. There is a need to reexamine the clinical spectrum and severity of P. vivax malaria especially in the light of thrombocytopenia. We also recommend formulation of separate guidelines for management of P. vivax with platelet count <1 lac/cumm looking at the severity profile of such patients.The trend of disease with


Antibiotic therapy during pregnancy may be beneficial and impacts positively on the reduction of adverse pregnancy outcomes. No studies have been done so far on the effects of daily Co-trimoxazole (CTX) prophylaxis on birth outcomes. A phase 3b randomized trial was conducted to establish that daily CTX in pregnancy is not inferior to SP intermittent preventive treatment (IPT) in reducing placental malaria; preventing peripheral parasitaemia; preventing perinatal mortality and also improving birth weight. To establish its safety on the offspring by measuring the gestational age and birth weight at delivery, and compare the safety and efficacy profile of CTX to that of SP.Pregnant women (HIV infected and uninfected) attending antenatal clinic were randomized to receive either daily CTX or sulfadoxine-pyrimethamine as per routine IPT. Safety was assessed using standard and pregnancy specific measurements. Women were followed up monthly until delivery and then with their offspring up to six weeks after delivery.Data from 346 pregnant women  and 311 newborns (CTX = 166 and SP = 145) showed that preterm deliveries ; still births , neonatal deaths , and spontaneous abortions  were similar between study arms. The low birth weight rates were 9% for CTX and 13% for SP. There were no birth defects reported. Both drug exposure groups had full term deliveries with similar birth weights (mean of 3.1 Kg). The incidence and severity of AEs in the two groups were comparable.Exposure to daily CTX in pregnancy may not be associated with particular safety risks in terms of birth outcomes such as preterm deliveries, still births, neonatal deaths and spontaneous abortions compared to SP. However, more data are required on CTX use in pregnant women both among HIV infected and un-infected individuals.NCT00711906.Clinicaltrials.gov  Malaria is one of the most important causes of morbidity and mortality worldwide, with children and pregnant women being the most severely affected groups Anaemia, malnutrition and HIV infection are also common events in malaria endemic areas and contribute to LBW. HIV infection in pregnancy is associated with an increased risk of malaria infection and higher parasite densities The World Health Organization (WHO) recommends a package of interventions for the prevention and control of malaria in pregnancy. This comprises intermittent preventive treatment (IPTp), use of insecticide treated materials (ITMs) Cotrimoxazole (CTX) has been used for treating malaria in children; recently, its daily use by non-pregnant HIV-infected adults was associated with 70% reduction in the incidence of clinical malaria A clinical trial comparing the protective efficacy of daily CTX versus IPTp-SP in pregnant women was carried out in southern Zambia. Its primary objective was to test the hypothesis that CTX prophylaxis is not inferior to SP prophylaxis in reducing placental malaria. The secondary objectives were to evaluate efficacy of CTX prophylaxis in preventing malaria peripheral parasitaemia, perinatal mortality and in improving birth weight; to establish the safety on the offspring by measuring the gestational age and birth weight at delivery, and also to compare the safety and efficacy of CTX prophylaxis to that of SP based on these parameters. The study participants were stratified by HIV status in order to follow the national preventive guidelines, taking note of the fact that pregnant women with CD4 count <200 cells/µl  are given daily CTX and should not receive SP-IPTp. However, at the time of implementation and thanks to the coordinated efforts for malaria control, malaria transmission had become extremely low to the extent that the study was prematurely stopped because it would not have had sufficient power to meet the original objectives. However, before interrupting the study, several pregnant women were included and received either daily CTX or IPTp-SP in a 1∶1 ratio. This provided the opportunity to report the safety of daily CTX in an area of low malaria endemicity.Choma district is located in the Southern Province of Zambia. Though malaria continued to be a key focus of public health services in Choma as is the case in Zambia, there has been large scale up of antimalarial interventions and considerable progress in control of disease in recent years The changing epidemiology was documented in Livingstone, less that 200 km from Choma, where the number of malaria cases declined from 8000 per quarter between 2004 and 2007The study was initiated in Choma area in January, 2009, shortly after a large observational study that was recruiting pregnant women with history of clinical malaria and receiving antimalarial treatment had just been concluded. The screening for the trial  included peripheral blood smear analysis. After 6 months of patient recruitment a review of the data on the screening smear results revealed that only 4 of the 421 screened patients had a malaria infection. This prompted the study director in liaison with entire investigational team to inform the ethical review committee at the Tropical Diseases Research Centre (TDRC) and the National Ethical Review Committee, the sponsor as well as the Zambia Medicines Regulatory Authority of the extremely low malaria prevalence in the study area. As the risk of malaria infection in pregnant women was much lower than expected, the trial would not have had sufficient power to provide the required answer, i.e. non-inferiority of the CTX arm in preventing placental malaria as compared to SP intermittent preventive treatment. The issue of changing the study site was discussed also with the DSMB that received and reviewed on a quarterly basis (unless differently requested by the DSMB members) the updates on screening and recruitment and on losses to follow-up, as well as the quarterly SAE reports routinely sent also to the Ethical Committees. In addition, Serious Adverse Events were also reported on a case-by-case basis, unless differently requested by the DSMB members, in order to allow them to promptly comment on specific events. The decision to move the study site to another location known to have high malaria transmission was taken by the research team in consultation with the DSMB, ethics committees, the Regulatory authority and the sponsor.Pregnant women between 16 and 28 weeks of gestation attending the antenatal clinic of the Shampande Health Centre or Choma Hospital in Choma District, accepting to be tested for HIV and willing to participate in the study were enrolled. The inclusion criteria were absence of symptoms consistent with malaria at the time of recruitment , willingness to deliver at the health facility and willingness to adhere to study requirements, e.g. monthly visits to the antenatal clinic. Exclusion criteria were history of allergy to sulpha drugs, history or presence of major illnesses likely to influence pregnancy outcome such as diabetes, severe renal or heart disease or active tuberculosis; intent to move outside the study catchment area before delivery, severe anaemia (Hb<7 g/dl) and previous history of unfavourable pregnancy outcome . Recruitment was conducted from February to September, 2009.http://clinicaltrial.gov/ct2/show/NCT00711906.The study was sponsored by the Institute of Tropical Medicine, Antwerp. It was approved by the Institutional Review Board at the Institute of Tropical Medicine, Antwerp, and the Ethics committees at the University of Antwerp and at the Tropical Diseases Research Centre, Ndola, Zambia. These bodies also approved the Amendment 1.0 of 25 March 2009, which was issued to align the study to the new management practices for prevention of mother to child transmission (PMTCT) in Zambia  if their CD4 count was <350 cells/µl). All patients were informed about the study before they were asked to sign the informed consent. Each study participant was assigned a unique patient code that was used for all subsequent forms in the study, so that confidentiality of all subjects was guaranteed. A no fault study insurance was taken to indemnify possible damage linked to a subjects participation in the study. The trial was registered in the Clinicaltrials.gov registry, identifier: NCT00711906, URL: This was a phase 3 randomized open label clinical trial whose planned primary endpoint was placental malaria.  is shown in All HIV negative and HIV positive with CD4 count ≥200 cells/µl were randomized to either three monthly doses of SP–IPT  or daily CTX prophylaxis . Therefore, the study intervention and randomization did not vary by HIV status in these two groups of women. Nevertheless, HIV positive pregnant women with CD4 count <200 cells/µl and complying with all other entry criteria were recruited in an observational cohort as they had to be given 2 tablets of CTX prophylaxis daily and antiretroviral treatment as per standard guidelines. In Zambia CTX prophylaxis for these women is part of the routine ANC care since November, 2003, in accordance with the WHO policy At enrolment, a standardized questionnaire to collect demographic information, history of malaria episodes, past medical and obstetric history was administered. Other risk factors (such as smoking and drinking) that may affect pregnancy outcome were directly recorded in the Case Record Form (CRF). The socioeconomic status was also assessed on the basis of some key household parameters and a physical examination was performed. Laboratory tests at baseline included thick and thin blood smears for the diagnosis of malaria infection and measurement of haemoglobin. IPTp-SP administration was directly observed while CTX intake was supervised only for the first dose, the rest being taken at home until the next monthly visit when participants were reviewed.The randomization was stratified by HIV status. Eligible women were randomized to one of the two arms according to a pre-defined randomization list prepared at ITM, Antwerp. Participants were assigned sequential study numbers which were matched with numbered envelopes containing the arm allocation that were opened by the study nurses only after recruitment of the study subject. There was no blinding as each of the study drugs was openly administered.Women attended the antenatal clinic monthly for assessment of efficacy and safety parameters. Safety parameters included adverse events (AEs), serious adverse events (SAEs) and concomitant medications. During the monthly visits, physical examination, including foetal viability, was performed, information on bed net use was recorded, the study drugs provided, and compliance assessed by means of drug accountability. A blood sample for haemoglobin measurement , blood film for malaria parasites and a filter paper for later molecular biological assessments were collected (for efficacy assessment). Urine was analysed for glucose, proteins and blood using a dipstick urine test. At delivery, similar information as that collected at monthly visits was collected, including any AEs, SAEs and use of concomitant medication. In addition, a placenta blood sample for a thick blood film and later molecular analysis (filter paper) was collected from the maternal surface of the placenta. A biopsy was also obtained.Women and their babies were seen at one and six weeks post-delivery. Pregnancy specific assessments included rates of stillbirth (>28 weeks gestation), neonatal mortality (≤28 days of birth), maternal mortality (up to 6 weeks post delivery), spontaneous abortion (≤28 weeks gestation), stillbirth, preterm delivery (≤37 completed weeks), incidence of low birth weight, gestational age at delivery  using Macro 3.0 , a CFR21Part11 compliant software with an in built audit trail, password control and electronic signature. Each individual involved in this process  had a unique password-protected database user profile.The eCRF comprised a data entry interface which was an exact but electronic copy of the source document (SD) and incorporated pre-programmed checks and warnings for inconsistencies, omission and commission errors. Data were entered with single data entry and then verified by the investigators who confirmed it with an electronic signature. A study monitor performed several external monitoring visits throughout the study, during which partial source data verification (SDV) was done. In case of inconsistencies, queries were raised in the eCRF. All findings were discussed with the entire study team at the end of each visit, so that corrective actions could be taken, and similar errors could be prevented in the future. Similarly, the investigators based at TDRC also performed SDV to resolve inconsistencies and answer queries raised by monitors and TDRC based data managers along with the study team on a monthly basis. In addition, a manual review of the database was done on key variables such as safety- and endpoint data. The system was set up on offline laptops and had a facility for uploading and saving data at a central server at the ITM. The data were extracted into SAS 9.2  for formatting and statistical analyses.Statistical analyses were based on the intention-to-treat principle (ITT). For continuous variables, tests of the normality of their distributions were done. Descriptive statistics means, medians, modes and standard deviations were determined as appropriate. Frequency and percentage distributions were computed for discrete variables. Additionally, asymptotic 95% confidence intervals (95% CI) for means of continuous variables were determined. Clopper-Pearson exact 95% confidence intervals were determined for percentage distributions of count variables. Missing data were assumed ignorable and thus all statistical analyses included only complete cases. Maternal and foetal outcomes such as still births and neonatal deaths; spontaneous abortions (≤28 weeks gestation); preterm deliveries (<37 completed weeks); neonatal mortality (within 28 days after birth) and birth weight, including LBW , were outcomes of interest.For each of these variables, the treatment arms were analyzed per stratum (i.e. HIV status).Four hundred and twenty one pregnant women were screened, 346 met the entry criteria and one additional patient was recruited among those whose baseline visit was at delivery . GestatiBaseline characteristics including age, weight, height and gestational age were similar between treatment arms . Most ofMost pregnancy outcomes were spontaneous vaginal deliveries at gestational age of ≥37 weeks, with over 89% of the total deliveries between the 2 arms . There wNo congenital malformation was detected. There were 24 (8%) LBW babies, 15 9.3%) in the CTX group and 9 (6.3%) in SP group . The mea% in the A total of 61 AEs were observed in the 346 enrolled patients and there was no difference between study arms . Most AEThirty three AEs were defined as SAEs  and inclThere were only 2 patients on the CTX arm and 1 on the SP arm who had malaria positive peripheral smears at the end of follow up of the 347 pregnant women recruited. During the screening phase no patients were declared ineligible due to confirmed clinical malaria . Among the women screened, there were only 4 out of the 421 that were found to have malaria infection.Among the placenta blood samples collected on filter paper and analysed by PCR, only 1 sample out of 214 was positive for malaria. Placenta histopathology was also performed and only one sample out of 214 had malaria pigment. This positive sample by histopathology was the same as the placenta blood positive by PCR analysis.For all HIV-infected adults, including pregnant women, with CD4 cell counts below a given threshold, WHO recommends the use of CTX to prevent opportunistic infections The trial was aimed at determining the protective efficacy of daily CTX against malaria, both in HIV infected and uninfected pregnant women. Nevertheless, after starting the trial, it became obvious that the risk of malaria infection in the study area had decreased substantially, to the extent that that the trial could not answer the primary question on the protective efficacy of CTX prophylaxis. Therefore, the trial was prematurely terminated though a substantial number of pregnant women had already been recruited. This provided the opportunity of investigating the safety of CTX prophylaxis in an area of extremely low malaria transmission.Short term treatments with CTX (10–14 days) have been associated with birth defects when administered during the first trimester The rate of preterm deliveries using the LMP did not differ between the CTX (combined group of HIV negative and positive mothers) and SP arm. Similarly, the occurrence of preterm deliveries, stillbirths, neonatal mortality, spontaneous abortions and low birth weight among the HIV uninfected and infected with CD4 count ≥200 cells/µl women did not differ between study arms. For both study arms and irrespective of HIV status, the majority of the infants had birth weights consistent with full term delivery. These results are comparable to the findings from a cohort study conducted in the same study area After stopping this trial, a similar trial  was set up in an area of high malaria transmission and data are currently being analysed. Therefore, more information on the safety and efficacy of CTX against malaria will be made available in due course.Exposure to CTX during the latter part of pregnancy may not be associated with increased safety risks when compared to SP. However, considering the increasing resistance to SP, which is the drug currently used for IPTp, and the need to prevent opportunistic infections in HIV infected pregnant women by administering daily CTX, there is need of exploring the possible role CTX may have in preventing malaria in pregnant women, both HIV infected and uninfected.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


Alterations of blood coagulation are thought to be involved in malaria pathogenesis. This study had the aim to investigate changes of blood coagulation under the standardized conditions of controlled human malaria infection.Plasmodium falciparum sporozoites were intravenously (n = 24) or intradermally (n = 6) injected into 30 healthy volunteers. Twenty-two participants developed parasitaemia. Serial blood samples before and during prepatent period and at parasitaemia, diagnosed by microscopic assessment of thick blood smear, were obtained. Biomarkers of blood coagulation  were determined.In a clinical trial aseptic, purified, cryopreserved P. falciparum parasitaemia, 72.7 % of volunteers had peak thrombin generation 10 % above their baseline. Overall, peak thrombin generation was 17.7 % higher at parasitaemia compared to baseline . There were no significant changes of other coagulation parameters.At first detection of P. falciparum malaria, suggesting a hypercoagulable state may be induced, even when parasite density is low.The thrombin generation potential, an in vitro blood coagulation test, which reflects an individual´s global coagulation status, was increased by 17.7 % at very early stages of The online version of this article (doi:10.1186/s12936-015-1079-3) contains supplementary material, which is available to authorized users. Plasmodium falciparum. Leading causes of death in patients with malaria infection are severe anaemia and cerebral malaria. Moreover, in a review of imported malaria cases requiring intensive care unit admission it was reported that about 10 % of severe malaria cases develop disseminated intravascular coagulation (DIC), a serious coagulopathy associated with poor survival .Results of the thrombin generation assay, D-dimer, prothrombin fragment 1 + 2, vWF, ADAMTS 13-Activity and sP-selectin at baseline did not differ between those subjects who later developed parasitaemia (n = 22) and those who did not (n = 8) develop parasitaemia . Overall, peak thrombin generation was 17.7 % higher at the time of first detection of P. falciparum parasitaemia, compared to baseline. Lag time was significantly shorter at the time of malaria. There was no significant difference in D-dimer, F1 + 2, ADAMTS 13-Activity or sP-selectin.  had peak thrombin generation 10 % above their baseline. There were no differences in thrombin generation between those subjects who were symptomatic (n = 9) and asymptomatic [n = 13; median (25th–75th percentile): 211.4 nM (173.3–248.6) vs. 229.1 nM (159.3–295.6); Furthermore, changes of coagulation parameters during the incubation period, i.e. at the time before microscopically detectable parasitaemia, were investigated. No significant changes in coagulation parameters measured 1–3 days (median: 2 days) before microscopically detectable parasitaemia in comparison to baseline values were present (data are not shown but can be provided upon request).p = 0.028).Figure P. falciparum infections. The study showed that the coagulation potential of infected volunteers, measured by an in vitro thrombin generation assay, was increased at the time of first detection of parasitaemia, a time at which only 9 of the 22 subjects had symptoms or signs of malaria.The present study was done in the context of a project that established a standardized, CHMI protocol by IV administration of PfSPZ, and aimed was to investigate changes in blood coagulation induced by The thrombin generation assay is a global coagulation test that gives information on the potential of an individual to generate thrombin, which reflects the overall haemostatic capacity . ThrombiHigh thrombin generation is associated with hypercoagulable states, such as obesity , and incP. falciparum-infected erythrocytes can cause loss of endothelial protein C receptor (EPCR). EPCR physiologically enhances conversion of protein C to activated protein C (APC); APC is an anticoagulatory protein and decreased APC due to loss of EPCR consequently leads to the generation of high levels of thrombin [P. falciparum malaria who did not respond to standard treatment [P. falciparum specifically increases thrombin generation potential at the same time as parasites can be first detected by highly sensitive thick blood smear microscopy. However, it also has to be mentioned that in one-third of study participants no change in thrombin generation was observed.The coagulation system is increasingly recognized to play an important role in malaria . Obstructhrombin . Accordireatment ; howeverreatment . Our in P. falciparum infection, before other changes in coagulation parameters are observed.Of note, increase in peak thrombin generation was observed at the first time of microscopically detected parasitaemia in comparison to basic values in our study. Furthermore, lag time, which reflects time until start of clotting, was shorter at first diagnosis of malaria. This observation shows that the thrombin generation assay can capture coagulatory alterations at early stages of No significant changes in levels of the fibrin degradation products D-dimer and prothrombin fragment 1 + 2 were found in early stage malaria in our study, although these parameters indirectly reflect in vivo thrombin generation. The in vitro thrombin generation potential might be a more sensitive parameter for subclinical coagulation alterations, whereas other parameters might indicate coagulopathy in more advanced or severe malaria cases. In the current study, patients were treated immediately with anti-malarial drugs as soon as first microscopically detectable parasitaemia occurred.P. falciparum infection: a study of CHMI induced by bites of P. falciparum infected mosquitos found decreased blood platelet counts and elevated levels of vWF at very early stages of malaria infection [versus bites of infected mosquitoes), time point of the assessment during the course of the disease or the severity of the infection. In concordance with previous studies ADAMTS13-activity and sP-selectin remained unchanged in early malaria in this study [In the current study, also no changes in vWF, ADAMTS13-Activity, sP-selectin or platelet count during early stages of malaria infection were observed. These results are in part contrary to previous studies investigating alterations of coagulatory parameters in nfection , which wis study , 28. Howis study .P. falciparum infection remains to be studied in appropriate populations, since in the present study parasitaemia was several orders of magnitude lower than in severe or even uncomplicated malaria.In the current study, alterations of the thrombin generation potential were not observed before microscopic detection of parasites in peripheral blood. Measurement of coagulation parameters in malaria, such as thrombin generation, might therefore not be relevant prior to a microscopically detectable parasitaemia, although the routinely achieved limit of detection of microscopy is usually at least tenfold higher than in this study . WhetherP. falciparum infection and might have a role in advanced infections and the development of complications.In conclusion, the thrombin generation potential, an in vitro haemostatic test reflecting an individual´s coagulation status, was significantly elevated at early stages of malaria, reflecting a procoagulant status. The thrombin generation potential shows that the coagulation system reacts early during a


The scale-up of malaria control interventions has resulted in substantial declines in transmission in some but not all regions of sub-Saharan Africa. Understanding factors associated with persistent malaria transmission despite control efforts may guide targeted interventions to high-risk areas and populations.Household malaria surveys were conducted in Nchelenge District, Luapula Province, in northern Zambia. Structures that appeared to be households were enumerated from a high-resolution satellite image and randomly sampled for enrollment. Households were enrolled into cross-sectional (single visit) or longitudinal (visits every other month) cohorts but analyses were restricted to cross-sectional visits and the first visit to longitudinal households. During study visits, a questionnaire was administered to adults and caretakers of children and a blood sample was collected for a malaria rapid diagnostic test (RDT) from all household residents. Characteristics associated with RDT positivity were analyzed using multi-level models.A total of 2,486 individuals residing within 742 households were enrolled between April 2012 and July 2015. Over this period, 51% of participants were RDT positive. Forty-three percent of all RDT positive individuals were between the ages of 5 and 17 years although this age group comprised only 30% of study participants. In a multivariable model, the odds being RDT positive were highest in 5–17 year olds and did not vary by season. Children 5–17 years of age had 8.83 higher odds of being RDT positive compared with those >18 years of age ; there was an interaction between age and report of symptoms, with an almost 50% increased odds of report of symptoms with decreasing age category .Children and adolescents between the ages of 5 and 17 were at the highest risk of malaria infection throughout the year. School-based programs may be effective at targeting this high-risk group. During the past two decades, malaria control has been a focus of the international public health community. In 2007, malaria eradication was endorsed by the Bill & Melinda Gates Foundation and later supported by the World Health Organization (WHO) and Roll Back Malaria (RBM) Partnership as a worldwide goal ,2. ZambiMalaria transmission varies across climatic seasons, ecological zones, neighboring villages, and even between neighboring households –10. HousDespite recent reductions in malaria infection, illness, severe disease and death recorded across Zambia –17, malaUnderstanding individual, household and environmental factors associated with increased malaria risk is necessary to inform and optimize control strategies. Multi-level statistical techniques and spatial analysis are increasingly used to describe and assess risk factors for malaria ,20–22. Tst to March 1st.Nchelenge District is located in northern Zambia in the marshlands of Luapula Province along Lake Mweru, and shares an international border with the Democratic Republic of the Congo. Luapula Province has the highest prevalence of malaria in children younger than five years in Zambia in 2015, 38% by microscopy and 56% by malaria rapid diagnostic test (RDT) . NchelenThe study procedures have been described elsewhere . Briefly®). The study was approved by the Institutional Review Boards of the Tropical Diseases Research Centre in Ndola, Zambia and the Johns Hopkins Bloomberg School of Public Health.All individuals living at a selected residence were eligible to participate. Informed written consent was obtained from adults over the age of 16 years and parents or guardians of children younger than 16 years. A questionnaire was administered to collect demographic information, knowledge and beliefs regarding malaria transmission and prevention, history of recent malaria and anti-malarial treatment, care-seeking behavior, and the use of insecticide-treated nets (ITNs). A blood sample was collected by finger prick for a malaria rapid diagnostic test (RDT) . Individuals who tested positive were offered treatment with artemether-lumefantrine . A handheld Android tablet was used to record each household’s coordinates. A digital elevation model (DEM) for the area with 90 m resolution was obtained from the Shuttle Radar Topography Mission (SRTM) version 3. The DEM was processed in ERDAS Imagine 2011. The ArcHydro Tools module of ArcGIS was used to develop a stream network and classification. This module uses elevation values to determine water flow direction and accumulation to build a stream network, which is assigned to a stream order based on the Strahler classification. The Strahler classification assigns an order value of 1, 2, 3, etc. based upon the hierarchy of tributaries. Each beginning segment of a stream or river within a stream network is a first-order or category 1, and where two first-order streams come together they form a second-order stream. When two second-order or category 2 streams meet they form a third-order stream. The degree of slope was also derived from the SRTM image.All structures that appeared to be households in the study area were enumerated based on satellite imagery. The sum of these structures located within 500 meters of a study household was calculated as a measure of population density. The Euclidean distance from each enrolled household to the nearest road, nearest health facility and Lake Mweru were also calculated. A binary variable denoting households near the lake or interior was generated based on spatial location, with households less than 3 km from the lake considered near. The distance from Lake Mweru to the most distant site within the study area was 17 km.The Normalized Difference Vegetation Index (NDVI) was calculated using LandSat 5 data and is calculated as: (near infrared—visible infrared) divided by (near infrared+visible infrared) (μm). The values correlate with density of vegetation. NDVI was explored as a continuous variable but a binary NDVI variable was used for the statistical models due to lack of variation in the study area. Rainfall data from a HOBO Micro Station  was used to generate a binary variable for season. The HOBO is a four-sensor data logger designed to measure rainfall, temperature, and relative humidity at hourly intervals.RDT positive individuals were compared to RDT negative individuals for individual level characteristics. Household level characteristics were explored comparing households with no RDT positive residents to those with any RDT positive residents. Proportions were tested using the chi-square test and means were compared using Student’s t-test. The effects of individual and household level characteristics on malaria risk were analyzed using multi-level logistic regression models. All Euclidean distance variables were multiplied by -1 to show increased risk in relation to closer proximity to the ecological variable. Multi-level models with regression inference based on generalized estimating equations (GEE) were used to account for the data hierarchy and clustering of individuals nested within households. For analysis, environmental variables were considered at the same data level as households but labeled separately for purposes of interpretation. Two-way interaction terms were generated to assess seasonal variation in the effects of each environmental risk factor. Variables with p values ≤0.1 in univariate models were eligible for inclusion in the final multivariate model and only retained if at the stricter 0.05 significance level. Statistical analyses were carried out using SAS software version 9.3 .A total of 2,486 individuals residing in 742 households were visited between April 2012 and July 2015. Parasite prevalence as determined by RDT was 38% in 2012 and increased to 52% in 2015 . DespiteSeveral individual level factors were associated with RDT positivity in univariate analyses. A higher proportion of RDT positive individuals were 5 to 17 years old (43%) compared to children younger than 5 years (24%) and adults older than 18 years (28%) . RDT posSeveral household level factors were associated with having at least one RDT positive resident compared to households with no RDT positive residents. As expected, households comprised of a higher number of residents were more likely to have at least one RDT positive resident (median 4 vs. 2 household members), reflecting the higher number of individuals in the household at risk . ReporteIn a multi-variable GEE analysis, several individual and household characteristics were associated with RDT positivity. Compared to adults over the age of 18 years, children younger than five years had 3.39 higher odds  of being RDT positive, and children 5 to 17 years of age had almost nine times higher odds  of being RDT positive . As expeSeveral household level factors were associated with RDT positivity in the multivariable GEE model. The median elevation in the study area was 951 meters . As household elevation increased by 10 meters, the odds of RDT positivity decreased 15%  . HousehoDespite the rollout of malaria control interventions, Nchelenge District experiences intense perennial transmission of malaria. This study identified school-age children as a high-risk population and identified several household and environmental factors that may increase transmission risk. School-age children were the most likely to have malaria but also less likely than young children to experience symptoms, presumably due to the acquisition of clinical immunity ,26. ProxThere are several reasons malaria control efforts may be ineffectual in this setting. The recent rise in reported cases could be attributed to increasing insecticide resistance, population movement across borders from high transmission areas, shifts in biting behavior of mosquitoes to outdoors or earlier times, or increased use of RDTs for parasitological confirmation ,16,28. AInterestingly, the odds of a positive RDT were higher closer to roads. This may be because erosion along dirt roads created puddles that are ideal for breeding sites, or may be indicative of travel and increased risk of imported malaria. A study in Kenya found that water pooled along roads, creating potential breeding sites . TestingIn addition to environmental factors associated with RDT positivity, some individual and household level characteristics were also significant. Age of the participant was highly associated with RDT positivity, in particular among children aged 5 and 15 years. Children in this age category are at increased risk of malaria and are least likely to use interventions such as LLINs ,32. PrioP. falciparum. In such a high transmission setting, some people may be antigenemic after parasite clearance, leading to over-estimation of parasitemia by RDT [This analysis has some limitations, mainly that it is reliant on RDTs to detect infection with a by RDT . Accordia by RDT . Lastly,Despite seasonal rainfall, perennial transmission of malaria continues in Nchelenge District. In resource constrained settings, targeted control interventions and novel strategies are necessary to reduce transmission –41. Firs