Plasmodium parasites causing disease in humans. Although not included in the World Health Organization criteria for severe Plasmodium falciparum malaria, severe thrombocytopaenia has been increasingly mentioned as an indicator of P. vivax malaria severity.Thrombocytopaenia is the most frequent malaria-associated haematologic alteration observed with all five P. vivax malaria in a 37-year old man from Pakistan who presented with severe thrombocytopaenia (5 × 109/L). He was admitted to the intensive care unit and initially treated with a 1-day course of intravenous quinine followed by oral chloroquine and primaquine. The patient’s platelet count increased as early as 4 hours after treatment inception and the clinical course was favourable and uneventful.Here, it is described a case of imported P. vivax malaria, suggests that the prognostic role of severe thrombocytopaenia is ambiguous in absence of severe haemorraghic complications and its use as diagnostic criterion of malaria severity may lead to overestimation of severe P. vivax malaria cases.This case report, along with a review of published cases focusing on the relationship between thrombocytopaenia and severe P. vivax malaria should be considered indicative of severe malaria.Due to the lack of high quality studies it is at present unclear if severe thrombocytopaenia in the setting of  Plasmodium vivax is responsible for nearly half of all malaria cases diagnosed outside sub-Saharan Africa and it exhibits the widest geographical distribution of human malaria parasites with an estimated 2.49 billion individuals living in areas at risk of infection [Plasmodium vivax malaria has been long known as “benign tertian malaria” as opposed to the “malignant tertian or sub-tertian malaria” caused by P. falciparum [P. vivax as a cause of severe and even fatal malaria [nfection . Plasmod malaria –7. For t malaria .P. vivax malaria that are increasingly reported in the literature, partly because of the lack of a clear definition of the severity criteria, and partly from the possible interference of concomitant morbidities and infections on the clinical presentation and outcome [However, conflicting issues emerge from cases of severe  outcome .P. vivax malaria severity.Low platelet counts are commonly encountered in all types of malaria, and values lower than 60,000/μL have been reported in 29–46 % of patients affected with vivax malaria . HoweverP. vivax malaria characterized by severe thrombocytopaenia along with a review of similar cases reported in the literature. Furthermore, it is discussed whether severe thrombocytopaenia should be considered a reliable biomarker of P. vivax malaria severity.Here, it is described a case of imported 9/L; total white cell count, 6.3 × 109/L ; platelet count, 14 × 109/L. With the exception of d-dimer concentration (9376 ng/dL), the other coagulation parameters were within the normal range. Hyponatremia (127 mmol/L) was present as well as raised values of C reactive protein (126 mg/L), total bilirubin (3.3 mg/dL), and lactate dehydrogenase (371 U/L). Serum concentrations of glucose, liver enzymes, urea and creatinine were within normal ranges. An ultrasound scan of the abdomen showed no hepatosplenomegaly and a normal biliary tract. Legionella and pneumococcus urine antigens were negative. A thin blood film showed “malaria parasites” that were not identified at the species level by the laboratory technician. Due to lack of anti-malarial drugs and the expertise for the diagnosis and therapy of malaria, the ED physician contacted the Luigi Sacco hospital in Milan for assistance. Ten hours after presentation at the first ED, the patient was finally admitted to the intensive care unit of Luigi Sacco hospital with a provisional diagnosis of “severe malaria”, based on the results of the laboratory tests (severe thrombocytopaenia and raised bilirubin levels) and the presence of hypotension. On physical examination, when admitted to the ICU, the patient was conscious, fully oriented to time and place with high fever (40 °C) and had severe headache. The pulmonary, cardiovascular and abdominal examinations were reportedly unremarkable. Neither signs of bleeding nor skin petechiae were detected. No previous history of malaria episodes was elicited and no underlying chronic diseases were present. The patient was treated with a loading dose of intravenous quinine (1200 mg). At treatment inception the platelet count was 5 × 109/L, without haemorrhagic manifestations. Examination of the peripheral blood smear by the haematologist did not reveal platelet clumping and a blood sample obtained with sodium citrate confirmed the finding of low platelet counts thus excluding a diagnosis of pseudothrombocytopaenia. In the absence of bleeding the haematologist judged as unnecessary the use of platelet transfusions. Thick and thin blood smear examinations were diagnostic for P. vivax infection with schizonts, gametocytes and ring forms being detected and a very low parasitaemia (0.1 %). Plasmodium vivax monoinfection was subsequently confirmed by DNA polymerase chain reaction performed on a stored blood sample obtained on admission. Blood and urine cultures were obtained but gave negative results. Serology for HIV, CMV, dengue virus, leptospirosis, syphilis, Brucella and typhoid fever were negative.A 37-year old male from Pakistan arrived in Italy in July 2013 to attend a professional course. After few days, he developed high grade fever (40 °C) associated with chills, headache and one episode of vomiting. He was visited by a primary physician who prescribed him cefixime 400 mg/day and acetaminophen. After 4 days, due to the persistence of fever and the onset of productive cough and asthenia, cefixime was changed to levofloxacin (500 mg/day). However, the fever did not resolve, and therefore the patient was taken to the nearest emergency department (ED). At presentation, he was febrile (39.5 °C), tachycardic (121 beats per min) and hypotensive (90/50 mm Hg); there were no meningeal signs and the Glasgow Coma Score was normal (15). Oxygen saturation while breathing ambient air was 99 %, the respiratory examination and a chest radiograph were both normal. Laboratory investigations were as follows: haemoglobin, 15.9 g/dL; red blood cell count, 5.54 × 109/L, 16 × 109/L, 20 × 109/L, 32 × 109/L, respectively, after 4, 10, 18 and 24 h). The following morning an infectious diseases consultation was requested which suggested an interruption of intravenous quinine (after two doses administered). In consideration of the stable clinical condition and the diagnosis of P. vivax malaria the patient was transferred to the Infectious Diseases ward where oral chloroquine was started to complete the treatment. Parasite clearance was observed within 48 h after hospital admission as well as improvement or complete disappearance of all accompanying signs and symptoms . After completing a 3-day course of chloroquine treatment, and documentation of normal glucose 6-phosphate dehydrogenase activity levels, the patient was started on oral primaquine (15 mg of base in a 26.3 mg tablet per day for 14 days) and discharged the fifth day of hospitalization in good clinical conditions and with platelet counts within the normal range (223 × 109/L).Platelet counts showed a rapid increase soon after the infusion of quinine  is a frequent haematological finding in all types of malaria and it is observed in 29–93.3 % of patients with P. vivax malaria [Thrombocytopaenia  in patients with vivax malaria have been published in the medical literature from 1993 to 2014 (the majority after 2006) , 24–51. i.e., disseminated intravascular coagulation and gastrointestinal tract haemorrhage) [Information regarding the relationship between vivax malaria and thrombocytopaenia adopted as a criterion for assessing malaria severity may be also extracted from 17 clinical studies  , 58.TablP. vivax and P. falciparum infections were not ruled out in 14 out of 17 studies [P. vivax malaria had concomitant dengue infection and these had a higher probability to present with haemorrhagic manifestations and jaundice [It is noteworthy that mixed  studies , 63–66.  studies , 60–66. jaundice .i.e., anaemia, hypotension and elevated creatinine levels) with odds ratios (OR) of 3.47 and 5.44, respectively [P. falciparum malaria (OR 6.03 vs 3.73 for P. vivax). However, the mortality risk for patients with severe thrombocytopaenia was higher among patients without malaria (7.9 %) than among those with P. falciparum (2.1 %) or P. vivax (1.5 %) malaria [P. falciparum and P. vivax malaria [Other studies have focused on the prognostic role of thrombocytopaenia in vivax malaria. Leal-Santos and coworkers in a cross-sectional study conducted in 186 patients from the Mato Grosso region showed that mean platelet volume and platelet distribution width (PDW) were significantly associated with the presence of warning signs of severe and complicated malaria , but in only four of the seventeen patients who underwent autopsy death could be exclusively attributed to P. vivax malaria, resulting in a CFR of 0.002 % [P. vivax malaria was 16-fold lower than the one observed for P. falciparum malaria in the same period  [P. vivax malaria resulted in a CFR of 0.12 per 1000 infections [This issue has been addressed in one study only which was conducted in Brazil and reported the autopsy findings of 17 patients who died of  malaria . The aut 0.002 % . Of notefections . Of noteP. vivax malaria observed in the UK over 27 years (1987–2013), seven deaths were registered, giving an overall mortality of 0.05 %. The median age of deceased patients was 72 years, while no deaths occurred in the 9927 patients aged less than 50 years [P. vivax malaria conducted by the TropNetEurope and the GeoSentinel network, reporting respectively 618 and 278 patients (either travellers or immigrants), no deaths were recorded among patients affected by this species of Plasmodium [Moreover, in a retrospective analysis of 12.769 cases of imported asmodium , 74.P. vivax malaria seems to be different in endemic areas as opposed to what is observed in cases of imported disease in Europe or USA. In endemic areas, the risk of death is low and influenced by several factors, such as very young age, severe anaemia at presentation, malnutrition, pregnancy, coexisting comorbidities and concurrent infections [P. vivax malaria is considered, the risk of death is generally absent or negligible and principally influenced by old age. Another important issue that should be considered when comparing malaria mortality is the lack of high level supportive care in many countries where malaria is endemic a factor that explains the better outcome observed in non-endemic countries where high-quality healthcare is available [P. vivax malaria should not be overlooked.Thus, based on available data, the mortality risk of fections –71, 75. vailable . DespiteP. vivax malaria severity cannot at present neither be discarded nor confirmed and it deserves to be addressed in well conducted prospective studies in both endemic and non-endemic countries.In conclusion, given the lack of high quality studies and the fact that several confounding factors cannot be ruled out in the majority of case reports and studies published so far, the role of severe thrombocytopaenia as an indicator of


Plasmodium falciparum has lasted for more than 100 years, with considerable progress in the identification of a number of vaccine candidates. The post-genomic era offers new opportunities for an expedited search using rational vaccine design and prioritization of key B-cell epitopes involved in natural acquired immunity.The search for a vaccine against malaria caused by Malaria vaccine candidate genes that have reached clinical trial were searched on an evolutionary relationship tree, to determine their level of lineage-specificity. Ten other genes with similar protein features and level of lineage specificity to the vaccine candidates were randomly selected, and computationally evaluated for the presence of B-cell epitopes. The protein fragment with maximum probability of putative epitopes were synthesized and used in an ELISA experiment to determine the presence of antibodies to these peptides, in the serum of malaria patients and healthy malaria uninfected inhabitants from a malaria endemic region (Bolifamba), alongside with a vaccine candidate EBA-175.Two peptide fragments of 25 and 30 amino acid length from PF3D7_1233400 and PF3D7_1437500 respectively, coded as PF4-123 and PF4-143 were shown to contain B-cell epitope(s). Total IgG antibodies to these peptides were not significantly different between sick and healthy participants, but cytophilic antibodies to these peptides were significantly higher in healthy participants (p < 0.03). Total IgG to the vaccine candidate EBA-175 was significantly higher in sick participants than in healthy participants, likewise cytophilic antibodies (p < 0.04). Antibodies to the peptides PF4-123 and PF4-143 correlated negatively  to parasite load. Total IgG antibodies to EBA-175 showed a negative correlation to parasite load (r = −0.144), which was not significant (p = 0.276). Duration of stay in Bolifamba also negatively correlated with parasite load  and total IgG to PF4-143 was significantly associated with prolonged duration of stay in the locality of Bolifamba, Cameroon .The present study has identified two genes PF3D7_1233400 and PF3D7_1437500 containing peptide fragment (PF4-123 and PF4-143) with B-cell epitopes that are correlated with naturally acquired immunity to malaria. A pipeline has been developed for rapid identification of other B-cell epitopes involved in naturally acquired immunity. Plasmodium adaptation to its host [The global burden of malaria is still alarming despite wide spread use of anti-malarial drugs, treated bed nets and indoor residual spraying. In 2014, 3.3 billion people were still at risk of malaria infection and 584,000 people died of malaria in 2013 [Plasmodiumfalciparum. Naturally acquired immunity to malaria is known to increase with age and duration of stay in an endemic region [P. falciparum extract were significantly higher in adults than in children among inhabitants of Bolifamba, Cameroon [Current malaria vaccine candidates in clinical trials face the serious problem of being polymorphic . To addrc region . Childrec region  and, thec region . Thus, ic region  by opsonc region . Thus, aCameroon . In thatP. falciparum infection, repeated cycles of parasite invasion of red blood cells (RBCs) quickly amplify blood-stage parasite load and aggravate malaria symptoms. EBA-175 is an antigen involved in merozoite invasion of RBCs which is known to bind to glycophorin A on human erythrocytes during the invasion process [During  process . EBA-175 process , 13. How process . In Came process . HoweverBlood samples used in this study were collected from Bolifamba, a malaria endemic locality on the eastern slope of Mount Cameroon, as reported in previous studies , 17. TheThe study was approved by the University of Buea Faculty of Health Sciences-Institutional Review Board, reference number 2013/144/UB/FHS/IRB. Administrative clearance was obtained from Ministry of Public Health Regional Delegation for the South West, reference number R11/MPH/SWR/RDPH/PS/108/263. All the participants who took part in the study were informed about the purpose of the study and signed the informed consent form. Consent of minors was obtained from their parents or guardians.Two ml of venous blood was collected from each participant and serum extracted from the blood was stored at −20 °C in 50 % glycerol, until used. Thick blood film smears were made on slides, air-dried and stained with 5 % Giemsa for quantification of parasite load by microscopy with the ×100 (oil immersion) objective.−30) [The study design shown in Fig. −30)  for an a−30)  with def−30) .Fig. 1FlThe set of 10 genes was then assessed for B-cell epitopes. The Bepipred Linear Epitope Prediction method in Immune Epitope Data Base (IEDB) makes use of hidden Markov model and a propensity scale was used to predict B-cell epitopes on the selected protein sequences . Only th2O2 for 30 min in the dark and the optical density was read at 450 nm with an Emax Precision Microplate ELISA reader . Five European naïve sera were included in the experiments as negative controls.Checkerboard titration was used to determine the working antigen concentration and serum dilution for each antigen prior to screening of the entire serum sample collection. The vaccine candidate EBA-175 was obtained through MR4/BEI Resources, NIAID, NIH: EBA-175 RII-NG, MRA-1162, and used at a concentration of 4 μg/mL in carbonate buffer, pH 9.6 to coat 96-well, flat bottom, high-binding polystyrene ELISA plates  with 100 μL/well, overnight (18 h) at 4 °C. Blocking was done with 0.2 % casein in PBS-Tween20 (0.05 %) at 200 μL/well for 1.5 h at room temperature. Washing was performed three times between each step using PBS-T, followed by incubation with serum in duplicate at 1:100 dilution in 1 % non-fat skimmed milk-PBS-T at 37 °C. Total IgG antibodies to EBA175 was detected by incubation with 1:10,000 dilution of goat-anti-human IgG (Fc specific)-peroxidase  in 1 % non-fat skimmed milk-PBS-T for 1.5 h at room temperature. Plates were developed in the dark with 1 mg/mL o-phenylene diamine  in 70 mM citrate-phosphate buffer, pH 4.2 containing HCytophilic antibodies (IgG1 and IgG3) are important mediators of malaria parasite clearance in humans . IgG1 anThe peptide ELISA was carried out using the same procedure as above, with some modifications. Coating was done with 4 μg/mL of peptide in carbonate buffer, at 37 °C overnight, while the ELISA plates were being rotated at 2.8 rpm using an adapted device placed inside the incubator . Blocking was performed for 1 h using 150 μL of 0.2 % casein in PBS-T. Serum was diluted at 1:50 in 1 % nonfat skimmed milk-PBS-T and incubated for 2 h 20 min at 37 °C. The procedure for measurement of IgG1 and IgG3 antibody subclasses to the peptides were as in the measurement of EBA-175 IgG1 and IgG3 antibody subclasses with the same modifications as in measurement of total antibodies to the peptides, and the serum was used at 1:30 dilution. All peptides that did not show an antigen concentration dependent and serum dilution dependent optical density during the checkerboard titration were eliminated from the ELISA experiments.The data were analysed using IBM SPSS statistics version 21.0. The Mann–Whitney U non-parametric test was used to compare differences in antibody responses between study groups and the Spearman’s rank correlation test was used to test for significance of correlation between log of parasite load and antibody responses or duration of stay in Bolifamba. The data was graphically presented using SPSS and Microsoft Excel.Plasmodium (45.5 %) or proteins that are unique to P. falciparum (45.5 %). Ten additional P. falciparum genes with unknown functions, similar evolutionary profiles and evidence of a putative surface localization,  were identified. Of these, two genes were selected from the set of genes unique to P. falciparum, four from the set of genes unique to Plasmodium genus and four from the set of genes common to Plasmodium, Piroplasms, Toxoplasma and Neospora  antibody responses for each of the three antigens  , and was higher among sick children compared to healthy children (p = 0.04), but there was no significant difference between sick adults and healthy adults (p = 0.3). Total IgG to PF4-123 showed no significant difference between healthy and sick participants (p = 0.99). However, healthy adults had a significantly higher antibody response than sick adults (p = 0.04), contrary to what was obtained in the children’s subgroup, where sick children had a significantly higher antibody response compared to healthy children (p = 0.03). Total IgG response to PF4-143 was not significantly associated with health status (p > 0.1) whether in adults or children.IgG1 subclass response to EBA-175 and PF4-123 was significantly higher in adults than in children (p < 0.01), likewise, they were significantly higher in healthy adults than in healthy children and in sick adults than in sick children (p < 0.05) , likewise in healthy adults than in sick adults (p < 0.02) and in healthy children than in sick children (p < 0.02). Contrary to IgG1 response to EBA-175, which was significantly higher in sick participants than in healthy participants (p = 0.03), there was no significant difference between sick and healthy adults and between sick and healthy children.IgG3 subclass response to all three antigens  was significantly higher in adults than in children (p < 0.02), and in healthy adults than in healthy children (p < 0.021)  and in sick children compared to healthy children (p < 0.01), but there was no significant difference between sick adults and healthy adults. Contrary to EBA-175, PF4-143 was significantly higher in healthy participants, compared to sick participants (p = 0.013) and in healthy adults compared to sick adults (p = 0.023), however there was no significant difference between healthy and sick children. PF4-123 was not significantly different between healthy and sick participants, or between healthy children and sick children but it was significantly higher among healthy adults compared to sick adults (p < 0.01).Total IgG antibody response to all antigens  had a negative correlation to increasing parasite load Fig. . NotablyGenerally, healthy adult participants in this study had lived for a significantly longer period of time in Bolifamba than sick adult participants (p < 0.001). Among the sick adults, duration of stay in Bolifamba negatively correlated with parasite load  Fig. . IncreasPlasmodium led to reduced symptoms and decreased parasite density [P. falciparum were also protective in P. vivax [Bioinformatics analysis of current vaccine candidates revealed that they are strongly species-specific. This is in agreement with results from the old practice of using induced malaria as a therapy for neurosyphilis, in which second and third inoculations with homologous or heterologous strains of the same species of  density . The genP. vivax . This isP. vivax . The othIn this study, there was no significant difference in the total IgG response to EBA-175, between sick adults and healthy adults. This is in agreement with previous work in Cameroon . HoweverAnopheles funestus, which caused a series of malaria epidemics . This work has also developed a pipeline to identify additional potentially protective B-cell epitopes against malaria. The approach can be applied to other infectious diseases.The demonstration of possible malaria protective properties by antibodies to PF4-123 and PF4-143 in this study opens the gateway for assessing the feasibility of monoclonal antibody therapy to malaria. Likewise a cocktail of peptides which elicit protective antibodies to malaria might be used to immunize people and re-establish naturally acquired immunity to malaria, which is typically lost with progressive elimination of malaria in various localities . This typidemics . This woThis work has identified two genes PF3D7_1233400 and PF3D7_1437500, which encode peptide fragments containing B-cell epitopes, PF4-123 and PF4-143, respectively, that are involved in naturally acquired immunity to malaria.


Background. Asymptomatic parasitemia is common even in areas of low seasonal malaria transmission, but the true proportion of the population infected has not been estimated previously because of the limited sensitivity of available detection methods.Methods. Cross-sectional malaria surveys were conducted in areas of low seasonal transmission along the border between eastern Myanmar and northwestern Thailand and in western Cambodia. DNA was quantitated by an ultrasensitive polymerase chain reaction (uPCR) assay  to characterize parasite density distributions for Plasmodium falciparum and Plasmodium vivax, and the proportions of undetected infections were imputed.Results. The prevalence of asymptomatic malaria as determined by uPCR was 27.5% (1303 of 4740 people tested). Both P. vivax and P. falciparum density distributions were unimodal and log normal, with modal values well within the quantifiable range. The estimated proportions of all parasitemic individuals identified by uPCR were >70% among individuals infected with P. falciparum and >85% among those infected with P. vivax. Overall, 83% of infections were predicted to be P. vivax infections, 13% were predicted to be P. falciparum infections, and 4% were predicted to be mixed infections. Geometric mean parasite densities were similar; 5601 P. vivax parasites/mL and 5158 P. falciparum parasites/mL.Conclusions. This uPCR method identified most infected individuals in malaria-endemic areas. Malaria parasitemia persists in humans at levels that optimize the probability of generating transmissible gametocyte densities without causing illness. Plasmodium falciparum and Plasmodium vivax infections in populations living in malaria-endemic regions of western Cambodia and the Thailand-Myanmar border  is the total area under the density function between 0 and 48 hours , and that count was multiplied by the adjustment factor corresponding to the value of µ , with the truncations corresponding approximately to the lower and upper limits of accurate quantitation; (2) estimation of the mean m and standard deviation s of the untruncated distributions of log10-transformed parasitemia values, using the maximum likelihood method; and (3) estimation of the tail of the distribution corresponding to the lower densities' truncated segment from the log-normal distribution with parameters .For each data set, the following were performed: (1) analysis with the Kolmogorov–Smirnov test to determine whether the log-transformed data conformed to a truncated normal distribution with parameters , and the Oxford Tropical Research Ethics Committee .Of the 7 villages selected for more-detailed studies, 3  are in Cambodia, with 2151 inhabitants surveyed between June 2013 and June 2014. The 4 remaining villages  are along the Thailand-Myanmar border, with 2589 inhabitants surveyed between May 2013 and June 2015. Of the population included in the census, 83% participated in the surveys in the Cambodian villages and 67% participated in villages along the Thailand-Myanmar border. The median age of the participants was 21 years; 37% were <15 years old, and 51% were male. The age distributions were similar in the different villages .P. falciparum only, 1553 (63.9%) were P. vivax only, 52 (2.1%) were mixed, and 596 (24.5%) had indeterminate parasite species .In total, 4740 participants were tested on 16 662 occasions, with a median of 5 tests/participant . Malaria parasitemia was detected by uPCR testing on 2432 occasions in 1303 participants: 231 infections (9.5%) were P. falciparum, 48 P. vivax, 3 mixed, and 79 indeterminate infections). The parasite density distributions were log normal and unimodal . After these exclusions, the uPCR-measured parasitemia levels had geometric means of 9801 parasites/mL  for P. falciparum, 11 919 parasites/mL  for P. vivax, and 476 parasites/mL  for the indeterminate species infections.Distributions fitted to the quantitated parasite densities resulted in exclusion of 122 infections with a density of <30 parasites/mL and 31 with a density of >30 000 000 parasites/mL  within the detection limits. Among P. falciparum infections, 15.3% had parasite densities of 30–999 parasites/mL, and 11.2% had densities of ≥1000 parasites/mL (P = .031). To examine how this affected the estimates of undetected parasitemias, PFP was estimated by method 1, where PFP = 0.1214 for all densities, and by method 2, where PFP = 0.1534 for densities between 30 and 1000 parasites/mL and PFP = 0.1124 for densities between 1000 and 30 000 000 parasites/mL.Overall, PFP = 0.1214, for the 1000 simulated corrected data sets the mean estimated log density parameters were m = 2.86 , with standard deviation s = 2.16  for P. falciparum and m = 3.34  and s = 1.60  with an asynchronous parasite age distribution for P. vivax (scenario A). These estimates were only slightly different for synchronous P. vivax parasite age distributions (scenario B): m = 3.25  and s = 1.64 , respectively , the estimates changed slightly for P. falciparum but not for P. vivax; over the 1000 corrected data sets, for scenario A m = 2.64  and s = 2.56  for P. falciparum and m = 3.35  and s = 1.59  for P. vivax, and for scenario B m = 3.26  and s = 1.63 . For both methods, the distributions of the log10-transformed parasite densities in all the corrected data sets could be assumed to be normally distributed .With the uniform P. falciparum and 4529 parasites/mL  and 4598 parasites/mL  respectively for P. vivax with scenario A and 4454 parasites/mL  and 4513 parasites/mL  respectively for P. vivax with scenario B  of those with P. falciparum. The proportions truncated at the upper ends of the distributions were 0.5%  for P. vivax for both scenarios A and B and 1.6%  for P. falciparum. Analysis with different PFP ratios above and below parasite densities of 1000 parasites/mL had little effect on the estimates, with an estimated 11.5%  or 13.3%  of P. vivax infections, respectively, being undetected and 29.8%  of P. falciparum infections being undetected. This results in an overall true distribution of asymptomatic infections in these populations as follows: P. falciparum, 13%; P. vivax, 83%; and mixed, 4%.After correction for the indeterminate parasitemias, the imputed corrected median (IQR) geometric mean parasite densities for detected infections were 5158 parasites/mL  and 4627 parasites/mL  for P = .001). The risk of fever increased with parasitemia density .Fever  was detected on 73 occasions in 68 participants who did not complain of symptoms. Participants in Cambodia had a higher risk of fever than those on the Thailand-Myanmar border  were consistent with the proportions in the range where species identification was possible, then the geometric mean densities for all detected asymptomatic P. falciparum and P. vivax infections were similar, at approximately 5 parasites/µL .In malaria-endemic areas of western Cambodia and the Thailand-Myanmar border, >85% of asymptomatic P. vivax malaria, as parasite genomes in merozoites in developing schizonts and those free in plasma (<0.01% of concomitant intraerythrocytic numbers) and phagocytosed by leukocytes may be quantitated [P. falciparum infections, a significant proportion of the parasites detected by uPCR in asymptomatic individuals are likely to be gametocytes. In this study, the potential errors related to schizontemia and synchronicity of infection in P. vivax infection were examined and found to contribute little to the estimates of geometric mean parasite density or undetected proportions, and measurement errors from free DNA or merozoites were minimized by red cell separation. If the distribution of parasite densities in asymptomatic individuals living in low-transmission settings is visualized as an iceberg, only the top of which is revealed by conventional microscopy or rapid diagnostic tests (RDTs), then high-volume uPCR reveals most of the iceberg and thereby allows prediction of the proportion of individuals who have parasite densities below the level of detection.The uPCR DNA–based measurement can overestimate parasite densities slightly, particularly in ntitated . DNA assntitated , 22. As These data also suggest that the majority of infected individuals should be detected by widely used methods involving PCR analysis of capillary blood specimens on filter paper, which typically contain only 5–10 µL of blood –25. HoweP. vivax malaria, persistence is enhanced by relapse, which occurs in the majority of infections in Southeast Asia [Asymptomatic malaria infections in low-transmission settings may be harbored in an individual for long periods in a quasi-steady state –33. If tast Asia –37. Someast Asia , 38, 39.P. falciparum infections, compared with P. vivax infections, in this study may reflect the currently lower transmission of this species in the study areas and, thus, the greater probability that an individual was being sampled while their infection was being eliminated, whereas P. vivax infections were acquired both from inoculation and more-frequent relapse [P. falciparum and 8 genomes/µL for P. vivax in identified infections [P. falciparum, approximately 650 genomes/µL; for P. vivax, approximately 140 genomes/µL). Overall, it is likely that more of the “iceberg” of the parasite density distribution in asymptomatic people will be detectable in the microscopy or RDT detectable ranges in settings of high transmission, compared with settings of low transmission.Are these parasite density distributions from areas of low, unstable malaria transmission in Southeast Asia relevant to higher-transmission settings, where a much larger proportion of asymptomatic individuals have parasitemias detectable by microscopy? Immunity to blood stage parasites is greater in higher-transmission settings, and splenic function is augmented and, thus, clearance of parasitized erythrocytes is likely to be more rapid, so the net result of higher inoculation rates will be a truncated lower end of the parasite density range, with proportionally fewer densities below the limit of uPCR detection. This is because, with a median sporozoite inoculum of approximately 10 and liberation of approximately 35 000 merozoites from each hepatic schizont, the median blood parasite density following hepatic schizogony of each inoculated brood in an adult  approximates 70 parasites/mL (which is above the current level of uPCR detection). The greater proportion of very low-density  relapse . In suchfections . HoweverThese data suggest that uPCR assessment of parasite densities in population cross-sectional surveys provides accurate characterization of the true prevalence of malaria in the community in all transmission settings. If the density distributions reported here are general properties of malaria, then the regional and total global burden of malaria parasites can be calculated, and these data used to estimate accurate selection pressures provided by drugs and other interventions.


HbS) is the gene known to confer the strongest advantage against malaria morbidity and mortality. Multiple HbS effects have been described resulting in protection against parasitaemia and reduction of severe malaria risk. This study aimed to explore HbS protection against severe malaria and Plasmodium falciparum parasitaemia in Angolan children exhibiting different severe malaria syndromes.Haemoglobin S  and 319 uninfected controls, attending a central paediatric hospital in Luanda. Severe malaria syndromes were cerebral malaria (n = 130), severe malaria anaemia (n = 30) and hyperparasitaemia (n = 128). Quantitative trait locus analysis was carried out to study HbS association to parasite densities.A case–control study was designed with 430 malaria cases  and corroborated by transmission disequilibrium test (P = 4 × 10−3). High parasite density protection conferred by HbS was detectable within severe malaria patients (P = 0.04). Stratifying severe malaria patients according parasite densities, it was found that HbS was highly associated to hyperparasitaemia protection (P = 1.9 × 10−9) but did not protect non-hyperparasitaemic children against severe malaria complications, namely cerebral malaria and severe malaria anaemia. Many studies have shown that HbS protects from severe malaria and controls parasite densities but the analysis further suggests that HbS protection against severe malaria syndromes was at a large extent correlated with control of parasitaemia levels.Previously reported HbS confers resistance to hyperparasitaemia in patients exhibiting severe malaria syndromes and highlights that parasitaemia should be taken into account when evaluating HbS protection in severe malaria.This study supports the hypothesis that  HbS) is very well recognized as the human single gene conferring the strongest advantage against falciparum malaria .Kimbundu, the predominant ethnic group in Luanda was the most represented in the sample (50.7 %) followed by the Umbundu group 13.7 %). This ethnic representation did not differ in UM, SEV and UIF patient groups  was significantly under-represented in SEV patients (3.7 %) as compared to UM patients (10.9 %) and to UIF controls (15.1 %)  to children with SEV (P = 0.002)  Table . These a%) Table . The tra P = 0.00  were lower in presence of HbS  and non-hyperparasitaemic , irrespective of other observed clinical complications.To determine whether the protection against SEV afforded by HbS allelic frequency was higher in non-hyperparasitaemic children suggesting that HbS protects SEV patients from developing hyperparasitaemia  exhibited concomitantly different malaria clinical complications. These overlapping malaria syndromes corroborate the notion that severe malaria cases often represent clinical syndromes rather than clinical isolated entities [HbS protection against one clinical condition faces possible confounding by co-occurrence of other infection parameters. Strikingly, by contrasting allelic and genotypic HbS frequencies in severe malaria patients stratified either for parasitaemia values or by other causes of clinical severe malaria , it was found that protection against severe malaria was at a large extent attributable to resistance to hyperparasitaemia. Although with limitations due to the reduced number of HbS homozygotes the analysis supports the notion that control of parasite density was quantitatively dependent on the number of T alleles.The mechanism of oversial –27. Studentities . In thisHbS showed to control quantitatively parasitaemia in clinical malaria both in presence or absence of uncomplicated malaria cases. This result suggests that HbS contributes to resistance to expansion of blood stage parasite irrespective of malaria clinical presentation. Indeed, these results sustain the debate around the effect of HbS protection against hyperparasitaemia and its mechanisms. An experimental cerebral malaria (ECM) study in mice expressing sickle haemoglobin concluded that the protective effect exerted against lethal ECM was irrespective of parasite load [APOE ε4 children for Plasmodium spp. densities compared to non-APOE ε4 children and concluded a suggestive epistatic interaction between APOE and HbS genes such that sickle cell trait only had an effect on parasite density in APOE ε4 children [Furthermore, ite load . Meanwhichildren .HbS protection effects here identified.The predominant occurrence of severe malaria in children under 5 years of age in the study sample is in line with what is reported in other areas of stable malaria , 30. It HbS allele frequency in the uninfected control group was 15.1 %, similar to that of the HapMap Yoruba (Nigeria) sample where the frequency is 12.5 %, though HbS frequency may exceed 20 % in other African populations [HbS allele against severe malaria syndromes (OR = 0.15) as compared to uncomplicated malaria (OR = 0.50). Studies in Kenya reported similar genetic effects on protection against uncomplicated malaria (OR = 0.50) and against severe malaria (OR = 0.17) [HbS allele was evaluated in case–control supplemented with TDT [The ulations . Case–co = 0.17) . This st = 0.17) . Another = 0.17) . It is wwith TDT  and was HbS protection in severe malaria. Nevertheless, the mechanism of HbS protection against hyperparasitaemia was not addressed in this study leaving open the possibility that HbS may partially accelerate the process of immunity acquisition against P. falciparum [The results of the present study reinforce the notion that parasitaemia levels are to be taken into account on evaluation of lciparum .HbS does not significantly reduce the risk of developing clinical forms of severe malaria syndrome not entailing hyperparasitaemia and that the risk of co-occurrence of cerebral malaria or severe anaemia and hyperparasitaemia is higher in non-carriers. Thus, this study supports the hypothesis that HbS confers resistance to hyperparasitaemia in patients exhibiting severe malaria syndromes.Although these findings should be evaluated with caution due to sample size, they suggest that


G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these  in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of  G6PD, residing in the X chromosome, are found among African populations: mutations causing A- deficiency can lead to serious clinical outcomes (including hemolytic anemia) but also confer protection against severe malaria. Epidemiological studies have used some of the genetic markers that cause A- deficiency to establish who is protected from severe malaria, with differing results. Whether females, with one or two copies of mutant genes, males with one copy, or both genders are protected is uncertain. This uncertainty is due to G6PD and malaria phenotypic complexity and misclassification, and to genetic differences between populations and the limited numbers of genetic markers  considered. In this study we analysed more than 30 G6PD genetic markers in 506 Tanzanian children with severe malaria and 477 without malaria. We found that only females with one normal and one mutant copy of the gene (heterozygotes) were protected from severe malaria. Further, we established that the G6PD gene is under evolutionary pressure with the likely mechanism being selection by malaria. Our work demonstrates that studies of severe malaria and G6PD enzymatic function across African populations require, in addition to complete and accurate G6PD phenotypic classification, the identification and analysis of the full repertoire of G6PD genetic markers.Glucose-6-phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Numerous genetic variants of  P. falciparum gametocytes, thereby blocking transmission to mosquitoes [G6PD locus , with more than 150 single nucleotide polymorphisms (SNPs) reported [G6PD gene locus in enzyme deficient individuals. The G6PD and the Inhibitor of kappa light polypeptide gene  that have been characterised ,3. An unsquitoes . Howeversquitoes . Cytochesquitoes , but arepathways ) loci ovpathways . The regelements . The gen complex , and new complex ,9.G6PD polymorphisms [Despite these limitations, genotyping of the 202A/376G G6PD A-allele  has beeactivity), 16 was G6PD locus, how this relates to enzyme function, and how it varies between regions and ethnic groups, if genetic epidemiological studies are to provide robust and reproducible findings. A recent study in Mali using 58 SNPs across the G6PD gene found differences in core haplotypes and their frequencies between Dogon and Fulani ethnic groups [Further understanding is required of the true extent of genetic diversity within the c groups . The latc groups . Whilst G6PD and surrounding loci (IKBKG and CTAG1A/B), including the 202, 376, 542, 680 and 968 A- deficiency polymorphisms , and severe malaria. The work is set within a case-control study  conducted in an area of intense malaria transmission in the Tanga region in northeastern Tanzania [G6PD gene and protection from severe malaria in female heterozygotes but not in hemizygous males. Very high linkage disequilibrium across this locus allowed us to distil this SNP diversity into just 4 G6PD alleles, ranging in frequency from ∼6% to >60%, and 8 common genotypes (>1%), 2 of which are associated with protection from severe malaria.Here we investigate associations between 68 SNPs within the Tanzania . To compG6PD alleles that confer resistance to severe malaria in this population and reveals a potentially important role of female heterozygotes in maintaining the high frequency of G6PD polymorphisms in malaria endemic populations.In summary, this study identifies specific Of the severe malaria cases (n = 506), many had severe malarial anaemia 48.6%) or acidosis (57.5%) phenotypes . Compare.6% or acThe G6PD202A and G6PD376G A- alleles were among the 29 SNPs retained with minor allele frequency (MAF) in excess of 1% . Both G6G6PD alleles confirmed that a combination of haplotypes 1 and either 2 or 3 were protective  compared to a double haplotype 1 (wild-type) genotype (The correlation between the 29 SNPs was high (linkage disequilibrium D’ median (IQR): all subjects 0.987 (0.811–0.997); female controls 0.988 (0.731–0.998)). Similarly, LD was high across this region in the trio parents ; female only: 0.998 (0.995–0.999)) and children (0.998 (0.996–0.999)) . This higenotype . This reTajima’s D metric was applied to assess if the excess number of heterozygous alleles led to evidence of balancing selection in the G6PD gene. There was very strong evidence of balancing selection across all groups . The magnitude of effect is at the extreme positive tail of an observed negatively centred African population distribution [G6PD on severe malaria in females (P = 0.34), nor males (P = 0.98). Similarly, no evidence of epistasis between alpha thalassaemia and G6PD .It is possible that the greater protective effects of haplotypes 2 and 3, could be due to the presence of more mutations (≥6), leading to a possible compound heterozygous advantage effect. The number of heterozygous genotype calls in female controls was greater than in cases . The ribution , where pribution . This reribution , and conribution ,18. Suchribution . There wP. falciparum development is hindered in G6PD deficient red cells [P. falciparum 6-phosphogluconolactonase mRNA in parasites from G6PD-deficient children [G6PD (and surrounding loci) SNPs (n = 68) in a genetic association study, within a Tanzanian case-control setting, we have established a set of new G6PD alleles associated with protection. These SNPs need to be further investigated to assess their effect on enzyme function in light of potential use of primaquine for malaria elimination. After validation, these SNPs may be used to identify G6PD-deficient individuals in studies of primaquine efficacy.Although G6PD A- deficiency is known to protect against severe malaria in African populations, the underlying genetic mechanisms are not well understood. ed cells , slowinged cells , lower achildren , and impchildren . By usinFREM3, the major histocompatibility complex, and the sickle cell trait loci [Further, we have shown that the protective effect of G6PD deficiency is limited to female heterozygotes. This is entirely consistent with heterozygote advantage and balancing selection, relying on alleles at modest frequency and avoiding fixation, where protection provided by this G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications, such as neonatal jaundice and haemolytic crises. In female heterozygotes, random inactivation of one of the two X chromosomes results in some cells with normal enzyme and others with mutant enzyme , 28, 29,ait loci .G6PD locus with the presence of multiple functional SNPs, each of which may separately modify an individual’s enzyme status and susceptibility to malaria. Until very recently, almost all-large association studies genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376 for A- deficiency), and this approach has been too blunt to capture the full picture. However, analysis of 58 G6PD SNPs has demonstrated major G6PD haplotypic differences between sympatric ethnic groups in Mali [G6PD and surrounding regions, with multiple populations and ethnic groups, leading to a more complete map of G6PD that would guide future evolutionary and association studies.Hitherto, there has been much uncertainty about the relationship between G6PD status and susceptibility to malaria, due in part to G6PD and malaria phenotypic complexity and misclassification, and potentially also from the genetic complexity of the  in Mali  and geno in Mali  would haG6PD requires further correlation of enzymatic activity with full sequences of G6PD and surrounding loci, set within large severe-malaria case and control studies. There have been no such studies to date. A recent study of four G6PD deficiency polymorphisms  and associated enzymatic activities for 110 sequenced genes in African Americans [A surprising association result is that the G6PD376 mutation is potentially more influential than G6PD202 and haplotypes that contain the 376G with or without the 202A mutation appear to be similar in terms of protective effect on heterozygotes. The 202A mutation is thought to have a more severe effect on enzyme function than the 376G mutation  andmericans  but inclmericans  in 1,828G6PD locus, we have identified alleles associated with protection from severe malaria in Tanzania, driven by a balancing heterozygous advantage mechanism. Further work should extend the mapping of diversity at this genomic region, and identify how the resulting mutations relate to enzyme function, and how they vary between region and ethnic group. In doing so, genetic epidemiological studies are likely to provide robust and repeatable data, which may be used to develop interventions, and improve malaria disease control.In summary, through a much better understanding of the true extent of genetic diversity within and around the P. falciparum malaria is intense (50–700 infected bites/person/year) and perennial, with two seasonal peaks [P. falciparum parasites in children aged 2–5 years in the study area was recorded as 88.2% in 2002 [The study was conducted in the Teule district hospital and surrounding villages in Muheza district, Tanga region. In this region, mortality in children under 5 years of age is 165 per 1000 (Tanzanian census 2002) and transmission of al peaks . The com in 2002 .P. falciparum malaria parasites. Parasite infection was initially assessed by rapid diagnostic test (HRP-2—Parascreen Pan/Pf) and confirmed by double read Geimsa-stained thick blood films. Residence and ethnic group of both parents was recorded from information provided by the caregiver for each child [Severe malaria cases (n = 506), aged six months to ten years, were recruited during a one-year period between June 2006 and May 2007, with patent parasitaemia, and fulfilling any one of the following eligibility criteria; history of 2 or more convulsions in last 24 hours, prostration (unable to sit unsupported if <9 months of age or drink at any age), reduced consciousness , respiratory distress, jaundice, severe anaemia (Hemocue Hb < 5g/dL), acidosis (Blood lactate ≥ 5 mmol/L), hypoglycaemia (blood glucose < 2.5mmol/L). Cases were defined as having had cerebral malaria if their Blantyre coma score was less than or equal to 3 on presentation or early during admission. Participants with co-existing severe or chronic medical conditions  unrelated to a severe malarial infection were excluded. All cases were confirmed as having ch child .Controls (n = 477) were recruited matched on ward of residence, ethnicity and age using household lists during a four-week period in August 2008. Study participants resided in 33 geographical wards  surrounding Muheza town in the Tanga region. The participants had a median age of ∼2.6 years, and were predominantly from seven ethnic groups see . BecauseTo complement the case-control collection, we collected samples anonymously from 60 healthy parental and child trios  during 2007 and 2008 from lowland villages near the West Usambara mountains in the Tanga region of Tanzania, which ranges from high to medium levels of malaria transmission. No malaria phenotypic data is available on these individuals, but their genotypic profiles were used to provide validation data of the genetic aspects of the case-control study.Approximately 3ml of venous blood was collected from participants into EDTA vacutainers. A blood film was prepared and haemoglobin levels measured by hemocue. Children in the control group with haemoglobin levels of <11g/DL were referred to the nearest health facility; those with a positive blood film were treated in line with Tanzanian national treatment guidelines and excluded from the genetic analysis. Samples were spun at 5000rpm for 5 minutes and the plasma removed and stored for future analysis. DNA was extracted and purified from the blood cell pellet using a nucleon kit , and both promoter and enhancer histone marks, with a number of different binding proteins and regulatory motif changes (compbio.mit.edu/HaploReg)).All analyses involving SNPs were stratified by gender. Genotypic deviations from Hardy-Weinberg equilibrium (HWE) in females were assessed using a Chi-square statistical test. SNPs were excluded from analysis if they had at least 10% of genotype calls missing, more than 2% of males genotype calls were  called heterozygous, or if there was a distortion from HWE in female controls (HWE Chi-square P<0.00001) . On thisD-prime (and R-square) metrics [haplo.stat library was used to implement haplotype analysis. The Tajima’s D metric was used to quantify evidence of balancing selection based on the allele frequency spectrum [Tajima's D indicates purifying selection and/or population size expansion, while positive values may indicate balancing selection. Values greater than +2 or less than -2 are likely to be significant [Case-control association analysis using SNP alleles or genotypes was undertaken within a logistic regression framework, and included age and ethnic group as covariates. In this approach we modelled the SNP of interest assuming several related genotypic mechanisms  and reported the minimum p-value from these correlated tests. Epistatic effects between polymorphisms were considered by inclusion of statistical interactions in these models. The haplotypes of females were inferred from genotypes using an expectation-maximization algorithm . Haploty metrics . Performspectrum . A negatnificant .All DNA samples were collected and genotyped following signed and informed written consent from a parent or guardian. Ethics approval for all procedures was obtained from both LSHTM (#2087) and the Tanzanian National Institute of Medical Research (NIMR/HQ/R.8a/Vol.IX/392).S1 Table(DOCX)Click here for additional data file.S2 Table* with minor allele frequencies (MAF) in excess of 1%** b36_153411172, b36_153412566, b36_153412620, b36_153412734, b36_153412861, b36_153413455, rs72554665, b36_153413799, b36_153414077, b36_153414378, G6PD968, b36_153414531, b36_153414709, b36_153414937, b36_153415014, G6PD680, rs5986875, b36_153415799, rs5030868, rs5030872, b36_153415904, b36_153416019, b36_153416656, b36_153416679, b36_153417405, b36_153417417, b36_153424232, b36_153426313, b36_153427408, b36_153427466, rs5986992, b36_153429686, and rs5986997 had MAF<1% and were excluded from association analysis.(DOCX)Click here for additional data file.S3 Tablers33950507 (HbC)- G, b36_153412566-C, b36_153412620-C, b36_153412734-G, b36_153412861-G, b36_153413455-A, b36_153413678-C, b36_153413799-G, b36_153414378-G, G6PD968-T, b36_153414531-C, b36_153414709-C, rs598699-G, b36_153414937-T, G6PD680-G, 36_153415799-G, b36_153415828-G, G6PD542-A, b36_153415904-C, b36_153416019-C, b36_153416656-G, b36_153416679-A, b36_153417405-A, b36_153417417-A, b36_153424232-T, b36_153426313-G, b36_153427466-T, rs5986992-C, b36_153429686-G, rs5986997-C are all fixed; b36_153411172-G, b36_153415014-A, rs5986875-A, and b36_153426354-C all had allele frequencies less than 1%.(DOCX)Click here for additional data file.S4 Table* rs5986990, rs2515905, rs2515904, G6PD376, G6PD202, rs762515, rs762516; LCL lower confidence interval, UCL upper confidence interval(DOCX)Click here for additional data file.S1 FigThe horizontal dashed lines represent a p-value cut-off of 0.006. Some SNP results are not presented because of statistical model non-convergence due to low numbers of cases and low minor allele frequency.(DOCX)Click here for additional data file.S2 FigD’, Bottom right R-square; black = 0 -> white = 1)(Top left (a) All cases and controls(b) Female controls(c) All parents in the Trio study(d) Female parents in the Trio study(DOCX)Click here for additional data file.


Anopheles mosquitoes was initially considered. However, this diagnostic hypothesis was ruled out due to negative entomological survey and the absence of additional cases.Four cases of airport malaria were notified for the first time in Tunisia during the summer of 2013. All patients were neighbours living within 2 km of Tunis International Airport. They had no history of travel to malarious countries, of blood transfusion or of intravenous drug use. Although malaria transmission had ceased in Tunisia since 1980, autochthonous infection by local Plasmodium falciparum. Clinical presentation was severe (important thrombocytopaenia and parasitaemia), because of relatively important delay in diagnosis (average of seven days). This indicates the need to consider malaria while examining airport employees or people living near international airports presenting with fever of unknown origin. It also stresses the need for effective spraying of aircrafts coming from malarious areas.All cases were caused by  Malaria has been eliminated from Tunisia in 1979 , mainly 3, haemoglobin at 16 g/dL and thrombocytopaenia . Antibiotic therapy was prescribed. On 11 July, because of the persistence of fever, a second biological assessment was required and showed a thrombocytopaenia at 35,000/mm3. Blood smear performed to control thrombocytopaenia and check the presence of aggregated platelets revealed intra-erythrocytic trophozoites of Plasmodium falciparum with a parasitaemia at 1%. The patient was treated with Lumartem® (20 mg artemether/120 mg lumefantrine). He received a total of six doses (4 tablets per dose) over a period of three days, as recommended. The patient is a student, living within 2 km of Tunis international airport.A 27-year-old man presented on 3 July 2013 with fever (40°C), myalgia and headache of two days standing for which he received paracetamol without improvement. The biological assessment showed WBC at 4,230/mm3 with thrombocytopaenia  and abnormal liver function findings . Serological tests for influenza A and B virus were positive and Tamiflu was prescribed. On 11 July, malaria was suspected based on the diagnosis of case 1, and microscopic examination of blood smears confirmed the presence of P. falciparum with parasitaemia at 12%. The patient was treated for seven days with intravenous quinine dihydrochloride®  8 mg/kg quinine base every eight hours after an initial loading dose of 17 mg/kg.A 25-year-old friend and neighbour of case 1 presented on 6 July 2013 to the emergency department with fever, headache and myalgia accompanied by epistaxis. The diagnosis of angina was accepted and the patient was treated with amoxycillin. On the next day, the patient’s general condition deteriorated with vomiting, fever, and shivering. Laboratory investigations revealed leukocytopaenia at 2,300/mm3), leukocytes 4,600/mm3, C reactive protein at 163 mg/l, and abnormal liver function transaminase . In light of the two recent cases diagnosed in the same area, malaria was rapidly suspected and a microscopic examination of blood smears confirmed the presence of P. falciparum with a 0.5% parasitaemia level. The patient was treated with Lumartem®.A 21-year-old man, living within 200 m from cases 1 and 2 was admitted to a private clinic with vomiting, shivering and muscular pain of one week’s standing. He had received paracetamol and metoclopramide (Primperan®). On 13 July, laboratory tests revealed thrombocytopaenia . Microscopic examination of blood smears was prescribed and revealed P. falciparum (parasitaemia = 0.01%). The patient was treated with Lumartem®.A 21-year-old man presented on 7 July with fever, myalgia, shivering and vomiting that had begun two days earlier and persisted despite treatment with paracetamol. Oral antibiotic therapy (amoxicillin/clavulanate) was prescribed. After an initial improvement, the patient's symptoms recurred with pain in the right hypochondrium. Consequently, he was admitted to hospital on 12 July, where physical examination revealed a slight spleen enlargement. Laboratory findings showed thrombocytopaenia at 38,000/mmP. falciparum in all cases. The genomic characterization of isolates was performed in the Malaria National Reference Centre Paris, France. It was performed on DNA extracted from blood samples by using the QIAamp DNA Blood kit  according to the manufacturer's recommendations. Five P. falciparum microsatellite loci  located on different chromosomes were genotyped using the method described by Musset et al. [P. falciparum in all extracts. Treatment led to rapid improvement in all cases and recovery was confirmed by negative control blood smears . As soon as the 1st patient was diagnosed, active screening of febrile individuals living in the area (Berges du lac) was carried out for several months without detecting new cases of malaria.History taking revealed that all cases lived in the same residential area within a radius of 2 km, south east of Tunis Carthage International Airport. They had no history of travel to malaria endemic areas or of blood transfusion. Repeated interviews eliminated also intravenous drug use. Three of the four patients were close friends. PCR testing confirmed t et al. . GenotypAnopheles specimens were captured.An entomological survey was conducted in July 2013 during four days in the patients’ neighbourhood. Adult mosquitoes were caught using CDC light traps and larval stages were collected in available water collections. No Plasmodium parasite transmission. All patients had never travelled to malaria endemic areas and had no history of blood transfusion. The hypothesis of infection through intravenous drug use was also ruled out by careful history taking. Although eliminated from Tunisia since 1980, infection by local mosquito bites was initially considered as it had previously occurred in Algeria, Greece, Italy and United States [Anopheles representing potential vectors [The four airport malaria cases diagnosed in Tunisia during the summer of 2013 concerned citizens born after the stop of d States -8. In fa vectors . HoweverAnopheles mosquitoes transported by aircraft from a malaria endemic country. In fact, Tunis international airport is linked by regular flights to many West-African capitals where malaria transmission is common such as Abidjan, Bamako, Dakar, Nouakchott and Ouagadougou. Moreover, the local conditions, especially the temperatures in June 2013; corresponding to the supposed period of contamination , were auspicious for the survival and the activity of imported mosquito vectors [All four patients were geographically located in the same restricted district just 2 km distant from Tunis International Airport. Onset of symptoms occurred in a short period of less than one week. By ruling out infection by local mosquitoes, exchange of contaminated needles and blood transfusion, it was concluded that the transmission occurred through the bites of infected  vectors .Airport malaria is not an exceptional event. Between 1969 and 1999, 89 cases were reported in Europe mainly in France, Belgium, Italy, the Netherlands, Spain, Switzerland and UK -15. MostP. falciparum isolates obtained from patients showed that a similar strain was involved. This finding could suggest several infections caused by the same mosquito taking an interrupted blood meal. Although rare, such event had been previously reported in 1999 with three malaria cases induced [Genotyping of the four  induced . Bites bP. falciparum aetiology and the naïve immune status of the patients, the observed clinical and biological presentations were severe . Therefore, malaria should be considered in case of high fever of unknown origin particularly in airport employees or people living near international airports.Malaria is generally unsuspected by clinicians in patients from non-endemic countries who have never travelled to malarious areas. Hence, all four cases were diagnosed relatively late. Because of this delay (average of 7 days), Anopheles gambiae in Brazil and Egypt [Beyond the possibility of airport malaria cases, new direct flights between Tunisia and some sub-Saharan African countries increases the risk of invasion by highly competent vectors, as has happened with nd Egypt ,22. Thisnd Egypt . Preventnd Egypt . All potThe occurrence of airport malaria in Tunisia stresses the need for sensitization of clinicians for considering malaria in febrile individuals even when they have not travelled to an endemic area. Beside, this report highlights the importance of efficient disinfection of aircraft arriving from endemic areas.Written informed consent was obtained from the patient for publication of this Case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Malaria during pregnancy remains a serious public health problem. The aim of this study was to determine the prevalence and possible risk factors for malaria in pregnant women attending antenatal clinic at two primary health facilities in Bobo-Dioulasso.Plasmodium falciparum parasitaemia. Hemoglobin concentration was measured. The associations between age, parity, gestational age, schooling, number of ANC visits, use of IPTp-SP, use of insecticide-treated nets (ITN) and anemia with the occurrence of P. falciparum malaria infection during pregnancy were analyzed through logistic regression.We conducted a cross sectional study from September to December 2010 in two primary health facilities located in the periurban area of Bobo-Dioulasso. Pregnant women attending antenatal clinic (ANC) were included in the study after signing informed consent. For each participant, the social-demographic profile, malaria and obstetric histories were investigated through a questionnaire. Peripheral blood was collected and thick and thin blood smears were prepared to check P. falciparum. The hemoglobin concentration mean was 10.5 ± 1.7/dL and was significantly lower in pregnant women with malaria infection (9.8 g/dL ±1.6) than in those who had no malaria infection (10.6 g/dL ±1.7) (P < 0.001). Multivariate analysis indicated that, education , parity [primigravidae  and secundigravidae ], and anaemia  were significantly associated with P. falciparum malaria infection. The use of IPTp-SP was not associated with P. falciparum malaria infection.During the period of study, 105 (18.1%) out of 579 pregnant women were infected by P. falciparum malaria infection is common in pregnant women attending antenatal clinic and anaemia is an important complication. The results show that the use of IPTp-SP does not reduce the risk of malaria incidence during pregnancy.The online version of this article (doi:10.1186/s12879-014-0631-z) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria infection during pregnancy . Morregnancy . In Burkregnancy .P. falciparum infection assessed by microscopy was 24% in urban area ,13],[13]P. f,[13]P. fFactors influencing malaria prevalence in pregnant women include maternal age, parity, use of prophylaxis, nutrition, host and parasite genetics and level of anti-parasitic immunity  with conP. falciparum malaria infection and possible associated risk factors in pregnant women attending antenatal clinic in the periurban area of Bobo-Dioulasso.Understanding the epidemiology of malaria during pregnancy facilitates decision on control strategies.This study aimed to assess the prevalence of A cross sectional study was performed from September to December 2010 in Lafiabougou and Kua two primary health facilities both located in the periurban area of Bobo-Dioulasso (Burkina Faso). In this area, the high transmission season of malaria lasts 4 months from August to November. The entomological inoculation rate (EIR) was about 63 infective bites per habitant per year "unpublished observations".All pregnant women presenting for their routine antenatal clinic visits were included in the study after explaining to them the purpose of the study and signing informed consent. Each participant was evaluated only once. Data were obtained through a questionnaire and included the following information: social-demographic profile, malaria history, obstetric history including parity, gestational age. Gestational age was calculated from the first day of bleeding of the last menstrual period. Only two pregnant women refused to participate to the study because their husband did not allow them to participate to the study.Blood samples were taken by finger prick for checking both malaria parasite and hemoglobin concentration. Blood smear and thick drop assays were stained with 10% Giemsa dye. Smears were fixed with May Grunvald for three minutes before staining with Giemsa for 20 minutes. Parasite density was determined by counting asexual forms of the parasite per 200 leukocytes assuming 8,000 leukocytes/μL of blood. A slide was considered negative if no parasite was found after counting 500 leukocytes. All the slides were double-checked blindly and for discrepant results a third consensus reading was performed.Hemoglobin concentration was determined by the hematic acid method using a hemoglobinometer  and were classified as anemia (<11 g/dL), severe anemia (<8 g/dL) and normal (≥11 g/dL) .The sample size calculation was based on an assumed prevalence of malaria in pregnant women attending antenatal clinic and benefiting from IPT-SP of 13.8% , a preciData were double entered in EpiData 3.1 software and analyzed by using Stata 12 software.P. falciparum malaria infection has been estimated with a 95% confidence interval (CI). Univariate analyses were performed by using the Pearson Chi-square or Fisher's exact tests to compare proportions for categorical variables. Comparison between continuous variables with normal distributions including age and hemoglobin concentration was done by the Student's t-test or the Anova test. The Wilcoxon rank sum test and the Kruskall-Wallis test were used to compare continuous variables with non-normal distributions (parasite density and number of ANC visits).The prevalence of P. falciparum malaria infection categories) was considered as the dependent variable. The independent variables included age, schooling, gestational age, parity, use of IPTp-SP and insecticide-treated net (ITN) during pregnancy, number of ANC visits and anaemia. Variables were categorized as follows: age ; parity as primigravidae, secundigravidae, multigravidae (≥3); gestational age , number of ANC visits . To investigate the association between the several independent variables and malaria, we began by performing simple logistic regressions with each independent variable. Next, we applied multiple regression models to control possible confusion. Variables exhibiting statistically significant associations (p < 0.05) or with important epidemiological meanings were included. These co-variables were kept in models, independent of their significance, in univariate analysis due to their possible relevance in the final results; thus, we could analyze their possible influence when considered together with the other variables.Simple and multiple logistic regression models were also used as described below. The variable malaria . A written informed consent was obtained from all pregnant prior to their enrolment in the study. For illiterate pregnant women, the informed consent discussion process was witnessed by an impartial individual. In those cases, the informed consent form had been signed with a thumbprint.All women with anaemia or positive for malaria have been treated orally with folic acid plus ferrous and quinine 300 mg (24 mg/day until 7 days), respectively.P. falciparum parasitaemia was 18.1% (95% CI = [15.1-21.2]) and the geometric mean of the parasite density (GMPD) was 2254 parasites/μL (95% CI = [1755-2894]). The geometric mean of the density of P. falciparum parasitaemia was significantly lower in pregnant women who had used IPTp-SP (P <0.001). The haemoglobin concentration mean was significantly lower in pregnant women with malaria infection (9.8 g/dL ±1.6) than in those who had no malaria infection (10.6 g/dL ±1.7) (P <0.001).The baseline characteristics of the 579 pregnant women involved in the study are summarized in Table The proportion of women receiving the recommended 2 or 3 doses of IPTp-SP increased (p < 0.001) with the number of ANC visits attended , parity [primigravidae  and secundigravidae ], and anaemia  were significantly associated with .2), pariP. falciparum malaria infection and possible associated risk factors in pregnant women attending ANC in the periurban area of Bobo-Dioulasso. The prevalence of maternal peripheral P. falciparum infection assessed by microscopy was 24% in urban area [This study aimed to assess the prevalence of ban area  and variban area -9],13],[13]P. f,[13]P. fban area  could beban area  even thoP. falciparum malaria.This agrees with previous findings in India . Ho. HoP. fain India . Other ain India ,23],, but but33] bP. falciparum is common in pregnant women attending antenatal clinic in Bobo-Dioulasso and that anemia is an important complication associated with P. falciparum infection. Lack of formal education and parity even adjusted for age are main risk factors for malaria. The use of IPTp-SP was not associated with P. falciparum malaria infection.Our results suggest that Prevalence of maternal peripheralP. falciparuminfection from 2004 to 2011 in Burkina Faso. The data provided show the prevalence of maternal peripheral P. falciparum infection reported in Burkina Faso, organized by type of study, period of study, location, pregnant women's data including age and parity. (PDF 13 KB)Additional file 1:


Plasmodium falciparum. Most of the severe malaria symptoms were observed among children infected with P. falciparum and P. vivax. The study showed that significant number of the children developed severe life threatening malaria complications. This calls for prompt early diagnosis and effective treatment of patients to reduce mortality and complications associated with malaria in the study site.Despite rigorous effort made to control malaria for more than a century, it is still among the main public health problems in least developed regions of the world. Majority of deaths associated with malaria occur in sub-Sahara Africa among biologically risked groups. Thus, this study was designed to assess the incidence of severe malaria syndromes among children in Pawe Hospital, Northwest Ethiopia. Children seeking medication for malaria infection in Pawe Hospital during the study period were recruited. Sociodemographic characteristics, physical, hematological, and clinical features of complicated malaria were assessed following standard parasitological and clinical procedures. A total of 263 children were found malaria positive. Among these, 200 were infected with Plasmodium species  , and hyperpyrexia (body temperature > 40°C) . These severe malaria symptoms were highly prevalent among children less than five years. But other severe malaria symptoms were not significantly different between children of age groups < 5 and > 5 years infected withAlthough the prevalence of malaria has been showing declining pattern in some parts of Ethiopia , in the  Plasmodium species, P. falciparum and P. vivax, are important parasites in malaria related problems in Ethiopia, P. falciparum was the most predominant species accounting for about 76% of all malaria infections among children assessed in this study.Although the two P. vivax strengthens the fact that this parasite is no more mild; rather it could be responsible for some life threatening complications among children in malaria endemic regions [Malaria is a deadly disease to all humanity. But the most vulnerable  are infants and young children (due to their underdeveloped immunity) and pregnant women . In pregnant women it causes increased risk of abortion, stillbirth, premature delivery, and low-birth weight infants besides mortality , 23. Thu regions , 24–26.The observed severe malaria complications were much lower than the earlier reports made from Papua New Guinean, Ghana, and Yemen –29. ThisMost of the children enrolled in this study were found in age group ≤5 years. In this age category higher load of parasitemia, respiratory distress, and incidence of severe anemia were observed. This might be due to the development of poor immunity against the disease  although P. falciparum infection due to intense hemolysis (destruction) of infected RBCs due to higher parasitemia caused by the parasite. Unlike other Plasmodium species, P. falciparum infect all types of RBCs found at different stages of development (from immature young to old RBCs). With additional hemolysis of noninfected RBCs by host immunity, the likely occurrence of severe anemia in P. falciparum infected children is high. However, due to selective preference to only young RBCs by P. vivax, it appears that the number of haemolysed RBCs during P. vivax infection is minimal. Thus, the incidence of severe anemia associated with P. vivax might occur as a result of rigor inflammatory reactions due to proinflammatory response and cytokines activation [ P. vivax infection [ P. vivax is much higher (~32) than P. falciparum (~8) [ P. falciparum and P. vivax positive children strengthens the fact that P. vivax was one of the risk factors associated with incidence of severe anemia among children.Usually anemia is a hallmark oftivation  and lessnfection , 35. On rum (~8) . FurtherAlthough bed net is unquestionable tool for prevention of malaria vector and widely used in most malaria endemic areas of the country, in the current study its distribution and utilization among the biologically risked groups, supposed to get priority in the family, were very limited and only few were sleeping under bed net. This situation might be among the most important factors aggravating the highest prevalence of malaria in the study area, when the current trend of malaria infection dramatically dropped in most malaria endemic areas of the country. P. falciparum were observed in significant number of children assessed in this study. P. vivax caused severe malaria complications documented in the current study strengthen the fact that this parasite is no longer benign. Thus, early detection of infected cases and implementation of effective treatment should be in practice to reduce mortality and morbidity associated with malaria in the current study area.Severe malaria symptoms associated with


Papio anubis)-Plasmodium knowlesi (P. knowlesi) H strain model of placental malaria, experiments were carried out to determine placental cytokine profiles underlying the immunopathogenesis of placental malaria. Four pregnant olive baboons were infected with blood stage P. knowlesi H strain parasites on the one fiftieth day of gestation while four other uninfected pregnant olive baboons were maintained as uninfected controls. After nine days of infection, placentas were extracted from all the eight baboons through cesarean surgery and used for the processing of placental plasma and sera samples for cytokine sandwich enzyme linked immunosorbent assays (ELISA). Results indicated that the occurrence of placental malaria was associated with elevated concentrations of tumour necrosis factor alpha (TNF-α) and interleukin 12 (IL-12). Increased levels of IL-4, IL-6 and IL-10 and interferon gamma (IFN-γ) levels were detected in uninfected placentas. These findings match previous reports regarding immunity during PM thereby demonstrating the reliability of the olive baboon-P. knowlesi model for use in further studies.Malaria parasites are known to mediate the induction of inflammatory immune responses at the maternal-foetal interface during placental malaria (PM) leading to adverse consequences like pre-term deliveries and abortions. Immunological events that take place within the malaria-infected placental micro-environment leading to retarded foetal growth and disruption of pregnancies are among the critical parameters that are still in need of further elucidation. The establishment of more animal models for studying placental malaria can provide novel ways of circumventing problems experienced during placental malaria research in humans such as inaccurate estimation of gestational ages. Using the newly established olive baboon ( Placental malaria causes adverse pregnancy outcomes like abortions, pre-term deliveries, still births, low birth weights (LBW), intrauterine growth retardation (IUGR) and reduction in gestational age. Furthermore, PM causes increased maternal and foetal mortality and morbidity .Immunological mechanisms underlying the drastic pregnancy consequences during PM have been reported in human and animal model studies . Many questions concerning PM immunological mechanisms that lead to problematic pregnancies have not been clearly addressed due to these limitations . One possible solution of avoiding these difficulties is the expansion of the number of feasible animal models, especially non-human primates (NHP) like olive baboons through extensive characterization experiments.Different challenges are usually experienced during human PM studies such as inaccurate gestational age estimation, skepticism among study subjects, financial, ethical and moral obstacles (Steketee P. knowlesi-olive baboon (Papio anubis) PM model has increased the number of animal models available for placental malaria experiments .The use of the recently established P. knowlesi  whose entire genome has been sequenced  thus allowing the function of drug and vaccine candidate molecules to be screened using P. knowlesi genes .This novel model of pregnancy/placental malaria makes use of the non - human primate and human malaria parasite P. knowlesi parasite infects humans , is phylogenetically close to P. vivax and is highly virulent in the olive baboon system. Olive baboons are readily available and have immunopathophysiological and host-pathogen interactions that are similar to those in humans  making them ideal candidates for studying PM and or pregnancy associated malaria (PAM).The P. knowlesi model for pregnancy malaria studies. Therefore malaria investigations in NHP like olive baboons can be expected to provide insights and results that reflect what could happen in the human PAM and or PM conditions.The above factors support the reliability of the components of the baboon-et al., 2007). Despite extensive research in the area of PM and or PAM immunology, immunological mechanisms that render pregnant women susceptible to PM still require further clarification.Placental malaria involves the preferential accumulation and sequestration of malaria parasites in the placental tissue where they induce pro-inflammatory immune responses that have been suggested to promote drastic PM incidences  of four pregnant olive baboons with PM were compared with those of four controls uninfected olive baboon placentas.P. knowlesi PAM and or PM model in future investigations.Findings communicated in this study provide new insights and results that are largely consistent with those documented from other animal model and human PM experiments, thereby increasing the relevance of the olive baboon-Papio anubis) originally captured from Kajiado District of Kenya with an average weight of 10 Kg were used in these experiments. All baboons were trapped and maintained in the quarantine facilities at the Institute of Primate Research, Karen, Kenya, for not less than three months. Examination of the baboons was done to confirm that they were free of hemoprotozoan, gastrointestinal parasites and Simian Immunodeficiency Virus (SIV) before inclusion into the study.Eight adult female olive baboons  at the Animal Resources Department’s (ARD) baboon colony facilities.ad libitum.All experiments were performed in accordance with the institutional guidelines of IPR as outlined by the IPR’s Institutional Scientific and Ethical Review Committee (ISERC). Malaria infected baboons were transferred to the IPR biocontainment facility where each baboon was housed individually in a squeeze back cage, 0.6 x 0.6 x 0.68 meters high. They were maintained on a commercial non-human primate diet , supplemented with fruits, vegetables and additional ascorbic acid. Mineral salts and water were provided The biocontainment facility was inspected for proper lighting, ventilation, drainage, temperature, foot bath sterilisation and sprayed with insecticide daily according to IPR standard operating procedures (SOP). For all invasive procedures, the baboons were anaesthetised with ketamine hydrochloride (10 mg/kg body weight).Papio anubis) were maintained in the olive baboon colony facility of the IPR in the company of an adult male olive baboon for mating to occur. Ultrasonography was used to confirm pregnancy status and gestational levels of the olive baboons. Four of the olive baboons were infected with P. knowlesi blood stage, from parasites cultured overnight, on gestation day 150 (when the placentas were mature enough for experiments).The eight adult female olive baboons  and sera samples for IFN-γ, IL-4, IL-6, IL-10, IL-12 ELISA.in vitro propagation of malaria parasites was done using methods described previously . Briefly, in vitro cultures were set-up using cryopreserved P. knowlesi parasites previously isolated from P. knowlesi infected olive baboons. Prior to culture, vials containing the parasite were removed from liquid nitrogen (-196°C) and quickly thawed at 37°C in a water bath andtransferred into 50 ml centrifuge tubes (Becton Dickinson). Subsequently, an equivalent volume of 3.5% NaCl (at room temperature (RT)) was added and mixed with the parasites before centrifugation at 800×g,  for 10 min at 24°C.The The supernatants were aspirated and 1/2 original volumes of 3.5% NaCl added before repeating the centrifugation. RPMI 1640  with 10 % heat inactivated olive baboon serum  was added, mixed and centrifuged again as before. RPMI-10  was then added and mixed with the parasites for a final washing step by centrifugation as before. The parasite pellets were transferred into culture to a starting erythrocyte PCV of 2.5%.P. knowlesi parasites  were used to initiate blood stage malaria infections in experimental baboons. 1.0 x 106 parasites/ml were resuspended in incomplete RPMI 1640 (Invitrogen). Each of the 8 sedated olive baboons received 1 ml of inoculum.The complete culture medium consisted of 2.5% baboon erythrocyte packed cell volume (PCV), 20% baboon serum, 15 µg/ml gentamycin solution and the rest RPMI 1640 (Invitrogen). All olive baboon sera for use had been heat inactivated previously at 56°C. One hundred to two hundred microlitres of culture were used for thin smear preparation (for determination of parasitaemia). Cultures were mixed gently and transferred into T25 culture flasks. The flasks were gassed, tightly capped and transferred to an incubator (37°C). Overnight cultured et al., 2003).The parasites were injected through the femoral vein of the olive baboons’ left legs via butterfly needles from 1 ml tuberculin syringes. Parasites were immediately flushed into the olive baboons’ bodies using 5 ml saline solution for every baboon. Following infection, the olive baboons were carefully transported to the biocontainment facility and maintained in cages .Caesarean deliveries were performed as detailed in the IPR SOP in order to obtain intact sterile placental tissue from olive baboons which could otherwise be consumed by them. Abdominal incisions were executed with minimum bleeding and light pressure manipulations resulted in the eventual expulsion of the placentas .The placentas were then placed in sterile dishes for extraction of placental blood. Each of the placentas was first rinsed once with 2% penicillin–streptomycin (P/S) in phosphate buffered Saline (PBS) .Multiple incisions were made on the maternal side of the placenta and blood was squeezed out under sterile conditions. Thin placental blood smears were prepared and air dried for 5 minutes then fixed in 100% methanol. Fixed slides were stained for 10 min in 10% Giemsa solution and used for microscopic observation at ×100 magnification. At least 2000 red blood cells (RBC) were counted in every parasitaemia determination session. This was done by counting erythrocytes from a quarter of each light microscope field and multiplying by four. Counting was done with the aid of manual lab counters. Parasitaemia was calculated as follows: Parasitaemia % =  × 100 . The blood samples were left at RT for 2 hours to allow for coagulation to take place. The blood samples were then subjected to overnight temperatures of 4°C. The blood was then spun at 850×g for 10 minutes at 24°C .et al., 2006), heparinised blood samples were centrifuged as above and the separated plasma samples were stored at -20°C .Clear sera were sucked off and divided into 10 ml aliquots. Sera samples  were stored at -20°C until use. For preparation of placental plasma samples for the TNF-α ELISA, a cytokine with a short half-life . Ninety-six well flat bottomed ELISA microtiter plates  were coated with 50 µl/well of cross reactive antihuman/baboon cytokine  capture monoclonal antibody (Becton Dickinson) at a final concentration of 5 µg/ml.These assays were performed essentially as described previously  followed by incubation at 37°C for 1 h. After washing the plates six times in ELISA washing buffer (0.05% tween in PBS (PBST (0.05%)), undiluted samples of either sera or plasma and recombinant cytokine standards were dispensed in duplicate (50 µl/well) and the plates incubated for at 37°C for 2h.Standards were serially diluted by transferring 50 µl from well to well, commencing with a neat concentration of 500 pg/ml. The plates were then washed as before and detector mouse biotinylated antihuman/baboon cytokine  monoclonal antibodies (Becton Dickinson) at dilutions of 1:2000 (50 µl/well). This was followed by incubation at 37°C for 1 h, and washed as before. Streptavidin Horse-Radish Peroxidase  diluted 1:2000 was added 50 µl/well and incubated at 37°C for 1 h before a furthersix washes. Colour development was achieved by adding 50 µl/well of Tetramethylbenzidene (TMB) substrate  and optical densities were read using a Dynatech MRX ELISA reader at 630 nm filter setting after incubation at 37°C for 15 min.P < 0.05 were considered significant.Mean values of cytokine concentrations in PM-infected olive baboon placentas were compared with the respective mean values of cytokine concentrations in malaria free olive baboon placentas using non parametric Mann-Whitney U analysis. Probability values of P. knowlesi parasites  and plasma (TNF-α) samples extracted from malaria-infected and malaria-negative olive baboons Figures .P < 0.05) in sera samples from P. knowlesi infected placentas (21.2 pg/mL) than concentrations in sera from uninfected placentas (68.3 pg/mL). TNF-α levels in plasma samples from malaria infected placentas (88.3 pg/mL) were significantly increased (P < 0.05) compared to levels in plasma samples from malaria negative placentas (17.9 pg/mL). TNF-α and IL-12 were detected at significantly (P < 0.05) lower levels in uninfected placentas in comparison with malaria infected placentas. Concentrations of IL-12 (58.3 pg/mL) were significantly increased (P < 0.05) in sera samples from malaria positive placentas compared to concentrations in sera samples from malaria negative placentas (24.9 pg/mL).The mean concentrations of IFN-γ cytokine were significantly lower (P < 0.05) in sera samples from P. knowlesi infected placentas (30.6 pg/mL) than concentrations in sera from uninfected placentas (86.3 pg/mL). IL6 levels in sera samples from malaria infected placentas (24.4 pg/mL) were significantly reduced (P < 0.05) compared to levels in sera samples from malaria negative placentas (57.5 pg/mL). Concentrations of IL-10 (31.6 pg/mL) were significantly reduced (P < 0.05) in sera samples from malaria positive placentas compared to concentrations in sera samples from malaria negative placentas (72.5 pg/mL).The mean concentrations of IL-4 cytokine were significantly lower (Papio anubis) infected with P. knowlesi H strain parasites. Parasite-mediated interference in the pregnancy-sustaining placental anti-inflammatory immunomodulation has previously been implicated as a mechanism that leads to PM-related complications .This study was designed to determine cytokine  immune responses that take place in the placental unit following PM infection in olive baboons . In that study which involved TNF-α mRNA quantification, increased expression in the placenta was also associated with IUGR. TNF-α was also previously confirmed as a trigger agent of abortions in the malaria infected primate host .Our current findings suggest that there is a possible PM disease promotion role by TNF-α in the non-human primate host. Increased presence of TNF-α in malaria infected placentas was documented previously  and the current detection of elevated TNF-α expression in sera derived from PM infected placentas are findings that are in tandem with previous results . TNF-α is also a critical factor during murine PM that acts by inducing placental coagulopathy . In the present study, TNF-α levels in malaria infected placentas were 5 times higher than in malaria free placentas.The previous reporting of occurrences of abortions in the et al., 2010c).Reduced quantities of IFN-γ detected in the malaria infected placentas could have conferred increased susceptibility to PM. Significantly lower levels of the IFN-γ cytokine were reported to occur in the peripheral circulation of malaria-infected pregnant olive baboons compared to malaria-infected non-pregnant ones . In that study, intervillous blood mononuclear cells (IVBMC) from PM-negative multigravid women experienced elevated production of IFN-γ compared to IVBMC from PM-negative primigravid and secundigravid and PM-positive multigravid women following in vitro cytokine assays. IFN-γ was recently reported to occur at significantly higher concentrations in uninfected women with PM compared to PM positive women . Our results confirm that the down-regulation of IFN-γ is strongly associated with presence of PM in the malaria infected host and that increased IFN-γ cytokine production in the placenta may be important for the onset of the protective responses against placental malaria. This report reveals that increased IL-12 values are associated with PM in olive baboons either as a risk factor or as a result of parasite-driven skewing. In accordance with our results, IL-12 producing monocytes were previously found to be significantly increased in P. falciparum infected placentas, compared to malaria free placentas .In human studies, higher concentrations of IFN-γ within the maternal foetal unit was previously implicated as critical protective factor against PM . During pregnancy, the immune system may be biased towards antibody mediated immunity rather than towards cellular responses and this immunomodulation associated with successful pregnancies . The significantly higher levels of IL-4 measured in uninfected baboon placentas is a finding that supports this phenomenon.In this article, we surmise that a reduced IL-4 level is associated with the incidence of PM in the non-human primate (olive baboon) host. IL-4 was reported to be higher in concentration in uninfected placentas compared to infected placentas . Significantly higher levels of IL-6 were measured in malaria - free placentas than in malaria infected placentas . Control uninfected baboon placentas were discovered to have significantly elevated sera IL-10 levels. In another study , elevated production of IL-10 was detected in malaria-free placentas compared to malaria-infected placentas. Our findings are in agreement with these previous results, indicating that increased levels of IL-10 are may be needed for protective responses against PM. The data presented here provide new insights into the cytokine concentration changes that take place in the non-human primate model of PM.Decreased expression of IL-6 and transforming growth factor–b1 were found in malaria-infected placentas compared with uninfected placentas  were maintained in uninfected placentas.Uninfected olive baboon placentas expressed significantly higher levels of IL-4, IL-6 and IL-10 compared to malaria infected placentas, indicating that higher levels observed in this panel of cytokines (Diouf P. knowlesi parasites in infected baboon placentas is therefore revealed in these experiments as a factor that leads to the onset of an inflammatory immune response in the placental unit. This could potentially be detrimental to foetal development.This phenotypic polarization in the malaria free placentas became altered towards the TNF-α and IL-12-dominated cytokine panel following placental malaria. The presence of P. knowlesi-olive baboon model of pregnancy/placental malaria is suitable for further malaria immunology studies.Further information from these experiments has shown that IFN-γ has a crucial protective role against PM since reduced levels of this cytokine were detected in the malaria infected placentas. Cytokine responses in the placentas were significantly altered following the onset of PM leading to a shift in immunity towards IL-12 and the induced innate TNF-α domination. This communication is an addition to the available knowledge base required in the development of immunological control measures. Immunological parameters associated with placental malaria in this NHP model were found to correspond with those described in the human situation indicating that the