Plasmodium falciparum, which is acquired in successive pregnancies. In some studies , grand multigravidae  are more susceptible to poor birth outcomes associated with malaria compared to earlier gravidities. By comparing peripheral cellular responses in primigravidae (G1), women in their second to fourth pregnancy (G2–4) and grand multigravidae we sought to identify key components of the dysregulated immune response. PBMC were exposed to CS2-infected erythrocytes (IE) opsonised with autologous plasma or unopsonised IE, and cytokine and chemokine secretion was measured. Higher levels of opsonising antibody were present in plasma derived from multigravid compared to primigravid women. Significant differences in the levels of cytokines and chemokines secreted in response to IE were observed. Less IL-10, IL-1β, IL-6 and TNF but more CXCL8, CCL8, IFNγ and CXCL10 were detected in G5–7 compared to G2–4 women. Our study provides fresh insight into the modulation of peripheral blood cell function and effects on the balance between host protection and immunopathology during placental malaria infection.Immunopathology of placental malaria is most significant in women in their first pregnancy especially in endemic areas, due to a lack of protective immunity to  Plasmodium falciparum infection is characterised by the accumulation of infected erythrocytes (IE) in the intervillous spaces of the placenta. Immunopathology of pregnancy-specific malaria is largely confined to women in their first and second pregnancy due to a lack of protective immunity, and results in increased risk of severe anemia and low birth weight (LBW), especially in highly endemic areas Placental malaria due to P. falciparum infections decline The variant surface antigen VAR2CSA displayed on the IE surface mediates binding to chondroitin sulphate A (CSA) expressed on the placental syncytiotrophoblast and in placental blood, representing the principal target for opsonising antibody. Antibodies against VAR2CSA block placental sequestration and opsonise IE for phagocytic uptake. These antibodies develop with gravidity, and are associated with protection against LBW and severe maternal anaemia Successful pregnancy outcome in the context of malaria infection depends on appropriate regulation of pro-inflammatory and anti-inflammatory cytokines to achieve equilibrium between immunity and the risk of inflammation-associated severe malaria. Studies of malaria-infected placentas confirm that pro-inflammatory cytokines and β chemokines are secreted by intervillous macrophages and monocytes in response to IE We hypothesised that differences in cytokine responses between multigravidae and grand multigravidae might explain the greater susceptibility of grand multigravidae to malaria. Peripheral blood mononuclear cells (PBMC) obtained from primigravidae (G1), women in their second to fourth pregnancy (G2–4) or their fifth or later pregnancies (G5–7) were exposed to unopsonised IE and IE opsonised with heat inactivated (HI) autologous plasma, and culture supernatants were collected for measurement of cytokines and chemokines. Using a phagocytosis assay to measure opsonising antibody levels, higher levels of opsonising antibody were present in plasma derived from multigravid compared to primigravid women. PBMC from G5–7 women secreted lower levels of IL-10, IL-1β, IL-6 and TNF but higher levels of CXCL8, CCL8, IFNγ and CXCL10 in response to IE than gravida 2–4 women. Our study sheds new light on the role of peripheral blood cells derived from pregnant women in the immune response to malaria.The study was approved by the Institutional Review Board of the PNG Institute of Medical Research, the PNG Medical Research Advisory Committee  and Melbourne Health Human Research Ethics Committee, Melbourne. Witnessed, written informed consent was provided by all participants.P falciparum infection by PCR only . Exclusion of these women did not significantly affect the results. Women then received insecticide-impregnated bed nets and sulphadoxine-pyrimethamine (SP) and chloroquine, or repeat doses of SP and azithromycin. Only PBMC from pregnant women without current malaria infection were analysed in this study, and consisted of PBMC obtained from 33 primigravid and 34 multigravid women. The multigravid group was further divided into women in their second to fourth pregnancy  or five or more pregnancies . Maternal clinical characteristics are outlined in Peripheral blood and plasma samples were collected between 2009–2011 as part of studies on malaria prevention in pregnancy in Madang Province, PNG. Geographic location of the study site, annual rainfall and malaria transmission have been previously described g, 10 min) and plasma collected and stored at −80°C. Blood cells were resuspended in RPMI 1640 (Gibco) at room temperature and transferred to a 15 mL tube before PBMC isolation by density gradient centrifugation using Histopaque (Sigma). Plasma was clarified  and inactivated at 57°C for 45 min.Sodium heparin vacutainers containing venous blood were centrifuged  and thawed gently when required as previously described The laboratory-adapted 4) were added to opsonised IE  in 96-well plates, and incubated at 37°C for 40 min. Uningested IE were removed using FACS Lysing Solution (BD Biosciences) as described Phagocytosis assay to measure levels of opsonising antibody was performed using the non-adherent human monocyte cell line THP-1 maintained as described 8 per mL and added immediately at a ratio of 3 per PBMC (6×105 or 6 μL).IE were left unopsonised or opsonised with 9% HI autologous plasma for 30 min at room temperature as described previously 6 per mL and 100 μL was added to U-bottom 96-well plates  and cells were rested for 1 h at 37°C. Unopsonised IE, opsonised IE or an equivalent number of uninfected erythrocytes (E) were added to PBMC at a 3∶1 ratio and 1% phytohaemagglutinin  was used as a viability control. After 48 h incubation (established using a time course pilot study), culture supernatants from replicate wells were pooled and clarified , aliquoted and stored at −80°C. Supernatants harvested from resting PBMC and PBMC exposed to E or PHA were assessed for IL-6 secretion using ELISA (MABTECH) prior to completion of the multi-plex assay. Ten of 83 PBMC were considered to have been contaminated during purification and were excluded from further analysis (if IL-6 response to media/IL-6 response to PHA or E) ×100% ≥75% . A further 2 samples were unresponsive to PHA and 3 yielded insufficient cell numbers and were therefore excluded. A range of cytokines and chemokines in culture supernatant were tested using multi-plex assay  including IL-1β, IL-1Ra, IL-6, IL-10, IFNγ, CXCL10, CCL3 and TNF. CXCL8 and CCL8 were measured by ELISA (R&D Systems).Vials of PBMC were thawed rapidly in a 37°C water bath, washed twice in warm cell culture medium and cell number and viability were determined in a haemocytometer using trypan blue exclusion. Cell viability was always 80–95%. PBMC were resuspended at 2×1010 scale due to high inter-sample variability. Where constitutive cytokine production exceeded that of IE-induced cytokine a value of zero was assigned; this occurred for IL-1Ra (2 samples), CXCL8 (5 samples) and CXCL10 (14 samples). For CCL3, nine samples out of 68 were higher than the standard curve and were assigned the highest standard curve value of 21,878 pg/mL. Comparison between three gravidity groups  was performed using a non-parametric One Way ANOVA: Kruskal-Wallis test and the Dunn's Multiple Comparison post-test. For comparison between two paired groups (e.g. G1 IE and G1 IE-IgG) a non-parametric Wilcoxon matched-pairs signed rank test was performed. Statistical analysis was carried out using Prism 5.0 software (GraphPad Software) and significance was defined as a probability value below 0.05.Resting PBMC cytokine production was subtracted from IE-induced cytokine to eliminate the contribution of constitutive production and data is presented on a logA significant difference in maternal age at enrolment of G1 (mean (SD) 20.8 (2.7); years) compared to G2–4 (27.4 (5.2) and G5–7 (33.5 (3.6)  was obseLevels of opsonising antibody were assessed using a THP-1 phagocytosis assay in which CS2 IE were opsonised with HI plasma derived from 33 primigravid and 34 multigravid women who were further divided into secundigravidae to gravidae 4 (G2–4) and grand multigravidae (G5–7). Significantly higher levels of opsonising antibody were detected in plasma obtained from G2–4 (p = 0.002) and G5–7 (p = 0.02) compared to G1 . There wWe examined constitutive cytokine secretion by resting PBMC not exposed to IE and for most cytokines analysed no significant difference was recorded (data not shown). Less IL-10 (median (IQR) 3 (2–6) versus 6 (3–14) pg/mL; p<0.05) was detected in resting PBMC derived from G1 compared to G5–7.Opsonisation of IE resulted in increased TNF, IL-1Ra and CCL3 secretion in G1, G2–4 and G5–7 . FollowiLess IL-10 was detected in G5–7 compared to G2–4 PBMC in response to IE (174 (57–355) versus 459 (387–796) pg/mL; p<0.01) and plasma-opsonised IE (97 (40–236) versus 382 (290–646) pg/mL; p<0.01) . IL-10 lIn contrast to IL-10, IL-1β and TNF, there was more CXCL8 secreted by G5–7 compared to G2–4 PBMC in response to IE  versus 10,637  pg/mL; p<0.001) and plasma-opsonised IE  versus 9,657  pg/mL; p<0.001) . SecretiNo variations in the level of IL-1Ra and CCL3 secretion were observed between women in the 3 groups .Our results demonstrate that PBMC from grand multigravidae are characterised by a distinct cytokine and chemokine response to pregnancy-specific IE.P. falciparum IE. Our methodology examined cytokine and chemokine secretion from PBMC derived from malaria exposed pregnant women without current malaria infection by microscopy. Thus we investigated potential immune responses, and how gravidity influences these, and not the actual response to current malaria infection. The majority of studies conducted to investigate changes in cytokine and chemokine secretion caused by placental malaria or associated with increasing gravidity involve measuring circulating peripheral or placental plasma levels, mRNA expression using PCR, or protein levels by histological techniques using fixed placental tissue. The major limitations to these approaches are that plasma cytokine levels may not reflect the ability of peripheral blood cells to secrete cytokines in response to infection, and that measurements taken during acute infection provide limited information about protective responses, as participants are already infected. Our study used an in vitro approach to investigate the capacity of circulating PBMCs derived from women without current malaria infection to respond to CS2 IE expressing VAR2CSA, therefore reflecting baseline cellular responses in otherwise healthy uninfected women. It would be of interest to compare responses in individuals with current malaria infection . A low prevalence of malaria in our study cohort precluded this comparison. One limitation of our model was that peripheral PBMC may not be fully representative of those found in the placenta, and future studies of placental blood cells are indicated. Limited PBMC yield prevented us from identifying the cytokine-and chemokine-producing populations in these women. In children from PNG, most of the early IFNγ response to unopsonised IE is from γδ or αβ T cells This study explored the role of gravidity in modifying the early cellular immune response of pregnant women to The effects of modulation of the immune response by the syncytiotrophoblast This study identified significant gravidity-dependent differences in the pattern of cytokine production in response to CS2 IE. PBMC derived from grand multigravidae secreted significantly lower levels of IL-10, IL-1β and IL-6, and somewhat lower amounts of TNF than cells from women in their second to fourth pregnancy. By contrast, PBMC derived from grand multigravidae secreted higher levels of CXCL8, CCL8, IFNγ and CXCL10 compared to women in their second to fourth pregnancy in response to IE. For most cytokines and chemokines, levels secreted by G5–7 women were similar to those from G1. This observation is consistent with the high susceptibility to placental malaria of women in their first pregnancy Higher levels of opsonising antibody to parasites expressing VAR2CSA were present in plasma derived from G2–4 and G5–7 women compared to G1 providing further evidence for the role of antibody-mediated immunity in modulating the prevalence and intensity of malaria infection in successive pregnancies. This result is in agreement with previous studies published in our laboratory showing IgG opsonic activity in serum is gravidity dependent in pregnant women in Malawi PBMC obtained from G5–7 secreted significantly lower levels of the key immunoregulatory Th-2 cytokine IL-10 compared to primigravidae and G2–4. Both decreased P. falciparum malaria.Changes in IFNγ with placental malaria vary by study Increased chemokine production by monocytes and macrophages may be an important trigger for accumulation of monocytes, neutrophils and other cells in the placenta leading to increased phagocytosis and parasite clearance. Levels of the neutrophil chemotactic factor CXCL8 were higher in G5–7 compared to G2–4 suggesting increased potential for chemotaxis and phagocytosis of IE, which might limit malaria infection. Placental malaria infection has previously been associated with elevated placental CXCL8 mRNA expression and plasma concentrations; the latter were also associated with placental monocyte infiltration Among other chemokines measured, high levels of CCL3 secretion were detected, which did not differ with gravidity. Previous studies have demonstrated that placental malaria infection was associated with elevated CCL3 mRNA levels which correlated with monocyte density in the placental intervillous space Our results demonstrate that PBMC from grand multigravidae are characterised by a distinct cytokine and chemokine response to pregnancy-specific IE. Compared to cells from gravida 2–4 women, they showed decreased production of pro-inflammatory cytokines IL-1β, IL-6 and TNF and the regulatory cytokine IL-10, and increased production of IFNγ and the downstream protein CXCL10, and of chemokines CXCL8 and CCL8. In combination with the acquisition of pregnancy-specific opsonising antibodies, which are rare in first pregnancy, these findings are more in keeping with an increased maturation of the immune response in grand multigravidae. The chemokine and cytokine responses suggest early engagement of T cells and NK cells (and possibly neutrophils) in the response to IE, rather than marked inflammatory macrophage responses, which are seen in the placenta in early pregnancies. Rather than indicating susceptibility to placental infection or its consequences, these responses indicate that grand multigravidae display a distinct peripheral cellular response with counter-regulatory and feedback mechanisms in place.


Plasmodium falciparum malaria . In this study, we wanted to test the hypothesis that P. falciparum infections during early infancy results in elevated EBV viral load which results in diminished EBV-specific T-cell immune responses over time.Endemic Burkitt’s lymphoma, the most prevalent childhood cancer in Equatorial Africa, is a rapidly growing B-cell malignancy that is ultimately fatal if untreated. Two co-factors are linked to the etiology of this pediatric cancer: Epstein-Barr virus (EBV) infection, and sustained and intense exposure to 6 PBMC was determined by scanning with ImmunoSpot Reader and Software.Infants were enrolled from two rural sites in Kenya: Kisumu District where malaria transmission is holoendemic and risk for eBL is high and Nandi District where malaria transmission is limited and the risk for eBL is low. Finger prick blood samples were taken through 2 years of age to measure EBV viral load, EBV antibodies, and malaria parasitemia. Venous blood samples were collected at 12, 18 and 24 months of age and PBMC were isolated and stimulated with peptides for both EBV lytic and latent antigens. After 2.5 days of stimulation, IFNγ ELISPOTS enumerated EBV-specific T cell responses, and the number of SFU/10When we compared EBV lytic and latent IFNγ T cell responses at 12, 18 and 24 months of age, we saw that although children in Kisumu were able to mount an IFNγ response against EBV lytic peptides, the magnitude of that response declined significantly by 24 months of age. In contrast, the magnitude of the response did not decline in the Nandi cohort. We also observed higher overall viral loads in infants from Kisumu suggesting that the apparent loss of EBV-specific IFNγ response to lytic antigens in the Kisumu children may be associated with these higher viral loads.P. falciparum malaria contributes to loss of EBV-specific immunity by inducing the collapse of an antiviral IFN-γ mediated CD8+ T cell response.We found that by 2 years of age, there was a significant difference in the capacity of children living in a malaria holoendemic region compared to malaria sporadic region to maintain a T cell response to EBV lytic antigens. This suggests that


Plasmodium vivax is a rare parasite in most parts of Africa, it has significant public health importance in Ethiopia. In some parts of the country, it is responsible for majority of malaria associated morbidity. Recently severe life threatening malaria syndromes, frequently associated to P. falciparum, has been reported from P. vivax mono-infections. This prompted designing of the current study to assess prevalence of severe malaria complications related to P. vivax malaria in Ethiopia.Although, P. vivax mono-infection. Parasitological data were collected following standard procedures. Hemoglobin and glucose level were measured using portable hemocue instrument.The study was conducted in two study sites, namely Kersa and Halaba Kulito districts, located in southwest and southern parts of Ethiopia, respectively. Children, aged ≤ 10 years, who visited the two health centers during the study period, were recruited to the study. Clinical and demographic characteristics such as age, sex, temperature, diarrhea, persistent vomiting, confusion, respiratory distress, hepatomegaly, splenomegaly, hemoglobinuria, and epitaxis were assessed for a total of 139 children diagnosed to have P. vivax malaria was a risk factor for incidence of anemia (P < 0.05) in the two sites.Median age of children was 4.25 ± 2.95 years. Geometric mean parasite count and mean hemoglobin level were 4254.89 parasite/μl and 11.55 g/dl, respectively. Higher prevalence rate of malaria and severe malaria complications were observed among children enrolled in Halaba district (P < 0.001). However, severe parasitemia was higher (72.4%) among children who visited Serbo health center (Kersa district). Male children had significantly higher risk of malaria infection , while female had higher risk to anemia . The observed number of anemic children was 43%, of which most of them were found in age range from 0–3 years. Furthermore, P. vivax associated severe malaria complications observed in this study was lower than those reported from other countries. However, incidence of severe malaria complications in one of the sites, Halaba district, where there is highest treatment failure to first line drug, could have significant impact on national malaria prevention and control activities. Plasmodium vivax is the second important parasite of human malaria widely perceived as causing mild and self-limited illness. Unlike P. falciparum, it has wider geographical distribution. Though the public health importance of this parasite is overshadowed by P. falciparum, it is important parasite outside Africa, mainly in Asia and South America. It causes more than 390 million clinical cases per year[P. vivax, previously thought benign parasite, is recently reported to cause life threatening complications among children from endemic regions such as Indonesia, India and Brazil[per year. P. vivad Brazil-4. Some d Brazil-9.P. vivax is a chief risk factor for severe anemia among young children in most vivax-endemic areas[ic areas,10. Thouic areas-13.P. vivax infection in east Africa, particularly in Ethiopia is higher. In some areas of the country, the prevalence rate exceeds even 70% of total malaria infections. This was previously accounted to high Duffy blood group positivity of most population of the country[Unlike other countries in Africa, prevalence of  country, but rec country,16.P. vivax malaria is highly prevalent in some parts of Ethiopia and its risk to drug resistance is increasing[P. vivax associated severe malaria complication is almost none. Thus, it is rational to assess the current status of P. vivax related severe malaria complications in order to estimate the associated burden among biologically risked groups, children, in the endemic areas.Although creasing-20, to tP. vivax malaria complications in areas where there is intense P. vivax transmission  and lesser transmission (Kersa). Geographically, Kersa district is located between altitudes ranging from 1,740- 2,660 meter above sea level (masl), which is relatively high land, where as Halaba district is found within altitudes of 1554–2149 masl  were recorded on pre-designed case record form by trained health professionals working at the two health centers. Accordingly, body temperature of each child was measured using digital thermometer; clinical symptoms such as fever, headache, hyperpyrexia, epistaxis, persistent vomiting, impaired consciousness, respiratory distress, hemoglobinuria, splenomegaly, and hepatomegaly were assessed. Children with at least one or more symptoms of severe malaria complications set by WHO were claData from laboratory tests were collected by experienced laboratory technicians working in the health centers. Accordingly, a drop of blood sample was collected on clean glass slide from lancet pricked finger to prepare thin and thick blood smears in duplicate per patient for microscopic examination. Thick and thin blood smears were stained with 10% Giemsa , while thin smears were fixed in methanol prior to Giemsa staining. Malaria parasite was identified by observation of the smears and the morphological appearance of the parasite in the infected RBC under oil immersion objective. Parasite load was calculated after counting asexual parasites per 200 white blood cells (WBC), assuming mean WBC count is 8,000/μl. Each blood smear was examined by experienced laboratory technician in the health centers and then re-checked by certified laboratory technician at Jimma University. The degree of parasitaemia was graded as mild, moderate, and severe, when a count was between 1–999 parasite/μl, 1000-9999/μl, >10000/μl, respectively, following method described by Cheesbrough.P. falciparum malaria[From the same pricked finger, few drops of blood samples were taken for measurement of hemoglobin (Hb) and blood glucose concentration (Glu) using handheld portable hemoglobin and glucose analyzer Hemocue™ . Hypoglycemia was considered when blood glucose concentration was < 40 mg/dl. Children with Hb level < 11 g/dl were considered anemic. Briefly, level of anemia was classified as severe, moderate and mild, when Hb concentration < 5 g/dl, between 5 and 8 g/dl, and between 8 and 11 g/dl, respectively, following the WHO anemia classification for severe  malaria. Hyperpy malaria.U test. Median was considered over mean for non-normally distributed variables. In all analysis, significance level was considered at 95% confidence interval.Data was analyzed using SPSS statistical software (version 16.0). Descriptive statistical tests were used for analysis of some clinical, demographic and parasitological data. Associations between variables were evaluated using Pearson correlation test and presented using matrix plot. Odd ratio was employed to describe strength of associations between variables in groups. Independent variables were compared using Mann–Whitney The study was ethically approved by Ethical Review Committee of College of Natural Sciences, Jimma University. Written consent/assent was obtained from guardians of the study participants prior to data collection.P. vivax and P. falciparum, respectively. Based on the inclusion criteria, a total of 139 children with P.vivax who fulfilled the inclusion criteria  were retrospectively analyzed. The rest (17/156) were excluded from the study due to prior medication, lack of consent from their guardians, and few due to admission to Tb clinic. The prevalence of P. vivax malaria was significantly different (P < 0.05) at Halaba district and significantly higher among children aged ≤ 5 years (P < 0.01). Sample composition in terms of sex and age did not differ significantly (p > 0.05) between the two study sites. Even if the overall malaria prevalence shows a declining trend across the country, the number of malaria patients is still high in Halaba district  were positive for malaria infection. Among these, 32.64% (156/478) and 67.36% (322/478) were infected with Based on the observed clinical and demographic characteristics, median age of the children was 4.25 ± 2.95 years. Male children had significantly higher risk of malaria infection . At the time of admission, about 39% (54/139) and 21% (29/139) of the children had vomiting and diarrhea, respectively. Though most children, 94.2% (131/139) had a history of fever for the past 48 hours, only 55.4% (77/139) of them were febrile and had axillary temperature ≥ 37.5°C during enrollment. The median Hb and Glu concentration were 11.55 (4.7-15.2 g/dl) and 103 (36–170 mg/dl), respectively, while the geometric mean parasite count  and average body temperature were 4254.89  and 37.43 (36.5-40.7°C), respectively  but had positive correlation to hemoglobin levels. Accordingly, as age of children increased the parasite count decreased, while the level of Hb increased . However, there was no significant differences  between Hb level and parasite count  fulfilled at least one of the WHO criteria for severe malaria. Some ofP. vivax malaria was a risk factor for incidence of anemia (P < 0.05).The frequency of incidence of anemia  was significantly higher (P < 0.05) among children within the age range of 0 - 3 years than other categories (Table U test. The result showed that Hb level was significantly different (P < 0.01) between the two age categories, but parasite count didn’t show significant difference (P > 0.05). Parasite counts of all children with severe anemia were found in the range of 1000–9999 parasite/μl.Severe parasitemia was observed in 41 children with almost similar proportion in all age groups except for children < 3 years  who was found to have higher parasitemia. Hb level and parasite count of children in the age category of 0–3 years was compared with those in the highest age category (>6 years) using Mann–Whitney P. vivax and the rest were accounted to P. falciparum .The prevalence rate of malaria observed in this study was 31.9%, which was much lower than the very recent report from Nigeria (81.9%). This miMost of the children enrolled in this study were found in age group ≤ 5 years. In this age category higher load of parasitemia and incidence of severe anemia, but lower concentration of hemoglobin was observed. This strengthens the fact that children in this age group who live in holo or hyperendemic areas are biologically risked group. This isP. falciparium. However, due to selective preference to only young RBCs by P.vivax, it appears that the number of hemolyzed RBC during P.vivax is minimal. Thus, the incidence of anemia might occur as a result of rigor inflammatory reactions due to pro-inflammatory response and cytokines activation[P. vivax infection[P. vivax is much higher (~32) than P.falciparum (~8)[Percentage of anemic cases documented in this study (43%) was higher than the earlier 34% reported from children and 30.5tivation and lessnfection. On the  higher ~ than P.frum (~8).P.vivax associated severe malaria complications documented in this study (13.67%) was less than those report from Eastern Sudan (18%) from among children admitted to hospital due to severe P. vivax malaria[Frequency of n Sudan 1% from am malaria. In agren Sudan 1% from am malaria.P.vivax malaria was documented[With regard to sites, the numbers of children with severe malaria complications were higher among those children who visited Halaba Kulito health center, the site where the highest (13%) treatment failure to chloroquine (the first line drug) by cumented. Siliva-cumented elaboratcumented. On the cumented.P.vivax malaria parasite had already developed resistant to the first line drug, chloroquine, in one of these study sites, underestimation of complications caused by P. vivax malaria could have significant impact on current malaria control and prevention activities being under taken in the districts. Thus, it calls for special attention and frequent surveillance by concerned bodies in order to achieve the sought national malaria reduction plan.As The authors declare that they have no competing interest.Both authors equally involved in all phases of the study including designing of the study, data collection and monitoring, data analysis, and write-up of the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/637/prepub


Plasmodium vivax was diagnosed in Greece in a young woman residing in the Eastern Attica region. The source of infection could not be identified. No other autochthonous malaria cases have been described in the Attica region since 1974. This was a sporadic case with no evidence of further local transmission, and no more cases have been reported in Attica up to now, two years later.In August 2009, one case of autochthonous malaria due to  In non-endemic countries, malaria cases are mostly imported (from travelers or immigrants), but blood transfusion malaria, or malaria in transplant recipients, or even cases of "airport malaria" can occasionally be seen . Greece Toxoplasma, Leishmania) serology was negative, and throat swab PCR for H1N1 antigens was also negative. Based on serology , there was no evidence of systemic disease. Ultrasonography of the upper abdomen showed enlarged liver and spleen . The initial Giemsa-stained peripheral blood smear was negative for Plasmodium. In a second peripheral blood smear, obtained on the third hospital day, Plasmodium parasites were identified. The patient was started on mefloquine. When the smear was re-examined by an experienced microbiologist, the parasite was identified as Plasmodium vivax according to morphological characteristics of trophozoites, immature/mature schizonts, and gametocytes , because of high fever (up to 40.3°C) of abrupt onset with chills for eight days. She also reported sore throat, headache, remarkable weakness and sweating with unpleasant smell. Fever was almost periodic, being higher every second day. Anti-pyretic drugs were causing lysis with excess sweating. Her past medical history was unremarkable. She denied travelling abroad and had no history of blood transfusion. Upon admission, patient was in good condition, and physical examination revealed mild hepatosplenomegaly. There were multiple skin lesions suggesting insect bites, especially on lower limbs. During the first 48 hours of hospitalization, she had fever up to 40°C, symptomatically treated with paracetamol. Initial blood evaluation showed normocytic, normochromous anaemia , thrombocytopaenia , with a normal leukocyte count. Liver function tests yielded slightly abnormal results: AST 28 u/L, ALT 71 u/L (reference range < 40), LDH 943 u/L (reference range 230-450), and total bilirubin 1.2 mg/dL . Kidney function tests and coagulation study were normal. ESR was 58 mm, and CRP 139 mg/L (reference range < 5). Chest x-ray and urinalysis were normal. Two sets of blood cultures and urine culture were negative. Viral , bacterial (brucella) and parasitic  through the mandatory notification system. This is the only case of autochthonous malaria in Attica (Athens city and suburbs region) before 2011. It was caused by The most likely explanation of the few reported autochthonous cases is limited transmission of the disease to nearby residents or workers by local mosquitoes (vectors), infected from undiagnosed imported malaria cases or asymptomatic gametocyte carriers infected outside Greece. This is attributed to the large number of documented and non-documented  immigrants from endemic countries of Asia and Africa. Also, climate change, causing high temperatures during summer time, is favourable for mosquito growth .P. vivax, Plasmodium falciparum and Plasmodium ovale have been reported in other European countries, such as in Spain, France, Italy and Germany. In Spain, a case of malaria was reported from the Aragon region in 2010 [Isolated autochthonous cases caused by  in 2010 , and the in 2010 . One cas in 2010 ,15. In I in 2010  and one  in 2010 . In Germ in 2010 . Such caDuring 2010 and 2011, attention was paid to mosquito-borne infections in Greece because of West Nile virus outbreaks and KEELPNO has responded by enhancing its surveillance system and intensifying mosquito control. Efforts to destroy mosquitoes using insecticides are currently undertaken by the Greek government .Although malaria risk in Greece remains extremely low, and the country has implemented control measures, individual measures to prevent mosquito bites, such as using insect repellent when outdoors during the peak-biting period for mosquitoes (dusk and dawn), seem reasonable. Also, health practitioners must include malaria in the differential diagnosis, and try to rapidly identify and report suspected malaria cases to respective authorities. Diagnosis is based on microscopy (blood smears) and/or serology. When there is a high suspicion index for malaria, repeated peripheral blood smears should be examined, drawn every six to 12 hours for a minimum of three days, preferably upon fever or chills .The authors declare that they have no competing interests.CL and ML were attending the patient. KT was the microbiologist that identified the parasite species. IK and SP carried out the epidemiological study. All authors read and approved the final manuscript.


This review is focused on childhood specific aspects of malaria, especially in resource-poor settings. We summarise the actual knowledge in the field of epidemiology, clinical presentation, diagnosis, management and prevention.These aspects are important as malaria is responsible for almost a quarter of all child death in sub-Saharan Africa. Malaria control is thus one key intervention to reduce childhood mortality, especially as malaria is also an important risk factor for other severe infections, namely bacteraemia.In children symptoms are more varied and often mimic other common childhood illness, particularly gastroenteritis, meningitis/encephalitis, or pneumonia. Fever is the key symptom, but the characteristic regular tertian and quartan patterns are rarely observed. There are no pathognomonic features for severe malaria in this age group. The well known clinical  and laboratory  features of severe falciparum malaria in children, are equally typical for severe sepsis.Appropriate therapy  – possibly a drug combination of an artemisinin derivative with a long-acting antimalarial drug - reduces treatment duration to only three days and should be urgently started.While waiting for the results of ongoing vaccine trials, all effort should be made to better implement other malaria-control measures like the use of treated bed-nets, repellents and new chemoprophylaxis regimens. The epidemiology of malaria in children is difficult to assess as most of clinical symptoms are non-specific and most of the cases occur in settings where no routine testing is available.Malaria remains a leading cause of ill health. More than 40% of the world's population (approximately 3 billion people) are exposed to malaria in 108 endemic countries. It caused between 655 000 and 1.240.000Plasmodium falciparum one of the leading causes of child death from infectious diseases worldwide, according to new data it may be responsible for up to 24% of total child death in sub-Saharan Africa.All taken together this makes the infection with the species The epidemiology of falciparum malaria has been changing over the past 10 years, with declining numbers of clinical cases reported in different parts of the world. In Africa, malaria deaths have been cut by one third within the last decade; outside of Africa, 35 out of the 53 countries affected by malaria, have reduced cases by 50% in the same time period. In countries where access to malaria control interventions has improved most significantly, overall child mortality rates have fallen by approximately 20%,4Use of artemisinin based combination therapies (ACTs) and increased coverage with insecticide-treated nets and indoor residual spraying have undoubtedly contributed to the falling number of cases. This improvement has been associated with a change in the observed age pattern of clinical malaria: in costal Kenya the mean age of children admitted to hospital with a positive malaria blood slide has increased from 3 years to 5 years.6P.vivax is the most prevalent of the five human malaria parasites outside Africa. It is mostly absent from central and west Africa because a high proportion of the population have the Duffy-negative phenotype, which prevents erythrocyte invasion by the parasite. In other tropical regions of the world, P.vivax coexists with other Plasmodium species and mixed infections are common. Because transmission rates are low in most regions where P.vivax is prevalent, affected population do not achieve high levels of immunity to the parasite and people of all ages are at risk of infection, although children are more often ill.P.vivax is getting more and more chloroquine resistant in Asia8The clinical manifestations of malaria, the severity and course of a clinical attack depends on the species and strain of the infecting plasmodium parasite, as well as the age, genetic constitution (ethnicity),P.vivax and P.ovale and every 72 hours for P.malariae. P.falciparum infections are usually asynchronous, resulting in nonperiodic febrile episodes, at least during the first days of illness.10The malaria paroxysm results from the lysis of parasitized red blood cells and release of merozoites into the circulation at the completion of asexual reproduction. The paroxysm is characterized by fever and chills accompanied by constitutional symptoms, alternating with periods of fatigue but otherwise relative wellness. Although periodicity of the paroxysm in primary attacks is thought to be pathognomonic for malaria species, this periodicity may take several days to become established, may not occur at all in asynchronous infections, or may be modified by previous immunity or treatment. In patients with previous malaria who are partially immune, merozoite release by erithrocytic schizonts and the accompanying febrile paroxysms are synchronous: approximately every 48 hours for P.falciparum) and may hamper treatment with oral anti-malaria drugs.In children symptoms are varied and often mimic other common childhood illness particularly gastroenteritis, meningitis/encephalitis, or pneumonia. Fever and headache may be the sole symptoms, or gastrointestinal symptoms may predominate. Fever is the key symptom, but the characteristic regular tertian and quartan patterns are seen in < 25% of children; however, children are more likely to have high fever (>40°C), which may also lead to febrile convulsions. Nausea and vomiting are also common , hypoglycaemia (<2.2 mmol/l), elevated aminotransferases, severe anaemia (Hb <5 g/dl), and high parasitaemia (defined as >5%–10% infected erythrocytes or more than 500 000 infected erythrocytes per microliter).It is important to remember that there are no clinical features that are pathognomonic for severe malaria. The well known clinical  and laboratory  features of severe falciparum malaria in children, are equally typical for severe sepsis. Leukocytosis does not allow for discrimination either, as it has been described in up to 20% of young children with severe malaria.16Since severe malaria is a multisystem, multi-organ disease, children frequently present with a combination of the classical clinical phenotypes: cerebral malaria (CM), severe malarial anaemia (SMA), respiratory distress, and hypoglycaemia.P.falciparum parasitaemia in who other causes of encephalopathy have been excluded. Children with CM may develop focal neurological signs, decerebrated or decorticated posturing due to raised intracranial pressure, decreased level of consciousness or coma, behavioural changes, hallucinations, and seizures. Seizures can be protracted or multiple and may be followed by a long postictal state or they may be difficult to recognize if they present only by conjugate eye deviation, nystagmus, oral automatisms, salivation, and hypoventilation. Although most children with CM regain consciousness within 48 h and seem to make a full neurological recovery, approximately 20% die and up to 10% have persistent neurological sequelae.The former two, CM and SMA, are the most common complications of malaria in children. Cerebral malaria is defined by WHO as unrousable coma in a patient with Malarial retinopathy  is highly specific,Increasing evidence shows involvement of the angiopoietin-Tie-2 with retinopathy and mortality in paediatric cerebral malaria.19P.falciparum parasitaemia) is more common in children than in adults. While mortality of SMA is low in asymptomatic children (approx. 1%), the presence of respiratory distress and metabolic acidosis is often (up to 30%) associated with a fatal outcome. According to the world malaria report 2011, the fatality rate for high risk populations approaches 40%.Severe malarial anaemia  is a clinical sign of metabolic acidosis, and has emerged as a powerful independent predictor of fatal outcome in falciparum malaria. It can be misinterpreted as cardiac failure and circulatory overload, especially if associated with severe tachycardia.Blood lactate values are thus among the most useful predictors for stratifying the risk of fatality in children. However, measurement of blood lactate values requires specialized and costly equipment seldom available in African health centers.18Hypoglycaemia (blood glucose concentration < 2.2 mmol/l) is also associated with a poor outcome in children with malaria as in other severe childhood infections. Clinical evidence suggests that hypoglycaemia in African children with severe malaria results from impaired hepatic gluconeogenesis rather than from quinine-induced hyperinsulinaemiaP.malariae).The kidneys may also be affected by, among others blackwater fever , acute tubular necrosis, and immune complex glomerulonephritis , spontaneous bleeding, disseminated intravascular coagulation, hypotension, cardiovascular shock and multi-organ failure.Tropical splenomegaly syndrome, also known as hyperreactive malarial syndrome, is rarely seen in children, as it usually occurs after repeat exposure to malaria. It is characterized by gross splenomegaly, high antibody levels of Plasmodium species, hypergammaglobulinaemia (mainly IgM), clinical and immunological response, and regression of splenomegaly over several months after antimalarial therapy.11Pregnant women are more likely than others to be inoculated with and infected by malaria parasites and are more prone to severe forms, making adverse outcomes particularly common in primigravida women and their offspring.Besides the mother, Malaria can infect also the placenta and the fetus, leading to low birth weight through intrauterine growth retardation and/or prematurity. Estimates for malaria induced low birth weight range from 7.8–45.3 of every 1000 live births and the associated mortality risks during the first month of life is about 40 times that of babies with normal birth weight.21The parasite concentration dependent correlation between maternal peripheral blood parasitaemia, placental, and umbilical cord-blood  parasitaemia has long been known.22P.vivax. That congenital malaria is not seen more frequently is due in part to the effective barrier function of the placenta. Although congenital malaria develops in 0.1% of immune and 10% of no immune mothers in endemic areas, placental infection occurs in as many as one third of pregnant women. In endemic areas, distinguishing malaria acquired congenitally from that acquired by post-natal transmission from mosquitoes is difficult.All four types of human malaria can be transmitted congenitally, but the disease most often is associated with The onset of symptoms is insidious and usually occurs at 2 to 8 weeks of age. The typical malaria paroxysm is usually absent, with the infant presenting instead more sepsis like symptoms:Interestingly, new evidence suggests that a subset of those vertically affected infants is also at higher risk of malaria infections later in life.24Imported Malaria occurs in children in many non-endemic countries. Over 1000 imported cases in children are diagnosed in Europe each year.The differential diagnosis of fever in a patient with recent international travel history is broad. Common causes of fever by symptom complex are indicated in Although headache and fever may be severe in malaria, there is usually no neck stiffness or photophobia resembling that in meningitis. Although myalgias may be prominent in older children, they are not usually as severe as in dengue fever, and the muscles are not tender as in leptospirosis or typhus. In addition, malaria is not usually associated with a rash, helping to distinguish malaria from meningococcemia, typhus, leptospirosis, Rocky Mountain spotted fever, enteric fever, dengue fever, and viral hemorrhagic fevers. However, malaria may also coexist with other infections. The importance of malaria is underscored by a recent analysis from the GeoSentinel surveillance network: malaria was found to be the illness with the highest hospitalization rate (69%) in children after international travel.269/L) is characteristic of malaria and occurs in 50–70% but, unlike adults, usually is not associated with bleeding, even at very low counts. However, severe infections may be accompanied by prolonged coagulation profiles. Thrombocytopenia is associated with high parasitaemia levels, lower age, low Hb levels, increased MPV and platelet aggregate flag.27Haematological and biochemical parameters are often abnormal: normochromic normocytic anaemia occurs in 31–100% of cases, although the need for transfusion is rare (<2%). Thrombocytopenia  to normal, but it may be increased  in severe infections. In fact leukocytosis was strongly found to be associated with younger age, deep breathing, severe anaemia, thrombocytopenia and death - irrespective of bacteraemia.28Jaundice (30–50%) and raised liver enzymes (25–40%) are also relatively common but not usually associated with an adverse outcome.In severe malaria, metabolic (lactic) acidosis, hypoglycaemia, hyponatraemia, and hypocalcaemia may be presenting. Hypoglycaemia occurs frequently with falciparum malaria: it's thought to occur as a result of parasite consumption of glucose, inadequate hepatic gluconeogenesis, and treatment with quinine. When present in children before treatment, hypoglycaemia is associated with a poor prognosis. Hyponatraemia may occur as part of the syndrome of inappropriate secretion of antidiuretic hormone in some patients. Serum creatinine and blood urea nitrogen may be elevated transiently or may rise significantly with acute renal failure. Malaria stimulates a polyclonal increase of immunoglobulins associated with rapid production of malaria-specific antibodies and reduced complements levels. CSF is usually normal.WHO guidelines recommend prompt and accurate parasitological confirmation of malaria diagnosis by optic microscopy or rapid diagnostic tests based on lateral flow immunochromatography as part of an effective disease management,As in adults, the gold-standard diagnosis of malaria rests on demonstration of the parasite in peripheral blood smears of a febrile child. Both thick and thin blood smears should be examined: the thick smear has the advantage of concentrating the parasites and thus increasing diagnostic sensitivity. The thin smear allows for positive identification of the malaria species.In malaria endemic regions, there is a tendency to treat all fevers as malaria, particularly in high risk groups such as young children. As a result it is not uncommon for malaria to be clinically diagnosed and treated without microscopic confirmation or despite a negative blood smear.The results of a retrospective study conducted in Uganda among children up to 15 years of age, with a diagnosis of malaria, provide evidence that children who did not have microscopy performed or had a negative blood smear had a higher risk of death than those with a positive blood smear. The higher mortality in children diagnosed and treated for malaria without microscopic confirmation is likely due at least in part to misdiagnosis and a lack of treatment for conditions other than malaria. These results argue for microscopy or rapid diagnostic testing of all children admitted with a presumptive diagnosis of malaria and evaluation of other causes of disease in children with negative results.12P.falciparum schizonts and pigment deposits in peripheral polymorphonuclear leukocytes may indicate severe malaria.11In addition, in falciparum malaria, the percentage of parasitized red blood cells, the presence of Serological tests provide confirmation of past malaria infection, but they don't help in the diagnosis of acute infections for treatment purposes. Furthermore, due to persisting maternal antibodies, their use in infants and young children is even more limited.P.falciparum; however, their sensitivity and specificity depend on the pre-test probability making it difficult to express clear recommendations. In a recent study from Burkina Faso,designed to evaluate the accuracy of a rapid diagnostic test on the diagnosis of malaria infection and of malaria-attributable fever during low and high transmission season, the overall performance of the sensitivity of the test was below the WHO-recommended threshold of 95%. During the rainy season, almost 90% of febrile children below 1 year, and almost 85% of those between 1 and 4 years, had a positive rapid test, but a positive malaria test result had a Positive Predictive Value of only 82% and 69%, respectively.Additional diagnostic tests for malaria include but are not limited to rapid dipstick tests and polymerase chain reaction. There are now commercially available whole-blood rapid dipstick tests for falciparum malaria that are based on the qualitative detection of the histidine-rich protein 2 antigen of Unfortunately even a negative test result could not rule out malaria in either group, with a residual probability of 16% and 10%, respectively. This raises concern especially for infants between 6–11 months, because, even at very low parasite densities (the most likely to go undetected by the rapid diagnostic tests), fever is almost invariably attributable to malaria.30a posteriori diagnosis and epidemiological research.10Although polymerase chain reaction techniques for the detection of malaria have good sensitivity and specificity, they are technically more complex, and require sophisticated and expensive equipment, so they are usually performed in reference laboratories and reserved for Treatment of malaria depends on the (presumptive) identification of the species of Plasmodium causing the infection, knowledge of the presence of resistant organisms in the area in which the malaria was contracted, national guidelines, antimalarial availability, individual patient factors and whether the malarial illness is categorized as either uncomplicated or severe.P.falciparum infections can cause rapidly progressive illness and death.For falciparum malaria, the urgent initiation of appropriate therapy is especially critical, because In endemic areas children with uncomplicated malaria, low parasitaemia, no vomiting and who maintain their nutrition and hydration orally may be treated with oral antimalarials, on an outpatient basis.10P.vivax can not be excluded.11Where possible and feasible, a thick and thin blood smear, FBCs, electrolytes, blood glucose, and renal and liver function tests should be performed on all patients hospitalized, as well as testing for glucose-6-phosphate dehydrogenase (G6PD) deficiency, if Drug combinations, rather than monotherapy, are now seen to be the best solution for treating malaria, but the primary problem with using drug combinations in Africa is cost: in much of the continent people have malaria several times a year, and treatment cost – including procurement of drugs - could be prohibitive both for governments and households.13P. falciparum and chloroquine-resistant P.vivax infection. The main artemisinin-based combination therapies (ACTs) are artesunate combined with either mefloquine or amodiaquine, artemether combined with lumefantrine, and dihydroartemisinin with piperaquine. The artemisinin derivatives are safe and well tolerated by young children, and so the choice of ACTs will be determined largely by the safety and tolerability of the partner drug.Artemisinin and its derivatives are now a standard component, due to their high plasmodium killing rates and their capacity to target both, sexual and asexual stages they prevent both, clinical deterioration and transmission. Combination of an artemisinin derivative with a long-acting antimalarial drug reduces treatment duration to only 3 days. Artemisinins are generally safe and well tolerated, and are recommended by WHO as first-line treatment for Sulfadoxine-pyrimethamine should be avoided in the first weeks of life because it competitively displaces bilirubin with the potential to aggravate neonatal hyperbilibinaemia. Furthermore the correct dosing in young children still needs to be defined [32]. Primaquine should also be avoided in the first month and in children known to have severe G6PD deficiency.Alternatives such as clindamycin and doxycycline may also be given, but only in children >8 years because of risk of dental hypoplasia and permanent teeth discoloration. With these exceptions there is no evidence for specific serious toxicity for any of the other currently recommended antimalarial treatments in infancy.29P.malariae, P.ovale and P.knowlesi, also ACT seem to be equally effective.Chloroquine remains recommended for the treatment of infections caused by P.vivax and P.ovale infections, patients recovered from the first episode of illness may have additional attacks, or relapses, after months or even years without symptoms, because these Plasmodium species have dormant liver stage parasites (hypnozoites) that may reactivate. Treatment with primaquine phosphate for 14 days should be included in the treatment of the first attack to eradicate the hepatic hypnozoites.In Of course, HIV-infected children should receive prompt, effective antimalarial treatment according to the WHO guidelines. While there is evidence that Lopinavir/ritonavir based antiretroviral treatment can lower the risk for malaria in children35With regard to paracetamol use, current WHO guidelines on the management of fever recommend it’s use in children with a temperature of 38.5°C or above. However, in a recent study there seems to be no statistically significant difference in temperature or other symptoms like vomiting or headache between children receiving paracetamol or placebo.36As already said earlier, the risk of death from severe malaria is greatest in the first 24 hours.Patients with severe malaria infections or those unable to take oral medications should thus be hospitalized and managed as any emergency: airway patency should be checked, and oxygen should be given. Intravenous access should be rapidly established to allow for lab work (including blood cultures when possible) and parenteral antimalarial therapy.If referral to a treatment facility able to administer IV treatment cannot be accomplished within 6 h, pre-referral treatment with intramuscular artesunate,38Indications for hospitalization include cerebral malaria, severe anaemia, haemoglobinuria, renal failure, pulmonary oedema, coagulopathy, severe thrombocytopenia, shock, high parasitaemia, metabolic acidosis, hypoglycaemia, intractable vomiting, dehydration, seizures, or altered level of consciousness.For parenteral treatment of severe malaria the cinchona alkaloids (quinine and – in the US quinidine) and the artemisinin derivatives  can be used.41Artesunate seems to offer additional advantages, ranging from ease of administration (no cardiac monitoring) to the reduction in the incidence of convulsions, coma, and hypoglycaemia developing after discharge.For children, artesunate 2.4 mg/kg BW IV or IM given on admission (time = 0), then at 12 h and 24 h, then once a day is the recommended treatment. Artemether, or quinine,is an acceptable alternative if parenteral artesunate is not available: artemether 3.2 mg/kg BW im given on admission then 1.6 mg/kg BW per day; or quinine 20 mg salt/kg BW on admission (IV infusion or divided im injection), then 10 mg/kg BW every 8 h; infusion rate should not exceed 5 mg salt/ kg BW per hour.Children with anaemia associated with severe malaria may require blood transfusion. Platelet transfusions for thrombocytopenia are generally not recommended because thrombocytopenia is not associated with bleeding problems in children.Blood glucose should be monitored every 4 h and haemoglobin and parasite count at least daily.Empirical parenteral antibiotic treatment with a third-generation cephalosporin or even a quinolone should also be given as co-infection with malaria and (multidrug resistant) gram-negative bacteria, like non-typhoidal salmonellae,43Regarding the duration of the antimalarial treatment, currently experts recommend to give parenteral antimalarials in the treatment of severe malaria for a minimum of 24 h, once started or until the patient is about to tolerate oral medication , before giving the oral follow-up treatment, which should consist in a full course of an effective ACT (artesunate plus amodiaquine or artemether plus lumefantrine or dihydroartemisinin plus piperaquine) or artesunate  or quinine (plus clindamycin or doxycycline). Where available, clindamycin may be substituted in children and pregnant women; doxycycline cannot be given to these groups. Regimens containing mefloquine should be avoided, if the patient presented initially with impaired consciousness, because of an increased incidence of neuropsychiatric complications associated with mefloquine, not only following cerebral malaria.44With regard to supportive treatment, bolus-fluid resuscitation should be used with caution. Recent evidence from Africa shows that fluid-bolus with either albumin or saline, as compared with control, increased the absolute risk of death for children with severe febrile illness and impaired perfusion at 48 hours by 3.3 percentage points and the risk of death, neurologic sequelae, or both at 4 weeks by nearly 4 percentage points. The risk was higher for children with a positive malaria test as compared to those with a negative malaria test and for those with <5g/dL haemoglobin as compared to those with higher haemoglobin levels.The two components of malaria prevention are reducing exposure to infected mosquitoes and chemoprophylaxis.All children  should take an appropriate antimalarial drug. The choice of which chemoprophylactic agent to use should be based on the presence of chloroquine-resistant or mefloquine-resistant strains in the specific area.Intermittent administration of a full therapeutic dose of an antimalarial drug (or a combination of drugs) at specified timepoints, whether or not parasites are present is known as Intermittent Preventive Treatment (IPT). IPT shows great benefit in areas of high transmission and for specific risk groups . Sulfadoxine-pyrimethamine is particularly suited for this approach as it has a long half-life. An analysis of pooled efficacy data from six randomized trials of sulfadoxine-pyrimethamine given to infants showed that compared with placebo, IPT had a protective efficacy of 30.3% against clinical malaria, and 38.1% against malaria associated hospital admissions, but no efficacy on mortality.A further development of IPT is Seasonal Malaria Chemoprevention (SMC). WHO is now recommending SMC for the prevention of falciparum malaria among children less than 5 years of age in areas with highly seasonal malaria transmission as the Sahel sub-region. A complete 3-day treatment course of amodiaquine plus sulfadoxine-pyrimethamine (AQ+SP) should be given to children aged between 3 and 59 months at monthly intervals, to a maximum of four doses during the malaria transmission season. SMC should not be given to children with severe acute illness or unable to take oral medication, to HIV-positive patients on co-trimoxazole, to a child who has received a dose of either AQ or SP drug during the past month or who is allergic to either drug. The recommendation is based on results from 7 studies on SMC (IPTc) showing a reduction of 75% in malaria episodes in children less than 5 years of age without significant side effects.48Key interventions currently recommended by WHO for the control of malaria in endemic areas are the use of insecticide treated nets (ITNs) and/or indoor residual spraying (IRS) for vector control, and prompt access to diagnostic testing of suspected malaria and treatment of confirmed cases.Community randomised trials in Africa have shown that full coverage with insecticide-treated nets can halve the number of episodes of clinical malaria and reduce all-cause mortality in children younger than 5 years of age. Initial fears that reducing malaria transmission might paradoxically increase child mortality through delayed acquisition of malarial immunity have not been realised. When used by pregnant women, insecticide-treated nets can lead to substantial reductions in low birth weight, placental parasitaemia, stillbirths, and miscarriages. The proportion of households in Africa estimated to own at least one insecticide-treated net rose from 17% in 2006 to 50% in 2011, with 24% of children younger than 5 years of age using an insecticide-treated net during 2008.1) DEET . 2) Picaridin KBR 3023 or Icaridin -1-piperidinecarboxylic acid 1-methylpropyl ester). 3) Oil of lemon eucalyptus (OLE) or PMD  commonly known as Citriodiol . 4) IR3535 .Use of topical insect repellent is an important component of the prophylaxis against arthropod bite vector borne diseases too. Rational repellent prescription for a child must take into account age, active substance concentration, topical substance tolerance, nature and surface of the skin to protect, number of daily applications, and the length of use in a benefit-risk ratio assessment perspective. The repellents currently recommended in the 2012 edition of the Yellow BookA table comparing these substances in detail has been published by F. Sorge.51no more than 30% DEET when used on children.52Efficacy and duration of protection for all these products are markedly affected by ambient temperature, amount of perspiration, exposure to water, abrasive removal, etc. In general, higher concentrations of active ingredients provide longer duration of protection, although DEET efficacy seems to reach a plateau between 30 - 50% covering > 6 hours. The American Academy of Pediatrics (AAP) recommends that repellents should contain In field trials the minimum concentration of each of the four agents required to be effective for 3 hours against most arthropods is 20% , products with <10% active ingredient may offer protection only for 1 or 2 hours.The common side effects of these agents described so far are usually mild local irritative dermatitis and allergy. Prolonged exposure to DEET has been related to more severe side effects, namely neurotoxicity. A comprehensive evaluation of the DEET health effects on humans including the rare reports of adverse effects associated with dermal application in children, can be found on the ATSDR website.53The European Parliament amended its directive on biocide products in 2010,However, national recommendations on the use of DEET in children differ in the lower age limit, with the Canadian Recommendation not giving any age restriction,The most detailed guideline (with respect to age classes and number of daily applications) has been published by the Groupe de pédiatrie tropicale of the Société française de pédiatrie,Citriodiol (at 20–30%) and Ir3535 (20%) may be safely used once daily in children between 6 – 12 months and twice daily between 12 months and 12 years. Above 24 months IR3535 may be used at a concentration as high as 35%. For children aged more than 12 years, adult guidelines apply.International recommendations regarding the use of topical repellents in children for the prophylaxis of arthropod borne diseases are limited to short/medium term usage . But the use of repellents is prolonged or chronic among the majority of children living in subtropical regions where these vector borne diseases are endemic. The toxicity of topical repellents used continuously for longer time periods has not been assessed in pediatric age groups. So if the lenght of expected exposure is over 3 months, other preventive measures are to be preferred.Whatever repellent is used, once returned in a safe indoor environment, don’t forget to wash the skin with soap and water to remove any repellent. Do not spray repellent directly in the face, nor on cuts, wounds or otherwise irritated skin. Furthermore use of products that combine repellent (DEET) with sunscreen is discouraged, as sunscreen should be applied first (and may be repeated).Clothing, hats, shoes, bed nets, mesh jackets, and camping gear can be treated with permethrin for added protection. Make sure infants and children don’t put treated items in their mouth!All newborns and infants in their first months are protected best from mosquitoes by using an infant carrier draped with mosquito netting with an elastic edge for a tight fit or make sure to tuck the bed net firmly under the mattress.


Placental infections with Plasmodium falciparum are associated with fetal growth restriction resulting in low birth weight (LBW). The mechanisms that mediate these effects have yet to be completely described; however, they are likely to involve inflammatory processes and dysregulation of angiogenesis. Soluble endoglin (sEng), a soluble receptor of transforming growth factor (TGF)-β previously associated with preeclampsia in pregnant women and with severe malaria in children, regulates the immune system and influences angiogenesis. We hypothesized that sEng may play a role in development of LBW associated with placental malaria (PM). Plasma levels of sEng were measured in women (i) followed prospectively throughout pregnancy in Cameroon (n = 52), and (ii) in a case-control study at delivery in Malawi (n = 479). The relationships between sEng levels and gravidity, peripheral and placental parasitemia, gestational age, and adverse outcomes of PM including maternal anemia and LBW were determined. In the longitudinal cohort from Cameroon, 28 of 52 women (54%) experienced at least one malaria infection during pregnancy. In Malawi we enrolled two aparasitemic gravidity-matched controls for every case with PM. sEng levels varied over the course of gestation and were significantly higher in early and late gestation as compared to delivery . Circulating sEng levels were higher in primigravidae than multigravidae from both Cameroon and Malawi, irrespective of malarial infection status . Peripheral parasitemia in Cameroonian women and PM in Malawian women were each associated with elevated sEng levels following correction for gestational age and gravidity . Increased sEng was also associated with the delivery of LBW infants in primigravid Malawian women (p = 0.017); the association was with fetal growth restriction (p = 0.003) but not pre-term delivery (p = 0.286). Increased circulating maternal sEng levels are associated with P. falciparum infection in pregnancy and with fetal growth restriction in primigravidae with PM. Plasmodium falciparum infections of the placenta referred to as placental malaria (PM) P. falciparum infection every year Low birth weight (LBW) is a well documented effect of malaria infections in pregnancy LBW resulting from maternal malaria is associated with placental inflammation. This includes monocyte infiltration of the placental intervillous space Soluble endoglin (sEng) is involved in both regulation of immune function and angiogenesis. It exerts these effects by binding and interfering with the activity of transforming growth factor (TGF)-β, which is a regulatory cytokine with multiple isoforms  and pleiotropic effects Elevation of plasma sEng levels has been shown to precede the onset of preeclampsia The prospective study in Cameroon was approved by the Institutional Review Board, Georgetown University and the National Ethical Committee, Ministry of Public Health, Cameroon. The cross-sectional study in Malawi received ethical approval from The College of Medicine Research and Ethics Committee in Blantyre, Malawi. Written informed consent was obtained from each study participant. In Cameroon, if written informed consent was unable due to illiteracy, documented verbal consent was obtained  signed that the woman had consented). This consent approach was specifically approved by the relevant review bodies.P. falciparum in Yaoundé is low with ∼13 infectious bites/person/year P. falciparum asexual parasites at monthly visits (up to seven visits per woman) spanning all three trimesters of pregnancy. Women who were blood-smear positive for malaria were prescribed antimalarial treatment according to the Cameroon Ministry of Health's policy. Peripheral plasma samples, thin and thick blood smears, intervillous space (IVS) blood and placental impression smears were collected at delivery, and infant weight was recorded. Levels of sEng were measured in samples from all study participants who satisfied the following inclusion criteria: primigravidae (first pregnancy) or multigravidae (≥3 pregnancies) with live singleton birth, minimum of three peripheral plasma samples from different gestation points available for testing, and PM status determined at delivery. A total of 244 peripheral plasma samples from 52 women were tested for sEng. Participants infected with Plasmodium species other than P. falciparum were not included in this study.During 2001–2004, a cohort of pregnant women was prospectively followed over the course of pregnancy in Yaoundé and Ngali II, Cameroon P. falciparum asexual parasites present in the placental blood, as assessed by smear. For each case, two controls that were equal in age (±2 years) and gravidity, and were negative for malaria parasites by both peripheral and placental smear, were enrolled. All gravidities were included in this study population (n = 479) with multigravidae being defined as more than one prior pregnancy. Peripheral EDTA plasma samples were collected and stored at −80°C until testing. Mononuclear cell infiltration was assessed by microscopy, as previously described P. falciparum infection but no sign of placental infection (n = 21) were not included in the sEng analysis.To further evaluate the association of sEng levels with PM, and with poor clinical outcomes of PM, a second study population was also tested: Between 2001 and 2006, pregnant women who attended the Queen Elizabeth Central Hospital (QECH) were invited to participate in the study following delivery of a live singleton newborn. QECH is a referral hospital in urban Blantyre where malaria transmission is seasonal with an average of 1 infective bite per person per year th centile for growth, after Poor clinical outcomes include LBW (<2500 g), anemia (<11 g Hb/dL), growth restriction . All samples were tested with the investigators blinded to group and outcome.Continuous variables are reported as median [interquartile range (IQR)] and analyzed by Mann-Whitney U test. Categorical data were analyzed by Pearson's chi-square test or Fisher's exact test, as appropriate. A mixed linear multivariate model was applied to analyze the prospective study, with gestational age, gravidity, presence of peripheral parasites, and placental malaria infection as fixed variables, and study participant as a random variable. End of gestation (delivery), primigravidae, absence of peripheral parasitemia and absence of placental malaria infection were set as reference points for the fixed variables, respectively. Two-factor effects of log-transformed data  were analyzed by 2-way ANOVA; between-group effects were analyzed by Bonferonni post-tests. Correlations between continuous variables were analyzed by Spearman's correlation or partial correlation on log-transformed data to control for the effect of additional variables. All P values are two-sided. Statistical analyses were performed using Prism 4.03  or SPSS .Characteristics of the prospective study population are listed in Characteristics of the cross-sectional study population are listed in Since sEng is secreted by the placental syncytiotrophoblast and has anti-angiogenic effects on endothelial cells that might be important for physiologic placental vascular remodelling during gestation Gravidity is an important factor in the susceptibility to PM, with primigravidae being more susceptible to both infection and to sequelae resulting from infection In the Cameroonian study population, the presence of peripheral parasitemia during pregnancy was associated with an increase in maternal sEng levels when gestational age and gravidity were taken into account . Among women with placental malaria infection, sEng was positively correlated with placental monocyte infiltration ; however, the relationship was strongest in primigravidae with PM .In the Malawian study population, 51 (17.5%) of the primigravid women and 9 (5.5%) of the multigravid women delivered LBW infants. Among primigravidae only, the median maternal sEng level was higher in mothers of LBW infants than in mothers of normal birth weight infants : PM, p = 0.008; LBW, p = 0.006; Stratification of primigravid participants by malaria infection and birth weight illustrates that both PM and LBW outcomes were associated with increased peripheral sEng levels at delivery  A third way that sEng may mediate fetal growth restriction is by dysregulating erythropoeisis. We observed an increase in sEng levels with maternal anemia in primigravidae, and previous reports have associated anemia with LBW The mechanism(s) by which sEng may mediate growth restriction in PM will be more thoroughly informed by a prospective study that involves serial blood sampling together with ultrasound measurements of fetal growth to assess how changes in sEng at different points in gestation related to fetal development. It would also be informative to measure TGF-β alongside sEng; however, circulating TGF-β must be measured in platelet poor plasma, which was not collected from the current study populations.HIV infection status was unknown in our study participants, but the prevalence of HIV infection in pregnant women was 7% and 30% in Cameroon and Malawi, respectively, during the periods of recruitment To our knowledge, this is also the first study to describe elevated sEng levels in first pregnancies. Primigravidae are known to be more susceptible to malaria infection and sequelae associated with malaria P. falciparum infection in pregnancy and with fetal growth restriction in primigravidae with PM. These changes are not sufficient for sEng on its own to be a useful biomarker of these conditions, but its potential in combination with other biomarkers deserves further study. Our findings are compatible with the hypothesis that sEng may play a role in the pathogenesis of PM-related fetal growth restriction.In conclusion, we found that maternal circulating sEng is increased with


Plasmodium falciparum malaria during pregnancy.Blood platelet levels are being evaluated as predictive and prognostic indicators of the severity of malaria infections in humans. However, there are few studies on platelets and P. falciparum malaria (cases) and healthy pregnant women (controls).A case–control study was conducted at Gadarif Hospital in Eastern Sudan, an area characterized by unstable malaria transmission. The aim of the study was to investigate thrombocytopenia in pregnant women with p < 0.001. However, there was no significant difference in the platelet counts in patients with severe P. falciparum malaria (N = 12) compared with those patients with uncomplicated P. falciparum malaria (N = 48), 68, 000 /μL vs. 61, 000 /μL, respectively, p = 0.8. While none of the control group had thrombocytopenia , it was found that 6/12 (50%) and 27/48 (56.2%) (p <0.001) of the patients with severe malaria and uncomplicated malaria had thrombocytopenia, respectively. Pregnant women with P. falciparum malaria, compared with the pregnant healthy control group, were at higher risk  of thrombocytopenia. Two patients experienced bleeding, and there was one maternal death due to cerebral malaria where the patient’s platelet count was only 28,000/μL.The median (interquartile) platelet counts were significantly lower in patients with malaria (N = 60) than in the controls (N = 60), 61, 000  vs. 249,000 /μL, respectively, P. falciparum malaria is associated with thrombocytopenia in pregnant women in this setting. More research is needed. It has been estimated that, each year, 30.3 million African women become pregnant in malaria endemic areas . PregnanPlasmodium falciparum form, can cause thrombocytopenia, where there is an abnormally low number of platelets, and activation of the coagulation cascade [P. falciparum infections [P. falciparum infection during pregnancy [P. falciparum malaria parasites. The area is characterized by unstable malaria transmission; P. falciparum is the sole malaria species in the area [Malaria, especially the severe  cascade ,10. Henc cascade . Howeverfections -14. Therregnancy . The curthe area , and thethe area .P. falciparum malaria confirmed by blood film examination. Controls were afebrile healthy pregnant women attending for routine antenatal care without any illness, history, or symptoms  suggestive of malaria. In addition, the controls had parasite negative blood films. After signing an informed consent form, the obstetrics history was taken from cases and controls using pre-test questionnaires. Pregnant women with a hypertensive disorder of pregnancy , or pregnant women using aspirin or heparin were not included in the case or control groups. The cases were examined thoroughly for signs of severe P. falciparum infection, and those with one or more criteria of severe malaria [P. falciparum malaria and were treated with the first-line anti-malarial treatment (artesunate-sulfadoxine-pyrimethamine) as out-patients according to the Sudanese National Malaria control policy [A case–control study was conducted at the antenatal clinic in Gadarif Maternity Hospital in Eastern Sudan during the rainy/post rainy season of June-November 2011. Cases were pregnant women with  malaria  were adm malaria . Women wBlood films were prepared and stained with Giemsa, and 100× oil immersion fields were examined. All the slides were double-checked blindly and only considered negative if no parasites were detected in 100 fields. Densities (parasite per μL of whole blood) were then calculated on the basis of the WBC count of the individual subjects. An average of 500 WBCs were counted for each subject before parasite densities were estimated. Thin blood films were used for parasite species identification.Two mL of blood were collected from each woman in the case and control groups. The blood was collected into ethylenediaminetetraacetic acid anticoagulant through venipuncture and kept on a roller with constant mixing. The complete blood count was determined using a Sysmex automated hematology analyzer in the lab within one hour of collection. Sysmex accuracy was determined on a daily basis using manufacturer-provided samples with known cell counts.t-test and ANOVA were used to compare the means between two or three of the groups, respectively. Mann–Whitney and Kruskal-Wallis H tests were used to analyze medians. A Pearson correlation test was performed between the platelets and parasite counts. Univariate and multivariate analyses were used, with thrombocytopenia  as the dependent variable, while age, parity, gestational age, and temperature were the independent factors. Odds ratios and 95% confidence interval were calculated, and a P value < 0.05 was considered significant.Data were entered in computer using SPSS (version 16.0 for Microsoft Windows) and double-checked before analysis. Continuous variables were checked for normality, and normally distributed data were described by the mean (standard deviation) and non-normally distributed data by the median (interquartile range). Categorical data were compared using a Chi-square test or a Fisher's exact test, as appropriate. Student's The study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum, Sudan.P. falciparum malaria during the study period, and an equal number of healthy pregnant women were assigned to the control group. Out of the 60 P. falciparum-infected women, 12 were diagnosed as severe P. falciparum cases that exhibited various clinical manifestations, including, severe anemia (3), hypotension (9), jaundice (3), cerebral malaria (1), hyperparasitemia (5), bleeding (2) and more than one criteria (7).Sixty pregnant women presented with While there were no significant differences in the mean values (SD) for the age, parity, gestational age, temperature, or the biochemical tests between the severe malaria, uncomplicated malaria and the control groups, the hemoglobin values were significantly lower in patients with severe malaria Table . The parp < 0.001, of the patients with severe P. falciparum malaria and uncomplicated P. falciparum malaria had thrombocytopenia, respectively  and 27/48 (56.2%), P. falciparum malaria, the platelet count ranged from 17,000 to 311,000/μL. The median (interquartile range) of the platelets was significantly lower in patients with malaria  than in the controls, 61,000  vs. 249,000  (controls), p < 0.001. However, there was no significant difference in the median (interquartile range) of the platelets in patients with severe malaria compared with those with uncomplicated P. falciparum malaria, 68,000  vs. 61,000 , respectively, p = 0.8, Figure In patients with p < 0.001) . One patient died during resuscitation, after presenting with cerebral malaria and minor bleeding from the gums. This patient’s hemoglobin and platelet counts were 5 g/dl and 18, 000, respectively.The univariate and multivariate analyses found that malaria was the only risk factor for thrombocytopenia  Table . There wP. falciparum malaria in Africa. The main findings of the current study were that the platelet counts in pregnant women with P. falciparum malaria were significantly lower than in the controls, and there was no difference in the platelet counts in women with severe P. falciparum malaria vs. in the women with uncomplicated malaria. One noteworthy finding was that pregnant women with malaria had a 10 times higher risk of thrombocytopenia than did the controls. In women with P. falciparum malaria infections, there was no significant correlation between parasite and platelet counts. Recently, Tan et al. observed that pregnant women living at the Thai-Burmese border were more susceptible to thrombocytopenia  than their non-pregnant counterparts; the median platelet counts 134,000  in this study were significantly lower in pregnant women with P. falciparum malaria and Plasmodium vivax malaria, with the first malaria episode increasing the risk of thrombocytopenia [To the best of our knowledge, this is the first published data on thrombocytopenia in pregnant women infected with ytopenia -24P. falciparum parasitemia [P. falciparum-infected erythrocytes, and this process might lead to pseudo thrombocytopenia [In the current study, no correlation was found between the blood-stage parasite numbers and the platelet counts. This is in accordance with a previous observation, where it was shown that the pattern of thrombocytopenia and parasitemia did not have a linear relationship among malaria-infected Nigerian children . Howeverasitemia . Thromboasitemia -28. Platytopenia . In endeytopenia .In the current study, two patients presented with nasal bleeding and bleeding of the gums, and one of those who presented with cerebral malaria died. There were no episodes of bleeding or maternal death in our recent study on severe malaria among pregnant women in eastern Sudan . ThromboP. falciparum malaria. Nonetheless, Saravu et al. recently found that non-pregnant adult patients with severe P. falciparum malaria had a statistically significant lower platelet count than patients with uncomplicated malaria [In the current study, no significant difference was found in the platelet counts when patients with severe malaria were compared with those who had uncomplicated disease. This might be related to the small sample size in this study because only 12 patients presented with severe  malaria . One of P. falciparum malaria is associated with thrombocytopenia in pregnant women in eastern Sudan. More research is needed.The authors declare that they have no competing interests.MBA and IA carried out the study and participated in the statistical analysis. GKA and DAR participated in the clinical work and statistical analysis. MIE and DAR conducted the laboratory work. All the authors have read and approved the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/12/10/prepub