Plasma angiopoietin (Ang)-2 is associated with disease severity and mortality in adults and children with falciparum malaria. However the mechanism of action of the angiopoietins in fatal malaria is unclear. This study aimed to determine whether the expression of Ang-1 and Ang-2 and their receptor Tie-2 in cerebral endothelial or parenchymal cells was specific to cerebral malaria (CM), correlated with coma or other severe clinical features, and whether plasma and CSF levels of these markers correlated with the clinical and neuropathological features of severe and fatal malaria in Vietnamese adults.Immunohistochemistry was performed for Ang-1, Ang-2 and Tie-2 on post-mortem brain tissue from fatal malaria cases and controls. Quantitative ELISA for plasma and cerebrospinal fluid levels of Ang-1, Ang-2 and Tie-2 was done to compare fatal cases with surviving patients from the same study.Plasmodium falciparum-infected erythrocyte sequestration in the brain. However Ang-1 and Ang-2 expression in neurons was significantly correlated with the incidence of microscopic haemorrhages. Plasma levels of Ang-2 and Ang-2/Ang-1 ratio were associated with the number of severe malaria complications and were significant and independent predictors of metabolic acidosis and fatal outcome.Immunohistochemistry revealed significant differences in expression in endothelial and parenchymal cells compared to controls. However there was no significant difference in expression of these markers on endothelial cells, astroglial cells or neurons between CM and non-cerebral malaria cases. Immunostaining of Ang-1, Ang-2 and Tie-2 was also not associated with versus non-cerebral cases. Activation of the Ang-Tie-2 pathway in severe malaria is therefore related to acidosis, number of severity criteria and outcome, but is not a specific event in the brain during cerebral malaria.The independent prognostic significance of Ang-2 and the Ang-2/Ang-1 ratio in severe malaria was confirmed, although immunohistochemistry in fatal cases did not reveal increased expression on brain endothelium in cerebral  Plasmodium falciparum malaria, is characterized clinically using a series of criteria  , O, 2000) .The pathogenesis of CM is complex and multifactorial. A fundamental pathophysiological question in the genesis of coma in CM is how parasitized red blood cells (PRBC), remaining within the vascular space, influence parenchymal function to induce coma and death. Neurological dysfunction in severe malaria is significantly related to the sequestration of PRBC in the cerebral microvasculature, and resultant microvascular obstruction -7. Both The endothelium, which is the critical interface between PRBC and the brain parenchyma, is considered to be central to the pathogenesis of CM. During severe malaria infection, there is systemic activation of EC including the cerebral EC of the BBB ,10. ActiThe Ang-Tie-2 ligand-receptor system is a key regulator of the maintenance of functional integrity in both quiescent and activated endothelium . In mostin vivo studies have implicated the dysregulation of angiopoietins in the pathogenesis of severe and fatal malaria. In adults with cerebral and non-cerebral severe malaria, plasma Ang-2 has been associated with reduced endothelial nitric oxide bioavailability and mortality with Ang-2 a better predictor of death than venous lactate [Recent  lactate . In chil lactate -29. NeveThe therapeutic use of angiopoietins has been investigated in experimental murine models of brain ischemia, where Ang-1 inhibits BBB breakdown, stimulates the recruitment of neuroblasts and promotes behavioural recovery . The AngTherefore, in this study, the site and expression levels of the proteins Ang −1 and −2 and their receptor Tie-2 were examined in brain tissue of patients who died from malaria, to determine whether there were patterns of expression specific to cerebral malaria. Levels of the circulating forms of these proteins were examined in plasma and CSF of a subset of these patients in order to correlate circulating angiogenic protein levels and their expression patterns in the brain with the clinical manifestations of severe malaria.versus quinine for the treatment of severe malaria in Vietnam  [This retrospective case–control research study used archival brain, plasma and CSF specimens of fatal and non-fatal malaria patients, who were studied in a large double-blinded trial of artemether Q trial) . RelativA total of 81 adults were included in this clinicopathological study . Brain sections from 18 normal controls from the UK were also used. For the malaria patients, brain specimens were collected at autopsy within 24 hours (median 7 hours), fixed in 10% formalin, then embedded in paraffin and processed using standard methods as previously described . Clinicag for 3 min, then aliquoted and stored at −80°C until use. CSF was taken when the procedure was clinically indicated, aliquoted neat without any additives, spun and frozen at −80°C until use [Plasma samples from 62 out of 63 severe malaria cases were used for the quantitation of the angiogenic proteins by ELISA. CSF samples were available from only 12 severe malaria cases. Venous blood samples were taken immediately after admission into EDTA anticoagulant. Plasma samples were generated by centrifugation at 5,000 ntil use .The cases used in this retrospective study had subsequent extensive clinical, biochemical and neuropathological investigations ,33-38. TImmunohistochemistry (IHC) was performed for three different markers of Ang-Tie-2 pathway including the Ang-1 protein, Ang-2 and Tie-2 receptor. Staining was performed on 4 mm sections of formalin-fixed paraffin embedded brain blocks cut onto coated immunoslides (SupaFrost), from all malaria and control cases. The sections were dewaxed in xylene, rehydrated in graded alcohol series and then underwent microwave antigen retrieval in Tris-EDTA pH 8 for 10 min. The primary antibodies used were: Ang-1 , Ang-2  and Tie-2 . Detection of bound antibody was visualized using the high-sensitivity Novolink™ Polymer detection system . Negative controls comprised sections immunostained as above with omission of the primary antibody.Three areas of the brain from each case were examined including cortex, diencephalon and brain stem, to determine any site specific variations in the patterns of immunoreactivity. With each brain area, the pattern and intensity of staining were examined in three different cellular components including vessels, neurons and astroglial cells.2 was converted to a semiquantitative Vascular Expression Score (VasExp) as follows: Ang-1; low = 0–20 vessels/cm2, moderate = 20–40 vessels/cm2, high ≥40 vessels/cm2: Ang-2; low = 0–200 vessels/cm2, moderate = 200–400 vessels/cm2, high ≥400 vessels/cm2: Tie-2; low = 0–10 vessels/cm2, moderate = 10–20 vessels/cm2, high ≥20 vessels/cm2. Both scores were used in histopathological and clinicopathological correlations where continuous or semiquantitative categorical measurements where most appropriate.Vessels were considered positive for Ang-1, Ang-2 and Tie-2 when any parts forming the vessel including ECs, basement membrane, pericytes or smooth muscles were stained. The number of positive vessels in 150–300 fields in each slide at x200 magnification was used to calculate the number of positive vessels per centimetre squared. The continuous variable of number of positive vessels per cmAng-1 and Ang-2 expression on neurons and astroglial cells was classified in a semi-quantitative scoring system of both the number of cells and the intensity of expression. Cell numbers were classified according to a percentage score as follows: score 0 = percentage of stained cells 1% or less, score 1 = 1-10%, score 2 = 10-50% score 3 ≥50%. The intensity score was defined as: score 0 = no staining, score 1 = weak staining (+/−), score 2 = moderate staining (+), score 3 = strong staining (++). The neuronal expression (NeuExp) of Ang-1 and Ang-2 was represented as the sum of the percentage score and intensity score (NeuScore) and also divided into two groups of “low”  and “high” neuronal expression . Astroglial expression (GliExp) of Ang-1 and Ang-2 was classified based on a similar system combining the percentage score with the intensity score into “low”  and “high” astroglial expression . However, due to much lower expression of Tie-2 on neurons and astroglia cells, it could not be quantitated using the same scoring system as Ang-1 and Ang-2. Rather than using both the number of cells and the intensity of staining, the semi-quantitative scoring system for Tie-2 expression on neurons and astroglial cells was based on the number of cells staining as follows: “low” neuronal or astroglial expression (low NeuExp or GliExp) = percentage of stained cells 1% or less and “high” neuronal or astroglial expression (high NeuExp or GliExp) = percentage of stained cells >1%.Plasma and CSF concentrations of Ang-1, Ang-2 and Tie-2 were measured by quantitative ELISA , according to the manufacturer’s instructions, each sample tested in duplicate. Concentrations were interpolated from 5-parameter-logistic (5-PL) standard curve fitting, using the manufacturer’s recombinant human Ang-1, Ang-2 and Tie-2 proteins.A total of 62 plasma samples and 12 CSF samples from patients of severe malaria were examined, all in duplicate. Twenty-two of the 62 plasma samples and 12 CSF samples were from fatal malaria cases included in the immunohistochemical analysis. The other 40 plasma samples were from severe malaria patients in the same AQ trial, selected randomly, including fatal cases (n = 10/40) and survivors (n = 30/40).P value less than 0.05 taken as being statistically significant. Continuous variables were tested for normal distribution with the Kolmogorov-Smirnov test, . Thus data were summarized with median and interquartile values, and non-parametric tests, including Kruskal-Wallis, Mann–Whitney U test, and Wilcoxon matched-paired signed-ranks test were used to compare groups where appropriate. Correlations between continuous variables were tested by Spearman’s rank correlation.Analyses were performed using Stata/SE 9.2 , with a For IHC expression score data: due to the correlated nature of data obtained from different areas of the brain within the same patient (three areas of the brain from one patient), intraclass correlation, which affected the standard errors of the estimates, were taken into account by using clustered robust standard errors (one patient as one cluster) with univariate logistic or ordered logistic regressions where appropriate.The prognostic value of plasma angiopoietins, Tie-2 and other biomarkers was assessed by calculating the area under the receiver operating characteristic curve (AUROC). The AUROCs were compared nonparametrically by DeLong’s method.Multivariate analyses were conducted by either linear regression or logistic regression, depending on the nature of the dependent variable. Stepwise techniques were used manually in the light of clinical judgments where appropriate.Ang-1, Ang-2 and Tie-2 staining could be found in the cytoplasm and/or nucleus of a range of cells, including neurons, astroglial cells, ECs, pericytes, and perivascular smooth muscle cells, in the brain sections of malaria and non-malaria patients  and of malaria cases only is presented in Additional files Because the circulating levels of Ang-1, Ang-2 and Ang-2/Ang-1 ratio have been reported to be clinically informative biomarkers for CM, we examined the expression patterns of proteins Ang-1, Ang-2 and their receptor Tie-2 in the brain tissue of patients dying of CM and compared them to NCM. No significant differences were found between the immunostaining patterns of Ang-1, Ang-2 and Tie-2 on all three cellular components including neurons, astroglial cells and vessels in CM cases compared to NCM Figure . HoweverP < 0.001). Ang-1 expression on vessels did not differ between malaria and non-malaria cases.The incidence of high Ang-1 expression on neurons and astroglial cells was greater in malaria cases , compared to non-malaria cases  (P = 0.002). Ang-2 astroglial expression was lower in malaria compared to non-malaria (P = 0.078); which became statistically significant after adjusting for age and sex (P = 0.038).The incidence of high Ang-2 expression on neurons was greater in brain sections of malaria cases (65.22%), compared to non-malaria cases (33.33%) . The vascular expression of Tie-2 was significantly higher in malaria cases (high VasExp 40.58%), compared to non-malaria cases (low VasExp 11.1%) (P = 0.049). There was a trend of increased incidence of Tie-2 neuronal expression in malaria compared to non-malaria (P = 0.059).Reduced expression of Tie-2 was observed in astroglial cells of malaria cases, all slides of malaria cases (100%) did not express Tie-2 on astroglial cells, compared to 77.78% of non-malaria cases  was associated with the presence of microscopic haemorrhages (P = 0.013).Within the brain sections of malaria cases in this study, 40.6% showed haemorrhages. Petechial haemorrhages (88.9%) were more common than ring haemorrhage (11.1%). No significant difference in the incidence of haemorrhages was found between CM and NCM in this series. There was no correlation between the vascular or astroglial expression of Ang-1, Ang-2 or Tie-2 and the incidence or number of haemorrhages per cmP = 0.003, P <0.001, respectively).Taking all clinical groups  into account, high NeuExp of Ang-1  and Ang-2  was significantly associated with the presence of haemorrhage  and the expression of any of the angiogenic proteins in either vessels, astroglial cells or neurones.P = 0.0005, Wilcoxon matched-paired signed-ranks test). However this group was too small to determine any association between CSF levels and either plasma levels of the same marker, or clinical outcome.Sixty-two plasma samples of malaria patients were included. Only a limited number of CSF samples were available from cases where IHC had been performed on the corresponding case n = 12). Although this did not provide enough power to determine the association of the CSF levels of the angiogenic markers with the clinical groups or parameters, the CSF levels of Ang-1, Ang-2 and Tie-2 had not been previously examined in malaria patients. Median and inter-quartile range of CSF and plasma levels of Ang-1, Ang-2 and Tie-2 are shown in Table 2. AlthouP = 0.002 and P = 0.029, respectively). Ang-1 and Tie-2 concentrations were not associated with fatal outcome (Table The level of Ang-2 and the ratio of Ang-2/Ang-1 (plasma samples collected at time of admission) were significantly higher in patients with fatal outcome than survivors  curves were plotted, and the areas under the ROC (AUROC) were compared. ROC curves of other laboratory measures previously shown to be important prognostic markers of death in severe malaria were also plotted and compared with those of Ang-2 and Ang-2/Ang-1 ratio. These included plasma creatinine, lactate and TNF. Ang-2 has an area under curve (AUROC) of 0.727 (95%CI = 0.6-0.85), and Ang-2/Ang-1 ratio has an AUROC of 0.662 (0.522-0.802). The prognostic values of Ang-2 and Ang-2/Ang-1 ratio were not significantly different from each other or those of plasma creatinine, lactate and TNF  and lactate  were independently associated with fatal outcome. Ang-2 and lactate remain significant prognostic markers after adding into the model other clinical and biochemical parameters, including age, sex, presence of shock, presence of hypoglycaemia, admission Glasgow coma score (GCS), admission haematocrit and parasite count.High plasma levels of Ang-2 were significantly associated with the presences of several complications of severe malaria including fatal outcome, jaundice, anaemia, acute renal failure and hypoglycaemia Table . High AnThe presence of cerebral malaria was not related to the plasma concentrations of Ang-1, Ang-2 and Tie-2; however, patients with “pure” cerebral malaria  had significantly lower Ang-2 concentration and Ang-2/Ang-1 ratio than the other patients   Table . This inSpearman’s rank correlations were calculated between plasma concentrations of Ang-1, Ang-2 or Tie-2 and a number of clinical and biochemical markers in malaria patients at admission, including GCS, respiratory rate, haematocrit, white blood cell count, platelets, parasite count, liver function tests, creatinine, glucose, electrolytes, lactate, TNF, and arterial blood gas measures. The summary of correlations of some important markers of malaria severity was presented in Table 2). Moreover, plasma Ang-2 concentration was strongly positively correlated with CSF concentrations of quinolinic acid and picolinic acid, which have been previously shown to be strongly associated with acute renal failure in this group [Plasma Ang-1 concentration was positively correlated with the admission levels of platelets. This finding may reflect platelet production and release of circulating Ang-1 . Howeveris group . There wP = 0.47).Interestingly lactate and pyruvate levels, that could also contribute to metabolic acidosis, were not correlated with Ang-1, Ang-2 and Tie-2 levels. This finding suggested that Ang-2 was correlated with metabolic acidosis in a manner independent of lactate levels  of the variance in Ang-2  value of serum Ang-2 and have not studied the kinetics during severe disease. Lactate levels fall rapidly after treatment but in contrast remain high or rise in patients with fatal outcome despite treatment ,45. DataThe effects of Ang-1, Ang-2 and Tie-2 signalling on neurons and glial cells have not been as well studied as endothelium. It was notable that expression of Ang-1, Ang-2 and Tie-2 differed in neurons and glial cells in fatal malaria cases compared to controls. Recent studies indicate that Tie-2 signal transduction might have neuroprotective and mitogenic effects on neuronal cells. Ang-1-Tie-2 signalling promotes neural outgrowth from dorsal root ganglion cells , supportversus NCM patients. The strong correlation between plasma Ang-2 levels and Ang2/Ang1 ratio with metabolic acidosis and increasing number of clinical severity criteria suggests an association with widespread endothelial activation in severe disease, even if this does not have a specific effect on BBB function in the genesis of coma. However the Ang-2-Tie-2 pathway could be studied using in vitro models of BBB function in the presence of sequestered, malaria-infected erythrocytes to investigate this further. Given the association of plasma Ang-2 with acute renal failure in this and other series, and the relationship with metabolic acidosis in the multivariate analysis, further studies should examine the role of the Ang-Tie-2 pathway in regulating blood flow and microvascular pathology in the kidney in adult severe malaria patients.Despite the finding that Ang-2 is associated with poor outcome, the immunohistochemical staining and ELISA data provided no evidence for direct involvement of this pathway in the genesis of coma by increases in CM CM: Cerebral Malaria; NCM: Non-Cerebral Malaria; BBB: Blood–Brain Barrier; Ang-1: Angiopoietin-1; Ang-2: Angiopoietin-2; vWF: von Willebrand Factor; EC: Endothelial Cell; WPB: Weibel-Palade Body; GCS: Glasgow Coma Scale; ELISA: Enzyme-Linked Immunosorbent Assay; WHO: World Health Organization; CSF: Cerebrospinal Fluid; TNF: Tumor Necrosis Factor; SGOT: Serum Glutamic Oxaloacetic Transaminase; SGPT: Serum Glutamic Pyruvic Transaminase; BUN: Blood Urea Nitrogen.The authors declare that they have no competing interests.PP carried the laboratory experiments, prepared and analysed the data obtained, drafted and finalized the manuscript for submission with contributions from authors. IM helped supervise and perform the laboratory experiments, analyse results and reviewed the manuscript. NTHM, NPJD, NHP, TTH, NJW, GDHT collected samples and data for the study. NJW, NPJD, TWY, NMA, GDHT conceived of the study, helped analyse results, wrote and reviewed the manuscript. All authors have read and approved the final manuscript.Clinical details of malaria cases used in the immunohistochemical study.Click here for fileClinical details of control cases in the immunohistochemical study.Click here for fileAngiogenic markers expression in different cellular component across brain areas .Click here for fileAngiogenic markers expression in different cellular component across brain areas .Click here for file


Plasmodium falciparum malaria remains a major cause of mortality throughout in the tropical regions of the world. Pthophysiologic mechanisms of anemia in malaria is such as direct invasion of Red cells, anemia of chronic disease hypersplenism, Hemophagocytic syndrome and erythrophagocytosis, dyserythropoirsis, immune haemolysis and cytokine deregulation anemia of chronic disorder is characterized by moderate to mild normocytic, normochromic anemia along with microcytic hypochromic cells. Malaria occurs predominantly in children in the first three years of life. The purpose of this study was Demographic study of malaria during 2008 to 2012 in YazdThis study was an analytic-descriptive and manner descriptive study. All episodes  of disease from 2008 to 2012 which were documented in Yazd Central Health Service were carefully studied and reported. A total of 206 confirmed reported malaria patients from 2008 to 2012 were studied; Plasmodium (P). vivax species was mostly, 187(90.78%) and Plasmodium (P). Malaria species was Lesley, 1(0.49%). The mean age groups, accommodation with local malaria and years of reported outbreaks of different strains of the parasite conducted by Fisher exact Test, showed no significant difference, but the mean of foreigner immigrants of outbreaks of different strains of the parasite conducted by Fisher exact Test, showed significant difference .Although malaria has been designed on elimination program in Iran, but in the province of Yazd is reported imported malaria and its importance in causing anemia and other blood disorders is not negligible. Anemia is a condition that develops when the blood does not contain enough healthy red blood cells. These cells are the main transporters of oxygen to the organs in the body. Symptoms of anemia - like fatigue - occur because the organs aren't getting enough oxygen. There are many types of anemia. All Of them are very different in their causes and treatments. Iron-deficiency anemia, the most common type, is easily treated with dietary changes and iron supplements. However, some types of anemia may present lifelong Failure of health. There are more than 400 types of anemia, which are divided into three groups: Red blood cells can be lost through bleeding, which can occur slowly over a long period of time, and can often go undetected, decreased or faulty red blood cell production The body may produce few blood cells or the blood cells may not work properly. In either case, could lead to anemia. Red blood cells may be decreased due to abnormal red blood cells or a lack of minerals and vitamins needed for red blood cells to work, properly and excessive destruction of red blood cells -3. Malaria has been recognized as a severe and life-threatening illness for thousands of years. It still is one of the most common diseases affecting humans worldwide. The major impact of the disease is almost nearly on the developing countries, with the heaviest burden in Africa. Almost half of the total world population is exposed to the risk of contacting malaria. To summarize, malaria is the leading cause of death and disease in many developing countries. According to the World Health Organization’s World Malaria Report 2011 and the Global Malaria Action Plan, 3.3 billion people worldwide live in areas at risk of malaria transmission in 106 countries and territories. In 2012 malaria led to 216 million clinical episodes, and 655,000 deaths. An estimated 91% of deaths in 2010 were in the African region, followed by 6% in the south-east Asian region and 3% in the eastern Mediterranean region (3%). 86% of all deaths worldwide are children .Over 40% of the world’s populations are at risk of contacting malaria, which is endemic in 91 countries, mostly developing countries. Malaria has long featured prominently in the grey area between parasitological and hematological. Despite of intensive worldwide efforts to reduce its transmission, malaria remains the most serious and widespread protozoan infection of humans. In a classical European textbook of hematology published in the 1930s ,6. MalarPlasmodium falciparum. Anemia in this infection is caused by a variety of patho physiologic mechanisms, and in areas where malaria infection is endemic, co-morbidities like other parasitic infestations, iron, foliate and vitamin B12 deficiency, deficiency of other nutrients, and anemia, which is aggravated by anti-malarial drugs both through immune and non-immune mechanisms, is important considerations. In different endemic areas, β-thalassemia, α-thalassemia, Hb S, Hb E, G6PD deficiency, or ovalocytosis in different proportions interact with this infection. Finally, aberrant immune response to repeated or chronic falciparum malarial infection may produce tropical splenomegaly syndrome, a proportion of which show clone proliferation of B lymphocytes. Cooperation between chronic malarial infection and infection with E-B virus infection in producing Burritt's lymphoma is well known. Anemia involves red blood cell lyses due to parasite invasion, as well as mechanisms of intravascular hemolysis and decreased erythropoiesis. Exchange or blood transfusion is mainly recommended in the management of these patients. Hemorrhagic complications in severe malaria are relatively rare despite prominent thrombocytopenia and dysfunction in the coagulation pathway. Numerical, as well as functional changes in the white blood cell are less dramatic than other blood cell series, but still, remain a significant index of disease progression and ultimate prognosis. The province of Yazd is located exactly at the center of Iran, closed to two endemic provinces of Kerman and Sistan and Baluchistan; where generate 79% of total malaria cases in Iran. Therefore imported malaria incidence in this area is imported and is due to the presence of workers come to Yazd for work mostly as construction workers. The purpose of this study was Demographic study of malaria during 2008 to 2012 in Yazd  or foreign origin. Yazd province which is the host of local and foreign immigrants may be faced contacting malaria. Plasmodium falciparum malaria remains a major cause of mortality throughout the tropical world. Hematological abnormalities are considered a hallmark of malaria, bearing an impact on final outcome and representing indices of prognostic and follow-up value, which include severe anemia, coagulation disturbances, leukocyte numerical or functional changes and spleen involvement. Anemia is a common complication in malarial infection, although the consequences are more pronounced with  in Yazd .The study has been approved by the Ethic Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran. The present descriptive (study retrospective) was conducted to indicate epidemiological feature of Malaria in the province of Yazd, Iran. Therefore, all episodes from 2008 to 2012 documented in Yazd Central Health Service (CHS) was carefully studied and reported. Malarial patients were Iranian and foreigner people but all had resided in Yazd. In general, all suspected malarial patients are referred to central health service located in the city of Yazd and checked by physicians working in different clinics or hospital. Statistical AnalysisThe data was analyzed using SPSS Version 17 statistical software. Chi-square test was used for data analysis of qualitative variables, and values were compared using independent Fisher- exact Test. Differences were considered significant at P-values of less than 0.05. Informed consent was taken from patients and parents. Data showed that 90.78% of isolated species was P (P). Vivax, 7.28% species was Plasmodium (Lavrania).falciparum, 0.1% P (P). Malariae and 1.45% were mixed species. The mean age groups, accommodation with local malaria and years of reported outbreaks of different strains of the parasite conducted by Fisher exact Test, showed no significant difference  . 179 86. A few o. Yazd province which is the host of local and foreign immigrants may be faced contacting malaria. Imported malaria is limited in Yazd but since the province is highly active in industry and construction projects, it has become the host of thousands of male refuges from the endemic malarial areas of Afghanistan and south-eastern of Iran. Malarial anemia is an enormous public health problem in endemic areas and occurs predominantly in children in the first 3 years of life. Anemia is due to both a great increase in clearance of uninfected cells and a failure of an adequate bone marrow response. Iran is among the countries located in the Middle East with low malaria endemicity and in its some areas, there is the risk of malaria transmissionAlthough imported malaria does not depend on the age groups, but in this study, like most previous studies, the active age groups showed the highest rates of infection to imported malaria, and this is because being more susceptible to disease reservoirs and vectors . Like alBased on the result of this study, although in the province of Yazd malaria is reported as immigration  but its importance in causing anemia and other blood disorders is not negligible, Physicians, especially those in malaria- prone areas,Should be aware of the varied manifestations and maintain a high index of suspicion for the disease so that the diagnosis and treatment are timely and morbidity and mortality minimized.


The clinical manifestations of severe malaria are several and occur in different anatomical sites. Both parasite- and host-related factors contribute to the pathogenicity of the severe forms of the disease. Cytoadherence of infected red blood cells to the vascular endothelium of different organs and rosetting are unique features of malaria parasites which are likely to contribute to the vascular damage and the consequent excessive inflammatory/immune response of the host. In addition to cerebral malaria or severe anaemia, which are quite common manifestation of severe malaria, clinical evidences of thrombocytopenia, acute respiratory distress syndrome (ARDS), liver and kidney disease, are reported. In primigravidae from endemic areas, life threatening placental malaria may also be present.In the following pages, some of the pathogenetic aspects will be briefly reviewed and then data on selected and less frequent manifestation of severe malaria, such as liver or renal failure or ARDS will be discussed. P. falciparum infections, are due to severe anaemia or cerebral malaria, but different clinical manifestations also exist and vary in severity and outcome, depending on the parasite species, the organ involved and the access to care.Malaria is a life-threatening disease which in 2010 killed more than 600,000 individuals, mainly children under 5 years of age, and pregnant women.P. falciparum differs from other human malarial species in that infected red blood cells (IRBC) do not remain in the circulation for the entire life cycle. After 24–32 hours, when young parasites mature from the ring to the trophozoite stage, IRBC adhere to endothelial cells in the microcirculation of various organs. This phenomenon, termed “sequestration”, is believed to occur mainly to avoid splenic removal of IRBC. Sequestration causes microcirculatory obstruction, impaired tissue perfusion and inflammatory cells activation and it is linked to the severity of the disease.At schizonts rupture, from 4 up to 36 daughter merozoites, depending on the Plasmodium species, are released into the circulation and invade fresh RBC to perpetuate the asexual life cycle. At the same time, a large amount of toxins and parasite products are also released and cause the activation of the innate immunity, the release of inflammatory mediators and the symptoms associated with the malaria attack, such as fever.A combination of mechanical circulatory stress due to sequestration and excess inflammatory response contribute to the most severe manifestations of malaria including, but not limited to, cerebral malaria or anaemia. The present review will summarise some of the pathogenetic aspects of severe malaria and then it will report on selected and less frequent manifestation, such as renal failure or acute respiratory distress syndrome (ARDS).in vivo has been related exclusively to P. falciparum.in vitro to some endothelial cell lines and placental cryosections has also been seen in reticulocytes infected with P. vivax.5Cytoadherence, the ability of parasites to adhere to the vascular endothelium, was recognized as early as 1892 by Marchiafava and Bignami.Parasite sequestration is thought to be the pathological base of the severe manifestation of malaria, including cerebral malaria.7Sequestration is mostly mediated by mature parasite forms, approximately 20 hours after RBC invasion. The parasites produce new proteins that are exported to the IRBC surface and increase the adhesiveness of IRBC to the endothelium. During their 48-hour life cycle, the parasites can remain sequestered for 24 hours in the deep microvasculature. In this manner, they evade clearance by the spleen, and make the diagnosis more difficult since they are not seen in the peripheral blood.P. falciparum has been attributed to different class of molecules of parasite origin and ligands present on the human endothelium. Among those, the P. falciparum histidine-rich protein (PfHRP) and the erythrocyte membrane protein 1 (PfEMP1), have received significant attention. PfHRP is related to the establishment of knobs, symmetric membrane arrangements which appear on the surface of infected RBC, while PfEMP1, a multimeric protein encoded by the var (variant) gene10Sequestration of Box 1P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a multimeric protein encoded by a family of roughly 60 variant (var) genes.4var gene at a time, maintaining the remaining var genes in a transcriptionally silent state. Epigenetic factors control the exchanges in the expression of the var gene generating antigenic variation of the IRBCs surface.4Each individual parasite expresses a single PfEMP1 is localized in the Maurer’s clefts, from where it traffics to the erythrocyte membrane, and on the IRBC surface. Together with knob-associated histidine-rich proteins (HRPII), PfEMP1 forms protrusions, known as knobs.133PfEMP1 is crucial for malaria virulence and pathogenesis.134PfEMP1 mediates the adhesion to the endothelium, so parasites avoid destruction by the immune system and the spleen, and also their detection in the peripheral blood. The multiple adhesion domains are localized in the extracellular portion of PfEMP1 which is recognized by host receptors. There are approximately 11 receptors involved in adhesion of PfEMP1 to the endothelium. CD36, ICAM-1, chondroitin sulphate A (CSA) and trombospondin are particularly important for sequestration; whereas CR1, the complement receptor is involved in rosetting.135Sequestration is also seen during gestational malaria, when parasites adhere to the placenta. PfEMP1 is again the main adhesion receptor which adheres to the trophoblastic villous endothelium mainly through chondroitin-4-sulfate (CSA) and other sugars such as glycosaminoglycans and possibly hyaluronic acid (HA). As discussed later, malaria in pregnancy can be severe for mothers and induce fetal death especially during the first pregnancy, when women usually lack sufficient immunity against CSA-binding parasites.14Rosetting is one of the forms of cytoadherence of late stages IRBC to non-parasitized red blood cells and/or platelets.19P. falciparum, P. vivax, and P. ovale are all able to form rosettes,P. falciparum have been associated with severe malaria, and especially in African children they may enhance microcirculatory obstruction.Saimiri sciureus.In vitro, it has been found that TNF-α enhances the expression of ICAM-1,31The immune response to the parasite is complex and not completely understood, and it is essentially both species and stage specific.Malaria is an important cause of morbidity, but not everyone infected with the malaria parasite becomes seriously ill or dies. In areas of stable endemicity, repeated exposure to the parasite leads to the acquisition of specific immunity, which restricts serious problems to young children; malaria in older subjects causes a relatively mild febrile illness. However, individuals with no previous experience of malaria become ill on their first exposure to the plasmodium parasite. They develop a febrile illness which may become severe and in a proportion of cases may lead to death.36Immunity to malaria is provided by innate mechanisms, as we summarized above, and subsequently by the development of acquired immunity. Following repeated infections, people living in malaria endemic areas gradually acquire mechanisms, which helps limiting the inflammatory response to the parasite that causes the acute febrile symptoms. Sterilizing immunity is never achieved. Parasites have evolved to maintain a balanced relationship with their human hosts. In this sense they can partially escape from the host effector mechanisms, while hosts are able to develop partial immunity against the parasite. This type of immunity requires repeated infections, takes years to develop and usually lasts shortly. Natural acquired immunity is called premunition since low parasite burden often persists in the presence of circulating antibodies to the various stages, in the absence of clinical disease. In children whithout circulating antibodies to the parasite, premunition is lower. The poor and slowly developing immune response to malaria is partly due to parasite immune evasion strategies: antigenic polymorphism, shedding of parts of parasite proteins, cross-reactive antigen epitopes of developmental stages, prolonged exposure to endemic malaria and limited immunogenicity of antigens. In stable endemic areas, a heavy burden of morbidity and mortality falls on young children. Children born to immune mothers appear to be relatively immune to malaria for a period.38Anaemia is one of the most common causes of morbidity and mortality in malaria infection particularly in pregnant women and in children.40The potential mechanisms contributing to malarial anaemia can be divided into two categories: increased destruction of parasitized and un-parasitized erythrocytes  and decrease of RBC production . Co-infection with bacteremia, HIV-1 and hookworm, malnutrition and repeated malarial infections in holo-endemic countries may also contribute to decrease haemoglobin levels.43Parasitized red cells rupture caused by plasmodium cycles and clearance of deformed parasitized and un-parasitized erythrocytes are the principal causes of malarial anaemia. Phospholipid asymmetry, membrane rigidity and reduced deformability are the mechanisms involved in the premature removal of un-parasitized red cell.46Dyserythropoiesis plays an important role in the pathogenesis of anaemia; examination of bone marrow from children with severe anaemia showed hypercellularity, mild to normal erythroid hyperplasia and abnormal features of late erythroid progenitors. Hemozoin and its phagocytosis by bone marrow macrophages has been proposed to cause dyserythropoiesis either through direct accumulation in the bone marrow and generation of reactive toxic species or activation of the innate immune response.42The immune response is central in the pathogenesis of malarial anaemia; parasitized red cells, hemozoin and malarial antigens activate monocyte and lymphocyte response. Pro-inflammatory and anti-inflammatory mediators, including TNF-α, IFN-γ, IL-23 and IL-1, chemokines and growth factor are produced and contribute to anaemia. On the contrary, IL-12 and IL-10 seems to be protective cytokines since low levels are found in severe malarial anaemia.48Erythropoietin (EPO) levels are increased during malaria anaemia, but erythroid progenitors response is not adequate, particularly during chronic malaria infection, resulting in low reticulocytosis.Pro-inflammatory cytokines play an important role also in iron delocalisation pathway of malarial anaemia. TNFα induces re-localisation of ferroportin, an important protein abundant in the reticulo-endothelial system that mediates macrophage iron release and intestinal iron absorption. Relocalisation of ferroportin induces decrease of iron absorption and release from macrophage cells;50STAT6, a member of signal transducer and activator of transcription family proteins, seems also to be involved in malarial anaemia through the activation of the regulatory cytokines, in particular IL-4 and IFN-γ, resulting in erythropoietic suppression during blood stage malaria.53falciparum and P. vivax infections. Incidence is high both in children and in adults.P. falciparum and P. vivax malaria had lower platelet count, higher von Willebrand factor (VWF) concentration, lower ADAMTS13 activity and ADAMTS13 antigen concentrations.Thrombocytopenia is very common in malaria, usually during the early stage of P. P. falciparum malaria mortality rate was 35,4% and mortality was principally due to shock, acute renal failure, seizure and ARDS.P. falciparum infection, case reports due to P. vivax,P. ovaleP. knowlesiP. vivax caused three maternal deaths in a cohort of 221 patient in India during one year of observation.P. knowlesi, the fifth plasmodium species infecting humans, mostly in Southeast Asia, may cause life threatening diseases, as well.P. knowlesi infection was respiratory distress.75Deep breathing, respiratory distress and pulmonary oedema are some of the clinical feature defining severe malaria according with WHO classification.et al. showing that patients frequently have subclinical impairment of lung function, such as small airways obstruction, impaired alveolar ventilation, reduced gas exchange and increased pulmonary phagocytic activity.et al. in DBA/2 mice infected by P. berghei ANKA 77 observed that 60% of mice had dyspnea, airways obstruction and hypoxemia; pleural effusion, pulmonary hemorrhagies and oedema were also found as pathological findings, demonstrating the role of inflammation in ALI development. Moreover, high levels of vascular endothelial growth factor (VEGF) were found in mice with ALI, supporting the importance of increased vascular permeability in malaria respiratory failure. Another studyP. berghei K173, showed that proteins and inflammatory cells mainly CD4+ and CD8+ lymphocytes, neutrophils and monocytes accumulate in the lungs of infected mice. These results were confirmed by P.E. Van den Steen et al.,79Little is known about the pathogenesis of malaria associated ALI/ARDS. Inflammatory mediators and increased endothelial permeability may play an important role, while parasite sequestration may take a minor role. Pulmonary manifestations of uncomplicated malaria were analysed by Anstey P. falciparum are based on in vitro models or post mortem observations. It was shown in vitro that P. falciparum merozoite proteins could increase endothelial permeability, while P. falciparum IRBC did not show the same properties suggesting that the effects of the parasite on the pulmonary endothelium are probably mediated by the activity of Src-family kinases.P. falciparum -IRBC occurs in the lungs, even if to a lesser extent than in the brain, skin or intestine.80Most of the studies with P. falciparum and could be partially attributed to the sequestration and rosetting of infected RBC in the pulmonary microcirculation;P. vivax malaria.et al. suggested that the pathogenesis of lung injury by P. falciparum include cytokine-induced damage or direct effects of sequestration of parasitized erythrocytes, while ARDS caused by P. vivax may be due principally to dysregulation of cytokines production.P. knowlesi infection suggests parasite-specific effects that increase pulmonary capillary permeability,72Different pathological presentations of ARDS were observed among the different species of human malaria: the greatest severity and frequency of cases are due to P. falciparum malaria varied from 5.3% to 62% and from 2.5% to 21%, respectively, in different reports,P. vivax infection.According to the World Health Organization liver dysfunction is an uncommon occurrence in malaria, while jaundice is not unusual. The incidence of jaundice and liver dysfunction in P. falciparum infection and is more frequent in low transmission areas;P. vivax.P. malariae causes chronic and progressive glomerulopathy, known as quartan malaria nephropathy (QMN); only few cases of MARF caused by P. malariae has been published.103Kidneys in malaria are involved in two different manners: acute and chronic diseases. Acute renal failure (ARF) is one of the most challenging diseases in tropical countries and malaria plays an important epidemiological role.P. malariae infection and renal involvement was only coincidental.P. falciparum acute renal failure is more common in adults; its pathogenesis is complex and it includes mechanical and immunologic factors, volume depletion, hypoxia, hyperparasitemia and other factors.P. falciparum malaria;107Quartan malaria nephropathy (QMN) is frequently seen in African children and it is clinically associated with oedema and hypertension; urine analysis shows often proteinuria and microhematuria.P. falciparum and P.vivax infections, probably due to the sequestration of parasitized red cells in the skeletal capillaries and consequent vessels occlusion.110Rarely, acute renal failure has been associated to rhabdomyolysis in Black water fever (BWF), a rare but severe complication of severe malaria, is characterized by fever, intravascular haemolysis with haemoglobinuria, dark urines and often acute renal failure.P. falciparum infection, less common in P. vivax malaria; P. falciparum and P. vivax placental mixed-infection can occur.P. vivax placental malaria may lead to the same adverse events as P. falciparum infection, even if with milder consequences.124Malaria during pregnancy is associated to high morbidity and mortality both for the mother and the child.In high endemic areas, while adults are less susceptible, pregnant women are commonly susceptible to malaria infection because pregnancy causes a transient depression of cell mediated immunity. Age under 25 years and primiparity are both risk factors for developing placental malaria;125P. falciparum and P. vivax are sequestered within the placenta and they accumulate in intervillous spaces; trophozoite and schizont forms also accumulate in the placenta.126Alterations of materno-fetal blood exchange are the basis of placental malaria. During infection, parasitized red cells both from P. falciparum parasitized erythrocytes adhere to chondroitin sulphate A expressed in placenta; thus, placenta selects for strains of P. falciparum with a CSA-binding phenotype. Primigravidae who have not been exposed to P. falciparum CSA-binding phenotype are still susceptible to placental infection, while multigravidae have developed antibodies during the first successful pregnancies.P. vivax parasitized erythrocytes can also cytoadhere to the placenta but their mechanism is not clarified, yet.124var2csa genes compared to non-pregnant women. The multiplicity of var2csa-type genes confers to P. falciparum parasites a greater capacity for antigenic variation and evasion of immune response.var genes, different from var2csa, were found in 67% of placental isolates, revealing the importance of other adhesion molecules during pregnancy.129Molecular studies demonstrate that IRBC binding to chondroitin sulphate A (CSA), expressed on the apical membrane of the placental syncytiotrophoblast epithelium, is mediated by VAR2CSA antigen, a variant of the PfEMP1 family proteins. There are multiple genes encoding for different VAR2CSA antigens; a study performed in Cameroon shows that the parasites infecting pregnant women living in high transmission areas have an increased copy number of Complement activation may play an important pathogenetic role during placental malaria. C5a, a factor derived from the complement cascade, is increased in peripheral blood and placental blood of pregnant women with malaria compared with pregnant women without malaria infection.132The pathogenicity of severe malaria infection is complex and it is regulated by both parasite and host factors. Cytoadherence of IRBC to the vascular endothelium and rosetting are unique features of malaria parasites which are likely to contribute substantially to the vascular damage and the consequent excessive inflammatory/immune response of the host. This can occur in many different organs, a feature that can partly explain the complexity of the clinical manifestations occurring in severe malaria. In this context, adequate clinical management of malaria patients requires first an accurate diagnosis, then appropriate antimalarial treatment, associated with adjunct supportive therapies which need to be adapted to the different clinical presentation of the disease.


Malaria within the Greater Mekong sub-region is extremely heterogeneous. While China and Thailand have been relatively successful in controlling malaria, Myanmar continues to see high prevalence. Coupled with the recent emergence of artemisinin-resistant malaria along the Thai-Myanmar border, this makes Myanmar an important focus of malaria within the overall region. However, accurate epidemiological data from Myanmar have been lacking, in part because of ongoing and emerging conflicts between the government and various ethnic groups. Here the results are reported from a risk analysis of malaria slide positivity in a conflict zone along the China-Myanmar border.Surveys were conducted in 13 clinics and hospitals around Laiza City, Myanmar between April 2011 and October 2012. Demographic, occupational and educational information, as well as malaria infection history, were collected. Logistic models were used to assess risk factors for slide positivity.Plasmodium vivax infections were younger than those with Plasmodium falciparum. Furthermore, males were more likely than females to have falciparum infections. Patients who reported having been infected with malaria during the previous year were much more likely to have a current vivax infection. During the second year of the study, falciparum infections among soldiers increased signficiantly.Age patterns in These results fill some knowledge gaps with regard to risk factors associated with malaria slide positivity in this conflict region of north-eastern Myanmar. Since epidemiological studies in this region have been rare or non-existent, studies such as the current are crucial for understanding the dynamic nature of malaria in this extremely heterogeneous epidemiological landscape. While mo Africa ,5. Thereo Africa ,7, meanio Africa .Within the GMS, the malaria distribution is tremendously heterogeneous. While some countries are aiming to eliminate or eradicate malaria in the near future, some are struggling to control malaria. Furthermore, malaria heterogeneity in each GMS country is reflected in the clustering of malaria along international borders. The uneven distribution within a country often requires that limited control resources are targeted towards these hotspots. These efforts in turn rely heavily on accurate knowledge of malaria epidemiology.Myanmar has the heaviest malaria burden in the GMS and, with its unique geographical location, it plays an important role in regional malaria transmission. In the east it borders Thailand, where decreased sensitivity to artemisinins has recently been detected , whereasEven within Myanmar malaria is distributed unevenly and the most malarious regions border China, Thailand and India. These same sub-regions tend to also have high proportions of ethnic minorities who are frequently at the margins of society and lack adequate health care . High sePolitical instability and military conflicts have likely exacerbated the malaria situation in these regions. Myanmar is extremely diverse ethnically, with around 135 different self-defined ethnic groups living within its borders, and several of these groups have been involved in active military conflicts with the ruling government over the last half century . In addiOver the past two decades, particularly since the launch of the Mekong Malaria Initiative by the WHO, intensified malaria control efforts have greatly reduced malaria mortality and morbidity in a number of GMS countries . These aShortly after the present study began in 2011, an armed conflict erupted between the Kachin Independence Army and the Myanmar government, making wide-scale epidemiological sampling and surveillance challenging at best. However, medical staff was maintained at 13 clinics along the China-Myanmar border and demographic data, blood slides, and questionnaires were collected from febrile patients who presented at the clinics. The aim of this study was therefore to identify risk factors associated with slide positivity among febrile clinic attendees in these 13 clinics. Slide positivity studies can subsequently help inform clinical decision making and provide at least some information about the epidemiological situation in the catchment area .The study area included 13 clinics and hospitals in and around Laiza city, Kachin State, Myanmar along the China-Myanmar border . Multiple logistic regression models were used to test for statistically significant differences between sex, age groups, occupation  were found to be infected with malaria parasites by microscopy Table . All fouum Table . PlasmodP. vivax and P. falciparum cases for the period of April-October in 2012.Trends in case numbers from these clinics varied greatly over time, but basically followed the rainy season Figure , which tP. vivax and P. falciparum infections . Interactions between age and sex were also tested. The males in the age groups 15 to 24 and 25 to 44 were significantly more likely to have positive vivax slides, when compared to females of the same age groups. The same interaction term in the falciparum logistic model did not reveal any significant combinations.P. vivax infections. However, soldiers were less likely than farmers to have P. falciparum positive slides . This pattern changed drastically over the study period , as the aforementioned conflict emerged, and an interaction between study year and occupation revealed a statistically significant increase  in falciparum slide positivity in soldiers during 2012  of vivax infection when compared to those with no recent history of malaria infection. There was no statistically significant effect of recent infection history for falciparum infections. However, a comparison of only falciparum and vivax positive patients indicated that those with recent malaria infections had about half the odds  of falciparum infection.The demographic characteristics of individuals who reported having a history of malaria infection (having been infected in the last year) were further investigated using a logistic model  accounted for approximately 7% of all infections , are both parsimonious with transmission occurring in or near the village, where children spend the majority of their time. Furthermore, this study showed that individuals with a history of infection had about three and a half times the odds of having a vivax positive slide. Febrile patients with a history of malaria infection had slightly older age profiles (aged 15 through 34) and were disproportionately male. It is possible that these individuals are migrants who are at increased risk of infection; anecdotally, patients who reported having travelled in the previous two weeks were also more likely than others to report previously having been infected. However, we are cautious in these interpretations since patients with reported infection histories only made up approximately 4% of the patients included in this study. It is also possible that these are actually recrudescence rather than true re-infections, however it is impossible to differentiate the two from slides alone. If these are true re-infections, this lends further support to the idea that certain subgroups within the population are at greater risk of infection .Age-dependent patterns of malaria were observed for both For falciparum infections, slide positivity peaked between ages 15 and 34, the highest being in the 15 to 24 age group. Males were also at increased risk of falciparum infection, however an interaction between sex and age in the logistic model did not reveal any significant interactions. Regardless, this pattern may be the result of a number of different factors. For example, if falciparum occurs at lower transmission intensity than vivax, it is possible to see an average age of infection that is slightly older in comparison to vivax infections. Conversely, there is evidence that as transmission intensity increases, the age of peak morbidity decreases -28. ThisAt least one study has also suggested that in regions with both vivax and falciparum infections, immunity to vivax may develop more quickly, leading to an older age pattern in symptomatic falciparum when compared to symptomatic vivax . HoweverP. falciparum cases among soldiers  during the second year of this study  are valid. Future research will take a more detailed look at migration in this region. Also it is important to note that among all febrile patients who presented at the clinic, only about 10% actually had malaria infections. This means that most of these patients had a health condition, with fever as a symptom, but that only a small fraction of them received treatment from the malaria clinics. While this study is not able to address this problem, the conditions surrounding the conflict in this region also make crowd diseases a potential public health threat and future efforts should be directed at diseases other than malaria alone.Regardless, this study provides novel information about malaria slide positivity and associated risk factors for a conflict region in Kachin State, along the China-Myanmar border. Economic under-development and poor health infrastructure are probably responsible for the persistent levels of malaria endemicity in this and other similar areas, whereas recent military conflicts further aggravate healthcare delivery in this region. Thus, as a result of these problems, accurate epidemiologic and demographic data from this region are scarce. Such data are important not only for public health efforts in this region but also for the overarching GMS since Myanmar is thought to act as a reservoir for falciparum malaria and potentially as a conduit through which drug resistant malaria can spread . Given tGMS: Greater Mekong Subregion; WHO: World Health Organization.The authors declare that they have no competing interests.ZY, GZ, ML, GY, LC, and YW conceived and planned the study. NL, ZY, GA, JD, ML, and YW carried out field surveys and microscopic diagnoses. QF and YW confirmed diagnoses in the laboratory. NL, DMP, SAM, QZ GY, and YW, analysed the data. NL, DMP, SAM, LW, and YW wrote the paper. All authors helped in editing and revising the paper and all authors read the final version and approved of its submission.Plasmodium vivax cases by occupation over time. Description: Area stacked chart indicating the number of cases of P. vivax cases, and the occupational groups to which they are attributed, over the study period.Click here for filePlasmodium falciparum cases by occupation over time. Description: Area stacked chart indicating the number of cases of P. falciparum cases, and the occupational groups to which they are attributed, over the study period.Click here for fileThe allocation of malaria cases by occupation groups listed in the original questionnaire. Description: The second column indicates the way that the original occupational groups were grouped to avoid problems associated with small numbers.Click here for fileThe distribution of malaria cases by occupation groups used in analyses. Description: This figure indicates the distribution of malaria cases among the occupation groups that were used in the main analysis ). Description: The data provided here are the results of the main logistic regression model (shown in Additional file Click here for fileLogistic model showing predictors of reported bed net usage. Description: The data provided here are the results of an analysis into demographic risk factors associated with a patient reporting having used a bed net within the previous month.Click here for fileLogistic model showing predictors of having recently travelled. Description: The data provided here are the results of an analysis into demographic risk factors associated with a patient reporting having travelled within the previous two weeks.Click here for fileLogistic model results for risk factors associated with malaria slide positivity. Description: The data provided here represent the results of the main statistical analysis of the demographic risk factors associated with slide positivity for P. vivax infections and P. falciparum infections.Click here for fileLogistic model for history of malaria infection. Description: The data provided here are the results of an analysis into demographic risk factors associated with a patient reporting having had a previous malaria infection within the previous year.Click here for file


Building on previous acceptability research undertaken in sub-Saharan Africa this article aims to investigate the acceptability of intermittent preventive treatment of malaria in infants (IPTi) in Papua New Guinea (PNG).A questionnaire was administered to mothers whose infants participated in the randomised placebo controlled trial of IPTi. Mothers whose infants participated and who refused to participate in the trial, health workers, community reporters and opinion leaders were interviewed. Men and women from the local community also participated in focus group discussions.Respondents viewed IPTi as acceptable in light of wider concern for infant health and the advantages of trial participation. Mothers reported complying with at-home administration of IPTi due to perceived benefits of IPTi and pressure from health workers. In spite of patchy knowledge, respondents also demonstrated a demand for infant vaccinations and considered non-vaccination to be neglect. There is little evidence that IPTi has negative impacts on attitudes to EPI, EPI adherence or existing malaria prevention practices.The degree of similarity between findings from the acceptability studies undertaken in sub-Saharan Africa and PNG allows some generalization relating to the implementation of IPTi outside of Africa: IPTi fits well with local health cultures, appears to be accepted easily and has little impact on attitudes towards EPI or malaria prevention. The study adds to the evidence indicating that IPTi could be rolled out in a range of social and cultural contexts. Intermittent preventive treatment (IPT) of malaria involves the administration of treatment doses of an anti-malarial drug at predetermined intervals, regardless of parasitaemia or symptoms. IPT during pregnancy (IPTp) is linked to ongoing routine antenatal care and IPT for infants (IPTi) is delivered through the Expanded Programme of Immunization (EPI) .Various studies in sub-Saharan Africa have shown that IPTi with sulphadoxine-pyrimethamine (SP) given at the time of routine vaccinations in the first year of life reduces the incidence of clinical malaria by between 20% and 59% , and by Plasmodium falciparum and Plasmodium vivax), such as South-East Asia or Oceania, it is crucial to ensure that IPTi is socially and culturally acceptable in distinct social and cultural settings outside of sub-Saharan Africa.Although the acceptability research published to date provides a comprehensive insight into community responses to IPTi delivered in sub-Saharan Africa, there are no published data on the acceptability of IPTi outside of sub-Saharan Africa. Given that IPTi has the potential to reduce the malaria-related morbidity and mortality for infants in other regions with a significant level of malaria mixed endemicity , this article aims to:1. Describe knowledge, perceptions, experiences and responses relating to IPTi and EPI of trial participants, community members, and local health care providers2. Identify and understand the mutual interactions between perceptions of, attitudes to and experiences with EPI and IPTi.3. Identify and understand local barriers to the acceptance of and long-term adherence to IPTi.4. Identify wider socio-cultural, national and regional factors that affect, or may affect, the implementation or acceptability of IPTi.The acceptability research undertaken in PNG was carried out alongside a randomized placebo-controlled clinical trial of IPTi, which was run by the Papua New Guinea Institute of Medical Research (IMR) and carried out under the auspices of the IPTi Consortium with the financial support of the Bill and Melinda Gates Foundation. The trial tested the following drug regimens: single dose of SP associated to three days of either amodiaquine or artesunate given four times at three, six, nine and 12 months following the PNG EPI schedule. Each dose of the three-dose regimen was given on consecutive days: trial staff gave the first dose; the second and third doses were given to mothers to administer to their infants the following days. The study drugs were administered in collaboration with local health facilities responsible for operating Mother and Child Health (MCH) clinics, which, due to the scattered nature of settlements in PNG, deliver EPI through monthly village outreach services.Although the trial commenced in two areas of PNG, East Sepik Province and Madang Province, in 2008, the East Sepik site was closed due to a lower than excepted malaria incidence. The acceptability research therefore focused upon Madang Province, a mainly coastal lowland (0-500 m) with a high year-round malaria transmission rate. The study was carried out about 60 km north of Madang town.PNG is renowned for its linguistic and cultural diversity, and in the study area six languages coexist. However, this contrasts with the lack of socio-economic differentiation in rural areas. In Madang province, subsistence farming of a range of crops  is the main livelihood activity. In addition, both men and women migrate to Mandang town, which is one of the few urban centres in PNG.The methods employed in this study were based on those used in the acceptability studies carried out in sub-Saharan Africa -11. In PRespondents were drawn from four villages within the IPTi trial recruitment area. Initial sampling was based on convenience, e.g. mothers who were willing to be interviewed were recruited through study clinics. As the study developed theoretical sampling was used: participants were recruited on the basis of the research team's developing understanding of the field and the questions that emerged from the ongoing analysis of already collected data. The numbers of questionnaires, interviews and FGDs , approval number 0807.A number of key factors have emerged from the data that influence the acceptability of IPTi in positive or negative ways. These factors relate to local attitudes towards infant care and vaccinations, the clinic environment, the IPTi regimen and the nature of the clinical trial context.The interviews with mothers whose infants participated in the study indicate that there is common awareness of and demand for EPI vaccinations. Although the mothers were unable to name all the diseases that vaccination prevents (56% [119/213] could name some), they knew that vaccination prevented diseases or provided protection (91% [194/213]), were positive towards vaccination and often distressed by the possibility that their children may miss out on vaccination. Although over 20% (47/213) of mothers stated that vaccination provides prevention against malaria, it is unclear to what extent this was due to confusion with the IPTi study. Vaccinated children were viewed as healthy; indeed keeping the child healthy was cited as the reason for vaccinating children by over 40% of mothers (87/213). There was debate about whether vaccination provided complete or partial prevention for diseases, with general opinion leaning towards partial prevention: vaccination gave some protection, but complete prevention depends on other factors; the disease would develop more slowly giving mothers chance to go to the clinic; or, the child could be sick again in the future.Reports of vaccination-related side effects were minimal (2% [5/213] of participant mothers reported side effects), and descriptions focused on mild fever or passing distress. Although relatives and neighbours were said to complain occasionally of children crying after vaccination, mothers were able to dismiss these complains and they did not prevent them from vaccinating their children. Children receiving multiple injections during a single clinic visit was said to cause concern amongst mothers and relatives. Sometimes relatives questioned mothers about the number of injections that the child had received and said that two was too many. One mother reported getting angry after seeing blood drip down her child's arm after vaccination.In spite of the concerns about multiple injections, very few mothers reported factors preventing them from vaccinating their children . However, in the qualitative interviews, other factors mentioned included: health workers reprimanding mothers for missing previous appointments; difficulties in meeting the costs levied at the clinic; and, laziness.Although the data suggest that mothers are responsible for the healthcare of the children, taking them to the clinic and caring for them in the home, fathers often play the role of ultimate decision-maker, to whom women have to seek permission to travel to the clinic. Fathers also take a more prominent role in emergencies. Whilst respondents did not report that husbands prevented them from vaccinating their children, there were scattered reports, offered by mothers, of other women's husbands preventing their wives from participating in the IPTi trial. However, one health worker suggested that this might be an easy excuse to avoid refusing directly. The women who participated in the trial reported having made the decision to participate with their husbands, who agreed because they saw the benefits.The data revealed only scattered accounts of the use of non-biomedical means of preventing disease: two mothers reported using "paw paw" seeds to prevent disease, and one mother specifically mentioned preventing malaria; one mother referred to herbs more generally; and, a health worker described the use of amulets for spiritual protection by members of the local Catholic church. Herbal remedies were more popular for curing rather than preventing: respondents described a range of remedies for mild diseases such as cough, cold, diarrhoea and fever. Mothers, however, preferred that serious diseases be dealt with in the clinic.Two-thirds of mothers interviewed knew that the IPTi trial involved malaria prevention (67% [143/213]) and almost three-quarters knew they were participating in research (74% [157/213]). However, the vast majority (87% [186/213]) were unsure about the names of the drugs used, and only four named Fansidar (the brand name for SP) as one of the study drugs. Nevertheless, the mothers were almost universally content with their experiences of the trial: the IPTi drugs, the outreach clinics and the free treatment. Mothers reported a decrease in malaria amongst the participating infants and commented more generally on the health of the infants, comparing it favourably with that of other children who did not participate. They also reported that study participation had brought broader benefits as they travelled less often to the clinic and had more time to do other activities such as work. Indeed, mothers commonly enrolled more that one of their children in the study. Trial staff were considered diligent and caring, compared to other health staff who were rude, slow to deal with mothers, and did not give proper care. One mother conceded that this might be due to the health workers' heavy workload. A common concern expressed by respondents was what would happen when the IPTi trial came to an end in the area.Reports of side effects from the IPTi drugs were also rare: two mothers referred to the child's body being weak, but that they quickly recovered; a third, described how her child was seen by one of the trial doctors after suffering a reaction to the trial drugs. In none of the cases was the mother discouraged from continuing her participation.IPTi was delivered in three doses at four intervals to infants who participated. Two doses were left with the mother to give to the child the following days. Fifteen percent of mothers (31/213) reported that they sometimes did not give the IPTi drug as instructed. The majority, who reported having followed the instructions, did so out of the belief that the drugs would help the child and not doing so would be akin to neglect or due to pressure from health workers.Respondents reported that during the trial and particularly the recruitment phase there were reports that blood sampled from participants was being sold by the local research institute. People with less experience of research, such as men, the elderly and the residents of remote villages, were said to spread these rumours. Indeed, the majority of participant mothers surveyed reported that the blood was used for diagnostic purposes . One woman, who refused to participate in the IPTi trial, did so because she believed that the blood would be used for transfusion, something that she disagreed with. Although there were concerns from two participants about their children losing blood and 16% (33/213) of mothers were unsure about the purpose of the blood sampling, many mothers paid little attention to these complains. The majority of the mothers who refused to participate or dropped out from the study did so because of the rumours associated with blood sampling and not because of IPTi per se.There is no evidence that IPTi had a negative impact upon EPI adherence. Given the support that the staff employed by the clinical trial gave to the local healthcare system, in the local clinics and at monthly outreach visits to the communities, it is likely that EPI adherence increased during the trial. Furthermore, all participant mothers were positive about their participation.Participant mothers perceived IPTi as offering complete prevention from malaria: 98% (208/213) of mothers stated that after IPTi their children would not get malaria. This finding is however contradicted by the extent of bed net usage: over three-quarters (77% [163/213]) of participant mothers reported using bed nets as a means of malaria prevention. There was however debate about the effectiveness of bed nets. The new bed nets that were being handed out to trial participants were considered effective at repelling the mosquitoes, in contrast to the less useful older nets. One health worker attributed the popularity of bed nets, to previous campaigns of mosquito eradication that had left a strong message about the importance of preventing mosquito bites.In a similar fashion to acceptability studies undertaken in Africa , the datThe presence of the IPTi trial in PNG did not negatively affect participants' attitudes towards EPI vaccinations, nor did it influence their other malaria prevention practices, as was the case in the sub-Saharan African sites -11. HoweOne of the main determinants of acceptability is whether recipients and other community members perceive the intervention as performing as they think it is supposed to, and whether the benefits are perceived to outweigh the disadvantages. In the case of the clinical trial of IPTi in PNG, participant mothers understood that IPTi was intended to prevent malaria in infants and they identified a positive effect upon the health of participating infants and perceived a decrease in malaria incidence. In a similar fashion to the clinical trial sites, where acceptability studies have been undertaken  partBecause, in clinical trial settings, it is difficult to isolate the advantages and disadvantages linked to trial from those linked to the intervention, it is challenging to distinguish what precisely motivates mothers to attend clinic and adhere to treatment, and this makes it difficult to assess how they would respond in a more routine implementation setting. However, given the similarities with the response in the intervention sites in Africa it seems likely that the response in "natural" settings will not be very different. In the intervention studies with SP in Malawi and Ghana, 'IPTi was simply an unnoticed addition to an already routinized EPI' .During the PNG trial of IPTi participants had greater opportunity for non-adherence than in the other clinical trials of IPTi: mothers were given the two last doses of the trial drug to administer themselves at home. Although perhaps inflated by desirability bias, the majority of mothers reported having always given the doses as instructed, motivated by the benefits they perceived that the previous doses had proportioned their child (or children). Although a single dose drug regimen for IPTi would be ideal, in order to avoid potential non-adherence, the results from this study suggest that if they perceive benefits, mothers are prepared to administer drugs for prevention unsupervised. Data from other acceptability studies, where health workers sometimes provided IPTi drugs for mothers to administer at home, despite being instructed not to, suggest that some mothers did give the drugs correctly. Other mothers forgot and some, given that they thought the drug was to cool the post-vaccination fever, only gave if the child had fever ,11. The Respondents' concerns about the care that their infants would receive when the trial ends highlight the broader issue of the feasibility of implementing IPTi in PNG and in other countries with resource-poor health systems. Research in sub-Saharan Africa has, however, shown IPTi to be a cost-effective intervention  and piloThe data collected in PNG illustrate additional, striking similarities to those from acceptability studies undertaken in sub-Saharan Africa. For example, mothers in all sites were familiar with vaccination as prevention yet uncertain regarding particular diseases prevented and the nature of the prevention. Also the key barriers to attending clinics, mainly distance and transport and treatment costs, were common in sub-Saharan Africa and PNG. There were also common complaints about  health staff that criticize and chastise mothers in the clinic . The datA clinical trial is not the ideal context to investigate the acceptability of any intervention as many of the factors that affect a community's response to the intervention itself are influenced by the presence of the trial. However, in the case of IPTi in PNG, it was the only way of collecting acceptability data early enough to be able to make recommendations for improving access and long-term adherence during roll out. Using a range of methods (including direct observation of behaviour) and interviewing a range of respondents, enabled the triangulation of findings and ensured their reliability.The findings presented complement a broader programme of social science research undertaken in sub-Saharan Africa . The incThe authors declare that they have no competing interests.CP analysed the data and wrote the manuscript. LS trained the fieldworkers. LS, SP, IM, PS and NS planned and supervised data collection. SL and LS carried out data collection. MG and RP designed the IPTi acceptability research programme and the data collection tools. All authors provided comments on the draft manuscript and approved the final version.