BackgroundMalarial infection is a major cause of concern, both worldwide and in Pakistan. Gametocytes are the sexual forms of the parasite that are essential for transmission. They fuse inside the mosquito to develop sporozoites. Gametocytes of the plasmodium parasites, which cause the infection, differentiate into male and female gametocytes. These gametocytes constitute the sexual stage of the malaria parasite and are essential in transmission of the disease from human to vector Anopheles. Gametocytes are affected by factors such as host immunity, drug treatment, reticulocytemia, anemia, low levels of asexual parasitemia and stress to the parasite. The aim of this study was to observe the hematological parameters, age and gametocyte carriage in an area of seasonal malaria transmission.MethodsThe study was conducted at Aga Khan University Hospital (AKUH) Laboratory over the period of one year and 294 patients with uncomplicated malaria were recruited. Patients infected with Plasmodium falciparum  or Plasmodium vivax (P. vivax) malaria and no co-morbidities were included in the study.ResultsGametocytemia was highest during the period of July to November, with P. vivax, 267 (90.8%), predominating compared to P. falciparum, 27 (9.2%). P. vivax gametocytes were observed from May to October and P. falciparum gametocytes were observed from July to December. Low hemoglobin in females and low platelet levels were observed. The mean platelet count was significantly lower in cases of P. vivax having gametocytes compared to P. falciparum with gametocytes. Higher parasitic index was associated with lower platelet count. The most significantly altered parameters were hemoglobin, hematocrit, white blood cell (WBC), and platelet count. Hemoglobin and platelets were significantly lower during the malaria season in study participants, both male and female.ConclusionIn conclusion, infection with P. falciparum and P. vivax modulates significant changes in hematological parameters in populations living in malaria endemic regions. In the malaria season males were more frequently affected by malaria with thrombocytopenia. Gametocyte carriage remains unaffected by seasonal changes thus ensuring parasite transmission during the dry season. Malaria is one of the leading causes of morbidity and mortality worldwide, with nearly half of the world population classified as ‘at risk’ in 2015. According to the World Health Organization (WHO), there were around 212 million malaria cases reported and an estimated 490,000 deaths caused by malaria in 2015 worldwide [Gametocytes of P. falciparum are sequestered away from the peripheral circulation for maturation, and evidence has been presented for the development of gametocytes in the hematopoietic system of the human bone marrow . It has While there is much information about the asexual forms of the parasite, not much is known of the sexual forms and the process of gametocytogenesis. Knowledge of factors inducing or affecting gametocytogenesis/gametogenesis may help facilitate actions which could disrupt the malaria transmission cycle. The conversion of asexual to sexual stages depends upon factors such as host immunity, drug treatment, presence of reticulocytes (in vitro), anemia, low levels of asexual parasitemia and stress to the parasite . HoweverPresence of anemia is identified as a risk factor for gametocytaemia . FactorsHost immunity also plays a significant part in gametocyte production. Gametocytogenesis was induced in vitro for P. falciparum using lymphocytes and serum, acquired from P. falciparum-infected children . TherefoGametocyte carriage has also been shown to differ in different age groups and seasons. Gametocytemia is reported to be greater in the younger age groups as compared to the older, especially those aged <16 years . IncreasThis study aims to document and compare the changes in hematological parameters of the malaria-infected patients, along with the variations in gametocyte carriage and malaria transmission during different seasons. This can help in a better understanding of malaria transmission and prognosis and according to a study conducted, these hematological variations when used in combination with other clinical and microscopy methods can improve malaria diagnosis and treatment .In this study the changes in the hematological parameters, age and seasonal variation of gametocyte carriage were observed in an endemic area.The study was conducted at Aga Khan University Hospital (AKUH) Laboratory, Karachi, Pakistan. A total of 294 patients infected with P. falciparum or P. vivax malaria were included. Patients who were malaria negative and refused to consent were excluded.The study was approved by the Ethical Review Committee of Aga Khan University Hospital and conducted in accordance with the Good Clinical Practice of Declaration of Helsinki. Informed consent was obtained from enrolled patients.Peripheral blood (1 ml) was obtained from the participants for detection of the malaria parasite. Thick and thin smears were prepared from each patient’s blood, and Giemsa staining performed, to quantify and identify the species of the parasite respectively. The malaria positive blood films were reviewed by an expert. Parasitemia was calculated using 100x oil immersion microscopy. The parasites were counted against 200 white blood cells (WBCs) and the parasite density was calculated with the help of the total leukocyte count (TLC). Participants were stratified into three groups based on age less than 15 years, 16-40 years and greater than 40 years.Data was analyzed using SPSS Statistics for Windows, Version 21.0 . Arithmetical means and medians were calculated, where applicable, for all continuous baseline variables. Mann-Whitney U test and ANOVA test, if the assumption was found, then Kruskal-Wallis test were used to compare differences between different groups. Multivariate binary logistic regression was applied to compare the cause and effect among hematological parameters and demographics variable. p-value <= 0.05 was taken as significance at two-sided p-value.In this study of 294 malaria-infected patients, 207 were males (70.4%) and 87 were females (29.6%). Gametocytemia with P. vivax, 267 (90.8%), was observed to predominate compared to P. falciparum, 27 (9.2%). P. vivax gametocytes were observed from May to October and P. falciparum gametocytes were noted from July to December.The mean hemoglobin (Hb) and hematocrit (HCT) levels in females were observed to be low whereas, for males they were in the low-normal range Table .On comparison of the different age groups, the Hb and HCT levels of the patients less than 15 years were found to be the most significantly affected Table . A normaThere was a significant difference in all hematological parameters between P. falciparum and P. vivax, except for the platelet levels. The HCT (p = 0.000), Hb (p = 0.000) and red blood cell (RBC) (p = 0.000) levels were all higher in P. vivax-infected patients while the WBC (p = 0.000) and platelet (p = 0.090) levels were greater in those suffering from P. falciparum infection Table . Binary Gametocyte density for P. vivax peaked earlier, around July-August, while that of P. falciparum came later, around September-October. On the other hand, the mean Hb, HCT, RBC and WBC levels were found to be similar in both the rainy and dry seasons but, the mean platelet levels were lower in the rainy season as compared to the dry season Table .Bar charts represent gametocyte carriage of both P. falciparum and P. vivax detected in over a year period. Percent gametocytemia detected monthly are indicated. Average monthly temperatures recorded are plotted and indicated as red line. High gametocytes carriage was noted during malaria season from June to October when temperatures were higher (above 30°C). Gametocytemia was seen to increase as the temperature increased and was high from May to November as shown in Figure The two most common malaria species, Plasmodium falciparum  and Plasmodium vivax (P. vivax), have overlapping areas of distribution in Pakistan. Even though there is a higher incidence of P. falciparum infections throughout the world, our study showed a higher incidence of P. vivax species (90.8%) compared to P. falciparum (9.2%). These findings are consistent with the infecting patterns of this region -19. We hIn this study, the changes in hematological parameters of the malaria-infected patients were observed. The platelet counts were found to be significantly low in all age groups, for both genders. On the other hand, the female patients were diagnosed with anemia. Anemia was common in female patients compared to male .The majority of the Pakistani population suffers from anemia, especially iron deficiency anemia, and all age groups, from infants to elderly, are affected . Anemia There have been different observations regarding the changes in WBC count due to malaria. A study conducted in Kenya stated that malaria causes leukocytosis, and this was associated with a worse prognosis . WhereasMalaria incidence usually has a seasonal repetition because the life span, population and rate of survival of the Anopheles mosquito depend upon climatological elements. Pakistan is a mesoendemic region which means there is a regular seasonal transmission which occurs under normal rainfall conditions . In thisTherefore, strict preventive measures should be taken in the monsoon season, when gametocytemia is at its peak, to drastically reduce the incidence of malaria.This study also indicates a greater percentage of malaria infections in males than in females. In the Pakistani society, males are more exposed to outdoor activities, thus they are susceptible to mosquito bites. Karachi, where this study was conducted, is an industrial city and the waste water can provide breeding grounds for mosquito larvae, predisposing males to malaria infection -30.Malaria causes changes in hematological parameters which are not always consistent. Anemia and thrombocytopenia are the two most common hematological observations with malaria but the WBC count may vary. Malaria transmission is greatest during the monsoon season but malaria infections in dry seasons can also be seen and it is essential to take preventive measures in order to reduce malaria incidence. One of the limitations of this study was that we only included hospitalized patients and sample size was relatively smaller.In conclusion, infection with P. falciparum and P. vivax modulates significant changes in hematological parameters in populations living in malaria endemic regions. The mean platelet count was significantly lower in cases of P. vivax having gametocytes compared to P. falciparum with gametocytes. Higher parasitic index was associated with lower platelet count. The most significantly altered parameters were hemoglobin, hematocrit, WBC, and platelet count. Hemoglobin and platelets were significantly lower during the malaria season in study participants, both male and female. In the malaria season, males were more frequently affected by malaria with thrombocytopenia. Gametocyte carriage varies seasonally and may persist at low levels during the dry season.


Plasmodium falciparum in Cameroon. Gametocyte positivity was determined using three complementary methods: thick blood smear microscopy, RT-PCR and RT-LAMP, whereas exposure to the infection was assessed by enzyme-linked immunosorbent assay. Of 361 malaria endemic residents randomly included in the study , 87.8% were diagnosed with P. falciparum infection, of whom 45.7% presented with fever (axillary body temperature ≥37.5°C). Mature gametocyte positivity was 1.9% by thick blood smear microscopy and 8.9% by RT-PCR targeting the mature gametocyte transcript, Pfs25. The gametocyte positivity rate was 24.1% and 36.3% by RT-PCR or RT-LAMP, respectively, when targeting the sexual stage marker, Pfs16. Multivariate analyses revealed anemia as a common independent risk factor for both mature and all-stage gametocyte carriage, whereas fever and low anti-gametocyte antibody levels were independently associated with all-stage gametocyte carriage only. Taken together, the data suggest important differences in risk factors of gametocyte carriage depending on stage analyzed, with anemia, fever and low antiplasmodial plasma antibody levels representing the major contributing risk factors.Presence of mature gametocyte forms of malaria parasites in peripheral blood is a key requirement for malaria transmission. Yet, studies conducted in most malaria transmission zones report the absence of gametocyte in the majority of patients. We therefore sought to determine the risk factors of both all-stage and mature gametocyte carriage in an area with high stable transmission of  Plasmodium parasites, and 6 species  are responsible for malaria in humans , p<10−4, respectively) No association was found between gametocyte carriage and gender. On the other hand, gametocyte carriage increased with the onset of clinical symptoms. Indeed individuals with axiliary temperature ≥37.5°C are twice as likely to carry gametocytes  compared to non-febrile individuals   constitute risk factors for gametocyte carriage, univariate analyses were performed based on the proportion of gametocyte carriage in each analytic group. As shown in ctively) .As shown in Pfg27 and the ookinete-specific surface protein Pfs25, respectively. As shown in P. falciparum total antigen . Positivity for all stage or mature gametocytes was also significantly associated with low antibody levels directed against the sexual stage-specific protein Pfg27  but not against the mature gametocyte/ookinete stage-specific protein Pfs25 . Taken together, the data suggest a strong association between host pre-exposure to the infection  and gametocyte positivity, with weakly immune individuals more likely to develop P. falciparum gametocytaemia.To investigate if gametocyte carriage also depended on host pre-exposure to the infection and/or gametocyte carriage, we determined the association between specific plasma antibody levels and positivity for either all stage or mature gametocyte carriage. All tested antigens were differentially recognized by the different plasma samples, with the highest reactivity achieved by using protein extracts . A median OD of 0.405 (range: 0.0807–2.1057) and 0.2762 (range: 0.0499–1.8104) was obtained when using the gametocyte-specific protein, Pfg27 antigen. As shown in Pfg27 protein were independently associated with all stage gametocyte positivity, whereas only anemia and low antibody responses against the P. falciparum protein extract were associated with mature gametocyte carriage. Together, the data suggest important differences between the risk factors for all stage and mature stage gametocyte carriage with anemia constituting the only common factor.To identify the demographic, hematological and serological risk factors that associated independently with gametocyte carriage in the population, multivariate analyses was done using young age, fever, anemia and low antibody responses against the P falciparum protein extract or sexual stage-specific P. falciparum infected subjects in our analysis. Furthermore, we used a combination of three gametocyte detection techniques  to identify active carriers of P. falciparum gametocytes in the study population. Exposure to gametocyte carriage was also investigated using an ELISA-based method. Together, our data show an extremely low prevalence (1.9%) of mature gametocyte carriage in the study area when using light microscopy. This finding is similar to those reported in other regions in Cameroon by Songue et al (0%), Van der Kolk et al (4.4%), and Sandeu et al (8.9%) using light microscopy as diagnostic method [Pfs16, revealed a gametocyte prevalence three-fold (24.1%) higher than the Pfs25 RT-PCR. Considering that the Pfs16 transcript is expressed at a two-fold higher level in mature gametocytes when compared to the Pfs25 transcript [Pfs16 target was due to the presence of early stage gametocytes  in the majority of infected blood samples. Indeed, Plasmodium gametocytogenesis is known to initiate in mid-trophozoite stage parasites leading to the release of sexually committed merozoites that infect and develop within circulating red blood cells as sexually committed rings before maturing through older gametocyte stages (stages I-V) [Pfs16-based RT-LAMP and Pfs25-based RT-PCR) were negative for P. falciparum infection as determined by light microscopy and PfExp1-based RT-LAMP. We interpreted such observations as corresponding to individuals who have recently cleared the infection either naturally or following antimalarial drug administration. This is consistent with several previous reports indicating that gametocyte positivity can proceed for several weeks after the clearance of asexual parasites, depending on the treatment or host immunity [This study aimed to determine the risk factors of gametocyte carriage in a high stable malaria transmission zone in Cameroon. We employed a recently developed RT-LAMP method  to incluc method –38. The anscript , it is ages I-V) . Howeverges I-V) ,42. Apprimmunity –45Pfs16-based RT-LAMP, justifying our use of RT-LAMP in the association studies. Consistent with findings elsewhere, young age, anemia [Pfs16 marker or the mature gametocyte marker Pfs25  in Plasmodium gametocytogenesis has previously been demonstrated and thought to be related to the release of stress factors following massive red blood cell lyses that may induce Plasmodium gametocytogenesis [Of the identified risk factors, only anemia and low IgG antibody response against the ogenesis . Similarogenesis . MeanwhiPlasmodium gametocytogenesis.In conclusion, gametocyte rates in the study population were comparatively high, and depended on test sensitivity and the gametocyte stage being targeted. On the basis of gametocyte positivity in the study population, we identified anemia as a common risk factor for both all stage and mature stage gametocyte carriage. Importantly, we identified low anti-Plasmodial antibody responses as major contributing factors to mature gametocyte carriage whereas low antibody responses to sexual stage antigens may associate strongly with gametocytogenesis and the initial release of early stage gametocytes into the peripheral blood. The observed negative association between the anti-gametocyte plasma antibody levels strongly support ongoing efforts to develop novel malaria transmission-blocking vaccines targeting S1 Table(DOCX)Click here for additional data file.


Plasmodium falciparum infections compared to young children in the area of Lambaréné, Gabon as an indicator for semi-immunity.Malaria remains a public health issue, particularly in sub-Saharan Africa with special features of seriousness in young children and pregnant women. Adolescents and adults are reported to have acquired a semi-immune status and, therefore, present with low parasitaemia. Children are understood to present with a much higher parasitaemia and severe malaria. It is a concern that effective malaria control programmes targeting young children may lead to a delay in the acquisition of acquired immunity and, therefore, causing a shift in the epidemiology of malaria. Prevalence and parasitaemia were explored in adolescents and adults with Plasmodium falciparum infections were determined either by rapid diagnostic test (RDT) or by microscopy. Descriptive analysis of data on parasite densities, anaemia, and fever are presented.A cross-sectional study was conducted at the Centre de Recherches Médicales de Lambaréné (CERMEL) during a 6-month period in 2018. Symptomatic patients, of all ages were screened for malaria at health facilities in Lambaréné and Fougamou and their respective surrounding villages in the central region of Gabon. P. falciparum positive RDTs was high among adolescents (68%) and adults (44%), compared to young children (55%) and school children (72%). Out of 274 participants assessed for malaria by microscopy, 45 (16%) had a parasite count above 10,000/µl of which 9 (20%) were adults.1589 individuals screened were included in this analysis, including 731 (46%) adolescents and adults. Out of 1377 assessed, the proportion of P. falciparum infections in adolescents and adults associated with high-level parasitaemia similar to that of young children. Adolescents and adults seem to be an at-risk population, suggesting that malaria programmes should consider adolescents and adults during the implementation of malaria prevention and case management programmes with continuous care, since they also act as reservoirs for P. falciparum.This study shows a high rate of  Plasmodium falciparum infections in endemic regions, including pregnant women, infants, children under 5 years of age, and the non-immune migrants, as well as travellers.Malaria remains a leading cause of morbidity and mortality in sub-Saharan Africa despite the considerable progress made since the year 2000 –3. The WP. falciparum infections and is sequentially reflected first by a reduction of severe forms of malaria, then by a reduction in parasite density, but adults still can become infected and often remain asymptomatic [P. falciparum antigen variants and also the fact that the immune mechanisms change with age [There is evidence of age-dependent protection against severe malaria acquired by residents of high endemic malaria areas , 6. Partptomatic , 8. Age-with age . Consequwith age .Plasmodium transmission [P. falciparum infections and its parasite density in the adolescent and adult population living in a rural endemic area of Gabon.There is little known about the epidemiology of malaria and parasite level in the adult population in high transmission regions in comparison to what has been published about children, especially in Gabon. The prevalence of parasitaemia in the adult population remains an important issue because they act as a community reservoir for ongoing smission . There ismission . In Gabosmission , 14. Stusmission . This reP. falciparum malaria, which is perennial with little seasonal variation [P. falciparum show high levels of resistance against chloroquine and sulfadoxine-pyrimethamine [This prospective, cross-sectional analysis was done between January and June 2018. The data was retrieved from malaria diagnostic activities carried out on all patients seeking for health care at the Centre de Recherches Médicales de Lambaréné (CERMEL) in Gabon. Study participants came from Lambaréné, Fougamou, and surrounding villages, in the central region of Gabon, within the equatorial rainforest and highly endemic for ariation , 17. Locethamine .P. falciparum infections and those positive by either rapid diagnostic test (RDT) or microscopy were invited through an informed consent process to participate in the respective clinical trials. All studies excluded pregnant and lactating women as well as those not consenting. This report included all individuals with a positive RDT or microscopy who consented to one of the clinical trials.Patients of all ages were routinely screened for Demographic data including reported age, date of birth, gender, and place of residence were collected after an informed consent was obtained from the participants or their parents, or legal representatives in case of minors. Axillary temperature and haemoglobin levels were systematically recorded.For this prospective analysis, participants were grouped into the following age categories: young children: ≤ 5 years of age, school children: 6 to 12 years of age, adolescents: 13 to 18 years of age, and adults: > 18 years of age.https://www.hemocue.com) or with an automated haematology analyzer . Here, anaemia was defined as haemoglobin level < 10 g/dl, irrespective of the age and gender [Haemoglobin levels were assessed either with a Hemocue device  was used. A positive test was confirmed with a second RDT of the same brand read by at least two different health care professionals. Thick blood smear microscopy slides were prepared according to the WHO method or the Lambaréné method as described elsewhere and stained with a 20% Giemsa solution for 20 min [The malaria diagnostics used were both RDTs and microscopy. For the RDT, the Paracheck Pfr 20 min . The resP. falciparum densities determined by microscopy were categorized into four groups, 0 parasites or “negative,” < 1000 parasites per µl or “low density,” 1000–9999 parasites per µl or “moderate density,” and ≥ 10,000 parasites per µl or “high density” [Asexual density” , 23.Data recorded on screening logs were entered in an Excel table, and then imported for analysis using Stata version 13.1 . The data is presented in tables with numbers and proportions for qualitative data while quantitative data like parasite density is presented as median and interquartile range (IQR). No hypothesis was tested, as this was a descriptive analysis.Of the subjects screened for malaria from January 9 to June 30, 2018, data was available for 1589 subjects included in this analysis as shown in Fig. P. falciparum  were positive for During that same month of January, data on haemoglobin level and body temperature were available for 256 and 264 subjects, respectively. The overall median (IQR) haemoglobin level was 10.7 9.3–11.9) g/dl, while for young children, school children, adolescents and adults the median (IQR) haemoglobin levels were 9.6 (7.9–10.8) g/dl, 10.7 (9.7–11.8) g/dl, 11.3 (10.2–12.4) g/dl, and 11.4 (10.0–13.2) g/dl, respectively. The young children age group was the most affected while all the other groups had proportions of anaemic less than 30% as shown in Table –11.9 g/dPlasmodium falciparum infection in adults is reported as being of low parasitaemia due to age-dependent acquired immunity [P. falciparum infection rate of 58%, with school children and adolescents bearing the highest proportions of infections, with adults as much infected as young children. The findings are similar to the prevalence found in adults in Libreville, the capital of Gabon, in 2012 [P. falciparum infections in adults was reported to be approximately 28% and 14%, respectively [P. falciparum infections in adolescents and adults. This high prevalence of P. falciparum reported by other studies could also be explained by a decrease in malaria control strategies, such as a reduction of the free distribution of insecticide-treated bed nets and the lack of re-impregnation of used bed nets [Children under five years and pregnant women in high malaria endemic regions are the most vulnerable to malaria and studies mainly focus on these populations. immunity , 24. Rarimmunity . This st in 2012 . This is in 2012 . The obsectively . These rbed nets , 27.P. falciparum infections was found in school children and adolescents in this study. This finding suggests a shift of malaria risk from very young children to school-age children, which is in line with the results reported by Mawili-Mboumba et al. who stated that children above 5 years of age would now become an at-risk population for malaria [P. falciparum transmission.The highest rate of  malaria . Substan malaria , 29. TheP. falciparum/µl while the proportion was much higher for school children (47%). Studies suggest that semi-immunity to P. falciparum develops with age based on cumulative exposure to the parasites, therefore, parasite densities are much lower in adults than in children [P. falciparum infections in the general population in Gabon.Proportions of people with high parasite densities in young children, adolescents, and adults were similar with 15%, 18%, and 20%, respectively, having a high parasitaemia of ≥ 10,000 children . This daP. falciparum infection. The majority of anaemic individuals had haemoglobin values between 6 and 10 g/dl with only a few participants having severe anaemia, similar to reports in Cameroon and Thailand [P. falciparum infections in adults in a high transmission area. One limitation to this study is the absence of a full parasitological diagnosis for all participants as the finding related to parasite density may be specific to the only month of January and not be generalized to the other periods of the year. Nonetheless, it was observed that the prevalence of malaria during the 6-month period assessed by RDT was similar to the 1-month period assessed by microscopy. Further studies are necessary to more extensively characterize P. falciparum infections and prevalence in adolescents and adults in Gabon as adolescents and adults may be at greater risk of malaria with the control programmes oriented at earlier life periods.Approximately one-third of the participants had anaemia, which may be related to the presence of a Thailand , 31. No Thailand . Other fP. falciparum due to the effective control programmes during their younger ages may have delayed their acquisition of the acquired immunity. Therefore, there is a need for malaria control programmes to assure a continuum of care that includes adolescents and adults of both sexes.Global efforts for control of malaria have been targeting infants and children below 5 years and pregnant women because they are described as the populations’ the most affected by the malaria burden in terms of morbidity and mortality. The children once targeted by control programmes mature to adolescence and then adulthood becoming vulnerable to malaria. Indeed, the reduced exposure to


Periostin is induced by IL-13 and has been studied as a biomarker of asthma. The present study explored the relationship between serum levels of periostin and exercise-induced bronchoconstriction (EIB) in asthmatic children.n = 56) and healthy controls (n = 30). We measured the levels of periostin in serum and performed pulmonary function tests including baseline measurements, post-bronchodilator inhalation tests, exercise bronchial provocation tests (BPTs), and mannitol BPTs.The study population consisted of 86 children 6–15 years old divided into an asthmatic group (n = 30), asthmatics with positive exercise BPT but negative mannitol BPT (n = 7), asthmatics with negative exercise BPT but positive mannitol BPT (n = 10), and asthmatics with negative exercise BPT and negative mannitol BPT (n = 9). Serum levels of periostin in asthmatic children with both positive exercise and mannitol BPT were significantly greater than those in asthmatic children with both negative exercise and mannitol BPT  and controls . Periostin levels were significantly correlated with both the maximum decrease in %FEV1 and mannitol PD15 value.The 56 asthmatic children were divided into four groups: asthmatics with positive exercise BPT and positive mannitol BPT (Serum levels of periostin in asthmatic children with both positive exercise and mannitol BPT were significantly greater than those in asthmatic children with both negative exercise and mannitol BPT and also greater than in healthy controls. Exercise-induced bronchoconstriction (EIB) is an acute phenomenon where the airways narrow as a result of physical exertion. Although EIB is not observed in all cases of asthma, a significant number of asthmatic patients experience exercise-induced respiratory symptoms, as exercise is one of the most common triggers of bronchoconstriction in these patients.2H2-induced airway inflammation,H2-targeted therapy.H2 cytokine family and a product of eosinophils, basophils, activated T cells, macrophages, and mast cells.The pathophysiology of EIB has been elucidated over the last two decades.We hypothesized that periostin levels would be higher in asthmatics with EIB than in healthy children. Furthermore, as we reported previously that serum levels of periostin are associated with airway hyperresponsiveness (AHR) to methacholine and mannitol,1 (PC20).1, medication adherence, and symptom levels.2-agonists on demand to relieve symptoms, with or without controller medications . The controls were healthy children matched by age and gender, who had applied for a routine health checkup or vaccination. They had no history of wheezing or infection over the 2 weeks before the study. Exclusion criteria included acute exacerbation of asthma requiring systemic corticosteroids during the previous 6 months and parenchymal lung disease evident in chest radiographs performed 4 weeks before the study. Of the healthy controls, those at any risk for atopy or subclinical eosinophilic inflammation were excluded using fractional exhaled nitric oxide (FeNO) of ≥20 parts per billion (ppb).Subjects were recruited from outpatient clinics at Hallym University Kangdong Sacred Heart Hospital, Seoul, Korea. We systematically recruited all new asthma patients at their first visits because of suspected asthma, and all diagnoses were verified by clinical examination, pulmonary function testing, and methacholine bronchial provocation tests (BPTs). The patients were newly diagnosed with asthma and had undergone maintenance therapy for 0.5–2 years. Asthma was defined as the presence of symptoms with less than 16.0 mg/mL inhaled methacholine, which induced a 20% decrease in FEVA schema of the study design is shown in ® Autocorr®; Smiths Medical PM, Inc., Waukesha, WI). The temperature in the laboratory was maintained at 22 °C, with humidity of 40–50%. Inspired air temperature and humidity were measured. The treadmill speed was increased until the heart rate was ∼85% of the predicted maximum ([220–age] × 0.9) and maintained for 6 min. Spirometry was performed 20 and 5 min before each exercise challenge and repeated 0, 3, 6, 10, 15, and 20 min afterwards. The results of exercise challenges were considered positive with a ≥15% decrease in FEV1 after exercise.Exercise challenges were conducted in accordance with American Thoracic Society standards1 values were recorded as prescribed by current guidelines.1 recorded after inhalation of a placebo capsule served as the baseline value. The challenge was completed when a ≥15% drop in FEV1 from baseline occurred, which was considered a positive response, or when the maximum cumulative dose of mannitol (635 mg) was administered. Asthmatics exhibiting a decrease in FEV1 of at least 15% from baseline after inhalation of ≤635 mg mannitol were enrolled in the positive-mannitol BPT group, and the others were placed in the negative-mannitol BPT group. For positive challenge results, the cumulative provocative dose causing a 15% drop in FEV1 (PD15 dose) was calculated by log-linear interpolation of the final two data points. The responses to mannitol were expressed as PD15 values.Dry powder mannitol  was administered according to the manufacturer's recommendations, and FEV®; Aerocrine, Solna, Sweden) that provided measurements at an exhalation flow rate of 50 mL/s expressed in ppb.FeNO was measured using a portable nitric oxide analyzer (NIOX MINO−1) was incubated overnight at 25 °C on ELISA plates . Then the ELISA plates were blocked by blocking buffer  overnight at 4 °C and then washed three times with washing buffer (0.05% Tween20 in PBS). To measure periostin levels, diluted (1/100–1/200) serum samples or recombinant periostin standards were added and incubated for 18 h at 25 °C. After washing five times, the peroxidase labeled SS17B mAb (50 ng mL−1) was added followed by incubation for 90 min at 25 °C. After washing five times to remove excess Ab, reaction solution  was added, followed by incubation for 10 min at 25 °C and then the reaction was stopped by adding the stop solution (0.7 N HCl). The values were calculated by subtracting the absorbance at 550 from the absorbance at 450 nm. Periostin concentrations in the serum were calculated simultaneously using the recombinant periostin proteins. The assay was performed in duplicate.Blood samples were obtained between 08:00 and 09:00. Periostin levels were measured by Shino Test Corp.  using an enzyme-linked immunosorbent assay (ELISA), as described previously.2 tests for categorical variables. Post hoc pairwise comparisons were performed using the Tamhane test. Numerical parameters with non-normal distributions  were log-transformed. Correlations between periostin levels, lung function, total IgE levels, eosinophil counts in peripheral blood (PB), eosinophil cationic protein (ECP) levels, and FeNO values were evaluated by calculating Spearman's rho. The effects of log-transformed periostin levels on the log-transformed maximum percentage change in FEV1 from baseline to after exercise, and mannitol PD15 data were analyzed by linear regression to allow adjustment for age, sex, atopy, and PB eosinophil count. The estimates obtained were regression slopes for log-transformed periostin levels against the log-transformed maximum percentage change in FEV1 from baseline to after exercise and mannitol PD15 value. The overall test performance of periostin for identifying asthmatic patients with positive exercise BPT and for identifying asthmatic patients with positive mannitol BPT was reviewed based on receiver operating characteristic (ROC) curve analyses. The overall accuracy of the test was measured as the area under the ROC curve (AUC). The prevalence of disease used in the analyses was estimated from the ratio of positive and negative cases in the dataset. The 95% confidence interval (CI) for test characteristics was calculated using MedCalc v.14.8.1 .The data were analyzed using SPSS ver. 21.0 . Continuous data are expressed either as means with standard deviations or as medians with interquartile ranges depending on the data distribution. Groups were compared using the Kruskal–Wallis test for continuous variables or χn = 30), asthmatics with positive exercise BPT but negative mannitol BPT (n = 7), asthmatics with negative exercise BPT but positive mannitol BPT (n = 10), and asthmatics with negative exercise BPT and negative mannitol BPT (n = 9). The demographic data and pulmonary function parameters of the subjects are summarized in A total of 90 subjects were recruited and took part in this study. The patient group consisted of 60 asthmatics and a control group included 30 healthy subjects. During the run-in period, four subjects with asthma dropped out because of a failure to discontinue controller medications due to exacerbation of asthma. Eighty-six subjects who finished the study were enrolled in the final analyses (56 asthmatics and 30 controls). The 56 asthmatic children were divided into four groups: asthmatics with positive exercise BPT and positive mannitol BPT  ratios were significantly lower in asthmatics than in healthy controls, while the bronchodilator responses were significantly greater  and in controls  (P = 0.039) and controls  (Biomarker levels are shown in < 0.001) . Serum l= 0.001) ; Fig. 2.P = 0.015), FeNO , and total IgE levels . There were no significant correlations between serum levels of periostin, age, and sex in any group. After adjusting for age, sex, atopy, and PB eosinophil level, the log (periostin level) was significantly associated with the log (maximum decrease in FEV1 after exercise) , and with the log (mannitol PD15)   .Table 2T2, cysteinyl leukotriene receptor (CysLT), and histamine, which are mediators that trigger airway smooth muscle contraction, sensory nerve activation, and mucus secretion. Mast cells and eosinophils also produce IL-13, a pleiotropic TH2 cytokine, which is also secreted by basophils, activated T cells, and macrophages.We investigated the relationship between serum levels of periostin and EIB in pediatric asthma patients. The inflammatory cells most commonly involved in the pathogenesis of EIB are mast cells and eosinophils.v integrin to induce secretion of proinflammatory cytokines, including TSLP.Several studies have shed light on the possible mechanism by which periostin may be involved in EIB. Masuoka et al.In addition to mast cells, eosinophils seem to play a major role in the pathogenesis of EIB. Peripheral blood eosinophil counts are associated with severity of EIB,H2-driven inflammatory response, but the relationship between these distinct biomarkers may be complex and variable.H2 inflammation in asthmatic patients, and observed that while both FeNO and periostin levels were consistently low in eosinophil-low patients, FeNO showed a greater overlap between eosinophil-low and eosinophil-high subjects. In the present study, periostin levels were significantly correlated with both PB eosinophil and FeNO levels. We found that not only FeNO levels but also periostin were associated with EIB in asthmatic children. After adjusting for age, atopy, and PB eosinophil count, serum levels of periostin were significantly associated with EIB.Serum levels of periostin, eosinophil counts, and FeNO levels all reflect a TEIB is frequently documented with asthma and reflects insufficient control of underlying asthma.There were several limitations to the present study. First, the sample size was small. Second, we could not discuss how our findings may be linked to poor asthma control, because we did not have a group of patients exhibiting frequent exacerbation of asthmatic symptoms. Third, periostin may not be a dependable biomarker in growing children because it is an extracellular matrix protein secreted by osteoblasts. However, the levels in our study subjects aged 6–15 years old were no higher than published values for adultsTo the best of our knowledge, this is the first controlled observational study of the relationship between serum levels of periostin and EIB in asthmatic children. In addition, we also assessed AHR by performing both exercise and mannitol challenge tests.Serum levels of periostin were significantly greater in asthmatic children with both positive exercise and positive mannitol BPT than in those with both negative exercise and negative mannitol BPT and controls. Therefore, periostin levels may serve as a clinically useful biomarker for identifying EIB in asthmatic children.All procedures were approved by the Medical Ethics Committee of Hallym University Kangdong Sacred Heart Hospital, Seoul, Korea, and all subjects and/or parents gave written informed consent.(IRB No. 2015-07-004).Not applicable.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Hallym University Kangdong Sacred Heart hospital Research Fund (grant number: 2016-09).This research was supported by The authors declare that they have no competing interests.Ju Hwan Cho, PhD: conception and design of the study, collection of the data and analysis and interpretation of the data, and preparation and revision of the manuscript.Kyubo Kim, MD, PhD: collection of the data, interpretation of the data and preparation of the manuscript.Jung Won Yoon, MD: design of the study, interpretation of the data and preparation of the manuscript.Sun Hee Choi, MD, PhD: conception and design of the study, interpretation of the data, and preparation of the manuscript.Youn Ho Sheen, MD, PhD: conception and design of the study, interpretation of the data, and preparation of the manuscript.ManYong Han, MD, PhD: conception and design of the study, interpretation of the data, and preparation of the manuscript.Junya Ono, MS: collection of the data, and analysis and interpretation of the data.Kenji Izuhara, MD, PhD: conception and design of the study, interpretation of the data, and preparation of the manuscript.Hey-Sung Baek, MD, PhD: conception and design of the study, collection of the data and analysis and interpretation of the data, and preparation and revision of the manuscript.All authors read and approved the final manuscript.


TLR4 gene in susceptibility towards malaria, clinical outcomes of the disease and responsible species of malaria.Toll-like receptors (TLRs) of the human immune mechanism play important role in the detection of invading pathogens. TLRs specifically recognize the pathogen-associated molecular patterns (PAMPs) from pathogens and start the effective response. Single nucleotide polymorphisms (SNPs) in the TLRs can mediate their functions. Present study evaluated the importance of rs4986790 polymorphism of P. vivax group and P. falciparum group on the basis of causative species of Plasmodium. Malaria samples were also divided into mild and severe malaria group based on clinical outcomes of the disease according to WHO criteria. Healthy individuals were placed in the control group. Whole blood was used for the isolation of DNA. Genomic DNA was isolated and amplification of targeted SNP was performed using allele-specific PCR.Blood samples of 228 malaria patients and 226 healthy volunteers were selected for the study. Sample collection was completed during Sep 2013 to Sep 2015 from different hospitals of Punjab, Pakistan. Patient’s samples were divided into P. vivax infection, OR: 0.5, 95%CI: 0.285–0.876, P=0.038.Results indicate the protective role of AA genotype against the susceptibility of TLR4 gene modulates the susceptibility towards P. vivax infection. AA genotype is found to be protective against the development of P. vivax infection in the local population of Pakistan.rs4986790 polymorphism of  Plasmodium. Malaria is transmitted through bite of an infectious Anopheles mosquito. Almost 60% of the Pakistani population lives in malaria-endemic regions of the country. Two species of Plasmodium were considered as the main agents for malaria infection in Pakistan; P. falciparum and P. vivax , TLR3, 4, 5, 6, 7 and 8 recognize nucleotides associated with pathogens . TLR 4 is an important receptor involved in the recognition of Plasmodium. It has been reported to identify glycosylphosphatidylinositol (GPI) from the Plasmodium parasite. Dysregulation in TLR signaling affects the inflammatory response and may contribute in development of chronic inflammatory diseases (Toll-like receptors (TLRs) present on the surface of immune cells recognize PAMPs (pathogen-associated molecular patterns) from pathogens. Upon recognition of PAMPs, specific immune response is initiated. the host . Howeverthe host . Almost Association of polymorphisms in certain TLRs with susceptibility to malaria has not been confirmed yet. Various factors like ethnicity, environment, pathogen, and host render it difficult to infer a certain association between susceptibility to malaria and TLRs polymorphisms. Various studies on different human populations will help to clarify the problem .TLR4 gene is located on chromosome 9, locus 9q32–q33. TLR4 consists of 4 exons and show maximum expression in lymphocytes, monocytes, neutrophils, and splenocytes  to guanine (G) at 896bp region. This substitution results in amino acid replacement of aspartic acid with glycine at position 299 (Asp299Gly). It has been reported to cause disruption in the extracellular domain of TLR4 signaling, dampened inflammatory response, lower levels of soluble adhesion molecules and acute phase reactants. The carriers of G allele were found hypo-responsive to inhaled LPS  were collected in EDTA vials and stored at −20 °C till analysis.Plasmodium parasite in the blood. Plasmodium parasite was detected via kit method . Kit method involves addition of 5ul blood sample into the sample well (S) of test cassette, followed by the addition of 3 drops of lysis buffer in well B. After 30 min the results were calculated. If line appears on control and Pv then there is P. vivax in the sample. If line appears on control and Pf then there is P. falciparum in the sample. If line only appears on control, then there is no Plasmodium species or malaria. Patient’s samples were divided into two groups based on causative species of Plasmodium; P. falciparum and P. vivax.Identification of malaria samples: Patients were confirmed on the basis of presence/absence of Criteria for severe and mild malaria groups: The malaria samples were also divided into severe malaria and mild malaria groups according to WHO criteria. Severe malaria-infected patients have neurological problems , severe anemia , hyperparasitemia, gastrointestinal symptoms, hypoglycemia (serum glucose less than 40 mg/dl), oliguria, acidosis with respiratory distress, jaundice, cardiovascular shock and diffuse hemorrhages. Rests of the samples were placed in mild malaria group.TLR4 gene: Genomic DNA was isolated from whole blood using vivantis blood DNA isolation kit (Cat# GF-BD-100). Allele-specific PCR technique was used for the amplification of rs4986790. Two forward primers F1: 5′CTACTACCTCGATGA3′, F2: 5′CTACTACCTCGATGG3′ and one reverse primer R: 5′ CAGCCATTTTCAAGA3′ was used for amplification reaction. PCR involves initial step of denaturation (94°C for 5 min), followed by 30 cycles of denaturation (94°C for 30 sec), annealing (45.3°C for 30 sec) and extension (68°C for 30 sec). Final extension was performed at 68°C for 12 min.Blood samples of healthy volunteers were collected as controls from the local population. Amplification of rs4986790 polymorphism of Detection of genotypes: Agarose gel electrophoresis following UV illumination was used for the detection of results. Agarose gel of 0.8% was used for DNA detection and 2% was used for the detection of PCR product. PCR product of 557bp with F1primer was read as AA, with F2 primer was read as GG and with both F1 and F2 primers was read as AG genotype. Samples, which gave amplification results only with F1 primer, depict the presence of A allele and AA homozygous sample. Samples, which gave amplification results only with F2 primer, show the presence of G allele and GG homozygous sample. Samples, which gave amplification results with both F1 and F2 primers, indicate the presence of both A and G alleles and AG heterozygous sample. PCR product was detected in comparison with the DNA Ladder  that was run on agarose gel.Hardy Weinberg Equilibrium (HWE) estimation and genetic frequencies, allelic frequencies, and differences in the frequencies were analyzed using Chi-square. SPSS software, ver. 18 for Windows  was used for analysis of Chi-square test and other nonparametric tests. The relationships of various genotypes with malaria groups were examined using odds ratio (OR) considering control group as the reference. Odds ratios were performed using an online calculator .P. falciparum group. Results of HWE estimation indicated that all groups (P<0.05) were deviant from HWE except P. falciparum group.P. vivax malaria. It showed 0.5 times decrease in the chances of P. vivax malaria . AG and GG genotypes had positive but marginal association with susceptibility to P. vivax malaria. AG genotype increased 1.775 times  and GG genotype increased 2.729 times the chances of P. vivax malaria . rs4986790 polymorphism did not depict any association with P. falciparum malaria. Association of malaria groups and alleles did not show any significant association with malaria groups.Some malaria-infected individuals develop severe malaria, while others only suffer from mild or uncomplicated malaria. This variation in infection response and disease progression may develop due to various environmental and genetic variations. Variations in immunity, age, genetic makeup of host and parasite may determine the clinical outcomes of disease. Resistance to infection mainly depends on the immune system of the host. Innate immune mechanism of humans has TLRs, which specifically recognizes various pathogens through PAMPs. These TLRs initiate the intracellular signaling which starts the expression of various effector molecules that mediates the adaptive immunity. TLR genes have polymorphic nature; genetic variations in these genes can affect the pathogenesis of different diseases –15.Genetic polymorphism in TLRs genes was reported to have association with different diseases like age-related macular degeneration (AMD), Alzheimer, atherosclerosis, and prostate cancer ,17.TLR4 and TLR9 polymorphisms have been studied for altering the susceptibility and resistance to various inflammatory and infectious diseases  . We did TLR4 . AlreadyTLR4 , 20, 21.We did not find the association of this polymorphism with severe or mild malaria. These results are in accordance , 23. TheP. vivax infection. G allele of rs4986790 has been associated with reduced inflammatory response to lipopolysaccharide  have been completely observed by the authors.


Anopheles mosquitoes. Methods that fail to capture these important factors in malaria risk will not accurately model risk of malaria infection or disease.Modelling risk of malaria in longitudinal studies is common, because individuals are at risk for repeated infections over time. Malaria infections result in acquired immunity to clinical malaria disease. Prospective cohorts are an ideal design to relate the historical exposure to infection and development of clinical malaria over time, and analysis methods should consider the longitudinal nature of the data. Models must take into account the acquisition of immunity to disease that increases with each infection and the heterogeneous exposure to bites from infected Plasmodium falciparum infection and disease were reviewed to assess trends in usage of the appropriate statistical methods. The study was designed to test the hypothesis that studies often fail to use appropriate statistical methods but that this would improve with the recent increase in accessibility to and expertise in longitudinal data analysis.Statistical methods applied to prospective cohort studies of clinical malaria or Of 197 articles reviewed, the most commonly reported methods included contingency tables which comprised Pearson Chi-square, Fisher exact and McNemar’s tests , Student’s t-tests , followed by Cox models  and Kaplan–Meier estimators . The longitudinal analysis methods generalized estimating equations and mixed-effects models were reported in 41 (20.8%) and 24 (12.2%) articles, respectively, and increased in use over time. A positive trend in choice of more appropriate analytical methods was identified over time.Despite similar study designs across the reports, the statistical methods varied substantially and often represented overly simplistic models of risk. The results underscore the need for more effort to be channelled towards adopting standardized longitudinal methods to analyse prospective cohort studies of malaria infection and disease.The online version of this article (10.1186/s12936-019-2885-9) contains supplementary material, which is available to authorized users. Plasmodium falciparum infection is one of the most common parasitic infection in humans. Individuals in high-transmission settings are exposed to bites of infected Anopheles mosquitoes and develop frequent infections. In early childhood, infections are almost always associated with symptomatic disease  Table . HoweverPlasmodium infection published over a 22-year period, the statistical methods substantially varied across articles despite that they reported analysis of the same general study design and outcome measurements. The most commonly used models for analysing count number of malaria episodes included the Poisson and negative binomial models, but these fall short with repeated measurements data as they cannot account for within-participant correlation of the episodes [In this review of statistical methods among articles analysing prospective longitudinal cohort data for clinical malaria episodes or episodes , 28, 29.episodes , 30–32, episodes . As hypoSome articles analysed the repeated time-to-disease episodes or infections but applied the Cox model  or the KWhen analysing repeated times-to-episodes malaria or infection, analytical methods should account for possible within-participant correlation of events as well as censoring over follow-up. Such appropriate methods include recurrent event survival models , 36. In This review has demonstrated that publications on prospective longitudinal malaria cohorts are increasing over time, a trend reflecting the growing understanding of the importance of longitudinal cohort data coupled with advancements in computer software developments for analysing such data , 22. OptThis review may have included more than one report using the same data, which may have introduced bias due to duplication of datasets. For example, the same data may have been analysed using inappropriate methods in first instance and then using appropriate techniques. Furthermore, some authors published more than one article and such articles may share similarities in the choice of analytical methods, but accounting for this was beyond the scope of this review as author background information was not collected. Future studies should consider testing whether more appropriate statistical methods improve or change understanding of malaria epidemiology by using among other factors, authors’ background information. Lastly, further studies may explore and compare how results from this study would compare with trends specifically from field trials.Cohort studies assessing risk of malaria infection and disease over time did not employ consistent analytic methods. The statistical methods varied substantially and often represented overly simplistic models despite similar designs, which may have led to biased outcomes and results that cannot be compared across studies. This review suggests that recent studies are adopting more appropriate statistical methods, though the statistical analyses are not uniform. These results underscore the need for more effort to be channelled towards adopting standardized longitudinal methods to model recurrent events for malaria infection and disease.Additional file 1: Table S1. List of reviewed articles in the study with details of year of publication, duration of follow-up, outcomes, sample size, population, location and analysis methods.


The present study aimed to evaluate the management of severe malaria at Gezira State hospitals in Sudan by assessing hospital readiness, health care provider knowledge and the care received by severe malaria patients.A cross-sectional descriptive study was performed to assess the severe malaria management practices at hospitals level in Gezira State. The study population included hospitals, health care providers and patients. Data was collected using checklists and structured questionnaires.A total of 20 hospitals, 158 health care providers and 370 patients were included in the study. Out of the total hospitals, 95% (19/20) were providing 24 h outpatient services, 65% (13/20) had ICU units, while triage system was found in only 35% (7/20) of hospitals. From all hospitals evaluated, 90% (18/20) were suffering from shortage of staff, especially doctors. About half of the health care providers (46.7%) did not receive severe malaria management training. The average knowledge score among health care providers was 55.4%. Microscopy was available in all hospitals (100%), while rapid diagnostic test, complete blood count and renal function test were available in 15 hospitals (75%). Fever was the most presenting symptom (97.8%) followed by repeated vomiting (51.4%), convulsion in children (24.3%) and prostration in adult (57.9%). Correctly diagnosed patients were 68.9%. Essential tests were done for only 11.1% of patients. Majority of patients (91.7%) were treated with quinine, 5.9% received artemether, while 2.4% were treated with artemether–lumefantrine. Those who received both the correct dose and dosing regimen were 53.8%. The overall compliance to guidelines was 2.2%.This study highlights the fact that management of severe malaria at hospital level was suboptimal with serious shortcomings in the different aspects of care particularly in specialized hospitals. Technical staff was inadequate, hospitals were anguish from defective emergency services, and most patients were not treated according to the national guidelines.The online version of this article (10.1186/s12936-019-2805-z) contains supplementary material, which is available to authorized users. In spite of the remarkable progress that has been made in the fight against malaria, it remains as an important public health problem in Sudan . There aThree levels of health care services exist in Sudan; the primary health care level (PHC), secondary and tertiary health care levels. Malaria diagnosis and treatment usually take place at primary health care level through community health workers (CHWs), medical assistants or doctors. Although there are no clearly defined and strictly implemented referral mechanisms between the three levels, severe malaria is usually referred from the family health units and centers to be treated at hospital level. At hospital level all cases are received and admitted at outpatient clinic for 24 h and then shifted to the ward. According to the national guidelines, management of severe malaria includes; general management to the general condition of the patient and specific treatment which either by giving quinine or artemether injection. It is worth mentioning that artesunate injection has been introduced recently in the treatment policy. Pre-referral treatment using artesunate suppositories or quinine IM is also recommend for cases referred from the lower levels [Prompt and effective case management is one of the key factors in malaria control. Management of malaria, as revealed in many studies, is not satisfactory largely due to poor adherence of health care providers to national policies, which is often associated with weak knowledge of care providers and poor infrastructures at health facility level . This stThis was a cross-sectional descriptive facility-based study conducted to assess the severe malaria management practices at hospitals level in Gezira State, Sudan using questionnaires and checklists. The study was carried out during the peak malaria transmission seasons, autumn–winter 2017. The study population includes general, specialized and rural hospitals providing severe malaria management; health care providers directly responsible for making decisions of patients care; and patients admitted to hospitals and diagnosed as severe malaria.Out of the total hospitals in Gezira State, twenty hospitals fulfilled the inclusion and exclusion criteria were included in the study. For health care providers, 158 were selected using the equation for calculating sample size for a finite population. Regarding severe malaria patients a sample size of 370 patients was estimated, assuming 70% of patients with severe malaria are appropriately treated, at 95% confidence level, with a tolerable error of 0.05% and 15% non-response rate. Hospitals were selected from the list of hospital in the state. All hospitals meeting the inclusion and exclusion criteria were included in the study. As a result 20 hospitals were selected; among them eight were general, seven specialized (four paediatrics and three obstetrics) and five rural hospitals. These hospitals were distributed across all localities in Gezira State. The health care providers were selected using non-probability sampling from those available at these hospitals during the study period till satisfaction of the sample size. Health care providers selected were those responsible for making the decision on patient management, namely doctors at different professional levels. Similarly, for collecting data from severe malaria patients; all those admitted with sever malaria during the period of study were enrolled, till reached the total number required. Patients were interviewed when they are in a good condition and eligible to respond or otherwise the caregivers respond on behalf of them, as well as reviewing patients’ records.Considering the fact that Gezira State has eight localities, a supervisor from each locality was nominated, informed about the purpose of the study and trained by the researcher on how to conduct the data collection. The supervisors selected two data collectors from each hospital and trained them on how to use the questionnaire and check lists and how to approach target groups. The process started in each hospital by meeting with hospital manager, explaining the purpose of the study and taking permission to start the data collection in the hospital. The readiness of these hospitals, the knowledge of health care providers working for them and care received by the patients were assessed using the specific tools designed for the purpose of this study.Data was collected using three types of data collection tools adapted from the World Health Organization (WHO) hospital assessment tool, results and lessons learnt from implementation of WHO assessment tool and the checklist for assessing management of severe malaria –8. The fAfter collection, data was reviewed for completeness, verified and cleared by the researcher. The data was entered in Microsoft excel and analysis was carried out using SPSS (version 23). Descriptive data was summarized in percentages, frequencies and ratios and presented in tables and charts.Availability of essential equipment: availability of one piece or more from each type of equipment; (ii) Presence of triage system: sorting out of patients attending at hospital outpatient clinics by dedicated technical staff; (iii) Correctly diagnosed patient: when there was documented fever (or history of fever) with a positive malaria test and at least one feature of severe disease; (iv) Correct anti-malarial medicine prescribed: administration of parenteral quinine, artemether or artesunate; (v) Correct anti-malarial drug dose and dosing regimen: intravenous quinine 10 mg/kg every 8 h or intramuscular artemether 3.2 mg/kg on day 1, followed by 1.6 mg/kg; (vi) Appropriately treated patients: if patients received the correct anti-malarial medicine, at the right dose and dosing regimen; (vii) Compliance to guidelines; detection/eliciting severe malaria features (at least one); (viii) Conducting essential tests: malaria test, blood glucose and haemoglobin), prescription of correct AMDs, prescription of correct dose and dosing schedule (regimen), giving supportive treatment (I.V fluids) and follow-up done; and (ix) good referral practice: the patient referred with referral note and received pre-referral anti-malarial treatment. To evaluate the knowledge of health care providers through the three clinical scenarios, the responses of participants were evaluated. Equal score was given for each case scenario and for the three sections of each case scenario. The total score of each case scenario is 12°, with 4° for every section in the scenario . Based on the overall score and since sufficient knowledge is mandatory for dealing serious problems like severe malaria, the knowledge of health care providers was classified as acceptable if the score was 90% or above, otherwise it was considered as unacceptable. Severe malaria is defined as a medical emergency which requires hospitalization, and the clinical manifestations include impaired consciousness , prostration, multiple convulsions, deep breathing and respiratory distress, acute pulmonary oedema. The laboratory findings include severe anaemia (Hb < 5 g/dl), hypoglycaemia (< 40 mg/dl), metabolic acidosis, hyperparasitaemia , hyperlactaemia (lactic acidosis); and renal impairment.The assessment in this study was conducted based on the following explanations; (i) Ethical approval was obtained from the Sudan Medical Specialization Board (SMSB). Permission was taken from Gezira State Ministry of Health, Sudan and from the manager of each hospital. Due to the fact there is a cultural barrier to obtain written consent; verbal informed consent was obtained from health care providers and patients or caregivers prior to completing the questionnaire and interview at the selected hospitals. All measures to maintain confidentiality and anonymity of data were put in place.In this study 20 hospitals in Gezira State were assessed for their readiness and 158 health care providers were interviewed. In addition, the care received by 370 patients admitted at these hospitals during the period of the study was reviewed.Out of twenty hospitals; five general, seven specialized and eight were rural hospitals. The result revealed that most of hospitals  were having 24 h outpatient services and 85% (17/20) of hospitals containing an emergency room for admission of patients in the first 24 h. The intensive care unit (ICU) for the management of critically ill patients was found in 65% (n = 13) of hospitals. Triage system for sorting-out of critically ill patients was found in only 35% (7/20) of hospitals.Hospitals’ readiness was assessed in terms of availability of staff, essential equipment, laboratory services and medical supplies. In regard to hospital staff adequacy, doctors were reported to be inadequate in the majority 90%, 18/20) of hospitals. Nurses and laboratory technicians were found to be adequate only in 25% (5/20) of hospitals. Sphygmomanometer was found in all hospitals, while thermometer in only 40% (8/20) of hospitals  participated in the study. About half of them (46.7%) reported that they did not receive any training on severe malaria management. In response to the three hypothetical case scenarios; paediatric, adult and pregnant women, the average score for all health care providers was found to be 55.4%. The average score of each type of health care providers was in all scenarios was 51.8%, 57.8%, 60.4%, and 44.5% for house officers, medical officers, registrars and consultant, respectively. The performance of health care providers varied across the different sections of the three case scenarios with a mean score of 59%, 57.9%, 49% and 33% for knowledge about; features of severe malaria, investigations, treatment and follow-up, respectively. Those who scored within the acceptable level of knowledge (≥ 90%) about features of severe malaria were 19% for, investigations were 15%, and treatment and follow-up were 2%. The overall score of health care providers within the acceptable level of knowledge in all case scenarios was only about 2%.A total of 370 participants were included and two-third (66.4%) of them were children; about one-third of them were under 5 years, while one-third (33.6) were adults. Around 56% were females, and about 59% of participants were urban population. The median of duration of stay at hospital was 2 days.Out of the total participants, 56.2% were referred to hospital from other health facilities, 39.4% were given a referral note and 20.9% received pre-referral anti-malarial treatment. Good referral practice was found in only 11.1% of referred patients. From those who reported that they were brought in a critical condition (17.1%), 82.9% were given a priority in receiving care. Only 30% of patients reported having received the medical care within 30 min of their attendance.Most of patients were asked about their age (70%), fever or history of fever (93.8%) and continuous vomiting (58.6%). However, many common serious manifestations of severe malaria were not regularly checked. Repeated convulsions; prostration and difficult breathing were elicited in only in 39.2, 36.5% and 28.6%, respectively. Weight was measured in around two-third of children, temperature in 63% and blood pressure in 34.1% (Table Microscopy was requested for the majority of the patients (94.9%) to confirm malaria, 98.5% revealed positive result. Blood glucose level was requested for 27.8% of patients out of them 12.7% were hypoglycaemic. The haemoglobin level was ordered for 49.5%, almost half of them (46.8%) were anemic and 5.6% were severely anemic. Correctly diagnosed patients were 68.9%. The majority of patients were treated mostly with quinine (91.7%), 5.9% received artemether, while 2.4% were treated with artemether–lumefantrine. Around two-third of patients (71.6%) received the right dose of anti-malarial drug, whether quinine or artemether, in 57.3% the dosing regimen is also correct. About one-fifth of patients (18.4%), all of them received quinine; the drug was prescribed twice a day instead of three times a day. Those who received both the correct dose and dosing regimen were 53.8%. The follow-up at least for temperature, blood pressure and respiratory rate was done for 60%. Overall, those who were appropriately treated, according to guidelines, were found to be only 2.2%. The majority of patients (90.8%) paid for at least some of the medicine prescribed. Around two-third of them (66.8%) obtained the supplies through out of pocket payment, while those who paid for anti-malarial drugs were 15.9%.The findings of this study revealed that management of severe malaria at hospital level was substandard. Major defects were detected in the general setup and readiness of hospitals, availability of supplies and the level of care provided. This study revealed that the triage system, as one of the parameters that indicate quality and inclusiveness of services provided, was lacking in most of the hospitals. This is particularly important since many patients seek medical advice usually after development of complications and deterioration of their condition. In a study conducted Sudan, 73% of those who sought medical advice did so after the first day of start of the illness . In addiThe current status of hospitals demonstrated that almost all hospitals are suffering from clear shortage of staff,  and equipment. Since severe malaria is a medical emergency with multisystem disorders, full set of laboratory services is required in every hospital that deals with severe malaria patients.Although malaria microscopy and blood glucose tests were consistently available in all hospitals, other important test such renal function and complete blood count were lacking in a quarter of hospitals. These findings were also reported from studies conducted in similar settings , 11. ABGConsidering availability of severe malaria drugs, though the majority of hospitals showed availability of quinine in its different forms at the time of study; all anti-malarial drugs, including oral continuation therapies, experienced stock out during the last 3 months before study. The problem of availability of drugs and supplies could be attributed to the weak, fragmented supply system that delivers the commodities from the central level down to the facility level and also lack of accountability. This result may explain the fact that many patients paid for anti-malarial drugs and other supplies.In this study, knowledge of doctors about severe malaria management was far below expectations. The average knowledge across the three different scenarios; paediatric, adult and pregnant women was below the acceptable level. Unexpectedly, the consultants, together with house officers, have shown the lowest scores. While the level of knowledge about manifestation of severe malaria and investigation was relatively adequate, the awareness about management and follow-up appeared to be the weakest part. These observations corroborate previous report from Angola, where the average knowledge of health care providers was found to be only 5.6% . All theWhen patient’s care was evaluated, systematic checking of severe malaria manifestations was found to be far below the required level. Apart from fever, the other symptoms were not checked for in many patients. Rapid/difficult breathing and generalized body weakness (prostration) although they are associated with poor prognosis and need special attention , 14, wasIn this study, microscopy, a gold standard test, was used to confirm malaria in most of the cases. However, in previous studies from Swaziland and Zimbabwe, RDT was used to confirm malaria in the majority of cases , 15. AlmThe majority of patients in the present study were treated with the recommended anti-malarial drug for severe malaria and similar findings were observed in the previous studies from some countries in the region , 16, 19.The present study highlights the fact that management of severe malaria at hospital level was suboptimal with serious shortcomings in the different aspects of care. Those who were appropriately treated for severe malaria with all six parameters properly done from patient assessment to follow-up, the overall compliance with guidelines, were found to be only 2.2%. Similarly, in Uganda, only 16.9% of patients were considered treated appropriately for severe malaria .Ministry of Health (MoH) needs to review hospitals’ readiness and setup and ensure availability of equipment, essential laboratory tests and adequate, continuous supply system.MoH should develop better strategies to improve health worker adherence to guidelines; through expansion of training programmes on severe malaria and referral guidelines including pre-referral treatment for hospital staffs  and lower level staffs.It is also important to ensure the quality of trainings being provided by reviewing and updating the training materials and methods.MoH and hospital directorates has to establish malaria mortality audit at hospital level with the aim of improving clinical practice related to malaria and reduce malaria deaths.It is necessary to accelerate the introduction of artesunate injection as a treatment of choice for severe malaria management and review the implementation of free malaria treatment policy.Additional file 1. Checklist for each health facility, questionnaire for health care providers and patients/caregivers.


Plasmodium vivax TRAP (PvTRAP), it has never been explored so far in the Amazon region, so results are conflicting. Here, we characterized the (IgG and IgG subclass) antibody reactivity against recombinant PvTRAP in a cross-sectional study of 299 individuals exposed to malaria infection in three municipalities  from the Acre state of the Brazilian Amazon. In addition, the full PvTRAP sequence was screened for B-cell epitopes using in silico and in vitro approaches. Firstly, we confirmed that PvTRAP is naturally immunogenic in the cohort population since 49% of the individuals were IgG-responders to it. The observed immune responses were mainly driven by cytophilic IgG1 over all other sublcasses and the IgG levels that was corelated with age and time of residence in the studied area (p < 0.05). Interestingly, only the levels of specific anti-TRAP IgG3 seemed to be associated with protection, as IgG3 responders presented a significantly higher time elapse since the last malaria episode than those recorded for IgG3 non-responders. Regarding the B-cell epitope mapping, among the 148 responders to PvTRAP, four predicted epitopes were confirmed by recognition of antibodies . Nevertheless, the frequency of responders against these peptides were low and did not show a clear correlation with the antibody response against the corresponding antigen. Moreover, none of the linear confirmed epitopes were located in the binding regions of PvTRAP in respect to the host cell ligand. Collectively, our data confirm the PvTRAP immunogenicity among Amazon inhabitants, while suggesting that the main important B-cell epitopes are not linear.Thrombospondin-related adhesive protein (TRAP) is essential for sporozoite motility and the invasion of mosquitoes' salivary gland and vertebrate's hepatocyte and is, thus, considered a promising pre-erythrocytic vaccine candidate. Despite the existence of a few reports on naturally acquired immune response against  Plasmodium genus. Two species are considered the main etiological agents: Plasmodium falciparum and P. vivax  has been considered a promising candidate antigen. TRAP is a highly conserved transmembrane Plasmodium protein belonging to the TRAP/Micronemal protein 2 family (TRAP / MIC2). It is required for sporozoite motility and, in conjunction with the circumsporozoite (CS) protein, is essential for sporozoite invasion in the salivary gland of the mosquito and vertebrate hepatocytes  and P. vivax (PvTRAP) loci of clinical isolates revealed a small sequence heterogeneity , Guajará (n = 87), and Mâncio Lima (n = 88). Fifty-three samples from individuals living in non-endemic areas from Rio de Janeiro were considered the control group. Samples and survey data were collected from June 2016 to August 2016. Written informed consent was obtained from all donors and the study was reviewed and approved by the Oswaldo Cruz Foundation Ethical Committee and the National Ethical Committee of Brazil (CEP-FIOCRUZ CAAE 46084015.1.0000.5248).A cross-sectional cohort study was conducted involving 352 individuals from three different endemic areas of Acre : CruzeirIn order to evaluate the possible influence of epidemiological factors on humoral immunity against PvTRAP, all donors were interviewed. The survey included questions related to personal exposure to malaria, such as years of residence in the endemic area, recorded individual and family previous malaria episodes, use of malaria preventive measures, presence/absence of symptoms, and personal knowledge on malaria transmission. The volunteers answered the questions and all epidemiological data were stored in Epi-Info  for subsequent analysis.P. vivax and/or P. falciparum at blood smears were subsequently treated using the chemotherapeutic regimen recommended by the Brazilian Ministry of Health.Peripheral blood samples were collected by venipuncture in heparin tubes. After centrifugation , the plasma was collected and stored at −20°C and transported to our laboratory. Thin and thick blood smears of all donors were examined for malaria parasites. Parasitological evaluation was done by examination of 200 fields at 1,000× magnification under oil-immersion and a research expert in malaria diagnosis examined all slides. All donors found positive for P. vivax TRAP  was cloned into the pHLsec vector (2) under standard cell culture conditions. Five days after transfection cells were discarded and media was filtered through 0.22 μM disposable filters. The secreted protein was purified from the supernatant by Nickuel Sepharose affinity chromatography , using the Äkta Start chromatography system and eluted with Imidazole 500 mM. Finally, eluted protein was dylazed using Slide-A-Lyzer™ cassette (Fisher Scientific) against 1X PBS.For the expression and purification of PvTRAP protein, the codon-optimized gene of c vector , which iAntibody binding to PvTRAP was evaluated on plasma samples by enzyme-linked immunosorbent assay (ELISA). Briefly, MaxiSorp 96-well-plates  were coated with PBS containing 1.5 μg/ml of recombinant PvTRAP. After overnight incubation at 4°C, the plates were washed with PBS-Tween and blocked for 1 h at 37°C. Plasma samples diluted 1:100 in PBS-Tween containing 5% non-fat dry milk (PBS-Tween-M) were added in duplicate wells for each individual. After 1 h at 37°C and three washings procedures, specific antibodies were detected by peroxidase-conjugated anti-human IgG  and followed by the addition of o-phenylenediamine and hydrogen peroxide. Optical density was accessed at 490 nm using a SpectraMax 250 ELISA reader . Results for total IgG were expressed as reactivity indexes (RIs), which represent the mean optical density of each tested sample divided by the mean optical density of 10 non-exposed control individuals' samples plus 3 standard deviations. Subjects were scored as responders to PvTRAP if the RI of IgG against the recombinant protein was higher than 1. Additionally, IgG subclasses were evaluated on responders by the same method, using peroxidase-conjugated goat anti-human IgG1, IgG2, IgG3, and IgG4 .http://ailab.ist.psu.edu/bcpred/predict.html), BepiPred1.0 (http://www.cbs.dtu.dk/services/BepiPred-1.0/), and ABCpred (http://crdd.osdd.net/raghava/abcpred). BepiPred 1.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html). VaxiJen is the first server for alignment-independent prediction of protective antigens. It was developed to allow antigen classification solely based on the physicochemical properties of proteins without recourse to sequence alignment. Bacterial, viral and tumor protein datasets were used to derive models for the prediction of whole protein antigenicity, showing prediction accuracy from 70 to 89% , we used a combination of three prediction algorithms: BCpreds  solid-phase chemistry (R197−H227), 2332.61 g/mol (peptide PvTRAPE237−T258), 3487.59 g/mol (peptide PvTRAPP344−G374), and 2333.55 g/mol (peptide PvTRAPE439−I460).After a consensual analysis of the hemistry   was performed. MaxiSorp 96-well-plates  were coated with PBS containing 0.5 μg/ml of each peptide, followed by overnight incubation at 37°C. The plates were washed and blocked with BSA 4% for 1 h and 30 min at 37°C in the humid incubator. After this, the individual plasma samples were diluted 1:100 in PBS-Tween containing BSA 2% in duplicate wells. After 2 h at 37°C in the humid incubator, and three washings with PBS-Tween, bound antibodies were detected with peroxidase-conjugated goat anti-human IgG  and followed by the addition of o-phenylenediamine and hydrogen peroxide. Optical density was identified at 490 nm using a SpectraMax 250 ELISA reader . As made for recombinant protein, the results for each peptide were expressed as reactivity indexes (RIs), which were calculated by the mean optical density of an individual's tested sample divided by the mean optical density of 10 non-exposed control individuals' samples plus 3 standard deviations.p < 0.05 was considered significant.All statistical analyses were carried out using Prism 5.0 for Windows . The one-sample Kolmogorov–Smirnoff test was used to determine whether a variable was normally distributed. The Mann–Whitney test was used to compare RIs of IgG against recombinant PvTRAP between studied groups. Differences in proportions of the RI of IgG subclasses and epidemiological parameters were evaluated by Fisher's exact test and associations between antibody responses and epidemiological data were determined by Spearman rank test. A two-sided We used a principal component analysis (PCA) to detect the variation pattern of Anti-PvTRAP total IgG, age, time of residence in endemic area, time since last infection response, and number of previous malaria infections. Our objective was to reduce dimensionality of multivariate data to detect the variables that explained the variance structure of the data. Each axis of PCA, hereafter named Principal Component (PC) represents the amount of variation associated to this axis. Each variable studied in the PCA has a loading value indicating its contribution to each PC. In the graph, individuals are represented as points located between principal component 1 (PC1) and principal component 2 (PC2). Variables are represented as arrows and its longitude represent its contribution to the variation of data. Additionally, closer angles between variables (arrows), represents more correlated variables, and angles closer to 90° represent independence between variables .To evaluate total IgG responses to PvTRAP peptides and compare with PvTRAP recombinant protein, we constructed a two-dimensional heatmap with hierarchical clustering grouping individuals with similar responses and the responses for peptides and recombinant PvTRAP. We calculated Z-scores from transformed total IgG values for PvTRAP peptides and recombinant protein: Z-score = [/population PvTRAP peptides/recombinant protein total IgG standard deviation]. Clustering was performed from Z-scores using Euclidean distance metrics and Ward as the linkage algorithm. PCA was constructed using vegan package  and a twP. falciparum and P. vivax malaria during their entire life, which are the two prevalent species in Brazil. The number of past infections reported by individuals also varied greatly, ranging from 0 to 48 (mean = 8.62 ± 10.24) and the time elapsed since the last infection (TLI) varied from 0 to 240 months (mean = 33.5 ± 57.1). Finally, at the time of blood collection, 118 (39.5%) individuals were naturally infected with Plasmodium sp. The frequency of P. vivax infections (24.4%) was significantly higher than that of P. falciparum infections . Collectively, the parameters evaluated indicated that the studied population are naturally exposed to malaria, but have different degrees of exposure and/or immunity.The studied population is composed of 299 individuals living in three close municipalities of the Brazilian Amazon. The majority was composed by adults and all individuals were naturally exposed to malaria infection . The ageP. vivax in Brazilian amazon individuals, we assessed the IgG reactivity against the recombinant antigen. Firstly, we confirmed that PvTRAP is immunogenic in naturally exposed individuals from the Brazilian Amazon, as 148 (49%) individuals presented specific antibodies against the recombinant PvTRAP , IgG3 (p = 0.0021), and IgG4 (p < 0.0001). Thirdly, in relation to magnitude of antigen-specific IgG subclass responses, a wide distribution of RIs was observed, which ranged from 0.28 observed in IgG2 to 18.33 in IgG1. However, the overall mean of specific subclass RIs indicate that IgG1 cytophilic antibodies against PvTRAP (mean = 1.53 ± 1.91) was also significantly higher (p < 0.0001) than all other subclasses , and IgG4 (mean = 0.76 ± 0.60).To verify if PvTRAP is a target of naturally acquired humoral response against t PvTRAP  with no ividuals . Among rividuals , follower = 0.190; p = 0.001) and TREA . Antibody levels and the number of previous infections were orthogonal variables and we also did not observe correlation . A similar result was observed in the PCA for anti-PvTRAP IgG reactivity index and time elapsed since the last malaria episode  .In order to identify the contribuition of epidemiological data on the variation observed in humoral response to PvTRAP, data was matched with the magnitude and frequency of responses to PvTRAP. Firstly, we noted that the distribution of individuals into ordination space was homogeneous, without evident groups into it. Age and TREA were highly correlated, and previous malaria infections as well as time since last malaria infection were negatively correlated between them. However, only age and TREA was related to RIs against PvTRAP IgG to data variation. The nature of variables relationship was confirmed by calculating the correlation between the anti-PvTRAP IgG reactivity index and age (= 0.398) . Regardi= 0.398) . This fiR197−H227, PvTRAPE237−T258, PvTRAPP260−K279, PvTRAPP344−G374, and PvTRAPE439−K454)  presented Vaxijen score higher than threshold value (0.6) and were considered antigenic epitopes selected to experimental validation. Regarding the localization of predicted antigenic linear B-cell epitopes, epitopes PvTRAPE237−T258; PvTRAPP344−G374, and PvTRAPE439−K454 were localized in repeat region of TRAP, while epitope PvTRAPR197−H227 was inserted in thrombospondin type I repeat (TSR) domain  . Among t) domain .R197−H227, PvTRAPE237−T258, PvTRAPP344−G374, and PvTRAPE439−K454).To validate predicted antigenic epitopes of PvTRAP, we tested by ELISA the plasma from responders to the recombinant protein, against selected epitopes  presented low frequencies of responders . Beside, peptide R197-H227 presented the lowest magnitude of response (median = 1.25 interquartile range = 1.13–1.47) when compared to PvTRAPE237−T258, PvTRAPP344−G374, or PvTRAPE439−K454 .Among the 148 responders to PvTRAP recombinant protein, as showed in p = 0.0175; mean = 8.35 ± 9.60, p = 0.005, respectively).In order to investigate the protective role of antibodies against linear epitopes of PvTRAP, we compared epidemiological data from individuals who recognized at least one of the linear epitopes with data from those who did not recognize linear epitopes. Interestingly, individuals who were responders to linear epitopes presented a lower mean of the time elapsed since the last malaria episode (mean = 23.97 ± 49.78) and a higher number of previous malaria episodes (mean = 13.35 ± 14.10) when compared to non-responders . The majority of individuals reported a prior exposure to parasite , 30. SelP. vivax vaccine candidates, such as AMA-1  of malaria parasites growth in vitro, associated with the acquisition of clinical immunity to malaria . It is important to mention that two extracellular portions of PvTRAP (Domain II and repetitive region) are crucial for initial host cell adherence and stabilization of adhesion/deadhesion during gliding mobility of sporozoites . In addition, the B cell epitopes confirmed in our study are not preserved following PfTRAP.Whilst there is strong evidence showing that specific antibodies are associated with cumulative exposure and protection against vivax malaria , 49–51, rozoites  and are lciparum . In our h CelTOS  and CSP h CelTOS , 55, andh CelTOS . Indeed,In conclusion, this study describes the naturally acquired antibody response against PvTRAP in three endemic municipalities of the Brazilian Amazon. The IgG immune response was associated with exposure and mainly mediated by cytophilic IgG1 antibodies. A significant proportion of IgG3 responders presented a higher time elapsed since the last malaria episode, which could indicate the participation of anti-PvTRAP specific antibodies in protective immune response. Lastly, beyond the validation of four linear B-cell epitopes within PvTRAP full-length sequence, the low response observed against the peptide epitopes could suggest that the functional Domain II of PvTRAP present conformational epitopes. Therefore, more studies need to be addressed to this unexplored target.All datasets generated for this study are included in the manuscript/The studies involving human participants were reviewed and approved by Oswaldo Cruz Foundation Ethical Committee and the National Ethical Committee of Brazil. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.AM, JL-J, LP-R, and AR-S: conceptualization. AM, JL-J, RR, and JS-A: formal analysis. AM, IS, and RR: investigation. AM, RR, IS, LB-C, and BB: methodology. BB, LP-R, PT, AR-S, CL-C, and RS: sampling and resources. AM, JL-J, and RR: writing—original draft. LP-R, CD-R, CL-C, AR-S, and PT: writing—review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.