Although microscopy is a standard diagnostic tool for malaria and the gold standard, it is infrequently used because of unavailability of laboratory facilities and the absence of skilled readers in poor resource settings. Malaria rapid diagnostic tests (RDT) are currently used instead of or as an adjunct to microscopy. However, at very low parasitaemia , the test line on malaria rapid diagnostic tests can be faint and consequently hard to visualize and this may potentially affect the interpretation of the test results. Fio Corporation (Canada), developed an automated RDT reader named Deki Reader™ for automatic analysis and interpretation of rapid diagnostic tests. This study aimed to compare visual assessment and automated Deki Reader evaluations to interpret malaria rapid diagnostic tests against microscopy. Unlike in the previous studies where expert laboratory technicians interpreted the test results visually and operated the device, in this study low cadre health care workers who have not attended any formal professional training in laboratory sciences were employed.Finger prick blood from 1293 outpatients with fever was tested for malaria using RDT and Giemsa-stained microscopy for thick and thin blood smears. Blood samples for RDTs were processed according to manufacturers’ instructions automated in the Deki Reader. Results of malaria diagnoses were compared between visual and the automated devise reading of RDT and microscopy.The sensitivity of malaria rapid diagnostic test results interpreted by the Deki Reader was 94.1% and that of visual interpretation was 93.9%. The specificity of malaria rapid diagnostic test results was 71.8% and that of human interpretation was 72.0%. The positive predictive value of malaria RDT results by the Deki Reader and visual interpretation was 75.8 and 75.4%, respectively, while the negative predictive values were 92.8 and 92.4%, respectively. The accuracy of RDT as interpreted by DR and visually was 82.6 and 82.1%, respectively.There was no significant difference in performance of RDTs interpreted by either automated DR or visually by unskilled health workers. However, despite the similarities in performance parameters, the device has proven useful because it provides stepwise guidance on processing RDT, data transfer and reporting. Malaria case management, consisting of early diagnosis and prompt effective treatment, remains a vital component of malaria control and elimination strategies . Since 2For a long time, good quality microscopy has been regarded as a gold standard method for diagnosis and confirmation of malaria parasite infections , 3. HoweAlthough fast and simple in concept, RDT performance in practice requires health workers to be trained on aspects of RDT test performance so that they are able to correctly interpret results and properly record them . Conside®, ParaHIT, OptiMAL-IT®, SD Bioline and CareStart™. The SD Bioline Malaria Antigen Pf/pan , that detects PfHRP2 (Pf) and pLDH (pan), is the RDT that has been recommended by the Tanzanian National Malaria Control Programme (NMCP) for malaria diagnosis countrywide since 2012 [In Tanzania, RDTs have become a solution for malaria diagnosis at rudimentary health care facilities such as dispensaries and health centres where microscopy is almost impossible . Initialnce 2012 . The intnce 2012 , 25. Fornce 2012 .Since RDTs are not able to detect parasitaemia infections at the lowest detection limit  which is the parasitaemia level commonly observed in low malaria transmission settings, more sensitive detection methods are required to accurately detect malaria. At very low parasitaemia, the RDT test line is usually very weak and can be falsely interpreted as a negative test as reported in various studies conducted in Tanzania , 26, 27.This cross-sectional study was conducted at Mgambo health facility in Tanga region. Tanga region located in north eastern Tanzania is a malaria endemic area with two main malaria transmission seasons every year, the high malaria transmission seasons (HMTS) and low malaria transmission seasons (LMTS). The HMTS starts from April and runs through August, which is during and immediately after long rain season (April to June), and the LMTS starts from October and runs through December, which is the short rain season of the year. The study was conducted during the end of the rainy season  during the high malaria transmission period in Tanga region.Study patients included military staff and civilians of all sexes and ages visiting the outpatient department at the military dispensary of Mgambo National Service Camp presenting with a fever (in the past 24 h) likely attributable to malaria. All outpatients attended the facility during HMTS in 2014 were eligible to participate to the study.Prior to recruitment, all outpatients or parents/guardian of sick children were informed about the study and requested to participate. They were asked for voluntary written informed consent prior to participation; two informed consent forms were filled by a participant or guardian and counter-signed by health personnel. One copy of the informed consent was kept within study file while the second copy was given to the participant. Thereafter, patients who gave informed consent were enrolled into the study.Three military laboratory attendants volunteered for and trained for 2 days in the preparation and interpretation of RDTs per the guidelines of the NMCP in place at the time when this study was conducted. These health care workers were further trained for 2 days on how to use the DR for malaria diagnosis. Additionally, two laboratory assistants (who had attended 1 year laboratory foundation course) who were employees of Tanzania Peoples defense forces at National Service camp, were trained for 1 week on preparation of thick and thin blood smears, fixing of thin smears and staining of the smears.P. falciparum) and pLDH (pan) was used for malaria diagnosis alongside microscopy. The DR provides automated sequential procedures to guide personnel on how to conduct the RDT test. The device prompts the user to insert the labeled RDT in a drawer for taking pictures of the RDT to recognize patient identification (ID) numbers before the test; thus, no patient names were entered in the device. After capturing an image and recognizing the brand of the RDT, the device prompts the user to remove the RDT from the device to proceed with taking blood samples from the patient. About 5 µl of blood sample was collected from a finger prick using a standard sampler (provided by the RDT manufacturer) and added to the blood well of the RDT cassette. Four drops of buffer were immediately added to the second well of the RDT cassette. After the incubation period  as guided by the automated timer in the device, the user was prompted to insert the RDT in the device for the second time to capture an image of the RDT test which was then analysed and interpreted by the DR. The user was also prompted to input his/her own visual interpretation of the test results for both assays, i.e. P. falciparum and the pan-malaria line. The test results interpreted by the DR were not displayed on the device because the user was masked to the DR results. Both visual and device interpretation test results along with the image of the RDT and patient de-identified bio data were directly uploaded to the study web-portal.In this study, 05fk60 SD Bioline Malaria Antigen Pf/pan  RDT that detects PfHRP2  were stained using 10% working solution of fresh Giemsa for 30 min. The stained smears were examined by experienced senior microscopists at Tanga Research Centre of the National Institute for Medical Research (NIMR) who was blinded of the results of the RDT interpreted visually and by the DR. To confirm positive films, asexual parasites were counted against 200 white blood cells (WBCs) while sexual parasites were counted against 500 WBCs. Assuming that each microlitre (µl) of blood contains 8000 WBCs parasites, densities were calculated by multiplying the parasite counts by 40 for asexual and 16 for sexual parasites. A smear was regarded to be negative after examining 200 high-power fields and not detecting parasites. Malaria species were detected and differentiated using thin blood smears depending on the appearance of the parasite, size and shape of the surrounding red cells, the presence of dots, stippling and pigment [From the same finger prick, thick and thin blood smears were prepared as guided by a standardized template; a slide with one frosted end was placed on top of the template and 6 μl of blood was immediately added to the slide on a large circle marked on the template which was then spread to cover the circle. Similarly, 2 μl blood was added at the edge of a small circle of the template for the thin film, which was then smeared as per standard operating procedures . The pre pigment . Other cMicroscopy data were entered into Microsoft Access Data base. The data were double entered by two different data clerks, checked for consistency, validated and cleaned. Each patient’s record was identified by unique identification number which later was used to link microscopic data with patient’s de-identified demographic information and RDT results downloaded from secured web-portal. Data from RDT and the patients’ demographic information were entered by touching the screen interface in the DR and uploaded through mobile networks to a secured web-portal. The web-portal was accessed only by authorized project staff using password-protected. Data from RDT were downloaded to Microsoft Excel, checked, cleaned and then transferred to STATA software  for analysis. This data was merged with microscopy results, whereby all mismatched or uncorrected data were removed, including dropping of duplicate data to prepare the master file. Descriptive statistics with mean, standard deviations and proportions were used to summarize demographic information of the study participants Chi square was used to assess the associations between categorical variables. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) was used to compare automated RDT analysis and visual human interpretation against microscopy as reference test. A contingency table was used to calculate the indices where sensitivity as TP/TP+FN, specificity as TN/TN+FP, PPV as TP/TP+FP, NPV as TN/TN+FN and accuracy of the test as TP+TN/N. In these formulas N = number of individuals tested; TP = true positive . The sensitivity was presented as proportion with 95% confidence intervals. P-value was considered significant at P < 0.05.A total of 1293 among 1423 outpatients with axillary temperature ≥ 37.5 °C, reported fever in the past 24 h and/or other symptoms suggestive of malaria were recruited in the study. A total of 130 patients did not participate in this study of which 30 did not consent, 64 had no blood smears prepared together with RDTs and 36 had smears of low quality to carry out further analysis. The median age for study participants was 21 (Interquartile range 21–23) years  and 59.9% (775/1293) as interpreted by DR and visually, respectively. Malaria positivity rate by microscopy as a reference test was 48.4% (626/1293) with a geometric mean parasite density of 3399 (95% CI = 2872–4022) asexual parasites/μl  of all malaria species found and two samples were mixed infections of P. falciparum and Plasmodium malariae  were falsely diagnosed as negative by automated DR and 6.4% (40/624) by visual interpretation , in one smear positive sample P. malariae was falsely diagnosed as negative by both DR and visual interpretation. Out of the 667 smear negative samples, 28.2% (188/667) were falsely diagnosed as positive by DR and 27.6% (184/667) by visual interpretation  and that of human visual interpretation of RDTs was 93.5% (95% CI 91.6–95.5). The specificity of RDTs in malaria diagnosis as interpreted by the device was 71.8% (95% CI 67.3–74.3) and that of human visual interpretation of RDTs was 72.4% (95% CI 67.3–74.3). The PPV of RDT interpreted by the DR and human was 75.8% (95% CI 72.0–78.2) and 76.1% (95% CI 72.3–78.4), respectively. The NPV of RDT interpreted by the DR and interpreted visually were 92.8% (95% CI 89.9–94.7) and 92.2% (95% CI 89.8–94.6), respectively. The accuracy of RDT as interpreted by DR and visually was 82.6 and 82.1%, respectively. There was no significance difference between the PPV, NPV, the accuracy of RDT interpretation by automated DR and visual interpretation by a semi-skilled laboratory staff.Table In this study, automated analysis by DR and visual interpretation of RDTs by unskilled health care workers against microscopy was compared. This comparative study in interpreting RDTs between unskilled cadre of health workers and automated DR for routine outpatients is the first to be done in rural military health facilities. Whereas previous studies conducted in Tanzania, Colombia and Uganda , 29, 32 P. falciparum which is the dominant malaria species in Tanzania.In this study, the sensitivity, specificity, PPV, NPV, and accuracy RDTs as interpreted by the automated DR  were similar to the interpretations which were visually done by unskilled health care workers  with no significant differences. These findings are consistent with the previous studies conducted in Colombia, Tanzania and Uganda , 29, 32 Comparing this study with other recent studies that did not use DR for interpretation of test results in Tanzania, in this study the sensitivity of HRP2-based RDTs of about 94% which is lower than the > 95% recommended by the WHO . Two stuThe observed high sensitivity and low specificity of RDT should have been affected by high malaria transmission in the study area because high transmission is characterized by high false positive results and hence high sensitivity and low specificity as reported elsewhere . In thisThe reported specificity of about 72% found in this study is lower compared to what has been reported in other studies presumably due to the reported higher false positivity rate (28%), which could be due to persistence of PfHRP2 in the blood stream for several weeks after successful treatment leading clearance of parasites as reported elsewhere , 37. MorP. falciparum parasitaemia. In contrary, in Central African Republic, although the tests performed less well in cases of low parasitaemia, they reported sensitivity of > 95% at > 500 parasites/μl [The sensitivity of RDT interpreted by automated DR and visually had a direct relationship with parasite density. The lower the parasite density  among blood samples, the lower the sensitivity of the RDT interpreted by the device (69.2%) and visually (69.2%), while the higher the parasite density  among blood samples, the higher the sensitivity (100%) of the RDT interpreted by the device and visually. At the highest parasite density level , higher sensitivity (99.4%) was also registered although slightly lower by 0.6% compared to the maximum (100%) detection limit. This finding is in concordance with previous studies done in Tanzania , 39 thatsites/μl  as oppossites/μl .In this study, a false positivity rate of 28% was found by RDTs as interpreted by both the device and visually by unskilled health care workers. Such a high discordance rate could be attributed to the presence of persistent malaria antigens in the blood due to unreported recent self or hospital treatment with anti-malarials and hence leading to false positives which, has also been reported in other studies that used HRP2-based RDTs , 45. Thepfhrp2 or/and pfhrp3 gene as also reported by other studies [In this regard, high false-positivity rate accompanied with low specificity can result into over diagnosis of malaria among non-malaria febrile patients. If a patient is found to be positive by HRP2 RDT simply due to history of recent malaria treatment, that result should be investigated more thoroughly and other possible causes of non-malaria fever should be investigated for proper treatment. However, the reported high false positive rate can be valid only if microscopy was performed very accurately (taking into account that PCR was not performed to correct microscopy results), otherwise, some reported false results in this study could be correct diagnosis as also reported in recent studies that RDTs are becoming more sensitive than microscopy performed inaccurately especially in low malaria transmission settings , 47. Add studies –51. The  studies . In thisUnlike other studies that employed polymerase chain reaction (PCR) for resolving discordant results of RDTs compared to that of microscopy, this study only used microscopy to confirm RDT results. Thus, dependence on microscopy to confirm RDT test results may have impacted these results because microscopy test results are likely to be affected by transmission intensity, prevalence of infections and parasite density as reported elsewhere .The performance agreement of the DR compared to visual interpretation conducted by unskilled health care workers in interpreting RDTs does suggest that the device can be used alone or concurrently with microscopy for malaria diagnosis. The use of this DR device at the point of care not only interprets RDT results, but has the added advantage to provide stepwise procedures for RDT testing, data transfer and timely reporting as also studied in Kenya and Uganda , 52. Mor


In Madagascar, the last study on sickle cell disease (SCD) was done in the early 1980s. The country is known as endemic for malaria and respiratory infections. The main objective of this study was to estimate the prevalence of SCD; the secondary objective was to evaluate its association with malaria and respiratory infections.This is a cross-sectional study which was carried out in a rural village in the south east coast of Madagascar between May 2011 and November 2013. Participants were children aged between 2–59 months presenting with fever measured by axillary temperature ≥37.5 °C at inclusion. Genotyping of haemoglobin S was done by PCR and malaria was diagnosed by Rapid Diagnostic Test. Research for viral and atypical bacterial respiratory pathogens was performed on nasopharyngeal swabs. Uni-and multivariate polytomous logistic regression was done to assess associations between microbiological results and SCD status, with HbAA phenotype as reference.p = 0.3). Rhinovirus (22.5%), adenovirus (14.1%), and bocavirus (11.6%) were the most common respiratory pathogens detected. After univariate analysis, patients with SCD were more frequently infected by parechovirus (p = 0.01), while patients with SCT were more prone to RSV A or B infection (p = 0.01). After multivariate analysis, HbAS phenotype was associated with higher risk of RSV A and B infection compared to HbAA , while HbSS phenotype was associated with higher risk of parechovirus infection  compared to HbAA, independently of age, gender, period per quarter, and the other viruses.A total of 807 children were analysed. Prevalence of SCD among febrile children was 2.4%  and that of SCT was 23.8% . There was no difference in the prevalence of malaria infection according to haemoglobin status (The prevalence of SCD among under-five children presenting with fever was high in the study population. No association was found between SCT and malaria but few viruses, especially parechovirus, seem to play an important role in the occurrence of pneumoniae among SCD patients. During its 59th World Health Assembly held in 2006, WHO recognized sickle-cell disease (SCD), an inherited disorder of haemoglobin as a priority of public health. A resolution was adopted to develop and strengthen efforts for its prevention and management. Sickle-cell anaemia, one of the most common forms of SCD, is due to a point mutation within the sixth codon of the β-globin chain. The produced abnormal variant – haemoglobin S (HbS) – is responsible for chronic haemolytic anaemia and vaso-occlusion which are the underlying causes of the clinical presentation of SCD. Individuals who express the homozygous form (HbSS) manifest the disease, while those with the heterozygous form (HbAS), also known as sickle-cell trait (SCT), are usually asymptomatic carriers.Initially limited to the sub-Saharan Africa, the Middle-East and some parts of India , SCD hasIn area where malaria is endemic, its relation with SCT was often studied, in particular the high degree of resistance to severe and complicated malaria in sickle cell trait , 9. MalaFrom May 2011 to November 2013, a cross-sectional study was conducted in the rural village of Ampasimanjeva in the south east cost of Madagascar aiming to identify blood-borne protein biomarkers that can differentiate the causes of unexplained acute febrile illness in children. The study site is known for its high endemicity to malaria, acute respiratory infections and SCD. The main objective of this study was to estimate the prevalence of SCD; the secondary objective was to evaluate its association with malaria and respiratory infections.This is an observational cross-sectional study with prospective data collection of children aged between 2 to 59 months presenting with fever (axillary temperature ≥37.5 °C according to WHO criteria). The study was conducted from May 2011 to November 2013 in the rural village of Ampasimanjeva located in the south east cost of Madagascar. Ampasimanjeva is 320 km away from the capital city Antananarivo with a total inhabitant estimated to be 22,000 according to the last census. The east coast has hot and humid subequatorial climate and an annual high cumulative rainfall of 4,000 mm per year. Transmission of malaria in this region is reported to be strong and intense all over time. A standardized questionnaire on socio-demographic and clinical data was fulfilled for each individual. The Ampasimanjeva community hospital is a Hospital District Centre receiving each year around 950 children between 2 and 5 years old. Acute Respiratory Infections were the main cause of morbidity accounting for 35% of consultation all ages combined.Ethical approval for the study was obtained from the National Ethical Committee of the Ministry of Health of Madagascar (authorization 019-CE/MINSAN as of 09/04/2010). Individual written informed consent was provided by the parents of all study participants.For each child, venous blood was collected with safety blood containers on dry and spray-dried EDTA. The samples were transported in a refrigerated container at +4 °C to the Centre d’Infectiologie Charles Mérieux for analysis. Transport of samples was organized twice a week.I site and cleaved in 2 fragments (189 and 93 bp). The homozygous profile (HbSS) responsible of the disease has no restriction site and only one fragment of 282 bp is observed. The heterozygous profile (HbAS) also known as sickle-cell trait combines restriction and no restriction site and reveals 3 fragments .After DNA extraction  of the EDTA blood samples, the mutation responsible for the generation of HbS was identified by Restriction Fragment Length Polymorphism (RFLP) assay of PCR-amplified DNA  . The RFLRapid Diagnostic Test (RDT) of malaria was performed on site using CareStart™ Malaria  and parasite density was estimated microscopically from the positive samples as previously described .Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type b as previously described  compared to those with HbAS [−1.2 SD ] or HbAA [−1.5 SD ] phenotypes  were due to both P. falciparum and P. vivax, and the others were negative . There was no association between haemoglobin status and malaria infection  positive results of which 273 Giemsa-stained thick smear tests were done. The microscopic results showed that 197/273 (72.2%) infections were caused by S. pneumoniae was very high  and reached all (100%) children in the HbSS group compared to the HbAS  and HbAA  groups.Overall, 698  were positive for at least 1 bacterial pathogen and 575  for at least 1 viral pathogen. Co-detection of at least two viruses or both bacteria and virus was observed in 186  and 527  of cases, respectively. Carriage rate of n = 172), adenovirus , bocavirus , respiratory syncytial virus A or B , and human metapneumovirus A and B  were the most common respiratory pathogens detected; Chlamydia pneumoniae and Mycoplasma pneumoniae accounted for < 2.0% of cases  compared to the HbAA  and HbAS  patients (p = 0.02). Similar results were observed with human coronavirus 229E where patients in the HbSS group yielded high positive result  compared to the HbAA  and HbAS (n = 0) groups (p = 0.07)  compared to HbAA  and HbSS (n = 0) patients (p = 0.01). Viral coinfection were frequent in the HbSS group  compared to the HbAA  and HbAS  groups (p = 0.03).Parechovirus was most frequently detected from respiratory sample in HbSS patients n = 2, 1.1% compp = 0.01), and the risk of parechovirus infection which was higher in HbSS compared to HbAA groups  [Table OR = 1.9; 95% CI: 1.2–3.1, p = 0.01), independently of age, gender, period per quarter, and parechovirus infection. HbSS compared with HbAA phenotype remained associated with higher risk of parechovirus infection , independently of age, gender, period per quarter, and RSV A and B infection.Univariate polytomous regression disclosed the risk of respiratory syncytial virus A and B which was 2-fold more higher in HbAS compared to HbAA groups . All of them recovered well without complication.Our study shows that prevalence of SCD was 2.4% and that of SCT 23.8% among children aged 2–59 months presenting with fever and living in high endemic area for malaria. To our knowledge, this study is the first to document the prevalence of HbSS in febrile young children. Although no similar age group is available for comparison, previous results report high carrier rate of 29.6% to 31.9% , 17 fromOur results demonstrate a significantly lower mean weight-for-height Z-score for patients with SCD than those of normal children and individuals with SCT. Previous studies from other developing countries have reported a state of undernutrition among patients with SCD , 19 thatStudies reporting gender differences in SCD incidence are rare and mainly focus on the occurrence of crises or comorbidities , 22. HowP. falciparum positive result (36.6%) has been found among the studied population. Overall, we did not find any statistical difference between malaria-infected children in the HbAS group compared to the normal phenotype. However, as the age increases, the frequencies of children in the HbAS and HbSS groups who experienced malaria increases. The absence of malaria infection in the patients with SCD up to 2 years of age may have several causes including an early death due to malaria or other infections.Previous studies have shown that SCT protects against severe forms  and mildS. pneumoniae, a common colonizer of the nasopharynx in healthy children. Here we report a very high pneumococcal carriage rate among the febrile children studied. Strikingly, all children (100%) with homozygous sickle cell disease in our study were carriers. In previous reports, a much lower rate was found in the US (12%) [S. pneumoniae in the development of the disease in the majority of patients is hypothetical due to the absence of healthy matched control group. It should be mentioned that 1.9% of the patients had bacteraemia with S. pneumoniae during the course of infection but its causative role has not been proven up as up to 5% of febrile children may also develop asymptomatic or occult bacteraemia [Individuals with SCD are strongly susceptible to encapsulated bacterial organisms such as US (12%)  and UganUS (12%)  during steraemia .Picornaviridae, was significantly associated with a high risk of infection in SCD patients in our study. Parechovirus infections are usually asymptomatic or lead to a mild gastro-intestinal or respiratory diseases. Only two patients had parechovirus detected, both presenting with symptoms of pneumoniae, suggesting that larger prospective study is required to better describe the real impact of parechovirus infection among SCD population. Conversely, RSV A or B belonging to the family Paramyxoviridae, was significantly associated with a high risk of infection in SCT patients compared to the normal population whereas no virus was detected in SCD patients. RSV A and B are a major cause of lower respiratory tract infection during infancy and childhood and were associated with acute chest syndrome similar in severity to influenza infection in febrile children with SCD [The overall prevalence of viral infection/detection among the study population was high independent of haemoglobin status. However, among the 23 viral and atypical bacteria pathogens studied, there was no statistical difference in prevalence between the three groups for 20 of them and include HRV the most common respiratory virus detected or influenza virus A (H1N1)/pdm09 known to increase disease severity in children with SCD . Parechowith SCD . The reaDetection of respiratory viruses has been facilitated by the recent widespread availability of nucleic acid amplification techniques. In a study carried out previously in Ampasimanjeva to describe the aetiology of a clinically defined cohort of children with fever and acute respiratory infections, the most representative pathogens were human metapneumovirus (hMPV) for pneumonia, human parainfluenzavirus (hPIV) for other acute lower respiratory infections, respiratory syncytial virus (RSV) for flu-like illnesses and human adenovirus (HAdV) for upper respiratory tract infections . HoweverI may be found in other hemoglobinopathies. However, according to one recent publication [Our study was limited by the fact that the genotyping method used may not be totally specific to detect haemoglobin S as the presence or the absence of the restriction site Ddelication , HbS seeOur findings show that the prevalence of SCD among under-five children presenting with fever was high and a better knowledge of the role and distribution of viral and atypical bacteria pathogens in the study population has been found. This study has helped moving towards a better understanding of the epidemiology of SCD, and we hope it will trigger further research in that direction, for the following reasons. Firstly, as SCD is a major public health concern in Madagascar, a large prospective study on the general population to estimate the true magnitude of the disease should be done. Secondly, it is important to document data about the clinical course and mortality associated to SCD in regions where it is endemic. Thirdly, study involving increasing laboratory capacity should be conducted to help a better understanding of SCD epidemiology, as well as other hemoglobinopathies. Finally, conclusions from these studies will be used for awareness-raising among national authorities in order to strengthen the national control program.


Plasmodium falciparum (Pfsch07/03) was used in ELISA to measure and compare seroprevalence and magnitude of IgG antibody responses from 2000 to 2012.Coordinated scaled-up malaria control interventions have substantially contributed to the dramatic decrease of malaria-related morbidity and mortality in several endemic countries, including Senegal. However, the impacts of a given malaria control intervention on vector and parasite populations, acquired immunity, and disease burden remain very poorly documented largely due to the lack of continuous surveys. This study took advantage of the sera bank established as part of the Dielmo longitudinal project to investigate the dynamics of IgG antibody responses that accompanied the epidemiological changes resulting from malaria control interventions. Schizonts crude extract of a local strain of The prevalence of Pfsch07/03 IgG antibody responses progressively decreased from 97.25% in 2000 to 57.3% in 2012. The prevalence of Pfsch07/03 antibodies categorized between three different age groups  revealed increased seroprevalence with age ranging from 47.19 to 62.67 and 89.45%, respectively in  old age groups. A marked drop in seroprevalence was observed after 2008 and was significant in the younger (<7 years) and intermediate (7–15 years) age groups, unlike older individuals aged >15 years (p = 1.00).The study revealed a substantial contribution of all malaria control interventions to the decrease of IgG antibodies responses to Pfsch07/03 throughout prevention of human-mosquitos contacts, or reduction of parasite biomass. The present study demonstrates the wider potential of sero-epidemiological analysis in monitoring changes in malaria transmission resulting from a given malaria control intervention. Plasmodium falciparum malaria has been recorded during the last decade [Owing to several combined strategies deployed by national malaria control programmes in malaria endemic countries, significant decrease in mortality and morbidity attributed to t decade . In Senet decade , 3.P. falciparum disease over recent years have remained very poorly defined probably due to a lack of continuous surveys.To guide strategies to eliminate malaria from endemic areas, a better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and disease burden is needed. Moreover, the impacts of implemented strategies deserve to be monitored overtime to anticipate the consequences of changing malaria epidemiology . In mostIn Dielmo, an area of intense and perennial malaria transmission situated in the centre of Senegal  a longitIt has been shown that antimalarial antibodies play an important role in the efficacy of anti-malarial drugs in younger children more susceptible to the disease . ImmunogP. falciparum extracts between 2000 and 2010 showed a dramatic decline in seropositivity in Dielmo and Ndiop [In light with this, a recent evaluation of the relationship between changes in malaria transmission and antibody responses to crude nd Ndiop , two Sennd Ndiop , 7. In bnd Ndiop .P. falciparum antigens in relation to changing malaria epidemiology in Dielmo from 2000 to 2012.To gain further insights on the impact of malaria control interventions on anti-malarial antibodies responses, the study closely investigated the dynamics of IgG antibodies responses to crude The study was conducted using sera collected from cross-sectional sampling in 2000, 2002, 2008, 2010 and 2012 from inhabitants of Dielmo village. The sera were withdrawn from the collection of archived biological specimens established as part of the Dielmo cohort study described elsewhere , 10. ThiBlood samples were collected at the end of the rainy season and sera were separated from blood pellets and stored at −20 °C until use for immunological studies. Informed consent for the study was obtained from the individuals, and/or their parents or legal guardians. Sociodemographic characteristics for the donors of these sera are given in Table When follow-up programme of the Dielmo villagers was initiated in 1990, Dielmo was a malaria holoendemic area due to the presence of a small stream where anopheline mosquitoes bred all year round , 6, 11, P. falciparum strain 07/03 (Pf07/03) isolated from a malaria-infected patient in Dielmo have been detailed previously [The adaptation and in vitro maintenance of the eviously , 12, 13.eviously . After cNon-infected red blood cells were processed as described above and used as control in the ELISA to discriminate IgG antibodies responses to Pfsch0703 antigens from anti-erythrocytes antibodies (autoantibodies).ELISA Maxisorp plates  were used to optimize the dilution by dose effect. The optimal dilution was 1/300 and this was used for coating. Plates were coated with 100 μl of water-soluble crude extracts of Pfsch07/03. Non-infected red blood cells lysate was diluted in PBS and distributed into some wells on each plate as a control. The coated plates were incubated overnight at 4 °C and washed. Then, 200 μl of blocking buffer (2% BSA in PBS with 0.05% Tween) was added to each well and the plates incubated for 1 h at 37 °C. Serum samples were diluted at 1/200 in a dilution buffer (1% BSA in PBS with 0.05% Tween) and 100 μl of diluted serum was distributed into each well.2O2 (100 μl) and the reaction was stopped by addition of 4 N H2SO4 (50 μl/well). Between each incubation phase, the ELISA plates were washed extensively with PBS-0.05% Tween. The optical density (OD) at 450 nm was read in a BIO-RAD Microplate Reader (iMark). The threshold for positivity was defined as an OD ratio >2 (OD sample/OD naive serum). Inter-assay variations for positive controls did not exceed 20%.Negative and positive controls were included on each plate: positive controls were from Dielmo/Ndiop hyperimmune individuals and negative controls were from European individuals who have never been exposed to malaria. Polyclonal goat anti-human IgG conjugated to peroxidase at a dilution of 1/6000 in the dilution buffer (1% BSA in PBS-Tween 0.05%) was then added. Bound peroxidase was detected with orthotoluidine/HSeroprevalence between age groups and years were compared using Pearson Chi-2 or Fisher Exact test. Non-parametrical test, Wilcoxon and Kruskall Wallis were used for analysis of level of antibody responses. The threshold of significance was fixed at 0.05. Thus a p value of 0.05 or less was considered as significant. The statistical analyses were performed using R software (version 3.0.2).P. falciparum parasite strain (Pf07/03). The characteristics of the study population are summarized in Table A total of 1235 selected sera samples were distributed as follows: 218 in 2000, 186 in 2002, 269 in 2008, 288 in 2010 and 274 in 2012. The samples were obtained from inhabitants of Dielmo aged 3.4 to 90.9 years old and were tested for antibody responses to crude schizont extract from a locally adapted The prevalence of Pfsch07/03 antibodies progressively decreased from 97.25% in 2000 to 57.3% in 2012  for the five study periods. In 2000, the prevalence of Pfsch07/03 antibodies was high in all age groups (>95%) and was surprisingly highest in the youngest age group (100%) although the differences between age groups were not significant (p = 0.781) , seroprevalence progressively decreased from 2000 to 2010 and no reactivity was observed in 2012  that was significant in the younger (<7 years) and intermediate (7–15 years) age groups but not in the older one (>15 years) (p = 1.00).The IgG antibody levels to Pfsch07/03 and their distribution were compared between categorized age groups for the different study periods. For any given period, Pfsch07/03 IgG antibody levels progressively increased with age  (p = 0.006) and (7–15 years) (p = 0.0001) age groups contrasting with the (>15 years) age group for which a non-significant increase (p = 0.92) of mean IgG antibody level was recorded Fig. a. The meP. falciparum antigens and this was particularly apparent in the older age group  in the (<7 and 7–15 years) age groups, for all year-to-year comparisons except between 2002 and 2008. The profile observed in older individuals (>15 years group) was broadly similar to that of the intermediate group with a unique non-significant decline recorded between 2008 and 2010 (p = 0.921). The data suggest that interventions such as the shift from CQ to SP + Am in 2003 and the deployment of ACT in 2005 have somewhat impacted on the magnitude of IgG responses to While the magnitude of Pfsch07/03 IgG antibody responses in positive responders (OD ≥ 2) progressively decreased from 2000 to 2012 for the intermediate age group (7–15 years), the profiles were different for the younger and older age groups  and high (89.45%) seroprevalences to Pfsch07/03 antibodies respectively in children and older adults could be the reflect of more previous exposures to malaria infection of older adults. Further support to this is the significant decrease in seroprevalence observed in younger individuals compared to older ones, probably reflecting a delayed acquisition of immune responses in younger individuals originated from disrupted exposure to malaria following introduction of LLINs in 2008. Moreover, there is a general agreement that the persistence of clinical immunity acquired during early childhood is dependent on sustained exposure, thus antibody responses decreases when exposure to malaria is discontinued . By contSimilar to the prevalence of IgG antibody responses, the mean levels of Pfsch07/03 antibodies decreased substantially from 2000 to 2012 in almost all age groups despite two noticeable exceptions recorded between 2008 and 2010: a non-significant increase of mean IgG antibody levels in older individuals (>15 years) and an increase in mean antibody level in younger infants (<7 years). The decrease of mean IgG antibody level observed between 2000 and 2008 suggested that all implemented first line treatments contributed somehow to the reduction of available parasite biomass for mosquitos’ blood meal uptake that could have led to limited infectious contacts between infected mosquitoes and human hosts. In fact, it has been shown that EIR decreased from 482.6 to 155.3 infected mosquito bites/person/year from 2000 to 2008 in Dielmo, leading to a reduction of malaria prevalence from 70.6% in 2000 to 12.3% in 2008 in children and from 34.8% in 2000 to 9.0% in 2008 in adults [Anopheles gambiae s.l. infective bites, which in a community whose immunity had meanwhile declined [P. falciparum malaria antigens.The observed shift in the mean levels of Pfsch07/03 IgG antibodies in the younger individuals in 2000 (corresponding to the intermediate (7–15 years) age group in 2002 and 2008 and older (≥15 years group in 2010) revealed an additional interesting feature of malaria transmission and seroprevalence: the percentage of positive responders became progressively low from 2000 to 2008 in all age groups but increased significantly in the younger children (<7 years in 2010) before becoming null afterward in 2012. While the progressive decrease of the percentage of positive responders could be suggestive of a loss of immune responses to Pfsch07/03 antigens that adults had had when they were younger as a result of limited human-parasite contact, the increase seropositivity observed in younger individuals in 2010 could be explained by the rebound in malaria morbidity as previously reported . In factdeclined , 25, ledThe study revealed that all malaria control interventions implemented in Dielmo substantially contributed to the decrease of Pfsch07/03 IgG antibody responses either by preventing human-mosquitos contact, or by reducing parasite biomass. The present study demonstrates the wider potential of sero-epidemiological analysis in monitoring changes in malaria transmission resulting from a given malaria control intervention.


Infants are thought to be protected against malaria during the first months of life mainly due to passage of maternal antibodies. However, in high transmission settings, malaria in early infancy is not uncommon and susceptibility to the infections varies between individuals. This study aimed to determine malaria morbidity and infection during early childhood in rural Burkina Faso.Plasmodium falciparum infections were detected by rapid diagnostic test and/or light microscopy (LM) and quantitative PCR (qPCR).Malariometric indices were determined over 1-year follow-up in a birth cohort of 734 infants living in Nanoro health district. Clinical malaria episodes were determined by passive case detection at peripheral health centres while asymptomatic malaria infections were identified during  4 cross-sectional surveys at 3, 6, 9 and 12 months of age. P < 0.0001). Prevalence of asymptomatic infections was similar at 3, 6 and 9 months of age (17.7–20.1%) and nearly 1.6 times higher at 12 months (31.3%). Importantly, gametocyte prevalence among the LM-positive study population was 6.7%, but increased to 10% among asymptomatic infections. In addition, 46% of asymptomatic infections were only detected by qPCR suggesting that infants below 1 year are a potential reservoir for sustaining malaria transmission. Both symptomatic and asymptomatic infections showed marked seasonal distribution with the highest transmission period (July to December) accounting for about 89 and 77% of those infections, respectively.In total, 717 clinical episodes were diagnosed by qPCR over 8335.18 person-months at risk. The overall malaria incidence was 1.03 per child-year and increased from 0.27 per child-year at 0–3 months of age to 1.92 per child-year at 9–12 months of age. Some 59% of children experienced at least one clinical episode with a median survival time estimated at 9.9 months, while 20% of infants experienced the first episode before 6 months of age. The majority of the clinical episodes were attributable to microscopic parasitaemia (84.2%), and there was a positive correlation between parasite density and age (Spearman’s rho = 0.30; These findings indicate high and marked age and seasonal-dependency of malaria infections and disease during the first year of life in Nanoro, calling for intensified efforts to control malaria in rural Burkina Faso. Despite the scaling-up of malaria control measures in sub-Saharan Africa (sSA), malaria morbidity and mortality remains particularly high in some areas, especially among young children and pregnant women . In 2015Malaria burden among children under 5 years is well documented in sSA –8 with tAlthough children in early infancy are thought to be protected from malaria by maternal antibodies acquired in utero –26 and fIn Burkina Faso, malaria in infants remains the first cause of attendance to health centres . EpidemiThe aim of the current study was to investigate malaria infections and morbidity during the first year of life and assess variability in susceptibility between individuals in the rural health district of Nanoro in Burkina Faso. Using a highly sensitive quantitative polymerase chain-reaction (qPCR), the incidence of clinical episodes in a longitudinal survey and the age-specific prevalences of asymptomatic infections through cross-sectional surveys were determined. In addition, these results were compared with parasite detection by expert light microscopy (LM) to determine the prevalence of sub-microscopic infections in both symptomatic and asymptomatic patients. Finally, differences in susceptibility to malaria between individuals in early childhood were characterized.Plasmodium falciparum and the disease puts a significant burden on the population, especially in children under 5 years old and pregnant women. In 2016, health facilities in NHD reported 91,154 clinical malaria episodes, of which 9992 cases (11%) and 41,076 cases (45%) occurred among 0–1 year infants and 1–5 years children, respectively  using bivariate logistic regression models. Spearman correlation coefficient was used to estimate the correlation between parasite densities and age of occurrence of clinical episodes. The difference in median values  of parasite densities between (i) symptomatic and asymptomatic infections and (ii) infections occurring at 0–6 and 6–12 months, was analysed using the non-parametric Wilcoxon matched-pairs test. Out of the initial 761 infants (739 singletons and 11 pairs of twins) enrolled, 669  completed 1-year follow-up. Among the 92 (12.1%) infants who did not complete the follow-up, 7 (5 singletons and 2 twins from different mothers) died within 4 weeks after birth and 6 other singletons died before 12 months. In addition, 41 were lost to follow-up and 38 withdrew their consent mainly due to relocation out of the study area. Infants that were excluded from the analysis were the 7 neonates who died before 4 weeks of age due to the short duration of the follow-up and the 20 live twins owing to the risk of mixed samples and data from twin pairs.Finally, 734 infants were included for the present analysis Table . The meaIn total, 717 clinical episodes of malaria were diagnosed by qPCR out of 1522 fever cases (47.11%) over 8335.18 person-months at risk, resulting in an incidence of malaria episodes during the first year of life of 1.03 per child-year. The proportion of clinical episodes increased from 7% (50/717) at 0–3 months to 43.8% (314/717) at 9–12 months  experienced at least one clinical episode, and 201 (27.4%) experienced more than one episode. The mean number of clinical episode was 1.6 (range 1–4) per child. Nearly two-thirds (65.6% [284/433]) of the first malaria episodes occurred during the period of 6–12 months of age as compared to one-third (34.4% [149/433]) during the period from birth to 6 months. The survival analysis shows that the 6-months survival rate was 80%, while half of the infants (50% [367/734]) experienced the first clinical episode by 9.9 months. Severe malaria episodes represented 1.5% of total clinical cases. The frequencies of danger signs of severe malaria were: incoercible vomiting (63.6% [7/11]), inability to drink or suckle (36.4% [4/11]), impaired consciousness (27.3% [3/11]), respiratory distress (18.2% [2/11]), and severe anaemia (18.2% [2/11]).P < 0.001), with a median increasing from 1293.75 [IQR 127.00–7606.75] parasites/µL for 0–3 months to 24,822.50  parasites/µL for 9–12 months of age .Overall, parasite densities by qPCR correlated with age .The majority of clinical episodes presented with parasite densities detectable with LM . The distributions of sub-microscopic and microscopic parasitaemia according to age is presented in Fig. The prevalence of asymptomatic infections detected by qPCR were similar at 3, 6 and 9 months of age  but higher at 12 months  . As shown in Fig. P < 0.001.By qPCR, the overall median parasite density was 347.8 [IQR 1.94–3534] parasites/µL, which was significantly lower than that of clinical episodes  of asymptomatic infections were only detected by qPCR. The ratio of sub-microscopic:microscopic infections decreased from 0.9 to 1.2 at 3 and 6 months surveys to 0.6 at 12 months surveys . The lowest frequencies of malaria episodes were observed in May and June   Fig. a. Infantly) Fig. . Overallly) Fig. b. The evly) Fig. .Fig. 4MoP. falciparum gametocytes were detected in 29 blood smears from 28 individuals (9.7%). In total, 49 different gametocyte carriers were detected at both cross-sectional and longitudinal survey, resulting in an overall gametocyte prevalence of 6.7% (49/734) among the study population. The median gametocyte density was significantly higher in asymptomatic infections than in clinical episodes .The proportion of clinical episodes carrying gametocytes among LM-positive samples was 3.6% (22/603), with the 22 cases occurring in different individuals. Out of the 300 LM-positive asymptomatic infections, P. falciparum infections, which was characterized by an overall incidence of clinical malaria of 1.03 per child-year, an age-specific prevalence of asymptomatic infections ranging from 17.7 to 30.4% by cross-sectional survey, and a significant number of cases occurring during the first 6 months of life.This study describes malaria infection and morbidity during the first year of life in NHD, a rural area of Burkina Faso where the seasonal malaria chemoprevention (SMC) in children and SP–IPTi were not implemented at the time the study was conducted, though adopted by NMCP in 2014 . The finThis high prevalence and incidence of infections and disease is in agreement with previous studies in the country , 34 and Plasmodium infections between 0–6 and 6–12 months, about 29% of infants experienced a clinical episode and/or asymptomatic infection during the first 6 months of life. On the other hand, the observed increase of malaria morbidity with age is in agreement with findings of a pooled analysis, which included data from different epidemiological settings in sSA [The acquisition of maternal antibodies due to transplacental transfer and the presence of HbF in early infancy –28 are cs in sSA .The majority of clinical episodes had parasite densities detectable by LM (84%), while 46% of asymptomatic infections carried parasite densities only detectable by qPCR, indicating that parasite levels in peripheral blood are associated with clinical manifestations in infants. Moreover, in agreement with previous reports , parasitA marked seasonal distribution of malaria cases and infections was also observed, with the highest transmission period (coinciding with the wet season from July to December) accounting for 89 and 77% of all clinical episodes and asymptomatic infections respectively. In this regard, strategies such as SMC in children aged 3–59 months (from August to November)  can haveIn this study, 1.5% of infants developed severe malaria, which was lower than that estimated in peripheral health centres (4.2%) between 2014 and 2016 in the central-west region of the country , 56. AltOverall, the number of malaria episodes and asymptomatic infections vary among the study population with some infants not developing a malaria episode despite carrying parasite infections (asymptomatic infections) at one or several time points during the follow-up, suggesting a potential control or tolerance of malaria infections by some infants , 31. UnfVariation at individual level could be partially explained by differential benefit of the protective effect of maternal antibodies as their levels may vary from one newborn to another , 41, 59.Despite substantial efforts made in malaria control during this current decade in Burkina Faso, this study showed a high burden of malaria infections, with marked age and seasonal-dependency, during the first year of life in Nanoro. The high rates of asymptomatic infections (with a gametocyte carriage of 10% among LM-positive cases) and of sub-microscopic infections suggest that infants under 1 year old are a potential reservoir for sustaining malaria transmission. Intensified efforts to control malaria in rural Burkina Faso are urgently needed in order to reduce the disease burden in susceptible populations.


Patients presenting with severe falciparum malaria in a Bangladeshi tertiary hospital had higher total parasite burden, estimated by parasitemia and plasma PfHRP2, than uncomplicated malaria patients despite shorter fever duration. This suggests that higher parasite multiplication rates (PMR) contribute to causing the higher biomass found in severe disease. Compared with patients without a history of previous malaria, patients with previous malaria carried a lower parasite biomass with similar fever duration at presentation, suggesting that host immunity reduces the PMR. Informed consent was obtained from all patients, or their relatives if patients lacked capacity. Studies were approved by the local ethics committee and the Oxford Tropical Research Ethics Committee. Patients were enrolled if they had a blood slide positive for asexual eviously . The foln adults .Continuous variables were compared between groups using the Mann-Whitney U-test. Correlations were assessed using Spearman’s rank correlation. Nonlinear relationships were assessed using the method of fractional polynomials. An alpha of <.05 was used as the threshold for statistical significance. All analysis was performed using Stata version 14 .P = .01)  and were both significantly higher in the patients with severe malaria (P > .05) (P < .01 vs constant only or linear untransformed model), indicating an increase followed by a plateau of PfHRP2 with fever duration  of the patients with severe malaria died. Median fever duration was 7 days  in severe and 7.5 days  in uncomplicated malaria (P = .01) . The odd malaria . ParasitP > .05) , B and Cduration . No signP < .001) but was not associated with shorter fever duration . Peripheral blood parasitemia was lower in patients with previous malaria compared with those presenting with a first episode . Stratifying by disease severity, this difference was significant in patients with severe malaria  but not uncomplicated malaria . Age but not sex was a significant predictor of malaria severity  but not death , but this effect was not independent of a marker of biomass (reflected by log(PfHRP2) in plasma). Overall, an episode of previous malaria was associated with lower plasma PfHRP2 .In a multivariate logistic regression model for malaria severity including prior malaria, log pfHRP2, fever duration, and log age, only log pfHRP2 , fever duration , and age  remained significant predictors (McFadden’s Early diagnosis and treatment of falciparum malaria prevent severe disease and death. It is often assumed, therefore, that patients develop severe malaria because of delays in treatment. However, this study, conducted in an area of low seasonal transmission, suggests that a more fulminant disease process is present in severe disease. Patients with severe disease had higher plasma PfHRP2 and parasitemia yet slightly shorter durations of illness compared with adults presenting with uncomplicated falciparum malaria. A history of previous malaria was associated with a lower odds ratio for severe disease and lower estimated total parasite burden, but not a shorter duration of fever.These observations indicating a higher PMR in severe disease are consistent with a previous study from Asia in which the PMR measured ex vivo was higher for parasites from severe as opposed to uncomplicated malaria . HoweverIn Bangladesh, malaria transmission is seasonal . PreviouLimitations of this study include that patients or their attendants may report fever duration inaccurately, which could obscure a relationship with biomass. Consistent with this being reported relatively accurately, a previous study at this hospital observed the expected association between the duration of symptoms prior to admission and the distance from the hospital . PatientIn summary, intrahost parasite dynamics are different in adults with severe and uncomplicated malaria—patients with severe disease appear to have had a higher PMR and generally present with a larger biomass than those with uncomplicated malaria.


Plasmodium spp., and of designing vaccines against chosen antigens found on the parasite's surface. Given the extraordinary resources exhibited by Plasmodium to escape these traditional strategies, which have not been able to free humankind from the scourge of malaria despite much effort invested in them, new concepts have to be explored in order to advance toward eradication of the disease. In this context, amyloid-forming proteins and peptides found in the proteome of the pathogen should perhaps cease being regarded as mere anomalous molecules. Their likely functionality in the pathophysiology of Plasmodium calls for attention being paid to them as a possible Achilles’ heel of malaria. Here we will give an overview of Plasmodium-encoded amyloid-forming polypeptides as potential therapeutic targets and toxic elements, particularly in relation to cerebral malaria and the blood–brain barrier function. We will also discuss the recent finding that the genome of the parasite contains an astonishingly high proportion of prionogenic domains.The main therapeutic and prophylactic tools against malaria have been locked for more than a century in the classical approaches of using drugs targeting metabolic processes of the causing agent, the protist  Anopheles mosquito inoculates during a blood meal sporozoites of the malaria parasite, the apicomplexan protist Plasmodium spp., which migrate through the skin into the circulation and then to the liver. In a few minutes, sporozoites invade hepatocytes, where they will transform into merozoites that enter the circulation to invade red blood cells (RBCs) [Malaria infection starts when a parasitized female s (RBCs) . Merozois (RBCs) . Even ths (RBCs)  which lePlasmodium species are particularly high, especially in the proteome of the Plasmodium falciparum sporozoite. Several Plasmodium IUPs have been examined as potential targets of interest for protective immune responses because repeats present in many of them are highly immunogenic showing strong reactivity with antibodies. However, antibodies raised against these repeats do not mediate protection in front of the parasite, probably because IUPs adopt more ordered structures when interacting with different ligands, and antibodies induced by such regions may recognize a variety of antigen conformers and thus react poorly with the antigen on the parasite surface. The structural plasticity of IUPs, which allows promiscuous binding interactions, may favor parasite survival both by inhibiting the generation of effective high affinity antibody responses and by facilitating the interactions with host molecules necessary for attachment to the vascular endothelium and for the invasion of host cells. Amyloidogenesis is undergone by a diverse group of evolutionarily unrelated proteins from different organisms, all sharing permanent or transiently disordered structural regions with propensity to form β-sheet aggregates in their complete or fragmented forms.Sequence analysis has shown that apicomplexan parasite proteomes are enriched in intrinsically unstructured proteins (IUPs) , which cP. falciparum, merozoite surface protein 2 (MSP2) is unusually hydrophilic and contains tandem sequence repeats characteristic of IUPs [P. falciparum is high, and the MSP2 isoforms 3D7 and FC27 have been shown to elicit significant antibody responses in clinical studies [in vitro [in vitro into fibrils with amyloid-like properties [Among several merozoite surface proteins being assessed as potential components of a vaccine against  of IUPs . MSP2 is studies . Recombiin vitro . The foroperties   filaments anchored at their bases to the plasma membrane, as in other species of Plasmodium, such as P. knowlesi [Amyloid-like protein aggregates are associated with the surface of several microorganisms . Functioknowlesi  , we can possibly conclude that both MSP2 signal peptides are at least as amyloidogenic as the amyloid β peptide (Aβ). The N-terminus amyloidogenic region within the signal peptide is probably lost at the beginning of the protein transit through the endoplasmic reticulum (ER), whereas the C-terminus sequence within the GPI anchor signal peptide is excised later in the polypeptide processing. Because MSP2 is very abundant on the merozoite surface [Somewhat surprisingly, analysis of the peptide sequence of MSP2 isoforms 3D7 and FC27 with the amyloid sequence-predicting algorithms  FoldAmyl protein . In Tabl surface , these tin vitro assays to explore if both peptides formed amyloid-like structures in agreement with the theoretical predictions presented above. Two peptides derived from the MSP2 GPI anchor and signal sequences, SSNIASINKFVVLISATLVLSFAIFI-KKKK , and MKVIKTLSIINFFIFVTFNI-KKKK , both >95% purity according to chromatographic analysis, were incubated in phosphate buffered saline (PBS) at 37°C for 2–3 weeks. Since in both peptides the amyloidogenic region starts after or with a lysine , medium  and low immunoreactive . In each group, half of the samples were malaria positive by PCR. In addition, 11 sera from Equatorial Guinea patients with MSP1 and/or MSP9-specific IgGs were also examined. Malaria nonexposed control sera were used to determine the baseline signal; all serum samples were from blood donors that had provided informed consent and the procedure was approved by the clinical research ethical committee at the University Hospital of Getafe (Spain). The analysis of the recognition of the aggregated forms of MSP2 signal and anchor peptides by all sera did not show any values above the threshold in ELISA assays. Although one interpretation of this result is that such forms are not present in vivo, we cannot rule out the possibility that amyloidogenic peptide forms have low immunogenicity, as it has been described previously [Plasmodium might have evolved amyloid-forming regions in proteins that are at some time exposed to the plasma.As a first approximation to study the possible Plasmodium genome were related to some aspect of malaria pathophysiology, such as cerebral malaria (CM), the most severe neurological complication of falciparum malaria. Gene expression analysis suggested that CM and AD share common mechanisms of pathogenesis, as indicated by the accumulation of Aβ in brains of CM-susceptible (CM-S) but not in CM-resistant (CM-R) mice [Pathogenesis in Alzheimer's disease (AD) is linked to the accumulation of the highly amyloidogenic self-associating Aβ, whose oligomers, which are intermediate species in the assembly of fibrils, are powerful neurotoxins and might therefore be the key effectors of cytotoxicity . We deci-R) mice , in agre-R) mice . This re-R) mice . The ree-R) mice , has bee-R) mice . In addi-R) mice .Plasmodium to replicate while evading splenic clearance [Plasmodium-induced sequestration of infected and uninfected erythrocytes changes blood–brain barrier (BBB) function [Plasmodium merozoites from the pRBC, among which perhaps neurotoxic amyloids formed by MSP2 signal peptides. Amyloid structures have been described to bind glycosaminoglycans such as the chondroitin and heparan sulfates of cell-bound glycoproteins and proteoglycans, including those present on ECs of the BBB [P. falciparum E8B strain and free parasites showed significant adherence to confluent human brain microvascular endothelial cells (HBMECs) in an in vitro BBB model (Erythrocytes infected with mature stages of the malaria parasite bind to the endothelial cells in the capillaries of tissues in a phenomenon known as sequestration, which allows learance . pRBCs clearance . There ifunction . Entry ofunction . pRBC bi the BBB . ErythroBB model . AlthougPlasmodium-encoded protein unequivocally related to amyloid structures of potential pathological importance in malaria. However, recent data strongly suggest that in the short term, we will witness an important increase in the number of works dealing with the role in Plasmodium cell biology of a particular type of amyloids. When amyloid fibrils grow and divide with high efficiency they can propagate, become infective and transmit aggregative folds and are then termed prions [Plasmodium proved that they might encode abundant hydrophilic low-complexity protein fragments corresponding to species-specific, rapidly diverging regions that could form nonglobular domains helping the parasites to evade the host's immune response [P. falciparum the proportion of putative prions rises to an astonishing 10%. One in four proteins in this organism contains asparagine repeats, whose functions are still unknown [Plasmodium forces the organism through drastic changes in its environmental temperature, including cyclical fever episodes that can last several hours and exceed 40°C. Because heat shock increases the propensity of proteins to misfold and form aggregates, if only a fraction of the abundant prion-like domains in the malaria parasite aggregates during heat shock, this would certainly lead to the pathogen's demise. However, Plasmodium is able to survive with one of the most aggregation-prone proteomes in nature. This is due to the presence of specialized chaperones which prevent aggregation and are essential for parasite survival within RBCs [Plasmodium suggests that they might provide certain advantages, but the functional implications of such an amount of aggregation-prone proteins are still unclear. Identifying their so far unknown roles and mechanisms of action can provide new unexpected targets for future chemotherapeutic and prophylactic antimalarial approaches.MSP2 has been the first d prions . While pd prions . There aresponse . Analysiresponse . In most unknown , but arehin RBCs . AlthougPlasmodium proteome of amyloidogenic peptides suggests that these entities have a physiological function in the life cycle of the pathogen. Functional amyloids might have roles in the adhesion of the parasite to its host cells during invasion, or participate in some of the pathological effects of malaria related, for example, to blood–brain barrier alteration or the triggering of immune responses. However, Plasmodium-derived amyloids could also provide new prophylactic or therapeutic possibilities if we are capable of harnessing them for future innovative antimalarial strategies.The growing experimental evidence for the presence in the Plasmodium, MSP2, forms amyloid-like fibrils in vitro.At least one protein expressed by Plasmodium proteins might be released in the plasma.Amyloid-like structures formed by certain signal peptides of Cerebral malaria and amyloid-related Alzheimer's disease share common mechanisms of pathogenesis.Plasmodium-infected erythrocytes show adhesion to the blood–brain barrier according to in vitro assays.Plasmodium encodes one of the highest proportions of putative prion sequences.The genome of


Doppler velocimetry studies of umbilical artery (UA) and middle cerebral artery (MCA) flow help to determine the presence and severity of fetal growth restriction. Increased UA resistance and reduced MCA pulsatility may indicate increased placental resistance and intrafetal blood flow redistribution. Malaria causes low birth weight and fetal growth restriction, but few studies have assessed its effects on uteroplacental and fetoplacental blood flow.Colour-pulsed Doppler ultrasound was used to assess UA and MCA flow in 396 Papua New Guinean singleton fetuses. Abnormal flow was defined as an UA resistance index above the 90th centile, and/or a MCA pulsatility index and cerebroplacental ratio (ratio of MCA and UA pulsatility index) below the 10th centile of population-specific models fitted to the data. Associations between malaria  and abnormal flow, and between abnormal flow and birth outcomes, were estimated.Plasmodium falciparum (34 sub-microscopic infections), and 16 were Plasmodium vivax. Plasmodium falciparum infection before or at Doppler measurement was associated with increased UA resistance (adjusted odds ratio (aOR) 2.3 95% CI 1.0–5.2, P = 0.047). When assessed by ‘exposure’, P. falciparum infection was significantly associated with increased UA resistance  and a reduced MCA pulsatility index . Sub-microscopic P. falciparum infections were additionally associated with a reduced cerebroplacental ratio . There were too few P. vivax infections to draw robust conclusions. An increased UA resistance index was associated with histological evidence of placental malaria . A low cerebroplacental Doppler ratio was associated with concurrently measuring small-for-gestational-age, and with low birth weight.Of 78 malaria infection episodes detected before or at the ultrasound visit, 62 (79.5%) were P. falciparum infections impair fetoplacental and intrafetal flow, at least temporarily. Increased UA resistance has high specificity but low sensitivity for the detection of placental infection. These findings suggest that interventions to protect the fetus should clear and prevent both microscopic and sub-microscopic malarial infections.Both microscopic and sub-microscopic Trial Registration ClinicalTrials.gov NCT01136850. Registered 06 April 2010The online version of this article (doi:10.1186/s12936-017-1689-z) contains supplementary material, which is available to authorized users. The identification and management of risk factors for fetal growth restriction (FGR) is key to optimizing pregnancy outcomes. The incidence of low birth weight [(LBW) <2500 g], FGR and infant death are substantially higher in low- and middle-income countries (LMICs) , 2.Plasmodium falciparum and Plasmodium vivax infections are common during pregnancy in many LMICs, and are associated with LBW , MCAPI /time averaged velocity ), and ceFor each, z-scores were calculated, to adjust for variations in GA at the time of the study, because fetoplacental and cerebral blood flow vary with GA , 11. PreLastly, associations of measures indicating aberrant fetal growth with the Doppler measurements were evaluated. Specifically associations of Doppler indices with suspected SGA episodes at the time of scan, LBW, and placental malaria were investigated.Statistical analyses were performed using Stata 12.0  and Mathematica 9.0 . The majority of MCAPI and CPR measurements fell outside the previously published reference ranges,  with an exchangeable working correlation structure (binary outcomes) . AnalyseThe study cohort consisted of 396 singleton pregnancies that resulted in a congenitally normal live birth. The mean GA at enrolment was 19.6 weeks  were clinical.The 396 women were tested a total of 1565 times for malaria with either PCR or LM, 72 women tested positive for malaria infection in peripheral blood once, 13 twice and two women tested positive three times. Eighteen P. falciparum by LM or qPCR, and 27 infections were sub-microscopic. A total of 16 P. vivax infections were detected by either LM or qPCR .A total of 1179 peripheral malaria infection screening episodes preceded or coincided with Doppler scans. Overall, 59 were positive for Of 396 women, 361 had UA Doppler studies: 282 had one, 67 had two and 12 had three studies . MCA studies were performed on 298 women who had 370 MCA Doppler scans: 235 women were studied once, 55 women twice, seven women three times, and one woman four times. CPR data was available for 252 women, who had 303 paired UA and MCA studies. Of these, 206 women were studied once, 41 women twice and five women three times.The best fit equations (mean and standard deviations) for the relationship between UARI Eqs. , 1b, MCAThe Doppler velocimetry data and the respective regression curves and confidence intervals are shown in Fig. Tables P. falciparum parasitaemia before or at time of Doppler interrogation and a raised UARI measurement    . Malaria infections in the first 20 weeks of pregnancy were not associated with abnormal Doppler measurements.There was a significant association between the presence of P. falciparum parasitaemia was associated with reduced MCAPI ; a similar association was observed for sub-microscopic P. falciparum infections   Table . HoweverThere were 50 episodes of SGA  at the time of Doppler ultrasound in the cohort, 67 newborns (16.9%) had LBW and 46 out of the 308 histological analyses (14.9%) indicated placental infection (past and active infection).Table A reduced CPR was associated with SGA  and LBW , and similar results were observed when low CPR was defined as CPR <1.0  on flow was evaluated. Here, increased UARI and intrafetal flow redistribution were observed amongst women with microscopic or sub-microscopic P. falciparum infections. No such effects were observed for P. vivax infections or infections detected in early pregnancy  but their numbers were low. Documented episodes of increased UARI were associated with histological evidence of placental malaria at delivery.In this first Doppler ultrasound study from PNG to determine factors related to abnormal fetoplacental and intrafetal blood flow, peripheral P. falciparum infection can have deleterious effects on fetoplacental flow and intrafetal blood flow redistribution. Concurrent P. falciparum infection in early pregnancy was associated with increased UARI in two studies from Kenya [P. falciparum infection on placental vascular neogenesis, or potentially on uterine spiral artery invasion and conversion by reducing trophoblast motility and invasion capacity [P. falciparum infection may lead to more acute processes affecting placental function and perfusion, e.g., inflammation and placental thromboxane production. For the first time, the present research shows that falciparum malaria is associated with intrafetal blood flow redistribution, the diversion of blood flow from peripheral organs to more ‘essential’ organs, such as the brain. This can be a sonographic manifestation of growth restriction, and in clinical practice MCAPI is used to assess severity of suspected FGR in fetuses measuring SGA and who have normal umbilical artery Doppler studies [The present study contributes further evidence that om Kenya , 17, andom Kenya . An incrcapacity , 7. Alte studies . AlthougInterestingly, malaria infection was not associated with measuring SGA in pregnancy or at birth in this cohort . Most maP. falciparum [The present study observed stronger associations between malaria ‘exposure’  and Doppler indices than for malaria infections detected in peripheral blood at or before the Doppler study time point. The rationale for assessing associations of infections detected after ultrasound scan is that there is increasing evidence that in areas with declining malaria transmission a large number of infections remain undetected even by qPCR, in particular in pregnancy –48. In aPlasmodium vivax infections have been previously associated with LBW, and research indicates that such infections also affect umbilical artery flow [P. vivax infections and the study lacked power to demonstrate such relationships. There was no observable effect measure modification of the P. falciparum parasitaemia-blood flow relationship by maternal nutritional status or gravidity, in contrast to what has been demonstrated in other similar studies [ery flow , 49. The studies , 50.Plasmodium falciparum infection was associated with increased UA resistance in all analyses , yet the ‘exposure’ analysis revealed important additional associations, such as the impact of sub-microscopic P. falciparum infections on flow, with particularly strong impacts on the MCAPI and the CPR. Of note, a low CPR was the Doppler ultrasound measurement most strongly associated with LBW in this cohort. There is evidence to suggest that sub-microscopic P. falciparum infections can cause LBW, anaemia and possibly preterm birth [rm birth , 46. ThiIn contrast to other cohorts, gravidity, fetal sex  and MUACWomen with increased UARI measurement during pregnancy were more likely to have evidence of placental infection on histology. Although UARI had low sensitivity (0.26) for the detection of placental malaria, it had a good negative predictive value (0.87). Where ultrasound is available, further evaluation of its ability to predict placental malaria infection is indicated. The sensitivity of UARI could be improved by combining this measurement with other simple investigations that may help identify women with occult placental malaria .This study has some limitations. Firstly, the study was insufficiently powered to evaluate the effect of early pregnancy malaria and vivax malaria on fetoplacental and intrafetal flow. Secondly, the number of Doppler velocimetry measurements and malaria screening episodes was comparatively small, and thus episodes of abnormal flow or infection may have been missed. Only ~60 women had Doppler indices measured at two or more timepoints during pregnancy. In addition, only one measurement per assessment  was taken during each scanning episode, instead of the recommended three measurements . FurtherP. falciparum infections in pregnancy are associated with increased UARI and reduced MCAPI, which are indicators of placental insufficiency and fetal blood flow redistribution, respectively, and are both associated with FGR. Malaria prevention strategies for pregnant women must be able to clear all infections, including sub-microscopic ones. In malaria-endemic areas an abnormal UARI could indicate presence of placental infection, and women may benefit from presumptive treatment with a highly effective anti-malarial.In PNG, both microscopic and sub-microscopic


Concern exists about the safety of iron supplementation given to individuals in malarious areas. The possible unfavourable impact of iron supplementation on malaria might be less when slow-release iron compounds are used instead of ferrous salts, because no toxic non-transferrin bound iron is formed. The aim of this study was to determine the effect of iron supplementation using the slow-release iron compound iron polymaltose (IPM) on the acquisition of malarial parasitaemia.A randomized, placebo-controlled trial was performed in schoolchildren aged 5–18 years with mild or moderate anaemia on the Indonesian island Flores. Microscopic malaria-negative children were randomized to receive 8 weeks of IPM  or placebo . The primary outcomes were the occurrence of microscopically detectable malarial parasitaemia at week 4, 8, 12 and 16 after start of treatment and the proportion of participants with real-time (RT) PCR positive malarial parasitaemia at week 16.P = 0.01]. This higher rate was confined to iron-replete children. At the end of the study, 89% of the children in the IPM group had remained free from microscopically detectable parasitaemia vs 95% of children in the placebo group. The proportion of plasmodial RT-PCR positive children was similar in both groups at week 16 . When analysis was restricted to iron-replete children (serum ferritin ≥30 µg/l), there was a trend for a higher proportion being RT-PCR positive at week 16 in the IPM group compared with the placebo group . Erythrocyte microcytosis was an independent risk factor for microscopically detectable malarial parasitaemia.294 Children were assigned to the IPM group and 297 to the placebo group. Whereas IPM supplementation failed to increased haemoglobin or ferritin concentrations, the IPM group had a significantly higher rate of occurrence of microscopically detectable parasitaemia [hazard ratio 2.2, 95% C.I. 1.2–4.0; A short course of IPM should be used cautiously in anaemic children in malaria endemic areas, as it has limited efficacy in treating iron deficiency, while it increases the rate of microscopic malarial parasitaemia in those with replete iron stores.Trial registration ISRCTN 83091970. Registered 16 May 2012 (retrospectively registered)The online version of this article (doi:10.1186/s12936-017-1691-5) contains supplementary material, which is available to authorized users. Iron deficiency is highly prevalent in children in large parts of the world and is associated with anaemia and negative effects on immune function and cognitive development . There hPlasmodium vivax is more common, malaria transmission is generally less intense and clinical malaria is more frequently seen in older children.It is common practice among physicians in many parts of the world to prescribe a short course of iron in a therapeutic dosage to children with anaemia. Whether this practice is associated with an increased risk for malaria is unknown, especially in malaria-endemic areas outside sub-Saharan Africa where malaria caused by Slow-release iron compounds such as iron polymaltose (IPM) or sodium iron ethylenediaminetetraacetate (NaFeEDTA) are frequently used for iron supplementation instead of ferrous salts in Asian countries. These compounds may have a less unfavourable effect on malaria, as they do not produce toxic non-transferrin bound iron (NTBI) –9. HowevThe aim of our study was to investigate the effect of a short course of IPM on the risk for microscopic and real-time (RT) PCR positive malarial parasitaemia in anaemic school-aged children with a negative malaria blood slide on Flores Island, Indonesia. Including the number of PCR positive children is relevant as malaria parasite densities in (semi-) immune individuals are frequently below the microscopic detection limit. Secondary objectives were to determine the effects of IPM on haemoglobin (Hb) concentrations and iron status, to determine the influence of baseline iron status and mean cell volume (MCV) on the risk for malarial parasitaemia and the risk of children with submicroscopic malaria to develop microscopic parasitaemia.Plasmodium falciparum and Plasmodium vivax transmission in this area is stable enough to induce clinical immunity [P. falciparum, P. vivax, and Plasmodium malariae of 14.5, 13.2, and 1.9% respectively [Nangapanda village, Ende district, East Nusa Tenggara Province, Flores Island, Indonesia is a semi-urban village consisting of 18 sub-villages with an estimated population of 22,000 residents. It has a humid climate with an average yearly rainfall of around 1.800 mm and on average 82 rainy days. The rainy season lasts from November until April with a peak in rain during December to March. The primary general health care services are provided in a local Puskesmas . immunity . A populectively .This study was designed as a prospective double blind placebo-controlled randomized trial. It was conducted between July 2011 and January 2012. Children aged 5–18 years were recruited from twenty-one schools in Nangapanda area. Inclusion criteria were a negative malaria blood slide and mild anaemia, defined as a Hb level 8–11.4 g/dl in children below 7 years old, Hb level 8–11.9 g/dl for girls ≥7 years old and Hb 8–12.9 g/dl for boys ≥7 years old. These cut off levels were based on the 2011 criteria used by the Indonesian national government referral hospital Cipto Mangunkusumo. Exclusion criteria were clinical symptoms suggestive of malaria or any other underlying medical condition.®, Combiphar, Bandung, Indonesia) or placebo, both taken 6 days a week for 8 consecutive weeks. The iron(III)-hydroxide polymaltose complex tablets of 357 mg contain 100 mg of elemental iron. A weight-based dosage was used aiming at a daily dose of elemental iron of 6 mg/kg daily. Randomization  was performed using a computer-generated randomization list. The randomization list was created by researchers not involved in this trial, and the study doctors handed out the study medication using this list. Trial participants and laboratory personnel were blinded for treatment assignment; the study doctor was not blinded. The treatment phase started approximately 4 weeks after administration of albendazole and was followed by a follow up phase of another 8 weeks. Teachers handed out the study medication on a daily basis and documented compliance under supervision of the study clinician.Enrolled children were treated with the anthelminthic drug albendazole  400 mg OD for three consecutive days. Subsequently, they were randomized in a 1:1 ratio to receive either iron(III)-hydroxide polymaltose complex  and were reviewed by study clinicians at week 0, 4, 8, 12, and 16 following start of IPM or placebo. These study visits included measurement of body temperature and collection of venous blood, which was used for a full blood count, malaria slide  and real-time PCR detection of malarial parasitaemia and serum ferritin concentrations (latter two at week 0 and 16). The primary outcome measure was the detection of microscopically positive malarial parasitaemia at one of the four time points and the number of RT-PCR positive children at week 16. Secondary outcome measurements were the change in Hb and ferritin concentration and MCV value. Malaria slides were examined after the study had ended and results had therefore no clinical consequences for the participating children. With the exception of the full blood count, the other laboratory examinations were also performed after ending of the study. Children with any sign of illness during the study were referred to the Puskesmas.Plasmodium DNA isolation, prior to real-time PCR detection using Plasmodium-specific primers and species specific probes, as previously described [Presence of microscopic parasitaemia was examined by Giemsa staining of both thin and thick blood smears at the Department of Parasitology, University of Indonesia. An aliquot of 200 μl stored whole blood was used for escribed . Haemocyescribed . Iron deescribed .The study was approved by the ethical committee from the Faculty of Medicine, University of Indonesia (Ref: 96/PT02.FK/ETIK/2010) and all procedures followed were in accordance with the Helsinki Declaration. Parents or caretakers of all children provided written informed consent.P value <0.05 signified statistical significance.The sample size was calculated to detect a 1.5 hazard ratio (HR) in the IPM group with α = 0.05 and power of 90%. Baseline data are expressed as mean (standard deviation) or median (interquartile range), depending on whether data were normally distributed or as number (%). Differences in the characteristics between the intervention and placebo groups were evaluated by standard statistical methods. Overall cumulative probability to remain free of microscopic parasitaemia during the study period of 16 weeks was assessed by the Kaplan–Meier method and log-rank statistics using an intention to treat approach. Children were censored from the point at which they acquired the first episode of microscopic parasitaemia. Children presenting to the Puskesmas were also censored from the time of presentation. Differences in the proportion of RT-PCR positive children in the IPM and placebo group at week 16 were compared using Chi square tests. Exploratory subgroup analyses were also performed for participants separated on the basis of iron status and baseline MCV value. Unadjusted and adjusted HRs and 95% CI were calculated using Cox proportional hazards model with iron deficiency, age, sex and microcytosis as covariates. The first covariates were pre-specified, the latter (microcytosis) was added later after it was found to be independently associated with outcome. Data were recorded in MS Access  and exported for analysis in SPSS 21.0 . A P. falciparum PCR in the placebo group than in the iron group . Because of the low prevalence of submicroscopic parasitaemia at baseline, the PCR results for P. falciparum and P. vivax were combined (referred to as PCR Plasmodium) for further analyses. The proportion of children with absolute iron deficiency, defined as a serum ferritin level below 30 µg/l or 12 µg/l, was 26.9 and 8.6%, respectively. In contrast, microcytosis, defined as a MCV value below 75 fl, was present in over half (54.3%) of children. Using the Mentzer index (ratio MCV/RBC count below 13) as a screening test for thalassemia, this condition appeared common with an estimated overall prevalence of 23.0%.Figure A total of 13 children (10 from IPM group and 3 placebo group) were seen in between the regular study visits at the local health centre because of different clinical symptoms . They were not reviewed in a systematic way. Fever was among the symptoms in 4 children in the IPM group and one child in the placebo group. Because a final diagnosis was not established in these children and malaria diagnostics were not performed, they were censored from the time-point they presented to the local health centre.Table P = 0.01). The cumulative number of children developing microscopic positive parasitaemia between week 0 and 16 was 29  in the IPM group and 14  in the placebo group. At the end of the study, 89% of the children in the IPM group had remained free from microscopically detectable parasitaemia vs 95% of children in the placebo group. Seven children had fever (≥38 °C) at one of the four regular study visits time; three of these children had a positive malaria slide on the same time point. Exploratory analysis showed that when children were stratified by iron status, the increased rate of microscopic malaria in the IPM group was due to the increased rate in children who were iron replete (serum ferritin ≥30 μg/l) at baseline  of children in the IPM group and in 41  in the placebo arm (P = 0.47). Analysing only iron-replete children, we found that 41  out of 205 children in the IPM group and 28  out of 210 children in the placebo group (P = 0.07) were PCR Plasmodium positive. Overall, a higher proportion of children with microcytosis at baseline had a positive Plasmodium PCR at week 16 compared with children with a normal MCV value . However, in the subgroups of children with or without microcytosis, the proportion of Plasmodium PCR positive children was similar in the IPM and placebo arms.From the children with submicroscopic malaria at baseline (16 in the iron group and 18 in the placebo group), only 3 developed microscopically detectable parasitaemia during follow up (2 in the IPM group and 1 in the placebo group). At the end of the study, 5 children in the IPM group and 7 in the placebo group had remained PCR positive for The main findings of this study are that a short course (8 weeks) of therapeutic iron supplementation using the slow release iron compound IPM was ineffective in raising Hb or ferritin levels in anaemic Indonesian schoolchildren, whereas it did increase the risk for microscopically detectable plasmodial parasitaemia, as has previously been shown for ferrous salts in a fortification  and therWorldwide, the most common method of screening individuals for iron deficiency involves measuring haemoglobin or haematocrit. It should be taken into account, however, that iron deficiency anaemia only represents the end of the spectrum of iron deficiency. Recent iron supplementation studies in malaria areas were generally performed in children less than 5 years old from sub-Saharan Africa in whom anaemia and iron deficiency were common. The Indonesian children enrolled in our study were older, and although anaemia was also relatively common in the study area, it was mostly mild with relatively high iron stores (based on ferritin levels). In many low-resource areas, measuring iron status indicators such as ferritin is neither affordable nor logistically feasible. Instead of targeted iron supplementation based on iron status, supplementation using safer iron compounds that do not increase the risk for malaria would offer a valuable alternative. Unfortunately, the hypothesis that IPM would represent such an alternative, because it does not lead to the formation of NTBI in contrast to ferrous salts , 20, proIPM also had poor effectiveness in treating iron deficiency. This finding does not stand alone, as multiple studies have previously shown reduced or even absent effectiveness of IPM in correcting iron deficiency –13, 21. The mechanisms responsible for the higher incidence of malarial parasitaemia in those assigned to IPM remain speculative, especially since a clear effect of IPM on iron status was absent. It is also not easy to reconcile this finding with the fact that the increased malaria risk was confined to iron replete children. Moreover, only a minority of children with submicroscopic malaria at enrollment developed microscopically detectable parasitaemia and this risk was similar in the IPM and placebo groups. As reviewed recently, many questions still remain on how exactly iron deficiency protects against malaria and how iron supplementation may increase the risk for malaria . Direct P. vivax malaria is also common, and although the number of P. vivax cases was insufficient to analyse separately, its incidence appeared higher in the IPM group. This is consistent with two previous studies in children and pregnant women in which iron supplementation was associated with increased P. vivax burden [Whereas the Pemba trial showed an adverse effect of iron supplementation on malaria , anotherx burden , 34.The primary outcome in this study was microscopically or RT-PCR detectable plasmodial parasitaemia. It lacked sufficient power to examine clinical endpoints as the development of febrile malaria was rare and only few clinical events were noted. Only three children with a positive malaria slide had a concurrent fever at the same time point, suggesting that most of the children had asymptomatic parasitaemia. The observation that febrile malaria appeared rare is explained by the fact that the study was performed in semi-immune children from a moderate malaria transmission area. It can also not be excluded that the thirteen children who visited the local health centre had clinical malaria. However, fever was only reported by five of these children and many had symptoms suggestive for an alternative illness than malaria.Plasmodium vivax has a strong predilection for young erythrocytes, particularly reticulocytes, whereas P. falciparum also invades older and therefore more microcytic cells [P. falciparum favours invasion and replication in young erythrocytes as well [The data also showed that microcytosis was independently associated with the development of microscopic malaria. Although MCV has been included as a parameter in iron studies in malaria endemic regions –37, it wic cells . Recent  as well . One mayP. vivax separately. Malaria prevalence studies, conducted in the years prior to start of this study, had shown a prevalence of parasitaemia of 26.4% [This study has several limitations. First, the number of submicroscopic and microscopic malaria cases at baseline and during the study was lower than anticipated limiting the study power and the ability to analyse clinical malaria cases and of 26.4% . Second,A short course of IPM had limited efficacy in treating iron deficiency, but still increased the rate of occurrence of microscopic malarial parasitaemia. IPM is widely used in Asia, but the present findings add to the evidence that its use in anaemic children should be discouraged.