In sub-Saharan Africa, malnutrition and malaria remain major causes of morbidity and mortality in young children. There are conflicting data as to whether malnutrition is associated with an increased or decreased risk of malaria. In addition, data are limited on the potential interaction between HIV infection and the association between malnutrition and the risk of malaria.A cohort of 100 HIV-unexposed, 203 HIV-exposed (HIV negative children born to HIV-infected mothers) and 48 HIV-infected children aged 6 weeks to 1 year were recruited from an area of high malaria transmission intensity in rural Uganda and followed until the age of 2.5 years. All children were provided with insecticide-treated bed nets at enrolment and daily trimethoprim-sulphamethoxazole prophylaxis (TS) was prescribed for HIV-exposed breastfeeding and HIV-infected children. Monthly routine assessments, including measurement of height and weight, were conducted at the study clinic. Nutritional outcomes including stunting (low height-for-age) and underweight (low weight-for-age), classified as mild (mean z-scores between -1 and -2 during follow-up) and moderate-severe (mean z-scores < -2 during follow-up) were considered. Malaria was diagnosed when a child presented with fever and a positive blood smear. The incidence of malaria was compared using negative binomial regression controlling for potential confounders with measures of association expressed as an incidence rate ratio (IRR).The overall incidence of malaria was 3.64 cases per person year. Mild stunting  and moderate-severe stunting  were associated with a similarly increased incidence of malaria compared to non-stunted children. Being mildly underweight  and moderate-severe underweight  were not associated with a significant difference in the incidence of malaria compared to children who were not underweight. There were no significant interactions between HIV-infected, HIV-exposed children taking TS and the associations between malnutrition and the incidence of malaria.Stunting, indicative of chronic malnutrition, was associated with an increased incidence of malaria among a cohort of HIV-infected and -uninfected young children living in an area of high malaria transmission intensity. However, caution should be made when making causal inferences given the observational study design and inability to disentangle the temporal relationship between malnutrition and the incidence of malaria.NCT00527800.ClinicalTrials.gov:  Malaria is a leading cause of childhood morbidity and mortality in sub-Saharan Africa . AccordiFindings from studies evaluating associations between various measures of malnutrition and malaria have been contradictory. Some studies suggest a protective effect against malaria for wasted children  and chilThe role that HIV exposure and infection may have on potential associations between malnutrition and malaria in children has not yet been reported. This is a critical gap because HIV infection has been associated with an increased risk of both malnutrition ,16 and mTo assess associations between paediatric malnutrition and the incidence of malaria by HIV status, prospective data were collected from a cohort of HIV-unexposed, HIV-exposed and HIV-infected children living in Tororo, a rural area in Uganda with high malaria transmission intensity .This study was conducted in the district of Tororo, a rural area in south-eastern Uganda. Malaria transmission in Tororo is holo-endemic, occurring perennially with an entomologic inoculation rate estimated to be 562 infective bites per person-year . Data weSubjects were followed for all medical problems at a dedicated study clinic open seven days a week from 8 am to 5 pm. Parents (or guardians) were encouraged to bring their children to the study clinic whenever they were ill. After-hours care was available through the Tororo District Hospital. Children who presented with new medical problems underwent a standardized medical evaluation using algorithms to guide therapy for common illnesses that were developed locally by the study investigators. Medications with anti-malarial activity were avoided for the treatment of non-malarial illnesses. Monthly routine assessments were performed in the study clinic to ensure adherence with the study protocol. HIV-exposed children were re-tested for HIV by DNA PCR six to eight weeks after breastfeeding cessation. Those children who remained HIV-uninfected after breastfeeding cessation were then randomized to continue or discontinue TS prophylaxis as has been described elsewhere . HIV-infWeight and length/height measurements were taken at every visit to the study clinic at least once a month by trained study nurses in accordance with internationally recommended procedures . ParticiSubjects who presented to the study clinic with a documented fever (tympanic temperature ≥ 38.0°C) or history of fever in the previous 24 hours, had blood obtained by finger prick for a thick smear. If the thick smear was positive, the patient was diagnosed with malaria regardless of parasite density. Study participants ≥ 4 months and ≥ 5 kg were randomly assigned to receive either open label artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP) at the time their first episode of uncomplicated malaria was diagnosed. Study participants received the same treatment regimen for all subsequent episodes of uncomplicated malaria. Episodes of uncomplicated malaria in children < 4 months of age or weighing < 5 kg, as well as episodes of complicated malaria and treatment failures occurring within 14 days of initiating treatment were treated with quinine.Thick and thin blood smears were stained with 2% Giemsa for 30 minutes. A thick smear was considered negative if no parasites were seen after review of 100 high-powered fields. Speciation of parasites was based on readings of thin smears by a trained microscopist. A final diagnosis of malaria used in the analysis was based on a rigorous quality-control system that included re-reading of all blood smears by a second microscopist and resolution of any discrepancies between the first and second readings by a third microscopist within 24 hours of the initial smear reading.Assessment of infants HIV status prior to enrolment and at six to eight weeks after cessation of breastfeeding involved collecting a heel, toe or finger-prick blood sample on filter paper which was sent to a reference laboratory for HIV DNA PCR using Roche Amplicor HIV-1 DNA PCR test v1.5 kits .Data were double entered in Access , and statistical analysis was performed using STATA, version 10 . The observation period for each study participant began the day after enrolment and ended when a study participant was either prematurely withdrawn from the study or reached 2.5 years of age.Nutritional status was assessed using the following standardized anthropometric z-scores as per WHO guidelines ,28: heigMalaria incidence was defined as the number of new episodes of malaria per time at risk. Episodes of malaria occurring within 14 days of a previous episode were not considered incident events. Longitudinal associations between variables of interest and malaria incidence were estimated as an incidence rate ratio using generalized estimating equations with exchangeable correlation, negative binomial distribution, and adjusted for repeated measures. For longitudinal associations, measures of malnutrition were categorized into none, mild, and moderate-severe based on the cut-offs above for mean z-scores during observation periods of interest. Interaction among potential confounders was also examined by including interaction terms into the multivariate models. A p-value < 0.05 was considered statistically significant for all analyses.A total of 100 HIV-unexposed children, 203 HIV-exposed children, and 48 HIV-infected children were enrolled between August 2007 and April 2008  and then rose slightly to 30 months of age  and being prescribed TS chemoprophylaxis  were associated with a lower risk of stunting of any severity  of the study participants had experienced at least one episode of malaria. A total of 2,414 new episodes of malaria were diagnosed, resulting in an overall incidence of 3.64 episodes per person year. In multi-variate analysis, children with mild stunting  and moderate-severe stunting  had a similarly higher incidence of malaria compared to children without stunting (Table % of the This study was conducted in a cohort of young children living in an area of high malaria transmission intensity, where the incidence of malaria was over 3.6 episodes per person-year despite the use of long-lasting ITNs by all study participants and TS prophylaxis in majority of the study participants. Malnutrition was also very common, with 70% of study participants meeting criteria for mild stunting, and 38% of study participants being mildly underweight. HIV infection was associated with an increased risk of both stunting and being underweight, while breastfeeding and the use of TS prophylaxis were protective against stunting, but not being underweight.The main objective of the study was to evaluate associations between paediatric malnutrition and the incidence of malaria by HIV status. Stunting was associated with a modest 24% increase in the incidence of malaria after controlling for potential confounding factors, but the degree of stunting did not modulate this association. In addition, the association between stunting and an increased incidence of malaria was consistent between HIV-infected and uninfected children. In contrast, there was no statistically significant association between being underweight and the incidence of malaria.Interactions between malnutrition and HIV infection are complex and have been suggested to have negative feedback loops. Malnutrition is a significant risk factor for AIDS-related mortality and HIV-associated wasting often persists even with use of ART ,30. The Another novel finding of this study was that children taking TS prophylaxis had a significantly lower risk of stunting. A similar finding was reported in a randomized control trial of TS prophylaxis among HIV-infected adults in Côte d'Ivoire, suggesting that TS prophylaxis may improve nutritional status by the prevention of morbidity-related malnutrition or by other indirect mechanisms .Associations between measures of malnutrition and the risk of malaria appear to be complex as previous studies have reported a wide range of findings. One source of this complexity is due to differences in the indicators commonly used for malnutrition. Stunting is generally considered an indicator of chronic malnutrition, wasting generally reflects a recent and severe process, and underweight a reflection of a combination of factors. Earlier studies provide some evidence of a protective effect of stunting against malaria ,35 and tThere are several hypotheses as to why chronic malnutrition may increase the risk of malaria. It is now widely known that nutrition plays an important role in modulation of anti-pathogen immunity and most studies have demonstrated a deleterious effect of malnutrition on the appropriate immune response to infection ,38. In aStrengths of this study include the prospective, longitudinal study design and the assessment of HIV status. The mean of anthropometric measures of malnutrition taken over an extended period of follow-up were obtained and therefore better reflect the long-term nutritional status of a child compared to studies which rely on single measurements. In addition, malaria morbidity was analysed using an incidence measure of clinical disease, which best reflects the repeated nature of malaria in children residing in high transmission areas. Another strength is the inclusion of HIV-exposed and infected children, which has not been previously reported in studies evaluating associations between malnutrition and malaria. Limitations of this study include the lack of assessment of the temporal relationship between malnutrition and malaria. A number of studies have indicated a deleterious effect of malaria on nutritional status ,42. TherIn this study of young children living in a high transmission area, stunting was associated with an increased risk of malaria regardless of HIV status. Of note, the incidence of malaria was high in this cohort despite the use of ITNs and TS prophylaxis in a substantial number of children. Intervention studies aimed at improving nutritional status as a means of reducing the incidence of malaria in similar epidemiological settings deserve exploration.The authors declare that they have no competing interests.EA, TGS, JH, HW, AK, JT, MRK and GD contributed to the study design and oversight. EA, AG, WV, SY and GD contributed to the methodology, data analysis, interpretation of the results and drafting of the manuscript. All authors read and reviewed the final manuscript. All authors read and approved the final manuscript.


Plasmodium vivax has the potential to infect 2.85 billion individuals worldwide. Nevertheless, the limited number of studies investigating the immune status of individuals living in malaria-endemic areas, as well as the lack of reports investigating serological markers associated with clinical protection, has hampered development of vaccines for P. vivax. It was previously demonstrated that naturally total IgG against the N-terminus of P. vivax merozoite surface protein 1 (Pv-MSP1) was associated with reduced risk of malarial infection.Plasmodium vivax-infected individuals who did not develop clinical symptoms during a 2-months from those uninfected ones or who have had acute malaria. The acquisition of antibodies against Pv-MSP1 was also evaluated as survival analysis by prospective study over a year collecting information of new malaria infections in surveillance database.Immune response against Pv-MSP1 (N-terminus) of 313 residents of the Rio Pardo rural settlement  was evaluated in a cross-sectional and longitudinal follow up over two months (on site) wherein gold standard diagnosis by thick blood smear and rRNA gene-based nested real-time PCR were used to discriminate symptomless P. vivax-infected individuals (52-67%) showed immune recognition of the N-terminus of Pv-MSP1. Interesting data on infected individuals who have not developed symptoms, total IgG levels against the N-terminus Pv-MSP1 were age-dependent and the IgG3 levels were significantly higher than levels of subjects had acute malaria or those uninfected ones. The total IgG anti ICB2-5 was detected to be an important factor of protection against new malaria vivax attacks in survival analysis in a prospective survey (p = 0.029).The majority of P. vivax infected individuals and open perspectives for the rationale of malaria vaccine designs capable to sustain high levels of IgG3 against polymorphic malaria antigens.The study findings illustrate the importance of IgG3 associated to 2-months of symptomless in  Plasmodium vivax is the most widespread species of human malaria parasite, and risk of P. vivax infection is higher compared to Plasmodium falciparum[P. vivax being the most prevalent species (ca 85%) [P. vivax is currently associated with non-life-threatening malarial infection, recent studies have reported similar complications and pathogenic mechanisms frequently observed in malaria caused by P. falciparum[P. vivax as a less harmful parasite and raising the need for developing an effective vaccine.alciparum,2. In Br(ca 85%) . Althougalciparum-6. Drug alciparum,7-16 chaPlasmodium merozoite antigens have been proposed as targets for vaccine design [Pvmsp1 gene consists of six highly polymorphic domains  flanked by fairly conserved sequences  [In hopes of reducing malaria morbidity and mortality, e design . Amongste design . MSP1 ate design . MSP1 die design ,19,20. TP. vivax MSP1 (referred to as Pv-MSP1) humoral immune response has been shown to be mostly against the polymorphic domains [Many studies have indicated that MSP1 is highly immunogenic in natural malarial infections and often associated with parasite exposure -26. As s domains ,28,33-35 domains .Plasmodium vivax-infected individuals during a two-months follow-up at the Rio Pardo rural settlement , and performing humoral immune response analysis  against Pv-MSP1.The current study identified occurrence of symptomless At the Rio Pardo rural settlement Figure  an agricP. vivax was similar in both areas during the study period   and 48.6M-) negative or (M+) positive thick blood smear, for P. vivax or P. falciparum infection. Next, rRNA gene-based nested PCR assay was performed to identify P. vivax, P. falciparum or mixed  infections and the major were classified in minor subgroups: (PCR+/M-) positive mixed or P. vivax PCR and negative Giemsa-stained thick blood smears that showed no clinical symptoms of malaria during the two months of follow-up; (PCR+/M+) individuals positive for mixed or P. vivax PCR that were positive P. vivax for Giemsa-stained thick blood smears at cross sectional survey and had acute malaria. All subjects received anti-malarial drugs according to the practical guide for malaria treatment in Brazil [(PCR-/M-) negative individuals, who had no infection at cross sectional survey and individuals who were PCR positive or had malaria exclusively by P. falciparum were excluded from the study. Over 360 days, a prospective study was carried out and information about new malaria infection by P. vivax was collected through SIVEP-malaria database.In November 2008, a cross-sectional study was performed in order to collect blood samples to determine malarial infection by Giemsa-stained blood thick smears and real-time PCR and for serological studies to evaluate humoral responses of individuals of the riverine community. At this time, a standardized questionnaire containing demographic, clinical and epidemiological data  was applied by trained and calibrated interviewers. After that, a longitudinal follow up (on site) was performed over two months and the participants continued to be interviewed and examined. Based on the results of Giemsa-stained thick blood smears obtained at cross sectional, the participants were initially classified into major groups:  fusion proteins [P. vivax recombinant protein were purified using glutathione-sepharose 4B columns (Amersham Pharmacia), and protein concentration was determined by Bio-Rad Protein Assay Kit I  [The recombinant protein ICB2-5 (representing the N-terminus of Pv-MSP1) comprises three conserved blocks  and two variable blocks (Blocks 2 and 4) from the MSP1 protein of proteins -30,33. Aes, Inc) -30.Naturally acquired IgG antibodies against Pv-MSP1 have been characterized elsewhere by ELISA . Based oSimilarly, the positivity of serum to ICB2-5 was determined if the average OD of ICB2-5 was greater than ICB2-5 cut off. For each IgG subclass the ICB2-5 cut off was determined from same 20 healthy individuals who had never suffered malaria.IgG subclasses were determined by ELISA using specific monoclonal antibodies for each isotype  as described ,30. All P. vivax infection over one year follow-up period (November 2008 to November 2009) between the serology groups negative and positive for ICB2-5. Individuals who were found infected in the cross-section period using Giemsa-stained thick blood smears were excluded from this analysis. P-values <0.05 were considered statistically significant.Statistical analysis was performed using The R Project for Statistical Computing version 3.0.1. Normality was tested by Kolmogorov-Smirnov test. Logistic regression predicting positive vs. negative IgG anti ICB2-5 using non-infected subjects (PCR-/M- group) as reference was used to compare age, time of residence and time of last malaria attack. Differences in total IgG and subclasses levels were assessed between three groups  by one-way nonparametric Kruskal-Wallis test (first panel). Analysis to assess which group total IgG and subclasses levels were different was performed by non-parametric Dunn’s test for multiple comparisons (second panel). The significant differences (p < 0.05) were identified as letters “a” or “b”, intermediate level of antibodies was “ab”. The Kaplan-Meier survival analysis was performed to evaluate the probability of The study was approved by the Research Ethical Committee of the Federal University of Amazonas . Informed written consent was obtained from all participants. For those under 18 years old, parents were instructed about the objectives of the study and signed together with the participant an informed consent. All patients tested positive in the thick blood smear during the cross-sectional study were treated according to the anti-malarial treatment guidelines from the Brazilian Ministry of Health.From a census of more than 500 residents followed cross-sectional from September to November 2008, three hundred thirteen residents met inclusion criteria to participate of the current study. The epidemiological data from this human population were recorded through cross-sectional surveys and summarized in Table P. vivax, P. falciparum or mixed  infections performed through cross-sectional surveys were summarized in Table M-) and fifteen individuals had P. vivax malaria during cross sectional survey (M + P. vivax). Two individuals had acute P. falciparum mono-infection and were excluded  were excluded. From three hundred and eight individuals diagnosed by PCR, two hundred sixty eight individuals were uninfected at cross sectional because they were negative by microscopy examination and confirmed by PCR (PCR-/M-).After diagnosis by rRNA gene-based nested PCR assay for P. vivax infection was confirmed by microscopy examination and PCR in the fifteen individuals (PCR+/M+) of which one had acute P. vivax and P. falciparum infections. Twenty-five individuals whose diagnosis of P. vivax infections  was seen only in nested-PCR (PCR+/M-) showed no clinical symptoms of malaria over two-month follow-up  responded to ICB2-5, whereas among the subjects who did not have malaria infection until cross sectional survey (PCR-/M-), the frequency response was 32.0% (86/268). Importantly, frequency of responders to ICB2-5 from PCR+/M- was 52.0% (13/25) and slightly lower than PCR+/M+ .Acquisition of natural antibodies against the N-terminus of Pv-MSP1 showed from all samples tested, one hundred nine individuals responded with IgG anti ICB2-5 109/308), summarized in Figure 08, summaPCR+/M- group had more IgG anti ICB2-5 than uninfected individuals, whereas subjects with acute malaria had an intermediate level of antibodies “ab” . Analysing some epidemiological data, the presence of IgG antibodies ICB2-5 was solely dependent on the age (P = 0.003).Using logistic regression to assess antibody prediction into groups and epidemiological data Table , individPCR+/M- 84.6% (11/13) and PCR+/M + 62.5% (5/8) and reduced 44.2% (31/70) in the uninfected group  remaining the same in both groups 25.0% (2/8) in PCR+/M+ and 24.2% (17/70) in uninfected individuals (PCR-/M-). The frequencies of others subclasses were low.The subclass analysis was performed only those that had sufficient amounts of serum (N = 91) and numbers of tested sera for subclasses each group is shown in Figure PCR+/M-) were highlighted  or uninfected (PCR-/M-).When the subclasses levels were assessed between groups by one-way non-parametric Kruskal-Wallis test, increased levels of IgG3 anti ICB2-5 in infected individuals who did not develop symptoms  levels observed between groups were very low , which explains the higher frequency of responders to this polypeptide in endemic areas in Brazil [Despite that recombinant ICB2-5 contains long stretches of amino acids that are conserved among the n Brazil ,27,29, in Brazil . Howevern Brazil .PCR+/M- and PCR+/M+. As both groups represent respectively, contingents of infected persons with parasitaemia undetectable in thick blood smears and without clinical signs of malaria and those with patent parasitaemia and clinical symptoms, these data suggest that total IgG against the N-terminus of Pv-MSP1 may not be the only predictor of clinical protection. Nonetheless, total levels of IgG against the N-terminus of Pv-MSP1 were higher in individuals without clinical symptoms of malaria, corroborating with previous study [PCR+/M- group, in previous study IgG total levels were analysed separately to each subject [Investigation of total IgG response to the N-terminus of Pv-MSP1 revealed no significant differences between individuals of us study . Moreove subject .PCR+/M- in comparison to those had acute malaria, suggesting that higher levels of this subclass against the N-terminus of Pv-MSP1 could be related to absence of symptoms of malaria over two months of follow up. As parasitemia levels are detected only by real-time PCR and absence of symptoms of malaria does not allow determining since when these individuals were infected, it can be inferred that IgG3 dependent on continued exposure to the parasite could also be associated with this immune status.Analysis of antibody subclasses showed a stark difference in IgG3 levels of individuals from P. falciparum antigens, such as MSP142, MSP119, AMA-1, MSP2 and MSP3 [P. falciparum MSP1 (N-terminus) was associated with prolonged periods without malarial infection [Association between IgG3 antibodies and clinical protection from malaria was previously reported for and MSP3 ,23,40-44nfection . Moreovenfection . IgG3 renfection -49.Several studies have shown polymorphic antigens, including variants of the PfMSP1 Block-2, variants of the MSP-2, PvMSP5 frequently recognized by short-lived, species-specific antibodies -54. It iP. vivax[P. vivax incidence during the surveillance period  may be a potential candidate antigen to consider for malaria vaccine development. Based on study findings, optimal immunity may require sustained high levels of IgG3 against polymorphic malaria antigens, which may require special strategies that need to be factored into the vaccine development process.The immune status established by The authors declare that they have no competing interests.FGV carried out immunoassays. MESMA carried out expression of proteins. GCM, FOLV, LAS and LPS participated in the cross sectional study. PPO and LAMM carried out PCR. AASB performed the statistical analysis. WMM performed the prospective study accompanying the database. FTMC participated in the design of the study and manuscript. HAdP contributed reviewing data. MVGL contributed reviewing data and coordination. PAN participated in its design and coordination. All authors read and approved the final manuscript.


Plasmodium knowlesi is now established as the fifth Plasmodium species to cause malaria in humans. We describe a case of P. knowlesi infection acquired in Indonesian Borneo that was imported into Australia. Clinicians need to consider this diagnosis in a patient who has acquired malaria in forest areas of Southeast Asia. Plasmodium knowlesi is now recognized as a cause of potentially fatal human malaria in forest areas of Southeast Asia. We describe a case of P. knowlesi malaria acquired in Indonesia and imported to Australia.A 39-year-old man from Australia came to a suburban hospital in Sydney, New South Wales, Australia, with a 2-week history of morning fevers and mild headaches. His symptoms started 13 days after he left Indonesian Borneo . The patient had spent an average of 10 days per month for the past 18 months working adjacent to a forest area in South Kalimantan Province, Indonesian Borneo. The most recent visit was toward the end of the rainy season. He did not use any personal vector avoidance measures  or receive malaria chemoprophylaxis. The patient did not travel to any other malaria-endemic areas during this 18-month period.9 cells/L, reference range 150–450 × 109 cells/L), mild leukopenia , and unremarkable results for levels of hemoglobin , bilirubin , and creatinine .He did not have a remarkable medical history. On examination, he was febrile (38.9°C) and had a heart rate of 88 beats/min, blood pressure of 128/78 mm Hg, normal respiration rate, and oxygen saturation of 99% on room air. Physical examination was unremarkable. Laboratory investigations showed mild thrombocytopenia . A rapid diagnostic test result for histidine-rich protein 2 of P. falciparum was negative.Malaria parasites were seen on Giemsa-stained thick and thin blood films (parasitemia level 185 parasites/μL). Parasite morphologic features resembled those of hrocytes . Some paP. knowlesi in Malaysian Borneo, we conducted molecular studies to identify the species. Results of multiplex PCRs for P. falciparum, P. vivax, P. malariae, and P. ovale were negative .Given increased reports of The patient was treated with atovaquone/proguanil, 250 mg/100 mg, 4×/day for 3 days. His fever resolved and the thrombocyte count returned to the reference level within 48 hours. He did not show any complications.P. knowlesi was first described in Malaysian Borneo in 1965 after an unusual sequence of events  human malaria infections requiring hospitalization in Sarawak, Malaysian Borneo , trophozoites with a bird’s-eye appearance, mature trophozoites with a band appearance resembling P. malariae , and mature schizonts with a higher average merozoite count (16/erythrocyte) than in P. malariae (12/erythrocyte)  that cause human malaria is also present in P. knowlesi. Antibodies specific for pLDH of P. falciparum and P. vivax cross-react with pLDH of P. knowlesi  than in persons infected with P. falciparum (108 ± 59 × 109 cells/L) or P. vivax (118 ± 51 × 109 cells/L). Mean parasitemia level was 1,387 parasites/μL; 30.8% (33/107) of the case-patients had <500 parasites/μL. Severe infection was found in 7 (6.5%) of 107 patients, and the case-fatality rate was 1.8% (2/107) among hospitalized patients  and pig-tailed (M. nemestrina) macaques to humans by Anopheles latens mosquitoes  when humans visit forest or forest fringe areas. However, transmission does not seem to occur readily in villages .P. knowlesi infection that was acquired in Indonesia and imported to Australia. Fortunately, this patient had a low parasitemia level and mild disease. A high degree of clinical suspicion is likely to increase the number of P. knowlesi cases diagnosed in patients with malaria acquired in forest areas of Southeast Asia.We report a patient with


Malaria in pregnancy (MiP) is a major public health problem in endemic areasof sub-Saharan Africa and has important consequences on birth outcome.Because MiP is a complex phenomenon and malaria epidemiology is rapidlychanging, additional evidence is still required to understand how best tocontrol malaria. This study followed a prospective cohort of pregnant womenwho had access to intensive malaria screening and prompt treatment toidentify factors associated with increased risk of MiP and to analyse howvarious characteristics of MiP affect delivery outcomes.NCT00495508.Between October 2006 and May 2009, 1,218 pregnant women were enrolled in aprospective cohort. After an initial assessment, they were screened weeklyfor malaria. At delivery, blood smears were obtained from the mother,placenta, cord and newborn. Multivariate analyses were performed to analysethe association between mothers’ characteristics and malaria risk, aswell as between MiP and birth outcome, length and weight at birth. Thisstudy is a secondary analysis of a trial registered with ClinicalTrials.gov,number Overall, 288/1,069 (27%) mothers had 345 peripheral malaria infections. Therisk of peripheral malaria was higher in mothers who were younger, infectedwith HIV, had less education, lived in rural areas or reported no bed netuse, whereas the risk of placental infection was associated with morefrequent malaria infections and with infection during late pregnancy. Therisk of pre-term delivery and of miscarriage was increased in mothersinfected with HIV, living in rural areas and with MiP occurring within twoweeks of delivery.In adjusted analysis, birth weight but not length was reduced in babies ofmothers exposed to MiP .In this study, the timing, parasitaemia level and number ofperipherally-detected malaria infections, but not the presence of fever,were associated with adverse birth outcomes. Hence, prompt malaria detectionand treatment should be offered to pregnant women regardless of symptoms orother preventive measures used during pregnancy, and with increased focus onmothers living in remote areas. Despite numerous studies conducted over the last decades, malaria in pregnancy (MiP)remains an important public health problem that has proved difficult to tackle. Manystudies from areas with different malaria transmission patterns have investigatedthe consequences of MiP on both maternal health and birth outcomes. While theconsequences of MiP on maternal health are dominated by anaemia, data onmalaria-related maternal mortality are sparse . For theWhile reliable assessment of MiP is critical to elucidating its impact on birthoutcomes and infant health, it is problematic because many factors (some of whichare difficult to fully capture) are relevant to a complete understanding. MiP may beeither continuous or intermittent, depending on a woman’s exposure to vectors,level of immunity and possible co-infections , and on the efficacy of treatment and prevention interventions availableto her. Tools to measure parasite presence are limited by their sensitivity and byhow often women attend antenatal care services; hence MiP is often only partiallyobserved. To more fully evaluate the impact of MiP on both maternal and infantoutcomes, investigations must consider multiple aspects of malaria infection, suchas timing, frequency, intensity and severity of the infections, as well as thetreatment provided.Recent studies focused on one or a few features of malaria, such as timing and/orfrequency -15, or tMiP is thought to affect birth outcomes through two mechanisms, intrauterine growthrestriction (IUGR) and preterm delivery, which might - at least partially - explainthese discordant findings. It has been estimated that MiP in settings with stablemalaria transmission in Africa is potentially responsible for up to 70% of IUGR and36% of preterm delivery . The forIn recent years control of MiP has relied partly on intermittent preventive treatment(IPT), with WHO currently recommending at least two doses withsulphadoxine-pyrimethamine (SP) . HoweverThe study was conducted in Mbarara district, southwestern Uganda. Thispredominantly rural area lies at an altitude of about 1,500 m above sea leveland has moderate levels of malaria transmission . Betweenth week of gestation, we turned to apublished model that predicts gestational age from symphysis-fundal height (SFH)measurements and calibrated it using the data from the 16–20 week group [At baseline, a comprehensive assessment of the pregnant women’ssocio-demographic characteristics and health status was performed, including amedical and obstetrical history, clinical and obstetric examination, ultrasoundevaluation, blood smear and hemoglobin measurement. Estimated gestational age bywas determined by ultrasound in all women enrolled in the study between week16–20 of pregnancy (72% of the cohort). For the remaining mothers, i.e.,those recruited after the 20ek group , and thePlasmodiumvivax were treated with chloroquine. All women in the cohort receivedstandard supervised IPT with two doses of SP given at intervals of one month ormore during the second and third trimesters, as well as iron and folatesupplementation, antihelmintic treatment and insecticide-treated bed nets (ITN).All treatments were provided free-of-charge.After this initial assessment, the mothers returned to the clinic weekly for aclinical examination and malaria RDT. In case of positive RDT, malarialinfection was confirmed with a blood smear. Treatment of uncomplicatedfalciparum malaria included a random allocation of artemether-lumefantrine forthree days or quinine for seven days. Infections with only Plasmodium and malaria pigment.Placental histology was available only for a subset of the cohort (n=260).Placental malaria cases were classified according to the presence of parasitizederythrocytes, intervillous inflammation and haemozoin deposition [At delivery, blood smears were obtained from the mother, placenta, cord andnewborn to test for the presence of position ,35. NewbParacheck® RDTs were performed using a finger-prick blood sample andinterpreted according to the manufacturer’s instructions. Thick and thinblood smears were prepared and stained with Giemsa. Parasitaemia was calculatedby counting parasites against 200 white blood cells . Placental smears weretaken by incising a fresh placenta on the maternal surface halfway between thecord and the periphery, and were then examined for the presence of parasites andpigment .HIV testing and treatment was proposed to all participants and performedaccording to national guidelines , which iLow birth weight was defined as <2,500 g measured within 24 hours of birth;preterm as newborn gestational age <37 weeks at delivery; stillbirth as thedelivery of a non-living foetus ≥28 weeks gestation; and miscarriage asthe delivery of a non-viable foetus either at <28 weeks gestation or weighing<500 g.Various parameters of malaria exposure during pregnancy were described andanalysed for their temporal change and for their association with maternalcharacteristics or study interventions that may have affected MiPcharacteristics. Peripheral malaria was defined as the occurrence of a positiveperipheral blood smear. After a treated malaria episode, a subsequent episodewas considered a recurrence only after a minimum of 14 days, with at least onenegative blood smear during this period . PlacentThe risk of peripheral malaria infection was analysed with a mixed-effectsPoisson model ,38. SincThe number of IPT doses was introduced as a time-dependent covariate in the modelfor peripheral malaria risk. However, IPT was interrupted after the treatment ofa malaria infection, making the number of IPT doses an endogenous variable . Since dThe adverse outcomes evaluated in this study were stillbirth, preterm delivery,low birth weight and IUGR. IUGR was defined as a birth weight below the 10thpercentile of the birth weight-for-gestational age. Type I (symmetric) IUGR andtype II (asymmetric) IUGR were distinguished according to whether the Rohrerindex was above the 10th percentile of Rohrer index for gestational age or not.United States population-based references were used as standard ,41. The All analyses were performed using the open source statistical software R .NCT00495508.Written informed consent for study participation was obtained from allparticipants to the study. The study was approved by the institutional reviewboards of Mbarara University of Science and Technology, Uganda National Councilfor Science and Technology, and France’s “Comité de Protectiondes Personnes - Ile-de-France XI”. This study was registered withClinicalTrials.gov, number Of the 1,218 women enrolled in the cohort, 149 (12%) were excluded from thisanalysis because they were lost to follow-up before their pregnancy reached anoutcome  and to be at a moreadvanced stage of the pregnancy at enrolment . Of the 1,018 live births, 40 were excluded from theanalysis on birth weight and length  were observed at inclusion but no detectablemicroscopic parasitaemia were identified, while 24 (48%) were observed laterduring the follow-up period but with no previously documented infection.Peripheral malaria infection was associated with fever in only 16% of cases(n=62/361). The geometric mean (range) of parasitaemia was 1669 (24 –302 500) parasites/μL. There were 23 women who had malaria and were notin the trial. Of them, 13 (57%) received quinine, 6 (26%)artemether-lumefantrine and the information was missing for 4 patients(17%).A total of 304 (28%) women had one or more malaria infections detected byperipheral blood smear  during follow-up visits,resulting in a total of 361 peripheral malaria infections . Of the 242 (67%) infections recorded at inclusion, allinvolved In multivariate analysis, the risk of peripheral malaria was increased inmothers who were infected with HIV, were younger, primigravidae, had a lowereducation level, lived in rural areas and did not report bed net use atenrolment  andpigment in 17 (2.5%) cases. The presence of pigment was associated withdetectable parasites in 12 (7%) of the cases. Most of the infected placentas(17/20) came from mothers who had peripheral malaria detected during theirenrolment in the study; in the remaining three women, no peripheralmicroscopic or positive RDT was detected during pregnancy or at delivery.Conversely, almost all of the placental biopsies from mothers withoutmalaria detected during pregnancy had no haemozoin deposition norparasitized erythrocytes by histology (72/79). All eight malaria infectionsin mothers with detectable parasites, but no pigment in the placental smearwere observed during the third trimester. In adjusted analysis, theoccurrence of a placental infection was associated with the number ofperipheral malaria episodes, but not with parity  andpositively associated with parasitaemia level . No microscopic placental malaria was detected in womenwith a positive RDT, but negative blood smear.A total of 28 3%) miscarriages and 22 (2%) stillbirths were observed in thestudy cohort. In adjusted analysis, the risk of miscarriage or stillbirthwas significantly increased in mothers who were HIV-infected, living inurban areas or completed fewer follow-up visits  live-born pre-term deliveries were observed, with 45 (6%)occurring in the sub-group of mothers with ultrasound estimation ofgestational age. In adjusted analysis of both the full cohort data and thesubset with ultrasound, the risk of pre-term delivery was increased in womeninfected with HIV and in those with fewer follow-up visits Table . As withIn adjusted analysis, shortened gestation  was associated with the occurrence of malariainfection within the last two weeks of pregnancy Figure . A bordeIn total, there were 57 (7%) infants with low birth weight, 39 (6%) with type IIIUGR (asymmetric) and 89 (13%) type I IUGR (symmetric). In analysis adjusted forgender and for estimated gestational age at delivery, birth weight was reducedin primiparae, in mothers with low education level and in those who attended≤4 follow-up visits Table . By contAnalyses restricted to mothers with ultrasound-verified gestational age, or tothose with no or only one malaria infection, found qualitatively similarassociations, although sometimes with only borderline significanceFigure . Birth wThe novel features of this cohort study are the frequent, intensive malariascreenings (median of 21 screens per pregnancy) and the provision of treatment basedon the presence of parasite in the blood rather than on symptoms—practiceswhich differ markedly from those common in endemic Africa. Another strength of thisstudy is the accurate determination of gestational age for the majority ofpregnancies.Our results suggest that peripheral malaria infections during pregnancy, includingthose occurring late during gestation, contribute significantly to perinatalmorbidity. Malaria infection at the end of pregnancy and those with fever ratherthan other aspects of malaria exposure, were associated more specifically withmiscarriage or pre-term delivery. A similar association between malaria infectionswith fever and an increased risk of miscarriage has been reported in mothers with asingle malaria episode during the first trimester of pregnancy . LikewisAdjusting for gestational age at birth, we found that peripheral malaria infectionduring pregnancy was associated with lower birth weight, and that this associationwas consistently seen in both the full dataset and the subset of mother withultrasound examination. Furthermore, more severe birth weight impairment wasobserved after multiple malaria infections and in malaria infections with highparasitaemia, even when IPT and bed net use was reported. These findings underscorethe importance of implementing efficacious prevention, prompt diagnosis and highlyeffective anti-malarial treatment during pregnancy .In addition to primigravidity, a well-known risk factor for MiP ,4, it wain utero.A limitation of this study was that documentation of malaria infection began onlyafter the first trimester of pregnancy, resulting in left censored data. Women withmultiple infections were more likely to have been enrolled later during theirgestation and, therefore, early infections might have been missed. This couldexplain the absence of association in this analysis between MiP early duringgestation and low birth weight, in contrast to results from other studies ,14,15. APlacental malaria has been shown to be a key intermediate factor in the pathologicalpathway of malaria ,4,18,19.On the other hand, peripheral parasitaemia, which was associated with impaireddelivery outcomes in this cohort, can be detected by frequent screening, so thatprompt treatment can be given and adverse effects of the infection reduced. Sincepreventive efforts (IPT with SP and insecticide-treated bed net) still leave a largeproportion of women with parasitaemia, taking the opportunity to screen women whenthey present to antenatal care is a strategy that should be considered. However,diagnostics for MiP remain problematic, since pregnant women often have low levelsof parasitaemia and require diagnostic tools with greater sensitivity thanmicroscopy (and good specificity)—for example, the Loop-mediated isothermalamplification (LAMP) ,51. As mIn conclusion, this study shows that the timing, parasitaemia, symptoms and number ofperipherally detected malaria infections observed during pregnancy are associatedwith adverse outcomes. Prompt detection and treatment with an effectiveanti-malarial should be offered, irrespective of symptoms and use of otherpreventive measures in pregnancy. While frequent screening was associated withimproved birth outcome, reaching mothers living in remote areas to prevent lateattendance and low number of visits at antenatal care is essential, as they are morelikely to suffer from poor outcomes.The authors declare that they have no competing interests.PDB conducted the statistical analysis and wrote the paper. RM and PP designed thestudy, participated in the statistical analysis and manuscript drafting. ETparticipated in data collection, statistical analysis, and manuscript drafting. LWparticipated to the statistical analysis and manuscript review. CN and BTparticipated in the data collection, and manuscript review. YB participated in datacollection, data analysis, and manuscript review. AM performed the histologicalanalysis, interpreted the results and reviewed the manuscript. PG participated tothe study design and manuscript review. All authors read and approved the finalmanuscript.Appendix. Application of a multiple measures model usingsymphysis-pubis fundal height to predict gestational age in Ugandanpregnant women.Click here for fileSummary of the association between type I intra uterine growthrestriction and different parameters of malaria exposure duringpregnancy .Click here for fileSummary of the association between type II intra uterine growthrestriction and different parameters of malaria exposure duringpregnancy .Click here for file


Parasitological confirmation of malaria is now recommended in all febrile patients by the World Health Organization (WHO) to reduce inappropriate use of anti-malarial drugs. Widespread implementation of rapid diagnostic tests (RDTs) is regarded as an effective strategy to achieve this goal. However, the quality of diagnosis provided by RDTs in remote rural dispensaries and health centres is not ideal. Feasible RDT quality control programmes in these settings are challenging. Collection of information regarding diagnostic events is also very deficient in low-resource countries.A prospective cohort of consecutive patients aged more than one year from both genders, seeking routine care for febrile episodes at dispensaries located in the Bagamoyo district of Tanzania, were enrolled into the study after signing an informed consent form. Blood samples were taken for thick blood smear (TBS) microscopic examination and malaria RDT ). RDT results were interpreted by both visual interpretation and Deki Reader™ device. Results of visual interpretation were used for case management purposes. Microscopy was considered the “gold standard test” to assess the sensitivity and specificity of the Deki Reader interpretation and to compare it to visual interpretation.In total, 1,346 febrile subjects were included in the final analysis. The SD RDT, when used in conjunction with the Deki Reader and upon visual interpretation, had sensitivities of 95.3%  and 94.7%  respectively, and specificities of 94.6%  and 95.6% , respectively to gold standard. There was a high percentage of overall agreement between the two methods of interpretation.The sensitivity and specificity of the Deki Reader in interpretation of SD RDTs were comparable to previous reports and showed high agreement to visual interpretation (>98%). The results of the study reflect the situation in real practice and show good performance characteristics of Deki Reader on interpreting malaria RDTs in the hands of local laboratory technicians. They also suggest that a system like this could provide great benefits to the health care system. Further studies to look at ease of use by community health workers, and cost benefit of the system are warranted. Responding to the World Health Organization (WHO) recommendation that treatment for malaria cases should be administered only to cases with parasitological confirmation either by microscopy or rapid diagnostic tests (RDTs), many countries in sub-Saharan Africa (SSA) have adopted the use of malaria RDTs to improve case management and promote appropriate use of anti-malarial drugs . The appThe possibility of error in diagnosis using RDTs can be minimized through a careful selection process of RDTs, as recommended by WHO . Among oLarge-scale implementation of a diagnostic programme based on malaria RDTs requires the establishment of a comprehensive quality assurance (QA) strategy. This task can be particularly challenging considering that most RDTs are being used at point-of-care (POC) in health centres and dispensaries located in remote areas. Current recommendations by WHO include assessment of the competence of community health workers to perform RDTs, and to interpret and correctly use the results during supervisory visits, by direct observation with a standard checklist . HoweverRecently, the Fio Corporation  has developed a solution based on smartphone technology consisting of a mobile, battery-operated device (Deki™Reader), with a touch screen interface, that performs automated interpretation of RDTs  using proprietary digital image analysis software. Information related to the diagnostic event  is collected through the user interface, and after the RDT has been interpreted, the data along with a high-resolution image of the RDT are transmitted to a server using mobile-phone technology. At Fio cloud database (airFio™) the data are aggregated, analysed and presented in customized reports that can be accessed by authorized users in real time via a secure website portal.Plasmodium falciparum infections in an outpatient population in a malaria-endemic area of Tanzania.The use of new technologies, such as the one used in the present study, can help the large-scale implementation of RDT programmes by minimizing the chance of errors in diagnosis, providing expedited reporting of all diagnostic events, including those performed at remote POC locations, and allowing close monitoring of quality performance in the field. The objective of the present study was to assess the clinical diagnostic accuracy of the SD RDT when interpreted by the Deki Reader device and to compare it to the visual interpretation of RDTs by experts for detection of This was a study conducted in a prospective cohort of consecutive patients from January to March 2012 at Bagamoyo district in Tanzania. The district is situated north of Dar es Salaam and has an estimated population of 270,000 inhabitants, all of whom are considered to be at risk to develop malaria.Recruitment was from three peripheral health care dispensaries  located in the rural vicinity (approximately 30 km from the town of Bagamoyo), and from Bagamoyo District Hospital outpatient clinic in the urban centre of Bagamoyo. Subjects from both genders older than one year who were seeking routine care for malaria were enrolled into the study. A patient was enrolled once they were found to meet inclusion and exclusion criteria and had signed a study specific informed consent form.Before the conduct of the trial, the protocol was reviewed and approved by national (Ref: NIMR/R.8a/Vol.IX/l244) and institutional (Ref: IHI/IRB/No: 34) ethics committees and regulatory authority, TFDA (Ref: No: CEm57/180/04A/54).P. falciparum HRP2 and one that is pan-specific for pLDH for detection of other Plasmodium species.The malaria RDT used in the current trial was SD Bioline Malaria Antigen Pf/Pan . This is a lateral flow immunochromatographic test that contains a membrane strip encased in a flat plastic cassette. The strip is precoated with two antibodies: one that is specific for This mobile, rugged, battery-operated device  performs the following functions  and about three to four drops were collected in a heparinized microtube and sent to the reference laboratory of Ifakara Health Institute in Bagamoyo for preparation of thick blood smear (TBS) and a filter paper blood spot for polymerase chain reaction (PCR) testing, to be performed as quality control of microscopy. Technicians working at the reference laboratory were blinded to results of both visual and device interpretation of malaria RDTs performed at the site. Results of TBS microscopy examination and PCR (when applicable) were blinded to site technician who performed RDTs. At each of the study sites, the study technician collected blood from a finger prick for the processing of SD RDTs according to manufacturer’s recommendations. The processing of the RDT was guided by a job aid displayed on the screen of the Deki Reader device. The device also assisted technicians at the sites in keeping track of the incubation time for each RDT processed. Once the incubation period was completed, the study technician interpreted the RDT by visual inspection of the strip and captured the result using the touchscreen of the device. The study technician then immediately inserted the cassette into the device to perform the automated interpretation of the test. The results of the device’s interpretation of the RDT were concealed at all times, and were generated in all cases after the study site technician had entered results of her/his visual interpretation of RDT test results. All results of interpretation of RDTs, along with a high-resolution image of the RDT, and patient data collected were encrypted and automatically transmitted to a central cloud database specifically designed for the purpose of the trial.Patient management was performed according to routine local protocols, involving results of RDTs after visual interpretation by the study technician, and the opinion of the clinician at the site.TBS microscopy and PCR tests were performed at Ifakara Health Institute Reference Laboratory, Bagamoyo by technicians who were blinded to results of RDT interpretation by both expert study technicians and the Deki Reader.At the reference laboratory, blood smears were stained using 10% Giemsa solution for 30 minutes and examined under high-power objective. In positive smears, asexual and sexual parasites were counted against 200 and 500 white blood cells (WBCs), respectively. Parasite density was obtained by multiplying the parasite counts by 40 for asexual and 16 for sexual parasites . A smear was declared negative after examining 200 high-power fields.PCR was performed in all cases of discrepant results between expert visual interpretation of RDT, device’s interpretation of RDT and expert microscopy results (tiebreaker); and in a randomly selected subset of negative and positive samples . The PCR technique has high analytical sensitivity, allowing for qualitative and quantitative demonstration of parasites in clinical samples.P. falciparum beta tubulin target gene. DNA was extracted from dried blood spots by using QIAamp DNA mini kit (Qiagen), and PCR amplification was performed using Invitrogen QPCR SuperMix-UDG kit (Invitrogen). Real-time PCR amplification was done using MxPro3005 QPCR System ; forward (5’-TGA TGT GCG CAA GTG ATC C-3’) and reverse (5’-TCC TTT GTG GAC ATT CTT CCT C-3’) primers hybridize to the pathogen DNA. A probe (5’-TA GCA CAT GCC GTT AAA TAT CTT CCA TGT CT-3’) is included in the same reaction mixture. During PCR amplification, if the DNA of interest was present and amplified, the probe was cleaved and the reporter dye and quencher were separated leading to production of fluorescence. At the end of each PCR cycle, the resulting increase in fluorescence was detected by qPCR system as fluorescence units. As amplification continued, the cycle number at which detection of DNA products  was reached, and this information together with the standards  gave the value of parasitaemia.Molecular analysis involved extraction of parasite DNA from dried blood spots on filter papers. The DNA was used to determine the presence of Data were obtained from source documents at each of the participant sites and compared to information collected through devices and transmitted to the Fio cloud database. Data collected were monitored according to FDA regulations and GCP guidelines. There were three outcomes of interest for the purpose of the analysis in the current trial: a) visual interpretation of RDT: performed by laboratory technician at the site following visual inspection of the SD RDT at the appropriate time. This information was entered by the technician into the device; b) device interpretation of RDT: performed by the device, which was collected automatically by the device and transmitted to the database; and, c) microscopy results: performed on TBS by laboratory technician at reference laboratory and entered into the dataset manually. For the purpose of the analysis the result of TBS microscopy was considered the “gold standard”.Data collected at the Fio database were compared to data collected in the internal memory card of each Deki Reader device to ensure all data points were correctly transmitted to the cloud database. Data analysis was performed using JMP Version 8.0.2 and R Version 2.12.1 .The primary analysis was conducted using TBS microscopy as gold standard to determine diagnostic performance characteristics  and positive (PPV) predictive values, and overall diagnostic accuracy) of RDT interpretation by the Deki Reader and by visual interpretation. Correspondent 95% confidence intervals were constructed using the Wilson score method.The percentage of agreement in the interpretation of SD RDT between the device and visual (human eye) done by the expert laboratory technicians at the sites was calculated as percentage of negative agreement, percentage of positive agreement and overall percentage of agreement. Two-sided 95% confidence intervals were constructed using the Wilson score method.Plasmodium species (categorized as “P.f” and “Pan”), the statistical analysis was performed only in results of P. falciparum test line, due to the small number of infections expected by parasites other than P. falciparum.Although the RDT used in the present study is able to detect malaria infections caused by all P. falciparum.The prevalence of malaria among febrile patients presenting to a health clinic in the study area was estimated to be 10 to 15%, with greater than 95% of malaria cases caused by P. falciparum infection in the study area, the sample size was estimated to be between 1,200 to 1,500 subjects, under the assumption that it would provide approximately 120-225 positive results for P. falciparum. Assuming that the sensitivity and specificity of the interpretation of SD RDT is the same as the sensitivity and specificity of the human-eye interpretation this sample size would provide 95% confidence intervals within 5% of the point estimate.Based on this prevalence of Between January and March 2012, a total of 1,482 subjects were screened. Of the total, 1,346 (90.8%) met inclusion criteria after the pilot period and were included in the study and final analysis  and a specificity of 94.6% . The sensitivity of expert visual interpretation of RDTs was 94.7%  and the specificity was 95.6%   when a negative result was obtained, 96.9%  when a positive result was obtained, and an overall percentage agreement of 98.4%   discrepant results were truly false positive results.There were 29/1,346 (2.2%) discordant test results between the Deki Reader and the visual interpretation of RDTs. In eight of these cases, the software did not detect a control line, and therefore classified the test as “invalid” according to manufacturer’s recommendations. The visual interpretation of these eight cases was “negative” in six of them and “positive” in two. No re-testing of these samples was done. Out of the remaining 21 cases, in 15 the visual interpretation was reported as “negative”, and the device interpretation was “positive”; while in the other six cases, it was the other way around. In 10 (48%) of these 21 cases the discrepancy seems to have originated in a clerical error when entering the information into the device. Analysis of RDT images for these 10 cases that were transmitted to the portal and sorted in the database showed either the presence of a clearly visible control and test line(s) (and human expert interpretation was erroneously entered as “negative”) or a complete absence of test lines in the diagnostic strip (and human expert interpretation was erroneously entered as “positive”). The remaining 11 discrepant cases all correspond to RDTs visually interpreted as negative, in which the intensity of the test line was around the cut-off of the software, in some cases producing very faint lines that could have been missed by visual inspection . In three samples each of TBS positive and TBS negative results, PCR results were discordant  of discordant results between Deki Reader and visual interpretation in this study. A closer look at the discordant results showed that in about half the cases, the discrepancy was due to apparent clerical mistakes suggesting that device interpretation was correct; while in only nine cases (0.7%) was this due to a false positive result interpretation by the device  was tested to assess the performance characteristics of the Deki Reader. Considering the fact that different RDTs are available and used, the results cannot be generalize to other RDTs. However, the function of the Deki Reader is to interpret the presence or absence of a test line signal, independent of the analytes that are finally deposited on it. Hence, the performance of the automated reader is independent of the underlying immunochemistry and thus should provide similar performance characteristics for other malaria RDTs using different antigens and for RDTs used in diagnosis of other diseases in general.Despite the limitations of this study, the results obtained reflect the real situation of performance characteristics of the Deki Reader on interpreting malaria RDTs. More studies are recommended to provide further evidence of current findings and on the performance of the device using other types of RDTs. Further studies looking at feasibility of the use of the Fio system at community health worker level, cost benefit, suitability and acceptability by users, as well as involving RDTs for other diseases and other brands of malaria RDTs are warranted.The authors declare that they have no competing interests. SF and MC are employees of Fio Corporation.SS, SA and SF conceived and designed the study. SS, SA and SF wrote the manuscript. MC, CM, OJ and AM collected and monitored the data and provided comments on the manuscript. All authors read and approved the final manuscript.Discordant test Results between Deki Reader and Visual Interpretation of RDTs.Click here for file


Plasmodium acquired immunity.Intermittent preventive treatment in children (IPTc) is a promising strategy to control malaria morbidity. A significant concern is whether IPTc increases children's susceptibility to subsequent malaria infection by altering their anti-Plasmodium falciparum schizont extract were measured in Senegalese children (6 months-5 years old) who had received three rounds of IPTc with artesunate + sulphadoxine-pyrimethamine (or placebo) at monthly intervals eight months earlier. Potential confounding factors, such as asexual malaria parasitaemia and nutritional status were also evaluated.To investigate this concern, IgG antibody (Ab) responses to Plasmodium IgG Ab levels than the non-treated controls. When epidemiological parameters were incorporated into a multivariate regression, gender, nutritional status and haemoglobin concentration did not have any significant influence. In contrast, parasitaemia, past malaria morbidity and increasing age were strongly associated with a higher specific IgG response.Firstly, a bivariate analysis showed that children who had received IPTc had lower anti-P. falciparum seems to represent the main factor influencing anti-schizont IgG responses. Previous IPTc does not seem to interfere with this parasite-dependent acquired humoral response eight months after the last drug administration.The intensity of the contacts with  Malaria elimination is now considered a realistic goal for an increasing number of countries . It requPlasmodium immune responses. In Mozambique, sulphadoxine-pyrimethamine (SP) given at the age of three, four, and nine months did not significantly modify the development of naturally acquired antibody (Ab) responses to several Plasmodium falciparum antigens up to 24 months of age [Plasmodium IgG response in the Senegalese study [Additionally, immuno-suppression has been reported from experimental studies of artemisinin-derivatives . In the s of age . On the s of age . In the s of age  and SP is of age . In Ghans of age , malariase study , eight mse study , epidemiAnopheles larval breeding sites [P. falciparum asexual stage parasites/μl as the minimal threshold to consider a case as one of confirmed malaria. A cross-sectional survey was done in December 2002, in order to measure the impact of IPTc on the prevalence of children with parasitaemia, parasites resistant to sulphadoxine-pyrimethamine and anaemia at the end of the clinical follow-up period, and seven months later, in July 2003, before the beginning of the next rainy season. Anthropometric measurements were collected as previously described [Plasmodium asexual blood stages parasitaemia. Haemoglobin concentrations were measured in a HemoCue Machine® as previously described [The study population came from the community of Niakhar, situated in Central Senegal, 145 km east from Dakar, where regular demographic surveillance has been maintained since 1963 . It is ang sites . In 2002escribed . CapillaApproval of the study was obtained from the ethical review committees of the Senegalese government, the French Institute of Research for Development, and the London School of Hygiene and Tropical Medicine. Informed consent was obtained from parents or guardians before any child was included in the study.Plasmodium falciparum schizont extract was obtained from infected erythrocytes and coated on flat-bottom microtiter plates  at a concentration of 2 μg/ml. After blocking the plates with gelatin 0.5% in PBS, the plasma samples and a mouse biotinylated mAb to human IgG  were successively diluted in PBS-Tween 0.1% at 1:100 and 1:1000, respectively. After incubation with a peroxidase-conjugated streptavidin , ABTS (2.2'-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium, Sigma, St Louis, MO, USA) was added and optical density (OD) was measured at 405 nm. All sera were tested in duplicate wells coated with antigen together with a third single uncoated well to take into account non-specific bindings. A pool of positive sera was run in all the plates allowing adjustments for inter-plate variations. A negative pool  was also included in each plate.IgG antibodies were screened using a biotin-avidin amplified indirect ELISA. P. falciparum in December 2002 and in July 2003, haemoglobin concentration (g/dL) in July 2003 (continuous). Reference categories were as follows: females (gender), two years old class (age), normal , no malaria case (morbidity), no detected trophozoite (parasitaemia) and lowest value (haemoglobin). To account for the hierarchical structure of the data (groups of children sampled in 11 different villages), we included a random effect component at the village level in the regression model. Significance of the random effect was tested using a likelihood ratio test. Analyses were performed using the Stata software . The significance limit was P < 0.05.Comparisons between medians used the Mann-Whitney test. Multivariate hierarchical linear regression was performed to identify variables independently associated with antibody measurements. Since these were not normally distributed, optical densities were log transformed. Potential covariates entered in the model were: the treatment group (IPT vs placebo), age , sex, nutritional status  assesseThe 338 sampled children  lived in 11 villages (8-66 per village) and 158 (46.7%) received IPT in 2002. In this sub-sample, the overall malaria incidence in 2002 was 0.406 (placebo) and 0.089 (IPT) cases/child over the 3.5 months follow-up. Villages differed significantly in terms of parasite rates, ranging from 0 to 43.2% in December 2002 and from 4.8 to 42.9% in July 2003 .P. falciparum schizont in sera collected in July 2003 from children who had received either IPT or a placebo the previous year are shown in Figure Levels of IgG Ab against Plasmodium Ab levels  and at the beginning of the following transmission season (July 2003), was associated with a high Ab response, irrespective of the treatment received in 2002. Median IgG Ab levels were 5.2 (CI95%: 3.7-7.1) and 6.8 (CI95%: 5.4-8.0) times higher in parasitaemic children compared to blood film negative children at both cross-sectional surveys . Village of residence had a significant (P = 0.007) impact on antibody levels. The highest median specific IgG levels were observed in the villages closest to potential mosquito larval breeding sites (data not shown). No interaction was found between the covariates.Data were available from the 338 children except for information on parasitaemia for 48 children in December 2002 and 33 in July 2003 Table . When coPlasmodium immune response via two main distinct mechanisms, either by a direct immunosuppressor effect of the drugs or, indirectly, by reducing contacts between the treated child and Plasmodium thus impairing immunity development. There is already evidence to support the latter mechanism in the case of weekly chemoprophylaxis [P. falciparum is still susceptible to SP, a single dose of SP provides infants with a protection lasting from 4-6 weeks [Despite some debate on its efficacy, evidence is growing that IPT is a potentially valuable tool to control malaria in young children . It may phylaxis  and thisphylaxis ,31, neitphylaxis ) have shphylaxis . IPTi st-6 weeks  to 30-60-6 weeks  accordinPlasmodium schizont Abs compared to non-treated control children at the beginning of the next transmission season, eight months later. These findings are in agreement with the reduced anti-schizont IgG response measured in Ghanaian infants six months after a single dose of SP given alone [The present study showed that children less than five years who had received preventively the combination of SP + artesunate during the rainy season had a slightly lower level of anti-en alone  althoughen alone  versus sen alone ).Plasmodium IgG levels: age, village, past malaria morbidity and Plasmodium carriage. Age was a major determinant of specific IgG titres, especially among children older than four years. This is in agreement with findings in Northern Senegal where anti-schizont IgG responses increased progressively in one to five-year old children and then remained stable up to nine years of age [Plasmodium IgG responses in July 2003 than those who did not become sick. These observations suggest that release into the bloodstream of large numbers of schizont-derived Plasmodium stages during a malaria attack provides substantial antigenic stimulation to the immune effector cells. As reported in the two IPTi antibody surveys previously quoted [Plasmodium carriage at the time of blood sampling (July 2003) was associated with a strikingly higher anti-schizont IgG response. Increased humoral responses to various antigens in parasitized children compared to controls is a consistent observation [Plasmodium schizont Abs at a given time reflects a cumulative process of specific immunoglobulin production positively related to the level of contact between the host immune system and blood-stage parasites. It underlines the fact that Plasmodium carriage is the key factor for anti-schizont IgG production, irrespective of the preventive treatment received, although the possibility of some contributory effect from the antimalarial drugs used for IPT cannot be completely excluded.In a multivariate model, four co-variables significantly influenced anti-s of age . Age-asss of age  may expls of age  and in Gs of age , childrey quoted , Plasmodervation ,39, reflervation  may accoPlasmodium extract. Specific IgG Abs appeared to increase with the amount of exposure to Plasmodium asexual stages at the time of blood sampling but also to exposure during the previous 10 months. A more accurate analysis of the immune response, looking at different putatively protective isotypes and antigens [To conclude, administration of SP and artesunate used as sIPTc slightly decreased the humoral reactivity to a crude antigens , would bantigens . Monitorantigens .Haemophilus influenzae type b; IPT: Intermittent Preventive Treatment; IPTc: Intermittent Preventive Treatment in children; IPTi: Intermittent Preventive Treatment in infants; IPTsc: Intermittent Preventive Treatment in school-aged children; OD: Optical Density; PBS: Phosphate-Buffered Saline; sIPTc: seasonal Intermittent Preventive Treatment in children.SP: Sulphadoxine-Pyrimethamine.Ab: Antibody; EPI: Expanded Program on Immunization; Hib: The authors declare that they have no competing interests.DB, BG and FR conceived the study design. CS, BC and JFT coordinated the cohort follow-up. JBS, AMS and FF performed the bench work. DB and FR analysed the data and drafted the manuscript. All the authors amended the draft and approved the final manuscript.


Blackwater fever (BWF) is one of the severe forms of malaria. This complication was first described among non-immune European expatriates in the malaria endemic areas. Recently, resurgence of this form of malaria has been reported among the indigenous populations. The objective of this study was to investigate the risk factors among BWF patients.A case–control study was conducted between in four hospitals located in Kinshasa, Democratic Republic of Congo from January 2010 to December 2011. One hundred and twenty nine children were recruited with 43 (cases) and 86 (control).Plasmodium falciparum and Plasmodium malariae was observed between the two groups. Significant differences in haemoglobin, haematocrit, creatinine, urea and platelets levels were observed between the two groups (p < 0.001), but not for blood group and lactate dehydrogenase (LDH) level. Majority of the BWF cases occurred during the rainy season (88.4%). Treatment with quinine (95.3%) was significantly associated with cases (p < 0.001). Seven (16.2%) of the haemoglobinuric children developed acute renal failure.No significant difference in the gender and age distribution was observed between the case and control). The sex-ratio male to female in the case group and control group was respectively 1:1.0 and 1:1.1. The mean age was 8.62 years (SD = 3.84) in patients with haemoglobinuria and 8.55 years (SD = 3.77) in the control group. No difference in frequency of co-infection with Rainy season, low parasitaemia and quinine ingestion were the major risk factors significantly associated with haemoglobinuria. Acute renal failure was observed as the major complication of BWF. Plasmodium falciparum is the main species associated with complicated malaria cases such as blackwater fever (BWF) , p <0.001), while 59.3%, of the uncomplicated malaria group occurred during the same season , p = 0.015] Table . The mosAmong the haemoglobinuria patients, seven children (16.2%) developed acute renal failure (ARF), but only two children had peritoneal dialysis performed because of financial constraints. A conservative treatment was carried out for the remaining patients, with three of them developing anuric acute renal failure and succumbing with this complication (6.9%).In DRC, malaria is endemic, characterized by a high and perennial transmission. However, there are few reports on severe childhood malaria especially BWF. In the present study, the clinical and biological observations and the risk factors of BWF were evaluated for the first time in Central Africa. This case–control study has matched cases and controls for age, sex and location area. It suggests that haemoglobinuria in severe malaria is frequent in children above five years of age. The mean age of these patients in this series was 8.62 SD = 3.84) years. These findings are comparable with previous studies, which reported the high incidence of BWF in this age even in endemic stable areas where malaria immunity is acquired after six years .84 years,28. In NWhen BWF occurs among sufficient acquired malaria immunity patients, some questions must be addressed about the pathogenesis of this severe form of malaria and the immunity status of children who develop haemoglobinuria. The present study shows that children with haemoglobinuria have low parasitaemia compared to those without haemoglobinuria, [OR: 3.31 (1.41–7.78) with p = 0.005]. Many authors support that parasitaemia is low in BWF and sometimes may be absent ,30. TherPlasmodium falciparum remains the most prevalent species associated with the occurrence of severe malaria  in bi-variate analysis. This enzymatic defect is a known major risk factor for the occurrence of haemoglobinuria (BWF). This often occurs after taking oxidant products like quinine, mefloquine and others. Applying the logistic regression, results show OR: 0.39 (0.12–1.27). The high frequency of G6PD can suggest that G6PD may be high in Congolese population  and needIn addition, the results of this study show a significant association between quinine ingestion and the occurrence of haemoglobinuria [OR: 50.19 10.75–234.42) p < 0.001]. This is in line with previous studies that have reported repeated exposure to quinine as a risk factor for BWF , a low parasitaemia  and the ingestion of quinine  were the major risk factors associated with haemoglobinuria in this study. Among children with severe malaria, jaundice, severe anaemia and acute renal failure with elevated creatinine were the most common complications of BWF observed in the acute intravascular haemolysis compared to uncomplicated malaria. Immunology and genetics factors should be investigated to get more information about the risk factor of BWF in indigenous population in the endemic malaria areas.The authors declare that they have no competing interests.CNN, PMT, MNA and JMB, conceived, designed, deployed and directed the case–control study at the Department of Paediatrics at Kinshasa university hospital and wrote the manuscript. RLL carried out patient recruitment and follow-up, sample collection, storage and transport. KI and JV brought some precious corrections. PKK and PPA analysed data. AO edited the English and made corrections. All authors read and approved the final manuscript.