Understanding the factors which determine a household's or individual's risk of malaria infection is important for targeting control interventions at all intensities of transmission. Malaria ecology in Tanzania appears to have reduced over recent years. This study investigated potential risk factors and clustering in face of changing infection dynamics.Household survey data were collected in villages of rural Muheza district. Children aged between six months and thirteen years were tested for presence of malaria parasites using microscopy. A multivariable logistic regression model was constructed to identify significant risk factors for children. Geographical information systems combined with global positioning data and spatial scan statistic analysis were used to identify clusters of malaria.Using an insecticide-treated mosquito net of any type proved to be highly protective against malaria . Children aged five to thirteen years were at higher risk of having malaria than those aged under five years . The odds of malaria were less for females when compared to males . Two spatial clusters of significantly increased malaria risk were identified in two out of five villages.This study provides evidence that recent declines in malaria transmission and prevalence may shift the age groups at risk of malaria infection to older children. Risk factor analysis provides support for universal coverage and targeting of long-lasting insecticide-treated nets (LLINs) to all age groups. Clustering of cases indicates heterogeneity of risk. Improved targeting of LLINs or additional supplementary control interventions to high risk clusters may improve outcomes and efficiency as malaria transmission continues to fall under intensified control. Tanzania is heavily affected by malaria which is one of the leading causes of morbidity and mortality in the country , accountThe identification and quantification of heterogeneity in disease prevalence across a geographical range provides scope for targeting prevention and treatment interventions at high-prevalence or high-risk areas ,4. This A number of studies have used cluster analysis to identify spatial and temporal hotspots of malaria transmission in other parts of Africa -9. The eThis study aimed to investigate changes in malaria epidemiology in Muheza district by identifying significant household risk factors from individual, behavioural and house structural parameters. In addition, the study aimed to identify spatial clustering of malaria cases. It is intended that the study outcomes will inform targeting of interventions and treatment in the district and shed light on current epidemiological patterns.Anopheles gambiae s.l. has been shown to be the dominant vector in this region with abundance patterns strongly correlated with seasonal rainfall [The study was conducted within Muheza district in the Tanga region of North-East Tanzania . A 2001 census recorded the district population as 1,636,280 . Data werainfall . Malariarainfall  and assorainfall ,19.All children participating in the study were aged between 6 months and 13 years and resided in twenty one rural hamlets within five villages: Mlingano, Mwungano, Kwalubuye, Kibaoni and Misozwe. Villages were selected based on three criteria: that they had a population of approximately 400 residents and/or 100 children, that they were not involved with any other research programme and that they were within a logistically feasible distance of the research centre in Muheza town. Three state-recognised drug dispensaries were located within the study area: one in Misozwe and two in Kibaoni. Over 90% of all household heads in the study worked as farmers, mainly at the subsistence level.etrex legend®, Garmin International Inc. USA). Data were recorded using paper and personal digital assistants (PDAs) . Specific variables recorded were: individual-related: Age, gender, weight, height. Prevention behaviour-related: mosquito net use, type of mosquito net used , LLIN), frequency of mosquito net use, number of holes in mosquito net, proximity of penned livestock and structurally-related: house elevation, house volume, wall and roof construction material, presence and size of eaves and the number of rooms, doors and windows.Census data from 10,555 residents occupying 2,609 houses were collected during an enumeration round. Details were recorded of malaria prevention behaviours, socioeconomic status, house structure and location. GPS coordinates were taken from the front door of each residence using a GPS receiver , ranging from 0 to 6, was calculated as the sum of positive responses to questions concerning the ownership of the aforementioned items. Body mass index (BMI) was calculated as weight/height® Pf device, Orchid Biomedical Systems, India) to test for Plasmodium falciparum-specific histidine rich protein II. Thick and thin blood films were made for all children. Slides were stained with Giemsa, and double read at Teule hospital, Muheza. Asexual stage parasites were counted per 200 white blood cells (WBC) and gametocytes were counted per 500 WBC. Anti-malarials  were given if RDT was positive.Biometric measurements were taken from each child. Axillary temperature was taken using a digital thermometer and the parent/guardian was questioned about their child's recent history of fever. If axillary temperature was ≥37.5°C or a history of fever was reported then a rapid diagnostic test (RDT) was administered . All variables were analysed individually for an association with malaria risk using logistic regression. All variables showing evidence for a possible association with malaria risk (p < 0.15) were included in the preliminary main-effects multivariate logistic regression model. A stepwise backwards-elimination approach was then followed to exclude any variable that showed a lack of effect on malaria risk (p > 0.05). Models were multilevel to adjust for possible clustering of cases within village and households; this gave reduced weighting to each subsequent malaria positive child recorded from a village or household after the first. For multivariable analysis untreated ITN and no mosquito net use were combined, as was insecticide-treated ITN and LLIN use. Wald tests were used to analyse the effect of removing each non-significant variable from the model. Possible interaction terms were also considered before a variable was dropped.p-values for any clusters identified. The model was run with a maximum cluster size of 50% of the total population and p-values generated across 999 Monte Carlo replications.Spatial analysis was performed to look for possible clustering of cases across households. A Kuldorff spatial scan statistic was obtained using the Bernoulli model ,21 and SHouse locations were displayed using the GPS data and a geographical information system (GIS) . Data on administrative boundaries  were dowEthical approval for this study was granted from the London School of Hygiene and Tropical Medicine (LSHTM) ethics committee and the National Institute for Medical Research (NIMR) Medical Research Coordination Committee (NIMR/HQ/R.8a/Vol.IX/928). Each parent/guardian and their children were informed of the purpose of the study and the nature of the clinical prevalence work. Verbal and written consent was obtained from all study participants.P. falciparum prevalence was 14.5% (95% CI 12.7-16.3). 43.6% (95% CI 38.5-48.8) of children did not have a mosquito net, 50.6% (95% CI 45.4- 55.7) had an LLIN, 1.1% (95% CI 0.5-2.5) an insecticide-treated net and 4.7% (95% CI 3.1-7.0) an untreated net. Children in the younger age group (6 months-4 years) were more likely to sleep under an insecticide-treated mosquito net than children in the older age group (5-13 years) . There was no difference in the utilisation of mosquito nets between male and female children .A total of 1438 children were included in the study, 728 (51%) male, 702 (49%) female and 8 missing values. Overall Results of univariate analysis, after adjusting for possible village- and house-level clustering of cases, are shown in Table Multivariate logistic regression analysis with an adjustment for within village and household clustering indicated that there were three variables significantly associated with malaria risk Table .Risk of infection was significantly higher in children of five to thirteen years of age, compared to those aged six months to four years. There was strong evidence that sleeping under an insecticide-treated mosquito net was highly protective. The risk of malaria was lower for female than for males. There was no association between malaria risk and wall or roof construction material. There was no evidence of interaction between variables.Spatial scan statistic indicated two distinct spatial clusters of malaria cases Figure . In thesRisk factor analysis performed for this study identified a number of variables that, alone or in unison, would affect a child's risk of malaria. The child's age, gender and insecticide-treated mosquito net use were each associated with malaria risk. Use of insecticide-treated mosquito nets was highly protective against malaria whereas untreated nets were not. Older children, aged 5-13 years, and males had increased odds of malaria. Cluster analysis attempted to identify spatial hotspots of malaria where a child's risk of malaria was higher than expected for the study population as a whole. Two significant clusters of malaria were identified in two separate villages.Age remained an important factor within the model, with older age groups being exposed to a higher risk of malaria. This finding is of particular interest as historically in Tanzania prevalence has peaked in younger age groups -25. PartIn this study male children were at higher risk of malaria, possibly indicating that males were associated with high exposure behaviour, although insecticide-treated mosquito net coverage was not gender-skewed. Gender as a risk factor is likely to be linked to exposure, inherent and cultural factors and has inconsistent associations with malaria risk ,36. Due The protective effect of insecticide-treated mosquito net use shown in this study adds to the vast body of evidence supporting the efficacy and effectiveness of insecticide-treated mosquito nets for protection against malaria and other vector-borne diseases in this setting . In thisIncreased ability to target interventions in the Tanga region, a resource-limited setting, is vital. Identifying high-risk areas for disease allows policy to be tailored to give highest priority for LLIN distribution to those most at threat. An aggregation of cases within clusters may be somewhat driven by an elevated number of higher risk demographic groups residing within houses located in the clusters. Other factors not examined during this study but known to be important could also be rendering these areas as high risk ,38,43,44Identifying potential high-risk areas and the mechanisms by which the risk arises can have implications for the whole range of malaria-focussed activities, from increased surveillance to targeted interventions and treatment. In a low-income setting, such as rural Tanzania, any advances in the cost-efficiency and equitability of disease control are crucial.Malaria epidemiology in the Tanga region has not been static over recent years -14. ThisClustering analysis, when combined with knowledge of specific risk factors, will assist targeting of intervention measures to specific high-risk zones which could reduce costs, increase efficacy and improve the equity of control measures for the population in question. The ability to define high-risk areas for targeting of interventions in a newly emerging region of reduced transmission intensity is vital for local elimination of transmission and the rational distribution of control interventions.The authors declare that they have no competing interests.The study was designed by MK, PW and MR. PW, MK, GM and RM collected the data. PW, MK and MR drafted the manuscript. All authors read and approved the final manuscript.


The congenital muscular dystrophies (CMDs) comprise a heterogeneous group of heritable muscle disorders with often difficult to interpret muscle pathology, making them challenging to diagnose. Serial Sanger sequencing of suspected CMD genes, while the current molecular diagnostic method of choice, can be slow and expensive. A comprehensive panel test for simultaneous screening of mutations in all known CMD-associated genes would be a more effective diagnostic strategy. Thus, the CMDs are a model disorder group for development and validation of next-generation sequencing (NGS) strategies for diagnostic and clinical care applications. Using a highly multiplexed PCR-based target enrichment method (RainDance) in conjunction with NGS, we performed mutation detection in all CMD genes of 26 samples and compared the results with Sanger sequencing. The RainDance NGS panel showed great consistency in coverage depth, on-target efficiency, versatility of mutation detection, and genotype concordance with Sanger sequencing, demonstrating the test's appropriateness for clinical use. Compared to single tests, a higher diagnostic yield was observed by panel implementation. The panel's limitation is the amplification failure of select gene-specific exons which require Sanger sequencing for test completion. Successful validation and application of the CMD NGS panel to improve the diagnostic yield in a clinical laboratory was shown. COL6A1, COL6A2, and COL6A3 mutations, which manifest as Ullrich or Bethlem CMD; 2) recessive LAMA2 mutations, which result in merosin-deficient CMD (MDC1A); 3) recessive mutations in the POMT1, POMT2, POMGNT1, FKRP, FKTN, or LARGE genes, which manifest as purely muscular or syndromic conditions, such as Fukuyama CMD, muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), or CMD type 1C (MDC1C); 4) recessive mutations in the SEPN1 gene, which manifest as rigid-spine syndrome (RSS); 5) dominant lamin A/C mutations, which result in the congenital form of Emery Dreifuss muscular dystrophy; and 6) recessive ITGA7 mutations, which manifest as CMD with integrin A7 deficiency The main objective of human genetics is to identify the genetic variants underlying specific phenotypes and provide molecular diagnoses to guide clinical management Diagnosing a specific CMD subtypes may present a challenge and requires a multidisciplinary expertise . Currently, there are only a few centers with the expertise to recognize and delineate the wide range of overlapping clinical features Within the last 5 years, high-throughput sequencing technology, referred to as next-generation sequencing (NGS) has successfully identified mutations in novel genes for a number of conditions, including Sensenbrenner syndrome, Kabuki syndrome, and Miller syndrome In this study, we describe the development and validation of an NGS panel, using RainDance (as enrichment technology) and Applied Biosystems SOLiD3 (as sequencing platform), for comprehensive mutation detection in CMD genes, along with its diagnostic yield and clinical implementation in patients with confirmed diagnosis, serving as positive controls, and with clinically suspected CMD .POMT1 to 108× for POMGNT1, with an average of 50×. Despite the high mean gene read depth and target region coverage, several exons, including exon 1 of SEPN1 and all exons of POMT1 had no mapped reads The targeted next-generation sequencing panel was designed to amplify all exons of the 12 known CMD associated genes . The oveSEPN1, COL6A1, and POMT2 had very high GC content, namely, 87%, 73%, and 73%, respectively, specifically in their first coding exon, while POMT1 had an average of 56% GC content across all exons. In contrast, exons 34, 13, 6, 7, and 27, of COL6A1, COL6A3, FKTN, FKTN, and LAMA2, respectively, had very low GC content and consequently had no mapped reads in the NGS data.Of all the exons targeted, 49 consistently showed less than 20× average coverage across all samples, which could be due largely to sequence complexity, problematic library synthesis, or unusual GC content of the fragments . SEPN1, Known variants/mutations in the 5 variant-positive control samples re-detected by the CMD NGS test had coverage, Phred score, and allele percentages well above acceptable thresholds . This inCOL6A2, LAMA2, COL6A3, and COL6A2, respectively  to maintain an acceptable cost-benefit ratio. This was evaluated with the variant call data from known positive samples (CMD-8 to CMD-12) by including multiple parameters for variant filtering as illustrated in LAMA2 and FKTN yielded the highest percentages for the single-gene test, 64% and 35%, respectively. In contrast, panels 1 and 2, containing LAMA2 and FKTN, had call percentages of 54% and 94%. Generally, the total percentage call for the gene-by-gene approach was 17% compared to the higher total call percentage of 41% obtained by the 4 NGS panels. The low call percentage or mutation detection rate for individual gene tests is due mostly to incorrect presumption of genetic etiology and suspicion of the incorrect gene.Since the NGS CMD panel was made available at EGL, a number of tests have been ordered and yielded a higher call percentage than single-gene tests . Call peRecent studies have highlighted the potential of NGS in mutation detection POMT1 exons (19 exons) that failed amplification and hence had no coverage at all, most likely due to unusual GC content and sequence complexity. Amplification of exon 1 of most genes was problematic for similar reasons. The evaluation of NGS performance parameters is critical to offering such diagnostic strategies in a clinical laboratory. Our study design using negative  and positive controls (variant-positive samples CMD-8 to CMD-12) demonstrated the efficiency of the NGS technology in detecting all potential mutation types . The stuFollowing this successful validation, a clinical CMD NGS panel was launched at the Emory Genetics Laboratory (EGL) and has been used successfully by clinicians in CMD cases presenting with overlapping phenotypes, inconclusive biochemical studies, non-diagnostic brain or muscle MRIs. This expedited approach to molecular diagnosis avoids the diagnostic odyssey and cost associated with a serial gene testing approach. For example, on average the number of exons comprising a CMD gene is 25 (ranging from 1–65), costing around $2500 per gene for molecular analysis, clinical interpretation, and report issuance. Alternatively, the NGS-based sequencing panel offered for just $5000 includes comprehensive analysis of the current12 disease-associated genes. In addition, as more and more disease-causing genes are identified, they can be added to the panel without a significant increase in the overall cost, which is very unlikely to be the case for a gene-be-gene approach. In this study, the CMD panel approach convincingly showed better mutation detection or diagnostic yield compared to a single-gene analysis . The effAlthough whole-exome sequencing is making its way into clinical genetics, the high false-positive rate, long turnaround times requiring 3 to 6 months for analysis and interpretation alone, ethical challenges involving secondary findings, and the high test price when data and interpretation are included, may present prohibitive barriers to commercial application In conclusion, clinical CMD NGS panels offer cost-effective and more rapid turn-around molecular diagnostic testing than the conventional sequential Sanger sequencing of associated genes. A detailed step-wise approach to recommending NGS panel tests and diagnosing the disease subtype is illustrated as a flowchart . We antiCOL6A1, COL6A2, COL6A3, FKRP, FKTN, ITGA7, LAMA2, LARGE, POMGNT1, POMT1, POMT2, and SEPN1); Bethlem myopathy/Ullrich CMD sequencing panel in CMD-8 and CMD-10 ; merosin-deficient CMD Type 1A (MDC1A) panel in CMD-11 (sequencing of LAMA2); and muscle-eye-brain disease (MEB) panel in CMD-12 (sequencing of POMGNT1). Additionally, a variant-negative wild-type control sample (CMD-13) expected to lack any pathogenic variants/mutations was also included.To investigate whether an NGS panel can detect different types of mutations, 5 variant-positive samples , each with a different type of mutation , were included in this study. These 5 variant-positive samples have been previously characterized for pathogenic variants/mutations through the currently available Sanger sequencing panels: CMD comprehensive panel in CMD-9  were analyzed. To this end, Emory Genetics Laboratory staff were blinded to the identity, clinical phenotype and prior genetic testing results of these 20 samples. Written informed consent was obtained from all patients and approved by the Children's Hospital of Philadelphia IRB. The clinical features of each patient are summarized in http://frodo.wi.mit.edu/primer3/). All SNPs from dbSNP build 130 were filtered from the primer selection region. The in-house primer design pipeline performed an exhaustive primer design and selection across the 65-kb targeted region. The final library consisted of primer pairs for successful amplification of 383 amplicons with Tm ranging from 57 to 59°C and primer length ranging from 16 to 21 bp.A primer library for target amplification of all exons of the known CMD genes was designed using the manufacturer's design parameters (RainDance Technologies) and the Primer3 algorithm , 1.26 µl of MgSO4 (Invitrogen), 1.6 µl of 10 mM dNTP (Invitrogen), 3.6 µl of 4 M Betaine (Sigma), 3.6 µl of RDT Droplet Stabilizer (RainDance Technologies), 1.8 µl of dimethyl sulfoxide (Sigma), 0.7 µl 5 units/µl of Platinum High-Fidelity Taq (Invitrogen), and nuclease-free water to bring to a final reaction volume of 25 µl, per the RainDance protocol. These samples were then subjected to emulsification using the RDT1000 instrument (RainDance Technologies) to generate individual PCR droplets. Droplets for each sample were automatically dispensed as an emulsion into separate PCR tubes and transferred to a standard thermal cycler for PCR amplification. Samples were cycled in an Applied Biosystems GeneAmp 9700 thermocycler as follows: initial denaturation at 94°C for 2 min, 55 cycles at 94°C for 15 s, 54°C for 15 s, 68°C for 30 s, final extension at 68°C for 10 min, and a 4°C hold.After PCR amplification, an equal volume of RDT 1000 Droplet Destabilizer (RainDance Technologies) was added to each emulsion PCR tube, vortexed for 15 s, and centrifuged at 13,000 g for 5 min. The oil from below the aqueous phase was carefully removed from the sample. The remaining sample was then purified using a MinElute column (Qiagen) following the manufacturer's recommended protocol.The ends of the amplicons were blunt end-repaired by adding the reagents to the purified DNA (diluted to 68 µl): 10 µl 10× Blunting Buffer (Epicentre), 10 µl 2.5 mM dNTP Mix (Invitrogen), 10 µl 10 mM ATP, 2 µl End-it enzyme mix (Epicentre), and sterile water to a total reaction volume of 100 µl. The reaction was incubated at room temperature for 30 min, and the DNA was immediately purified using Ampure XP beads (Agencourt). The amplicons were then concatenated using the NEB Quick Ligation kit according to the manufacturer's protocol. DNA was purified using Ampure XP beads and eluted in 105 µl of low TE. An Agilent 7500 Bioanalyzer chip was run to confirm the concatenation of PCR products. The sample was then fragmented and processed as described in the manufacturer's standard SOLiD3 workflow (Applied Biosystems).LAMA2, COL6A1, COL6A2, COL6A3, FKTN, POMGnT1, POMT1, POMT2, FKRP, LARGE, ITGA7, and SEPN1. Samples were prepared by fluorescence sequencing on the ABI 3730XL DNA analyzer with BigDye Terminator chemistry and the BigDye XTerminator purification kit (Applied Biosystems). Individual sequences were aligned against reference sequences (downloaded from NCBI) using Mutation Surveyor v3.30 software (SoftGenetics) and analyzed for variations or mutations.Exon-specific primers were designed to amplify individual exons along with 20–50 bp of flanking intronic regions on either side for


Sickle cell trait (HbAS) is the best-characterized genetic polymorphism known to protect against falciparum malaria. Although the protective effect of HbAS against malaria is well known, the mechanism(s) of protection remain unclear. A number of biochemical and immune-mediated mechanisms have been proposed, and it is likely that multiple complex mechanisms are responsible for the observed protection. Increased evidence for an immune component of protection as well as novel mechanisms, such as enhanced tolerance to disease mediated by HO-1 and reduced parasitic growth due to translocation of host micro-RNA into the parasite, have recently been described. A better understanding of relevant mechanisms will provide valuable insight into the host-parasite relationship, including the role of the host immune system in protection against malaria. Plasmodium falciparum, has been a major cause of morbidity and mortality throughout human history. As a result, malaria has exerted extraordinary evolutionary pressure on the human genome and appears to have selected for multiple genetic polymorphisms that provide protection against severe disease . In Kenyan children, protection afforded by HbAS against symptomatic malaria increased from 20 % in children less than two years old to a peak of 56 % by age ten years . In a reP. falciparum appear to be increased in HbAS compared to HbAA individuals. The mean lymphoproliferative response to affinity-purified P. falciparum soluble antigens [P. falciparum in HbAS children. However, it is unclear whether this is a cause or effect of the protective effects of HbAS. The lymphoproliferative response is suppressed during and after acute malarial infection in HbAA individuals [Cell mediated responses to antigens -81 was fantigens -85 but aantigens ,83. Thusividuals ,81,86-89P. falciparum proteins found no increase in the magnitude or breadth of the P. falciparum-specific IgG response [vs HbAA children living in areas of low malarial transmission in Burkina Faso [Investigators have also found evidence for an enhanced humoral response in subjects with HbAS. Increased levels of gamma globulin were found in HbAS compared to HbAA children ,91. Howeresponse . One stuina Faso , but notina Faso , possiblin vitro[In contrast, higher levels of IgG directed at PfEMP-1 family proteins, which are located on the surface of the red blood cell, have been found in individuals with HbAS in a number of studies. On study found HbAS individuals had a higher IgG response to the infected red blood cell in vitro. In The in vitro, Gabon [in vitro, and a lin vitro, HbAS chin vitro. Howeverin vitro,98,102 pin vitro,35,41,46in vitro. Higher in vitro,35,46. APlasmodium berghei infection. They also reported decreased expansion of cytotoxic CD8 T cells that they hypothesized may also contribute to protection against severe disease. Protection was shown to be present irrespective of parasite density. Although the majority of human studies have shown that HbAS is, in fact, associated with lower parasite densities during symptomatic malaria [In addition to direct antiparasitic effects, HbAS may confer protection against severe malaria by limiting pathogenesis independent of effects on pathogen load. A recent study using a well-established mouse model of sickle cell disease implicated increased levels of hemeoxygenase-1 (HO-1), an enzyme that breaks down free haem, in the mechanism of protection against cerebral malaria . The aut malaria -31,33,46Plasmodium falciparum also likely inflicts oxidative damage on the HbAS red blood cell. Chronic low levels of oxidized haem in sickled red blood cells may induce HO-1, leading to host tolerance in severe disease. Oxidized haem may also interfere with the formation of an actin cytoskeleton, leading to altered PfEMP-1 expression on the infected red blood cell surface membrane and reduced cytoadherence. Reduced cytoadherence may lead to decreased endothelial activation and decreased inflammation implicated in the pathogenesis of severe malaria. In addition, it could also lead to increased splenic uptake of infected erythrocytes. Within the spleen, increased antigen presentation may lead to accelerated development of protective immune responses, including antibodies to PfEMP-1, potentially augmented by biochemical protection against high parasite densities that might otherwise dampen an effective response. Higher levels of antibodies directed against PfEMP-1 and possibly other surface proteins may enhance opsonization and phagocytosis of infected red blood cells and further destabilize the cytoadherence properties of infected red blood cells. While there are some data to support all of these mechanisms, it is still unclear which ones are relevant in vivo. Together, however, it is clear that relevant mechanisms lead to better control of parasitaemia in HbAS children and protect against both uncomplicated and severe malaria. Improved understanding of the mechanisms of protection derived from this single point mutation will give further insight into the host-parasite relationship including how P. falciparum interacts with the human immune system.It is likely that both biochemical and immune mechanisms contribute to the protection afforded against falciparum malaria by the HbAS genotype. HbS clearly induces biochemical changes in the red blood cell that may affect parasite metabolism and growth. Plasmodium falciparum erythrocyte membrane protein 1; PSE: Parasite schizont extract; RESA: Pf155/ring infected erythrocyte surface antigen.AMA1: Apical membrane antigen 1 (AMA1); CSP: Circumsporozoite protein; EBA175: Erythrocyte binding antigen 175; GLURP: Glutamate-rich protein; HbAA: Normal adult haemoglobin; HbAS: Sickle cell trait; HbC: Haemoglobin C; HbS: Sickle haemoglobin; HO-1: Hemeoxygenase-1; MSP1: Merozoite surface protein 1; MSP1: Merozoite surface protein 2; PfEMP-1: The authors declare that they have no competing interests.LG performed a comprehensive review of the primary literature and drafted the manuscript. SP, PJR and BG reviewed and identified additional primary literature, directed the organization of the manuscript and edited the manuscript. All authors read and approved the final version of the manuscript.


Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil.19) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay.The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-1+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses.The prevalence of activated CD419 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.The results presented here shows that PvMSP-1 Plasmodium species responsible for natural infection of human, Plasmodium vivax has the widest geographical distribution, being the second leading cause of malaria [P. vivax malaria cases have been reported worldwide [P. vivax accounts for around 85% of clinical cases [Malaria remains a serious public health problem causing high levels of morbidity and mortality in malaria-endemic regions. There were an estimated 219 million cases of malaria and 660,000 deaths in 2010 [al cases .P. vivax merozoite surface protein-1 (PvMSP-1) is a promising candidate.Since an effective malaria vaccine has long been envisaged as a potential tool for malaria control, two important points for its development are the identification of antigens that elicit the relevant immunological machinery and the correlation between the resulting immune system products and the clinical and/or parasitological protection induced. In this context, several antigens are being evaluated in clinical trials. To date, one candidate vaccine is currently being assessed in Phase 3 clinical trials and approximately 20 others in Phase 1 or Phase 2 trials [Plasmodium species, being synthesized in a precursor form during schizogony. Post-translational proteolytic processing of the MSP-1 precursor molecule generates different fragments . The 42 kDa fragment is processed to a 33 kDa and a 19 kDa fragments, leaving a membrane-anchored 19 kDa fragment (MSP-119) on the parasite surface after its internalization in the erythrocyte [MSP-1 is the most abundant and best-studied blood-stage antigen . MSP-1 ithrocyte -15.Saimiri monkeys [19 is a target for protective immunity against asexual blood stages of malaria parasites [19 antibodies and it is also dependent of CD4 T cells [The potential of PvMSP-1 as a vaccine candidate is based on previous studies that reported that it is highly immunogenic under natural conditions of exposure -24 and t monkeys . Severalarasites -28. This T cells ,29,30.19 in individuals naturally exposed to P. vivax or Plasmodium falciparum infections in a malaria-endemic area in the north-eastern Amazon region of Brazil.Given the cumulative data supporting the potential of PvMSP-1 as a malaria vaccine, and the substantial data generated through studies in human indicating that both humoral and cellular immune responses are needed to protect against malaria, the present study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1The study was carried out in Paragominas , Pará State, in the Brazilian Amazon. The samples were collected in 2004. The individuals were studied by means of a questionnaire, whereby all relevant information, including personal and epidemiological data, were collected.Vacutainer® EDTA tubes  from 35 individuals with uncomplicated malaria at the Hospital Municipal de Paragominas (HMP). Blood samples from 17 individuals living in Paragominas but with no history of current or previous malaria episodes were included in the study as non-infected control individuals. Blood collection was performed at the day of diagnosis and patients were treated, according to the Brazilian Ministry of Health standards for malaria therapy, immediately after blood sample collection.Written informed consent was obtained from all volunteer donors and 10 ml of venous blood samples were drawn in Thin and thick blood smears were examined for identification of malaria parasite and determination of parasitaemia by two expert malaria microscopists from HMP and from the Laboratory of Malaria Research  which is a reference centre in malaria diagnosis for the Brazilian Ministry of Health. Blood smears from all subjects were stained with Giemsa and examined under 1,000-fold magnification. Parasitaemia was determined by counting parasites in reference to 200 white blood cells in thick blood films, and the number of the blood parasites per millilitre was calculated.Nonendemic control blood samples from five individuals of the laboratory staff  who had neither history of malaria nor contact with a malaria transmission area, were included in the study as 'Rio controls’. The study was reviewed and approved by the Fundação Oswaldo Cruz and Instituto Evandro Chagas Ethical Committees.g and, after removal of plasma, the corresponding volume of RPMI-1640  medium containing 15 mM glutamin (Sigma), 10 mM Hepes (Sigma), 200 U/ml penicillin (Gibco), 200 μg/ml streptomycin (Gibco), 3 mg/ml gentamicyn (Sigma) and 2 g/L sodium bicarbonate (Sigma) was added. Subsequently, PBMC were isolated by density gradient centrifugation (Fycoll-Hypaque) and were washed twice in serum-free RPMI 1640 medium (Sigma). The cells were cryopreserved according to the method described by Ichino and Ishikawa [g.The blood samples were centrifuged for 10 min, 400 × Ishikawa . Brieflyex-vivo) by cytometry flow using 7-aminoactinomycin D (7-AAD). This rapid and sensitive method allows the discrimination of live cells from apoptotic or necrotic cells [The number of viable cells was verified immediately after thawing of PBMC  was cultivated in vitro according to the method described by Trager and Jensen [P. falciparum crude extract was obtained from infected erythrocytes with parasitaemia above 6%. Parasitized erythrocytes with a predominance of schizonts were washed three times with PBS. The lysis of infected erythrocytes was done by addition of 0.1% saponin and gentle shaking for 15 min. The lysates were ultrasonicated in the presence of 1 mM phenylmethylsulfonyl fluoride and centrifuged 7,000 × g for 15 min at 4°C in order to eliminate the cellular debris.The PSS1 d Jensen . The P. 6 cells/ml. The samples were then incubated for 10 min at 37°C. Five millilitres of RPMI-1640 at 4°C were added and the samples were incubated for 5 min in ice-bath. After incubation, the cells were washed three times with RPMI-1640 and then ressuspended with 1 ml RPMI-1640 (Sigma) medium containing 15 mM glutamin (Sigma), 10 mM Hepes (Sigma), 200 U/ml penicillin (Gibco), 200 μg/ml streptomycin (Gibco), 3 mg/ml gentamicin (Sigma), 3,7 g/l sodium bicarbonate (Isofar), 100 mM piruvate (Sigma),14 mM β-mercaptoetanol (Sigma) and 10% of inactivated foetal calf serum (FCS) (complete medium). The PBMC were distributed 2.5 × 105 cells/well in triplicate in 96-well flat-bottom microtiter plates  in final volume of 200 μl of complete medium alone or in the presence of 5 μg/ml phytohaemagglutinin (PHA), 10 μg/ml P. falciparum crude extract or PvMSP-119 (kindly provided by Dr. Mauricio Rodrigues and Dra. Irene Soares) and, then, incubated at 37°C in 5% CO2 for 48 or 96 h. After culture, the cellular proliferation was analysed using a FACSCalibur flow cytometer.Cellular proliferation was analysed by using the vital staining carboxyfluorescein diacetate succinimidyl ester . The PBMC were ressuspended in 1 ml of PBS plus 0,01% bovine serum albumin  at 37°C. Two microlitres of a stock solution of CFSE (5 μM) were added in 1 ml of cellular suspension containing up to 1x105 cells in 50 μl PBS containing 2% foetal calf serum and 0.1% sodium azide (azide PBS), followed by incubation for 20–30 min at 4°C. After two washes with PBS, the cells were resuspended in 200 μl of azide PBS. The cells were then fixed with a 2% paraformaldehyde solution and maintained in the dark until the analysis in a FACSCalibur flow cytometer. At least 10,000 events were analysed.The determination of cellular activation was done by using monoclonal antibodies against CD25, CD4, CD8 and CD21. The protocol consisted of the addition of 5-10 μl of the optimal antibody dilution to 5 × 10Maxisorp) were coated overnight at room temperature with 50μl of 4μg/ml PvMSP-119 protein in 0,05 M carbonate-bicarbonate buffer, pH 9.6. Plates were washed three times with PBS containing 0,05% Tween 20 (PBS/T20). Uncoated sites were blocked for 2 h at room temperature with 200μl of PBS/T20 containing 5% powdered-milk. After incubation, 50μl of the plasma sample diluted 1:50 in powdered-milk-containing PBS/T20 were added and the plates were incubated for 2 h at room temperature.Microtiter 96-well plates (Nunc Sigma) peroxidase conjugated, in optimal antibody dilution in PBS, was added. Plates were incubated for 1 h at 37°C. After washing the plates with PBS/T20, 50 μL of a solution containing 1 mg/ml of OPD (Sigma) and 15 μl of 30% H2O2 in citrate-phosphate buffer pH 5.0 were added. After incubation for 10–15 min at room temperature in the dark, the reaction was stopped with 50 μl/well of H2SO4. The absorbance was read at 405 nm in a spectrophotometer .Plates were washed thrice with PBS/T20 and 50μl/well of mouse anti-human IgE, IgM, IgG or IgG subclass  plus two standard deviations from controls that never visited malaria endemic areas. To standardize the OD data obtained in different experiments, OD index was calculated for each immunoglobulin determination as the ratio of the observed OD to the cut-off values. A sample with an OD index > 1.0 was considered positive.The cytokine levels in plasma and supernatant cultures samples were measured by ELISA using reagents from BD Biosciences Pharmingen, USA. Briefly, 100 μl of the capture monoclonal anti-human TNF, IFN-γ or IL-10 antibodies in optimal dilutions were used to coat 96-well plates for 14 h at 4°C. After washing and blocking, 100μl of plasma samples diluted 1:2 or supernatant of the cultures were added to duplicated wells and incubated for 24 h at 4°C. After the samples were washed, biotinylated anti-human cytokine were added and the plates were incubated for 1 h at room temperature. The presence of bound antibodies was detected using streptavidin-peroxidase  for 30 min at room temperature, followed by the addition of 2,2′-azinobis (3-ethylbenzthiazolinesulfonic acid)  and 30% hydrogen peroxide  as the substrate. The reaction was stopped with 20% sodium dodecyl sulphate , and the absorbance was read at 405 nm in a spectrophotometer . A standard curve was constructed for each cytokine by using different dilutions of human recombinant cytokines.p values less than 0.05 were considered significant.For unpaired analyses, the nonparametric Mann–Whitney test was used to determine the significance of differences in plasmatic concentrations of cytokines between patients with acute malaria infection and control individuals and also among patient antibody responses. The Spearman rank correlation coefficient test was used to evaluate the correlation of epidemiological and immunological data. Student’s t-test was used to analyse differences in mean values, and chi-square analysis was applied to compare the prevalence of positive responses. Spearman rank coefficient test was also used to analyse the variable correlations. P. falciparum (n = 12) or P. vivax (n = 23) infection and from individuals (n = 17) living also in downtown Paragominas but with no history of current or previous malaria episodes (control group).Blood collection was performed at the day of diagnosis, before malaria treatment. Patients sought health care at Brazilian health services 6.8 ± 6.9 days after onset of symptoms. Throughout the study period, a total of 35 samples were collected from uncomplicated malaria cases  with P. falciparum and 75 to 5,000  parasites/μl for P. vivax.The malaria group consisted of 6 female and 29 men with age ranging from 12 to 67 years old (31 ± 14 years old). Twenty six percent denied prior malaria infection and 64% reported 4.7 ± 6.0 previous malaria episodes during life. The individuals from the malaria group claimed living in malaria endemic area for 19 ± 13 years. All of them presented, at time of blood sampling, symptoms and positive thick blood smears, with a parasitaemia ranging from 50 to 3,000  parasites/μl for Individuals from the control group claimed living in malaria endemic area for 18 ± 13 years and corresponded to 10 women and seven men with average age of 35 ± 7 years. All individuals were negative for malaria parasites as assessed by thick blood films.P. falciparum and P. vivax malaria patients.The viability of mononuclear cells after thawing was analysed and the percentages observed in malaria patients (78.1 ± 12.5%) were similar to those found in healthy individual (78.7 ± 19.6%). Likewise, no difference was observed when comparing the viability of PBMC from ex-vivo PBMC revealed that, as expected, CD4+ T cells were found more frequently than CD8+ and CD20+ cells, in both malaria and control individuals , using an anti-CD25 monoclonal antibody.ex-vivo analysis, PBMC from malaria individuals presented higher levels of cellular activation (27.2 ± 14.2%) than control individuals (18.2 ± 11.9%). No difference in cellular activation was observed when comparing P. falciparum (24.7 ± 16.8) and P. vivax (31.4 ± 18.3) infected individuals  than CD8+ T cells (3.2 ± 3.3%). The same result was observed when comparing P. vivax  and P. falciparum  infected individuals. However, no difference was observed in the levels of cellular activation of CD4+ or CD8+ between P. falciparum and P. vivax infected individuals  or PSS1 crude antigen , higher levels of cellular activation were observed in malaria group than in controls individuals. The same result was observed when comparing P. vivax  and P. falciparum  infected individuals. However, no difference was observed in the levels of cellular activation between P. falciparum and P. vivax infected individuals   Figure .p = 0.0186; r = - 0.3958) in malaria individuals.In malaria patients, no correlation was observed between levels of plasmatic TNF or IFN and age, the number of previous malaria infection, time of residence in malaria endemic area, parasitaemia or the time elapsed between first symptoms and malaria diagnosis. However, a negative correlation was detected between plasmatic levels of IL-10 and the number of previous malaria infection  or without stimulus . No statistical difference was observed between cytokines levels from supernatant cultures of P. falciparum- or P. vivax-infected patients. In malaria patients, a positive correlation was observed between the levels of TNF obtained from supernatants of cultures stimulated with PvMSP-119 and the time of living in an endemic area .In the 96 h supernatant culture as represented in Figure 19  or with PSS1  and the time of residence in endemic area. Positive correlation was also observed between the number of previous malaria infection and the levels of IFN-γ when PBMC were stimulated with PvMSP-119 .Regarding to IL-10, positive correlation was observed between the levels of this cytokine obtained from supernatants of PBMC stimulated with PvMSP1-19 was noticed. The frequencies of malaria patients presenting antibodies against PvMSP-119 were 63% (22/35), 80% (28/35) and 51% (18/35) for IgG, IgM and IgE class, respectively . Besides the IgM frequency, these patients presented also higher levels of anti PvMSP-119 IgM antibodies (p = 0.004) . The frequency and the levels of IgG, IgM and IgE antibodies against PvMSP-119 were higher in patients infected with P. vivax than in patients infected with P. falciparum  of malaria patients presenting antibodies to IgG, IgM, IgE type and IgG isotypes against PvMSP-1) Figure . No corr Figures .19 or PSS1 crude antigen. The prevalences of IgG1, IgG2, IgG3 and IgG4 subclasses in IgG-positive malaria patients were, respectively, 64% (14/22), 86% (19/22), 14% (3/22) e 91% (20/22). PvMSP-119-induced antibodies were predominantly of non-cytophilic subclasses . The levels of anti-PvMSP-119 IgG4 antibodies were higher than the levels of IgG1, IgG2 and IgG3 antibodies  ; anti-PvMSP-119 IgM antibodies and IFN-γ levels in P. falciparum-malaria patients  and; anti-PvMSP-119 IgE antibodies and TNF in both P. vivax- and P. falciparum-malaria patients .Positive correlations were observed between anti-PvMSP-119 was evaluated in individuals naturally exposed to P. vivax and P. falciparum infections in a malaria-endemic area in the north-western Amazon region of Brazil.In the present work, the profile of acquired cellular and antibody immune responses against PvMSP-1It is well known that suboptimal cryopreservation could result in a significant decrease of cell number and viability, causing alterations on the cellular phenotype and on the immune response to specific antigens. To avoid troublesome events, a protocol to successfully recovery PBMC after freezing and thawing was previously standardized . Thus, iex-vivo PBMC revealed that CD4+ T cells were found more frequently than CD8+ and CD20+ cells, in both malaria and control individuals. In malaria individuals, a higher prevalence of activated CD4+ than CD8+ T cells was observed, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. It can be speculated that CD4+ T cells were the most stimulated cells due the nature of antigenic stimulus because CD4+ T cells are activated by the parasite in blood stage, while CD8+ T cells are most often activated during the hepatic stage of parasite life cycle [The phenotypic analysis of fe cycle .19 of P. falciparum and Plasmodium chabaudi chabaudi have already been demonstrated [19 after 96 h culture. This low proliferative response may occur because the majority of activated cells undergo activated-induced cell death, an active cell suicide mechanism of widespread biological importance that constitutes the physiological response of normal cells to activation and is believed to control the number of antigen-stimulated cells during the immune response [Low proliferative responses in the presence of MSP-1nstrated ,38. The response .in vitro studies have shown that IL-10 suppresses the expression of malaria parasite-induced production of TNF by PBMC [Anti-inflammatory cytokines are involved in a feedback mechanism to regulate the expression of pro-inflammatory cytokines, and prevent the pathological effects that may result from their continuous secretion. Previous  by PBMC . In factParasite clearance seems to be related to IL-10 and IFN-γ levels. Here, an intense secretion of IFN-γ and IL-10 as detected by dosage of plasmatic levels, were shown in malaria patients. However no correlation was found between parasitaemia and IFN-γ or IL-10 levels. The high levels of IL-10 and IFN-γ are in agreement with previous clinical reports that IL-10 is up-regulated in concern with IFN-γ ,42, suggP. vivax and P. falciparum infected individuals. However, besides plasmodial species, other factors, like the number of previous malaria infections, may influence the levels of this and other cytokines. In fact, a negative correlation between number of previous malaria infections and IL-10 levels was found. In addition, previous studies have shown that an adaptive type 1 regulatory CD4+ cells have been identified as the main source of IL-10 in experimental murine infection with Plasmodium yoelii[, while in P. chabaudi infections in mice, an activated effector TH1 cells were major IL-10 producers [IL-10 levels were similar in um yoelii, while iroducers . Underst19-stimulated PBMCs from malaria patients showed higher TNF response than those recorded in both PSS1-stimulated and non-stimulated PBMCs. The production of TNF in recall to PvMSP-119 may suggest a protective role of PvMSP-119 immune response because TNF has been related to parasite clearance.High production of TNF in malaria individuals is related to the development of severe malaria. In the present work, lower than expected plasmatic TNF levels  were detected in malaria patients. Interestingly, PvMSP-119, independently of the reported number of previous malaria attacks or the time of residence in a malaria-endemic area, indicating that this protein is immunogenic in natural conditions of exposure and seems to be independent of the time of exposure. The high immunogenicity of PvMSP-119 may be reflex of its limited polymorphism, because independent groups demonstrated that PvMSP-119 is conserved [19 is the single fragment that remains on the parasite surface during red cell invasion through glycosylphosphatidylinositol (GPI) anchor [A great majority of studied individuals presented antibodies against PvMSP-1onserved ,46,47. A) anchor  and stud) anchor .19 antibodies were not detected in the sera of five individuals from malaria group . The absence of anti-PvMSP-119 antibodies could be related to immunosuppression observed in humans in malaria infection [19-specific B cells were present, although circulating antibodies titers were undetectable. Alternatively, the lack of PvMSP-119 antibody response may be due the genetic restriction of immune response that has been described to several plasmodial recombinant proteins and synthetic peptides [19 was found [Anti-PvMSP-1nfection ,50, howepeptides ,51-55. Has found .Plasmodium antigens in the same population as well as the same Plasmodium antigen in different populations can induce different antibody profiles, it was also evaluated the frequency and levels of IgG, IgM and IgE class and IgG subclass against PvMSP-119. IgM antibodies were the most prevalent and the one with the highest levels. PvMSP-119-induced IgG antibodies were predominantly of non-cytophilic subclasses. Different data have been reported for individuals living in other Brazilian-endemic areas with different levels of exposure where the PvMSP-119-induced antibodies were predominantly of IgG1 subclass [P. vivax[1 to the Th2 pattern of immune response modulating the IgG subclass expression. Independently of its origin, the differences between antibody profile reported in the present study and the aforementioned underline the importance of conducting immunoepidemiological studies in different malaria endemic areas where transmission intensities and human genetic background are different.Considering that different subclass ,23. Diffsubclass . In thissubclass  as well [P. vivax. In addi[P. vivax. One oth19 antibodies and number of previous malaria episodes was observed. This finding may reflect that people living for a longer period of time in the region may have acquired some degree of clinical immunity after experiencing a number of infections, therefore, reporting less episodes of clinical malaria in the more recent years. Interestingly, only the levels of IgE antibodies were inversely correlated with the number of previous malaria episodes. Thus, it can be supposed that higher levels of IgE antibodies may have an important role against clinical malaria. Similar results were already reported that shown that high levels of malaria specific IgE were associated with reduced risk for subsequent clinical malaria episodes [Associations between antibody responses with time of residence and/or number of previous episodes have been commonly reported to several malaria antigens ,58,59. Hepisodes .19 were higher in individuals infected with P. vivax than P. falciparum or who had reported the last malaria episode due P. vivax. Similar results were reported elsewhere where sera from subjects who had had P. vivax in their last malaria clinical episode presented higher levels of antibodies when compared with those whose last malaria episode was due to P. falciparum[In this study the frequency and the levels of IgG, IgM and IgE antibodies against PvMSP-1alciparum.P. falciparum studied in this work reported previous infection with P. vivax. This may explain the recognition of PvMSP119 antigen by antibodies from individuals infected with P. falciparum. However, the similarity between PfMSP1 and PvMSP1 antigens may also explain, at least in part, the recognition of PvMSP1 by patients infected with P. falciparum, although a recent study has shown that sera from immunized mice with PfMSP119 or PvMSP119 failed to cross-react with heterologous antigen [The individuals infected with  antigen .19 was able to induce a high cellular activation leading to production of TNF, emphasizing the high immunogenicity of PvMSP-119 in naturally exposed individuals and therefore its potential as a malaria vaccine candidate.In conclusion, the results presented here shows that PvMSP-1The authors declare that they have no competing interests.19; JMS enabled and facilitated the performance of experiments in Paragominas; CTDR reviewed the manuscript and MFFC conceived the study, participated in its design and coordination and reviewed the manuscript. All authors have read and approved the final manuscript.EKPR designed the study, carried out the experiments, performed the statistical analysis and drafted the manuscript; PRRT, VEV and LRPR participated in its design, carried out the experiments and reviewed the manuscript; MMR and ISS produced and provided the PvMSP-1


Plasmodium vivax congenital malaria case in Guatemala and the first case in Latin America with genotypical, histological and clinical characterization. The findings show that maternal P. vivax infection still occurs in areas that are in the pathway towards malaria elimination, and can be associated with detrimental health effects for the neonate. It also highlights the need in very low transmission areas of not only maintaining, but increasing awareness of the problem and developing surveillance strategies, based on population risk, to detect the infection especially in this vulnerable group of the population.This is a report of the first  Congenital malaria (CM) has been an often neglected consequence of malaria in pregnancy. It occurs when malaria parasites cross the placenta either during pregnancy or at delivery -3. TherePlasmodium falciparum and from sub-Saharan Africa [Congenital malaria has been predominantly reported for n Africa -7. The pn Africa ,6,8. Then Africa -12. Littn Africa ,14. MorePlasmodium vivax congenital mono-infection on the neonate’s health have not yet been reported. This is the first P. vivax CM case from Guatemala and the first case in Latin American in which a comprehensive clinical, histological and genotypical investigation has been carried out.In Latin America, reports on CM have been scarce ,15,16; aP. falciparum has been reported in the FBC area . Malaria transmission is sustained in the population by the temporal succession of the main vectors, Anopheles albimanus, Anopheles vestitipennis and Anopheles darlingi[On September 2010, a 21-days-old female baby with a two-day history of fever was admitted to Fray Bartolomé de las Casas (FBC) District Hospital. FBC is a rural low-transmission malaria endemic area  in Northern Guatemala. FBC was a high-risk area where malaria transmission has been greatly reduced over the last years due to a scale-up of control interventions. Long-lasting insecticide-treated nets (LLITNs) and the improvement of the case management system implemented from 2006, resulted in a dramatic reduction in malaria parasite incidence from 91/1,000 population in 2006 to 1.9/1,000 population in 2010. Since 2009, no case of  darlingi.st day of admission, 24,000 platelets/μL on the 2nd day of admission), and leucopaenia (white blood cells 3.5x103/μL) with relative lymphocytosis (66.8%). Yet, the newborn did not show any signs or symptoms of haemodynamic failure nor evidence of bleeding throughout admission or after discharge. Biochemical and microbiological examinations were not available in the hospital. Based on initial suspicion of neonatal sepsis, the baby was started on intravenous (IV) ampicillin along with IV fluids and antipyretics. The thick blood smear examination showed P. vivax infection , and then she was started on anti-malarial treatment with oral chloroquine (CQ) . The neonate was treated with CQ only, following national guidelines in Guatemala that do not recommend the administration of primaquine to infants less than six months. Clinical progress was adequate and the baby was discharged after treatment completion. National guidelines also recommend therapy with ¼ tablet of CQ 150 mg base every 21 days until the patient is six months of age, so the infant kept on with CQ prophylaxis until that age after treatment completion. Follow-up during household visits two and eight months after hospital discharge was also favourable.On admission physical examination of the baby revealed paleness, hepatosplenomegaly (1 cm), dusky skin appearance and desquamation, and a body temperature of 36.7°C which increased to 38.9°C in the next 24 hours. Neurological, respiratory and cardiac examinations were unremarkable. Main laboratory findings were anaemia (Hb 7.2 g/dL), severe thrombocytopaenia  visit, at 16 weeks of gestational age, in a multi-centre descriptive study –PregVax study- aimed to determine the burden and impact of This is a retrospective congenital malaria case report that mainly builds on the hospital clinical data available during its management. Molecular analyses, which were available due to the mother’s participation in the main study and carried out after the detection of CM infection, allowed the confirmation of the malaria case.Plasmodium infection was not detected in the mother, either actively or passively, during pregnancy, neither by microscopy nor by polymerase chain reaction (PCR) at any of the ANC at 16, 21 and 26 weeks of gestational age. Delivery was at term (40 weeks), and the birth weight was 2,839 g. At delivery, the mother reported fever within the last 24 hours. Physical examination showed an axillary temperature of 38°C, paleness, and laboratory tests showed thrombocytopaenia  and haemoglobin of 12.4 g/dL. Plasmodium vivax asexual parasites  were detected in peripheral blood . Cord blood parasitaemia determination is not routinely done at this health facility. In this case, cord blood parasitaemia determination was done later after birth at the study referral laboratory as part of the study objectives.Immediately after birth, physical examination of the newborn revealed no clinical abnormalities. No Plasmodium vivax infection was detected by conventional PCR for specific P. vivax merozoite surface protein 1 gene (Pvmsp1) in all samples except in the newborn’s blood at the time of birth . Species-specific primers and probes were used as previously described by Veron et al. [DNA extraction (Purelink TM Genomic DNA Kit – Invitrogen) was done on one whole blood-spot on filter paper from each of the following compartments: maternal peripheral blood at delivery, placental and cord blood, and neonate’s blood at the time of birth and during hospital admission. n et al. , selecteP. vivax microsatellites (MS) were amplified from P. vivax positive samples as described in Karunaweera et al. [2, 200 μM of each deoxynucleotide triphosphate, 5 picomoles of each primer and 2.5U of FastStar Taq polymerase (Roche). To determine the amplicon length variation, PCR products were analyzed by CEQ 8000 Genetic Analysis System (Beckman Coulter) using CEQ 8000 software for fragment analysis. Identical allelic profiles were obtained at the seven MS loci tested , confirming that the same genotype was present in all compartments.Seven a et al. . BrieflyP. vivax congenital malaria in Guatemala and the first one from Latin America with clinical, histological and genotypical characterization. Co-infection with P. falciparum was excluded by molecular diagnosis, and the same parasite genotype in maternal blood at delivery, in placental and cord blood and in peripheral blood of the 21-days-old neonate was detected by microsatellite genotyping.This is the first reported case of P. vivax, but only three were reported as CM cases [Plasmodium species was excluded. Most of the information on CM comes from high malaria transmission areas and indicates that CM is rarely symptomatic [Several reports have been published from Latin America on malaria infection during the neonatal period ,22,23 moCM cases ,15,16. Iptomatic .P. vivax infection, however, the possibility of a new infection, although very unlikely, it cannot be totally ruled out.In the case outlined here, the onset of symptoms occurred 21 days after birth. The possibility that the neonate’s infection was acquired in the short period after birth from an infected mosquito bite is very low given the low malaria transmission in the FBC area. The microsatellite genotyping showing identical allelic profiles at all MS loci tested does reinforce the vertical transmission of the P. vivax infection and/or by the absence of a placental binding phenotype of P. vivax parasites [The physiopathology of CM is not well known. It has been hypothesized that, although the placenta functions as an effective barrier, maternal micro-transfusions into the foetal circulation, and direct penetration of the parasite through the placental villi could lead to vertical transmission of malaria . In thisarasites .In a malaria pre-elimination context, as is the case of the FBC area, the level of acquired anti-malarial immunity in the population may decreased and waned dramatically leading to increased susceptibility and risk of more severe forms of malaria when infection occurs. This may be particularly frequent and serious in vulnerable groups of the population, such as pregnant women and neonates. In the current case, the baby presented with severe thrombocytopaenia and anaemia, both being potential life-threatening haematological complications. This report highlights the need of establishing epidemiological and clinical surveillance strategies as malaria control programmes are reducing transmission to near-elimination levels, to effectively and promptly detect infections ,27. The P. vivax malaria occurs in very low malaria endemic settings, and it may be detrimental for the infant’s health. These findings emphasize the relevance in very low transmission areas of not only maintaining, but even increasing clinical and epidemiological awareness of this preventable and treatable disease in pregnancy and in the neonate. They also highlight the need for establishing active surveillance strategies in the areas that are in the pathway towards malaria elimination.Mother-to-child transmission of The case study protocol was approved by the Universidad del Valle de Guatemala Ethics Review Committee. Written informed consent was obtained from the mother for her participation and that of her baby for the publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.All authors have made substantial contributions to the investigations presented in this manuscript. MEC supervised all data collection and participated in drafting the manuscript, AB drafted the manuscript, and NP conceived and designed the report. MM and CS carried out the molecular and genotyping studies. AB, NP, AM, MD and CM revised the manuscript. All authors have read and approved the final manuscript.


The objective of this study was to evaluate the prevalence of self-diagnosis of malaria and other febrile illnesses in Bo, Sierra Leone.All households in two neighboring sections of Bo were invited to participate in a cross-sectional survey.A total of 882 households (an 85% participation rate) that were home to 5410 individuals participated in the study. Of the 910 individuals reported to have had what the household considered to be malaria in the past month, only 41% were diagnosed by a healthcare professional or a laboratory test. Of the 1402 individuals reported to have had any type of febrile illness within the past six months, only 34% had sought a clinical or laboratory diagnosis. Self-diagnosis of influenza, yellow fever, typhoid, and pneumonia was also common.Self-diagnosis and presumptive treatment with antimalarial drugs and other antibiotic medications that are readily available without a prescription may compromise health outcomes for febrile adults and children. The overuse or inappropriate use of antimalarial medications and antibiotics is a growing concern in many parts of the world, including Sierra Leone, a post-conflict country in West Africa. In Sierra Leone, these drugs are dispensed by government-run clinics and hospitals, private healthcare facilities, licensed pharmacies, and through the informal sector . BecauseEasy access to antimalarial drugs and other antibiotic agents may compromise patient health outcomes. If the medications purchased for an infection are inappropriate — such as antibiotics taken for a viral infection or chloroquine taken for a chloroquine-resistant case of malaria — or if medications are not taken in the appropriate dosage and for an appropriate length of time, self-diagnosis and treatment may contribute to prolonged illness, more severe morbidity, and an increased risk of mortality. A study in Bo district in 2008 found that only 48.3% of malaria-positive patients at a hospital that offered free care completed the full course of prescribed antimalarial treatment .Shigella dysenteriae in 1999-2000 [Staphylococcus aureus among children during a 2008-2009 study in Freetown [Furthermore, misuse of antimicrobial agents can contribute to the emergence of drug resistance, which is a growing concern in Sierra Leone. A 2002-2003 study found treatment failure among more than half of pediatric malaria patients treated with chloroquine, and treatment failure was also frequently observed for sulphadoxine-pyrimethamine (SP) [999-2000  and drugFreetown . Sierra Freetown .Over-diagnosis of malaria, in particular, may be common. Even clinicians can find it difficult to diagnose malaria accurately solely based on symptoms. A study in the Bo district in 2005 found that 82% of children suspected by clinical officers to have malaria based on physical symptoms such as fever, splenomegaly, and vomiting tested positive for parasitemia by a Paracheck-pf® rapid diagnostic test (RDT) and 18% did not . BetweenSampling Strategy. All households within the Kulanda Town and Njai Town sections (neighborhoods) of the city of Bo were eligible for participation. The research laboratory has previously mapped all of the buildings within these sections and conducted a household census, in 2010, to identify which structures were residential ones [Data Collection. Each interview began with questions about the household's environmental characteristics, such as building materials and access to utilities, and about household demographics, such as the age and sex of each current resident. Students at boarding school, adults working in another town and not sleeping at the residence in Bo, and others who spent at least 6 months of the past year living elsewhere were not considered to be current household members.Then a series of questions were asked about febrile illnesses experienced by household members. These questions were developed in consultation with residents of the Kulanda Town / Njai Town sections to ensure clarity, and asked about all household members of all ages. First, the household representative was asked whether anyone currently living in the household had been ill with what the household considered to be malaria in the past one month. If malaria was reported to have occurred, follow-up questions asked where those with malaria were diagnosed  and whether they were tested for malaria by a laboratory. A second set of questions asked whether anyone currently living in the household had a febrile illness earlier in 2012 . Follow-up questions asked about the frequency and duration of febrile illnesses; symptoms associated with these fevers ; and whether the febrile person was examined by a clinician and/or had a laboratory test to determine the cause of the fever. A final set of yes/no questions asked whether the household considered anyone currently living in the household to have ever had any of more than a dozen listed communicable and non-communicable conditions, and whether those diagnoses were made by a doctor or were self-diagnosed.Data Management and Analysis. Responses were entered by the interviewers directly into a Filemaker Pro 12 relational database on a password-protected tablet computer. Households were identified by a number linked to a map stored at the research laboratory; these codes were random and not related to the geographic coordinates of the map. Data were analyzed using the statistical software program SPSS (version 20). Proportions, means, and standard deviations were used to describe the variables. Chi-squared tests were used to compare rates in independent populations, such as different age groups.Ethical Considerations. Adults ages 18 and older were interviewed after providing informed consent, which was documented with a signature or a thumbprint. The consent form and study materials were available in both English and Krio, the local language in Sierra Leone. No compensation or other incentive was offered. To protect the confidentiality of information shared with the research team, no names or addresses were entered into the database. The data entered into each tablet computer were deleted daily after the data files on the tablets were downloaded to a password-protected desktop computer in a locked and guarded research facility. The research protocol was approved by the Sierra Leone Ethics and Scientific Review Committee and by Njala University (Sierra Leone), the Liverpool School of Tropical Medicine (UK), the U.S. Naval Research Laboratory (USA), and George Mason University (USA).The 882 participating households contained 5410 individuals, with a mean household size of 6.1 persons. The households reported a somewhat diverse set of socio-environmental characteristics. While 1186 (66.7%) of the 1778 beds reported to be located in the participating homes were said to have bednets, 328 (37.2%) of households reported having no bednets. In total, 877 (99.4%) of households reported seeing a rat in the house in the past month, 860 (97.5%) reported seeing cockroaches in the house in the past month, 677 (76.8%) had a tile or concrete rather than dirt floor, 546 (61.9%) had a trash bin in the home (of which 153 covered the bin), 353 (40.0%) had electricity, and 155 (17.6%) had a drinking water source within 50 meters of the home.In total, 675 (76.5%) of the 882 households reported at least one case of malaria (as defined by the household) in the month prior to the survey, with a total of 910 (16.8%) of the 5410 individuals reported to have had malaria during that time period. However, 540 (59.3%) of these 910 individuals were presumptively diagnosed by the ill person or a household member, and only 370 (40.7%) were diagnosed following laboratory testing.A total of 1402 (25.9%) of the individuals within participating households were reported to have had any type of febrile illness  within the past six months. The rate of fever reported differed by age group and by sex, with young children (those 0 to 4 years old) having the highest rate (p<0.001) and females reporting more fevers than males (p<0.001) . Only 33Self-diagnosis of several other conditions was common . All of We found that the majority of febrile illnesses in Bo, Southern Province, Sierra Leone, are self-diagnosed without clinical examination or laboratory testing, including the more than half of suspected malaria cases that are treated presumptively without any clinical diagnostics. The fact that households with greater numbers of individuals were more likely to report that at least one household member had been tested for a febrile illness supports the validity of reports about diagnosis and testing rates, since a greater number of persons in the household increases the probability of a severe illness occurring for at least one resident. The validity of the survey instrument is also supported by the low numbers of households reporting uncommon diseases such as bacterial meningitis unless these conditions were diagnosed by a doctor.These results are similar to those from studies in other parts of West Africa which have found that more than half of adults self-diagnose fevers and self-medicate for what they consider to be malaria , 13. Of This high rate of self-treatment is concerning, since it is likely that a significant proportion of these presumptive cases are treated inappropriately (as per the introduction to this paper). Some people who would benefit from antibiotic and supportive therapy may not be receiving adequate care, and many people who purchase medication for their self-diagnosed malaria or other conditions may be taking drugs that are ineffective for their condition. Febrile individuals who self-treat may experience disease complications and increased treatment costs resulting from delayed access to appropriate medications and other therapy . Taking When tests are available at a reasonable cost, testing before treating may significantly reduce the inappropriate use of antimicrobial medications. For example, a cohort study of children ages 1 to 10 years in Uganda found that only 32% of fevers were caused by malaria, and the researchers concluded that a test-before-treating approach in that study population would reduce the use of antiparasitic drugs by two-thirds . The redTesting does not have to occur in a clinical setting. In rural Sierra Leone, free malaria testing by community health workers (CHWs) has increased access to diagnosis for thousands of households . Those wBesides the direct benefits to patients, expanded use of testing before treatment may slow the further development of drug resistance by reducing the proportion of the population accessing pharmaceutical agents without prescriptions tailored to their actual diagnoses. Patients who have seen a clinician and have a confirmed diagnosis may be more likely than others to complete a full course of an appropriate antibiotic or antimalarial, especially if their treating clinicians counsel them about the importance of compliance with prescribed treatments . Community-based behavior change communication processes may also help to promote healthy use of pharmaceuticals by households and communities, but when these public health structures are not in place the burden of health education typically falls on clinicians.Limitations. This study had several limitations that require a conservative interpretation of the findings. No laboratory tests were conducted to confirm the reported causative agents for participants’ febrile illnesses, so we do not know how often their self-diagnoses were accurate. Additionally, participants were not asked about their use of antibiotic and antiparasitic drugs, such as what medications they preferred to take when febrile or where they procured these medications. Because these questions were not part of the survey, we have limited information about whether the self-treatment used by febrile participants was appropriate for their illnesses.This study provides evidence that self-diagnosis and self-medication for malaria and other febrile illnesses is common in Bo, Sierra Leone. In order to better understand the implications of self-diagnosis and presumptive treatment on patient health outcomes, we recommend that further studies evaluate the types of infections common in this population to see how well clinical laboratory results match self-diagnoses. Future research should also examine the pharmaceutical access and use habits of local residents to see whether appropriate courses of medication are being taken by those with and without prescriptions from a clinician. Understanding the knowledge, attitudes and beliefs, and health practices and behaviors of residents regarding diagnosis and treatment of fevers may contribute to improved health services, policies, and practices.


Plasmodium vivax and multidrug-resistant Plasmodium falciparum malaria. Little has been reported in the peer-reviewed literature regarding the burden of severe malaria (SM) in Cambodia.Despite recent malaria containment and control efforts leading to reduced incidence, Cambodia remains endemic for both Medical records for all patients admitted to the Battambang Referral Hospital (BRH) with an admitting or discharge diagnosis of SM from 2006 to 2009 (suspected SM cases) were reviewed. Those meeting the case definition of SM according to retrospective chart review and investigator assessment of probable cases, based on published national guidelines available at the time, were analysed for trends in demographics, mortality and referral patterns.Of the 537 suspected SM cases at BRH during the study period, 393 (73%) met published WHO criteria for SM infection. Despite limited diagnostic and treatment facilities, overall mortality was 14%, with 7% mortality in children 14 and under, but 19% in adults (60% of cases). Cerebral malaria with coma was relatively rare (17%), but mortality was disproportionately high at 35%. Mean time to hospital presentation was five days (range one to 30 days) after onset of symptoms. While patients with delays in presentation had worse outcomes, there was no excess mortality based on treatment referral times, distance travelled or residence in artemisinin-resistance containment (ARC) Zone 1 compared to Zone 2.Despite limitations in diagnosis and treatment, and multiple confounding co-morbidities, mortality rates at BRH were similar to reports from other countries in the region. Interventions to improve access to early diagnosis and effective treatment, combined with modest improvements in intensive care, are likely to reduce mortality further. Patients referred from Zone 1 did not have excess mortality compared to Zone 2 ARC areas. A steep decrease in SM cases and deaths observed in the first half of 2009 has since continued, indicating some success from containment efforts despite the emergence of artemisinin resistance in this area. By definition, patients with severe malaria are in critical condition and require emergency therapy with parenteral drugs . UntreatThrough implementation of a successful national malaria control programme, officially reported malaria incidence in Cambodia decreased by more than 50% between 2001–2010, from 11.03 cases per 1,000 in 2000 to 4.07 cases per 1,000 in 2010. Malaria mortality steadily decreased over the last decade from 5.2 deaths (in 2001) to 0.98 deaths per 100,000 in 2010 [In 2007 an estimated 2.13 million Cambodians  living within 2 km of an endemic forested area were at risk of malaria, with 42,124 confirmed cases. There were 2,648 severe cases admitted to referral hospitals, of which 219 died. Data from the National Center for Parasitology, Entomology and Malaria Control, Cambodia (CNM) in 2007 indicated that Battambang Province had the second highest case-fatality rate (CFR) nationally (17.4%) – double the national CFR  were assessed. The operational utility of published guidelines for the diagnosis and management of severe malaria were also evaluated. The authors report here the first data on the burden of severe malaria at the government referral hospital in Battambang, Cambodia’s second largest city, and the first area in the world to report artemisinin-resistant malaria .The study was conducted at the BRH, located in Battambang City in Battambang Province, Western Cambodia Figure . BRH is Plasmodium falciparum and/or Plasmodium vivax) presenting with one or more criteria of severe disease [Medical records for all patients admitted to the BRH from January 2006 to June 2009 with an admission and/or discharge diagnosis of severe malaria (irrespective of confirmation by slide or rapid diagnostic test) were reviewed to analyse morbidity, mortality, demography and referral patterns. Since no separate log for severe malaria cases existed, all hospital records for the study period were searched in order to identify the severe malaria cases. Individual patient records were reviewed by a study physician, and data systematically extracted onto case report forms. Discharge diagnoses were reviewed independently for adherence to the Cambodian national diagnosis and treatment guidelines in use at the time. Severe malaria was defined as a complicated case of malaria infection  condition. The retrospective hospital record review was carried out in compliance with the Declaration of Helsinki 2000, following protocol approval by the Walter Reed Army Institute of Research IRB and the National Ethical Committee for Health Research (NECHR) in Cambodia. Personal identifying information was not collected on the case report forms.Data were translated where needed from French and/or Khmer into English, and transcribed from medical records onto paper case report forms, and analysed using SPSS 12.0.0 for Windows. Double data entry was performed using Microsoft Excel to minimize transcription errors. Descriptive statistical analyses were performed for demography, referral patterns, symptoms, signs, admitting laboratory parameters, complications with respect to survival outcomes, and case management data. Fisher’s exact test (two-sided) was used to compare proportions from non-parametric tests when the expected values were less than five. Mortality risk was expressed with odds ratios (OR) and 95% confidence intervals (95% CI). Multiple logistic regression analysis was used to calculate adjusted odds ratios (aOR) to evaluate associations of mortality with presenting signs and symptoms, and complications occurring during hospital admission. Variables with crude OR significant at the level of p <0.2 in univariate analyses were included in a multivariate analysis.Between January 2006 and June 2009, a total of 537 records from suspected severe malaria cases were reviewed from patients admitted or discharged from BRH with a diagnosis of severe malaria. After review by study physicians, 393 patients met published Cambodian severe malaria case definitions and were considered to be ‘probable severe malaria cases’ , 33 P. vivax (12.4%); and 15 documented mixed infections (5.7%) positive for both P. falciparum and P. vivax , with the majority dying (38 or 9.7%) in hospital. The majority were male (224 or 57.0%), aged between five and 30 (median age 18 years), and were school-aged children (107 or 27%) or farmers (101 or 26%). Most patients (92%) were self-referred, arrived by private vehicle, and resided in Battambang Province , as well as one spontaneous abortion and three stillbirths . The only pregnant P. vivax case survived with normal delivery at term during hospital admission.Out of 55 total deaths reported, there were 36 deaths among the 265 smear-positives , versus 19 deaths among the 128 smear-negatives . Lowest case fatalities were found in the five to 14 year age group (6.2%), while most fatalities were 15 to 30 years old (19%) or older than 50 (19%). The under-five mortality rate was 13%. Occupations with the highest CFR included labourers (23%), government workers (20%), farmers (19%), and housewives (29%). A higher death rate was found in referred patients (19.2%), in particular those referred from government health facilities, versus patients who came directly to the hospital (8%). There were two fatalities among the 13 pregnant severe malaria cases , headache (63%), dark urine (56%), chills (53%), and impaired consciousness (42%). There were 65 patients who presented in a coma (17%). The only presenting symptoms, adjusted for age group  that remained significantly associated with a fatal outcome after a multiple logistic regression analysis were coma , pallor , dyspnea  and dark urine  (Table Laboratory assessment at BRH was limited and included microscopy for parasitaemia 94%), white blood cell count (92%), haematocrit (63%) or haemoglobin (6%), platelets (48%) and plasma glucose (32%). Assessment of liver enzymes (3%) were done infrequently, and other commonly used tests including blood urea nitrogen, creatinine, bilirubin, albumin, plasma lactate and bicarbonate were unavailable. Higher parasitaemia based on the qualitative scoring system used was not associated with higher mortality. Although severe anaemia was common, with approximately 29% of patients having haematocrit values less than 15% at initial presentation, mean presenting haematocrit values between non-survivors and survivors were similar (25.2% versus 24.0%), and no patient had a presenting glucose level less than 40 mg/dL. Non-survivors had higher mean leukocyte counts than survivors . Of 191 patients tested, the majority had a platelet count greater than 150,000/μL, with the mean platelet count significantly lower in non-survivors   of suspected severe malaria cases received parenteral therapy on admission which included either an anti-malarial drug alone (238 or 61%), or a combination of anti-malarial and an antibiotic 107 or 27%)  after onset of symptoms. No clear relationship was observed between treatment delay and outcome. Overall CFR of patients originating from ARC Zone 1 districts was lower than those from Zone 2 districts (11% versus 15%) Table . For theP. falciparum, and an increasing predominance of P. vivax malaria. The study provides baseline information on the burden of severe malaria in a referral hospital near the epicentre of global anti-malarial drug resistance (WHO Zone 1) during the 2.5 years preceding the Artemisinin Resistance Containment Project and supports other preliminary evaluations reporting success in containment and reduction of mortality rates [P. falciparum subpopulations have recently been demonstrated in Cambodia [This is one of the few studies to describe the presentation and epidemiology of severe malaria in Western Cambodia, a low transmission setting with multidrug-resistant ty rates . These ity rates . Followity rates . Other lCambodia  and futufalciparum malaria infection as a risk factor for bacteraemia in African children [On review of the hospital records, it was often apparent that malaria was a co-existing infection of another serious condition, such as meningitis, sepsis and gastrointestinal haemorrhage which is not unexpected, given the recent evidence of children . BacteriDespite substantial limitations, diagnostic and treatment guidelines were reasonably well adhered to where possible at this facility, likely contributing to an overall CFR of 14.2% which was intermediate compared to other reports from the region. CFR in other retrospective studies in the region have varied from 1.8% in Thailand , 6.3% inAlthough life-threatening malaria complications can affect patients of all ages, disease presentation and mortality have been found to be closely related to transmission intensity and age of the patient . In a loIn addition to diagnostic and prognostic limitations, challenges in management included lack of mechanical ventilation units, renal haemodialysis, centrally administered pressors, exchange transfusion capabilities or real time haemodynamic and metabolic data to guide resuscitation. Limited peritoneal dialysis was available at times, but rarely employed. As a result, renal failure and pulmonary oedema, while rare at this facility (reported in only 7% and 2% patients),were associated with the highest CFR in this group.Recent descriptions of treatment-seeking behaviour, cultural practices and socio-economic barriers have revealed causes for delays in appropriate malaria treatment in Cambodia, representing an important potential area for intervention to reduce mortality . SeveralRoughly 90% of all patients discharged with a severe malaria diagnosis at BRH received intramuscular artemether on the day of admission, which at the time of publication remains the current national standard of care for severe malaria in Cambodia. A five to seven-day course is used, followed by treatment with mefloquine, but is sometimes unavailable, prompting patients to obtain drugs from private-sector pharmacies risking counterfeit or substandard treatment. Parenteral artesunate was not available in Cambodia during the study period. Parenteral artesunate currently remains the most effective treatment remaining for severe malaria , and wasP. vivax in tropical regions [P. vivax in Southeast Asia and the Pacific have been high in several reports, accounting for up to 25% of severe malaria cases. While some have shown reduced risk of severe disease attributable to P. vivax and/or mixed infection in low transmission settings [P. vivax is typically thought of as a relatively benign disease, 14.5% of patients meeting the case definition of severe malaria at BRH during the study period had slide proven P. vivax, and there were four documented deaths. There were no deaths reported among 17 patients with documented mixed infection. HIV-1 infection has been reported to be an important risk factor for severe malaria and death [There has been recent recognition of the substantial proportion of severe malaria cases attributable to  regions . Observesettings , others settings . Though nd death , but theThe study’s retrospective design placed limits on accurately interpreting the underlying disease burden. Accurately defining the terms ‘impaired consciousness’ and ‘prostration/coma’ retrospectively were challenging. WHO defines impaired consciousness as a ‘rousable mental condition’, and prostration as the inability to sit upright without support or drink. However, varying terms were used by clinicians at BRH, including descriptions such as “somnolence”, “obnubilation”, “agitation”, “delirium”, “irritation”, “pre-coma”, and in some cases a Glasgow Coma Score (GCS) was given. In the retrospective analysis, the study team adopted the term ‘coma’ for those found to have either a clearly documented coma state, or understood to have an unarousable coma based on GCS scores. Limitations on diagnostic capabilities at this facility leaves open the possibility that cases and/or complications may have been missed. The study does not reflect cases that never made it to medical attention, died out of hospital, or were treated at local facilities, in the private sector or at home. As such, the in-patient data are only representative of the referral centre and not of the overall severe malaria burden for this region of Cambodia. Likewise, the non-availability of important diagnostic measurements may have resulted in under-reporting of complications.A large proportion of patients were slide-negative on admission, though nearly 50% of these patients still responded to anti-malarials alone. In such cases, preliminary microscopic diagnosis in hospital may have been obscured or simply not performed due to pre-treatment out of hospital prior to transfer. Many cases were diagnosed by rapid diagnostic kits or simply treated on clinical grounds. Patients with negative malaria smears in this cohort had higher crude mortality rates at 16% versus 11% in the patients with high parasitaemia, possibly due to delays in diagnosis and appropriate treatment, or earlier parenteral therapy in the higher parasitaemia patients. In this setting, a high index of suspicion for severe malaria remains warranted and may prove lifesaving. However, an empiric treatment approach to severe malaria should not preclude investigation of co-morbidities, which were common.BRH continued to have a significant number of severe malaria cases between 2006–2009, suggesting that interventions to improve access to early diagnosis and treatment of malaria remain priorities for this region. Diagnostic ambiguities were common in this setting, with multiple confounding co-morbidities and limited patient management resources. The key to preventing mortality remains early detection, treatment and referral where necessary, and maintaining a high index of suspicion for severe malaria cases is essential. This includes not only adherence to guidelines, but consideration of the individual patients’ risks based on local endemicity, clinical history, pre-hospital care, and severity of presenting illness syndromes. Co-administration of broad-spectrum antibiotics in this resource-poor setting with frequent co-infection and septicaemia may improve outcome. A subset of technologically appropriate core guidelines for diagnosis and management of severe malaria may be useful in this setting, and monitoring of the patient’s renal and acid–base balance through simple chemistry panels may be a useful and cost-effective addition to management at this and similar facilities. The drop in both numbers of severe cases and mortality during the period immediately following this study suggests some improvement in malaria control, likely to due to large-scale containment efforts in the region.The authors declared that they have no competing interests.DS, CL, AT, DB, JL, YS, NS, KL, ST, MF, DW and CMC conceived, designed and supported the study. NB, SN, SC, CL, DS and YS collected the data. AT, CL, DS, SK, TW, DC and SJ analysed the data. DS, AT, DB and CL wrote the paper. YS passed away during the writing of this manuscript. All authors read and approved the final manuscript.