Plasmodium falciparum and Plasmodium ovale, on two isolated hospital visits within a six-week period. The epidemiology of mixed infections, likely pathophysiology of his presentation, and the implications for malaria testing and treatment in returned travellers are discussed.Malaria is a common and potentially fatal cause of febrile illness in returned travellers. Endemic areas for different malaria parasites overlap, but mixed species infections are rare. An adolescent male returned from a trip to Ghana in late summer 2013. He subsequently presented with blood smears positive for two species of malaria parasite,  Plasmodium parasites with the largest burden of mortality and morbidity being due to Plasmodium falciparum. While Plasmodium species have different geographical distributions, there are areas of significant overlap. Thus, individuals may be infected with more than one species at any given time though this is rare in returned travellers , underscoring the rarity of this infection in returned travellers. Mixed P. ovale and P. falciparum infections are also uncommon in areas where malaria is endemic. A study that followed 830 children in Burkina Faso from 2007–2010 found study participants had an overall malaria prevalence of 71% over the three years, and among those who presented with malaria, the rate of P. falciparum and P. ovale mixed infection was 0.72% [Plasmodium ovale itself, again, was quite rare accounting for 1.08% of all recorded malaria infections.Mixed species malaria infections are rare in travellers, comprising 2.1% of the 1,140 cases of malaria logged by the GeoSentinel Surveillance network based on treating physician diagnosis between 1997 and 2002 [P. ovale malaria are consistently low, there is evidence to suggest that infection with this species may actually be more common than most studies suggest. A research group in Senegal followed 101 children and 105 adults over a four-month malaria season with regular thick blood films regardless of the presence or absence of signs and symptoms of infection [P. ovale infections were documented, 48.5% of children and 32.5% of adults had blood films positive for P. ovale at some point during the study period suggesting that while infection with P. ovale may be common, the majority of infections are subclinical.While reported rates of nfection . AlthougPlasmodium species have different life cycles and behaviours, accounting for the immediate and more severe presentation of P. falciparum malaria and delayed and milder course of P. ovale malaria in this patient. Plasmodium falciparum cycles through a transient liver phase, following which multiple schizonts rupture simultaneously and the infection proceeds to the symptomatic blood stage. This almost always occurs within two months of exposure. In contrast, P. ovale has a prominent liver hypnozoite phase. Parasites remain dormant in this stage and may persist for years. Presentation of P. ovale malaria is often delayed and hypnozoites rupture asynchronously producing intermittent symptoms of a milder nature. Specific treatment is required to target the hypnozoite phase to prevent relapse of infection. It is worth noting that the presentation of P. ovale and P. falciparum mixed infections cannot always be expected to follow this patient’s pattern. The timing of infection, variability of hypnozoite phase duration, interactions between species and other factors may play a role. A recent case report described a mixed-species infection where the patient presented first with P. ovale malaria with a subsequent delayed presentation of P. falciparum[The two alciparum.Plasmodium species during his recent trip to Ghana, with the presentation of P. ovale being either initially masked by the higher parasitaemia of P. falciparum or delayed while it remained in the hypnozoite phase. The possibility that this patient acquired his P. ovale infection during his trip to Kenya the year prior cannot be excluded. Although entomological data exists for co-infected mosquitoes, rates of confirmed mosquito co-infection are very low and most cases of mixed malaria infection are thought to be acquired from two separate inoculations [P. falciparum and Plasmodium vivax, specific testing for P. ovale is not available. Current assays developed to detect mixed malaria infections have suboptimal sensitivity for the diagnosis of P. ovale[It is likely that the patient contracted both ulations . Rapid dulations . Althoug P. ovale,11. An a P. ovale, which cPlasmodium species as described [The more sensitive malaria PCR, a costly test which is not routinely performed, is helpful in arbitrating incongruent results of microscopy and RDT, in identifying very low parasitemia infections, and in identifying those parasites with aberrant morphology on microscopy ,13,14. AP. falciparum infection, it may not have prevented P. ovale infection. There have been case reports of returned travellers receiving appropriate prophylaxis who nevertheless present with P. ovale malaria [P. vivax, another Plasmodium species with a hypnozoite phase [P. vivax prevalence as well as a terminal prophylaxis in long-term travellers to endemic areas of relapsing malaria [P. ovale, raising the possibility that while not likely, the patient described in this report may have contracted P. ovale during his previous trip to Kenya even though he took appropriate prophylaxis [While appropriate prophylaxis would likely have prevented  malaria ,16. Thiste phase ,18. How  malaria . Relapsephylaxis -23. His P. falciparum, P. ovale is almost universally chloroquine sensitive. Very few studies have attempted to determine the sensitivity of the blood stage of P. ovale to alternative therapies such as atovaquone/proguanil. One small study from 1996 treated seven adults presenting with Plasmodium malariae or P. ovale malaria with atovaquone and proguanil [P. ovale infection with atovaquone/proguanil [P. falciparum are also acceptable for P. ovale[P. ovale infection was treated with chloroquine for 48 hours to clear the blood phase, followed by a two-week course of primaquine to clear the hypnozoite phase.In contrast to roguanil . Each ofroguanil . The CenP. falciparum malaria, then returned one month later with milder symptoms and a blood smear positive for P. ovale. Mixed malaria infections are rare in returned travellers, and P. ovale itself is a rare cause of documented malaria that often has mild symptoms, delayed presentation, and may cause subclinical infections in the majority of cases. Compared to other species of malaria, little is known about this parasite’s actual prevalence and response to different anti-malarial therapies. This case highlights the value of accurate speciation of malaria blood films, and follow-up to ensure patients receive appropriate treatment including radical cure, if indicated, of the identified parasite. It also raises the possibility that more sensitive testing, such as PCR, may be useful in the setting of recurrent illness. It demonstrates the importance of considering malaria in the differential diagnosis of returned travellers with delayed symptoms, regardless of their use of prophylactic medications or previous malaria treatment. More generally, this case reinforces the importance of pre-travel physician counselling and adequate malaria prophylaxis for trips to endemic areas.In the summer of 2013, a paediatric returned traveller presented to SickKids with a rare, mixed-species malaria infection. He initially presented with more severe Written informed consent was obtained from the patient for 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.HS drafted and revised the manuscript. NA and SM revised the manuscript and SM provided overall supervision. AKB critically appraised and revised the manuscript. All authors read and approved the final version.


Plasmodium that are unlikely to be detected by conventional microscopy. Such submicroscopic infections only occasionally cause acute disease, but they are capable of infecting mosquitoes and contributing to transmission. This entity is frequent in endemic countries; however, little is known about imported SMM.Submicroscopic malaria (SMM) can be defined as low-density infections of The goals of this study were two-fold: a) to know the frequency of imported SMM, and b) to describe epidemiological, laboratorial and clinical features of imported SMM.Plasmodium included Field staining and microscopic examination through thick and thin blood smear. A semi-nested multiplex malaria PCR was used to diagnose or to confirm cases with low parasitaemia.A retrospective study based on review of medical records was performed. The study population consisted of patients older than 15 years attended at the Tropical Medicine Unit of Hospital Carlos III, between January 1, 2002 and December 31, 2007. Routinely detection techniques for SMM was diagnosed in 104 cases, representing 35.5% of all malaria cases. Mean age (IC95%) was 40.38 years (37.41-43.34), and sex distribution was similar. Most cases were in immigrants, but some cases were found in travellers. Equatorial Guinea was the main country where infection was acquired (81.7%). Symptoms were present only in 28.8% of all SMM cases, mainly asthenia (73.3% of symptomatic patients), fever (60%) and arthromialgias (53.3%). The associated laboratory abnormalities were anaemia (27.9%), leukopaenia (15.4%) and thrombopaenia (15.4%). Co-morbidity was described in 75 cases (72.1%).Plasmodium vivax) in Europe may be speculated.Results from this study suggest that imported SMM should be considered in some patients attended at Tropical Medicine Units. Although it is usually asymptomatic, it may be responsible of fever, or laboratory abnormalities in patients coming from endemic areas. The possibility of transmission in SMM has been previously described in endemic zones, and presence of vector in Europe has also been reported. Implementation of molecular tests in all asymptomatic individuals coming from endemic area is not economically feasible. So re-emergence of malaria ( Plasmodium that are unlikely detected by conventional microscopy.Malaria is usually diagnosed using microscopic methods (thick or thin blood smears) or immunochromatography. These procedures are routinely used in clinical practice, but have limited sensitivity. Submicroscopic malaria (SMM) is defined as low-density infections of Several questions arise from this definition: Is SMM a real entity or “only” a laboratory finding? Is SMM a rare condition or a frequently neglected problem? And finally, can SMM play a role in malaria transmission or is it a problem only for individual patients? Since the development of polymerase chain reaction (PCR) techniques, attempts have been made to answer these questions. SMM has been associated to anaemia and low weight at birth in several studies with pregnant women-3, althoSMM is common in endemic countries and its prevalence is comprised between13% and 33% in some areas-10. The However, little is known about imported SMM, namely its relevance in terms of frequency, clinical manifestations, or laboratory abnormalities. In countries free of malaria like Spain or Greece, recent autochthonous cases-15, suggThis study has two objectives: a) assessing the frequency of imported SMM, and b) describing the epidemiological, laboratory and clinical features of imported SMM.In Spain, malaria is a reportable disease. Last autochthonous case was notified in 1961 and sincA retrospective study based on a review of medical records was performed. The study included those patients older than 15 years and diagnosed with malaria in Hospital Carlos III between January 1st, 2002 and December 31st, 2007. The study was approved by the corresponding Ethics Committee.Exclusion criteria were: a) Unspecified diagnosis methods; b) Medical records with lack of data (>25% items): epidemiological data (> 5 items), clinical data (> 5 items), or analytical data (> 7 items).Plasmodium spp. infection could be detected by conventional microscopy and/or using PCR, regardless of the presence of symptoms. A patient was defined as suffering from SMM if he/she produced a positive PCR test even if the examination of thick and thin blood smears by light microscopy was negative. Cases detected by conventional microscopy were considered as microscopic malaria. WHO criteria were followed to identify the severe malaria cases[A patient was diagnosed with malaria when ia cases.Cases of malaria were allocated into four groups following the classification criteria published by our study group,19: natiEvery patient underwent a complete clinical history and an exhaustive physical examination. They were routinely screened for human immunodeficiency virus (HIV), hepatitis , syphilis, intestinal helminthiasis, or protozoan infestation. The screening for filarariasis (symptomatic or symptom-free) was performed for natives, native-travellers, or residents in endemic zones having travelled to zones endemic for filariae.Plasmodium included Field’s staining and microscopic examination through thick and thin blood smears. For all patients we always collected one blood sample, independently of the presence of fever. If the sample was negative, a second blood sample was collected only in febrile patients. A semi-nested multiplex malaria PCR[Plasmodium spp. In this second reaction, infections with different human Plasmodium species yielded products of different sizes. A band of 269 bp indicates an infection by Plasmodium malariae; a band of 395 bp evidences a P. falciparum infection; a band of 436 bp suggests a Plasmodium ovale infection; and a band of 499 bp indicates a Plasmodium vivax infection. The mixed infections would show the corresponding bands.For every patient coming from zones endemic for malaria, routine detection techniques for aria PCR served tPatients with confirmed malaria (microscopic or submicroscopic) were treated according to the WHO guidelines at the time of the diagnosis. For each case, demographic, clinical, and laboratory data were documented . For univariate analysis of categorical variables, Pearson’s Chi-square test was used (Fisher test when needed). For continuous data, t-Student test was chosen to compare means between groups, except when the variances of the samples were not homogeneous. In this case (absence of homoscedasticity), the non-parametric Mann–Whitney test was used. A p-value p < 0.05 was considered significant.293 cases of malaria were included in the study. 189 cases (65%) were classified as microscopic malaria and 104 cases (35%) as SMM. TableTableP. falciparum infection in 171 cases of microscopic malaria (90.5%), and in 90 cases of SMM (86.5%) . Infection was caused by other Plasmodium species, such as P. ovale , P. vivax , and P. malariae .These data may be surprising, but further analysis revealed that only 10% of the patients suffering from microscopic malaria achieved good adherence to chemoprophylaxis (3 cases out of 30), compared to the 66.7% of patients affected by SMM (4 cases out of 6) (p = 0.008). Other parasitic infections were more frequent in the SMM group, but no differences where found concerning infections by HIV, hepatitis B virus (HBV) or hepatitis C virus (HCV). Malaria was caused by We found some relevant differences in clinical characteristics between microscopic malaria and SMM groups Table. Most miTableTableA total of 13 SMM cases were described in travellers (non immune). A total of 4 patients were symptom-free and did not show laboratory abnormalities. A good adherence to anti-malarial chemoprophylaxis was described in only one of these SMM cases. In the remaining nine symptomatic SMM patients, one suffered from anaemia and one from thrombocytopaenia. For those nine patients, good adherence to anti-malarial chemoprophylaxis was achieved in three cases. When good adherence was achieved the reported symptoms were asthenia (3 cases), arthromyalgia (3), fever (2), headache (1), and cough (1). The symptoms reported when no adherence to anti-malarial chemoprophylaxis was achieved were asthenia (5 cases), fever (4), headache (3), arthromyalgia (3), rash (1), ocular pain (1), diarrhoea (1) and liver enlargement (1).This study aimed to describe the frequency of imported SMM and its epidemiological, clinical, and laboratory features. Most of the cases of imported malaria reported in Europe are of microscopic malaria and a few reported cases are of SMM. Two main important reasons explain this fact: a) Malaria is suspected when symptoms are present and b) Diagnosis is usually made by microscopic examination but PCR is not routinely used. Examination for SMM has been progressively implemented in the daily care schemes of our hospital, based on several studies showing the high prevalence of SMM in some countries,9-11 or Although SMM is a clinical entity usually symptom-free, it can be associated to asthenia, fever or musculoskeletal pain. In addition, anaemia, leukocytopaenia or thrombocytopaenia were reported in more than 10% of the cases. Symptoms and laboratory abnormalities may be linked to other diseases when co-morbidity is present, but sometimes SMM is the only cause. SMM should be envisaged when conventional techniques for malaria detection gave negative results but some clinical or analytical abnormalities remain unexplained in patients coming from zones endemic for malaria.Negative microscopic tests can produce a positive PCR test in these cases: a) False positive of the PCR technique, or b) Low-density parasitaemias. In low-density parasitaemia, five possibilities have to be considered: 1) Infection in semi-immune people; 2) Anti-malarial treatment; 3) Anti-malarial chemoprophylaxis; 4) First stages of infection; or 5) False negative microscopic method .False positive (or PCR contamination) is a common problem, with rates reported by laboratories between 0.7% and 10%,23. InfeAnopheles atroparvus, Anopheles claviger or Anopheles maculipennis) has also been reported[Plasmodium vivax) in Europe can only be speculated.Data from the WHO Regional Office for Europe have shown that the number of imported malaria cases have not changed significantly in the last ten years (except in France). Autochtreported,28. SincSMM is a frequent condition that should be considered when some clinical or analytical abnormalities remain unexplained in a patient coming from zones endemic for malaria. Undetected and untreated SMM and the spreading of competent vectors might be the causes of a re-emergence of malaria in Europe.The authors declare that they have no competing interests.GRO, PR, MDH, ML and SP attended the patients, collected the data and drafted the manuscript. MS carried out the microscopic examination. JMR carried out the molecular genetic studies. All authors read and approved the final manuscript.


Malaria is a serious parasitic disease in the developing world, causing high morbidity and mortality. The pathogenesis of malaria is complex, and the clinical presentation of disease ranges from severe and complicated, to mild and uncomplicated, to asymptomatic malaria. Despite a wealth of studies on the clinical severity of disease, asymptomatic malaria infections are still poorly understood. Asymptomatic malaria remains a challenge for malaria control programs as it significantly influences transmission dynamics. A thorough understanding of the interaction between hosts and parasites in the development of different clinical outcomes is required. In this review, the problems and obstacles to the study and control of asymptomatic malaria are discussed. The human and parasite factors associated with differential clinical outcomes are described and the management and treatment strategies for the control of the disease are outlined. Further, the crucial gaps in the knowledge of asymptomatic malaria that should be the focus of future research towards development of more effective malaria control strategies are highlighted. Plasmodium falciparum is reported to cause the highest morbidity and mortality. Young children with naïve immune systems [P. falciparum disease severity ranges from severe and complicated, to mild and uncomplicated, to asymptomatic [P. falciparum on the human host across this range is critical for learning how to improve the management of the disease.Malaria remains a serious global health burden, with an annual incidence of 247 million cases and nearly one million deaths, most of which afflict children living in Africa . Of the ptomatic ,4. UnderP. falciparum malaria in high transmission regions typically reduces the period of risk for severe malaria, while in lower transmission regions infrequent exposure extends this period of risk [Generally, severe or complicated malaria has been at the core of epidemiological studies because it is the principal cause of malaria-related deaths. Researchers and clinicians have established diagnostic criteria based on the clinical manifestations upon disease onset, which has aided in forming an integrated approach to improving the management and treatment of severe malaria. Severe malaria is now defined by at least one of the following clinical manifestations: unrousable coma , convulsions, malarial anaemia, haemoglobinuria, hypoglycaemia, metabolic acidosis (associated with respiratory distress), acute pulmonary oedema, acute renal failure, jaundice, circulatory collapse, hyperparasitaemia, high fever electrolyte disturbance, and/or spontaneous bleeding . In area of risk .et al. [In contrast, individuals with mild or uncomplicated malaria typically present clinically with fever and perhaps one or more of the following symptoms: chills and sweats, headache, vomiting, watery diarrhea, anaemia, jaundice, and swelling of the spleen , but do not generally have any of the features identified in severe or complicated malaria . If propet al.  reportedDiagnosing asymptomatic malaria is not as straightforward due to the obvious lack of clinical manifestations and often subpatent (undetectable by microscopy) level of parasites . AsymptoFor more than two decades, researchers have investigated the development of two types of immunity which may result in asymptomatic malaria: 1) an anti-disease immunity that allows one to carry parasite loads without symptoms; and 2) an anti-parasite immunity that may be responsible for the suppression of parasite loads after a certain age, which is likely a factor of exposure-related clinical immunity -12. InteP. falciparum. However, like asymptomatic P. falciparum, asymptomatic Plasmodium vivax malaria has been reported in a range of endemic settings. For example, the low transmission setting of Temotu Province, Solomon Islands [P. vivax. Another Amazonia study reports that the prevalence of "symptomless" falciparum and vivax malaria infections are 4-5 times higher than the symptomatic ones, with a significant correlation of "symptomless" malaria with older age groups [Few reports are available on the study of asymptomatic malaria caused by species other than  Islands  and the  Islands  both repe groups . UnfortuPlasmodium malariae and Plasmodium ovale asymptomatic infection have also reported. A case report of transfusion-transmitted P. malariae infection from an asymptomatic donor [P. malariae can be harbored asymptomatically. Supporting this, another study also reported a case of malaria in a Grecian woman due to P. malariae whose illness was reactivated after decades of latency [P. ovale can also be asymptomatic [P. falciparum.Though rare, cases of ic donor  suggests latency . Infectiptomatic . HoweverThe purpose of this review is to: (1) critically examine how asymptomatic falciparum malaria is defined; (2) describe potential relationships to transmission dynamics; (3) speculate on the potential roles of putatively important parasite genes; (4) review human genes that may contribute to susceptibility or resistance to infection; (5) discuss the management and treatment of asymptomatic malaria; and (6) discuss the gaps in knowledge and highlight areas of future research.<37.5°C, and an absence of malaria-related symptoms [et al. [A major obstacle in the study of asymptomatic malaria is the lack of standard diagnostic criteria. For example, infected individuals may be in a pre-symptomatic period with parasitaemia, and present with clinical manifestations at a subsequent date . Alternasymptoms -30. Somesymptoms -33. Quansymptoms -33. Thou [et al. ,34-36 foP. falciparum infection has been shown to persist asymptomatically in semi-immune individuals for more than 18 months [P. falciparum infections can also persist inter-seasonally in regions with seasonal transmission [The transmission dynamics of malaria are complex. Even in endemic areas, transmission is not consistently stable; rather, it can be patchy and dependent on factors such as climate, the location of mosquito breeding sites, and areas of clustered human habitations, which serve as reservoirs of parasites for mosquito infection ,40. Asym8 months . Asymptosmission . For exasmission ,43,44. TP. falciparum parasitaemia is common among immune inhabitants [In areas with annual malaria transmission, asymptomatic abitants ,46 and aabitants . A positabitants ,34-36. Sabitants ,49. Highabitants -21. Two abitants ,51. ThouThe production of gametocytes within an infected human host is essential for transmitting the parasite to the mosquito. Understanding the variables that are involved in gametocyte production within the human host, such as the host immune response to parasites, anti-malarial drug treatment, and parasite genetic diversity, and correlating these with the presence or absence of symptoms, is layered with complexity. For example, some studies have indicated that the quantity of gametocytes may impact the level of mosquito infectivity ; howeverP. falciparum gametocytaemia and transmission potential [There is evidence that patients with severe anaemia have higher levels of gametocytes , which motential . The stuotential ,64.et al. reported that parasites from asymptomatic individuals in an endemic region of western Kenya were more infective to mosquitoes than parasites from symptomatic individuals. The authors concluded that this was a function of both the greater gametocyte abundance in asymptomatic individuals and a higher quality of infectious gametocytes [A study in Thailand investigating mosquito infectivity differences between febrile and asymptomatic patients reported that febrile patients were more infective than asymptomatic individuals to mosquito hosts . This waetocytes ,67.These observations of asymptomatic malaria in low and high endemic areas highlight the importance of carrying out active surveys for the identification of asymptomatic carriers. The treatment of the infective parasite reservoir of asymptomatic individuals may be an important intervention strategy, and if this reservoir is greatly reduced it will provide a positive impact on the intervention of disease transmission.P. falciparum infections are symptomatic while others are asymptomatic, but parasite factors are likely to be involved [var (variant antigen receptor) gene family.It is not clear why some involved . Malariaet al. reported cerebral malaria as the cause of death in aparasitaemic individuals in autopsies following effective antimalarial treatment [P. vivax- and P. falciparum-infected asymptomatic individuals tend to have low and submicroscopic parasite densities [The precise relationship between parasite density and disease severity remains unclear, as the density of sequestered parasites and circulating parasites varies greatly depending upon the stage and synchronicity of the infection . In genereatment . A studyensities . Anotherensities .A study investigating the relationship that parasite density has on platelet count showed that malaria-infected children with thrombocytopenia (decreased platelet count) were younger, had higher parasitaemia, lower hemoglobin levels, an increased mean platelet volume, and exhibited platelet aggregation . SimilarP. falciparum and its association with the development of clinical symptoms. There is strong evidence that immunity to malaria is specific to the particular strain eliciting the host response, enabling an individual to resist infection by that particular strain, but not by heterologous ones; this has been termed 'strain-specific immunity' [Various studies have investigated the genetic diversity of mmunity' -76. The Other studies have sparked an investigation of some symptom-specific molecular characteristics based on polymorphisms of surface antigens that have been identified in isolates collected from asymptomatic, uncomplicated, and severe malaria cases across various geographic regions -79 see . Such pomsp-1 and msp-2 with clinical severity of disease. For example, a study conducted to examine the relationship between the genetic diversity of msp-1 block 2 of P. falciparum and clinical severity of malaria in Nigerian children showed that the presence of K1 and MAD20 alleles were significantly associated with asymptomatic malaria and consequently reduced the risk of developing symptomatic disease [et al. [msp-1 allele (K1) with a specific var gene (var-D) was overrepresented among patients with severe compared to mild disease, and this genotype combination was consistently observed in the most severe clinical cases. However, investigations of genotype associations with disease severity need to be expanded to include all categories of disease, including asymptomatic malaria. Further, genetic diversity should be longitudinally monitored to ensure that such clinical associations are maintained and not biased by cross-sectional selection of isolates.Several studies have investigated the association of  disease . Yet, it [et al.  found thmsp-2) according to clinical status [msp-2 was shown to be twice as likely to be found in symptomatic cases than in asymptomatic cases [msp-2 may be associated with the morbidity of malaria. A similar association was reported in Papua New Guinea, where the FC27 allele was linked to increased disease severity, however, this study did not consider asymptomatic cases [msp-2 and malaria symptoms [There are conflicting reports regarding the differential distribution of alleles within particular genes , which cluster on the cytoplasmic face of the knob membrane [An important aspect of membrane . KAHRP imembrane . In one membrane . StudiesP. falciparum have focused on the diverse var gene family, encoding various forms of erythrocyte membrane protein 1 (PfEMP1), which is involved in cytoadherence of RBCs (red blood cells) to endothelial cells [var multigene family is thought to facilitate evasion of the host immune response [var genes fall into three major groups: var A, var B, and var C. Emerging evidence suggests that specific expression of these groups has clinical relevance and that major differences in the transcription profile of var gene expression may differentiate disease severity [var group C transcript levels were increased in asymptomatic cases, whereas transcripts of var group A and B were abundant in patients with severe malaria [P. falciparum isolates from cerebral malaria patients are significantly more likely to transcribe var genes with DBLα1-like domains, characteristic of Group A and Group B/A (an intermediate group of var gene), than the isolates from patients with non-severe hyperparasitaemia  [var gene expression and malaria severity have yielded conflicting results [var gene transcription and the phenotypes displayed by circulating parasites, which may be different to those sequestered, as in severe malaria. Therefore, comparing the repertoire of genomic and expressed var genes with a particular malaria outcome will be helpful to understand the role of this gene family in asymptomatic malaria.Many gene expression studies in al cells . Switchiresponse ,92. Mostseverity ,93. For  malaria . Anotherdisease) . Recent  results ,93,95,96The formation of rosettes may influence the severity of malaria by causing distinct patterns of sequestration with different pathogenic consequences . Parasitet al. [et al. [et al. [Intraleucocytic malaria pigment found in neutrophils is suggested to be a better indicator of disease severity than the peripheral parasite count ,100. Ngu [et al.  confirme [et al.  also rep [et al.  expandedStudies on the association of the roles of host factors such as genes of the immune system, RBC polymorphisms or disorders, circulating levels of immunoglobulins and cytokines, and pregnancy should not be limited only to symptomatic malaria, but also include asymptomatic malaria. To date this has usually not been the case; the role of host factors in asymptomatic malaria largely remains a field ripe for inquiry.i.e., an association demonstrated for one ethnic group may not be the same for a population that differs both in genetic background as well as frequency of exposure to infection [Significant correlations between SNPs (single nucleotide polymorphisms) in regulatory or coding regions and severe malaria have been reported for several immunologically relevant genes, including cluster for differentiation-40 ligand (CD40L), Fc gamma receptor II (FcγRII), complement receptor 1 (CR1), tumor necrosis factor α (TNF-α), interleukins -4, -12, and -13 , intracellular cell adhesion molecule-1 (ICAM-1), CD-36, platelet endothelial cell adhesion molecule-1 (PECAM-1), toll-like receptor (TLR), and mannose binding lectin 2 (MBL2)-115. Hownfection . Thus, dnfection . A studynfection . HoweverSeveral RBC polymorphisms, like G6PD deficiency ,119, hae- heterozygosity in females confers protection against all forms of malaria, including the asymptomatic form [- heterozygous female host must cycle between G6PD A- and G6PD wild type erythrocytes and may fail to adapt to the G6PD A- environment [However, research on G6PD polymorphisms has expanded in the last decade to include a range of disease outcomes. For example, G6PD Atic form . The mecironment .p = 0.05). Further evidence for such protective effects must not only consider the distribution of blood groups, but also other explanations, such as the anti-rosette formation effect associated with blood group antigens [The global distribution of ABO blood group antigens reflects natural selection by various pathogens. There is strong evidence that O blood group provides protection against malaria by a mechanism of reduced rosetting and sequestration , while oantigens .i.e., intense and constant over months and years) malaria transmission areas. This immunity acquired due to repeated exposure reduces the risk of both severe and mild malaria. Therefore, asymptomatic malaria could be a consequence of natural immunity. However, in low transmission areas in South America clinical immunity is found to develop in underexposed individuals, resulting in asymptomatic malaria [Different clinical outcomes during malaria infection may be due to differences in the host immunity level. Acquisition of natural immunity to malaria has been observed in high and stable  [P. falciparum malaria than in uncomplicated mild malaria [P. falciparum IgE levels have been associated with a reduced risk of developing clinical malaria [Malaria infection induces polyclonal immunoglobulin production the proportion of which determines protection against the blood stages of  G (IgG) ,134. Amo G (IgG) -139. Inf malaria ,141. The malaria . Additio malaria . This su malaria -146.Plasmodium parasites with soil transmitted helminthes or Hepatitis B Virus (HBV) has been reported in several studies. Helminth infections may alter susceptibility to clinical malaria by changing the T-helper1/T-helper2 (Th1/Th2) balance [Plasmodium influences the malaria burden by stimulating an increased inflammatory response. Plasmodium-infected individuals with HBV infection were more likely to be asymptomatic with lower levels of parasitaemia and a decreased inflammatory cytokine profile [One of the most challenging issues with studying malaria immunology is that individuals at risk for malaria infections are typically at risk for other parasitic or non-parasitic diseases. Distilling the immunological response induced by malaria apart from other infectious diseases is not straightforward. For example, co-infection of  balance ,148, the balance , which h profile . Thus, tAge is considered one of the most important factors that correlate with protective immunity in malaria endemic areas. Infections among non-immune individuals invariably result in clinical symptoms, and often lead to death in young children if untreated . Young cγ stimulate neutrophils in order to increase parasite destruction [In adult individuals, the acquired immunity or protection which is thought to keep their malaria asymptomatic or uncomplicated could be due to increased frequency of both type 1 and type 2 cytokine-producing T cells . Cytokintruction .P. falciparum Erythrocyte Membrane Protein-1), rifins, and stevors. These antigens are expressed on parasitized RBCs infecting the placenta and are most commonly referred to as Variant Surface Antigens of Pregnancy Associated Malaria (VSA-PAM) [Pregnant women are more susceptible to malaria infection, a phenomenon especially apparent during the first pregnancy . In succVSA-PAM) ,158. TheVSA-PAM) .et al. [et al. [Drug therapy has often been linked to the development of gametocytaemia, as environmental pressure caused by the targeted destruction of the asexual stage parasites stimulates the production sexual stage parasites (reviewed in ).Though et al. , which s [et al.  investigSince the transmission of malaria parasites from humans to mosquitoes requires the presence of gametocytes, any strategy that interferes with the development or persistence of gametocytes should help to interrupt transmission. In most malaria endemic areas, the majority of parasite carriers are asymptomatic  and thesP. falciparum. Treatment regimens containing artemisinin and/or its derivatives are reported to lower gametocyte carriage, and reduced infectivity among treated individuals [The identification and management of asymptomatic carriers has become a new and increasingly important challenge for malaria control programs. One challenge, in particular, is to reform the development of novel anti-malarial drugs to include those that explicitly target transmissible sexual stages. At the present, the majority of anti-malarial drug treatments on the market target the asexual blood stage of ividuals -165. Artividuals ,166,167.ividuals . The shoividuals .P. vivax malaria, actively clears submicroscopic P. falciparum gametocytes [P. falciparum gametocytes and asexual parasites detected by microscopy [Apart from selection for drug resistance, there is an additional risk of the persistence of submicroscopic gametocytes even after treatment, allowing for post-treatment malaria transmission . Primaquetocytes -172, butcroscopy  as well croscopy . As partet al. [i.e. the identification of vector breeding and resting sites, the impact of climate change and temperature variation on vector survival and capacity, and also insecticide resistance.The transmissibility of submicroscopic parasites within asymptomatic malaria infections may help to drive the persistence of malaria within endemic regions. The use of antimalarial drug treatment (particularly ACTs), in combination with insecticide treated nets (ITNs), long-lasting insecticidal nets (LLINs), and indoor residual spraying (IRS) is perhaps the most aggressive method for reducing the malaria burden in endemic regions. A recent study by Aregawi et al.  using ACThe history of research on asymptomatic malaria dates back to 1900 when Robert Koch first identified such cases in a study among patients in Papua New Guinea . Yet, asScaling up efforts to improve the characterization of asymptomatic malaria in endemic regions and establishing a standard case definition for this malaria disease outcome is a priority. To help facilitate this in field studies, longitudinal active case detection methods, improved epidemiology questionnaires, and advanced epidemiological modeling must become universal components in all epidemiological studies. Concerted efforts should also be directed towards the development of highly sensitive, cheap and easily obtained rapid diagnostic detection kits, which can be used to detect parasitaemia at submicroscopic densities . IdentifSecondarily, the underlying causes of asymptomatic malaria must be fully investigated. It is important to expand beyond the traditional association and correlation analyses to include more comprehensive molecular methods. If detection efforts are improved, then it will be possible to discriminate between the host and parasite roles in asymptomatic infection. Consequently, parasite population diversity studies will become more thorough, the association of parasite virulence genes will become more relevant, and the malaria field will have a better sense of how to interpret and manage different disease outcomes. Importantly, information on host genetics and immune responses in association with asymptomatic malaria are severely lacking, but could provide a wealth of knowledge on the management of disease and the roles that they play in mounting different responses to infection.The authors declare that they have no competing interests.DDL and PS mined the literature. DDL, PLS, NN, VLS, RCS and JMC wrote the manuscript. HJ contributed to initial discussions and planning. All authors with the exception of HJ (deceased) read and approved the final manuscript.Diagnostic criteria for defining malaria patients as asymptomatic. The list represents a snap-shot of some of the studies of asymptomatic infections world-wide.Click here for fileP. falciparum genes reported to be associated with different clinical outcomes of malariaList of . (DOC 78 kb).Click here for fileList of human genes reported to be associated with different clinical outcomes of malaria. A. Human gene polymorphisms B. Human blood disorders.Click here for file


Objective: We present 2 cases of trigeminal trophic syndrome treated by surgery. Methods: We performed reconstruction of the ala nasi using a nasolabial flap or paramedian forehead flap in combination with an auricular chondrocutaneous composite graft. Results: One case was successfully treated. However, ulceration recurred intermittently in the other case. Conclusions: Although trigeminal trophic syndrome is rare, we believe that plastic surgeons should have a raised awareness of this entity and familiarity with the treatment options. Trigeminal trophic syndrome (TTS) is a rare condition deriving from damage to the trigeminal nerve system. Patients of TTS develop skin ulcers in the trigeminal sensory region, most frequently at the nasal ala because of self-manipulation of the lesion in response to an altered sensation. The classic clinical appearance of TTS involves 1 or more crescent-shaped lateral nasal ala ulcerations. Differential diagnosis with other entities, such as skin neoplasms, vasculitis, infections, and granulomatous disease, is necessary. Furthermore, a positive neurologic history of the trigeminal system, such as stroke, treatment of trigeminal neuralgia, brain tumor, and postencephalopathic Parkinsonism, also is of importance. Recurrence of skin ulcers in TTS patients is common, and many therapeutic options have been reported, including behavioral modification, pharmaceutical intervention, installation of a protector, transcutaneous nerve stimulation, and surgical repair. Here, we report 2 patients of TTS treated by surgery.A 62-year-old woman was referred to us with ulceration of her left nasal ala, and xeransis of the nasal mucosa . She hadReconstruction of the ala nasi was conducted in a 2-stage fashion. In the first stage, a left paramedian forehead flap and an auricular chondrocutaneous composite graft from the right concha were transferred to cover the surface and inner lining of the alar defect, respectively. Two weeks later, the pedicle of the forehead flap was divided. Her course was uneventful after the second operation. However, 18 months later, she complained of a spread nostril and subsequent xeransis and crust formation on the nostril floor. As a result, she started scratching her nostril floor, and not the reconstructed ala nasi. Therefore, we narrowed the nostril by Z-plasty, and the ulcer healed spontaneously. Subsequently, she stopped scratching, and the ulceration has not recurred for 22 months .A 79-year-old woman was referred to us with a defect of her ala nasi and ulceration of the left nostril floor . She hadWe planned a 1-stage reconstruction using a nasolabial flap and an auricular chondrocutaneous composite graft from the concha, because she had dementia. Postoperative wound healing was uneventful, but 10 days after surgery, she started scratching the reconstructed nasal ala. The flap was immediately epithelialized by installing a protector, however, she continued to manipulate the reconstructed ala nasi and nasal ulcerations occurred intermittently . AlthougTrigeminal trophic syndrome was first described by Loveman5,The treatment options reported so far are behavioral modification, pharmaceutical intervention, installation of a protector, transcutaneous nerve stimulation, and surgical repair. Whether innervated flaps or noninnervated flaps should be used for surgical repair remains controversial. Abyholm and Eskeland4We reported our experience of 2 patients with TTS treated by surgery. Although TTS is rare, we believe that plastic surgeons should have a raised awareness of this entity and familiarity with the treatment options.


Plasmodium falciparum malaria. In this brief review, in keeping with the theme of this journal, we focus on the close and complex relationship betweeen this blood disease and this infectious disease. On one hand, heterozygotes for the sickle gene (AS) are relatively protected against the danger of dying of malaria, as now firmly established through a number of clinical field studies from different parts of Africa. In addition, experimental work is consistent with a plausibile mechanism: namely, that in AS heterozygotes P falciparum-infected red cells sickle preferentially and are then removed by macrophages. On the other hand, patients who are homozygous for the sickle gene and therefore suffer from sickle cell anaemia (SCA) are highly susceptible to the lethal effects of malaria. The simplest explanation of this fact is that malaria makes the anaemia of SCA more severe; in addition, in SCA there is often hyposplenism, which reduces clearance of parasites. From the point of view of public health it is important that in malaria-endemic countries patients with SCA, and particularly children, be protected from malaria by appropriate prophylaxis.Sickle cell anaemia is a major chapter within haemolytic anaemias; at the same time, its epidemiology is a remarkable signature of the past and present world distribution of  The history of sickle cell anaemia (SCA) lists several gold medals. First, it was for SCA that the term molecular disease was coined over half a century agoThus, the entire field of human molecular genetics is greatly indebted to SCA; at the same time, as far as haematology is concerned, SCA is a major chapter within haemolytic anaemias.Here we intend to discuss briefly one aspect of this condition that is eminently germane to the very name of this journal: we focus on where SCA, a blood disease, meets malaria, an infectious disease. The relationship is complex. Here we will try to briefly pinpoint what we know and what we don’t yet know about this two-way relationship: malaria has influenced greatly the epidemiology of SCA, and SCA affects the clinical course of malaria.Plasmodium falciparum, is highly lethal. Second, it is estimated to have been around in many parts of the world for several thousands of years, i.e. for several hundreds of generations: thus, malaria as an agent of natural selection seemed a better candidate than an infectious disease causing occasional epidemics even if associated with high mortality (such as plague or influenza). Third, deaths from malaria take place mostly in children, i.e. before reproduction, a critical criterion for effective selection. Last but not least, Plasmodia take on different forms in the course of their life cycle, but what causes disease are the intra-erythrocytic parasites: therefore in principle it is not surprising that if red cells are in any way abnormal , this may affect the chance of success of the parasite.That different persons may differ in how they respond to an infectious disease has been probably perceived for a long time. However, the first to formulate this notion in terms of Darwinian selection was J B S Haldane, who speculated that, depending on their genetic makeup, people would have a different risk of dying when they are confronted by a parasitic organism: so much so, that even if a gene offering protection against that parasite were otherwise harmful, its frequency would increase when a population was exposed to the parasite.strr – that makes them resistant to this antibiotic. If we now isolate one of the resistant bacteria we can grow up a new culture in which the entire population will be streptomycin resistant. It happens that the streptomycin-resistant bacteria do not grow quite as fast as the streptomycin-sensitive ones:strr gene is a great advantage; in the absence of streptomycin it is a disadvantage. Since bacteria are mostly haploid (i.e. they have only one copy of each gene), each one of them either has the strr gene or it doesn’t: there is nothing in between.Many fundamental experiments in genetics have been carried out in micro-organisms, and biological selection is a good example. Growing bacteria in a culture medium containing streptomycin is a very simple and certain way to select for the few bacteria, within the culture, that already had a gene – we can call it balanced polymorphism – had been already well characterized in DrosophilaSince we humans, like most animals, are diploid, we have in this respect more options. SCA is a disease of homozygotes (SS) – this is why we call the disease recessive – whereas heterozygotes (AS) are normal for most intents and purposes. The first test of Haldane’s hypothesis was carried out by A C Allison,Allison’s seminal work has been abundantly confirmed by numerous studies on much larger population samples validated by rigorous statistical analysis;in vitro studies that the rate of sickling of AS red cells that had been parasitized in vivo was significantly higher than that of non-parasitized red cells within the very same blood sample.These data from clinical epidemiologyin vitro culture studies that have shown normal growth of P falciparum in AS red cells and even in SS red cells,per se that hinders parasite development: it must be something downstream of the parasite cycle, such as phagocytosis of sickled cells.This mechanism is consistent with In fact, although it is often stated that the mechanism of protection against malaria of AS heterozygotes is not clear, over the past 40 years there has not been any evidence contrary to the sickling-phagocytosis model; and increased phagocytosis of AS parasitized red cells has been confirmed.P falciparum erythrocyte membrane protein 1 (PfEMP-1).P berghei malaria in mouse is very different from that of P falciparum in humans, and therefore it is difficult to know whether this interesting phenomenon observed in the former is relevant to the latter.The impaired cytoadherence seems to result from altered display on the red cell surface of the Acquired immunity is a major determinant of the clinical outcome of malarial infection. Several studies have suggested that AS heterozygotes have accelerated acquisition of immunity,If AS heterozygotes were protected from malaria through failure of infection, one might expect protection to be at least as effective in SS homozygotes, i.e. in patients with SCA: however, the mechanism is not failure of infection, and therefore it may not necessarily apply to homozyogotes SCA patients. They have a prototype congenital haemolytic anaemia and are susceptible to malaria, which is a prototype acquired haemolytic anaemia. Clinical experience has shown that, not surprisingly, this combination is highly dangerous.In an era of evidence-based medicine it is still not uncommon that hypothetical propositions are stated as facts. By contrast, it is quite remarkable that the protective effect of the Hb S gene against malaria is still portrayed as a hypothesis when it is, in fact, one of the best documented examples in the human species of balanced polymorphism, in which the severe disease of homozygotes (SS or SCA) is balanced by the advantage of AS heterozygotes.P falciparum malaria. If, as doctors, we have a professional obligation towards all of our patients, for those with SCA we have an added human obligation, if we consider that they carry the genetic burden that has helped human populations to survive in malaria-endemic regions of the world.Malaria and sickle cell anaemia are still major challenges to infectious disease medicine and to haematology respectively, and both are also major public health problems. One might have hoped that what we have learnt about the of advantage of AS heterozygotes with respect to malaria would enable us to protect from malaria mortality other people as well. That this has not yet happened is disappointing but perhaps not surprising, because the key is sickling of red cells, and this is a unique phenomenon. It cannot be a straightforward task to mimic sickling by a pharmacological approach in subjects who do not have Hb S, and in a way that would act selectively only on parasitized red cells. We can still hope that human imagination will evolve novel approaches that can match the power of mutation and selection in biological evolution. In the meantime SCA remains a source of great suffering to patients, especially in those developing countries where the numbers are staggering see . It is u


Plasmodium falciparum AMA-1 and CSP in malaria semi-immune adults and children.This trial was conducted to evaluate the safety and immunogenicity of two virosome formulated malaria peptidomimetics derived from The design was a prospective randomized, double-blind, controlled, age-deescalating study with two immunizations. 10 adults and 40 children (aged 5–9 years) living in a malaria endemic area were immunized with PEV3B or virosomal influenza vaccine Inflexal®V on day 0 and 90.No serious or severe adverse events (AEs) related to the vaccines were observed. The only local solicited AE reported was pain at injection site, which affected more children in the Inflexal®V group compared to the PEV3B group (p = 0.014). In the PEV3B group, IgG ELISA endpoint titers specific for the AMA-1 and CSP peptide antigens were significantly higher for most time points compared to the Inflexal®V control group. Across all time points after first immunization the average ratio of endpoint titers to baseline values in PEV3B subjects ranged from 4 to 15 in adults and from 4 to 66 in children. As an exploratory outcome, we found that the incidence rate of clinical malaria episodes in children vaccinees was half the rate of the control children between study days 30 and 365 .These findings provide a strong basis for the further development of multivalent virosomal malaria peptide vaccines.NCT00513669ClinicalTrials.gov  The development of an effective malaria vaccine is regarded as one cornerstone in the fight against this deadly disease and to achieve its eventual elimination P. falciparum sporozoite, has been the focus of numerous efforts to develop a pre-erythrocyte vaccine that aims at prevention of hepatocyte invasion by sporozoites P. falciparumThe circumsporozoite protein (CSP), the major surface protein of the IRIVs are reconstituted viral coats of influenza viruses lacking the infectious nucleo-capsid RNA but retaining their target cell surface binding and fusogenic activity In a Phase 1a clinical trial virosomally formulated UK-39 and AMA49-C1 was well-tolerated in malaria non-immune Caucasian volunteers For this Phase 1b trial, UK-39 and AMA49-C1 were coupled to the surface of lyophilizable IRIV formulations The protocol for this trial and supporting CONSORT checklist are available as supporting information; see www.ClinicalTrials.gov (NCT00513669).This study was conducted in compliance with ICH-GCP, the Declaration of Helsinki, and local regulatory requirements. The protocol and all related documents were approved by an independent ethics committee in Switzerland  as well as by the Institutional Review Board of the Ifakara Health Institute and the Medical Research Coordination Committee of the National Institute for Medical Research in Tanzania through the Tanzanian Commission for Science and Technology (COSTECH). The trial was registered at This was a prospective Phase 1b, single-center, randomized, double-blind, controlled, age-deescalating study. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see and A total of 50 healthy subjects were enrolled; 10 adult male volunteers 18–45 years of age and with a BMI of between 18 and 30, and 40 children of both sexes, 5–9 years of age with a mid-upper arm circumference (MUAC) >12 cm. All subjects, or legal representatives thereof, had given written informed consent. Exclusion criteria were: use of any investigational drug or vaccine within 30 days prior to study start or planned use during the study period; chronic immunosuppressant therapy within 6 months prior to study start; chronic medication; immunosuppressive or immune-deficient condition including HIV infection; history of allergic disease; acute disease at the time of enrolment (defined as the presence of a moderate or severe illness with or without fever); acute or chronic, clinically significant pulmonary, cardiovascular, hepatic or renal functional abnormality; acute or chronic diabetes; history of alcohol consumption and/or intravenous drug abuse.The study was performed at the Bagamoyo Research and Training Unit of the Ifakara Health Institute (BRTU-IHI) from January 2008 to March 2009. Subjects originated from the area around Bagamoyo town, on the Tanzanian coast, 70 km north of Dar-es-Salaam. Malaria transmission in this area is perennial and almost entirely due to P. falciparum. Insecticide Treated Bednets (ITN) are promoted through a national program. Artemether/lumefantrine  is currently the first line treatment for P. falciparum malaria in Tanzania.The test vaccine PEV3B was composed of 50 µg AMA49-C1 (PEV301T) plus 10 µg UK-39 (PEV302T) peptides formulated in virosomes in phosphate buffered solution pH 7.4 that was subsequently lyophilized. PEV3B lyophilisate was supplied in vials, and reconstituted with 0.6 mL water <4 hours prior to vaccination, of which 0.5 ml were injected. The comparator Inflexal®V is a commercially available virosomal influenza vaccine . The trial vaccines and comparator were administered i.m. in the left arm on day 0 and in the right arm on day 90 (±4 days). With respect to the first vaccination, two adults were immunized before the remaining 8 volunteers with a safety delay of 11 days . After aRandomization was computer-generated by an independent statistician. The block of 10 adults  was randomized separately. The 40 children were randomized in blocks of 8 . A randomization number (1 to 50) was assigned according to the sequence of assignment of the subjects to the study and after inclusion/exclusion criteria were confirmed by the investigator. Vaccines were provided as kits labeled with the randomization number. The trial vaccines and comparator were administered by an independent pharmacist and/or nurse, with the investigators having no access to the vaccination room. Syringes were covered using an opaque foil in order to ensure blinding of the subjects. Investigators and subjects remained blinded until the end of the study and database lock.The primary objective was evaluation of safety and tolerability of PEV3B in malaria semi-immune subjects. The secondary objective was to measure the magnitude and duration of antibody responses to the malaria specific antigens. Additional exploratory objectives included measuring vaccine-induced cellular immune responses (results to be presented elsewhere) and assessing incidence of clinical episodes of malaria during the follow-up period.The sample size of this Phase 1b study was determined by the requirement to demonstrate the safety and immunogenicity of the virosome-formulated synthetic peptides. A sample size of 50 volunteers is considered appropriate to estimate incidence rate of frequent AEs and assess immunogenicity with an acceptable accuracy, still allowing for dropouts. Since the target population for malaria vaccines are small children, the adult subgroup included to assess vaccine safety in malaria exposed populations was much smaller than the children group. The adult data are presented for comparison with the children data, but it was not intended to carry out detailed statistical analyses within the adult subgroup. The rationale for an unbalanced allocation of study vaccine and comparator within adult and children subgroups was the main focus on safety and immunogenicity of the experimental malaria vaccine and the established safety profile of the comparator. To rule out that recorded anti-malaria antibody titer increases are caused by cross-reactive immune responses to influenza antigens, the small number of subjects immunized with the comparator influenza vaccine was sufficient.st vaccination), on days 7 (±2), 90 , and day 7 (±2) after 2nd vaccination.Occurrence of solicited local  and solicited systemic  adverse events (AEs) within 4 days after both immunizations was assessed by the physician see also , and genSafety evaluation included the assessment of the incidence of clinical malaria episodes. A blood sample was taken for parasitological examination by microscopy from all subjects presenting with a history of fever or raised temperature (>37.5°C), irrespective of any other symptoms or sign.Causality of AEs was categorized as related, probably related, possibly related, unlikely related, and unrelated. All local solicited AEs recorded within 4 days after each vaccination were considered as related to the study vaccine.nd vaccination), 120 (±4), 180 (±7), and day 365 (±14). Procedures for analysis of antibody titers against synthetic peptides using ELISA were described previously Endpoint titers of anti-AMA49-C1 and anti-UK-39 IgG were measured by ELISA at baseline and on days 30 (±4), 90  population included all participants, who received the two vaccinations in the allowed intervals, attended all the scheduled blood sampling visits in the allowed timeframe, and for whom no major protocol violation was reported. The intention-to-treat (ITT) population included subjects who received at least one vaccination, and for whom at least one blood sample was taken.Anti-AMA49-C1 and anti-UK-39 IgG endpoint titers were measured by ELISA independently both at Swiss TPH and Pevion Biotech AG. Endpoint titers obtained at Swiss TPH were, on average, slightly higher compared to data from Pevion , which is an expected variation for repeated ELISA measurements. All statistical analyses were run on the data from each lab separately, as well as on the averaged data set (geometric mean). The identity of the lab, as well as averaging the data, had no influence on the results of the statistical analyses, and the results presented here are for the averaged data set. The exact Wilcoxon test was used to test for differences in endpoint titers between adults and children (at baseline) and between treatment groups (separately for adults and children for each sampling time point). Indices of antibody responses were expressed as the ratio of endpoint titers after immunization with reference to the baseline value (day 0). Wilcoxon's exact test was used to test for differences in indices of antibody responses between treatment groups .In addition to the above analyses, which were specified in a statistical analysis plan prior to final vaccine accountability and unblinding, the following post-hoc exploratory analyses were performed: 1) The area under the log (antibody titer)-curve above the baseline antibody titer (ΔAUC) was calculated as an integrated measure of antibody response for three intervals: days 0–30, 0–120, and 0–365. ΔAUC values for anti-AMA49-C1 and anti-UK-39 were compared between those subjects who were never tested positive for P. falciparum during the respective interval and those subjects who were tested positive at least once, and Wilcoxon's exact test was used to test for differences. 2) ELISA responders were defined as subjects who seroconverted (from an endpoint titer <50 to ≥50) or showed an at least 4-fold increase in endpoint titer versus baseline. Fisher's exact test was used to test for differences in the proportions of responders between treatment groups . 3) Spearman's rank correlations between anti-AMA49-C1 and anti-UK-39 endpoint titers were computed for each time point in each vaccine group.Time to first or only clinical malaria episodes  in children were compared using Kaplan-Meyer failure curves from 30 days post first vaccination until the end of the study (day 365). Difference between failure functions was assessed using the log-rank test, and the hazard ratio was assessed using Cox regression. This analysis had been specified in a statistical analysis plan prior to unblinding. Following the new recommendation of the WHO Malaria Vaccine Advisory Committee The participant flow is summarized in All subjects were included in the safety analysis. Solicited  and unsolicited  AEs occurring within 30 days after first and second vaccination are summarized in The only local solicited AEs (monitored from days 0–3 and 90–93) were pain at the injection site, of which all were Grade 1, required no action, and resolved without sequelae. With data combined across both vaccinations the proportion of children reporting pain at the injection site was lower for PEV3B compared to Inflexal®V  reported in the study were elevated temperature (>37.5°C) and headache; all of these occurred in children . ElevateAll unsolicited AEs (monitored from days 0–365) in this study were considered unrelated to the vaccine and resolved without sequelae.No statistically significant differences were observed between the children vaccine groups for the proportions of subjects with any AE within 30 days after vaccination, with any Grade 3 AE at any time during the study, or with any related AE at any time during the study  had serious AEs (SAEs), which were primarily uncomplicated or complicated malaria . Each of the SAEs required hospitalization and medication, all were considered unlikely related or unrelated to study vaccine, and resolved without sequelae.No differences in hematology or biochemistry laboratory values between the vaccine groups (adults and children) were observed over the course of the study (data not shown).The intention-to-treat population comprised 1 subject more than the per protocol population (1 PEV3B child was excluded due to a tetanus toxoid vaccine administered two weeks after second vaccination). Since the exclusion of this subject had no effect on the results of any statistical test, results presented in the following sections are for the intention-to-treat analysis.Within the adult and children subgroups there were no a priori differences in pre-vaccination anti-AMA49 and anti-UK-39 IgG endpoint titers between the vaccine groups . The preThe antibody endpoint titers for AMA49-C1 and UK-39 in the PEV3B groups increased in response to the vaccinations as measured 30 days after the first (day 30) and second (day 120) immunization, whereas in the Inflexal®V groups the endpoint titers for both antigens remained at background levels . HoweverIn PEV3B vaccinees, the average index of response for AMA49-C1 (ratio of endpoint to baseline titers) ranged from 4.6 (day 90) to 15.3 (day 120) in adults and from 3.7 (day 365) to 27.5 (day 120) in children. For UK-39, the index of response ranged from 4.0 (day 365) to 8.7 (day 30) among adults and from 5.4 (day 90) to 66.3 (day 120) among children . In bothAnti-AMA49-C1 and anti-UK-39 IgG endpoint titers were significantly correlated with each other in PEV3B children at all sampling points including baseline . For the 8 PEV3B adults, the correlation was not significant at any of the sampling points . In PEV3B vaccinated children, Spearman's rho was maximal at day 30 and day 120, indicating that the kinetics of antibody responses to AMA49-C1 and UK-39 in response to vaccination correlated with each other (data not shown).first ep  = 0.00514 for Inflexal®V and 0.00306 for PEV3B, p = 0.20; hazard ratio HRfirst ep and 95%-CI  = 0.58 [0.24–1.36])    of malaria from the first peak of endpoint titers (day 30) until the end of the follow-up period (day 365) tended to be lower in the PEV3B group than in the Inflexal®V group (67% versus 80% respectively). However, the estimated failure functions were not significantly different  . For the9–1.09]) . For the0–1.15]) . The faiThe number of clinical malaria episodes per child subject from day 30 after first vaccination until the end of the follow-up period (day 365) was significantly lower in PEV3B vaccinees compared to the control group . This corresponds to a reduction of 50% .For two adult subjects a clinical episode of malaria was reported – both in the PEV3B group. Thus the proportion of adults affected by malaria was lower than in children. This observation is in line with the increased disease resistance of the adult population to malaria, which therefore is not the target group for a malaria vaccine.Among PEV3B children, those diagnosed with P. falciparum parasitaemia  during the study periods from day 0–30, day 0–120 and day 0–365 showed a lower integrated antibody response for UK-39 compared to those who remained undiagnosed for P. falciparum parasitaemia . This diPhase 1a and 2a clinical trials conducted with virosomal formulations of UK-39 and AMA49-C1 in malaria non-immune Caucasian volunteers The two commercialized virosome-based vaccines  have already demonstrated the excellent safety profile of the virosomal antigen delivery system. In the present trial, local reactogenicity was minimal with only mild or moderate pain and no grade 3 AE related to the vaccine reported. All SAEs were considered unlikely or unrelated to the study vaccine and all resolved without sequelae. This excellent reactogenicity profile contrasts with results obtained with recombinant malaria proteins formulated in ASO2A, ASO1E or Montanide ISA 720 & 51, or DNA vaccines in viral vectors, which show much higher rates of grade 2 and 3 AEs PEV3B elicited IgG responses to both target antigens in semi-immune adults and children, with the highest titers generally observed 30 days after the second immunization. 275 days after the second immunization (day 365) endpoint titers against both target antigens were still higher than baseline titers. In children, the index of response to UK-39 tended to be stronger compared to that to AMA49-C1, which was primarily due to the lower baseline endpoint titers for UK-39. In adults, responses to both antigens were comparable.In this Phase 1b trial, malaria morbidity was not the primary outcome, thus the design was not powered to assess efficacy. However, the analysis of time to event for first or only episode of clinical malaria in children suggests that the PEV3B had a protective effect. There was no statistically significant difference in incidence rate of first or only clinical malaria episode for the period from 30 days after the first vaccination until the end of the study on day 365. For two separate intervals, from day 30 after first vaccination until second vaccination, and from day 30 after second vaccination until the end of the study, however, the reduction of incidence rate of first or only clinical malaria episode by 70% and 50% respectively was approaching statistical significance. These results, which look inconsistent at first glance, are explained by the significantly higher rate of malaria episodes in control subjects compared to PEV3B vaccinees. Multiple events in the same subject are not captured in a conventional time-to-first-event Kaplan-Meier graph. Data on the total number of episodes are a better measure for the disease burden from a public health perspective compared to first episode data In the children PEV3B group, subjects with at least one episode of P. falciparum parasitaemia in the study periods day 0–30 and day 0–120 showed a significantly lower average response in antibody titers against UK-39 for the corresponding period. For anti-AMA49-C1 the differences in response of antibody titers related to incidence of parasitaemia were not statistically significant. This may indicate that the induced antibody response to UK-39 is a key component of the possible protective effect of PEV3B, and raises the question whether there is a link to the pattern of differences in background antibody levels between the highly immune adult population and children. A close correlation between anti-UK-39 IgG titers assessed in ELISA, immunofluorescence and Western blotting analysis using sporozoites and sporozoite lysates, respectively, has already been demonstrated The most advanced subunit malaria vaccine to date, RTS,S, is currently tested in a phase III clinical trial (NCT00866619) and is based on a fusion protein of part of the CSP of P. falciparum clone 3D7 (amino acids 207 – 395) with the hepatitis B surface antigen Studies with RTS,S in an endemic area of Mozambique showed that vaccination of children aged 1–4 years induced partial protection against infection and clinical malaria including severe disease Since IRIVs represent a modular antigen delivery system, they are an ideal platform to evaluate individual antigens separately, which can subsequently be combined with other components to a multi-component subunit vaccine Checklist S1CONSORT checklist.(PDF)Click here for additional data file.Protocol S1Trial protocol.(PDF)Click here for additional data file.


Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe  Why do some malaria-infected children develop severe and lethal forms of the disease, while others only have mild forms? In order to try to find potential answers or clues to this question, we have here analyzed more than 1,000 different human proteins in the blood of more than 500 malaria-infected children from Ibadan in Nigeria, a holoendemic malaria region. We identified several proteins that were present at higher levels in the blood from the children that developed severe malaria in comparison to those that did not. Some of the most interesting identified proteins were muscle specific proteins, which indicate that damaged muscles could be a discriminatory pathologic event in cerebral malaria compared to other malaria cases. These findings will hopefully lead to an increased understanding of the disease and may contribute to the development of clinical algorithms that could predict which children are more at risks to severe malaria. This in turn will be of high value in the management of these children in already overloaded tertiary-care health facilities in urban large densely-populated sub-Saharan cities with holoendemic malaria such as in the case of Ibadan and Lagos. Plasmodium falciparum attributable to a third of the childhood deaths accounted in Nigeria Human malaria is a life-threatening disease causing an estimated 655,000 deaths in 2010 Parasitized red blood cells (pRBC) are specifically withdrawn from the peripheral circulation during severe malaria infection through binding to and activation of vascular endothelial cells, erythrocytes, leukocytes and platelets, which may obstruct the blood flow. It is known that increased micro-vascular congestion accompanies coma in cerebral malaria and the depth of coma is correlated to the extent of the sequestration of the pRBC Despite the current plethora of new technologies available for analyzing and profiling proteins in body fluids, the yield of validated biomarker molecules remains low Here, we have employed an affinity proteomics approach to study 1,015 human proteins with 1,132 antibodies in the peripheral blood of children from Nigeria with different syndromes from severe to uncomplicated malaria as well as non-diseased parasite-negative children from the same community. Protein markers of inflammation, endothelial activation, platelet adhesion, vaso-modulation, glucose metabolism, oxidative stress and muscle-damage were found in relation to severe malaria. In particular, three muscle derived proteins, creatine kinase, carbonic anhydrase III and myoglobin, were detected in the plasma of children with cerebral malaria suggesting deep lesions into their micro-vasculature including the vascular smooth-muscle cell-layer and extra-vascular striated muscles cells, concurrent with excessive sequestration of pRBC throughout the human body. As a consequence we have identified protein signatures that allowed the distinction between the different presentations of the disease with AUCs up to 0.90 in plasma samples from a verification cohort. The data contributes to a deeper understanding of the complex mechanisms that lead to severe disease and may serve as a basis for the development of novel diagnostic strategies that would enable the prediction of the severity of malaria.In order to explore the potential of the human proteins in plasma to predict disease status during malaria infections, we have conducted an extensive exploratory profiling approach using antibody suspension bead arrays . Using tFirst, a set of patients was selected and carefully matched and balanced to contain 356 childhood malaria patients and controls recruited during 2006 to 2011 in Ibadan, Nigeria. This set, denoted as “discovery cohort” in this study, included samples from patients suffering from uncomplicated malaria , severe malaria anemia  and cerebral malaria  and from parasite-negative community controls . In total, a list of 372 antibodies targeting 304 different human proteins was compiled and denoted as ‘targeted array’. This list comprised primarily plasma proteins associated with acute inflammation  where more than 40,000 antigen purified and protein microarray validated antibodies have been generated. The antibodies fulfill the quality criteria of having a concentration that is higher than 50 µg/ml and that the specificity is validated on planar protein arrays. These criteria were also true for the antibodies on the targeted array. These two ‘random arrays’, covering 760 antibodies were corresponding to 711 unique proteins, generating the total sum of 1,132 antibodies representing 1,015 unique proteins.The technical reproducibility of the assays was assessed in independent experiments  between any of the groups, i.e. UM, SMA and CM as well as healthy parasite-negative CC. From the ‘targeted array’, 29 human proteins were identified in plasma as discriminatory protein targets .By using self-organizing tree algorithm (SOTA) cluster analysis on candidate proteins from the ‘targeted array’, four clusters grouped proteins according to disease severity . A heatmA total of 17 proteins were found to discriminate malaria disease from controls. The set of proteins that was denoted ‘Malaria decreased’  containeThe 14 proteins denoted ‘Malaria increased’ were primarily inflammatory acute phase proteins and increased with severity of the disease. Notably, the most significant candidates differing between controls and malaria groups were major inflammatory components von Willebrand factor (VWF) and C-reactive protein (CRP). Additionally, lipopolysaccharide binding protein (LBP), serpin peptidase inhibitor, member 3 (SERPINA3) and orosomucoid (ORM) are known members of the malaria-induced acute inflammatory response , which has been reported to be strictly tissue specific and present at high levels in skeletal muscle and creatine kinase (CK), which is involved in energy homeostasis and specific to brain, muscle and heart tissues. These two candidates are associated to muscle tissue and indicate a linkage between muscle damage and CM.Overall for the ‘discovery cohort’, pathway analysis using the Ingenuity Pathway analysis software (Ingenuity Systems) outlined acute phase signaling as the most significant pathway affected in malaria disease  with an area under the ROC curve (AUC) of 0.84 alone . The besNext, Carbonic anhydrase III (CA3) alone discriminated CM from UM with a high AUC of 0.90 , UM versus CM (23 proteins) and SMA versus CM (8 proteins) comparisons were 0.72, 0.90 and, 0.86, respectively .To further verify the results technically, additional antibodies were used targeting other regions of the same protein. For example, von Willebrand factor (VWF) was repeated using the same antibody (HPA00282) and an additional antibody (HPA001815), with both showing comparable results in the different disease groups in the verification cohort and the discovery cohort  samples  from patCA3 was identified as the top candidate to differentiate CM from the other two malaria syndromes. In both the discovery and the verification analysis, two antibodies with distinct specificities to CA3 showed concordant performance  at the University College Hospital (UCH) in the city of Ibadan, Nigeria. Malaria-negative community control (CC) children were recruited from local vaccination clinics as well as during school visits across several Ibadan districts. This case-control study was divided in a discovery cohort that contains those patients recruited during 2006 to 2011 and a verification cohort made up of those recruited in the 2009 to 2012 period.P. falciparum malaria P. falciparum parasitaemia and PCV (Packed Cell Volume) greater than 20% who did not require hospital admission. Severe malarial anemia (SMA) cases were defined as conscious children with PCVs less than 16% in the presence of P. falciparum parasitaemia. Cerebral malaria (CM) cases were defined as children in unrousable coma for at least one hour in the presence of asexual P. falciparum parasitaemia with normal cerebrospinal fluid and PCV greater than 20%. Community controls were children that did not show any obvious symptoms of illness and seemed healthy. They were screened for parasite presence and were only included in the study if the Giemsa staining of both thick and thin blood films were negative for Plasmodium parasites. They were selected to match age and sex with malaria-infected patients. The clinical data was compiled for each patient and samples were collected as previously described Children were aged from 6 months to 13 years and were screened for parasite detection by microscopy following Giemsa staining of thick and thin blood films as performed routinely at UCH. Clinical definitions used were as defined by the WHO criteria for severe www.proteinatlas.org) consisting of more than 40,000 antigen purified and protein microarray validated antibodies.Antibodies were selected and acquired from the huge antibody collection within the Human Protein Atlas  and incubated with 1.6 µg antibody in a multi-well microtiter plate for 2 h. After the coupling reaction, beads were stored at 4°C in a protein containing buffer  supplemented with ProClin (Sigma-Aldrich). Before incubation with samples, the different bead identities were combined to create a 384-plex-bead array. Antibody coupling was confirmed with R-phycoerythrin (PE)-conjugated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch).For the generation of antibodies suspension beads, antibodies were diluted using a liquid handling system  and coupled to carboxylated magnetic microspheres  as previously described The data from single antibody and direct sample labeling assays was judged by technical replication of the experiment, profile concordance of several antibodies raised towards a common target protein, and biological replication of the analysis in new, independent samples.Biotinylation of plasma samples was performed as previously described Data analysis was performed using R language for statistical computing bi: random effect of the ith target.The data from the verification cohort was normalized using a linear mixed model1 penalization proposed by Tibshirani For the identification of protein signatures a logistic regression model was used. We used LText S1Supplementary Information. An extensive set of supplementary material is available with detailed descriptions and visualizations of the data and the identified proteins of interest with ten figures, four tables and methods.(PDF)Click here for additional data file.


Verbal autopsy (VA) procedures can be used to estimate cause of death in settings with inadequate vital registries. However, the sensitivity of VA for determining malaria-specific mortality may be low, and may vary with transmission intensity. We assessed the diagnostic accuracy of VA procedures as compared to hospital medical records for determining cause of death in children under five in three different malaria transmission settings in Uganda, including Tororo (high), Kampala (medium), and Kisoro (low).Caretakers of children who died in participating hospitals were interviewed using a standardized World Health Organization questionnaire. Medical records from the child's hospitalization were also reviewed. Causes of death based on the VA questionnaires and the medical records were assigned independently by physician reviewers and then compared. A total of 719 cases were included in the final analysis, 67 in Tororo, 600 in Kampala, and 52 in Kisoro. Malaria was classified as the underlying or contributory cause of death by review of medical records in 33 deaths in Tororo, 60 in Kampala, and 0 in Kisoro. The sensitivity of VA procedures for determining malaria deaths in Tororo was 61% (95% CI 44–78%) and 50% in Kampala (95% CI 37–63%). Specificity for determining malaria deaths in Tororo and Kampala was high (>88%), but positive predictive value varied widely, from 83% in Tororo to 34% in Kampala . The difference between the cause-specific mortality fraction for malaria as determined by VA procedures and medical records was −11% in Tororo, +5% in Kampala, and +14% in Kisoro.Our results suggest that these VA methods have an acceptable level of diagnostic accuracy for determining malaria deaths at the population level in high and medium transmission areas, but not in low transmission areas. The primary goal of the PMI is to reduce malaria-related deaths by 50% in fifteen target countries in Africa, including Uganda. However, providing accurate estimates of malaria-specific mortality in resource-poor settings remains a challenge Despite recent evidence that the burden of malaria is decreasing in many endemic areas Verbal autopsy (VA) is an indirect method of determining cause of death based on an interview with the caretakers of a deceased individual, which has been widely used to collect information on cause-specific mortality where medical information on deaths is incomplete Studies evaluating the validity of VA procedures in African children indicate that sensitivity of verbal autopsy for determining malaria-specific mortality may be lower than for other diseases, and may vary with transmission intensity We conducted a prospective study to evaluate the validity of VA procedures, in three different epidemiological settings in Uganda, using a questionnaire developed by the World Health Organization. The primary objective was to calculate the sensitivity, specificity, predictive values, and accuracy of cause-specific mortality fractions (CSMF) of VA procedures for attributing deaths to malaria as compared to review of medical records as the “gold standard” and to compare results between the different epidemiological settings.The study was approved by the Ugandan National Council for Science and Technology, the Centers for Disease Control and Prevention, and the ethics committees of Makerere University Faculty of Medicine, and the London School of Hygiene and Tropical Medicine. Permissions were obtained from district officials and medical superintendents at each hospital.The trial was conducted between June 2008 and September 2009 in five hospitals in three districts with different epidemiology including Tororo , Kampala , and Kisoro . Tororo and Kisoro districts are served by two hospitals; both were included from each district. MulagoHospital was chosen as the single hospital from Kampala because it is the main public hospital and serves all socio-economic groups. The estimated entomological inoculation rate is 562 in Tororo Prior to the study, staff in participating hospitals were trained, in accordance with standard hospital procedures, to help improve the quality of medical record keeping. Training emphasized the importance of documenting the full physical address and relevant history and physical examination. The importance of obtaining a blood smear for malaria parasites in all children who present with a history of fever or a documented fever was also emphasized.Study personnel monitored hospital admissions and deaths of children in participating hospitals. If a death was identified, the medical record of the deceased child was reviewed for the initial screening criteria including: 1) age <5 years, 2) residence within 60 km of the participating hospital, and 3) adequacy of the medical record . If the initial criteria were met, the household was scheduled for a visit approximately one month after the child's death. Study personnel located households based on the physical address with the help of local leaders. If a household could not be located within three months, the case was excluded. When a household was located, study personnel were introduced by the local leaders and condolence parcels containing sugar, salt, and soap were presented in accordance with local custom. Written informed consent was sought from the parent or guardian of the deceased child. Caretakers of deceased children who fulfilled the entry criteria were enrolled, and asked to identify an appropriate respondent(s) for the interview, defined as a person who was able and willing to provide an accurate account of the circumstances leading to the child's death. Verbal consent from the respondents was obtained prior to the interviews using an information sheet. Medical records from the child's hospitalization were acquired after written consent was obtained.Interviews were conducted by study personnel in the local languages using a standardized verbal autopsy (VA) questionnaire developed by the World Health Organization Two causes of death for each deceased child were derived; one from the hospital medical records and a second from the completed VA questionnaire. Different sets of physicians independently reviewed the medical records and VA questionnaires to determine cause(s) of death. Physicians were not blinded to the study sites. Cause(s) of death were determined based on the physician's clinical judgment and were classified as immediate, underlying, or contributory causes and coded according to the International Standard of Classification of Diseases (ICD-10) guidelines The primary objective of the study was to calculate the sensitivity, specificity, predictive values, and accuracy of CSMFs of VA procedures for attributing deaths to malaria  as compared to review of medical records as the “gold standard” in low/medium/high malaria transmission areas, and to compare the PPV of malaria deaths identified through VA between the different sites with variable malaria transmission. Comparisons were also made using two restricted definitions of malaria deaths: (1) considering all cases in which malaria was classified as the underlying cause of death only, and (2) considering only cases in which a blood smear result was documented, in which malaria deaths (classified as the underlying cause of death) were confirmed by a positive blood smear. For evaluation of all-cause mortality, and misclassification of malaria deaths, the restricted definition of malaria death (classified as the underlying cause of death without blood smear confirmation) was used.We tested the alternative hypothesis that a significant difference in PPV could be detected in sites with variable malaria transmission, estimating that the PPV would be 90% in high transmission, 70% in medium transmission and 50% in low transmission areas In Tororo, 160 deaths in children under five were recorded between September 2008 and June 2009; of these, 93 (58%) cases were excluded . All 67 The sensitivity of VA procedures compared to medical records for determining malaria deaths, when malaria was classified as the underlying or contributory cause of death, ranged from 50% in Kampala to 61% in Tororo . In KisoConsidering deaths in which malaria was classified as the underlying or contributory cause of death, the CSMF for malaria determined by VA procedures in Tororo was lower than that determined by medical records , indicatTo evaluate the accuracy of VA procedures for classifying non-malarial deaths, we determined the sensitivity and specificity of VA for other leading causes of death at each site. In Tororo, the leading causes of death were malaria and malnutrition; in Kampala, the majority of deaths were classified as “other” which included neonatal sepsis (26%), congenital malformations (14%), other neonatal causes of death (12%), tuberculosis (9%), septicemia (7%), anemia (7%), burns (6%) and others (19%); pneumonia was also a common cause of death . In KisoIn Tororo, five deaths due to other illnesses were inaccurately attributed to malaria , and 12 deaths due to malaria were inaccurately attributed to other illnesses  were misclassified as meningitis. In Kisoro, one-half of the false positive cases  were in fact due to pneumonia. However, no obvious patterns of misclassification were found in our study. It is possible to adjust for misclassification if the sensitivity and specificity of VA procedures is known The most significant limitation of this study was the quality of the “gold standard”, including the in-patient care provided and documentation in the medical records. Validation studies typically compare the sensitivity and specificity of VA diagnoses against causes of death established by medical records as the gold standard In addition, barriers to obtaining laboratory tests existed at most hospitals, limiting use of microscopy, as evidenced by the low numbers of cases with a blood smear result recorded. Despite these limitations, when the accuracy of VA procedures was compared to a more refined “gold standard”, in which malaria deaths (as the underlying cause of death) were confirmed by a positive blood smear, the results were similar, supporting the validity of our data.Our study had several other significant limitations. The proportion of cases excluded during screening was high, which may have resulted in selection bias. In Tororo, most cases were excluded due to inadequate medical records, while in Kampala, inadequate documentation of physical addresses and difficulty tracing households contributed to the exclusions. In addition, we were unable to reach our sample size targets in Tororo and Kisoro due to low numbers of deaths, which limited our ability to assess for variations in the PPV of VA procedures for determining malaria deaths between the sites, particularly in Kisoro where no malaria deaths were captured, limiting attempts at determining the accuracy of VA procedures at this site. However, despite not reaching the sample size target in Tororo, the observed difference in PPV of VA procedures for determining malaria deaths between Tororo and Kampala was significant. In this study, we relied on physician review to classify cause of death. Use of standardized algorithms to derive cause of death from VA questionnaires has been advocated as a way to systematically assign diagnoses, and to save time In summary, our results suggest that these VA procedures have a high diagnostic accuracy for determining malaria deaths at the population level in high and medium transmission areas, but not in low transmission areas. For other causes of death, VA provided moderate sensitivity and high specificity regardless of transmission level. As malaria transmission falls to very low levels, VA is likely to overestimate malaria-specific mortality and underestimate the impact of control interventions. Indeed, there is likely to be a “tipping point” in malaria transmission where VA procedures cease to be useful, but defining this threshold may not be possible due to methodological constraints. VA procedures appear to have a role in estimating malaria-specific mortality, but only in high and medium transmission areas; in low transmission areas, the utility of VA appears to be limited.