Plasmodium vivax malaria.Analyses of a case-control study among Afghan refugees in Pakistan find that a G6PD (glucose-6-phosphate dehydrogenase) “Mediterranean” type deficiency confers substantial protection against  Plasmodium vivax, is more difficult to control than P. falciparum because of the latent liver hypnozoite stage, which causes multiple relapses and provides an infectious reservoir. The African (A−) G6PD (glucose-6-phosphate dehydrogenase) deficiency confers partial protection against severe P. falciparum. Recent evidence suggests that the deficiency also confers protection against P. vivax, which could explain its wide geographical distribution in human populations. The deficiency has a potentially serious interaction with antirelapse therapies (8-aminoquinolines such as primaquine). If the level of protection was sufficient, antirelapse therapy could become more widely available. We therefore tested the hypothesis that G6PD deficiency is protective against vivax malaria infection.The most common form of malaria outside Africa, p = 0.001). Genetic analysis demonstrated that the G6PD deficiency allele identified (Mediterranean type) was associated with protection in hemizygous deficient males . The deficiency was also protective in females carrying the deficiency gene as heterozygotes or homozygotes .A case-control study design was used amongst Afghan refugees in Pakistan. The frequency of phenotypic and genotypic G6PD deficiency in individuals with vivax malaria was compared against controls who had not had malaria in the previous two years. Phenotypic G6PD deficiency was less common amongst cases than controls  0.06–0.52], G6PD deficiency (Mediterranean type) conferred significant protection against vivax malaria infection in this population whether measured by phenotype or genotype, indicating a possible evolutionary role for vivax malaria in the selective retention of the G6PD deficiency trait in human populations. Further work is required on the genotypic protection associated with other types of G6PD deficiency and on developing simple point-of-care technologies to detect it before administering antirelapse therapy.Please see later in the article for the Editors' Summary Plasmodium falciparum is responsible for most malaria deaths, P. vivax is the commonest, most widespread cause of malaria outside sub-Saharan Africa. Like other malaria parasites, P. vivax has a complex life cycle. Infected mosquitoes inject a parasitic form known as sporozoites into people where they replicate inside liver cells without causing symptoms. About 8–9 days later, merozoites (another parasitic form) are released from the liver cells and invade red blood cells. Here, they replicate rapidly before bursting out and infecting more red blood cells. This increase in the parasitic burden causes malaria's characteristic symptoms (debilitating and recurring chills and fevers). P. vivax infections are usually treated with chloroquine  but patients must also take primaquine, a drug that kills hypnozoites, a form of P. vivax that hibernates in the liver. Hypnozoites can cause a relapse months after the initial bout of malaria and make P. vivax malaria harder to control than P. faciparum malaria.Malaria is a parasitic infection transmitted to people through the bite of an infected mosquito. Although P. falciparum malaria—P. falciparum does not thrive in G6PD-deficient red blood cells. In areas where P. vivax malaria is common, Mediterranean and Asian variants of G6PD deficiency are more widespread than A− G6PD, so the question is, do these variants protect against P. vivax malaria? In this case-control study (a study in which the characteristics of people with and without a specific condition are compared), the researchers investigate whether G6PD deficiency protects against P. vivax infection in a population of Afghan refugees living in Pakistan.Some mutations (DNA changes) protect their human carriers against specific disease-causing organisms. These mutations occur at high frequencies in populations where these organisms are common. For example, the widespread distribution of mutations that cause a deficiency in an enzyme called glucose-6-phosphate dehydrogenase (G6PD) mirrors the distribution of malaria and the African (A−) form of G6PD deficiency, a type of G6PD deficiency that is common in people of African origin, is known to provide partial protection against severe P. vivax malaria during the previous two years and 743 refugees who had not had malaria over the same period. They measured G6PD activity in the participants' blood to detect “phenotypic” G6PD deficiency (reduced enzyme activity) and looked for the Mediterranean variant of the G6PD gene in the participants (“genotypic” G6PD deficiency). 5.7% of the controls but only 1.1% of the cases had phenotypic G6PD deficiency. Statistical analyses indicated that participants with reduced G6PD levels were about one-fifth as likely to develop P. vivax malaria as those with normal G6PD levels after allowing for other factors that might affect their susceptibility to malaria, an adjusted odds ratio (AOR) of 0.18. The genetic analysis indicated that the Mediterranean G6PD gene variant provided protection against P. vivax infection in men (AOR 0.12) and in women carrying either one or two defective copies of the G6PD gene (AOR 0.37); because the G6PD gene is on the X chromosome, men have only one copy of the gene but women have two copies.The researchers enrolled 372 Afghan refugees who had had P. vivax malaria infection in this population of Afghan refugees. Although further studies are needed to determine whether other G6PD variants protect against P. vivax malaria, these findings suggest that P. vivax malaria might be responsible for the retention of the G6PD deficiency trait in some human populations. In addition, these findings may have implications for the treatment of P. vivax malaria. Currently, in most places where P. vivax malaria is common, primaquine is not given routinely because primaquine can trigger red blood cell death (hemolytic anemia) in G6PD-deficient people and tests for G6PD deficiency are rarely available. These findings suggest that the risk of exposure to primaquine among people infected with P. vivax might be lower than previously assumed, because G6PD deficiency is less common among P. vivax-infected patients than among the general population. Nevertheless, these findings are unlikely to increase the use of primaquine immediately. Such an increase, the researchers suggest, will only occur if a simple test for G6PD deficiency is developed.These findings indicate that Mediterranean-type G6PD deficiency protects against http://dx.doi.org/10.1371/journal.pmed.1000283.Please access these Web sites via the online version of this summary at World Health Organization on malaria Information is available from the malaria (in English and Spanish)The US Centers for Disease Control and Prevention provides information on Wellcome Trust on all aspects of malaria, including a news item about G6PD deficiency protecting against severe P. falciparum malariaInformation is available from the malaria (in English and Spanish)MedlinePlus provides links to additional information on Plasmodium vivax malariaThe Malaria Vaccine Initiative has a fact sheet on Vivaxmalaria provides information about P. vivaxG6PD deficiency can be found on KidsHealth from the Nemours Children's Health SystemMore about  Plasmodium vivax, which causes up to 390 million clinical cases a year amongst a population at risk of approximately 2.6 billion P. falciparum and to its formation of a latent liver stage (the hypnozoite), which initiates secondary blood-stage infections The majority of malaria outside sub-Saharan Africa is caused by Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common genetic enzymopathy in humans with more than 200 variants identified to date P. falciparum. This hypothesis was confirmed in endemic areas of Africa where the African (A−) variant of G6PD deficiency protected against severe disease P. vivax infection or with P. falciparum. Genetic evidence for an evolutionary interaction was also demonstrated. Vivax malaria, often incorrectly assumed to be benign The concordance of the distribution of G6PD deficiency with maps of historical malaria led to the hypothesis that the trait is protective against P. falciparum. Ethnically, the population is Pashtun Using a case-control design, we examined the association of G6PD deficiency with vivax malaria, retrospectively, over 2 years of observation. The study was conducted in three Afghan refugee villages in North West Frontier Province, Pakistan . The refugee villages had populations ranging from 1,000 to 3,500 people. Malaria transmission was seasonal and hypoendemic n = 30), which confirmed that all tested cases were indeed infected with P. vivax.In November and December 2006, cases were selected from residents of the refugee villages who in earlier studies were diagnosed with vivax malaria Village health records were used to select community controls from the same population as cases. Controls had no known episodes of malaria during the same 2 years of observation. The record system contained information on every family, and each family had a health card on which every member was recorded by name. The registration was regularly updated for births and deaths during consultation in the village health clinics. If a malaria blood smear was taken, the slide result was recorded against the patient's record (even if it was negative). If the patient was parasitaemic, the treatment given was also recorded. The records are maintained by trained village health workers and clinic staff. This allowed the compilation of a list of all individuals in the villages who had not had malaria over the 2 years before the study. All residents from the same villages as cases who were more than 2 years old and had no history of malaria in their clinic record were therefore eligible for enrolment as controls.Controls were selected at random from the complete list of eligible village residents. A bottle was spun outside the house of each case in order to select a random house and if it contained an eligible control (of the same sex and within 5 years of age of the case), then the control was enrolled. If no suitable control was available, the bottle was re-spun until a suitable control was found from a house in the vicinity of the case. The selection was therefore controlled for the likely confounding factors of age and village (associated with risk of infection) and sex (associated with risk of G6PD deficiency) to ensure that disease and exposure risk was broadly similar between cases and controls. Once informed consent was given, the control was assessed by microscopy for current malaria parasitaemia, and if positive the control was not included. Because no controls had parasitaemia at the time of the survey, none were excluded on the basis of this criterion. Information on treatment-seeking practice was collected to identify those individuals who had sought medical care outside the village in the last 2 years; a restricted analysis, in which such individuals were excluded, was conducted as a check against potential bias resulting from malaria that was not recorded in clinic records. Children under the age of 2 years were excluded since they would not have been born at the start of the observation period.Blood samples were collected in EDTA tubes and immediately tested for phenotypic G6PD deficiency using a colorimetric test  according to the manufacturer's instructions. Samples found to be deficient were tested a second time. Each participant gave a blood sample stored on filter paper for genetic analysis. The filter papers were allowed to dry, marked with the participant's identifier and stored in individual bags. The samples selected for analysis were sent to the Department of Genome Sciences at the University of Washington, Seattle, USA.Ethical approval was obtained from the London School of Hygiene and Tropical Medicine Ethics Review Board and from the Pakistan Medical Research Council Committee on Bioethics. All participants gave written informed consent to participate. For the gene sequencing, ethical approval was given by the University of Washington Ethics Committee.The primary analysis examined the association between phenotypic G6PD deficiency and vivax malaria. A sample size was calculated for a 2∶1 ratio of controls to cases. A sample of 500 cases and 1,000 controls gave 80% power to detect a frequency of phenotypic G6PD deficiency of 1% amongst cases and 3.5% amongst controls We conducted univariate and multivariate analysis using unconditional logistic regression and adjusting for prespecified potential confounding factors between cases and controls; those factors included in the final unconditional multivariate logistic regression model for the primary outcome were age group, sex, tribe, and refugee village. In response to suggestions from the statistical referee we undertook an additional post-hoc analysis using conditional logistic regression, which was not part of the prespecified analytical plan. In this analysis each case was matched with the two controls identified in their village by bottle-spinning and was thus matched for village, sex, and age group. We included tribe as a potential confounding factor in the model.G6PD deficiency is an X-linked mutation so the secondary analysis examined the association between the deficiency (both phenotypic and genotypic) and infection separately for males and females. This analysis took the same approach as before and applied unconditional logistic regression and a post-hoc conditional logistic regression. The genotyped samples were used to assess the level of protection conferred by confirmed hemizygous deficiency in males and heterozygous and homozygous deficiency in females. The study was not powered specifically for the secondary analyses. Data was analysed using STATA v8 .5′ GTT TAT GTC TTC TGG GTC AGG GAT GG 3′ and 18396R: 5′ AGT GTT GCT GGA AGT CAT CTT GGG T 3′ from Saunders et al, Participant blood samples were examined for G6PD genotype in two phases. Initially, we selected half of the samples from males who were phenotypically deficient and the requisite number of male non-deficient samples (in an approximate 1∶2 ratio). We then amplified a 5.2 kb fragment using the primers 13125F: 2, pH = 9.0), 0.8 µM primer, and 1 µl Big Dye . Products of sequencing reactions were separated on an ABI 3130xl  and analyzed using Sequencher . The following primers were used for the Med allele: Medf: 5′ ATGATGCAGCTGTGATCCTCACTC 3′ and Medr: 5′ ATGAGGTTCTGCACCATCTCCTTG 3′.In the second phase we conducted PCR amplification of Med allele fragments on the 40% sample of the participants whose samples were available, in equal proportion between cases and controls. These comprised the same 1∶2 ratio of cases to controls and allowed examination of the frequency of the Med− allele between cases and controls. Med allele fragments were amplified in a 20 µl reaction volume containing: 1× PCR PreMix A, 0.2 µM each primer, 0.5 units FailSafe enzyme mix , and 2 µL of template DNA. Cycling conditions were: 94°C for 3 min; 36 cycles of 94°C for 30 s, 55°C for 30 s, 62°C for 60 s; 62°C for 5 min, 15°C for 5 min. Sequencing was done in a 10 µl volume, using 1–3 µl of PCR product, 1.7 µl 5× sequencing buffer for Big Dye  had either repatriated to Afghanistan or had been enrolled more than once in the previous studies (but were included only once in the present survey). All participants were ethnic Pashtuns.A total of 372 cases and 743 controls were enrolled into the study at a ratio of roughly 1∶2. 2 = 13.1, p<0.001)  was similar to the unmatched analysis (There was a lower frequency of phenotypic G6PD deficiency amongst cases (4/372 [1.1%]) than controls (42/743 [5.7%]), Chip<0.001) . Uncondip<0.001) . The pril groups . For theanalysis  althoughp = 0.004). Amongst females we did not detect evidence of an effect , the number of deficient females being too low to make a robust estimate (n = 2/215) .Because the G6PD gene is located on the X chromosome, sex-stratified analysis was also conducted. The frequency of deficiency in males (7.0%) was higher than for females (2.0%). Amongst males, G6PD deficiency was less likely in cases than in controls  . For the= 2/215)  and the n = 16) and homozygous deficient females (n = 2) in the sample were phenotypically deficient, and all genotypically G6PD normal males and females were phenotypically nondeficient. Of the heterozygous females 24/32 (75%) were phenotypically normal and 25% were phenotypically deficient. All individuals identified as carrying the mutant allele had a C to T change at nucleotide 563, which is characteristic of the Mediterranean variant and similar to alleles found in India A total of 418 samples were successfully sequenced to provide genotype data . As expep = 0.037)  . Amongst= 0.037) . Only th2 = 1.03, p = 0.69). Among cases, the frequency of the allele among males (1.6%) and females (7.7%) was much lower, and being protective against malaria these will differ from the gene frequency in the general population.The frequency of the allele for G6PD deficiency among male controls was 12.1% (15/124), and because males carry a single copy of the X-linked gene, this approximates the population frequency of the deficient allele . Among tTo examine evidence of possible misclassification of cases as controls, several factors that could have led to misclassification were considered using a restricted analysis against the primary outcome, as follows. Less than one-third of the controls reported using health facilities outside the village as their source of consultation for minor illness or malaria treatment and diagnosis . Also amongst controls, 26/743 (3.5%) had self-reported malaria, which was not recorded in clinic records over the period of observation and 5/743 (0.7%) had ambiguous clinic records. In addition, 202/727 (27.8%) of controls were family members of cases. The restricted analysis, in which all individuals who were in these categories were excluded, produced results that did not differ from the unrestricted analysis.The study demonstrated that G6PD deficiency, whether measured by phenotype or genotype, is associated with substantial protection against vivax malaria infection. Among heterozygous females the deficiency was associated with protection less often than in hemizygous males. In populations where G6PD deficiency has been measured it rarely occurs at a frequency greater than 15% P. vivax infection is supported by its geographical distribution and may explain why the more severe variants (Mediterranean and Asia) are seen in vivax-predominant areas. This study is supported by recently published findings The discovery that G6PD deficiency is protective against P. falciparum malaria in African variant G6PD-deficient children P. vivax has exerted considerable selection pressure on human populations. This pressure could come either through mortality—severe vivax malaria being much more common than previously realised P. vivax is known to cause anaemia and low birthweight in pregnancy P. vivaxMalaria is considered by some to be the single greatest source of selection pressure acting on human populations originating from endemic areas P. falciparum are known to be more sensitive to the oxidative stress produced by the parasite The discovered protection is biologically plausible. G6PD-deficient erythrocytes infected by P. falciparum being wrongly diagnosed as having P. vivax is unlikely in the present study because the highly trained research microscopists were routinely subjected to quality control through slide re-examination Potential shortcomings of the case-control design include misclassification of cases as controls at the selection stage. Misclassification will be limited because asymptomatic infections are rare (none were found in this study) and almost all treatment was given in village clinics. Any such misclassification is unlikely to explain the observed result because any dilution would underestimate the effect of G6PD deficiency on vivax infection. Similarly, misclassification due to patients with G6PD deficiency is heritable and therefore could be clustered at the household level and provide a source of sampling bias. However, when family members of cases were excluded from the analysis, the overall conclusions were unchanged; again, residual clustering would lead to an underestimate of the observed effect. Failure of laboratory determination of G6PD phenotypic deficiency is another potential source of error. The high concordance of genotypic and phenotypic deficiency suggests this is not a major factor in this study. It is probable that some controls with discordant results  were, in fact genuinely G6PD deficient for other variants that may exist at low levels in this population. The small proportion of discordant phenotypic and genotypic results makes this unlikely to be a significant source of bias in this study.In case-control studies the association between two factors does not necessarily prove the direction of causality except in the case, as here, of innate genetic conditions. Measured potential confounding factors had little effect on the outcome. We conclude that it is unlikely the apparent substantial protective effect of G6PD deficiency arose by chance or from bias in the study design.In public health terms, the current treatment practice in most settings where vivax malaria is common is not to use antirelapse therapy in proven cases of vivax malaria because of the risk of haemolysis when testing for phenotypic G6PD deficiency is not available. This restriction is one of the major barriers to effective control and eventual elimination because hypnozoites constitute the reservoir of infection. The findings presented here indicate that, assuming accurate diagnosis, the risk of exposure to primaquine amongst G6PD-deficient individuals with vivax malaria would be lower than previously assumed because the prevalence of G6PD in vivax-infected patients would be significantly lower than in the general population. It is important to examine the level of protection conferred by other types of G6PD variant occurring in other vivax-endemic areas. The risk of severe haemolysis among G6PD-deficient individuals administered with other regimens of primaquine is also unclear; literature on this topic is sparse. For example, in Mediterranean variant G6PD deficiency, haemolysis with primaquine can be severe and, on the basis of limited data, might be quite common if full 14-day courses are taken Under prevailing conditions, however, G6PD testing is rarely available and G6PD deficiency goes largely undetected, so our finding is unlikely to increase access to antirelapse drugs in the near future because of the continuing risk of haemolysis. Hence the hypnozoite reservoir will remain unchallenged and will continue to contribute to morbidity through relapse and transmission. For effective control and treatment, either a simple test for G6PD deficiency or an antirelapse drug that can be safely given to patients with G6PD deficiency is required. This study, however, shows that the probability of individuals with vivax malaria being deficient for G6PD may be significantly lower than in the general population from which they are drawn.


Plasmodium falciparum blood stages of individuals naturally exposed to malaria who live in endemic areas of Brazil in order to assess the prevalence of different specific isotypes and their association with different malaria clinical expressions.The development of protective immunity against malaria is slow and to be maintained, it requires exposure to multiple antigenic variants of malaria parasites and age-associated maturation of the immune system. Evidence that the protective immunity is associated with different classes and subclasses of antibodies reveals the importance of considering the quality of the response. In this study, we have evaluated the humoral immune response against P. falciparum blood stages, IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA, were determined by ELISA. The results were based on the analysis of different clinical expressions of malaria  and factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks. The occurrence of the H131 polymorphism of the FcγIIA receptor was also investigated in part of the studied population.Different isotypes against The highest levels of IgG, IgG1, IgG2 and IgG3 antibodies were observed in individuals with asymptomatic and uncomplicated malaria, while highest levels of IgG4, IgE and IgM antibodies were predominant among individuals with complicated malaria. Individuals reporting more than five previous clinical malaria attacks presented a predominance of IgG1, IgG2 and IgG3 antibodies, while IgM, IgA and IgE antibodies predominated among individuals reporting five or less previous clinical malaria attacks. Among individuals with uncomplicated and asymptomatic malaria, there was a predominance of high-avidity IgG, IgG1, IgG2 antibodies and low-avidity IgG3 antibodies. The H131 polymorphism was found in 44.4% of the individuals, and the highest IgG2 levels were observed among asymptomatic individuals with this allele, suggesting the protective role of IgG2 in this population.P. falciparum antibody pattern in different clinical expressions of malaria and showed that even in unstable transmission areas, protective immunity against malaria can be observed, when the appropriated antibodies are produced.Together, the results suggest a differential regulation in the anti- The acquisition of natural immunity to malaria is slow and requires repeated parasite exposure to be maintained . This imPlasmodium falciparum seems to depend on the proportion of specific cytophilic Ab relative to the proportion of non-cytophilic Ab [1 and IgG3 cytophilic Ab in endemic areas has been associated with either lower parasitaemia [4, may inhibit effector mechanisms by competing with cytophilic Ab and are considered nonprotective [2 is non-cytophilic, but could be correlated with protection in individuals carrying a specific allelic variant of monocytes FcγRIIA receptor that can bind IgG2 [Cooperation between monocytes and Ab appears to be crucial in acquired protective immunity . The Ab hilic Ab . The presitaemia  or a lowsitaemia ,12. Otheotective ,13. IgG2ind IgG2 .P. falciparum malaria than in uncomplicated malaria [IgE levels and IgE anti-plasmodial Ab are elevated in human and experimental malaria infections, but their role in protection and/or pathogenesis is not well-established in malaria -17. In f malaria ,21, whil malaria . Recent  malaria .Plasmodium yoelii [Contradictory results have been also found in relation to IgM Ab. Even though there are indirect epidemiological data suggesting that IgM Ab does not participate in protection ,24, somem yoelii ; (c) them yoelii . In termPlasmodium-infected subjects living in malaria endemic areas of Brazil: (a) subjects with non-cerebral complicated malaria, (b) subjects with uncomplicated malaria, and (c) asymptomatic subjects – investigating the distribution of P. falciparum blood stages-specific isotypic Ab IgG, IgG1, IgG2, IgG3, IgG4 IgM, IgE and IgA. The avidity of IgG subclasses, a parameter of humoral immune response quality that has received little attention in studies on malaria, was also evaluated in this study.The gradual development of natural immunity to malaria has been observed in areas where malaria transmission is high and stable. The great majority of the studies on the prevalence of different immunoglobulin isotypes and their role in malaria infection has been conducted in such areas, but little is known about areas with unstable malaria, such as the Amazon Basin of Brazil. The main objective of this work was to compare the humoral immune response in three groups of Venous blood samples were collected from 233 residents from two cities, Peixoto de Azevedo and Manaus, in the Amazon Basin of Brazil. Peixoto de Azevedo is in the state of Mato Grosso (PA/MT) and Manaus is the capital of the state of Amazonas (MA/AM). The equatorial and humid climate of both cities is characterized by a rainy season from November to April and a dry season from May to October. Malaria transmission is unstable, with an increase at the end of the rainy season. Informed consent was obtained individually from all participants before the blood sample was taken. This study was approved by the Committee of Ethics of the Faculty of Medicine of the University of São Paulo, of the Institute of Tropical Medicine of São Paulo (IMT/SP) and of the Foundation of Tropical Medicine of Amazonas (FMT/AM). A questionnaire containing vital statistics and reports of the number of previous malaria attacks was available from all individuals.® (Quantitative Buffy Coat) were prepared according to standard protocols for all subjects. Based on the clinical expression of malaria, three groups were defined: (a) 70 subjects presenting non-cerebral complicated malaria  (CM group); (b) 148 subjects presenting uncomplicated malaria  (UM group), and (c) 15 subjects with Plasmodium infection, but without malaria symptoms  (AS group). Non-cerebral complicated malaria was defined as either severe anaemia and/or high parasitaemia, hypoglycaemia < 40 mg/dL, serum creatinine level > 1,5 mg/dL. Uncomplicated malaria was characterized by a positive blood smear and fever without other causes of infections and no manifestations of severe malaria as described above. Ninety serum samples from malaria-naïve blood donors were used as negative control in the assays. Table At the time of blood collection, thick blood films and/or QBCP. falciparum blood stages antigen as described above [P. falciparum antigen diluted to 5 μg/mL in phosphate buffered saline (PBS) (50 μL/well) for 2 h at 37°C and subsequently for 18 h at 4°C in a moist chamber. Plates were saturated with 5% skim milk in PBS-Tw20 (200 μL/well) for 1 h at 37°C. Serum dilutions in 1% skim milk in PBS-Tw20 (serum diluent) (50 μL/well) were incubated for 40 min at 37°C in duplicate wells, 1:50 for IgG1, IgG2 and IgG3, 1:5 for IgG4. For IgE detection, IgG Abs were first depleted by treating them with a 1:20 serum dilution with a sheep anti-human IgG  (1:1). The biotinylated sheep anti-human IgE antibody (Sigma Chem. Co.) was diluted to 1:500 in serum diluent (50 μL/well) and biotinylated mouse monoclonal Ab specific to each IgG isotype were used at the following dilutions in serum diluent (50 μL/well): anti-IgG1 , 1:1,000; anti-IgG2 , 1:500; anti-IgG3 , 1:500; anti-IgG4 , 1:500. The peroxidase-labeled streptavidin was diluted to 1:1,000 (50 μL/well) in serum diluent and incubated for 40 min at 37°C. The enzymatic reaction was developed with hydrogen peroxide and tetramethylbenzidin (Sigma Chem. Co.) diluted in citrate-phosphate buffer (H2O2-TMB) (50 μL/well) for 30 minutes at 37°C, and the reaction was stopped with 25 μL/well of 2N H2SO4. Spectrophotometric reading at 450 nm was performed on a Titertek Multiskan (MKII)-MCC/340 spectrophotometer.IgG subclasses and IgE Ab were detected with ELISA using a Zwittergent-extracted ed above . Flat-boet al [The results were expressed as a reactivity index (RI) or concentration. The reactivity index (RI) was calculated as the ratio of the serum sample optical density to the cut-off optical density. Serum samples with an RI equal to or greater than 1.0 were considered positive. The cut-off was defined as the mean plus 2 SD of the optical density values obtained with 90 negative control sera. Standard curves were drawn by testing in each microplate six serial dilutions  of myeloma protein of any the four IgG subclasses (Sigma Chem. Co.) or IgE myeloma. Coating was done as described above with 50 μL/well of a mixture (1:1) of one κ and one λ myeloma protein of each isotype. Concentrations of antigen-bound Ig isotypes were calculated from standard curves using a four-parameter log-logit function, with the aid of the ELISA programme developed by Plikyatis P. falciparum IgG and IgG subclasses was assessed with an urea-elution based ELISA. Microplates were coated and saturated as described above. After serum samples were incubated, 8 M urea in PBS was added (100 μL/well) for 5 min to one of the duplicates, while PBS-Tw20 was added to the other duplicate. After washing, the assay was completed as described above. The avidity index (IA) was calculated as the ratio of the urea-treated serum sample optical density to the non-treated serum sample optical density, and multiplied by 100. All values higher than 50% were ranked as high avidity and values equal or lower than 50% were considered low avidity.The avidity of anti-P. falciparum IgG, IgM and IgA Ab when analysed as described above. Serum dilutions in serum diluent (50 μL/well) were incubated for 40 min at 37°C in duplicate, 1:100 for IgG and IgM, and 1:25 for IgA. The peroxidase-labeled sheep anti-human IgG, IgM and IgA were diluted to 1:10,000, 1:20,000 and 1:2,000, respectively, in serum diluent (50 μL/well) and incubated for 40 min at 37°C. The enzymatic reaction was developed with H2O2-TMB (50 μL/well) for 30 minutes at 37°C, and the reaction was stopped with 25 μL/well of 2N H2SO4. A spectrophotometric reading at 450 nm was performed on a Titertek Multiskan (MKII)-MCC/340 spectrophotometer.Slight differences were noted among anti-The concentrations of total human IgE and IgA were determined in 66 serum samples from malaria endemic areas using commercial nephelometry kits .et al [et al [Bsh1236I (FnuDII) enzyme. The H131 allele contains a BstUI site in the 3' region and the R131 allele contains two BstUI sites in the 3' and 5' regions. After BstUI digestion, the H/H131 genotype produces a 343 fragment, the R/R131 genotype produces a 322 fragment and the H/R131 genotype produces one of each fragment.FcγRIIA H/R131 polymorphism was determined in 126 DNA samples from the subjects by using an allele specific restriction enzyme digestion method . DNA extet al  and modi2-test. Antibody levels in the three malaria groups were compared using the non-parametric Kruskal-Wallis (KW) test and the Mann-Whitney (MW) test when two groups were compared. Correlations were assessed by Spearman rank test (Sp), using real or logarithmically transformed values. P values ≤ 0.05 were considered significant.Analysis was carried out using SigmaStat software . Antibody frequencies among the three malaria groups were assessed by a χP. falciparum blood-stage antigens of 233 Plasmodium-infected subjects presenting different clinical expressions of malaria: complicated malaria (CM), uncomplicated malaria (UM) and asymptomatic malaria (AS). The results are presented on Table IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE isotypes levels were tested in P. falciparum IgG1, IgG2 and IgG3 Ab were higher in the UM and AS groups than in the CM group . In contrast, levels of IgG4 were higher in the CM group than in the UM and AS groups (P = 0.002). Also, IgE levels were higher in CM but without significant differences . Levels of IgG, IgM and IgA Ab did not differ among the groups . Higher avidity indexes were observed for IgG, IgG1 and IgG2 antibodies in the UM and AS groups compared to the CM group   (Table P > 0.05). High-avidity IgG, IgG1, IgG2 and IgG4 Ab predominated in the UM and AS groups, while low-avidity Ab predominated in the CM group, although a significant difference was only noted for IgG1 Ab . Low-avidity IgG3 Ab predominated in the three groups, but with no significant differences (P > 0.05)  was compared with P. vivax (62.7%) infected patients ; and when P. vivax (62.7%) was compared with P. falciparum and P. vivax (92.6%) infected patients . On the other hand, no significant difference was observed for all other Ab classes and subclasses studied (P > 0.05).With regards to the P. falciparum were compared in the groups of subjects presenting different clinical expressions of malaria. Interactions of IgG1, IgG2 and IgG3 were higher among the UM and AS groups, while interactions of IgG4 and IgE were higher in the CM group. Higher IgG2/IgG4 and IgG/IgM ratios were observed in the UM and AS groups , IgG1  and IgG2 Ab . The number of previous clinical malaria attacks was positively correlated with IgG, IgG1 , IgG2  and IgG3 , and negatively correlated with IgG4 . Levels of IgG1, IgG2, IgG3 and IgG were significantly higher in subjects reporting more than five previous clinical malaria attacks than among those reporting one to five previous clinical malaria attacks or those undergoing their first malaria episode . Conversely, the levels of IgG4 and IgE were significantly higher in subjects undergoing their first malaria episode . No significant difference was observed in the levels of IgM and IgA Ab (P > 0.05).The influence of age and the number of previous clinical malaria attacks on the levels of the isotypes specific for P < 0.0001). The AI of IgG, IgG2, IgG3 and IgG4 Ab were not statistically different .The mean avidity indexes (mAI) of the IgG1 Ab increased significantly with the number of previous clinical malaria attacks: above five previous clinical malaria attacks (mAI = 71.7%), one to five previous clinical malaria attack (mAI = 52.2%) and the first malaria episode (mAI = 22.9%) ; only four subjects had normal total IgE levels. Of the 62 serum samples with high total IgE levels, 40 (64.5%) had anti-P. falciparum IgE Ab and 22 (35.5%) did not (P = 0.002). The median of the total IgA levels was 2.00 g/L, while normal levels are between 0.7 and 2.0 g/L. Three serum samples showed that total IgA levels were higher than normal, while four serum samples were below normal levels.Total IgE and IgA levels were investigated in 66 serum samples of the Plasmodium-infected subjects. Overall, the H131 allele was found in 44.4% of the subjects; 15.1% were H/H, 58.7% were H/R and 26.2% were R/R. IgG2 Ab levels were higher in the asymptomatic subjects with the H131 allele (78.6 mg/mL) than in those without it (38.1 mg/mL), but this difference was not significant. A significant difference was observed in IgG2 levels when asymptomatic subjects with the H131 allele were compared to symptomatic subjects without the H131 allele (4.5 mg/mL)  , uncomplicated (UM) and asymptomatic (AS) malaria. Factors related to prior malaria exposure such as age and the number of previous clinical malaria attacks were also analysed.In this study it was investigated how IgG and its four subclasses, IgM, IgE and IgA isotypes, work against P. falciparum and/or P. vivax. It is likely that, due to the fact that a great number of these subjects had suffered multiple malaria episodes in the past and as a result of the cross-recognition of antigens, the frequency of antibodies did not differ significantly among the different infections; in any case, all subjects were analysed.All the subjects were infected with P. falciparum merozoite surface protein (MSP) MSP antigens, MSP1-19, MSP2A, and MSP2B in individuals with severe malaria than in uncomplicated [The IgG, IgG1, IgG2 and IgG3 antibody levels and their respective frequencies of positivity were higher in the UM and AS groups than in the CM group. The isotype levels were positively correlated with the number of past malaria attacks and the IgG, IgG1 and IgG2 antibody levels were positively correlated to age. These results are in line with those of other authors who demonstrated that cytophilic antibodies IgG1 and IgG3 were related to uncomplicated malaria. More specifically, several studies showed that specific IgG1 and IgG3 antibodies could protect against malaria, while lgG4 antibodies probably do not protect against the disease ,12,32-36plicated . A recenplicated .There have been reports of asymptomatic malaria in these Brazilian regions, suggesting that a certain degree of natural immunity can develop ,40. ConsP. falciparum parasitaemia [In spite of the fact that the IgG2 antibodies did not participate in the mechanisms that caused opsonization, phagocytosis and ADCI as they do not bind to the FcγR receptors , the results showed a predominance of IgG2 antibodies among individuals from the UM group. For this, the R131H polymorphism in the FcγRIIA receptor gene was investigated. FcγRIIa is a low-affinity IgG receptor with an important role in neutralizing events of malaria parasites mediated by monocytes . Its abisitaemia  and agaisitaemia ,46. In csitaemia . This disitaemia . The R/Rsitaemia ,49.P. falciparum antigens were also associated to protection against malaria. Here, the levels of IgG2 Ab were higher in the asymptomatic subjects with the FcγRIIA H/R131 allele (78.6 mg/mL) than in those without this allele (38.1 mg/mL), but this difference was not significant. However, a significant difference was found in the levels of IgG2 of asymptomatic individuals with the FcγRIIA H/R131 allele and the symptomatic individuals without allele H131 (4.5 mg/mL) . The high levels of IgG2 that were found in AS individuals carrying the H131 polymorphism could suggest that IgG2 plays a role as a cytophilic antibody among this population.In the present study, the H131 allele was found in 44.4% of the subjects; 15.1% were H/H, 58.7% were H/R and 26.2% were R/R. The frequency of the H131 allele was very similar to that observed in a population of Burkina Faso (43%) , when hiin vitro; these antibodies are responsible for mediating the opsonization of infected erythrocytes [et al observed that the positive correlation of IgG4 with the risk of infection was stronger when IgG2 levels were low, suggesting that IgG4 could be neutralizing the cellular cytotoxicity mediated by monocytes or other effector cells that depend on IgG2. Therefore, IgG4 could block the protection provided by IgG2 [The IgG4 levels showed a negative correlation with the number of previous malaria episodes. In addition, the frequency of positivity and the IgG4 levels were higher among subjects from the CM group. The absence of malaria protection in IgG4 antibodies has been observed by other authors ,14. IgG4hrocytes . Aucan eP. falciparum blood stages antibodies among populations from the endemic area with stable transmission in Africa and unstable transmission in the Brazilian Amazon Basin, Ferreira et al noted a predominance of high avidity antibodies, particularly IgG1, in the clinically immune African subjects [P. falciparum antibodies could be initially produced during the intra-erythrocytic growth of the parasite, and that these antibodies react when parasitaemias are still low, with higher effectiveness against the severity of the disease. In the current study, low avidity antibodies of all the immunoglobulins predominated among subjects from the CM group, which could reflect the prevalence in this group of individuals with few previous malaria episodes. On the other hand, the predominance of low avidity antibodies could show that uncontrolled infection is related to an expression of immunodeficiency [P. falciparum [Another essential aspect of the humoral immune response is the functional affinity or avidity of the antibodies; this is the measurement of the force between the antigen and the antibody and it can reflect the quality of the antibodies involved in protection or in different phases of the illness. When studying the avidity of anti-subjects . Among pficiency . In geneficiency ,52 and ilciparum .P. falciparum IgE levels with a reduced risk of developing clinical malaria [High overall levels of IgE have been found in populations exposed to malaria , a fact  malaria ,56.P. falciparum [P = 0.07) for specific IgE found in the group of individuals with complicated malaria.On the other hand, other evidence suggests that IgE could play a role in the pathogenesis of malaria; for example: a) there is an increase of the levels of IgE among individuals suffering from severe malaria in comparison to uncomplicated malaria ,57-59; blciparum . Immunoclciparum ,61. Althlciparum ,63. HereThe frequencies of positivity and the levels of the IgM antibodies were higher among the primo-infected individuals than among those who claimed to have had previous bouts of malaria, decreasing with each additional previous episode. Primo-infected individuals were expected to have high IgM antibody levels, since IgM is the first class of antibody produced as a primary humoral response. This must also contribute to the predominance of IgM among subjects with complicated forms of malaria, since the majority of the primo-infected subjects were suffering from such forms of the disease.The frequency of positivity of IgA antibodies does not have a correlation with the number of previous malaria episodes, though the IgA levels decreased with each previous episode. These results were in line with previous results when the predominance of IgA antibodies among primo-infected subjects was observed Since IgA deficiency has been observed among certain populations , total IThe selection of the isotype and the antibody affinity maturation of an antibody are the results of a process of differentiating B cells that occurs in the germinative centers of the lymph nodes of an immune individual. However, functional affinity or avidity depends on the antigen and on the selection of cell clones that produce antibodies with better cynetics for binding to the epitope. The selection of isotypes is a complex phenomenon that depends on several different mechanisms, many of which are still not fully understood . The graP. falciparum antibody profile in different clinical expressions of malaria. Interactions of the antibodies IgG1, IgG2 and IgG3 were predominant among individuals from the AS and UM groups, while interactions between IgG4 and IgE prevailed among individuals from the CM group. The IgG2/IgG4 levels were higher in the AS and UM groups. The IgE/IgG levels were significantly higher among CM individuals than among those without clinical complications, which may suggest that the IgG and IgE antibodies reactive against the same epitopes could play exactly opposing functions during the infection. Therefore, the relationship between the antibodies IgG/IgE, IgG1-IgG2-IgG3/IgG4, IgG2/IgG4 and IgG4-IgE/IgG2 could reflect the balance between protection and pathogenesis in malaria.In conclusion, the work reported here suggests a differential regulation in the anti-These results show that even in populations from regions with unstable malaria transmission, a certain degree of naturally acquired immunity can develop when the right antibodies are produced. Also, the findings can contribute to a better understanding of the role of different isotypes in immunity to malaria and may provide new insights into the development of malaria control strategies, especially vaccination.The authors declare that they have no competing interests.FMSL made technical contributions to the entire work, including data interpretation of data and manuscript drafting. RRD, MVL and MGA contributed to patient selection and follow-up. AWF contributed to the conception of the study. MCAS assisted in the statistical analysis, interpretation of data and manuscript drafting. SLM participated in the conception, design, analysis and interpretation of data, and the final approval of the version to be published.


Plasmodium knowlesi is an important, but newly recognized, human pathogen. For the first time, post-mortem findings from a fatal case of knowlesi malaria are reported here.Zoonotic malaria caused by P. knowlesi infection was confirmed by nested-PCR. Macroscopic pathology of the brain and endocardium showed multiple petechial haemorrhages, the liver and spleen were enlarged and lungs had features consistent with ARDS. Microscopic pathology showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. Brain sections were negative for intracellular adhesion molecule-1. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis and endothelial cells in heart sections were prominent.A formerly healthy 40 year-old male became symptomatic 10 days after spending time in the jungle of North Borneo. Four days later, he presented to hospital in a state of collapse and died within two hours. He was hyponatraemic and had elevated blood urea, potassium, lactate dehydrogenase and amino transferase values; he was also thrombocytopenic and eosinophilic. Dengue haemorrhagic shock was suspected and a post-mortem examination performed. Investigations for dengue virus were negative. Blood for malaria parasites indicated hyperparasitaemia and single species The overall picture in this case was one of systemic malaria infection that fit the WHO classification for severe malaria. Post-mortem findings in this case were unexpectedly similar to those that define fatal falciparum malaria, including cerebral pathology. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain. These results suggest that further study of knowlesi malaria will aid the interpretation of, often conflicting, information on malaria pathophysiology in humans. Plasmodium falciparum on human life, particularly in African children [P. falciparum malaria is the lack of a permissive animal model and a comparator disease in man. With the recent discovery of severe malaria, caused by the zoonotic parasite Plasmodium knowlesi in the human population of Southeast Asia [The underlying pathophysiology of malaria is not well understood despite considerable and prolonged international research effort. This effort is largely driven by the need to reduce the impact of children ,2. Part ast Asia ,4, it isP. knowlesi causes asymptomatic low-grade parasitism. In contrast, hyperparasitaemia and death ensue in the well-used Rhesus experimental model [P. knowlesi malaria, including organ pathology in various animal models [P. knowlesi infections of humans were formerly missed because of morphological similarity with Plasmodium malariae [In the natural primate hosts (long and pig-tailed macaques), al model . Consequl models -8. Naturmalariae ,10. Now malariae ,4.P. knowlesi vertebrate host switch from Southeast Asian macaques to a full-blown emergence into the human population [P. knowlesi to assist strategies to reduce health-impacts if human-to-human transmission of this virulent pathogen is established.The pathophysiology of severe knowlesi malaria in humans is undescribed, but nonetheless important for several reasons. First, to provide improved guidelines for the diagnosis, treatment and management of severe knowlesi cases. Secondly, when severe knowlesi infection is compared with severe falciparum malaria, this can help clarify determinants of severe disease. Thirdly, it is possible that we are observing a pulation ,12. TherP. knowlesi biology and comparisons with the salient features made with what is known of the pathophysiology of severe falciparum malaria [The clinical, laboratory and, for the first time, post-mortem findings in a fatal case of knowlesi malaria are reported here. The histopathology in various organs is explained within the context of  malaria -16.A forty year-old male was brought to the Queen Elizabeth Hospital, Kota Kinabalu, Sabah (8.30 am) in a state of collapse. He was unable to give a history himself or to stand. On examination his blood pressure was unrecordable and oxygen saturations were recorded as low.The patient had no past medical history. He had spent two weeks in the jungles of Borneo before leaving for an urban setting. Ten days after leaving the jungle he experienced the onset of fever and body aches. Two days later, he sought treatment at a government outpatient clinic continuing to complain of fever and myalgia. A specific diagnosis was not made and he was able to work although he developed rashes the next day. He remained unwell for the next two days, when he presented in a state of collapse and had developed abdominal pain.Resuscitation measures were begun and the patient was immediately intubated, given adrenaline/atropine and sodium bicarbonate. On examination and after resuscitation measures his vital signs were: symmetrical air entry into lungs, BP 58/44 mm Hg, pulse rate 40-50 per minute with poor peripheral perfusion and cyanosis. Heart sounds were normal. He had generalized petechiae and his abdomen was tense and distended.Resuscitation measures continued for one hour, during which time "coffee grounds" were observed in the nasogastric aspirate. The patient became asystolic after one hour and although cardiopulmonary resuscitation was given for a further 20 minutes, there was no response. The patient was pronounced dead two hours after admission.Dengue haemorrhagic shock was suspected and a post-mortem examination was performed approximately 24 hours later.Plasmodium knowlesi, as a single species infection, was confirmed by nested-PCR [Laboratory results are summarized in Table sted-PCR . Post-moExternal examination showed a well-nourished adult male. The conjunctivae showed tinges of jaundice and the right eye had subconjunctival haemorrhages. There were multiple petechial haemorrhages on the body and venepuncture sites were associated with marked bruising. Coffee ground material was noted in the mouth. Internal examination revealed no tissue oedema or excess fluid in the body cavities.The external surfaces of the cerebrum were dusky. The cut sections showed multiple petechial haemorrhages. The cerebellum also showed petechial haemorrhages externally and on multiple cut sections Figure  and 2b. P. falciparum. Immunohistochemistry stained sections from the brain were Plasmodium anti-aldolase positive  were abundant although parasite bodies were obscured by haemozoin  pigment. Chemical removal of pigment and oil immersion  magnification revealed trophozoites that were discernibly bigger than those of e Figure  and negastaining .Many petechial haemorrhages (up to 600 μm diameter) arising from the rupture of the small vessels of the cerebrum and cerebellum were observed Figures  and 4b SAlthough sections from the spleen showed some autolysis, expansion of the red pulp and atrophy of the white pulp was noted. Germinal centers were not observed. Abundant pigment-containing macrophages and some haemophaghocytosis was evident in the red pulp and parasitized red cells were plentiful Figure . There wThere were many PRBC's in the liver sinusoids with haemozoin pigment in Kupffer cells and evidence of haemophagocytosis. The portal tracts and sinusoids had moderate chronic lymphoplasmacytic inflammation. Overall the liver was non-cirrhotic but with severe macrovesicular steatosis. No cholestasis, regional necrosis or thrombotic microangiopathy was observed Figure .The renal cortex showed dilated and congested blood vessels. Many PRBC were observed within glomerular capillaries with pigment deposition in the mesangium. There was no evidence of thrombotic microangiopathy . The tubules showed acute tubular necrosis and regeneration. There were a small number of eosinophilic intra-tubular casts Figure .Sequestration of PRBC's was evident in the small vessels of the heart Figure . EndotheThe adrenal gland appeared active with eosinophilic cytoplasm in the fasciculata layer with no evidence of PRBC sequestration or of parenchymal haemorrhage. Samples of lung, intestine or bone marrow were not available for histopathology examination.The overall picture was one of systemic malaria infection with multi-organ damage, particularly in the brain where there was much vascular rupture and petechial haemorrhaging.Fatal human knowlesi malaria has hitherto not been reported at post-mortem, so this report may increase our understanding of severe and fatal malaria whatever species cause these syndromes. Interestingly, vivax malaria is also becoming recognized as causing severe and sometimes fatal infection in a significant proportion of individuals, suggesting re-evaluation of the dogma that falciparum infection may be the only important cause of fatal disease -21.The WHO classification for severe falciparum malaria would have included this individual because he had several qualifying features including hyperparasitaemia (~10% infected erythrocytes), renal impairment, jaundice and ARDS, although coma was not prominent in the history ,4,22. CoThe beefy appearance of lung tissue in this case is consistent with respiratory distress syndrome, but could not be confirmed histologically. Changes in the kidney of some recovering areas of acute tubular injury are consistent with the observation of renal impairment reported in other severe cases of knowlesi malaria and an uDespite the absence of antecedent established coma, cerebral pathology in this case is very similar to that observed in fatal falciparum infections, confirming that it was important to exclude this co-infection. Although there was obvious accumulation/sequestration of infected erythrocytes in capillaries and venules, there were also some differences between these appearances and those seen with fatal falciparum infections. For example, there was no platelet clumping, no notable thrombi, and uninfected erythrocytes were also interspersed with infected cells. Neither was up-regulation of ICAM-1 expression detected in endothelial cells, suggesting that the mechanism of sequestration/accumulation of infected erythrocytes in knowlesi infections needs further investigation. In falciparum cerebral malaria, display of ICAM-1 by up regulated endothelial cells mediates adhesion to parasitized red cells .P. knowlesi (up to 16 per mature schizont) compared with P. falciparum (up to 36). Although up to 24 merozoites per schizont have been observed in Plasmodium vivax, parasitaemia is restricted to reticulocytes by limitation of invasion pathways in this species, rather than merozoite number.Hyperparasitaemia is a marker of severe falciparum malaria. It is also apparent in severe knowlesi infections , but is P. falciparum infections, all asexual developmental stages are seen in the peripheral circulation of knowlesi-infected patients [Hyperparasitaemia in falciparum malaria is often explained by protection of late stage parasites from the filtering action of the spleen. Unlike patients . HyperpaAs with many fatal cases of falciparum malaria, malaria pigment was evident in blood films and was present in circulating leucocytes (~40%), tissue sections and organ specific macrophages. Reticuloendothelial changes in both liver and spleen were associated with pigment, accumulation of red cells and laden macrophages, and some inflammation in liver portal tracts was observed. Circulating pigment has been variably implicated as an indicator of poor prognosis for falciparum malaria -25 and tP. falciparum, resulting in sequestration of all but early trophozoite stages in falciparum malaria. Cytoadherence is often implicated in malaria pathology and is mediated by the expression of variable var gene products (of the PfEMP-1 family) at the surface of infected host red blood cells with concomitant expression of post-capillary endothelial cell receptors [P. knowlesi infected cell agglutination mediated by variant surface antigens of P. knowlesi SICA var genes (orthologues of the PfEMP-1 family) [P. knowlesi, up-regulation of ICAM-1 was not detected, nor was there evidence for cytoadherence as parasitized cells were not marginalized and were interspersed with uninfected erythrocytes in smaller vessels.Among the malaria parasites of humans, cytoadherence is purportedly unique to eceptors ,30-32. A family) ,34. ImpoCerebral malaria and severe acute anaemia are often peculiar to falciparum infection while organ, respiratory and metabolic dysfunction are common with severe knowlesi, vivax and other forms of severe falciparum infection ,19,35-40P. knowlesi present with or develop severe symptoms and 1-2% of cases are fatal [P. knowlesi may emerge as a human pathogen beyond its current zoonotic manifestations [The need to develop knowlesi-specific diagnosis, treatment and management guidelines is urgent. A recent prospective clinical study in Sarawak Malaysian Borneo revealed that approximately 1:10 patients infected with re fatal . Complicre fatal ,4. Therestations ,12, specApproval to use this case for educational purposes was obtained from the Deputy Director for Health, Sabah State Department of Health, Ministry of Health, Malaysia.The authors declare that they have no competing interests.JCS collected and collated case information and details and obtained consent. JH performed the post mortem, provided gross pathology images and provided the autopsy report. PCD, ZSR, PC and AP performed PCR, Immunohistochemistry and sample processing. SK, SBL, ANZ and WKT prepared and reviewed the clinical, histopathology and pathology reports. SBL and BS provided descriptions and information for the manuscript. JCS and SK wrote the manuscript. All authors have read and approved the final manuscript.


The effectiveness of malaria chemoprophylaxis is limited by the lack of compliance whose determinants are not well known.The compliance with malaria chemoprophylaxis has been estimated and analysed by validated questionnaires administered before and after the short-term missions (about four months) in five tropical African countries of 2,093 French soldiers from 19 military companies involved in a prospective cohort study. "Correct compliance" was defined as "no missed doses" of daily drug intake during the entire mission and was analysed using multiple mixed-effect logistic regression model.The averaged prevalence rate of correct compliance was 46.2%, ranging from 9.6%to 76.6% according to the companies. Incorrect compliance was significantly associated with eveningness (p = 0.028), a medical history of clinical malaria (p < 0.001) and a perceived mosquito attractiveness inferior or superior to the others (p < 0.007). Correct compliance was significantly associated with the systematic use of protective measures against mosquito bites (p < 0.001), the type of military operations  and other individual factors (p < 0.05).The identification of circumstances and profiles of persons at higher risk of lack of compliance would pave the way to specifically targeted strategies aimed to improve compliance with malaria chemoprophylaxis and, therefore, its effectiveness. Non-immune civilians and military personnel traveling in malaria-endemic areas are at risk of getting malaria and may become clinically ill during or after their travel. Approximately 25-30 million travellers from non-tropical regions visit malaria-endemic countries annually, and about 30,000 cases of travel-associated clinical malaria occur each year . In the Plasmodium falciparum to most anti-malarial drugs and the resistance of Anopheles to insecticides may limit the efficacy of these prophylactic measures. Chemoprophylaxis is a key component of malaria prevention because none of the vector protection measures totally avoid mosquito bites during night activities. However, the effectiveness of chemoprophylaxis is limited by lack of compliance with drug intake, even if the regimen is adapted to the chemosusceptibility of P. falciparum [Malaria is an important threat to tourists, soldiers and employees travelling or working in endemic areas, particularly because of the potentially rapid onset of infection and the severity of the disease. Non-immune travellers should be protected from malaria by chemoprophylaxis and prophylactic measures against mosquito bites, including insecticide-impregnated bed nets (IIBN), repellents and insecticide-treated long-sleeved clothes and pants. In malaria-endemic areas, the use of most of these prophylactic measures is mandatory for non-immune soldiers of Western armies or for non-immune employees of most major international groups. The resistance of lciparum ,7.Many clinical malaria cases and outbreaks -11 have It is generally acknowledged that the lack of compliance with medications is unrelated to the lack of symptoms or risk perception. In fact, compliance determinants are not well known, especially for chronic diseases, such as HIV infection ,18, diabThe objective of the present study was to identify the determinants of compliance with malaria chemoprophylaxis among French soldiers on a short-term mission in malaria-endemic areas of tropical Africa.The present study was part of a larger prospective cohort study on the risks of vector-borne diseases among French soldiers carried out between February 2004 and September 2007. The study comprised 19 French military companies  serving a short-term mission (about four months) in five tropical African countries: eight companies in Côte d'Ivoire, four in Gabon, three in Chad, two in Central African Republic and two in Senegal. The soldiers were required to take chemoprophylaxis and apply protective measures against mosquitoes. These companies were selected to represent the various conditions that French soldiers experience in Africa. All members of the companies designated for the mission were eligible for the study and were invited to participate. Soldiers absent from the regiment on the dates of the surveys or who left the company between the survey and the departure or during the mission were excluded from the analysis.The protocol was approved by the ethical committee Marseille II . The informed consent of each participant was obtained at the beginning of the study after a thorough explanation of its purpose.Two self-administered questionnaires containing behavioural items were filled out by each soldier and validated by a member of the research team. The first survey was administered no earlier than 15 days before their departure, and the second was completed no later than 15 days after their return.The dependant variable was the level of compliance with malaria chemoprophylaxis during the mission as assessed retrospectively by the return questionnaire. "Correct" compliance was defined as "No missed doses" of daily drug intake during the entire mission, while "incorrect" compliance was defined as one of the three other possible responses: "Less than one missed dose per month," "One or more missed doses per month, but less than one missed dose per week" or "One or more missed doses per week".Independent variables that were considered potential determinants of compliance were recorded for each soldier either upon departure or return. These variables were related to individual characteristics or to the particular settings of the mission, as described below.i.e. the circadian rhythm of the soldiers: morning people waking up early and being more alert in the first part of the day, evening people being more alert in the late evening hours and preferring to go to bed late.Variables relative to the mission were the dates of departure and return, country and mission type, such asmilitary operation or training. Demographic and behavioural variables were age, gender, rank, usual morningness and eveningness (as determined using the reduced Horne and Östberg morningness-eveningness questionnaire -31), wake.g., birth of a child, illness or death of a relative or friend, or separation or announcement of a divorce). Other variables included the subjective perceived risk level during the mission associated with i) a dangerous situation, ii) the individual likelihood of acquiring clinical malaria, and iii) the severity of disease if malaria were contracted.Other variables pertained to the occurrence of important familial events during the mission . A global score was calculated by combining the frequency of use of each antivectorial measure during each activity with the time spent in each activity. Moreover, a global variable summarizing the use of all anti-vectorial protective measures  was constructed.Independent variables relative to the chemoprophylaxis were the drug itself (either doxycycline or chloroquine-proguanil combination with once-daily dosages), tolerance and usual time schedules of intake.i.e., themain vector-borne diseases experienced by the French Forces). Variables pertaining to medical events that occurred during the mission included clinical malaria, non-malarial fever, diarrhoea, boil, other severe infectious disease , perceived frequency of mosquito bites and perceived personal mosquito attractiveness.The variables incorporating medical antecedents were allergy to insect bites, usual cutaneous response to mosquito bites and medical history of malaria, leishmaniasis or dengue fever . Only participants who completed both questionnaires were considered for statistical analysis. Missing values affected less than 1% of the participants and were replaced using the single imputation method. When a question was not asked to one or more companies, the responses were coded as missing data for those companies.The confidence interval of correct compliance with malaria chemoprophylaxis was estimated by taking into account the clustered design of the study using the svy commands under STATA 9.0. Compliance with chemoprophylaxis was analysed as a dependant variable according to individual and company characteristics using a random effect mixed logistic regression model. The model was designed to take into account the intra-company correlations that could exist due to the sampling design (company effect as random effect). The logistic model was also adjusted using a generalized estimating equations (GEE) approach. Random effect and GEE regression models allow the estimation of company-specific and population-averaged effects, respectively .p-value less than 0.25 [p < 0.05) independent variables and their interactions in the final model. The statistical quality of the final model was assessed by looking at the adequacy between observed and predicted probabilities of correct compliance.First, a descriptive analysis of the independent variables was performed. A bivariate analysis was then conducted by entering each independent variable in a logistic regression model. Variables were retained for the multivariate analysis when their effect had a han 0.25 . A backwOf 2,901 eligible soldiers from 19 companies, 808 were excluded because they did not complete both questionnaires, leaving a sample of 2,093 subjects. The most common reason for not completing the questionnaires was the absence of the subject from the regiment at the time of the survey . Some soldiers who had planned to deploy to Africa and completed the first questionnaire did not travel and were replaced by others who had not completed the survey. Some soldiers returned to France before the rest of their company or were transferred to another regiment and were not present at the time of the second survey. The rate of refusal to participate in the study was lower than 5%.The descriptive characteristics of the companies are summarized in Table Assessment of compliance level with chemoprophylaxis was based on the self-reported occurrences of daily medication doses missed during the mission. "No missed doses" was indicated by 967 out of 2,093 soldiers, or 46.2% "correct" compliance. Between companies, the lowest proportion of correct compliance was 9.6%, and the highest was 76.6%.Among the other three responses corresponding to incorrect compliance, "less than one missed dose per month" was declared by 637 soldiers (30.4%); "one or more missed doses per month but less than one missed dose per week" was declared by 298 soldiers (14.2%); and "one or more missed doses per week" was declared by 191 soldiers (9.1%).The frequency of correct compliance was calculated according tothe following: variables related to the mission, demographics and behavioral characteristics; the occurrence of particular events during the mission and the perception of risks; use of anti-mosquito protective measures; use of malaria chemoprophylaxis; and medical history and occurrence of medical events during the mission. These data are shown in Tables p = 0.0074).The variables retained in the final mixed multivariate logistic regression model are shown in Table There was no significant interaction between independent variables. The company effect was significant in the final model. The OR estimates were similar for both the random effect and GEE regression models.Many studies have been conducted in order to identify factors that influence compliance with medications, most of which focused on treatments to cure, reduce or delay complications and symptoms caused by chronic diseases. Only a few studies have assessed compliance in the context of chemoprophylaxis. Recently, the concept of adherence has supplanted compliance, as adherence implies that the patient agrees with the prescribed recommendations rather than passively obeying them  . HoweverThe systematic use of protective measures against mosquito bites  was associated with correct compliance with chemoprophylaxis. Whereas previous studies showed no association  or even Among life events, only the birth of a child during the mission was associated with correct compliance. This could be explained by an increase in the level of responsibility felt towards the newborn, a greater maturity among those in a position to father a child or internal pressure from the family ,36, any The "morning-type" soldiers, as determined by both the reduced Horne and Östberg morningness-eveningness questionnaire and by sleep/wake hours during the mission, were more likely to have correct compliance. Similarly, "evening-type" soldiers were less compliant with chemoprophylaxis. Eveningness has been associated with certain personality traits and dimensions  -39 and iIt has been shown that travellers who perceived themselves at high risk of becoming ill with malaria after returning home  or traveSimilarly, individuals who perceived themselves to be either more or less potent mosquito attractors than others were more compliant with chemoprophylaxis. This unexpected significant association also persisted after adjusting for the other covariates. Individuals who considered themselves less attractive to mosquitoes could be less compliant because of the perception of a lower risk of contracting malaria. In contrast, those who thought that they attracted mosquitoes more so than others were expected to be more compliant with anti-mosquito measures and could have believed that these anti-vectorial measures were sufficient to protect them. However, no significant interaction was found between the effects of the perceived mosquito attractiveness and compliance with anti-mosquito measures or chemoprophylaxis. Other determinants induced by the military setting such as fatalism or coping with danger by avoidance, denial or vulnerability could explain this unexpected association.A previous medical history of malaria was associated with present lack of compliance. This suggests that having experienced an episode of clinical malaria was not a factor contributing by itself to the adoption of prophylactic behaviours . TherefoThe present study did not show any significant association between compliance and gender or age, although it has previously been shown that women and older travellers were more compliant ,35,45. HMilitary operation-type missions were associated with correct compliance, independent of the country or the army corps. In such conditions, the soldiers were probably more closely supervised by the command, so they were submitted to a higher pressure to comply with the chemoprophylaxis. Indeed, the good health of the soldiers, including the prevention of malaria, was an important concern of the command because of the operational nature of the mission: the global health of the group had to be preserved for a military purpose . CompliaAfter taking all these factors into account, there was still a significant group effect; that is, significant behaviour determinants associated with the companies that were not identified in the present study. Moreover, the rate of compliance was very heterogeneous among companies, meaning probably that individual behaviours were not independent within a company. This unconscious or conscious imitation was not explained by the previous determinants. The lack of isolated soldier limited the possibility of studying the group effect. Compliance with anti-malarial prevention measures appears to be the end result of a complex mixing of the perception of risks and of protective/curative interventions, the acceptance of these measures and their application in spite of discomfort; qualitative approaches could be appropriate for exploring these complexities .®: Medication Event Monitoring System) that records the date and time of bottle cap opening has been conducted to objectively assess the rates of compliance in the first company. This electronic monitoring is considered to be more accurate than questionnaires, diaries or counts of returned untaken tablets for the assessment of compliance [® as the reference), the answers to the questionnaire used in the present study had a 0.69 and 0.75 sensitivity and specificity, respectively, to the assessment of the correct compliance. These quality indexes were close to those previously found by others using only one question [In this study, the compliance was measured based on a self-administered questionnaire. This method could overestimate the rate of correct compliance, as previously shown for anti-viral therapy  or malarmpliance ,50. If cquestion ,51,52. Ta priori determinants likeeveningness, which are associated with compliance. The identification of circumstances and profiles of persons at higher risk of lack of compliance would allow the implementation of specifically targeted strategies of health information, education, and communication, in order to improve compliance with malaria chemoprophylaxis and therefore its effectiveness.The results of the present study are applicable to French soldiers travelling for a short-term mission in intertropical Africa because the companies were representative of the different malaria-endemic areas to which they could be sent. The prevalence of correct compliance with malaria chemoprophylaxis among soldiers was close to those reported elsewhere among civilian travellers ,28,35, aThe authors declare that they have no competing interests.NR performed the statistical analysis and wrote the article. VM participated significantly in the data management. LO, EOP and GT participated significantly in the data management and the conception of the questionnaires. JG participated in the statistical analysis. BP, CTT and AB contributed significantly in the preparation of the study. CR conceived the study, took part in the analysis of the data and the discussion and wrote the article. The final version of the manuscript was seen and approved by all authors.


Plasmodium falciparum infections are needed to facilitate a comprehensive understanding of malaria epidemiology in the complex Kenyan highland eco-epidemiological systems where malaria recently expanded, as well as the evaluation of control measures.The Kenyan highlands were malaria-free before the 1910s, but a series of malaria epidemics have occurred in the highlands of western Kenya since the 1980s. Longitudinal studies of the genetic structure, complexity, infection dynamics, and duration of naturally acquired P. falciparum isolates that were analyzed using 10 microsatellite markers.We followed a cohort of 246 children residing in 3 villages at altitudes 1430 - 1580 m in western Kenya. Monthly parasitological surveys were undertaken for one year, yielding 866 E = 0.787-0.816), with ≥83% of infections harboring more than one parasite clone. Diversity remained high even during the low malaria transmission season. There was no significant difference between levels of genetic diversity and population structure between high and low transmission seasons. Infection turn-over rate was high, with the average infection duration of single parasite genotypes being 1.11 months, and the longest genotype persistence was 3 months.Infection complexity and genetic diversity were high (HThese data demonstrate that despite the relatively recent spread of malaria to the highlands, parasite populations seem to have stabilized with no evidence of bottlenecks between seasons, while the ability of residents to clear or control infections indicates presence of effective anti-plasmodial immune mechanisms. Plasmodium falciparum malaria is presently among the most fatal diseases in the East African highlands, where an estimated 34 million people remain at risk . Variability data for the low versus high malaria transmission seasons were respectively as follows: Na = 12.4 vs 13.2; ne = 6.75 vs 6.52; He = 0.789 vs 0.793, and I = 2.0 for both seasons . AMOVA revealed no significant difference in genetic structure between the low and high transmission seasons  to 27 (Polyα). Genetic diversity was high throughout the year (HP < 0.05) in the low and high transmission seasons, respectively, with no significant association involving loci on the same chromosome. Moreover, no locus pair remained in LD after applying both the highly conservative Bonferroni correction and the less conservative Benjamini-Yekutieli false discovery rate (B-Y FDR) procedure for multiple comparisons [Only three and one out of 45 possible pair-wise tests showed significant LD , but no difference was observed in infection complexity between the two seasons (P = 0.641). The average number of P. falciparum clones per infection for the entire study period was 2.5 (range 1- 6).Data for December were not used for monthly infection complexity analysis because of a small number of infected samples . The prevalence of mixed-clone infections was high (83.3 - 98.5%), while the prevalence of single infections was generally low . There was a wide variation in infection durations estimated by individual microsatellite markers. The less polymorphic loci gave longer genotype persistence estimates. The highly polymorphic markers (e.g. Polyα and TA109) yielded statistically similar mean infection duration estimates to the multilocus estimation . The multilocus estimation showed the average duration of individual parasite genotypes to be 1.11 months, with the majority (92.2%) of infecting genotypes persisting for only one month range 1-3 months). Considering infections from individuals who fell sick and got malaria treatment at some point during the year versus those who never got treatment at all, the proportion of infections lasting more than one month was higher among individuals who received treatment than among those who were asymptomatic throughout the test period and never got treated  and there were no differences in genetic diversity observed between the low and high transmission seasons, contrary to what would be expected based on the substantial differences in EIR among the sites. A number of possible explanations may account for this observation. First the EIR data cited were obtained in different years; perhaps a clearer EIR picture could have been obtained with data obtained during the study period. Second, vector sampling for EIR estimates may have not represented all the areas where the children resided . Third, P. falciparum haplotype composition within this highland area. Moreover, the absence of genotypic linkage disequilibrium indicates that factors such as inbreeding/selfing, genetic drift, or the Wahlund effect were not sufficiently significant to cause changes in parasite genetic structure [The high infection incidence rate, high parasite population genetic diversity, together with the high level of among individual variability depicted by AMOVA suggests that there is substantial heterogeneity in tructure . The rectructure . In Kenytructure . Three rtructure . Interimtructure . We havetructure . Africantructure ,41, and tructure , necessiMore than 83% of the asymptomatic infections were multi-clonal throughout the 12-months study period. Similar estimates were obtained from earlier cross-sectional studies of symptomatic patients in the highlands of Kakamega and Kisii, and the low lands of Kombebwa; all in western Kenya . VariousP. falciparum parasitemias among individuals in this area for up to 12 months [P. falciparum parasitemia [P. falciparum observed in our study area thus appears to be a sequel of more frequent clearance and acquisition of infections, rather than their protracted persistence.Application of molecular genotyping to longitudinally-collected parasite samples provides high utility in studying infection dynamics, as it enable distinction between pre-existing and newly acquired infections. Without genotyping, we previously found detectable 2 months . The pre2 months  and for 2 months . The sig2 months . Moreoveasitemia ,46 by acIt is difficult to determine the impact of antimalarial drug treatment on infection clearance in our study. First, the study was carried out in 2006, when resistance to the most commonly used drug, sulfadoxine-pyrimethamine, was supposed to be high. Majority of samples obtained within one month after treatment were parasite-negative, with genotyping results showing only 2 out of 18 positive samples obtained within one month of treatment being recrudescent; the rest were new infections. Second, nearly half (43.4%) of the subjects who reportedly had clinical signs of malaria and took treatment at some point during the study period were parasite-negative at the times of our sampling, throughout the year; raising questions whether they were true malaria cases that adequately responded to treatment and never got re-infected. Because there are many causes of fever and other malaria-like symptoms, cases of presumptive treatment of fevers with antimalarial drugs abound in Africa, with clinicians ignoring laboratory results in many cases . OverallOne limitation of our study was that fever was not measured; hence we cannot rule out the possibility that some of the parasitemias were associated with clinical malaria. However, besides fever, clinical malaria is often associated with other symptoms, such as shivering, headache, muscle weakness, joint pain and vomiting. None of these symptoms were observed at the times of sampling, and the majority of the children did not report to have fallen sick afterwards or taken any antimalarial drug throughout the study period.The rapid infection turnover observed in our study area is typical of high transmission areas, and the observed longest single genotype duration is within the expected range (50 - 150 days) for areas of high transmission ,50. ThesP. falciparum population during the low and high malaria transmission seasons in this highland area [In a population having a constant effective size over the recent past, there is an equal probability that loci show excess or deficit of heterozygosity . Overalland area ,30. The and area .ne than Na suggesting the presence of adaptive, low-frequency alleles [P. falciparum populations in this area may be having a high potential for responding to environmental changes and pressures imposed by control measures.The observed high re-infection rates call for unrelenting malaria control efforts, which should not ignore the asymptomatic sub-population, if malaria is to be ultimately eliminated in this area. The existence of parasite populations that are largely sheltered within the asymptomatic sub-population in this highland area, together with a high prevalence of mixed infections pose a challenge to control efforts. First, asymptomatic parasitemias are often so low that they often escape detection by microscopy, the widely used tool for malaria diagnosis . Second, alleles ,52,57, mTaken together, these data demonstrate that despite the relatively recent spread of malaria to the highlands, parasite populations seem to have attained a high effective population size, while the ability of the majority of residents to clear or control infections indicates presence of effective anti-plasmodial immune mechanisms. Asymptomatic parasitemias may be playing a large role in maintaining the parasite burden in this area; they are not presently targeted for treatment. Since the high parasite population size, with high multi-clonality give a biological advantage to the parasites for their survival and spread ,54, the None of the authors has a commercial or other association that might pose a conflict of interest in this work.FNB: Carried out the molecular genetic studies, performed the statistical analyses, data interpretation and drafted the manuscript. YA: Conducted sample collection and helped with writing the manuscript. DAA: helped in writing of the manuscript. MB: Participated in data analysis and writing of the manuscript. AMV-Z: Participated in data analysis and writing of the manuscript. DMM: Participated in writing of the manuscript. AKG: Participated in the design of the study, facilitated, and conducted field sample collection. GY: Conceived the study, acquired funding, and participated in sample collection and in manuscript preparation. All authors read and approved the final manuscript.This study was funded by grants R01 AI050243, D43 TW001505, and R03 TW007360 from the National Institutes of Health of the USA.Our dear colleague, Dr David Maina Menge passed away on Saturday 29th May 2010, when the manuscript was still under revision. We dedicate this paper to him.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/283/prepub