Plasmodium falciparum malaria is a major cause of mortality among young children in western Kenya. The factors that lead to the age-specific incidence of this anemia are unknown. Previous studies have shown an age-related expression of red cell complement regulatory proteins, which protect erythrocytes from autologous complement attack and destruction. Our primary objective was to determine whether in a malaria-endemic area red cells with low levels of complement regulatory proteins are at increased risk for complement (C3b) deposition in vivo. Secondarily, we studied the relationship between red cell complement regulatory protein levels and hemoglobin levels.Severe anemia due to Three hundred and forty-two life-long residents of a malaria-holoendemic region of western Kenya were enrolled in a cross-sectional study and stratified by age. We measured red cell C3b, CR1, CD55, and immune complex binding capacity by flow cytometry. Individuals who were positive for malaria were treated and blood was collected when they were free of parasitemia. Analysis of variance was used to identify independent variables associated with the %C3b-positive red cells and the hemoglobin level.Individuals between the ages of 6 and 36 months had the lowest red cell CR1, highest %C3b-positive red cells, and highest parasite density. Malaria prevalence also reached its peak within this age group. Among children ≤ 24 months of age the %C3b-positive red cells was usually higher in individuals who were treated for malaria than in uninfected individuals with similarly low red cell CR1 and CD55. The variables that most strongly influenced the %C3b-positive red cells were age, malaria status, and red cell CD55 level. Although it did not reach statistical significance, red cell CR1 was more important than red cell CD55 among individuals treated for malaria. The variables that most strongly influenced the hemoglobin level were age, the %C3b-positive red cells, red cell CR1, and red cell CD55.Increasing malaria prevalence among children >6 to ≤ 36 months of age in western Kenya, together with low red cell CR1 and CD55 levels, results in increased C3b deposition on red cells and low hemoglobin. The strong contribution of age to C3b deposition suggests that there are still additional unidentified age-related factors that increase the susceptibility of red cells to C3b deposition and destruction. Plasmodium falciparum malaria is responsible for 1 to 2 million deaths per year, with most in sub-Saharan Africa [P. falciparum and tends to occur in children <3 years of age, whereas cerebral malaria occurs in areas where the annual inoculation rate is low, and it occurs in older children and adults [d adults .P. falciparum [The pathogenesis of severe anemia in malaria is complex. However, a number of observations suggest that the destruction of uninfected red cells is a significant contributor to, if not the major cause of, this anemia: uninfected red cells have a decreased life span in patients with lciparum  and the lciparum . Furtherlciparum . These olciparum ,10 and Clciparum , have belciparum ,13.In order to understand the nature of the susceptibility of red cells to complement activation during malaria infection we have investigated the expression of red cell complement regulatory proteins in persons living in a malaria-endemic area. Complement receptor 1  is a 200 kDa protein that is found on red cells and most leukocytes . It alsoP. falciparum infection and severe anemia have acquired deficiencies in CR1 and CD55, and increased C3b deposition [The other red cell complement regulatory protein that we have studied is decay accelerating factor  . It is aposition ,22. Exceposition .P. falciparum infection, we have studied the relationship between red cell CR1 and CD55 and age. Our studies suggest that the expression of red cell CR1 and CD55 are age-dependent, decreasing from birth to a nadir somewhere between 1 and 2 years of age and rising thereafter [To understand the age predilection of severe anemia during ereafter . This paereafter . To testThe study was cross-sectional in design and was approved by the Walter Reed Army Institute of Research Human Use Research Committee and by the National Ethical Review Committee, Nairobi, Kenya. It was carried out from October through December 2004 in Kombewa Division, an area off the shores of Lake Victoria within Kisumu District. Malaria is holoendemic in this region, occurring throughout the year with peak seasons during the long rains (March through August) and during the short rains (October through December) . The areP. falciparum malaria from Kisumu District, western Kenya, who participated in our studies from 1998 to 2001 [Enrollment was stratified into nine age groups selected based on the age distribution of children with severe anemia (Hgb ≤ 5.0 g/dl) and  to 2001 ,26,27: 0 to 2001 , and oth to 2001 , HIV inf to 2001 , and oth to 2001 ,31 can aA preliminary census was carried out within selected villages to identify potential participants. All individuals who met the entry criteria were invited to participate. On the days of recruitment individuals or parents with children self-reported to the clinic for enrollment in the study. Informed consent was obtained prior to any procedures. Individuals underwent a standard medical evaluation. Date of birth was confirmed by national registration card, baptismal certificate, or birth certificate. During the initial screening, blood obtained by finger prick was used to prepare thick and thin Giemsa-stained blood smears. If an individual was positive for malaria, he or she was treated with artemether/lumefantrine  and askeMalaria smears were read by trained and certified microscopists. All smears were read in duplicate by independent microscopists who were unaware of the status of the participants. Discrepancies were settled by a third independent microscopist. A smear required a minimum of 200 high power fields scanned prior to being considered negative. Once an individual was deemed well and asymptomatic, a 2.5 ml ethylenediaminetetraacetic acid (EDTA)-anticoagulated sample of venous blood was obtained. An aliquot was used for complete blood count determination using an automated hematology analyzer . The remaining sample was centrifuged and the plasma removed. The red blood cell pellet was then cryopreserved until used .Erythrocyte CR1 and CD55 levels were determined using frozen samples. In preliminary experiments, we observed no difference in the level of red cell complement regulatory proteins between fresh and frozen samples. All primary antibodies were titered to saturation. 10 μl of thawed erythrocyte pellet was washed twice in 1 ml of Alsever's buffer and resuspended in the same volume of buffer. Unless otherwise stated, all procedures were as previously described . The folFlow cytometry was carried out using a FACScan flow cytometer (Becton-Dickinson). Analysis was done using FCS Express v2.5 . Red cells were gated on the basis of their forward and side scatter characteristics using logarithmic amplification. The median fluorescence intensity (MFI) of each sample was measured using logarithmic amplification. The MFI values for CR1 and CD55 were normalized to the mean of the MFI of the red cell standard using the formulaCorrMFIs' and 'MFIs' are the corrected and uncorrected sample MFI respectively, 'MFIcmean' is the mean of all the MFI values of the standard control, and 'MFIc' is the mean of the control obtained in parallel with the sample. The number of molecules of CR1 per red cell was derived from a fluorescence standard curve created using cells with known CR1 numbers. Red cell anti-CD55 antibody binding capacities were derived from a standard curve created using beads of known antibody binding capacity  [where 'ers, IN) .50 μl of 49 mg/ml rabbit anti-BSA  and 3 μl of 5 mg/ml BSA-FITC  were added to 950 μl of RPMI1640 (Sigma-Aldrich). This combination was noted to be the point of equivalence in preliminary experiments. The mixture was incubated at 37°C for 1 hr and overnight at 4°C. The next day, the IC preparation of soluble and insoluble IC was aliquoted and stored at -20°C.All centrifugation steps were at × 500 g for 5 min. Rabbit polyclonal anti-C3a (negative control antibody) and anti-C3b (Accurate) were pre-adsorbed ×3 by adding a 1:50 dilution of antibody in phosphate buffered saline (PBS) pH 7.4 to an equal volume of packed pre-washed erythrocytes from the normal standard control. The cells were incubated for 1 hr at 37°C with constant rocking followed by centrifugation. The pre-adsorbed antibody was frozen at -20°C in single-use aliquots. 100 μl of pre-washed freshly thawed erythrocytes at 1% hematocrit in Alsever's buffer was added to wells of a 96-well plate and resuspended in 50 μl of pre-adsorbed rabbit anti-C3b, anti-C3a, or in PBS (unstained control), and incubated for 10 min at 37°C. After two washes in PBS, the cells were resuspended in 1:50 anti-rabbit PE (Sigma-Aldrich) for 30 min at room temperature, washed twice, and resuspended again in PBS. Acquisition was carried out as above. The %C3b-positive cells were calculated by Overton subtraction  of the bFor IC opsonization, 5 μl of stock IC or RPMI 1640 (unstained control) was incubated in a total volume of 100 μl containing 30% AB+ serum in wells of a 96-well plate. For a negative control, a separate set of wells contained IC plus 10 mM EDTA. Following incubation at 37°C for 30 min with constant rocking motion, 100 μl of 1% hematocrit suspension of freshly thawed red cells from each study participant or from a standard aparasitemic control in RPMI 1640 was added to each of the wells of the above 96-well plate. This was followed by further incubation for 30 min at 37°C. The erythrocytes were then washed twice in 200 μl of ice-cold RPMI 1640, resuspended in PBS containing 1% paraformaldehyde, and stored at 4°C until acquisition. After gating, the erythrocyte FITC fluorescence was measured using logarithmic amplification and the positive cutoff was set using the unstained cells. The percent of positive red cells (IC binding capacity) was calculated based on this cutoff. To control for day-to-day variation, the IC binding capacity was normalized to the mean IC binding capacity of the red cell standard used throughout using a formula similar to the one used for correction of CR1 and CD55 (above).th and 75th percentile, the line through the box represents the median, the whiskers the 10th and 90th percentile limits, and the dots are the outliers. Analysis of variance (ANOVA) was used to detect differences across age groups adjusting for factors and covariates as indicated in the figure legends. P values were adjusted for multiple comparisons using the Sidak correction. The independent samples t-test was used for comparisons of normal continuous data between two groups. The chi-square test was used to compare proportions across groups. The general linear model procedure was used to carry out univariate ANOVA and analysis of covariance (ANCOVA) to identify independent variables associated with the %C3b-positive red cells and the hemoglobin level. Age groups, red cell CR1, red cell CD55, parasite density, malaria status, and IC binding capacity were the independent predictor variables included in the models. %C3b-positive red cells were also used as an independent variable for the hemoglobin level. Categorical variables were transformed into dummy variables and the B coefficient for the last group set to '0' for reference. All tests were two-sided with α ≤ 0.05.Statistical analysis was carried out using SPSS v11.5 . The flow cytometry and parasite density data are presented graphically for each age group as box plots, where the box represents the boundaries between the 25Four hundred and two potential participants or parents of children who met the inclusion criterion signed the consent form and were screened. Of these, 344 met none of the exclusion criteria, attended the screening sessions, and agreed to provide blood samples Table . Blood sThe nadir of hemoglobin level occurred in the >12 to ≤ 18 month group Figure . For mulP. falciparum malaria and the parasite density both rose and peaked within this age group. However, the prevalence of P. falciparum malaria  had a high level of %C3b-positive red cells, but this was explained by relatively low red cell CR1 in these individuals. With few exceptions there were no significant differences in levels of CR1, CD55, and hemoglobin between malaria-treated and aparasitemic volunteers . For the adjusted analysis of samples from malaria-treated individuals, age was the only statistically significant factor followed by CR1, which showed a trend towards significance (P = 0.09). Of interest, parasite density was not important in either analysis. Lastly, in the adjusted analysis of samples from malaria-negative individuals, CD55 and IC binding capacity made the strongest contribution after age.We carried out analysis of variance to identify the factors most strongly associated with the %C3b-positive red cells and with the hemoglobin level in all the samples and in the subgroups of aparasitemic and malaria-treated participants Tables . For theFor the analysis of factors associated with the hemoglobin level Table , once agP. falciparum occurs principally in young children and is most common in areas of intense malaria transmission [Severe anemia due to smission ,5,38. Cesmission . Identifsmission . This pasmission . Our prismission ,42, we cP = 0.09), CR1 appeared to be more important than CD55 in protecting red cells from C3b deposition during malaria infection (Table The relationship between red cell C3b and red cell complement regulatory protein expression seemed complex Figures  and 2a. The presence of parasitemia seemed to be a more important contributor to C3b deposition than the degree of parasitemia. This is somewhat surprising since although parasite density peaked at an earlier age than parasite prevalence Figures  and 2c, It is interesting that increased C3b deposition was also seen on red cells of children with low red cell complement regulatory protein but without malaria infection, suggesting that there is a defect in the normal regulatory mechanisms during this age that prevent spontaneous complement activation. Alternatively, C3b on aparasitemic individuals may have been residual from recent previous malaria infections or infections with other agents. It is also possible that some or many of these individuals have levels of parasitemia below the limit of detection of microscopy.The question of whether parasite prevalence or parasite density is a contributor to uninfected red cell destruction has important ramifications for the understanding of the pathogenesis of severe anemia during malaria. Current belief is that natural acquired immunity does not sterilize but suppresses the level of parasitemia and, therefore, parasite density is a reflection of the level of immunity . Hence, The most important variables that influenced the hemoglobin level in this study were age, red cell CR1, %C3b-positive red cells, and red cell CD55 Table . InteresThe two ANOVA models Tables  and 4 suOur data suggest that children >6 to ≤ 36 months of age who develop malaria are at high risk of uninfected red cell destruction due to ineffective complement regulation. They have relatively low red cell CR1 that may be insufficient to control complement activation on cell surfaces. On the other side of this equation is the contribution of macrophage activation, which should also be considered. Macrophages can be activated by the malaria pigment hemozoin as well as by malaria GPI, leading to increased production of pro-inflammatory cytokines which can upregulate the expression of complement receptors leading to phagocytosis -50. In aThese results suggest that increasing malaria prevalence among children >6 to ≤ 36 months of age in western Kenya together with low red cell CR1 and CD55 levels result in increased C3b deposition on red cells leading to increased red cell destruction by phagocytosis. The strong contribution of age to C3b deposition suggests that there are still additional unidentified age-related factors that increase the susceptibility to C3b deposition on red cells and red cell destruction.ANCOVA: analysis of covariance; ANOVA: analysis of variance; DAF: decay accelerating factor; EDTA: ethylenediaminetetraacetic acid; FITC: fluorescein isothiocyanate; GPI: glycosyl-phosphatidyl-inositol; IC: immune complex; MFI: median fluorescence intensity; PBS: phosphate buffered saline.The authors declare that they have no competing interests.COO carried out flow cytometry to measure the red cell C3b. WO supervised the recruitment and evaluation of the study participants. CA supervised the measurement of clinical laboratory parameters. MMO carried out flow cytometry to measure red cell CR1, CD55, and IC binding capacity. JAS and WO designed the study and wrote the protocol. COO and JAS drafted the manuscript which was reviewed and approved by all.The pre-publication history for this paper can be accessed here:


Plasmodium falciparum allelic forms.Malaria transmission in Equatorial Guinea and its space-time variability has been widely studied, but there is not much information about the transmission of malaria on the small island of Annobon. In 2004, two transversal studies were carried out to establish the malaria transmission pattern on Annobon and analyse the circulating Plasmodium infection by microscopical examination and by semi-nested multiplex PCR. The diversity of P. falciparum circulating alleles was studied on the basis of the genes encoding for the merozoite surface proteins, MSP-1 and MSP-2 of P. falciparum.A blood sample was taken from the selected children in order to determine msp-1 gene were identified and 39 of the msp-2 gene. The variability of circulating allelic populations is significantly higher in the rainy than in the dry season, although the multiplicity of infections is similar in both, 2.2 and 1.9 respectively.The crude parasite rate was 17% during the dry season and 60% during the rainy season. The percentage of children sleeping under a bed net was over 80% in the two surveys. During the rainy season, 33.3% of the children surveyed were anaemic at the time of the study. No association was found between the crude parasite rate, the use of bed nets and gender, and anaemia. However, children between five and nine years of age were five times less at risk of being anaemic than those aged less than one year. A total of 28 populations of the three allelic families of the Based on the high degree of geographical isolation of the Annobon population and the apparent marked seasonality of the transmission, it is feasible to believe that malaria can be well controlled from this small African island. Plasmodium falciparum is a highly polymorphic parasite with a high antigens heterogeneity  in the dry season and 60% (50/83) [CI: 49%–71%] in the rainy season. In the first survey, prevalence of infection was significantly higher among males than females . This difference was not noted in the second survey  .Plasmodium have been described, although infections by Plasmodium vivax are anecdotical [P. falciparum, 96% (66/69). The mixed infections  diagnosed by microscopy in three percent of infections were not confirmed by PCR. The gametocytic index (percentage of studied population with gametocytes in blood) was 6% (7/112) during the dry and 14% (12/83) in the rainy season.In Equatorial Guinea, four species of human cdotical . The maiThe percentage of children sleeping under a bed net was over 80% in the two surveys. Furthermore, the number of girls that claimed to sleep under a bed net was significantly higher than the number of boys, both in dry  and rainy season . Children aged between five and nine were less protected by a bed net, although the difference with respect to the rest of the groups was not significant  . In the study conducted during the dry season, only 3.1% (3/98) of the children surveyed had a PCV percentage less or equal to 30%. PCV percentage could not be measured in 14 cases. With such a low sample size, the relation between the anaemia and the rest of the measured variables  could not be analysed. However, during the rainy season, 33.3% of the children surveyed (26/78) were anaemic at the time of the study. PCV percentage could not be measured in five cases.Table msp-1 gene were identified and 39 of the msp-2 gene. Out of these, 17 for the msp-1 gene and nine for the msp-2 gene were present in the two climatic seasons. Fifty-seven per cent of the MAD20 allelic families of the msp-1 gene were only present in the samples gather during the dry season and 66.7% of those of the msp-2 gene only in the rainy season. The only allelic form belonging to the RO33 family of the msp-1 gene was observed in the two surveys , while in the case of the msp-2 gene this number nearly triples (13/35).The variability of circulating allelic populations is significantly higher in the rainy than the dry season . During the dry season, the number of The multiplicity of infections (MOI) is similar during the dry and rainy season , the MOI average for the dry season is 2.2 (SD = 1.3), while it is 1.9 (SD = 1.0) for the rainy season.P. falciparum positive samples in the dry season was insufficient to carry out this type of analysis.In the case of the samples taken during the rainy season, no association was observed between age, gender, use of bed nets and anaemia, and MOI values over 1. The number of The transmission of malaria on the Island of Annobon is stable although mainly occurs during the rainy period. The number of diagnosed malaria cases between 2002 and 2003, in the unique health center of the island , was 660 (314 per thousand inhabitants) in the dry season and 1,370 (651 per thousand inhabitants) in the rainy season.A. gambiae s.l was identified as the malaria vector. It is necessary to perform an entomological longitudinal study on Annobon to identify the main malaria vector to verify the transmission pattern.Taking into account the results obtained in the transversal studies and the regional hospital registry, it could be hypothesized about a seasonal malaria transmission in the island. This markedly seasonal transmission pattern had not been observed in previous studies ,14, nor In Annobon, a significant difference was observed in the prevalence of infection between sexes  during the dry season. Furthermore, the prevalence of infection was higher in children between five and nine years old, but only during the rainy season. The CPR difference between sexes was not observed in studies conducted in other regions of the country, on the island of Bioko ,23 and mThe only malaria prevention strategy in Annobon is currently the use of insecticide-treated bed net. There are no distribution and reimpregnation points on the island, which means that all the bed nets come from the country's capital, Malabo. Despite this, the cover reached on the island is high (80% of the children surveyed said that they slept under a bed net), although some differences were noted between sexes  and age groups (five to nine years old less than other age groups). This would neither explain the differences found in the prevalence of infection, nor the reason for the seasonal differences. However, it is possible that the sample size calculated to dry season was insufficient to established differences between groups, because of the low CPR found in this season against preliminary data .On the other hand, the percentage of children surveyed with anaemia was significantly higher in the greater transmission period (rainy season). No seasonal differences in the prevalence of anaemia were observed in Bioko, nor were any associations found with variables such as the sex, age, place of residence and use of the bed net . On AnnoP. falciparum population variability, seasonal differences in the variety of circulating populations were observed. When analysing this difference in depth, it was observed that the majority of allelic forms of the RO33 and K1 families of the msp-1 gene are present in the two climatic seasons. However, only one third of the allelic forms of the MAD20 family (msp-1 gen) and 23% of the allelic forms of the msp-2 gene were present in the samples collected in both seasons. Therefore, it must be attributed the greater seasonal variability to the alleles of the msp-2 gene. The multiplicity of infection is high and similar in the two seasons and it is not associated with age, sex, the use of the bed net or suffering from anaemia.With respect to the analysis of the msp-2 alleles or on the multiplicity of infection [msp-1 gene populations. Furthermore, it was discovered that the allelic variability on these islands was lower than that observed in others countries with a lower transmission level.In other regions of Africa with a markedly seasonal transmission, such as is the case in Sudan, it has been observed that the diversity of genotypes and multiplicity of infection reduces significantly with the transmission . In the nfection . In a renfection , where tmsp-2 gene in island zones are available. During a study conducted on São Tomé Island, up to 43 msp-2 alleles were identified, although the variability between climatic seasons was not analysed [Few studies performed to determine the seasonal differences in the allelic diversity of the analysed .msp-2 gene allelic families.In the study conducted on Annobon, the influence of transmission intensity on the variability of the circulating parasite populations has been suggested, particularly for the msp-2 gene observed in rainy season.It is known that the genetic recombination occurs during the sexual phase of the biological cycle of the parasite, therefore one of the factors to be taken into account will be the infection rate in the vector. In Equatorial Guinea, the low anopheline density, compared to neighbouring countries , is offsP. falciparum allelic forms on the island, may suggest that the impact of bed net usage on transmission is low.In this way, it is logical to think that the introduction of efficient control measures would result in a reduction in the population heterogeneity of the parasite. In Annobon, 80% of the children studied said that they slept under a bed net. This high cover contrasts with the prevalence of infection and the differences found in terms of gender and age groups. On the other hand, no association was found between the risk of being anaemic and whether or not a bed net was used, which contradict the results obtained in other African countries , even foThis low protective efficiency of the bed nets may be due to biting habits of the vector species that are not compatible with this measure of protection, and poor state-of-repair and/or inappropriate use of the bed nets by the population. These are important aspects that must be studied in the future to learn more about the malaria transmission mechanisms on the island.P. falciparum allelic diversity in Annobon, it is conceivable that the lack of efficient control measures, a high entomological inoculation rates and crowded households could be related to this high allelic diversity.With respect to the P. falciparum resulted in a serious epidemic with many deaths [During the second half of the last century, it was believed that malaria could be eradicated. Good results were obtained in controlling the transmission of the disease in some regions of Asia, South America and Central America and on some African islands (Sao Tomé and Príncipe), and its eradication in the majority of the countries of the Mediterranean basin . On the y deaths . In 1999y deaths .The most recent experience to control and eradicate malaria was in Vanuatu, an island complex in the south-west Pacífic . In 1991Taking this experience into account, the high degree of geographical isolation of the Annobon population and the suggested seasonal nature of the transmission, it is conceivable that malaria could be eradicated in this small African island, as long as the sustainability of the malaria control programme can be ensured.JC was involved in the design of the survey, participated in the data collection and the interpretation of statistical analysis, and coordinated the draft of the manuscript. PB was involved in the molecular studies and helped to draft the manuscript. AL was involved in the molecular analysis. MD was performed the statistical analysis and interpretation, and drafted the manuscript. SN, LB, MO and JB participated in the collection of the data and blood samples. AB and GN participated in the design of the surveys and have given approval of the version to be published. All authors read and approved the final manuscript.


Plasmodium vivax re-emerged in 1993. Although the number of infections has been steadily decreasing, it is likely to continue to affect public health until it is eradicated. The aim of this study is to measure anti-circumsporozoite protein (CSP) antibody and compare malaria prevalence. As to understand the prevalence, an epidemiology study has to be conducted in the Republic of Korea.Escherichia coli transformed with a CSP gene-inserted pET-28a(+) expression vector. Microscopic examination was performed to identify malaria parasites.A total of 1,825 and 1,959 blood samples were collected in 2010 and 2011, respectively, from the inhabitants of Ganghwa and Cheorwon counties. The antibody titers of the inhabitants were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant protein purified from P < 0.01). The intensity of the immune responses of the inhabitants of Cheorwon, as measured by the mean optical density, decreased from 0.9186 ± 0.0472 in 2010 to 0.7035 ± 0.0457 in 2011 (P = 0.034), but increased in Ganghwa from 0.7649 ± 0.0192 in 2010 to 0.8237 ± 0.1970 in 2011 (P = 0.006). The immune response increased according to age .The annual parasite incidence (API) in Ganghwa decreased from 4.28 in 2010 to 2.23 in 2011, and that in Cheorwon decreased from 1.88 in 2010 to 1.15 in 2011. The antibody-positive CSP rate in these areas also decreased from 18.14% (331/1825) in 2010 to 15.36% (301/1959) in 2011. Pearson analysis showed a strong correlation between the API and the antibody-positive CSP rate in these areas (r = 1.000, The positive CSP-ELISA rate was closely related to the API in the study areas. This suggests that seroepidemiological studies based on CSP-ELISA may be helpful in estimating the malaria prevalence. Moreover, such studies can be used to establish and evaluate malaria control and eradication programmes in high-risk areas in Korea. Plasmodium vivax is the causative agent of relapsing benign tertian human malaria, the second most common type of human malaria, and it afflicts several hundred million individuals annually. This disease is a major public health problem in most tropical regions and many temperate countries, including the Democratic People’s Republic of Korea (DPRK) and the Republic of Korea (ROK) [ea (ROK) .et al. subsequently reported cases in two infected civilians [The first scientific documentation of malaria occurrence was published in 1913, although malaria had been prevalent throughout the Korean peninsula for several centuries . As a reivilians . Thereafivilians .Parasitaemia provides the basis for the classical method of measuring the endemicity of malaria. However, using data on only the incidence of parasitaemia may be insufficient to adequately determine the epidemiology of malaria in a given population. For example, when the incidence of malaria is low, mass blood surveys do not yield results commensurate with the work involved ,10. SeroThe annual parasite incidence (API) in Ganghwa county Figure A fluctuaEscherichia coli to produce antigen for the detection of anti-P. vivax CSP antibody levels in the inhabitants of Ganghwa and Cheorwon counties to evaluate CSP antigen in comparing the local malaria prevalence.In this study, the dormant form of CSP was expressed and purified from 2 that is covered with forested hilly mountains , agricultural land , building site , and others . The total population of Ganghwa county was 67,668 persons  living in 29,055 households in 2010 [2 that is covered with forested hilly mountains , agricultural land , building site , and others . The total population of Ganghwa county was 49,463 persons  living in 20,316 households in 2010 [The study was conducted in Ganghwa county of Incheon Metropolitan city and Cheorwon county of Gangwon province, ROK. Ganghwa county  contains 179 villages  in an area of 411 kmThe study locations are shown on the map in Figure Blood smears were prepared for microscopic examination. Sera were separated and stored at -20°C for antibody analysis. Informed consent was obtained from all individuals. All samples were collected using human protocols that were reviewed and approved by the Human Ethics Committee of Inha UniversityThin blood films were prepared to determine the infectivity of blood samples. The blood films were fixed with methanol and stained with Giemsa stain diluted with buffered water at pH 7.2 to emphasize the parasite inclusions in the red blood cells (RBCs). The fixed monolayer of RBCs in this procedure makes the morphological identification of the parasite to the species level much easier and provides greater specificity than that obtained with thick-film examination. Thin blood films are often preferred for routine estimation of parasitaemia because the organisms are easier to see and count with this method . To estiTo express the CSP gene, genomic DNA was extracted from the whole blood of a patient with malaria using a QIAamp Blood Kit . Polymerase chain reaction (PCR) was performed using AccuPower PCR PreMix , 50 ng of purified genomic DNA, and 40 pmoles each of forward  and reverse primer . The total volume was adjusted to 50 μl with distilled water. The thermocycler conditions were as follows: denaturation at 94°C for 5 min; 35 cycles of 30 s at 94°C, 30 s at 55°C, and 45 s at 72°C; and finally, incubation at 72°C for 5 min. All PCR products were analyzed on a 1% agarose gel, confirmed under a UV transilluminator, and purified with a NucleoSpin Extract Kit .P. vivax, the PCR product of the CSP gene was ligated into a pGEM-T Easy Vector  and transformed into E. coli DH5α. The PCR product containing E. coli DH5α was selected on ampicillin-containing medium [EcoRI digestion products after the plasmid was prepared with a Qiagen plasmid isolation kit according to the protocol supplied by the manufacturer. The CSP gene sequence was determined using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit FS  according to the manual supplied by the manufacturer. M13 reverse and M13 forward (-20) primers were used in the sequencing. Nucleotide and deduced amino acid sequences were analyzed using EditSeq and Clustal in the MegAlign program, a multiple alignment program in the DNASTAR package . The internet-based BLAST search program of the National Center for Biotechnology Information was used to search protein databases.To genotype the CSP gene of g medium . To confE. coli DH5α, a CSP gene fragment was amplified from a blood sample that was confirmed to be infected with the dormant type of P. vivax as described above with the exception of the addition of the Fex2 (5′-ggatccAAAAAGGATGGAAAGAAAG-3′) and Rex2 (5′-aagcttGACTTTTCATTTGGGGCA-3′) primers, which contain BamHI and HindIII sites on their 5′ ends, respectively. The amplified PCR products were digested with BamHI and HindIII, purified with a Qiagen gel extraction kit after being run on an agarose gel, and then integrated into the BamHI and HindIII cleavage sites of the pET-28a(+) expression vector . The resulting plasmid was subsequently used for the expression of a CSP-(His)6 fusion protein in E. coli. The transformants were confirmed by both gel electrophoresis of plasmid DNA after restriction enzyme digestion with BamHI and HindIII and DNA sequencing.To express the CSP gene in E. coli DH5α with isopropyl-1-thio-β-D-galactopyranoside (IPTG) [6 fusion protein was purified using immobilized metal ion affinity chromatography [Expression of the recombinant protein was induced in e (IPTG) . The CSPtography . The pur6 fusion protein was separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. After the transfer, the membrane was cut into strips and blocked for nonspecific binding with 3% skim milk for 12 h at 4°C. The membrane was then washed three times for 10 min each with 0.15% Tween 20-PBS. The strips were allowed to react with sera from patients with malaria or from uninfected individuals  for 4 h; they were then washed using the procedure described above. The membrane was subsequently incubated with diluted peroxidase-conjugated goat anti-human IgG secondary antibody  (Sigma) for 3 h at room temperature. For colour development, a solution containing 0.2% diaminobenzidine and 0.02% H2O2/PBS was applied to each well [The recombinant CSP-(His)ach well ,23.P. vivax. Briefly, the capture antigen solution  was placed on a 96-well plate  and incubated for 12 h at room temperature. The wells were aspirated and filled with blocking buffer  and incubated for 1 h at room temperature. After the wells were washed with 0.05% PBS-Tween 20 three times, human serum samples in blocking buffer at a dilution of 1:100 (vol/vol) were added to the wells. Four positive and four negative control serum samples were also added to each plate. After 2 h of incubation at room temperature, the plates were washed with 0.05% PBS-Tween 20 three times, and peroxidase-conjugated anti-human IgG  diluted in blocking buffer was then added. The plates were re-incubated for 1 h at room temperature. The reaction was stopped by washing the plates as described above. To develop the colour, 100 μl of 2.2′-azino-di-(3-ethyl-benzthiozoline-6-sulfonic acid) peroxidase substrate  was added, and the plates were incubated for 30 min. Absorbance was measured at 405 nm, and the cut-off value for positivity was defined as the mean + 3 standard deviations of the negative control samples. Negative sera were collected from volunteers among the staff of the Korea National Institute of Health (KNIH).Enzyme-linked immunosorbent assays (ELISAs) were used to determine whether the blood samples contained antibodies against the CSP VK210 of The annual parasite incidence (API) was calculated as the number of malaria-positive patients per 1,000 inhabitants for each of the study sites using microscopy: API =  × 1,000.P. vivax in a given year. The data were analyzed using SPSS software, version 17.0 . A P value of <0.05 was considered statistically significant. The correlation sizes were interpreted as none (0.0–0.09), small (0.1–0.3), medium (0.3–0.5), or strong (0.5–1.0) [The differences in the positive CSP rates between 2010 and 2011 were determined by a Mann–Whitney test. Data analyses were performed using GraphPad software . Pearson’s correlation analysis was performed to examine the relationship between seropositivity and the API of 0.5–1.0) .EcoRI digestion. The plasmid containing the PCR product was named pCS210 and was used for DNA sequence analysis. DNA sequencing revealed that the cloned CSP gene was 717 bp long and comprised 239 amino acids that were identified by DNASIS. The nonapeptide repeat unit, located between Region I (KLKOP) and Region II (PCSVT), showed the sequence GD(N)R(G)AD(G/A)GQP(A)A and was repeated 18 times.The CSP gene that was amplified by PCR from genomic DNA was analysed on a 1.0% agarose gel. Amplification of the CSP gene yielded an approximately 750-bp DNA fragment Figure A that, aBamHI and HindIII, and subcloned into the same restriction enzyme sites of expression vector pET-28a(+)  grown to logarithmic phase in liquid LB plus 100-μg/ml ampicillin to induce expression of the target protein. SDS-PAGE followed by Coomassie blue staining showed that the molecular weight of the CSP recombinant protein was 45 kDa under native purification conditions  Figure B. The reThe antigenicity of the CSP recombinant protein was determined by western blot. The sera of patients with malaria reacted positively Figure B. To detTwo large islands located west of Ganghwa, Gyodongdo island (Gyodongmyeon) and Seokmodo island (Samsanmyeon), were also surveyed Figure A. A totaP < 0.01). However, the seropositivity of CSP in 2011 showed a strong negative linear relationship with the APIs of 2010 and 2011   than in 2011 . However, Samsanmyeon had a higher positive rate in 2011  than in 2010 . The API for 2010 (4.28) was higher than that for 2011 (2.23). The APIs for 2010 (4.86) and 2011 (3.56) in Gyodongmyeon were higher than those for 2010 (3.50) and 2011 (0.44) in Samsanmyeon. The seropositivity of CSP in 2010 showed a strong positive linear relationship with the APIs of 2010 and 2011 . However, the seropositivity of CSP in 2011 showed a strong negative linear relationship with the APIs of 2010 (r = -0.926) and 2011 (r = -0.931), but again without statistical significance   and 45 of 611 (7.36%) study subjects in Cheorwon    . The corridor is heavily fortified on both sides of the buffer zones with land mines and barbed wire fences. Therefore, it is believed that these areas are exposed to mosquitoes. To estimate the prevalence of malaria exposure in these high-risk areas in Korea, CSP recombinant proteins were applied. It is a sporogony-stage protein that exists on the surface membrane of all plasmodium sporozoites. CSP has a central immunodominant region comprising short tandem repeat amino acid sequences that contain multiple copies of the immunodominant B-cell epitope [P. falciparum isolates of diverse geographical origins and of a smaller number of P. vivax isolates have been conserved within the species [P. vivax has been reported to be as long as 279 ± 41 days  [Our malaria research team is interested in diagnosing vivax malaria based on antibody detection. Although microscopic examination is the gold standard method for malaria diagnosis worldwide, it has disadvantages, including the need for trained experts, its time-consuming nature, and its tendency to miss cases of low parasitaemia levels. However, antibody detection can compensate for the disadvantages of microscopic examination. If an antibody detection method is applied in the field using a rapid diagnostic test kit, the method can be used without rigorous training and the time required for diagnosis will be reduced. Furthermore, most patients with malaria have antibodies against malaria parasites, even those with low parasitaemia levels, and this method can be applied on a large seroepidemiologic scale. In this study, the CSP of the vivax malaria antigen was used to seroepidemiologically evaluate its usefulness in understanding malaria transmission. The survey areas, Ganghwa and Cheorwon, are two re-emerging malarial outbreak areas in South Korea, and both areas are located within 10 to 15 km of the southern DMZ . The inc epitope . Because species . Interes species . These f52 days) . This fi52 days) . However52 days) . Even ifTo evaluate malaria transmission in a given geographical region, many factors, including temperature, mosquito density, vector capacity, climate, rainfall, and humidity, should be considered . ParasitAntibody detection using CSP-ELISA may provide useful information regarding malaria prevalence in certain areas and individuals. These serological methods are useful in identifying areas that require malaria control and evaluating the surveillance system in certain areas.The authors declare that they have no competing interests.TSK, SHC, YS, HIC, BKN, and HWL conceived and designed the study and contributed to the execution of the research. HWL and TSK wrote the manuscript. YS, BKN, and WON performed the statistical analysis. SHC, JHP, WON, SJH, WJL, SKL, YKP, PYC, SKA, JSK, and YYB collected the blood samples. SHC, YJK, PYC, SJH, WJL, and JHP performed CSP-ELISA. SWL (Eastside High School) who has been working ay the University of Florida involved in expression of CSP recombitant protein and performed ELISA. HIC also provided most of research funding for this study. All authors have read and approved the final manuscript.


As HIV becomes a chronic infection, an increasing number of HIV-infected patients are travelling to malaria-endemic areas. Association of malaria with HIV/AIDS can be clinically severe. Severe falciparum malaria is a medical emergency that is associated with a high mortality, even when treated in an Intensive Care Unit. This article describes two cases of HIV-positive patients, who returned from malaria-endemic areas and presented a parasitaemia > 5% of erythrocytes and clinical signs of severe falciparum malaria, both with > 350 CD4 cell count/μl, absence of chemoprophylaxis and successful response. Factors like drug interactions and the possible implication of anti-malarial therapy bioavailability are all especially interesting in HIV-malaria co-infections. Plasmodium falciparum imported malaria causes severe clinical episodes every year in European travellers. Between 2% and 16% of these infections are severe cases according to the World Health Organization (WHO) definition [finition . Currentfinition . Studiesfinition -9. Otherfinition . Therefofinition .P. falciparum malaria in two HIV-infected patients are presented and discussed, because of the possibility of improved diagnosis methods needed on admission or for follow-up anti-malarial therapy.Two clinical cases of imported 9 cells/L, N 89.1%, L 6.9% M 1.6%), moderate thrombocytopaenia , normal glucose levels (110 mg/dL), bilirubin , creatinine  and slightly increased aspartate transaminase  and an elevated C-reactive protein concentration of 157 mg/L . Malaria parasites were seen on Giemsa-stained thick and thin blood films with Field's stain in 7% of erythrocytes. Parasite morphology identified P. falciparum. A rapid diagnostic test (RDT) result for histidine-rich protein 2 of P. falciparum  was positive. Results of multiplex PCR assay, species-specific nested-PCR [P. falciparum. The patient was admitted to the intensive care unit with several signs of severe malaria  for monitoring. Patient was treated with quinine and doxycycline intravenous for seven days at the recommended doses in adults. The anti-retroviral therapy with tenofovir, emtricitabine and efavirenz was not interrupted. On day 2 the parasitaemia was 1%. The parasites were cleared after six days without recrudescence. The hospital stay lasted 13 days.A 47-year-old man from Spain presented in University Hospital La Paz in Madrid, Spain, with a two-day history of intermittent fever, headache and fatigue. The patient had returned a week earlier from a four-week trip to Equatorial Guinea. He did not take anti-malarial chemoprophylaxis during the visit. He was a category A3 HIV-infected patient with a CD4 cell count of 650/μL and HIV viral load of less than 20 copies/mL. He had been taking tenofovir, emtricitabine and efavirenz since 2008. On examination, he was febrile (38.7°C) and had a heart rate of 120 beats/min, blood pressure of 93/64 mmHg, normal respiration rate and oxygen saturation 95% on room air. Laboratory investigations showed normal haemoglobin concentration (14.8 gr/dL), normal cell count and leukocyte formula , moderate thrombocytopaenia , normal glucose levels (104 mg/dL), bilirubin , creatinine  and slightly increased aspartate transaminase  and ALT  and an elevated C-reactive protein concentration of 112 mg/L . Malaria parasites were detected by microscopic examination in 0.5% of his erythrocytes. The RDT  was positive for P. falciparum. The patient was treated with atovaquone/proguanil 250 mg/100 mg, every six hours for three days. On day 3, he was again febrile with P. falciparum parasitaemia nearly 8% of erytrocytes. His laboratory results showed a decreased haemoglobin concentration of 10.0 gr/dL and thrombocytopaenia more severe than 33 × 109 cells/L. Anti-malarial treatment was changed to intravenous quinine and doxycycline for seven days and was monitored in the intensive care unit for the first 24 hours. The anti-retroviral therapy with tenofovir, abacavir and darunavir/ritonavir was stopped, after initiating the malarial treatment with quinine. On day 6 he was cleared of parasitaemia. He was discharged after 10 days. The multiplex PCR assays performed on the sample collected on the day of admission confirmed that the patient was only infected with P. falciparum. Analysis of point mutations in the cytochrome b gene, related to resistance to atovaquone, and in the dihydrofolate reductase (DHFR) gene, related to proguanil resistance, show a single mutation in the cytochrome b gene (L283I) and four point mutations for the DHFR gene . He had not taken anti-malarial chemoprophylaxis. He had been diagnosed with HIV infection 10 years before. He was taking tenofovir, abacavir and darunavir/ritonavir with a CD4 cell count of 432/μL and undetectable viral load (less than 20 copies/mL). He was admitted to the emergency department with a temperature of 39.9°C but he was in good general condition with a normal respiratory rate, blood pressure of 147/88 mmHg, and normal oxygen saturation. The day of admission, laboratory investigations revealed: normal haemoglobin concentration , normal white cell count and leukocyte formula  and dihydrofolate reductase (dhfr) gene respectively. Isolates of P. falciparum collected by the European Network on Imported Infectious Disease surveillance were analysed for single-point mutations of the cytb gene and the results showed a level < 1% of these mutations [cytb gene showed the mutation L283I. This mutation, associated with V284K, shows a 76-fold increase in IC50 of atovaquone compared to non-mutant strains, but this effect must be the direct responsibility of V284K, as I283 has been observed in atovaquone sensitive parasites [P. falciparum malaria, but we do not have an answer to this question: could the HIV-infected patients have also a higher rate of atovaquone/proguanil treatment failure? No data about this point have been reported in HIV-infected patients. This question must be approached with caution. An explanation could be a delayed parasite clearance due to a slowness of action by atovaquone/proguanil, but the parasitaemia increased from 0.5% to 8% on day 3. On the other hand, treatment failure could be attributed to poor bioavailability of the atovaquone, proguanil or its active metabolite, cycloguanil. The biotransformation of proguanil could have been reduced. A possible interaction between atovaquone/proguanil and IP-based regimens could be presented [In the second case, the patient was initially treated with atovaquone/proguanil. Atovaquone-proguanil has shown high efficacy against multi- drug-resistant al drugs , such asutations . Atovaquutations . In the arasites . The preresented . HoweverP. falciparum malaria. However, it is not useful in the follow-up anti-malarial therapy, because it could positive HRP-2 antigen for three or four weeks after a successful therapy. The PCR-based methods are extremely sensitive with a detection limit of < 10 parasites/μl. Various authors have reported the development of a real-time PCR assay to detect P. falciparum, Plasmodium vivax and Plasmodium ovale in routine clinical diagnosis [In routine clinical practice, the PCR is performed on admission samples to confirm the results obtained by microscopy, as shown in this cases, and to avoid the limitations of the microscopy and RDT: misdiagnosis of mixed-species infections and low parasitaemia. The microscopic examination of Giemsa-stained thin and thick blood films remains the "gold standard" for the diagnosis of malaria and detects parasites at a density of 50 parasites/μl by an experienced microscopist . The RDTiagnosis . The risP. falciparum malaria, high parasitaemia, both severe clinical episodes with > 350 CD4 cell count/μl, absence of chemoprophylaxis and successful response. Factors such as drug interactions and the possible implication of anti-malarial therapy bioavailability are all especially interesting in HIV-malaria co-infections.In conclusion, this are two cases of HIV-infected patients with imported 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.SGB, CNSF, JMR, JRAL and AGA conceived this case report and wrote the paper. CNSF and JRAL participated in the medical care and follow-up of the patients. SGB, CNSF, drafted the manuscript and collect data. JMR was responsible for molecular detection and analysis of sequence data. All authors have read and approved the final manuscript.


Malaria in pregnancy is a major public health problem in endemic countries. Though the signs and symptoms of malaria among pregnant women have been already described, clinical presentation may vary according to intensity of transmission and local perceptions. Therefore, determining common signs and symptoms among pregnant women with a malaria infection may be extremely useful to identify those in need of further investigation by rapid diagnostic test or microscopy.Six hundred pregnant women attending the maternity clinic of Nanoro District Hospital, Burkina Faso were recruited, 200 with suspected clinical malaria and 400 as controls. Cases were matched with controls by gestational age and parity. Signs and symptoms were collected and a blood sample taken for rapid diagnostic test, microscopy and haemoglobin measurement. A multivariate model was used to assess the predictive value of signs and symptoms for malaria infection.The overall prevalence of malaria was 42.6% (256/600) while anaemia was found in 60.8% (365/600) of the women. Nearly half (49%) of the cases and 39.5% of the controls had a malaria infection (p = 0.03). The most common signs and symptoms among the cases were fever , history of fever  and headache . The positive predictive value for fever was 53% (95% CI:41–64), history of fever 58% (95% CI:37–63) and headache 51% (95% CI:41–61).Signs and symptoms suggestive of malaria are frequent among pregnant women living in areas of intense transmission. Common malaria symptoms are not strong predictors of infection. For a better management of malaria in pregnancy, active screening to detect and treat malaria infection early should be performed on all pregnant women attending a health facility. Plasmodium falciparum and MiP is predominantly asymptomatic and yet a major cause of maternal anaemia and low birth weight (LBW), the latter increasing the risk of infant death. Successful control of MiP may prevent 75,000-200,000 infant deaths every year [Malaria in pregnancy (MiP) is a major public health problem in endemic countries where 31 million pregnancies occur annually, resulting in approximately 23 million live births [ery year .The current strategy for clinical case management of malaria in pregnant women is based on the result of rapid diagnostic tests (RDT) or microscopy if available. Though the signs and symptoms of malaria among pregnant women have been already described in few settings, clinical presentation may vary according to intensity of transmission and local perceptions. Therefore, determining common signs and symptoms among pregnant women with a malaria infection may be extremely useful to identify those in need of further investigation by rapid diagnostic test or microscopy -7. FurthThe study was carried out at the Clinical Research Unit of Nanoro (CRUN), situated in the centre of Burkina Faso, 85 km from Ouagadougou, the capital city. Nanoro district is one of the five districts of the Centre-West health region. The main local language is Mooré, though French is the official language. The literacy rate is low for both men and women (about 23%) and there is a high migration flux among the youngsters toward the capital city and/or the border countries.Anopheles gambiae sensu stricto, Anopheles funestus and Anopheles arabiensis, and malaria is one of the most common reasons for attending a health facility [Plasmodium falciparum is the predominant malaria parasite. Malaria transmission is seasonal, during the months of August-December, and overlaps with the rainy season (July-October). The peak for malaria cases usually occurs in September-October.Malaria transmission is high with the entomological inoculation rate (EIR) in 2009 estimated at 50–60 infective bites/person/year . The more common vectors are facility . PlasmodSince February 2009, the CRUN has implemented a demographic surveillance system (DSS) involving about 54,000 individuals. Some important information such as socio-economic status, e g, characteristics of households, living conditions, health conditions, are collected through the DSS on a regular basis.The national anti-malarial drug policy was changed in 2005 from chloroquine to either amodiaquine-artesunate (AQ-AS) or artemether-lumefantrine (AL) as first-line treatments. Pregnant women received an insecticide-treated net (ITN) free of charge as soon as they complete two prenatal consultations or after delivery, while children receive theirs at each vaccination campaign. Nevertheless, a campaign for the promotion of insecticide-treated materials (ITM) targeting pregnant women and children under five years has been implemented since 2010 and nowadays an ITN is given to any pregnant woman attending for the first time during pregnancy an health facility.The study protocol was approved by the Institutional Ethic Committee of Centre Muraz (registration no. 005-2010/CE-CM). All pregnant women attending either the routine antenatal care (ANC) or the outpatient clinic were asked to participate in the study. During the visit and after having obtained a written informed consent, women were divided in cases or controls. Cases had at least one of the following signs and symptoms: temperature ≥37.5°C (measured by electronic thermometer) or history of fever in the previous 48 hours, headache, pallor, arthromyalgia, convulsions, vomiting, dizziness, malaise, fatigue, enlarged liver or enlarged spleen. Controls had none of them. For each case, two controls, matched by parity , gestational age  and seasonality (recruited within one month from the corresponding case). In order to capture possible seasonal variations in the clinical presentation, the study was conducted during both the low and high malaria transmission season.Capillary blood was collected for an HRP-2 RDT, haemoglobin and blood slide. Women with a positive RDT were treated with AQ-AS (standard adult dose) if they were in the second and third trimester of pregnancy while quinine was given for seven days to those in the first trimester (8 mg quinine base/kg every eight hours). Severe malaria cases were hospitalized and treated with parenteral quinine. Anaemia was treated according to the national guidelines with oral ferrous sulphate (200 mg) and folic acid (0.25 mg) daily for one month. All patients with malaria parasites and anaemia were promptly and adequately treated free of charge. Project staff was available 24 hours a day to identify women and to ensure adequate documentation and clinical management.10 for those <400/μl) [RDTs were carried out and results read according to the manufacturer’s procedures. All slides were read twice by two independent readers. Parasite density was estimated by counting the number of asexual parasites per 200 leukocytes in the thick blood film and assuming white blood cells (WBC) count of 8,000 parasites/μl. In case of discrepancy between the two readers  , a thirdThe sample size was calculated using the power calculation module for a two-sample comparison in order to detect with a two-sided significance level of 5% and 90% power important predictors of malaria infection in pregnant women. As there are no data available for the study area, the estimation was based on a study conducted in Mozambique [Double data entry, validation and cleaning were done using Epidata 3.1, and statistical analysis was performed with STATA.10 . Participants were categorized first as (i) cases and (ii) controls. The frequencies of signs and symptoms were compared between these groups. Odds ratio (OR) estimates and their confidence intervals (CI) were estimated in log binomial regression analyses. A P-value of ≤0.05 was considered statistically significant. The anaemia was classified as mild (Hb <11 g/dl), moderate (Hb 7–9 g/dl) and severe (Hb <7 g/dl). Parasite density was log transformed for the comparisons of mean parasite densities between cases and controls.Most recruited women were either in the second  or third  trimester of pregnancy, and the mean age was 27.6 years (95% CI:27.1-28.0). More than half of them had already had one to three pregnancies and most of them were married . The latter would not have been treated in case of clinical diagnosis. The mean (geometric) parasite density among infected women was not significantly different among cases  and controls  (p = 0.06). This is clearly illustrated by the Receiver Operating Characteristics (ROC) curve, with only 56% under the curve and no clear-cut off point distinguishing a case from a control Figure . More thFever, history of fever, headache, and dizziness each had a positive predictive value (PPV) around 50%, while for all the others the PPV was well below Table . The higvs 1.8/μl)(p = 0.003). Moreover, the mean (geometric) parasite density tended to be higher in women with vomiting , fever (5.7/μl vs 2.2/μl) (p = 0.07), and complaining of malaise .The occurrence of signs and symptoms was associated with higher parasite densities; in women complaining of headache, parasite density was significantly higher than in those without , fever  and headache  were or tended to be associated with malaria infection. In the multivariate model, fever and history of fever  also fever and history of fever and headache infection  were significantly associated with malaria infection  for IPTp. Moreover, it has been repeatedly reported that coverage of IPTp with at least two administered doses of SP is still relatively low, despite women attending antenatal clinics several times during their pregnancy ,11.P. falciparum malaria related MMR falling from an estimated 1,000/100,000 live births prior to weekly screening to zero in 2005 [In endemic countries, assuming that any pregnant woman attending an antenatal clinic is infected with malaria, regardless of symptoms, seems to be the right approach. This means that pregnant women should be either systematically screened for malaria infection and treated if positive or given SP at any time they visit a health centre, provided this is done at least one month apart. The latter has been recently recommended by the WHO Malaria Policy Advisory Committee and Secretariat  though i in 2005 .The prevalence of malaria infection was significantly higher in women with symptoms suggestive of malaria but none of them or no combination of any of them had a very high PPV. This is confirmed by the weak association between malaria infection and any of the signs and symptoms, underlying the importance of parasitological diagnosis for any suspected case. The parasite density in pregnancy is not related to being a symptomatic patient and does not increase the probability that fever is related to malaria.In places where transmission is high, malaria infection during pregnancy is often asymptomatic ,17. In MSeveral known risk factors such as maternal age ,19 and pMalaria-related signs and symptoms, e.g. fever or headache, although more frequent in pregnant women with a malaria infection, cannot be used to identify those in need of treatment as a large proportion of asymptomatic women can be infected. Any pregnant women attending a health facility should be tested regardless of symptoms for malaria infection, either by microscopy or RDT. This would be particularly useful for women in the first trimester of pregnancy as they cannot be administered SP as IPTp.The authors declare that they have no competing interests.The study was conceived by UDA and this paper drafted by MCT and UDA. It was conducted by MCT and HT with substantial contributions from JM. Data analyses were conducted by MCT, and supervised by AE and JM. AE, JBO, RTG, JPVG, HT and UDA participated in the overall running of the study, contributed to the interpretation of data, and gave critical review of the final draft. All authors read and approved the final version.


Malaria rapid diagnostic tests (RDTs) are increasingly used as a tool for the diagnosis of malaria, both in endemic and in non-endemic settings. The present study reports the results of an external quality assessment (EQA) session on RDTs in a non-endemic setting.After validation of antigen stability during shipment at room temperature, three clinical samples and a questionnaire were sent to clinical laboratories in Belgium and the Grand Duchy of Luxembourg using malaria RDTs. Participants were asked to report the results of the RDTs as observations (visibility of the RDT control and test lines) and interpretations (report as formulated to the clinician). In addition, participants were invited to fill in a questionnaire on the place of RDTs in the diagnostic strategy of malaria.Plasmodium falciparum (0.8%). Minor errors were related to RDT test result interpretation and included (i) reporting "RDT positive" without species identification in the case of P. falciparum and non-falciparum species (16.9% and 6.5% respectively) and (ii) adding incorrect comments to the report (3.2%). Some of these errors were related to incorrect RDT package insert instructions such as (i) not reporting the possibility of mixed species infection in the case of P. falciparum and Plasmodium vivax (35.5% and 18.5% respectively) and (ii) the interpretation of P. vivax instead of non-falciparum species at the presence of a pan-species antigen line (4.0%). According to the questionnaire, 48.8% of participants processed ≤20 requests for malaria diagnosis in 2009. During opening hours, 93.6% of 125 participants used RDTs as an adjunct to microscopy but outside opening hours, nearly one third of 113 participants relied on RDTs as the primary (4.4%) or the single tool (25.7%) for malaria diagnosis.A total of 128/133 (96.2%) of clinical laboratories using RDTs participated. Six three-band and one four-band RDT brands were used. Analytical errors were rare and included (i) not recognizing invalid RDT results (1.6%) and (ii) missing the diagnosis of In this non-endemic setting, errors in RDT performance were mainly related to RDT test line interpretations, partly due to incorrect package insert instructions. The reliance on RDTs as the primary or the single tool for the diagnosis of malaria outside opening hours is of concern and should be avoided. Plasmodium parasites in blood by antibody-antigen reactions on a nitrocellulose strip, which become visible as cherry-red lines. Different formats exist: two-band RDTs are mostly designed to detect Plasmodium falciparum. They display a control line and a test line, which targets either histidine-rich protein-2 (HRP-2) or P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH). Three- and four-band RDTs display a control line and two or three test lines, one targeting a P. falciparum specific antigen, a second line targeting antigens common to the four species, such as pan-Plasmodium-specific lactate parasite dehydrogenase (pan-pLDH) or aldolase, and in case of the four band RDTs, a third line which targets Plasmodium vivax-specific pLDH (Pv-pLDH).Malaria rapid diagnostic tests (RDTs) are increasingly used as a diagnostic tool in both malaria endemic and non-endemic settings. RDTs detect The use of RDTs is rapidly expanding: in 2007, more than 70,000,000 tests were performed and more than 80 brands were world-wide marketed . In malaThe present EQA session was organized among clinical laboratories in Belgium and the Grand Duchy of Luxembourg which had declared to be interested in EQA sessions on malaria rapid diagnostic tests when subscribing to the EQA session "Blood Parasites" or "Microbiology" organized by the Institute of Public Health, Brussels, Belgium. In a survey afterwards, the non-subscribing laboratories were addressed to ask whether they use RDTs or not in the diagnosis of malaria.P. falciparum, another with no evidence of Plasmodium and a third one with P. vivax  and examined by light microscopy using a × 500 magnification . Parasit.0 and exific PCR .In addition to the samples, a questionnaire on the performance of RDTs in clinical laboratories was prepared. This questionnaire was based on a previous survey performed in the UK  and addr®, Buchanan, Virginia United States), making it possible to monitor the temperature during transport and shipment. Upon arrival at ITM, the returned packages with the samples were stored at 4°C for a maximum of 48 hours and RDT testing was repeated. The test line results observed upon testing these returned aliquots were compared to those that had been obtained in fresh samples.For validation of the shipment at room temperature (in particular the antigen stability), a try-out session was performed among the members of the IPH referee committee, which consists of a panel of laboratory professionals in charge of piloting EQA sessions. Aliquots of the samples at ITM were retrieved, allowed to thaw and processed at room temperature. The samples were tested against a panel of RDTs available in Belgium, as listed in Table All RDTs were performed according to the instructions of the manufacturer, except that the transfer straws or loops supplied in the RDT kits were replaced by a transfer pipette . Readings were carried out at daylight assisted by a standard electric bulb by two subsequent blinded observers, and test line intensities were recorded . RDT kitThe questionnaire and the instructions were drafted in Dutch. They were translated into French  by a native French speaking professional and both versions were again checked by a professional not involved in the study preparations. Comments and feedback to the questionnaire and instructions raised during the try-out session were addressed during a next IPH referee committee meeting and a final version of the questionnaire was approved.i.e. the presence of control and test lines) and interpretations. Interpretations referred to the final diagnosis: participants were offered a free-text option and were invited to submit their answer formulated as a report to the clinician. Participants submitted the results on-line via the IPH-EQA website or sent them by post mail to IPH. The results were converted (answers through the website) or encoded (forms sent by post mail) in an Excel® database .Participants were asked to state the RDT brand they used and to report their results in terms of observations (e.g. phrasings such as "result depends on microscopy"), it was not included for analysis. For interpretation and scoring of the RDT results, major, minor and very minor errors were defined based on relevance and impact on patient care, they are as listed in Table For analysis, the results of the RDT tests, and answers to the questionnaire were reviewed and grouped. As the intended denominator consisted of the number of participants, only the first RDT was considered in case a participant used more than one RDT brand. Primarily, the interpretations of RDT results (report to the clinician) were considered. In case of an unexpected or incorrect result, the observations (results of control and test lines) were retrieved in order to distinguish between interpretative, analytical and clerical errors. In case a final result in terms of interpretations was not clearly stated . Mean and maximum temperatures during shipment were 23.6°C (ranges 22.6°C - 25.2°C) and 28.7°C (25.9°C - 32.1°C). The temperatures during shipment had exceeded 25.0°C for a median duration of 630 minutes . The formal EQA session was organized on February 22nd 2010, during winter season.The try-out EQA session was performed on June 24Results of the try-out testing were identical to those obtained by RDT testing of fresh samples for the three shipped samples and all the tested RDT brands. The results of Cypress and Ultimed  were in line with the expected results .The total number of laboratories subscribing to the Belgian EQA "Microbiology" in 2009 - 2010 was 183. A total of 128 subscribed to the session on malaria RDTs. When surveyed, 50 of the 55 non-subscribing laboratories declared not to perform RDTs as part of malaria diagnosis. In other words, 133/183 (72.7%) of clinical laboratories offering malaria diagnosis were using RDTs at the time of EQA, and 128 (96.2%) of them participated to the present EQA session on malaria RDTs.P. vivax-pLDH specific RDT), and another participant used Palutop as part of an internal evaluation procedure. The results of these additional RDTs were not considered for analysis.Table P. falciparum, parasite density of 53,024/μl). The expected result was: "P. falciparum, a mixed infection with P. vivax, Plasmodium ovale or Plasmodium malariae  cannot be excluded". Four participants did not give a final result in terms of interpretation and report to the clinician. Their observations of test lines were correct, but they were not included for analysis. The final denominator consisted of 124 participants. Only a single major error was observed: one sample was reported as negative. However, the observations of test lines reported for this sample were correct (presence of HRP-2 and pan-pLDH lines), suggesting an administrative error. Less than half of the participants scored this sample correct. One third committed the very minor error of not mentioning the possibility of a mixed infection with non-falciparum species. The reporting of only 'positive' without mentioning the presence of P. falciparum was observed in 16.9% of reports and was considered as a minor error.Table Plasmodium detected). The expected result was "negative" or "no Plasmodium antigen detected". Three participants did not answer a final result in terms of interpretation and report to the clinician. Their observations of test lines were correct, but they were not included for analysis. The final denominator consisted of 125 participants. Three major errors occurred. One participant reported "P. falciparum or mixed infection". The corresponding observations of the test line results were supporting this result, i.e. strong line intensities for both Pf-pLDH and pan-pLDH lines. Two other participants reported the absence of the control line for this sample but failed to report the test result as invalid. Twenty-four participants added a comment to the report: most comments were valuable adjuncts pointing to the need of repeating the RDT (and microscopy) in case of a negative test result and a persistent suspicion of malaria. Four participants added comments that were considered as not correct. One of these comments was raised by two participants, it stated - in case of negative result and persistent suspicion - "to repeat the RDT at the next peak of fever".Table P. vivax, parasite density 3,251/μl). The expected result was "P. vivax, P. ovale or P. malariae" or "non-falciparum malaria". In case of the four-band Palutop the correct result was: "P. vivax, mixed infection with P. ovale and P. malariae not excluded". Three participants did not answer a final result in terms of interpretation and report to the clinician. Their observations of test lines were correct, but they were not included for analysis. The final denominator consisted of 125 participants. None of the participants using Binax observed a test line and therefore all of them missed the expected diagnosis. Minor errors observed were the reports of simply "positive" or "pan-species" , without mentioning the absence of P. falciparum infection. The report of "P. vivax" in case of Palutop was considered a very minor error since the possibility of mixed infection was not mentioned. In case of CareStart, the answer "P. vivax" was of note since this brand does not include a P. vivax-specific test line.Table Table Table Tables To generate an idea about the parasite densities of patients presenting in Belgium and in the Grand Duchy of Luxembourg, archived data were reviewed at ITM. Table P. falciparum  and P. vivax  was striking. In sample 1, half of these errors occurred with Palutop and represented the vast majority (22/26) of reports obtained with this brand. For this reason, the package insert of this kit was analysed: for the combination of the "Pf" (HRP-2) and the "Pan" (pan-pLDH) lines, Palutop instructions mentioned "P. falciparum" without adding the possibility of a mixed infection with P. ovale or P. malariae. A similar omission was noted for the P. vivax interpretation: the possibility of a co-infection with P. malariae and P. ovale was not mentioned  and a regular laboratory kit. For the interpretation of a single pan-pLDH line, the former mentioned "pan-species" and the latter "s Figure , Table 6e.g. reporting "RDT positive" without species identification when this was possible based on RDT test line result). Errors in interpretations  were embedded in the package insert instructions of some RDTs. Nearly half (48.8%) of participants received ≤20 requests for malaria diagnosis in 2009. During opening hours, 93.6% of participants used RDTs as an adjunct to microscopy whereas outside opening hours, nearly one third of them relied on RDTs as primary or single tool for malaria diagnosis.The present study reports the findings of an EQA session on malaria RDTs in a non-endemic setting. Major analytical errors were rare. Minor errors occurred frequently and were related to interpretation or reporting of test results  was related to an administrative rather than to an analytical error; the unexpected single "P. falciparum" report for one out of 26 Palutop tests (sample 2) might also be related to a clerical error (mixing up specimens or laboratory forms) although the reported test line results were in line with the correct result. More of concern was the missed interpretation of invalid test results by two participants, which was considered as a major error: in the absence of a visible control line, test results are not reliable and the test should be repeated. Invalid test results occur rarely (<0.5% of samples tested) but consistently [In the present EQA session, analytical errors were rare. Two major errors were observed. The single missed diagnosis of istently -27 and lP. falciparum  and non-falciparum  or reporting "pan-species" without mentioning that P. falciparum was not involved  was considered as a minor error: all relevant information from the RDT results, in particular the presence or absence of the potentially fatal P. falciparum, should be exploited.Interpretation or reporting of RDT results was more subject to errors: simply reporting "positive" without mentioning the species in the case of Plasmodium antigen. Although this was a confirmed malaria-negative sample correctly identified by 97.6% of participants, it should be noted that false-negative RDT results may occur. Reasons for false-negative RDT results in a non-endemic setting are low parasite densities, polymorphisms in HRP-2 and the prozone effect. Low parasite densities are by far the most frequent cause of false-negative RDTs: for P. falciparum, they occur more frequently below the threshold of 100 parasites/μl, but at this density non-immune travelers can present with symptoms [P. falciparum samples with parasite densities below 100/μl can be expected in about 10% of samples. In such cases - and as raised by some participants as a comment to their report of sample 2 - repeated testing after 8 - 12 hours is advised, for up to four consecutive samplings [P. falciparum infections. Polymorphisms in HRP-2 may give rise to variations that are less likely to be picked up by current RDTs; they are geographically confined to the Asia-Pacific and South-American regions [P. falciparum and presents mostly as test lines with spuriously low intensity, although complete negative results do occur. It tends to occur in HRP-2 based but not in Pf-pLDH based tests [Sample 2 did not contain symptoms ,26-29. Famplings ,31. Of n regions -34. For ed tests .P. vivax in sample 3 was not considered as a major error in light of the ability of RDTs to detect non-falciparum species. In addition, EQA sessions are not designed as a side-to-side comparison of different brands nor as an evaluation of RDT test characteristics. However, it demonstrates the lower sensitivity of RDTs to detect non-falciparum species as compared to the detection of P. falciparum [P. vivax has been calculated to be 68.9% [P. vivax samples [P. vivax (91.0%), at the expense of a lower sensitivity for P. falciparum. Challenged to a panel of stored samples at ITM, SD Bioline, Palutop and CareStart showed overall sensitivities for the detection of P. vivax of 87.5%, 66.0% and 77.6% respectively [P. ovale and P. malariae, reported sensitivities have been even lower [P. falciparum, the sensitivity for the non-falciparum species is related to parasite density, with a threshold value at 500/μl [falciparum samples in the Belgian reference laboratory for malaria  was considered a minor error, as this has no impact on the short-term patient care: in case of an additional P. vivax or P. ovale species, the persistent liver schizonts have to be eradicated with primaquine treatment, but there is time to await this information from the reference laboratory. However, it was striking that this error as well as the error of not reporting the possibility of mixed infections in the case of P. vivax  were embedded in the package insert instructions of Palutop and OptiMAL. Likewise there were errors on interpretation in the Carestart package insert instructions, with incorrect labeling of the test lines in both versions. Manufacturers should be encouraged to revise and adapt their instructions where needed, especially with regard to test line interpretations.Not reporting the possibility of a mixed infection of The questionnaire confirmed the low critical volume in terms of requests for malaria diagnosis: assuming a 5-10% positivity rate for patients suspected of malaria, it is clear that laboratory technicians have little exposure to malaria-positive slides. Even in those laboratories with high numbers of requests, the number of performances per technician will be reduced by a high number of laboratory staff participating in the diagnosis. This is comparable to the situation in other countries, with about half of laboratories in the U.K. carrying out less than 100 malaria requests each year and about 10% of laboratories fewer than 10 . In FranThe number of participants (80.8%) replying that malaria RDTs had improved the diagnosis of malaria in their setting was much higher than revealed by a survey in the UK  . AlthougThe wider range with a tendency to lower scores for ease of use for Binax and, to a lesser extent, OptiMAL may be explained by the fact that these kits include more steps than the more recently released one-step kits such as CareStart and SD Bioline. In ITM experience as well as that of others ,38, partfalciparum species and low parasite densities), and also to assess signs of severity . It is important to ascertain reliable diagnosis of malaria during and outside office hours, and competent microscopy should be in reach at all times. To minimize the risk of microscopy errors and as an in-service training and feedback, all positive and doubtful samples should be submitted to the malaria reference laboratory.Of particular interest are the diagnostic strategies during and outside opening hours. Whereas the vast majority of participants used RDTs as a complement or adjunct to microscopy during opening hours, the reliance on RDTs as the primary or the single diagnostic tool during night and weekend shifts is of concern. In the UK-survey of 2006 a similar tendency was observed: less than 5% of 327 surveyed laboratories used exclusively a RDT during opening hours, versus 15-20% outside opening hours . The extThe policy of leaving the decision on the choice of RDT versus microscopy to the attending clinician may result in not performing the RDT (thereby not exploiting possible information generated by the RDT) or not performing microscopy. This policy is probably related to the reimbursement system of medical costs in Belgium: national health insurance reimburses only laboratory analyses that are explicitly requested by the clinician. Hospital-based diagnostic and treatment algorithms can guide the choice and priorities of laboratory tests, but for the individual patient however, the ultimate request of RDT, microscopy or both depends on the clinician's decision.The additional comment of five participants that they do not perform RDTs on follow-up samples is a correct policy. Indeed, HRP-2 may persist in the circulation for up to several weeks. Aldolase and pLDH depend on the living parasites and they rapidly decline after start of correct treatment, but their use is limited because they are also expressed by gametocytes . ConsequFrom the present EQA session, it is clear that RDTs are an essential part of malaria diagnosis in most diagnostic laboratories in Belgium and the Grand Duchy of Luxembourg. According to the results of the present EQA, it can be concluded that analytical errors in the performance of RDTs are rare. Errors are mainly related to the interpretation and reporting of RDT results, partly due to errors in the package insert. Laboratory staff has limited exposure to malaria positive samples in this non-endemic setting. Whereas during opening hours, RDTs are used as a complement or adjunct to microscopy, there are about one third of participants that rely on RDTs as the primary or the single diagnostic tool outside opening hours, which should be avoided.P: Plasmodium; Pan-pLDH: pan Plasmodium-specific parasite lactate dehydrogenase; PCR: Polymerase chain reaction; Pf-pLDH: Plasmodium falciparum-specific parasite lactate dehydrogenase; pLDH: parasite lactate dehydrogenase; Pv-pLDH: Plasmodium vivax-specific parasite lactate dehydrogenase; RDT(s): Rapid diagnostic test(s); WHO: World Health Organization.EDTA: Ethylene diamine tetra-acetic acid; EQA: External quality assessment; FIND: Foundation for Innovative New Diagnostics; IPH: Belgian Scientific Institute of Public Health; HRP-2: Histidine-rich protein 2; INRB: Institut National de Recherche Biomédicale; ITM: Institute of Tropical Medicine; The authors declare that they have no competing interests.PG, KV, MvE and JJ designed the external quality assessment. PG, MvE, and JJ carried out the try-out EQA session. KV and MvE carried out the formal EQA session. PG, PM, KV and JJ analysed and interpreted the results. All authors drafted and critically reviewed the manuscript and approved the final manuscript.


Abdisalan Noor discusses new research in <i>PLoS Medicine<I> that used model-based geostatistics to investigate the risks of anemia among preschool-aged children in West Africa that were attributable to malnutrition, malaria, and helminth infections. PLoS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.1000438Soares Magalhães RJ, Clements ACA (2011) Mapping the Risk of Anaemia in Preschool-Age Children: The Contribution of Malnutrition, Malaria, and Helminth Infections in West Africa. PLoS Med 8: e1000438. Ricardo Soares Magalhães and colleagues used national cross-sectional household-based demographic health surveys to map the distribution of anemia risk in preschool children in Burkina Faso, Ghana, and Mali.Anaemia is the most common blood disorder globally, is a major underlying cause of death among children and pregnant women in the developing world, and because it is most prevalent in the poorest communities, represents a useful marker of social and economic marginalization PLoS Medicine this week by Magalhães and Clements The article published in The application of model-based geostatistics to national sample survey data on anaemia is an important advance in our understanding of the geography of risk and will provide a more robust framework for computing disease burdens and attributable disability adjusted life years (DALYs). However, for ministries of health, which require disease mapping to guide control and prevention, it is less clear how these new maps will provide enough information on what must be done to reduce the anaemia burdens. Typically, these maps will tell us where the risks are high, moderate, or low, but without knowing the underlying multi-factorial—and most certainly interacting—causes of anaemia, it becomes difficult to determine which combination of interventions is most effective. The authors attempt to partly address this challenge by computing the anaemia PAF of malnutrition, malaria, and helminth infections. As recognised by the authors, PAF can be highly biased depending on whether important confounders are missing from the analysis. HIV infection, haemoglobinopathies, and several other contributors were not included in this study in estimating PAF. It is likely that the inclusion of these contributory factors would decrease the PAF of malnutrition, malaria, and helminth infections and therefore the cost-effectiveness of interventions to control these causes based on the current 60% PAF estimated in this study.The second limitation is one that is specific to the currency of the data used. In the past decade, specifically within the last six years, there has been substantial progress in governance, economics, and funding for control of tropical infections, as well as increased iron and micronutrient supplementation, which would have impacted on these anaemia risk factors in the study countries http://www.map.ox.ac.uk) could potentially address this challenge. The use of the malaria, helminth, and population distribution maps to define the burden of anaemia is novel, but these products are associated with uncertainties, and as yet there are no clear approaches to understanding their combined effect on the primary measure and whether they are additive or multiplicative. Future work must address these data and methodological challenges.More contemporary survey data, updated maps of malaria and helminth infections, and information on other important causal factors are therefore required to improve the value of the anaemia maps for efficient control planning. Data from national HIV/AIDS indicator surveys, recent work on mapping the distribution of sickle cell disease