Malaria and HIV-1 infection cause significant morbidity and mortality in children in sub-Saharan Africa. Recurrent malaria infection increases HIV-1 viral load in adults and increases the rate of progression of HIV-1 infection to AIDS. The effect of malaria on viral loads in children living with AIDS (CLWA) is not clearly known.Plasmodium falciparum malaria, which was treated with chloroquine (CQ) + sulfadoxine-pyrimethamine (SP) and the children followed up for 28 days. HIV-1 viral loads were measured over three time-points: at enrolment , during an episode of malaria, and at a visit about 8 weeks (range 6-19 weeks) after the malaria visit when the child had neither parasites nor any intervening malaria episodes . Primary analyses were restricted to children who on follow up had HIV-1 viral loads measured at the three relevant time-points.One hundred thirty five afebrile HIV-1 positive children having negative blood slides for malaria were recruited at Apac Hospital and followed up for one year. They were monitored for development of Malaria increased HIV-1 viral loadsignificantly in CLWA. Low parasitemia (200-4000/Cl) transiently increased viral load by 0.42 log , higher than that reported in adults. These patients’ viral loads returned to levels similar to those at baseline after treatment. In 13 patients with high parasitemia (>4000/Cl), the mean increase in viral load was 0.53 log (95% CI 0.14 to 0.51), p<0.0001, remaining significantly higher than at baseline after treatment i.e. mean difference (signed-rank test) in viral load “before” and “after” malaria was significant.Plasmodium falciparum malaria is associated with increasing HIV-1 viral loads in children, with some viral loads remaining significantly elevated several weeks after antimalarial treatment. Prolonged post-treatment elevation has important implications for the clinical course in pre-ART HIV-1 positive children and the potential for transmission in sexually active adults. Plasmodium falciparum remains a significant public health problem responsible for about 30% of all hospital admissions and 110,000 infant deaths annually in Uganda , of whom less than 15% have access to antiretroviral drug treatment (ART) [ therapy .We undertook a comparison of HIV-1 RNA concentrations at baseline , during malaria, and post-malaria , and determined the values of malaria-mediated increases in HIV-1 RNA.P. falciparum malaria on HIV-1 viral loads in Ugandan children living with HIV/AIDS but who are not yet on anti-retroviral Therapy. The study was done over a period of 18 months.Our goal therefore was to measure the effect of Pneumocystis jiroveci infections as per the existing National HIV care guidelines [The study site was Apac Hospital located in Apac District of Northern Uganda. Sixty percent of the district is swampy, while the rest of the terrain is savanna vegetation which provides ideal habitats for mosquito breeding. This site has one of the highest malaria transmission intensity in the world, with an Entomological Inoculation Rate of 1586  i.e.: SiP. falciparum infections increase HIV-1 viral loads CLWA; which in turn increases the rate of disease progression to AIDSin ART naïve children.Our hypothesis was that Pneumocystis jeroveci pneumonia and were not yet on ART. Ethical approval was obtained from the IRBs of Medical Biotechnology Laboratories, St. Francis Nsambya Hospital, Apac Hospital and the Uganda National Council for Science and Technology, and informed consent was elicited from the parents and guardians of all study subjects. In order to remove confounding factors known to affect viral load measurements in children, we excluded from the study children who had clinical features defining AIDS, a recent history of tuberculosis or demonstrable worm infestation, bacteremia, or suspected bacterial pneumonia or any other febrile illness. Children whose parents refused to give consent, and those who were not able to remain in the study for at least 9 months from enrolment were also excluded.The study population was ART naïve HIV-1+ children aged 18months to 12 years, with CD4 counts > 15% who were attending Apac hospital. These children all received cotrimoxazole prophylaxis for One hundred thirty five afebrile, malaria-free, and HIV-1 positive (by at least two rapid diagnostic assays) children who were 1.5 -12 years old were enrolled before the peak malaria transmission period (baseline). We used Abbott Determine and Unigold as the rapid HIV test kits. The children were followed up through the malaria season using a prospective cohort design to assess the effect of malaria on plasma HIV-1 RNA. Children who presented to the Outpatient Department with mild malaria or were hospitalized with severe and complicated malaria during follow-up received a standard dose of chloroquine (CQ)  plus a single dose of 1.25mg/kg pyrimethamine and 25 mg/kg sulfadoxine  on day 0; or quinine (10mg/kg), respectively, according to national guidelines. All doses were directly observed and if a patient vomited within thirty minutes of dosing, the medication was re-administered. Paracetamol was administered to all patients. The first dose of CQ+Fansidar was given at the clinic; the remaining 2 doses of CQ were given at home by parents/guardians.Monitoring: Mothers or guardians were instructed to follow a schedule of hospital visits that included a baseline visit (Day 0), routine visits every 8 weeks, interim visits when the children were ill, and visits 3, 7, 14, and 28 days after anti-malarial treatment. After 28 days, the children returned for their regularly scheduled routine visits until the end of follow up. Mothers were given a transport refund on each visit to the clinic.P. falciparum parasitemia during follow up, and had HIV-1 viral loads measured at the three relevant time-points. HIV-1+ children without malaria parasites at enrolment and who remained aparasitemic throughout the study served as a comparison group; they were followed up in parallel with HIV-1 RNA measurements at the 3 corresponding time points.HIV-1 viral RNA was measured over three time-points: at enrolment , during an episode of malaria , and a visit about 8 weeks after the malaria visit and the child had no parasites nor had any intervening malaria episodes . Primary analyses were restricted to HIV-1 positive children who were afebrile and parasite-free at baseline, had follow up visits, had at least one episode of Clinical evaluationAt enrolment, baseline demographic data was collected. At each hospital visit, a physical examination was carried out and a blood sample collected for complete leucocyte counts, malaria microscopy, and HIV-1 RNA load measurement. A history of fever or a sublingual temperature of > 37.5 °C was investigated by clinical examination, blood film examination for malaria parasites, differential WBC counts, were done. Four case definitions of malaria were used: any parasitemia, parasite density 10-20/High Power Field (HPF), parasitemia + fever, and parasite density V 20/HPF + fever. CD4 counts were done as per the schedule of the clinic. Milestone-defining CD4 counts; i.e. counts less than 15% of the total lymphocyte count, were flagged. These thresholds, which signal when to start or change ART, were based on reference interval data for infants on ART from other Ugandan settings and data extrapolated from pediatric AIDS children in neighboring Kenya .Laboratory analysesThin and thick blood films were made for each child at baseline, interim visits and post malaria treatment. The films were processed, Giemsastained, and examined for malaria parasites. A rapid microscopic examination of thick films was carried out at the hospital to confirm the clinical diagnosis of malaria  so that children with confirmed malaria were promptly treated with antimalarial drugs. The thin slides were examined under oil immersion at high magnification (100 X) to determine the parasite density. The number of asexual parasites was counted against 200 white blood cells (WBC) per high power field. The number of asexual blood stage parasites/µl was calculated as the mean number of parasites counted per high power field multiplied by 40 on the assumption of an average white cell count of 8,000 cells/ µl . Total lData analysisData was double-entered and validated using EpiInfo (version 3.5.1). The analysis was carried out using STATA software, version 7.0 . The study was designed to identify a minimum 0.5-log increase in mean HIV-1-RNA concentration during a clinical episode of malaria, with two-tailed Y= 0.05 and Z = 0.80. HIV-1 RNA was log10 transformed before analysis. Baseline characteristics of the population and characteristics between groups were compared with Pearsons X2 tests for categorical variables. All comparisons were 2-tailed. A multivariable analysis was also conducted to determine the impact of other non-malaria comorbidities on the observed effect. Clinical and demographic variables were evaluated in univariate repeated measures analysis to determine associations with log-transformed HIV-1 viral load.A total of 210 HIV-1 positive children seen at the pediatric HIV clinic were screened for malaria. 25 (12%) had a positive malaria blood smear at screening and were referred for treatment and excluded from the study. 135 fulfilled the inclusion criteria and were enrolled into the study.The primary reasons for exclusion were: presence of concomitant febrile illness i.e. a febrile child with two negative Blood Smears at least 6 hours apart, but has other foci of infection like pneumonia, flu, UTI, wound sepsis, GE or a neutrophilia. , not able to continue through the study period , AIDS defining illness , and malaria  .Of the 135 HIV positive children recruited, 120 patients had at least one malaria episode during follow up and received antimalarial treatment. This was a high incidence rate of about 11 cases of smear positive malaria per child-month of follow up time. Of these, 47 had viral load measurements at all three key time points. 15 patients did not get malaria. The baseline characteristics of patients who completed the study and were included in the analysis are shown in The median viral load of the patients used in the analysis was 55,000copies/ml . The aveWhen malaria was defined simply as parasitemia <=5/HPF, episodes were found to transiently increase viral load by 0.42 log . About 8 weeks (range 7-16) after treatment for malaria, the patient’s viral loads returned to levels similar to those at baseline. The effect on HIV viral load increased with the severity of malaria. In 13 patients with fever and high parasitemia (10-20/HPF), the mean increase in viral load was 0.53 log (95%CI 0.14 to 0.51), p = <0.0001 as compared to those with low parasitemia. However, it was noted that in cases with a high parasitemia, the viral load still remains significantly higher than at baseline even after treatment i.e. mean difference (signed-rank test) in viral load “before” and “after” malaria was significant. Baseline viral load was a strong predictor of load during malaria.We were not able to show data of the CD4+ counts of the children during the study because the counts were not done regularly by the hospital as we had anticipated. It was therefore difficult to have this data at comparable time lines.P. falciparum malaria infection affect plasma HIV-1 levels in children as had been demonstrated earlier in adults [The high malaria incidence in the study population may be attributed to the fact that recruitment was intensified during the two peaks of malaria transmission in Apac district. These findings suggest that n adults ,4,27. WhP. falciparum, has some antimalarial action [P. falciparum DHFR, and cotrimoxazole has been reported [There are a few limitations to this study. First, all the CLWA were on cotrimoxazole prophylaxis for PCP. Cotrimoxazole, by inhibiting the dihydrofolate reductase (DHFR) of l action -17. Cotrreported . AlthougSecond, the fact that the remaining chloroquine doses were given at home might have resulted in poor compliance and prolonged the “during malaria” period and as such prolonged the effect of malaria on the viral load. Third, infections with other pathogens such as tuberculosis, bacterial pneumonia, and helminthic infections might also have increased HIV-1 RNA concentrations. Although children with these and other infections were rigorously excluded the effect of subclinical conditions could not be completely removed and therefore the need for a multivariate analysis to determine the impact of these comorbidities.Fourth, there is concern over the wide range of ages of the study population. There might be some expected variation in the progression of disease over the range 5-12 years. However, the sample size was too small to allow for any reasonable stratification by age. The lack ofcomparable CD4+ counts makes it difficult to conclude whether the baseline immunological state has a bearing on the severity of the malaria and the log increase in viral load. Our assessment was limited in size and duration of the study. Furthermore, in attempting to provide optimal patient care through conducting monthly surveillance and encouraging mothers to bring children in during febrile episodes, together with the fact that these children were all on cotrimoxazole prophylaxis, our ability to assess the effect of high-density malaria was diminished because parasitemia levels reached very high significant levels in only four cases. The lack of facilities to diagnose HIV-1 infection early in children below 18months meant that this age group could not be included in this study. Although malaria is not common occurrence in this age group, due to maternal antibodies, a large amount of the antibodies are transferred through breast milk ,29 and yThe fact those in some children viral loads remain significantly elevated after anti-malarial treatment has important implications for the clinical course of HIV and its progression to AIDS in a setting where antiretroviral drugs are still not readily available. Since clinical malaria in children increases HIV-1 viral load as it does in adults ,27, thisThe fact those in some children viral loads remain significantly elevated after anti-malarial treatment has important implications for the clinical course of HIV and its progression to AIDS in a setting where antiretroviral drugs are still not readily available. Since clinical malaria in children increases HIV-1 viral load as it does in adults ,27, thisThe authors declared they have no conflict of interest.The primary author developed the concept of the study. The co-authors took part in the study design and assisted interpretation of the results. All clinical work and data collection for the study was done by the primary author. The writing of this paper was done by the Primary author assisted by the co-authors. The primary author of this paper did this study while working with St. Francis hospital Nsambya as a clinical research fellowunder Elizabeth Glaser Pediatric Foundation collaboration. Although he currently works with the US Centers for Disease Control and Prevention (CDC), the CDC had no role in the study design, implementation nor data analysis.


Although the mid upper arm circumference (MUAC) is used as a proxy to assess wasting in children, its validity abounds in controversies. This study therefore assessed the validity of MUAC as a measure of nutritional status among children aged 12-59 months in urban setting in western Nigeria. A cross sectional study of children aged 12-59 months in selected nursery schools in Moniya, Akinyele local government area, Oyo state was carried out between February to April 2010. The age, weight, length and MUAC, were obtained from the school children who were selected through total sampling. The sensitivity, specificity, positive predictive value, and negative predictive values of the MUAC were determined using weight for age as gold standard (underweight). Z-Scores for anthropometric indicators were calculated using EPI-NUT programme. Receivers operating curve was prepared to determine the optimal cut off of MUAC for the sensitivity and specificity. The overall prevalence of under nutrition among the respondents was 5.6%. The mean mid upper arm circumference was 15.47 ± 1.4 cm and appears to increase with age from 11.49 ± 3.0 at 12-23 months and peaks at age five with a value of 18.05 ± 3.5. At 13.5 cm cut off, the sensitivity of MUAC is 20%, and the specificity is 95.3%, with a Kappa of 16.7%. The receivers operating curve reveals an optimum cut off of 15.5 cm with optimal but improved MUAC sensitivity of 80% and specificity of 53.5%. Thus MUAC was a poorly sensitive indicator of under nutrition at a cut-off below 13.5 cm but highly sensitive at 15.5 cm. A higher cut off value is therefore for recommended for screening for acute malnutrition among under five children. The prevalence of under-nutrition in developing countries is very high. In Latin America, the prevalence of wasting ranges between 19-21% while stunting was about 20-25%. West Africa has prevalence rates of 17.4% while in Nigeria, some authors have documented a rate as high as 27.5% , and it The study was conducted at Moniya, Akinyele local government, Oyo State. Ibadan is made-up of five  urban LoThe study design was a descriptive cross sectional survey.Data collection. An interviewer administered data collection form was designed into which data such as age, weight, length, arm circumference, medical history and immunization history of each child was recorded. Measurements of MUAC (to the nearest 1.0 cm) were made using a non-stretch tape measure, nude weight of the child (to the nearest 0.1 kg) by a Salter bathroom scale and Supine length (to the nearest 0.1 m) was measured with a Holtain infantometer. Age was determined  by asking of both the child’s age and date of birth. Data was collected by the principal investigator and two trained research assistants.Data management. Data cleaning and editing of data collection form was done on daily basis. Data was analyzed using EPI-INFO computer package and statistical package for social sciences. The Z-scores for different nutritional indices, weight-for-age, height-for-age and weight-for-height were calculated in reference to WHO standards by using EPI-INFO programme. The sensitivity, specificity, positive predictive value, and negative predictive values of the MUAC was determined using weight for age, and weight for height indicators as gold standard respectively. The following anthropometric indicators were determined: weight for height (wasting); weight for age (underweight) and height for age (stunting). Descriptive analysis was used in summarizing demographic data. The chi-square test was used to evaluate association between categorical variables. The analysis was considered to show significant associations when the p value was less than 0.05. A receiver’s operating curve of MUAC and weight for age was plotted and hence, a more sensitive cut- off for MUAC was determined.Ethical issues. Ethical approval was sought and obtained from the UCH/UI ethics review committee. Informed consent was sought from the proprietors of the five nursery schools used and also the parents of each participant in the language they understood. It was made known to the participants’ parents and the school authority that they reserved the right to withdraw the respondent’s participation at any time they wish. Data was treated with absolute confidentiality.A total of 319 children were studied out of this, 49.8% were male and 50.2% of them were female with a mean age of 39.63 ± 11.98 months. Table The sensitivity of MUAC using weight for age as gold standard is 27.5%, and the specificity is 96.8%. The extent to which the agreement between MUAC and weight for age improves on chance agreement is 28.6% (Kappa agreement) Table A receiver operating curve was plotted to determine the optimal cut off and sensitivity and specificity for the MUAC, and was found to be a sensitivity of 80% and specificity of 53.5%. At these optimal reliability values, the cut off for MUAC is 15.5 cm Table  . As shown in the study, an optimal cut-off of 15.5 cm yielded a sensitivity of 80% and specificity of 53%. This might be a better cut-off as it is has a higher sensitivity and will therefore detect majority of those with malnutrition and yield better results from field studies. The 15.5 cm cut off is therefore recommended for adoption in Nigeria and further studies on the reliability and desirability of this new cut off is suggested.In conclusion, a cut-off of 15.5 cm for MUAC and is recommended for field screening for under nutrition in children in south-western Nigeria and similar areas. Further studies with larger sample size is recommended to confirm this finding.


Plasmodium falciparum malaria remains a leading cause of morbidity and mortality among African children. Innate immunity provides the first line of defence against P. falciparum infections, particularly in young children that lack naturally-acquired malarial immunity, such as the population examined here. Consistent with the fact that elevated interleukin (IL)-12 is an important component of the innate immune response that provides protective immunity against malaria, we have previously shown that suppression of IL-12 in African children is associated with the development of severe malarial anaemia (SMA). Since the role of IL12B variants in conditioning susceptibility to SMA remains largely unexplored, the association between a single nucleotide polymorphism , SMA (Hb<6.0g/dL), circulating IL-12p40/p70 levels, and longitudinal clinical outcomes in Kenyan children (n = 756) residing in a holoendemic falciparum malaria transmission area were investigated.P = 0.047, and AC: OR, 1.410; 95% CI, 0.953-2.087; P = 0.086) relative to wild type (AA). Although children with SMA had lower IL-12p40/p70 levels than the non-SMA group (P = 0.037), levels did not differ significantly according to genotype. Longitudinal analyses in the entire cohort (n = 756) failed to show any significant relationships between rs3212227 genotypes and either susceptibility to SMA or all-cause mortality throughout the three year follow-up.Multivariate logistic regression analysis in children with acute malaria (n = 544) demonstrated that carriers of the C allele had increased susceptibility to SMA (CC: OR, 1.674; 95% CI, 1.006-2.673; The rs3212227 is a marker of susceptibility to SMA in children with acute disease, but does not appear to mediate functional changes in IL-12 production or longitudinal outcomes during the acquisition of naturally-acquired malarial immunity. Plasmodium falciparum accounts for approximately 98% of the reported malaria cases in Africa and is a leading cause of morbidity and mortality among African children  with a peak incidence between 7-24 mos. of age . SMA was defined as Hb<6.0 g/dL (with any density parasitaemia) based on a previous longitudinal study examining the distribution of >14,000 Hb measurements in an age- and geographically-matched population in western Kenya  than the non-SMA (n = 98) group  Figure .P = 0.514, ANOVA; Figure To determine if rs3212227 genotypes were associated with functional changes in IL-12, circulating concentrations of log-transformed IL-12p40/p70 levels were compared across the genotypic groups. Levels of IL-12p40/p70 were comparable in the groups (P = 0.963) nor CC  genotypes were associated with development of SMA over the three year follow-up period. The relationship between genotypes and mortality were also examined by hierarchical logistic regression. The mortality rate over the three years of follow up was 7.1% (54/756). Compared to the AA reference group, mortality over the follow-up period was not significantly different for the AC  and CC  genotypes. Further exploration by both Poisson and Negative Binomial regressions also failed to identify any significant relationships between the genotypes and either susceptibility to SMA or mortality (data not presented).After determining the relationship between the rs3212227 locus and susceptibility to SMA in children with acute malaria, hierarchical logistic regression was used to investigate the relationship between carriage of the different genotypes and longitudinal outcomes: SMA and mortality. Genotypic distributions in the entire cohort (n = 756) were: 40.3% (AA); 41.4% (AC); and 18.3% (CC). Hierarchical logistic regression modelling revealed that relative to the AA genotype (reference), neither the AC , and also provides information about how polymorphic variants condition susceptibility to SMA and/or mortality throughout the critical phases of naturally-acquired immunity . We hypothesized that variation at rs3212227 locus may provide important information about the role of IL-12 in protecting against SMA.P. falciparum transmission regions, with cerebral malaria being a rare occurrence, this study specifically examined the role of rs3212227 in conditioning the development of severe anaemia and as such, it remains to be determined if results obtained here are applicable to areas with differing endemicities of malaria in which individuals develop severe disease manifestations apart from severe anaemia.Children with falciparum malaria underwent extensive clinical evaluations to rule out additional causes of anaemia so that they could be appropriately controlled for in the multivariate models. For example, since HIV-1 and bacteremia are some of the most common anaemia-promoting infectious diseases in African children -29, all IL-12 plays a critical role in modulating the immune response by promoting CD4+ T cells and NK cells to differentiate towards a Th1 phenotype for production of IFN-γ, an important component of cell-mediated immunity that helps eliminate intracellular pathogens . AccumulIL12A, IL12B, IL12RB1, and IL12RB2) to demonstrate a number of important associations between genetic variation and SMA, of which rs3212227 did not emerge as significant [Previous examination of the rs3212227 polymorphism in Tanzanian children demonstrated that homozygous C individuals had higher mortality from cerebral malaria, whereas no significant associations were found between rs3212227 genotypes and severe malaria (defined as a mixed phenotypic outcome) in children from coastal Kenya . Our recnificant . Howeverin silico analysis (using the web site http://www.cbil.upenn.edu/cgi-bin/tess/tess) which revealed that the region encompassing the rs3212227 had no major transcription factor binding site changes that would be affected by SNP variation within this region. Although the exact reason for the lack of a functional association between rs3212227 genotypes and IL-12p40/p70 levels reported here remains to be determined, it is possible that additional polymorphic variants, such as IL12Bpro [Since 3'UTRs contain regulatory elements that are highly conserved and control translational activity, variation within the 3'UTR can affect transcriptional stability and binding of translational proteins . A previIL12Bpro , or variIL12Bpro ,37.Since the mechanism through which IL-12 provides protection against malaria in murine models appears to be related to the ability of IL-12 to promote IFN-γ and tumour necrosis factor (TNF)-α production that, in turn, provide enhanced NO generation , we examIL12B gene (and other immunoregulatory genes that influence IL-12), to delineate the effects of genetic variation in cis-regulatory genes, and the impact of haplotypes on the host immune response to malaria.Recent data from our laboratory, obtained in the same population of children, demonstrated that multi-site loci provide more informative information about malarial disease outcomes than information obtained for a single locus ,40. ThesResults presented here demonstrate that the rs3212227 is an important marker of susceptibility to SMA, but variation at this particular locus may not serve as a functional mediator of IL-12p40/p70 levels. Given that similar results were observed in individuals with lepromatous leprosy  and ChagThe authors declare that they have no competing interests.JMO participated in the design and implementation of the genotyping assays, supervised the study and participated in writing the manuscript. EOR participated in genotyping experiments, data analyses and manuscript writing. PK participated in the genotyping assays and writing of the manuscript. SBA participated in the analyses and manuscript writing. TW participated in the analyses and manuscript writing. GCD participated in the analyses and manuscript writing. SK participated in the analyses and manuscript writing. JMV participated in the design, supervision of the study, and manuscript writing. CO participated in the analyses and manuscript writing. JBH participated in the design, analyses and manuscript writing. DJP conceived the study, participated in the design, implementation, analyses and manuscript writing. All authors read and approved the final manuscript.University of New Mexico Laboratories of Parasitic and Viral Diseases, Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya ; Center for Global Health, Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM, USA ; Department of Biochemistry and Biotechnology, Kenyatta University, Nairobi, Kenya (EOR); Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya (JMV); Department of Psychology, College of Charleston, Charleston, SC, USA (JBH).


Malaria remains one of the most important infectious diseases in pregnancy in sub-Saharan Africa. Whereas seasonal malaria chemoprevention is advocated as public health intervention for children in certain areas of highly seasonal malaria transmission, the impact of seasonality on malaria in pregnancy has not yet been investigated for stable, hyper-endemic transmission settings of Equatorial Africa. The aim of this study was to investigate the influence of seasonality on the prevalence of malaria in pregnancy in Gabon.Plasmodium falciparum infection.The study was conducted at a rural district hospital in Gabon between January 2008 and December 2011. At first antenatal care visits demographic data, parity, age, and gestational age of pregnant women were documented and thick blood smears were performed for the diagnosis of malaria. Seasonality and established risk factors were evaluated in univariate and multivariate analysis for their association with P. falciparum infection compared to low transmission seasons . Established risk factors including parity  and age  were significant risk factors for P. falciparum infection. High-risk groups including nulli- and primipara and younger women aged 13–17 years showed a disproportionately increased risk for malaria in high transmission seasons from 17% to 64% prevalence in low and high transmission periods, respectively.1,661 pregnant women were enrolled in this study. Participants presenting during high transmission seasons were at significantly higher risk for P. falciparum infection in pregnancy in the setting of central African regions with stable and hyper-endemic malaria transmission. The seasonal increase in malaria in pregnancy is most pronounced in high-risk groups constituted by young and pauciparous women. The evaluation of tailored seasonal prevention strategies for these high-risk populations may, therefore, be warranted.Seasonal variations lead to important differences in the risk for  Plasmodium falciparum during pregnancy in sub-Saharan Africa each year [An estimated 25 million women are at risk to become infected by ach year ,2. Adverach year ,4. The Wach year .P. falciparum[P. falciparum infection in pregnant women in Gabon [Malaria epidemiology in Gabon is characterized by stable, hyper-endemic transmission of more than 90% alciparum. In prevalciparum. The clialciparum. Malariain Gabon , howeverBased on the observation of high seasonality of malaria transmission in certain African regions including large parts of the Sahel, the concept of seasonal malaria chemoprevention has been developed for children . This coP. falciparum shows high resistance against chloroquine and increasing resistance against SP [This study was conducted in the town of Fougamou  in the rural province of la Ngounié in Gabon. The region is located in a hilly, rainforest covered area with predominance of subsistence farming as professional activity. The climate is equatorial and meteorological data indicate two dry seasons (December - January and June - September) and two rainy seasons  was chosen as cut-off point determining the risk-periods based on concordant annual trends during the study period: Low-risk periods were in April and between June and September (high-risk periods were May and October - March). Gestational age was calculated according to the reported first day of last menstruation. Ethical approval for this retrospective analysis of clinical data that were obtained from routine clinical records was provided by the Comité d’Ethique Régional et Independent de Lambaréné.All thick blood smears were defined as positive if any asexual forms of Data were imported for further analysis into a commercial statistical software package . Descriptive analyses were performed and a binary logistic regression model was used to compute the adjusted odds ratios (AOR) of independent risk factors for malaria in pregnancy. To minimize exclusion of data due to missing values binary logistic regression model was computed for two scenarios: including 1) participants with all data  available for analysis and 2) participants with data for parity, age and season. Since this sensitivity analysis demonstrated only minimal differences, the model with fewest exclusion is shown. Statistical significance was defined by a likelihood ratio with a p-value below 0.05. 95% confidence intervals (CI) were calculated by the exact binominal method.P. falciparum infections diagnosed at first antenatal care visit are also given in Table Between January 2008 and December 2011 a total of 1,661 pregnant women attended the hospital for their first antenatal care visit. Table p < 0.001, not shown in Table Parity, age and season were significantly associated with the risk for malaria computed by the binary logistic regression Table . MultipaP. falciparum infection with a prevalence of 23% (n = 40). In binary logistic regression for gestational age defined as an ordinal variable, the model showed a significant association of gestational age with a decrease in malaria positivity from the first to the third trimester  in August to 24% (n = 39) in January. In the course of the year malaria positivity declined from January to April to 12% (n = 17), increased thereafter to 16% (n = 19) in May for a further decline to 11% (n = 12) in June. Malaria prevalence increased again between September and November to 20% (n = 31). This pattern was comparable for all years during the study period. The variation in the prevalence of malaria in pregnancy depending on the annual season was statistically significant as assessed in logistic regression analysis .The dashed curve (drawn in grey) in Figure P. falciparum infection than the low-risk group (dashed curve). Highest prevalence was observed in January (64%) and lowest in September (17%), indicating a higher degree of variation in this high-risk group compared to the general study population.The solid curve and the dashed curve (drawn in black) in Figure P. falciparum infection in pregnancy occurs in a rural Central African community in Gabon. The finding of an AOR of 1.91 seems surprisingly high on the background of perennial, stable and hyper-endemic malarial transmission in Gabon [P. falciparum infection. Whereas these data confirm previous findings [P. falciparum infection may emphasize the need of effective prevention programs in this high-risk group for the prevention of malaria in pregnancy.This study demonstrates that marked seasonal variations in the prevalence of in Gabon . Besidesfindings ,10,17-22To date SP-IPTp is - besides the use of ITNs and case management - the recommended strategy of the WHO in most of sub-Saharan African countries to combat malaria in pregnancy . CurrentP. falciparum infections in the course of pregnancy can be drawn from this study. Similarly, data have been obtained from a hospital registry and potential problems in data accuracy can therefore not be excluded. Finally, the influence of concomitant intake of anti-malarial drugs cannot be ruled out. However, since all women presented for their first antenatal care visit, no major confounding by the intake of SP-IPTp is anticipated.Whereas our study aptly analyses risk factors and their association with malaria in pregnancy, several limitations of the study design need to be acknowledged. Firstly, this study is designed as a cross-sectional survey collecting data only at the first antenatal care visit. Therefore no robust estimate on the incidence of Even in Central African regions with stable, perennial and hyper-endemic malaria transmission, seasonality leads to important variations in the prevalence of malaria in pregnancy. These variations are even more pronounced in high-risk groups of young and pauciparous women. These findings evoke reflexions on the potential usefulness of more individualized prevention strategies based on a risk-benefit analysis, for low- and high-risk groups among pregnant women in sub-Saharan Africa.The authors declare that they have no competing interests.CGB, MJJ, RMZ and MR developed the idea for this paper. MJJ conceived the analyses and wrote the first draft of the manuscript. MJJ, CGB and MR drafted the final manuscript. BL conducted the binary logistic regression. All authors read and approved the final manuscript.


Plasmodium falciparum (Pf) antigens and age, asymptomatic parasitaemia, malaria exposure index  and immunity to febrile malaria during 10-months of prospective follow up.Although antibodies are critical for immunity to malaria, their functional attributes that determine protection remain unclear. We tested for associations between antibody avidities to 42 (MSP1) and Merozoite Surface Protein 3 (MSP3) were measured by Enzyme Linked Immunosorbent Assay in 275 children, who had experienced at least one episode of clinical malaria by the time of this study, as determined by active weekly surveillance.Cross-sectional antibody levels and avidities to Apical Membrane Antigen 1 (AMA1), Merozoite Surface Protein 1Pf malaria. There were no consistent prospective associations between antibody avidities and malaria episodes.Antibody levels to AMA1, MSP1 and MSP3 increased with age. Anti-AMA1 and MSP1 antibody avidities were (respectively) positively and negatively associated with age, while anti-MSP3 antibody avidities did not change. Antibody levels to all three antigens were elevated in the presence of asymptomatic parasitaemia, but their associated avidities were not. Unlike antibody levels, antibody avidities to the three-merozoite antigens did not increase with exposure to Pf-merozoite antigens are associated with either exposure or immunity to malaria.We found no evidence that antibody avidities to  The anti-parasitic and therapeutic effects of passively transferred malaria immune IgG, from immune adults to children or adults with malaria, provided strong evidence that antibodies are critical mediators of naturally acquired immunity to malaria 19-specific antibodies with multiple P. chabaudi infections in mice, suggesting that avidity maturation occurs in Plasmodium infections Pf-lysate-specific antibody avidities following resolution of clinical malaria in Brazilian adult patients, suggesting that human Pf infections are also associated with avidity maturation Achtman et al demonstrated increasing avidities of MSP1Pf-merozoite antigens are associated with age, asymptomatic parasitaemia, distance weighted local malaria prevalence (EI)  to three This study was approved by the Kenyan Medical Research Institute National Ethics Committee. Written informed consent was provided by the parents/guardians of all the children involved.The study was done at the KEMRI Centre for Geographic Medicine Research Coast situated at Kilifi District Hospital, Kenya. The hospital serves ∼500,000 people living in Kilifi District. The children investigated in this study were part of longitudinal cohort of 300 resident in Junju location, lying on the southern side of an Indian Ocean creek that divides Kilifi into North and South. This study site is predominantly inhabited by the Mijikenda people Pf transmission in Kilifi district P. falciparum parasitemia >2500 parasites per microliter) until the age of 13 years. We maintain extensive and detailed records of the numbers and dates of malaria experiences for each child, either from birth or from the time of recruitment.Although there has been a gradual decline in Pf transmission in Junju. Plasma was harvested and stored at −80°C.5 ml venous blood samples and blood smears were collected in a pre-season cross-sectional survey in May 2009, a time preceded by four months of minimal Pf-specific IgG antibody responses were quantified against recombinant Pf AMA1-FVO/3D7 ), MSP142 and MSP3, to which circulating IgG antibodies have been associated with clinical protection in our study population Pf antigens were provided by Dr. Louis Miller .Pf-infected red cells counted against 500 leukocytes and 1000 red blood cells, respectively, by expert microscopists.Thick and thin blood smears were stained with Giemsa and Antigens were coated onto flat-bottomed 96-well microtitre plates (MICROLON®) overnight at a concentration of 100 ng/ml in bicarbonate buffer. Coated plates were washed three times with Phosphate Buffered Saline (PBS) with 0.05% Tween (PBST), before blocking for 1 hour with 10% Foetal Calf Serum (FCS) in PBS.The plasma samples were diluted to 1∶500 in 0.3% PBST with 1 mM Ethylenediaminetetraacetic acid (EDTA) and 100 µl was added to each of the four wells allocated to each sample. A doubling dilution series from 1∶250 to 1∶32,000 of a pool of hyper-immune plasma from local adults, in duplicate wells on each plate, was used to produce standard curves and as a positive control. Plasma from seven non-immune European donors, also in duplicate wells on each plate, was used to generate a cut-off and as a negative control. Plates were incubated with the test/control plasma at room temperature for 2 hours then washed five times. 100 µl of 4 M guanidine hydrochloride, prepared in 0.05% PBST, was added to two out of four wells for each plasma sample, with 0.05% PBST alone added to the remaining two wells. After 10-minute incubation at room temperature the plates were washed four times. Alkaline phosphatase-conjugated Goat anti-Human IgG , plus substrate, were then used to assess the amount of IgG remaining bound in each well. Optical densities (OD) were read at 405/570 nm.Test samples with a mean OD less than two standard deviations above that of non-immune European controls (for MSP3) or less than four standard deviations above (for AMA1 and MSP1) were classed as negative for antibody to that antigen, and avidity indices were not calculated. A sigmoidal standard curve from the doubling dilution series of pooled hyper-immune adult plasma was used to convert mean OD (without guanidine) into an antibody level in Arbitrary Units (AU). Avidity indices were calculated as the ratio of guanidine-treated mean concentration to non-guanidine-treated mean concentration, multiplied by 100.This was calculated as distance weighted proportion of malaria infections (both asymptomatic and symptomatic) within 1 km radius of each child Pf malaria were determined by Cox regression analyses. Poisson regression models were fitted to determine whether the number of multiple malaria episodes were associated with antibody responses, age, and asymptomatic parasitaemia. For all tests, statistical significance was considered at the 5% level.Log transformed antibody and avidity data were analyzed using Stata  and GraphPad Prism for Macintosh . Multivariable linear regression was used to test the predictive value for age, asymptomatic parasitaemia, rates of previous clinical malaria attacks, and EI on levels and avidities. Associations between different continuous measures were determined by using linear regression. Wilcoxon rank-sum test was used where appropriate to compare median differences between groups for continuous variables. Associations between antibody responses (levels and avidities), age, and asymptomatic parasitaemia with the risk to the first (or only) episode of Pf parasitaemia.We tested samples from those children within the Junju cohort for whom we had documented evidence of at least one incident of malaria exposure since the start of surveillance in January 2005. From the cohort, 263 children had experienced at least one documented episode of clinical malaria by the cross-sectional sampling date in May 2009, rising to 275 children by the end of the follow up period 10 months later. The mean age at the sampling date was 6.2 years (standard deviation [SD] 2.46 years) . The meaTo be certain of substantial amounts of antibody, before attempting to determine avidity, we first screened all of the 275 plasma samples for the presence of antibody concentrations well above the mean levels of a panel of malaria-naïve control sera from the UK (see methods for positivity threshold). Of 275 children, 184 (67%), 218 (79%) and 130 (47%) children had antibody levels above the threshold of positivity for AMA1, MSP1 and MSP3, respectively. However, the chosen threshold for MSP3 antibody positivity was much lower than for AMA1 and MSP1 as only 29% of the children were positive at the mean + 4SD cut-off. These samples went on to be tested for antibody avidity indices. Antibody avidity indices were generally independent of the respective antibody levels .Pf transmission. These samples were randomly selected from the May 2010 annual cross-sectional-bleed (of Junju children and 50 adults), taken following administration of written informed consent (as stated in methods section). Antibody levels and avidities to AMA1 and MSP1 were measured for comparison between the groups.Separate sets of samples were taken from 15 randomly selected children and 15 adults  from the same malaria-exposed village and from 8 adults (“urban-adults”) living in a semi-urban area of Kilifi with no Antibody levels to AMA1, MSP1 and MSP3 were positively correlated with age . In contTo test whether the amount of previous malaria exposure may determine the cross-sectional levels and avidities of antimalarial antibodies, we checked for association with three markers of exposure: age, concurrent asymptomatic parasitaemia and the malaria exposure index (EI).Antibody levels to all three antigens were significantly higher in children with asymptomatic parasitaemia compared to uninfected children . In contWe also used the distance weighted local malaria prevalence for each child as a marker of the total malaria exposure at the individual level as described in Olotu et al Pf antigens.To further explore the apparent lack of avidity maturation with malaria exposure, we compared anti-AMA1, MSP1 and tetanus toxoid (TT) antibody avidities from 15 randomly selected children, 15 rural-adults, and 8 urban-adults. Compared with the avidity indices for antibodies specific to the tetanus toxoid vaccine antigen, avidities for the two malaria antigens (AMA1 and MSP1) were significantly reduced, irrespective of age , suggestPf transmission, but just prior to the subsequent two transmission seasons (10-months), were associated with the subsequent risk of malaria. For this analysis a malaria episode was defined as an axillary temperature of 37.5°C associated with Pf asexual parasitemia of over 2500 parasites per µl of blood. Two measures of malaria risk were analyzed: 1) time to the first, or only, malaria episode, and 2) incidence, including all episodes of malaria within the 10-month follow-up. The corresponding Cox and Poisson regression models controlled for age, EI and asymptomatic parasitaemia at the baseline. In multivariable analyses, both asymptomatic parasitaemia at the baseline and a higher EI were associated with shorter time to the first episode, as well higher incidence of clinical malaria  was for AMA1. However, this positive association was lost in multivariable analysis controlling for exposure and parasitaemia, suggesting that these two factors partially contributed to the association in univariable analysis. In contrast, anti-MSP1 antibody avidity was negatively, but weakly associated with age. Whilst antibody levels were elevated in the presence of asymptomatic parasitaemia, avidities were either reduced, in the case of MSP1, or unaffected, in the cases of AMA1 and MSP3. As expected from previous studies Pf. In contrast, antibody avidity did not increase with EI. Furthermore, the associations between avidity and EI were negative for AMA1 and MSP1.Sero-epidemiological studies have reported inconsistent associations between antibody levels to defined Pf parasites . Furthermore, the negative association between MSP1 antibody avidity and asymptomatic parasitaemia suggests that asymptomatic infections may interfere with avidity maturation.Classically, repeated exposures, with associated rounds of somatic hypermutation, are believed to pave the way for selection of memory B (and plasma) cells secreting antibodies of high avidity Pf merozoite antigens AMA1, MSP119 and MSP2 from the pre-season baselines following the transmission season in the Gambia Pf malaria will be required to clarify these issues.Few studies have associated antibody avidities with varying numbers of previous malaria episodes in humans. Reddy et al Pf antigen specific antibodies with immunity to malaria. Akpogheneta et al did not observe any associations between avidities for pre-existing antibodies and subsequent occurrence of clinical malaria Pf antigen among children with uncomplicated and asymptomatic malaria relative to children with complicated malaria, suggesting that the ability to make antibodies of high avidity in response to infection may be associated with immunity to severe malaria. Tutterow et al found that antibodies binding to VAR2CSA with high avidity were associated with reduced risk for placental malaria In our study, there were no consistent associations of antibody levels and avidities with the risk of malaria. However, baseline asymptomatic parasitaemia and high EI were associated with increased risk of clinical malaria. Few other studies have attempted to associate avidities for Pf parasites in our analysis, it is unlikely that it is the only possible explanation for the lack of association between antibody levels and immunity to clinical malaria. However, as we reported previously Because we controlled for heterogeneity of exposure to Pf malaria does not result in avidity maturation for the anti-merozoite antigens AMA1, MSP1 and MSP3. Instead, our data suggests that persistent exposure to malaria, or increased prevalence of asymptomatic infections, may interfere with avidity maturation. Alternatively, it may be that antibody avidity maturation takes place quickly and plateaus after the first few infections, making it difficult for longitudinal studies to observe maturation with increasing exposure. Significantly, neither the anti-malarial nor anti-TT antibody avidities were different between children and adults. More studies are required to determine to what extent exposure to Pf malaria interferes with avidity maturation, and how this phenomenon is associated with naturally acquired immunity to malaria. A greater understanding of what it takes to induce protective antibodies remains a critical technological challenge in the struggle to develop highly efficacious antimalarial vaccines.We conclude that, in contrast to antibody levels, persistent exposure to Figure S1Avidity index is independent of antibody-levels. The avidity indices for A) AMA1 B) MSP1 and C) MSP3 were plotted against their respective antibody levels. The level of correlation was tested by linear regression analysis.(TIFF)Click here for additional data file.


Lorenz von Seidlein discusses the study by Katherine Halliday and colleagues and explores the reasons why a school-based screening and treatment strategy for malaria might have failed.Please see later in the article for the Editors' Summary PLOS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.1001594Halliday KE, Okello G, Turner EL, Njagi K, Mcharo C, et al. (2014) Impact of Intermittent Screening and Treatment for Malaria among School Children in Kenya: A Cluster Randomised Trial. PLoS Med 11(1): e1001594. Katherine Halliday and colleagues conducted a cluster randomized controlled trial in Kenyan school children in an area of low to moderate malaria transmission to investigate the effect of intermittent screening and treatment of malaria on health and education.Falciparum malaria incidence is declining in many countries including in the formerly hyperendemic regions of sub-Saharan Africa PLOS Medicine, Halliday and colleagues describe a trial to evaluate intermittent screening and treatment in schools In this issue of The results cannot be attributed to methodological uncertainty, a frequent cause of negative study outcomes, since the trial was conducted in a large sample of schools according to the highest procedural standards. There was excellent follow-up of and adherence by study participants. Most likely, children found to be parasitaemic did benefit from early treatment but this outcome was not measured, as the investigators were interested in school-based rather than individual-based effects. Why did these individual effects not translate to school-wide outcomes? First investigators didn't treat all infections, rather only those of a sufficiently high density detectable by RDTs. Any child with a parasitaemia with a density below the RDT's detection threshold on that day remained untreated. The dimensions of this reservoir of infections with densities below the RDT's detection threshold are incompletely understood and are currently under investigation using new molecular tools. RDTs may require a parasite density of 100,000 parasites/ml Would the strategy have worked had the investigator screened and treated whole villages instead of school classes? Investigators in Burkina Faso have recently addressed precisely this question in a cluster randomised trial The evaluation of malaria elimination strategies requires considerable resources. Trials evaluating interventions to interrupt malaria transmission require populations living in a discrete geographic entity such as a village as unit of inference. To randomise villages in a statistically meaningful fashion involves tens of thousands of participants. Considering the time and resources required for such large undertakings, researchers use mathematical modelling to predict the outcome of interventions. Such models have been refined for more than a century and their popularity keeps growing despite the inherent complexity of vector borne diseases Plasmodium falciparum strains resistant to artemisinins are spreading through Southeast Asia It is easy to criticise retrospectively the failure of well-intended interventions and infinitely harder to predict success. But some of the more successful interventions could guide investigators and policymakers towards more promising strategies especially during an emergency. Increasing the coverage of populations at risk with effective interventions  should lead to the elimination and ultimately the eradication of falciparum malaria in the decades to come. That is if the pathogens and the vectors weren't constantly evolving. Mosquitoes resistant to the available insecticides are spreading through sub-Saharan Africa and Asia


Lymphocytopenia has frequently been described in patients with malaria, but studies on its association with disease severity have yielded conflicting results. The neutrophil/lymphocyte count ratio (NLCR) has been introduced as a parameter for systemic inflammation in critically ill patients and was found, together with lymphocytopenia, to be a better predictor of bacteraemia than routine parameters like C-reactive protein and total leukocyte count. In the present study, the predictive value of the NLCR and lymphocytopenia for severe disease was evaluated in patients with imported malaria.st 1999 and January 1st 2012 with differential white cell counts determined within the first 24 hours after admission were included in this retrospective study. Severe malaria was defined according to the WHO criteria. The performance of the NLCR and lymphocytopenia as a marker of severe malarial disease was compared back-to-back with that of C-reactive protein as a reference biomarker.All patients diagnosed with malaria at the Harbour Hospital between January 1A total of 440 patients  were included in the study. Lymphocytopenia was present in 52% of all patients and the median NLCR of all patients was 3.2. Total lymphocyte counts and NLCR did not differ significantly between groups. A significant correlation of total leukocyte count and NLCR, but not lymphocyte count, with parasitaemia was found. ROC analysis revealed a good negative predictive value but a poor positive predictive value of both lymphocytopenia and NLCR and performance was inferior to that of C-reactive protein. After complete parasite clearance a significant rise in total leukocyte count and lymphocyte count and a significant decrease in NLCR was observed.The NLCR was found to correlate with parasitaemia, but both lymphocytopenia and the NLCR were inferior to C-reactive protein as markers for severe disease in patients with imported malaria. The NLCR and lymphocytopenia are not useful as predictive markers for severe disease in imported malaria in the acute care setting. Plasmodium falciparum infection [Changes in blood cell counts are a well-known feature of malaria. Alterations in leukocyte counts are often less pronounced than in the other blood cell lineages, but in general total leukocyte counts have been found to be low to normal in malaria ,2. Lymphnfection . Studiesnfection ,3,4 and nfection  have beeet al. introduced the ratio of neutrophil to lymphocyte count as a parameter of systemic inflammation and stress in critically ill surgical and medical patients [Lymphocytopenia accompanied by a rise in neutrophil count is commonly seen in various infectious and non-infectious causes of systemic inflammation and stress -13. Zahopatients . The prepatients  and it wAlthough the NLCR is currently not routinely used as a clinical parameter, the above mentioned studies have demonstrated its value as an infection marker in critically ill patients. Unequivocal data concerning the predictive value of lymphocytopenia and the NLCR in malaria are not yet available.In non-endemic countries, where malaria is only seen as an imported disease, non-specialized hospitals often rely on rapid diagnostic tests for the diagnosis of malaria and generally lack experience in the examination of thick and thin blood smears to asses the parasite load. There is, therefore, still a need for simple and readily available parameters for the early identification of patients at risk of severe or complicated disease. The present study evaluated lymphocytopenia and the NLCR as predictive markers of severe disease in a large cohort of patients with imported malaria.st 1999 and January 1st 2012 and with differential white cell counts determined within the first 24 hours after admission were included.The Harbour Hospital is a 161-bed general hospital located in Rotterdam, The Netherlands. It also comprises the Institute of Tropical Diseases, which serves as a national referral centre. All patients diagnosed with malaria at our centre are included in the Rotterdam Malaria Cohort study. Demographic, clinical and laboratory data of all these patients are collected using a standardized form and stored in an electronic database. For the present observational cohort study, patients diagnosed with malaria between January 1Total and differential leukocyte counts were measured using an automatic cell counters. During the study period three distinct cell counters were subsequently used after careful calibration . Absolute numbers of lymphocytes and neutrophil subsets were obtained by multiplication of the absolute leukocyte counts with their respective differential leukocyte counts. Manual confirmation of automatic cell count results was performed when immature or aberrant leukocytes, erythrocytes or platelet clumps were detected and when cell count results differed substantially from normal values. Other available laboratory examinations included red blood cell counts, haematocrit, platelet counts, C-reactive protein levels, serum electrolytes, total bilirubin, serum creatinine and urea, sodium, potassium, liver enzymes, blood glucose and venous plasma lactate.Plasmodium vivax and Plasmodium ovale.The standard procedure to diagnose malaria comprised a Quantitative Buffy Coat (QBC) analysis, a rapid diagnostic test (RDT) for malaria antigens , and thick and thin blood smears using freshly collected blood specimens from finger pricks. The RDT and the QBC analysis were performed according to the manufacturer’s instructions. QBC capillaries were examined independently by two technicians by microscopic analysis of two complete rows of the region between the bottom of the capillary and the polynuclear leukocyte layer using an Olympus BX-60 fluorescence microscope equipped with UV-filter, 50x objective and 12.5x oculars . These two lanes represent about 100 microscopic fields (at 625 × magnification) and take an average examination time of 5 min. Subsequently, the polynuclear and mononuclear cell layer was screened for schizonts, gametocytes, malaria pigment and elderly trophozoites of Plasmodium parasites as well as Schüffner’s dots and Maurer’s clefts in infected erythrocytes. Thick and thin smears were examined with regular light microscopes at a total magnification of 1250x.Thick blood smears were stained with Field’s stain  and thin smears with Diff Quick stain . Both staining procedures have been optimized for optimal staining of Plasmodium detection methods were used throughout the whole study period.The same Patients were classified as having severe malaria if they met one or more of the WHO criteria for severe malaria, as published , either 9/L and leukocytosis was defined as a leukocyte count exceeding 10.0 × 109/L. Lymphocytopenia was defined as a lymphocyte count of less than 1.0 × 109/L and lymphocytosis as a lymphocyte count more than 4.0 × 109/L. Neutropenia was defined as a neutrophil count of less than 1.5 × 109/L and neutrophilia as a neutrophil count of more than 7.0 × 109/L. The neutrophil-lymphocyte count ratio was defined as the ratio of the absolute neutrophil count to the absolute lymphocyte count.Leukocytopenia was defined as a leukocyte count of less than 4.0 × 10P. falciparum during childhood. Patients who had been living in a malaria-endemic area for at least 2 years at the time of diagnosis were presumed semi-immune. Tourists from non-endemic regions who travelled to endemic areas were considered non-immune.The degree of immunity to malaria was estimated as previously described . Adult iData were not normally distributed (Kolmogorov-Smirnov test) and are therefore presented as medians and range. Univariate comparisons were performed using the Chi-Square test and the Kruskall-Wallis test with Dunn’s post-hoc tests (three groups), or the Mann–Whitney test and Fisher’s Exact test (two groups). Correlations with parasitaemia were calculated using Spearman’s rank correlation. For the comparisons of differential leukocyte counts before and after treatment, Wilcoxon’s matched pairs test was used.For clinical reference, the diagnostic performance of NLCR and lymphocytopenia for severe disease was compared to that of the classic biomarker serum CRP using receiver operating characteristic (ROC) analysis. The optimal cut-off point was identified using Youden’s index. The areas under the ROC curve (AUROCs) were compared to that of CRP in a pair-wise comparison by the method of Hanley and McNeil .Given its retrospective observational design, ethical approval of this study was not required, according to the Dutch Medical Research Involving Human Subjects Act.st 1999 and January 1st 2012 a total of 562 cases of imported malaria were seen. Differential leukocyte counts were available for 440 (78%) of the patients. Of these cases, 120 cases were caused by non-falciparum Plasmodium species: 88 by P. vivax, 27 by P. ovale, four by Plasmodium malariae and one by Plasmodium knowlesi. The majority of infections (320 or 72%) was caused by P. falciparum, including three patients who had a mixed infection; two with P. falciparum and P. ovale, one with P. falciparum and P. vivax. Sixty-one patients were classified as having severe malaria. All these patients met the severity criteria upon admission to the hospital, and no patients were re-classified as having severe malaria because of progression of disease during admission. Concomitant infection was present in 29 (7%) of patients and was more common in patients with severe malaria. None of the patients had positive blood cultures. The general characteristics of these patients are shown in Table Between January 1P. falciparum malaria and non-falciparum malaria (Table Leukocytopenia was present in 23% 100/440) of all patients but in only 11% (7/61) of patients with severe malaria  of all patients and was more frequently seen in patients with non-severe falciparum malaria (143/259 or 55%) than in patients with severe malaria (28/61 or 46%), and non-falciparum malaria (54/120 or 45%). Absolute lymphocyte counts did not differ significantly between the three groups.P. falciparum malaria and non-falciparum malaria (Table Neutropenia was found in 8% (36/440) of all patients and neutrophilia in 4% 17/440), but was more common in patients with severe malaria (7% or 4/61) compared to those with non-severe falciparum malaria and non-falciparum malaria . Neutrophil counts were higher in patients with severe malaria compared to those with non-severe 7/440, buP. falciparum (3.3) and non-falciparum (2.8) malaria, these differences were not significant.The median NLCR of all patients was 3.2. Although there was a trend towards higher values in patients with severe malaria (3.5) compared to those with non-severe s 0.304, p<0.0001) and parasitaemia and NLCR .Significant correlations were found between parasitaemia and total leukocyte count  but poor positive predictive value  a significant rise in total leukocyte count and lymphocyte count and a significant decrease in NLCR was observed. A significant decrease in neutrophil counts after treatment was only seen in non-severe malaria patients, but not in patients with severe malaria  patients with severe malaria and 114 patients with non-severe ia Table .In contrast to studies in bacterial sepsis, where lymphocytopenia and NLCR were found to outperform CRP in predicting the presence of bacteraemia, lymphocyte counts and NLCR did not allow for an accurate discrimination between malaria patients with severe disease and those without. Even though lymphocytopenia and NLCR had good negative predictive values, CRP was found to be a superior marker in back-to-back analyses.et al.[This lack of diagnostic power might partly be due to the fact that neutrophilia, while often marked in patients with bacterial sepsis, is not commonly seen in malaria. Some studies even report neutropenia . In the et al., an impoe.g. pooling in the enlarged spleens of patients [in vitro[Surprisingly for such a common phenomenon -7,21 thepatients  rather t[in vitro,24, poss[in vitro. Interes[in vitro,24,26, s[in vitro.In conclusion, the NLCR was found to correlate with parasitaemia, but both lymphocytopenia and the NLCR were inferior to CRP as markers of severe disease in patients with imported malaria in direct back-to-back comparisons. Although these parameters may have proven their usefulness in predicting bacteraemia, they are apparently not useful as predictive markers of severe disease in imported malaria in the acute care setting.The authors declare that they have no competing interests.MEvW contributed to data acquisition and analysis and writing of the manuscript. KV, MdeMM, PCW, MBM and JvH participated in data analysis and revising the manuscript. RK was responsible for collection of patient data and database management. PJvG participated in study design, data analysis and writing and revising the manuscript. All authors have approved the final version of the manuscript.


Plasmodium falciparum samples in an endemic field setting.Prozone means false-negative or false-low results in antigen-antibody reactions, due to an excess of either antigen or antibody. The present study prospectively assessed its frequency for malaria rapid diagnostic tests (RDTs) and P. falciparum high parasitaemia (≥ 4% red blood cells infected) were obtained from patients presenting at the Provincial Hospital of Tete (Mozambique). Samples were tested undiluted and 10-fold diluted in saline with a panel of RDTs and results were scored for line intensity . Prozone was defined as a sample which showed no visible test line or a faint or weak test line when tested undiluted, and a visible test line of higher intensity when tested 10-fold diluted, as observed by two blinded observers and upon duplicate testing.From January to April 2010, blood samples with P. falciparum, 92 (10.5%) had high parasitaemia and 76 were available for prozone testing. None of the two Pf-pLDH RDTs, but all six HRP-2 RDTs showed prozone, at frequencies between 6.7% and 38.2%. Negative and faint HRP-2 lines accounted for four (3.8%) and 15 (14.4%) of the 104 prozone results in two RDT brands. For the most affected brand, the proportions of prozone with no visible or faint HRP-2 lines were 10.9% (CI: 5.34-19.08), 1.2% (CI: 0.55-2.10) and 0.1% (CI: 0.06-0.24) among samples with high parasitaemia, all positive samples and all submitted samples respectively. Prozone occurred mainly, but not exclusively, among young children.A total of 873/7,543 (11.6%) samples showed Prozone occurs at different frequency and intensity in HRP-2 RDTs and may decrease diagnostic accuracy in the most affected RDTs. Plasmodium antigens in blood by antibody-antigen interactions on a nitrocellulose test strip. The targeted antigens include those specific to Plasmodium falciparum (histidine-rich protein-2 (HRP-2) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH)) and antigens common to P. falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae . RDTs combine a control line with one, two or three antigen-detecting test lines, and are referred to as two-, three- and four-band RDTs respectively.Currently malaria rapid diagnostic tests (RDTs) detect P. falciparum at densities above 100 asexual parasites/μl or > 0.002% of parasitized red blood cells (RBC). Most false-negative results occur at lower parasite densities. However, false-negative results have been reported also at high parasite densities. Part of those may be ascribed to genetic variations of the HRP-2 [P. falciparum parasite densities and dilution of the sample can trace and correct the effect [P. falciparum. Prozone was observed among 16 out of 17 HRP-2 RDTs but not among five Pf-pLDH RDTs [RDTs are being rolled out as an alternative to microscopic diagnosis in malaria endemic settings  and havehe HRP-2 , but thehe HRP-2 ,8. In RDe effect . In a reLDH RDTs . HoweverThe main aim of the present study was to assess the frequency of the prozone effect in a malaria endemic field setting. A subsidiary aim was the confirmation of the previous observation that HRP-2 RDTs, but not Pf-pLDH tests, are affected by prozone .P. falciparum. Transmission is perennial with peaks during and at the end of the rainy season (February - April)[The study was conducted in the emergency ward of the Provincial Hospital of Tete (PHT), located in Central Mozambique. In this area, malaria is predominantly caused by  - April),11.The PHT serves as a reference hospital for Tete Province . According to hospital statistics, yearly approximately 50.000 patients present themselves with clinical suspicion of malaria. Diagnosis is confirmed in about 20% of them (either by RDT or microscopy). For this study, all patients suspected of malaria and presenting at the emergency ward of the PHT were prospectively included on a 24 hours/7 days basis from January till April 2010.ICT Malaria, Paracheck-Pf and SD Malaria Antigen Pf FK50) were routinely used .Routine procedures for malaria diagnosis  at PHT are as follows: EDTA-anticoagulated blood is sampled and a full blood count is performed by an automated haematology analyzer . For children ≤ 5 years, malaria diagnosis is made on a thick blood film (TBF). For patients above five years of age, an RDT is performed and in case of a positive result a TBF is made for confirmation and determination of parasite density. TBFs are stained for 20 minutes at pH 7.2 using Giemsa 3.5% . According to the national malaria clinical guidelines, parasite density is scored on a semi-quantitative scale from 1+  to 5+  . During P. falciparum and scored as 4+ or 5+, parasite densities were quantitatively assessed: the number of asexual parasites was counted against 200 white blood cells (WBC) and converted to parasites/μl using the WBC count/μl. Hereafter, values were converted to % of parasitized RBC using the RBC count/μl. WBC and RBC counts were those provided by the haematology analyzer.For the purpose of the study, demographic data, presenting symptoms and clinical signs of severe malaria  were recSamples with a high parasitaemia, defined as ≥ 4% of parasitized RBC ,15, wereMalaria RDTs in cassette format were selected based on demonstrated diagnostic accuracy -18, use For each sample with high parasitaemia, a 10-fold dilution was made in saline. Both undiluted and diluted sample were assessed with each RDT brand and tests were run in duplicate and read by two observers. The second observer was blinded to the first observer's readings. Tests were performed according to the instructions of the manufacturer, except for the use of an automatic pipette  instead of the RDT kits' transfer device. Test line intensities were scored into five categories: none (no line visible), faint (barely visible), weak , medium  and strong (stronger than the control line) . When a Hexagon Malaria Combi and Malaria Pan/Pv/Pf Rapid device), not delivered in time for the prospective analysis, were assessed at the end of the study period with samples that had been stored at -20°C (maximum storage duration: 121 days).Two additional HRP-2 RDT brands . On a daily basis, 10% of TBFs were randomly elected and reread by a member of the study team who was blinded to the original result. All discordances were resolved by a third reader's reading. A photograph was taken from all RDT tests performed and the TBFs were stored for quality control.All RDTs were purchased in Belgium and shipped to Tete, except for According to the initial sample size calculation, a total of 5,700 patients suspected of malaria were required for reliable estimation of the frequency of prozone. This was based on the following assumptions and information from hospital statistics: malaria-attributable fraction during the wet season of 30%, prevalence of hyperparasitaemia of 10%, prozone frequency among hyperparasitaemia samples of 30% with 95% confidence intervals (CI) between 20% and 40%.Data were recorded in registers and on individual case report forms. Data were entered in Microsoft Access  and analyzed in Stata 11.1 . Differences between proportions were tested for significance using the Pearson's Chi-square test or, in case of small sample sizes, a two-tailed Fisher's exact test. Reproducibility and inter-observer reliability for line intensity readings were assessed using the kappa statistic for paired observers and percentage agreements. Differences between medians were tested using the Wilcoxon test. Lot variations for matched pairs of samples were assessed using the McNemar's test. The relation between line intensities of prozone samples across the parasite densities was assessed using the Cuzick's test for trend. A p-value < 0.05 was considered as significant.The study was approved by the Institutional Review Board of ITM, by the ethical committee of Antwerp University, Belgium and by the Comité Nacional de Bioética para a Saude (MoH), Mozambique. Patients, children's parents or guardians were informed in Portuguese or in the local language (Nhungue) and their written consent was required prior to enrolment.P. falciparum infection by thick blood film  were diagnosed with 1.6% wereHexagon Malaria Combi, Malaria Pan/Pv/Pf Rapid Device and CareStart Malaria pLDH): temperatures of 31°C and 32°C were registered for a cumulative period of 7.6 and 1 days respectively. Invalid results were observed for five brands and ranged between 0.2% and 0.7% of tests performed; upon repetition all tests performed well. For the HRP-2 line intensity readings, the overall agreement among the two observers ranged from 86.1% (Paracheck-Pf) to 97.1%  and kappa values ranged from 0.67  to 0.90 . The reproducibility of the RDTs among the duplicate tests in terms of HRP-2 line intensity ranged from 71.7%  to 97.4%  and kappa values ranged from 0.5  to 0.8 .Shipment and storage temperatures of RDTs ranged between 4.0°C and 23.5°C (mean 17.5 ± 8.7°C) and 23.3°C and 32.3°C (mean 28.9 ± 1.5°C) respectively. Storage temperatures exceeded the highest allowed temperature (30°C) for three RDT brands Prozone affected all six HRP-2 RDT brands in proportions ranging from 6.7% to 38.2% of samples tested. The two Pf-pLDH RDTs did not show any prozone positive sample. Table Paracheck-Pf) according to different denominators. As an example, the proportions of false negative or faint HRP-2 lines were 10.9%, 1.2% and 0.1% among the samples with parasite densities ≥ 4%, all P. falciparum positive samples and all suspected samples respectively. For the ICT malaria similar proportions were 14.1% (CI: 7.74-22.95); 1.5% (CI: 0.80-2.53) and 0.2% (CI: 0.09-0.29) respectively.Table Paracheck-Pf. Among the high parasitaemia samples, prozone occurred most frequently but not exclusively among the group less than five years of age. No significant relation between prozone and the presenting symptoms was found. However, cough was significantly more prevalent among the prozone positive group. Of note, clinical signs of severity were not associated with prozone, and, moreover, prozone occurred also in 10 (36%) patients without any sign of severe malaria. Among the laboratory values, the prozone group was significantly associated with lower hemoglobin levels and RBC counts. Parasite densities of prozone positive and prozone negative samples did not differ significantly.Table ICT Malaria, at parasite densities of 2.2%, 2.9% and 3.4% respectively).Table Malaria Pan/Pv/Pf Rapid Device: for 43 samples assessed by both lots, prozone was observed in 5 (11.6%) versus 22 (51.2%) samples respectively (p < 0.0005).Table P. falciparum in children in Africa [In 2009, an estimated 225 million cases of malaria occurred with 781.000 deaths, mostly due to n Africa  and are Some limitations are however to be mentioned. The wet season of 2009-2010 in Mozambique was characterized by irregular rainfall and long dry spells. Due to this low rainfall  and theParacheck-Pf and the two samples accounted for 1.1% of all malaria positive samples which is in line with the present findings. Similar observations affecting Paracheck-Pf or other HRP-2 RDT brands have been reported from endemic as well as non-endemic settings [There are only few original studies reporting on prozone in malaria RDTs ,29. Prozsettings -33.In line with previous findings, the present study demonstrated prozone in HRP-2 but not in Pf-pLDH based RDTs frequently used in field settings . CompareFrom the present results, it is clear that prozone occurred dispersedly among samples with high parasitaemia. Prozone with non-visible test lines occurred exclusively in samples with parasite densities above 8% and the frequency and intensity of prozone decreased below the 4% threshold. However, the association parasite density - presence of prozone among samples with parasite densities ≥ 4% was not straightforward. This may be ascribed to factors such as capillary sequestration of the parasites, variations in the antigen production during the cycle and strain differences . ProzoneParacheck-Pf and ICT Malaria), prozone with negative or faint HRP-2 test lines (the most dangerous situation) occurred in at least 1.2% of malaria-positive samples and 10.9% of samples with high parasitaemia. At such frequencies, the diagnostic accuracy may be affected and the impact on predictive values will depend on the malaria-attributable fraction of fevers and the proportion of high parasite densities: both factors are related to transmission intensity and pre-existing immunity of the affected population [P. falciparum prevalence rate in children aged two to ten years is actually estimated at 17% [Paracheck-Pf and ICT Malaria  respectively.For the two most affected RDT brand : Rapid Diagnostic Test(s); TBF(s): thick blood film(s); WBC: white blood cell; RBC: red blood cell; WHO: World Health Organization.Ag: Antigen; CI: confidence intervals; EDTA: Ethylene Diamine Tetra-acetic Acid; FIND: Foundation for Innovative New Diagnostics; HRP-2: histidine-rich protein-2; ITM: Institute of Tropical Medicine; NaCl: Sodium Chloride; MoH: Ministry of Health; The authors declare that they have no competing interests.PG, ADW, SM, EB and JJ designed the study; PG, AS, JS, ADW and EB organized laboratory and clinical supervision in Tete; PG, AS, JS, HC and OJV actively participated in the laboratory work in Maputo and Antwerp; PG, AS, ADW, EB and JJ assessed and interpreted the results. All authors contributed to the discussion of the results and the redaction of the manuscript, they all approved the final manuscript.


These markers can identify spatial variations in transmission patterns. Plasmodium falciparum or P. vivax was not detected by microscopy in cross-sectional surveys of samples from persons during the dry  and wet  seasons. Antibody responses against P. falciparum or P. vivax were detected in 17.9%  and 19.3%  of persons tested. Reactivity against P. falciparum was significantly different between 3 villages (p<0.001); clusters of seroreactivity were present. Distance to the nearest seasonal river was negatively associated with P. falciparum (p = 0.028) and P. vivax seroreactivity (p = 0.016). Serologic markers are a promising tool for detecting spatial variation in malaria exposure and evaluating malaria control efforts in areas where transmission has decreased to levels below the detection limit of microscopy.Areas in which malaria is not highly endemic are suitable for malaria elimination, but assessing transmission is difficult because of lack of sensitivity of commonly used methods. We evaluated serologic markers for detecting variation in malaria exposure in Somalia.  Plasmodium falciparum. Almost all areas where P. falciparum parasite prevalence is >50% in the general population are located in Africa  is difficult when mosquito numbers are low, sometimes below the detection limits of commonly used trapping methods  and 2 peaks in rainfall in April and August. Three moderately sized communities were randomly selected from census maps by using spatial random sampling techniques in Arcview version 3.2    sample and thick and thin blood smears. One-hundred high-power microscopic fields were examined and an additional 100 fields were examined if the first 100 fields were negative. RDT results were used for treatment with sulfadoxine-pyrimethamine and 3 doses of artesunate according to national guidelines. A second cross-sectional survey was conducted at the end of the wet season (August–September 2008) by using procedures identical to those described above, except that part of the fingerprick blood sample was placed on filter paper  as described by Corran et al.  in the 3 villages at the end of the wet season. Locally produced 250-mL dippers with a white surface were used. Five to 10 dips were made in the large water bodies and the presence of Anopheles spp. larvae was visually assessed and recorded.Presence of Filter paper samples were stored at 4°C with desiccant until processed. A 3.5-mm blood spot, equivalent to ≈3 μL of blood  and 1:2,000 for antibodies against apical membrane antigen 1 (AMA-1) by using described ELISA methods (19 (Wellcome genotype) and AMA-1 (3D7) were coated overnight at 4°C at a concentration of 0.5 μg/mL. Plates were washed by using PBS, 0.05% Tween 20 (PBS/T) and blocked for 3 h with 1% (wt/vol) skim milk powder in PBS/T. Positive controls (a pool of hyperimmune serum) and negative controls  were added in duplicate to each plate. The plates were washed and horseradish peroxidase–conjugated rabbit anti-human immunoglobulin G   was added to all wells. Plates were developed for 20 min by using an o-phenylenediamine dihydrochloride substrate solution. Reactions were stopped with 2 mol/L H2SO4. Plates were read immediately at 492 nm and optical density (OD) values recorded. For P. vivax, an identical protocol was used with MSP-119 (0. 5 μg/mL)  for each species . The seroconversion rate was estimated by fitting a simple reversible catalytic model to the measured seroprevalence by age in years by using maximum-likelihood methods. The serologic-derived annual EIR was then estimated by using the MSP-119 seroconversion rate and a calibration curve derived from determined values . Duplicate OD results were averaged and normalized against the positive control sample on each plate. A cutoff value above which samples were considered antibody positive was defined by using a mixture model as described , distance to the nearest enclosure of livestock (in 100 m), number of household members, number of houses in a 100-m radius, roofing material, wall material, floor material, travel history, recent or regular bed net use, and an indicator of household wealth. The household wealth index was calculated on the basis of principal component analysis on characteristics such as ownership of a television, radio, telephone, bicycle, motorbike, cattle, and access to electricity .For detection of spatial clusters in immune responses, age-adjusted logP. falciparum or P. vivax parasites were not detected on any of the examined blood slides  and August–September  2008. These surveys were characterized by a clear seasonality with no rainfall detected during November 2007–March 2008 and a median monthly rainfall of 114.5 mm in April–August 2008. None of the survey participants were positive by rapid diagnostic test, and d slides . AvailabP. falciparum  but not to P. vivax , after adjustment for age and village of residence. These 19 persons were excluded from further analyses.In August–September 2008, serum samples were collected from 1,128 persons in Xuunshaley (n = 271), Badahabo (n = 160), and Ceel-Bardaale (n = 697) . In the P. falciparum based on seroconversion rates for MSP-119 and AMA-1  of the persons tested showed reactivity against P. falciparum  and 19.3%  against P. vivax. There was a significant positive association between reactivity against P. falciparum and P. vivax (p<0.001). However, only 39.8% (66/166) of persons with antibodies against P. falciparum also responded against P. vivax antigens, and there was no apparent correlation between antibody titers against antigens of the 2 malaria species (p>0.58).All antigens tested showed a clear increase in seroprevalence with a person’s age . The datP. falciparum antibody prevalence was 9.4% (23/244) in Xuunshaley, 21.7% (30/138) in Badahabo (p = 0.001), and 20.4% (126/619) in Ceel-Bardaale (p<0.001)  in Xuunshaley, 21.0% (31/148) in Badahabo (p = 0.11), and 20.2% (131/648) in Ceel-Bardaale (p = 0.13) (p<0.001) . P. viva = 0.13) .P. falciparum seroreactivity was significantly increased in a cluster of 18 households (108 persons) in Ceel-Bardaale (p = 0.002) (P. vivax seroreactivity (p = 0.005).Age-adjusted = 0.002) . In Xuun>7 km apart and were therefore likely to have their own transmission characteristics. In all 3 villages, P. falciparum antibody prevalence increased with age  after adjustment for age and correlation between observations from the same household. Within the group of persons who had a positive antibody response against P. falciparum, the titer increased with age in Xuunshaley  and Ceel-Bardaale .Seroreactivity data were analyzed for villages separately because villages were with age . For CeeP. falciparum, P. vivax antibody prevalence increased with age in all 3 villages  after adjustment for age and correlation between observations from the same household. P. vivax antibody titer did not increase with age or any other factor in those persons who were seropositive. Household factors, socioeconomic factors, distance to the nearest livestock enclosure, and use of mosquito netting were not independently associated with immune responses against P. falciparum or P. vivax.Similar to villages . For CeeP. falciparum antibodies were detected in 22 children <5 years of age ; 10 had antibodies against P. vivax , and 2 of these children had antibodies against P. falciparum and P. vivax. Thirty children <5 years of age who responded to malaria antigens were from all 3 villages . Travel to areas in which malaria was highly endemic in the past 3 months was not reported for any of these children with antibodies against P. vivax, P. falciparum, or both. In children <5 years of age, a response against P. falciparum antigens was not related to a response against P. vivax antigens (p = 0.30).Although seroprevalence and antibody titers were higher in older age groups, seroreactivity was also observed in young children. 19 and AMA-1 seroprevalence data showed a clear increase in seroreactivity with age and evidence for variation in exposure to malaria between and within villages.We showed that serologic markers can be used to detect heterogeneity in malaria transmission in the Gebiley District of Somalia where malaria transmission occurs at levels too low to be detected by microscopy. None of the slides or rapid diagnostic tests showed parasite carriage in the population, and MSP-1P. falciparum transmission intensity in this area in Somalia was low (EIR <0.1 infectious bites/person/year). Because of the longevity of antibody responses, this estimate should be interpreted as an average EIR experienced over several years. The low EIR appeared to be supported by examination of breeding sites at the end of the wet season, which confirmed the presence of malaria vectors at a low density. We did not directly determine the EIR by sampling adult mosquitoes because the low density of mosquitoes would have required intensive sampling over different seasons   or P. vivax (n = 6). The presence of strong antibody responses (indirect fluorescent antibody titer >20) in children <15 years of age was used as evidence for active transmission of malaria in area of low endemicity in Middle America (Costa Rica) . Malaria should be considered as a plausible cause of febrile illness, particularly in an epidemic form. Low-intensity malaria transmission and the presence of malaria vectors make the area susceptible to malaria epidemics, which can have a high mortality rate in resource-poor areas (P. falciparum and, less markedly, P. vivax were lowest in Xuunshaley, which was furthest from seasonal rivers. Combined P. falciparum MSP-119 and AMA-1 antibody prevalence was 2× higher in Badahabo and Ceel-Bardaale than in Xuunshaley. SaTScan analysis indicated heterogeneity in malaria exposure at a microepidemiologic level. We observed 1 statistically significant cluster of persons with higher seroreactivity against P. falciparum and 1 with higher seroreactivity against P. vivax. In Ceel-Bardaale, where households were scattered along a delta of seasonal rivers, antibody prevalences to P. falciparum and P. vivax were negatively associated with distance to the nearest river. In several areas of higher endemicity, distance to the nearest body of water has been related to malaria incidence (We observed heterogeneity in seroreactivity within the study area. Although the 3 villages had low transmission intensity and showed no difference in microscopic parasite carriage, serologic markers showed variation in malaria exposure. Antibody prevalence against Our data indicate that serologic markers can be used to determine variation in transmission intensity at levels of malaria transmission that are too low for sensitive assessments by microscopy, RDT, or entomologic tools. The sensitivity of serologic analysis to detect small-scale differences in transmission intensity may prove extremely useful in evaluating malaria control programs in areas where conventional malariometric markers fail. It may also provide vital information on which areas are most likely to be receptive to transmission if malaria epidemics were to occur.