Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge.Thick blood films are routinely used to diagnose in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia.The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities.Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density. Plasmodium falciparum malaria. Density is usually assessed by thick films, either by counting parasites per microscope field, or by counting parasites per hundred white blood cells [Microscopy of thick blood films is the usual diagnostic test for od cells . Thick fSporozoite challenge experiments were conducted, where volunteers were exposed to experimental malaria challenge to assess candidate malaria vaccines -5. Treatin vitro studies.The sensitivity of thick blood films was studied using data obtained during these trials, compared this with quantitative PCR data, and further investigated these findings with Volunteers gave informed consent. Procedures were reviewed by OXREC (Oxford Research Ethics Committee), the local ethics committee, and were in accordance with the declaration of Helsinki (revised 1983). Twice daily blood samples were taken from day 6 until day 14, then daily until day 21. At least 100 high powered fields of a thick blood film were viewed and quantitative PCR performed on each sample. Volunteers were treated when a single parasite was seen by blood film, after the appearance of the parasite was confirmed by a second microscopist. Neither managing clinicians nor microscopists were aware of PCR data during the trial.Giemsa staining was used for the first two sporozoite challenge studies, and Field's stain in coplin jars for the later two studies. The thick film was air dried in both methods. For giemsa staining, the film was stood in 5% Giemsa for 30 minutes, then washed gently in tap water and air dried. Field's stain was applied by dipping the slide into Field's stain A for 3 seconds, then into tap water for 3 seconds (with gentle agitation), into Field's stain B for a further 3 seconds and then washing gently in tap water to remove excess stain. The slide was then air dried for at least 30 minutes. The lead microscopist held a post in the London School for Hygiene and Tropical Medicine Clinical Parasitology Laboratory, the UK national reference laboratory, and others at the Medical Research Council, the Gambia. The lead microscopist examined slides produced by serial dilutions, blind to source. The average thick film uses 10 μl of blood spread over one thousand high powered fields, so the 100 high powered fields routinely examined during views 1 μl of blood .P. falciparum was amplified by PCR and the increase in PCR product detected by binding the fluorescent dye SYBR Green I using the Rotor-Gene Real-Time PCR machine (Corbett Research), using l μl extracted DNA in duplicate. The increase in PCR product is quantitated by comparison with standard preparations of known parasite numbers.The PCR method is described elsewhere . Briefly870 paired blood films and PCR samples were examined from 80 volunteers with rising parasite counts.The poisson distribution was used to calculate the likelihood of sampling a parasite within the blood volume examined in microscopy, at given parasite densities identified by PCR. At low parasitaemias there was a discrepancy between the likelihood of diagnosis calculated by PCR readings, and the actual frequency of diagnosis at that density by thick film. For the thick films prepared between 100 and 1000 parasites per ml, the overall calculated likelihood of sampling a parasite in 1 μl was 26%. However, thick films had a sensitivity of only 9% (95% CI 4–15%) in this range. Between 1,000 and 10,000 parasites per ml, the calculated likelihood of a parasite being present was 84%, but the actual sensitivity of thick films was 29% (CI 19–39%). It was only above 10,000 parasites per ml when thick films had higher sensitivity (81% CI 65–97%), when the calculated probability of sampling was 99%.This surprising finding suggested that a significant number of parasites were not visualized on a thick film despite being theoretically present in the original blood sample used to make the film. Results did not vary according to staining protocol (Giemsa or Field stain) or by microscipist. A similar density threshold for reliable diagnosis of malaria by thick film examination is reported elsewhere .in vitro culture of Plasmodium falciparum was prepared at 5–10% parasitaemia. The parasite count was first accurately determined by a thin film (in duplicate). The culture was then serially diluted with uninfected, fresh whole blood. Red cell counts were made by Coulter™ counter for both the original culture and the uninfected blood used for serial dilutions. This allowed accurate calculation of predicted parasite numbers, without having to count thin films at each dilution. At each serial dilution, PCR analysis was conducted on 0.5 mls blood, and 2 thick films made, using exactly 10 μl. The whole film was read, blind to source.Experiments using serial dilution of a known parasite density were then conducted to extend this observation. An For the serially diluted parasite cultures, the parasite density measured by PCR and thick film was compared with that calculated from serial dilution Fig . AlthougIt is unlikely that cells or parasites are hidden during microscopy, since adequate preparation of the slide ensures visibility through all planes of focus. It is more likely that parasites are either washed off or lysed during staining, since PCR of the staining reagents to detect parasites was positive, and transfer of parasites from positive slides to negative slides during staining has been recognized for some time .in vitro cultures were synchronous.Thin film and thick film parasite density estimates have been compared in previous studies. Although thick films are more sensitive than thin films, they significantly underestimated the parasite density in some studies ,9,10, buThick films are considerably less sensitive than might be possible and underestimate parasite densities. In routine clinical work in non-endemic areas the loss in sensitivity makes thick film examination much more laborious, and a fixation method that prevented parasites being washed off would improve the sensitivity of the method. In malaria endemic areas, the loss in sensitivity is not critical, since semi-immune patients are likely to have a high parasitaemia if they present with febrile malaria. In epidemiological studies, parasite density thresholds are defined to distinguish febrile malaria from chronic parasitaemia ,13. A syExperimental Design: PB, FS, SGExperimental work: LA, FS, AHC.Writing of Manuscript PB, SG, AVSH.


Plasmodium falciparum outbreaks can occur in the coastal area of French Guiana, where the population is essentially non-immune. Two sporadic outbreaks were observed, including one with severe malaria cases. To characterize these outbreaks and verify previous observations of specific genotype characteristics in severe malaria in this area, all cases from each outbreak were studied.P. falciparum genotypes for six genetic loci were determined by PCR amplification from peripheral blood parasites. The msp1/block2 and msp2 genotypes were determined by DNA sequencing. Microsatellite and varD genotyping was based on size polymorphism and locus-specific amplification.The outbreak including severe malaria cases was associated with a single genotype. The other mild malaria outbreak was due to at least five distinct genotypes.varD/B-K1 msp1 linkage and its association with severe malaria in this area was confirmed.Two distinct types of outbreak occured despite systematic and sustained deployement of malaria control measures, indicating a need for reinforced vigilance. The  Plasmodium falciparum and 40 % due to Plasmodium vivax. French Guiana is an area with multidrug resistant P. falciparum malaria. Transmission occurs in isolated foci located in settlement pockets within the Amazonian forest and along the rivers. The main malaria-endemic areas are located along the Maroni and Oyapock rivers, which serve as natural frontiers with Surinam and Brazil, respectively [The annual malaria incidence in French Guiana has increased ten-fold during the last 30 years, reaching nowadays approximately 3%, with 60% due to ectively . As a reP. falciparum parasite population of French Guiana presents a remarkably low degree of polymorphism, with a clonal type structure [msp1/block2 allele that was called B-K1 and a particular var gene called varD in isolates from patients with severe P. falciparum malaria [The tructure . The par malaria .P. falciparum outbreaks were studied, one with five cases, including two cases of severe malaria and one death, the other with nine mild malaria cases. The isolates from each case were characterized for both outbreaks using a six-locus genotyping approach, including varD, msp1/block2, msp2 [To verify this association and better understand the possible cause of local outbreaks in the coastal area located away from the main endemic sites, two k2, msp2 , as wellk2, msp2 .P. falciparum malaria in health centres and sent to the laboratory for routine drug susceptibility phenotyping and genotyping analysis. The policy of the malaria control services is to get an in vitro susceptibility profile for each diagnosed P. falciparum malaria case. To this end, a 5 mL blood sample was systematically collected by venepuncture in EDTA tubes and maintained at 4°C during shipment to the laboratory. The samples were immediately processed for in vitro susceptibility assays and a 0.5 mL aliquot was frozen at -20°C for subsequent DNA preparation. The five Macouria isolates were collected in June and July 2001 from three mild  and two severe cases . Patient E57 had visited the endemic Macapa region in Brazil one month before the malaria attack. The other four Macouria patients did not report having left the area. The nine Matoury isolates, collected from February to June 2002 originated from mild malaria cases, none of whom reported a recent visit to an endemic area.The outbreaks occurred in Macouria and Matoury, both located on the French Guiana coast, an area with unstable, low transmission, away from the main endemic areas along the Maroni and the Oyapock Fig. . The intmsp1 block2, msp2 and varD, PCR was performed in a 50 μL final volume containing the DNA template, 1.5 mM MgCl2, 2 μM each primer, 200 μM dNTP, and 1.5 units of AmpliTaq Gold polymerase . The PCR conditions for msp1 block2 and msp2 were : one cycle at 95°C for 5 min, followed by 40 cycles at 94°C for 1 min, 56°C for 2 min, 72°C for 2 min, and final extension at 72°C for 10 min. The varD PCR conditions were: one cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 10 sec, 61°C for 1 min 30 sec, 72°C for 2 min, and one cycle at 72°C for 10 min. The PCR products were analysed by agarose gel electrophoresis and directly sequenced . Sequences were aligned using the Clustal W program, followed by manual analysis using the ED editor of the MUST package [DNA was extracted as described . Genotypet al. [2, 2 μM each primer, 200 μM each dNTP, and 0.5 Units Taq polymerase . The samples were subjected to 1 cycle at 94°C for 7 min, followed by 35 cycles at 94°C for 30 sec, 52°C for 30 sec, 47°C for 30 sec, 72°C for 30 sec, and a final extension step at 72°C for 15 min. The PCR products were analysed by 3% Metaphor agarose gel  electrophoresis and stained with ethidium bromide. Fragment size was calculated using the Taxotron software .Microsatellite typing was done using the C1M4, C3M27 and C4M69 loci, located on chromosomes 1, 3 and 4, respectively. The locus-specific primers described by Su et al.  were usemsp1/block2 and msp2 P. falciparum loci generated a single band for all isolates  repeats evolve at a faster rate than SNPs in the non-repeated domains [PCR amplification of the tes Fig. . Each PCus study . A smallus study , and a 3 domains . Numeroumsp2 alleles belonged to the 3D7-type family and were arbitrarily called 3D7a (591 bp), b (561 bp) and c (603 bp). The 3D7a allele was observed in all five Macouria isolates and in five of nine Matoury isolates. The 3D7b and 3D7c allele were observed in one and three Matoury isolates, respectively  carried the varD locus, none of which harboured the B-K1 msp1 allele . The Matoury 4 and Matoury 5 genotypes differed by the C4M69 microsatellite locus.French Guiana is a hypoendemic area, where the population has limited, if any, immunity. There are between 6,000–7,000 mild malaria cases and only between four and 20 severe malaria cases each year ,3. PrevivarD/B-K1 msp1 linkage and its association with severe malaria in this area. It is important to note that this association was previously detected in isolates collected in 1994-6, more than six years before the Macouria epidemic. Such a stability over time in a species with a high recombination rate [varD/B-K1 association was observed, as illustrated in Figure varD/B-K1 msp1 association was not strictly specific for severe malaria, as the same genotype was also observed in the non-severe Macouria cases. Whether the patients with mild malaria received an earlier treatment compared to those with severe malaria or were less susceptible due to their genetic or immune make up is unclear. The occurrence of two severe cases at one day interval in patients infected with this strain suggests a particular inherent virulence. Whether this is due to the particular msp1 and/or varD allele present is unknown. It may reflect physical association with another locus implicated in virulence. It is worth noting that isolate F52, which harboured varD together with the A-K1 msp1 allele, was collected from a patient with very high peripheral parasitaemia (20%), also considered a sign of severity [VarD is one of approximately 60 members of the P. falciparum var genomic repertoire [var gene is not synonymous with its expression. Expression of varD was demonstrated in a patient with fatal P. falciparum malaria in a previous study [varD expression in severe and non-severe malaria are warranted. Work is in progress to characterize the full varD gene sequence.These data confirm the ion rate  is consiion rate . The sevseverity . VarD ispertoire . The preus study , but couThe different genetic profiles of the isolates involved in the two outbreaks reveal distinct onset and dynamics scenarios. Genotyping strongly suggests that the Macouria outbreak was due to one single parasite strain, but the origin of this strain is uncertain. Transmission from E57, the first registered case, to the other cases is unlikely, due to the short delay between the clinical attack and the attack experienced by E61, E62 and E64 . E67 may possibly have subsequently transmitted the strain to E72, who experienced a malaria attack 37 days later. The diversity of the Matoury isolates indicates that the outbreak was certainly caused by several distinct strains. Matoury accommodates the largest airport in the country and consequently may serve as an occasional transmission focus for parasites originating in neighbouring endemic malaria areas. The possible cause and mode of transmission in this city at that time have not been identified.msp1/varD genotype with severe malaria, reinforcing the notion that some P. falciparum strains might cause more severe infections than others.The results point to two distinct types of outbreak in a region where malaria control measures are systematically deployed and sustained. Reinforced vigilance and rapid case notification are needed to ensure rapid deployment of vector control and personal protection measures to prevent such sporadic epidemics. The Macouria outbreak provoked two severe cases, including one death, a rare event in this area, where health facilities are well-equipped and treatment policy is regularly updated. Parasite genotyping confirmed the association of the B-K1 A. Lavergne and E. Legrand did the sequences analysis. B. Volney, C. Tournegros, D. Accombressi and L. Florent did the laboratory experiments. M. Guillotte and O. Mercereau-Puijalon adapted the microsatellite typing to field isolates. O. Mercereau-Puijalon did the database search. All authors participated in the analysis and interpretation of data. E. Legrand and O. Mercereau-Puijalon wrote the manuscript.


Malaria control strategies are designed as a solution for either the whole region or the whole country and are assumed to suit every setting. There is a need to shift from this assumption because transmission may be different from one local setting to another. The aim of this study was to assess the risk of clinical malaria given the village of residence among under-five children in rural north-western Burkina Faso.867 children (6–59 months) were randomly selected from four sites. Interviewers visited the children weekly at home over a one-year period and tested them for fever. Children with fever were tested for malaria parasites. An episode of clinical malaria was defined as fever (axillary temperature ≥ 37.5°C) + parasites density ≥ 5,000 parasites/μl. Logistic regression was used to assess the risk of clinical malaria among children at a given site of residence.Children accumulated 758 person years (PYs). Overall, 597 episodes of clinical malaria were observed, giving an incidence rate of 0.79 per PY. The risk of clinical malaria varied amongst the four sites. Taking one village as reference the odds ratio for the other three sites ranged from 0.66; 95%CI: 0.44–0.98 to 1.49; 95%CI: 1.10–2.01.Malaria control strategies should be designed to fit the local context. The heterogeneity of transmission should be assessed at the district level to allow cost-effective resource allocation that gives priority to locations with high risk. Functional routine health information systems could provide the necessary data for context specific risk assessment. Malaria remains a public health concern worldwide and especially in sub-Saharan Africa (SSA), where it is a major killer among children under five years of age -4. This There is evidence that malaria transmission risk varies even on the smallest scale. Wang et al have demonstrated inter-city variation of malaria prevalence among school children . In Dar The study was conducted in the town of Nouna and the villages of Cissé, Goni, and Kodougou. These four sites are part of the Nouna Demographic Surveillance System (DSS) area , which iA cohort of 867 children  aged 6 to 59 months, participated in the study. The children were selected from each site by means of cluster sampling of households using a sampling frame generated from the DSS database. The sample size was set to detect inter-site differences in the incidence of clinical malaria of at least 10% (if they exist), with 80% power and 95% confidence, and in anticipation of 15% loss to follow up. The Local Ethics Committee of Nouna approved the study.Four trained-interviewers, one based in each site, visited individual children at home every week from December 2003 to November 2004. This follow up period of 12 months covered one dry and one rainy season. At each visit, the interviewers measured the axillary temperature of the child and collected a blood sample (by finger prick) from any child with fever. In addition, they asked the mother/caretaker about the use of mosquito nets and collected data on housing conditions including the presence of animals, and potential breeding sites within a 30-metre radius of the house. This information was cross-checked by observation.A field supervisor regularly collected the forms and blood slides from the field and transported them to the Nouna Health Research Centre laboratory. Two laboratory technicians read the blood slides stained with Giemsa to determine the parasite count. In cases of non-agreement, a biologist read the slide. This happened with only 2% of the slides. Parasite density was estimated by counting parasites in 100 fields and equating the found value to 0.25 μl. The outcome of interest was clinical malaria episode, defined as axillary temperature ≥ 37.5°C plus a parasite density ≥ 5,000 parasites/μl. This case definition is similar to the one used by previous studies in this region .For ethical reasons, interviewers treated every child with fever for malaria, in accordance with national treatment guidelines. When fever persisted for two days or other symptoms surfaced, they referred the child to the nearest health facility. The project covered all related costs.Data entry clerks entered the data into a relational database developed in Microsoft Access. The data entry screen was similar to the data collection forms and included automatic consistency checks. At the field level, supervisors visited the interviewers and checked the filled forms before sending them for data entry. At the data entry level, a data entry supervisor randomly checked 5% of data entered each day. The critical proportion of error was set to the 5% level. Data was to be re-entered completely if the proportion of found errors was above 5%. However, this situation did not occur. Clean data were exported to STATA 8.1 for statistical analysis.Logistic regression was used to estimate the odds ratio (OR) of clinical malaria associated with residing in one of the four sites using the Goni site  as a reference. The multivariate model included a number of potential confounders and these are shown in Table The model was built as follows:πi is the predicted probability of having clinical malaria of the ith child; the odds of the same child will be β0 is the intercept and β1 ... β17 the regression coefficients of the independent variables (name following each coefficient). The odds ratio associated with a given coefficient is the exponential of the respective beta coefficient. Site_Cissé compared to Goni (reference) is the exponential of β1 (ORSite_Cissé = exp (β1)).Where p value = 0.420). Similarly, the age distribution did not differ between sites . Participants were almost equally distributed between the age groups except for the age group below 12 months (9.2%) (Table The 867 children were from 427 households and the average number of children per household was two. Kodougou had the largest mean number of children per household (2.5). Overall, females were more (52.5%) than males but the sex distribution across the sites was not significantly different  or due to migration out of the study sites (13) . This incidence was similar in all sites . Out of the 1,635 fever episodes, 597 were confirmed as being due to clinical malaria, giving an incidence rate of 787.6 per 1,000 PYs. The highest incidence rate was observed in Goni , followed by Kodougou , Cissé  and Nouna .The results of the logistic regression Table  showed tThere were differences in the risk for clinical malaria between the four sites as shown by the number of episodes per PY and the odds of clinical malaria given the site of residence. The largest incidence rate was observed in Goni, which had about 1.2 times higher incidence than Nouna and Cissé. The odds ratio of clinical malaria for this site was also higher even though the highest odds ratio was found in Koudougou. Such differences were observable despite the relative proximity of the four sites since they are within a 19–44 kilometre radius of each other.The lower incidence observed in Nouna and Cissé and Kodougou compared to Goni could be explained by differences in the ecological settings of these sites. Goni is located in a plain where there is seasonal rice farming which creates favourable habitats for mosquitoes. Kodougou is a village about 200 metres from a permanent river, which serves as a water source for traditional irrigation. Proximity to irrigated and flooded agriculture practices have been reported to be highly associated with malaria infection -14. The Regarding the results from the logistic regression, the pattern observed in the crude incidence rate of clinical episodes per 1000 PYs was maintained after accounting for variables that may influence this distribution. Compared to children living in Goni, those living in Kodougou were more likely to have clinical malaria while those from Cissé and Nouna were significantly less likely to have it. Differences in the ecological settings of the sites as discussed above could still explain these differences in risk. Other covariates showed different effects on the risk of contracting clinical malaria.et al [Older children were better protected than the young ones. The odds were much higher in the age group below 12 months. This was not surprising because children below 6 months have immunity acquired from the mothers, which they progressively loose, as they grow older. Reyburn et al  reportedStudies have suggested that ethnicity has a significant effect on the clinical malaria risk. Studies in this area have shown that the Peuhl  are less susceptible to malaria due to their reduced genetic susceptibility compared to other ethnicities ,18. HoweSelf-reported usage of a mosquito net was surprisingly not associated with a decrease in clinical malaria risk among children as has been extensively reported in other studies -24. The Among the factors related to the housing conditions, only the presence of farming activities within a 30-metre radius of the household was associated with increased risk of clinical malaria. The agriculture practice in this region is to dig small furrows between the crops to retain rainfall water for better ground infiltration. Such water bodies combined with the shadow provided by the crops create a suitable environment for vector breeding, thereby increasing the risk of clinical malaria.The rainy season, as expected is associated with an increase of clinical malaria episodes. In the region where the climate is mostly dry, rainfall, occurring during a specific period of the year, is the main source of breeding sites (open water bodies) for the malaria vector. In contrast, during the dry season, the hot and dry weather is unsuitable for vectors, thereby leading to high vector mortality and reduction in infection. These findings are consistent with those from other studies ,26The implication of these findings is that malaria control strategies should be designed to fit location-specific contexts. The distribution of the factors responsible for malaria transmission may lead to different malaria transmission patterns even within such a relatively small geographical entity. At the level of a health district or even for national malaria control policies, it would be very informative to know the risk difference between locations as small as a village. Scarce resources for malaria control could then be allocated accordingly. While all villages at risk should receive malaria control measures, high-risk villages can be identified and given priority.Conducting such studies at the village level in every health district is not only time consuming, but it is costly. Additionally there are very few such studies already available for use. It is, therefore, imperative that well functioning health information systems are established and utilized. In practice, this will require health districts to collect routine health information data series (for example over a three-year period) to assess which villages or localities are at the highest risk, accounting for seasonal variation. This risk assessment may then be utilized to decide to which localities malaria control efforts should be focused rather than distributing resources equally or otherwise based on other criteria.YY designed and coordinated the implementation of the study. He performed the statistical analysis and drafted the manuscript. CK and VL participated in statistical analysis, writing of the manuscript and interpretation of the findings. RS participated in the design and implementation of the study, and writing of the manuscript. All authors read and approved the final manuscript.The author(s) declare that they have no competing interests.


The acceptability and efficacy of a new kit with a new formulation of quinine alkaloids designed for the intra-rectal administration in the treatment of non-per os malaria was assessed in the peripheral health care system of Mopti, Mali.®) was administered by the intra-rectal route to children with presumptive non per-os malaria at six peripheral heath care centres. The children were then referred to two referral hospitals where standard inpatient care including intravenous route were routinely provided. A malaria thick smear was done at inclusion and a second malaria thick smear after arrival at the referral facility, where a more complete clinical examination and laboratory testing was done to confirm diagnosis. Confirmed cases of severe malaria or others diseases were treated according to national treatment guidelines. Cases of non per-os malaria received a second dose of intra rectal quinine alkaloids. Primary outcome was acceptability of the intra rectal route by children and their parents as well as the ease to handle the kit by health care workers.A single-arm trial was conducted from August 2003 to January 2004. An initial dose of diluted quinine alkaloids  and 92% of parents or guardians readily accepted the intra-rectal route; 84% of health care workers found the kit easy to use. At the peripheral health care centres, 32% of children had a coma score ≤ 3 and this was reduced to 10% at the referral hospital, following one dose of intra-rectal quinine alkaloids (IRQA). The mean time to availability of oral route treatment was 1.8 ± 1.1 days. Overall, 73% of cases were confirmed severe malaria and for those the case fatality rate was 7.2%.IRQA was well accepted by children, their parents/guardians and by the health workers at peripheral health facilities in Mopti, Mali. There was also a quick recovery from deep coma and a reduced case fatality rate in severe malaria. Clinical malaria can be severe right from its onset. This is strongly related to individual susceptibilities  and ofteIn developing countries such as Mali, West Africa, the health care system is organized to allow treatment of severe malaria at the hospital level. However, most cases of malaria occur in rural areas far away from any hospital. Although local care providers exist in most remote areas they are not trained to adequately manage severe malaria episodes. Combined with others factors ,5, this P. falciparum strains with decreased sensitivity to quinine were reported [Quinine is a critical drug widely used for the treatment of severe malaria . Despitereported , the druThe use of intra rectal route for administering quinine has a proven efficacy in the treatment of uncomplicated malaria in several studies when compared to intra-muscular or intravenous routes . Intra rThe intra rectal route is commonly used in some Malians villages by mothers for the treatment of others pathologies and by health workers to treat seizures with diazepam.i.e., community health workers), under the supervision of the health care personnel working at the primary contact point between population and health care services. In Mali, the first contact with health care services is organized to occur at the community-based health care centre. A step-wise approach was adopted to introduce quinine usage outside health care services. In a first step, this pilot study of the acceptability of the IRQA was designed, to assess how the peripheral health care personnel and the target population will accept the approach in the district of Mopti. In a second step would be evaluated the acceptability and efficacy of the intra-rectal route when used by ARs. The final step would be the gradual scale-up of the approach to reach national coverage.The Malian National Malaria Control Program (MNMCP) decided since 2002 to promote malaria case management within communities with the aim to allow earlier treatment of malaria clinical episodes. For severe malaria, the MNMCP chose an emergency one or maximum two doses of intra rectal quinine at periphery before referral. The doses would be given outside health care services by members of the communities  The results of the pilot study that assessed the acceptability of the intra rectal gluconate salt quinine given at periphery by health care agents as an alternative early pre-transfer treatment strategy of severe malaria in Mopti are reported in this paper.Anopheles gambiae s.l. is the main vector and at peak transmission the entomologic inoculation rate (EIR) can reach, 22 infective bites per person and per night [Plasmodium falciparum carriage rate was 40–80% and malaria accounted for 30% of overall cause of mortality i.e. the first child killer in the area [The study was conducted in the district of Mopti located in central Mali, an area of seasonal and intense malaria transmission during the rainy season from July to November. er night . Plasmodthe area .Health services are organized in three levels of care in the region of Mopti. At periphery, the first point of contact with population is the community-based health care centre, run by a nurse or a physician and equipped to provide clinical care with no laboratory support. At the second level, district hospitals served as referral structures for the community-based health care centres. Each district hospital covers a population close to 150,000–200,000 inhabitants and provide medical and surgical services including gynaecology and obstetric with clinical laboratory. At the third level, the regional hospital located in the city of Mopti, provides medical, pediatric, general and specialized surgical care  and has capacity for laboratory diagnosis and X-rays. Despite its regional vocation the regional hospital of Mopti (HRM) mainly served as referral centre for the city of Mopti and its surroundings. Our study focused on community-based health centres that refer usually to the HRM and to the district hospital of Sevare.The study was conducted among children attending six peripheral community-based health care centres in four villages , located at a maximum distance of 12 km from the city of Mopti. In addition two urban community-based health care centres both located in the city of Mopti were included. Children were given an initial dose of IRQA if they fulfilled the criteria for entry in the study and were either referred to the HRM in the case of children from Medina Coura and the two urban community health care centres, or to the referral health care centre of Sevare for children from Fatoma, Socoura and Soufroulaye.Before the study started, the personnel of the selected health care centres were trained by the MNMCP and the team of researchers to standardize their diagnosis of malaria and to use the IRQA. In addition, the study protocol and procedures were explained. The personnel were instructed to recruit cases from the population that routinely attended their centres for care and that fulfilled the entry criteria into the study.Children presenting spontaneously at peripheral health care centres and looking for care were eligible. Children were included if they weighed between 5 and 25 kg, were diagnosed on a clinical basis as a non-per-os malaria episode, and if their parents/guardians gave consent after receiving information about the study. The following exclusion criteria were applied: refusal of parents, association with others evident and severe clinical conditions such as severe malnutrition or meningitis, diarrhoea, pre-existing anal pathology such as prolapsus, rectitis, anal fissure, chronic diarrhoea, contra-indication to quinine and uncomplicated malaria episode.i.e., meningitis or acute respiratory infection). Children with P. falciparum positive slides and the criteria for severe malaria defined by the World Health Organisation (WHO) [After an initial clinical assessment, including weighing the children, blood was collected by finger prick for a malaria thick smear which was not read immediately. A dose of 20 mg/kg of diluted quinine was administered by rectal route. Children were observed for 30 minutes and in the case of early expulsion of the solution, they received a further half dose. Afterwards they were transferred to the referral health care structures, in Sevare or in Mopti. Study team offered transportation for parents who needed it. At this level a more complete clinical and laboratory assessment was done. Finger prick blood was collected for a second malaria thick smear, determination of blood sugar and haemoglobin level. Malaria smears were read then and children were classified into two groups: confirmed cases of malaria and other diagnosis of children severe febrile illness (on (WHO)  were con® is composed of 96.1% of quinine, 2.5% of quinidine gluconate, 0.63% of cinchonine and 0.67% of cinchonidine hydrochloride with a pH at 4.5. The mixture of alkaloids was constituted with the aim to reproduce as closely as possible, the composition of the natural bark of quinquina.Quinine was used in the form of an emergency paediatric kit provided by Sanofi-aventis, France. The solution of Quinimax®, a vial of 13.5 ml purified water and a round-ended syringe specially designed for rectal administration. The syringe was marked according to weight and was validated to ensure that the amount of diluted Quinimax® corresponded to a given weight of the child and had a content of quinine corresponding to the dose of 20 mg/kg for that specific weight. The overall content of the 4 ml vial was 500 mg gluconate alkaloids of quinine. Just before use, the content of the vial of Quinimax® was diluted with the 13.5 ml of purified water giving a final dilution of 28.6 mg/ml. Only the amount of diluted solution corresponding to the weight of the child was withdrawn from the vial into the syringe. The syringe was marked with kilogrammes instead of millilitres in order to avoid the need for a calculation of dose at peripheral level and to facilitate the use of the paediatric kit.The kit was constituted of a 4 ml vial of QuinimaxP. falciparum parasite in peripheral blood and one of the following criteria: haemoglobin ≤ 5 g/dl, parasite density ≥ 100,000/μl, blood sugar ≤ 40 mg/dl, respiratory distress, seizures in the last 24 hours, coma, or jaundice.Severe malaria was defined as episodes of fever associated with Severe febrile illness associated with persisting vomiting and inability to drink or eat, in the absence of evident clinical signs or symptoms for a specific diagnosis, was considered as a non per os malaria episode.Study primary outcome was the acceptability of the intra-rectal route by children, their parents or guardians and by health care centres personnel. Acceptability of children was assessed by the presence of particular reactions during drug administration, such as pain ; presence of particular reactions shortly (30 minutes) after drug administration: local pruritis, pain, false sensation of stools for older children/important screaming for younger children/for all children presence of diarrhoea, blood in stools, inflammation of anal mucosis. These particular reactions were assessed by health care centres personnel at periphery after first administration of IRQA and at the referral level after the second administration. Anal margin and stools aspects were directly observed.Parents or guardians acceptability was assessed by interview and asking them how they would feel if another administration of the intra rectal route was proposed to treat their children.Health care centre personnel acceptability was assessed by asking them to fill in the case report form whether it was easy or uneasy to prepare the solution for intra rectal administration, to read the marks on the syringe for dosing the amount of solution to be given and to administer/re administer (in case of early expulsion) the solution.Secondary outcomes were time to recovery of oral route and time to clearance of clinical signs/symptoms. These outcomes were measured twice a day for 7 days after inclusion into the study.Before the study started in June 2003, all clinical personnel involved in collecting data and administering IRQA were trained to comply with standardized study procedures for obtaining informed consent, clinical examination, laboratory testing, filling in of case report forms, administration of IRQA using the emergency paediatric kit. Subsequently clinical, behavioural and laboratory data collected during the study were noted directly into standardized case report forms. Case report forms were verified on a daily basis by the study supervisor and three monitoring visits were conducted during the study to ensure quality of the data and compliance with protocol.p-value was less than 0.05.Data from standardized case report forms were double-entered with Microsoft ACCESS and database was reconciled. Analysis was done using SPSS 11.0 . Descriptive analysis was done, computing percentages and means on main endpoints with 95 confidence intervals. Additional analyses included comparisons of proportions using Pearson chi-square and Yates corrections or Fischer exact tests where indicated. Mc Nemar chi-square was used for paired data. Means were compared using Student T test with adjustment to account for repeated measurements on the same individual. ANOVA was used to compare means. A difference was considered significant if The study protocol was submitted to and approved by the Faculty of Medicine and Pharmacy Institutional Review Board before the study commenced. Informed consent was obtained from parents/guardians of children prior to their enrolment. The same quality of care was offered to children in case the parents refused to be part of the study.P. falciparum. Thirty children with negative malaria smears were diagnosed with others diseases. Sex ratio was 1.13 for male. The mean age of all children was 36.9 months. The age of the youngest child was one month and the age of the oldest was 144 months. Mean weight of children was 11.8 kg with a minimum of 5 kg and a maximum of 23 kg. All children included received a first dose of IRQA. Early expulsion of the product after first administration occurred in 23.1% of the children and required re-administration of a half dose. A second dose was administered to 35.1% of the children at the referral unit. For these, the proportion of early expulsion increased significantly to 40.4% .Overall 134 patients were enrolled from June 2003 to January 2004. One child died before arriving at the referral centre; two others died, one forty minutes and the second three hours and twenty two minutes after arriving at the referral centre. Out of 133 children that arrived at the referral centre, 97 (73%) had malaria smear positive for ®, 10.6% of children had a specific reaction consisting of crying and contraction of back muscles prior to administration , oral drugs could be administered after 1.8 ± 1.3 days, the minimum being one day and the maximum seven days. Confirmed cases of malaria with positive thick smear that survived (n = 90) had oral route available after 1.6 ± 0.9 days, with a minimum of one day and a maximum of five days. The frequency of clinical symptoms such as coma with Blantyre score ≤ 3 significantly decreased at the referral health care unit after IRQA Table . OverallFrom August 2003 to January 2004 we assessed acceptability of IRQA as treatment of non per os malaria in the Mopti region, Mali. The framework of routine care offered at periphery according to the paediatric disease management policy of the Malian Ministry of Health was used.This implementation study showed that administration of IRQA at peripheral health care centres in Mali was accepted by 95% of parents and 67% of the children. In majority, the personnel (85%) found the kit easy to use. The most common adverse reaction after administration of IRQA was emission of liquid stools in 75.7% of children after the first shot. We observed a case fatality rate of 7.5%.Children presenting spontaneously at peripheral health care units were included. In such rural areas, attending the health care facility happened when all more affordable means to treat have been used unsuccessfully . This miThe diagnosis of severe malaria at periphery was based on symptoms and clinical signs only according to national guidelines. After referral, almost 30% of cases had negative blood smear and were diagnosed with others conditions.In few cases, inclusion criteria were not strictly observed and some children that had a history of diarrhoea were included. Abnormalities of anal margins were reported in these children.The study might have been biased to include children with less survival chances than cases of classic non per-os malaria, in which IRQA could be more efficacious and better tolerated. But the study population also reflects the reality of patients to whom IRQA will be administered in routine circumstances. In routine condition, the likelihood of strict adherence to definitions of non per-os malaria cases is rather low.IRQA was well tolerated. Liquid and semi-liquid stools were the most common adverse reactions observed. A quarter of patients had liquid stools after first administration and when they reached the referral care centre the proportion increased to three quarters. In previous trials much lower proportion of liquid stools were reported ,16. Suchi.e, to avoid cases of diarrhoea and lesion of the anal region. In summary the tolerance profile shown from this study favour the recommendation to further use IRQA in the treatment of severe malaria when the intravenous route is not available.In cases where abnormalities of anal margins were reported, the children had anal mucosis injured by previous episodes of diarrhoea. It emphasizes the need to respect contra indications of intra rectal route, Health care personnel at peripheral and regional referral health care unit levels unanimously considered the use of the kit easy. This will facilitate the subsequent large-scale implementation of the pre-transfer one or two doses of IRQA in remote and resource poor areas of Mali under the auspices of the National Malaria Control Programme.The sharp reduction in the severity of coma following one dose and the low case fatality rate observed in such biased study population confirmed the known efficacy of the IRQA. The role of confounding factors related to the single-arm design is balanced to some extent by the very high case fatality rate observed in untreated cases of severe malaria. Our observed case fatality rate,7.5%) is close to data reported from Niger . Higher .5% is clIn rural areas in Africa, the rectal route is becoming a promising alternative. It effectiveness doesn't require co -administration with fluids. When implemented at the periphery of a weak health care system, as in Mopti, it reduces the delay to an effective antimalarial treatment. Rectal arthemeter has also proven to be well tolerated and effective in the treatment of severe malaria . HoweverIn summary this study showed that IRQA is safe as long as contra-indications to its use are respected and efficacious for the initial treatment of severe malaria at peripheral health care level in Mali. The study supports the further implementation of the step-wise approach to introduce quinine usage outside health care services in Mali.OK Doumbo, MA Thera, V Lameyre and JP Ducret designed the study. OK Doumbo, MA Thera, V Lameyre and M Sacko developed the protocol. MA Thera directed the overall conduct of the study and the data analysis. F Keita and OB Traore conducted the study in the field and contributed to data analysis. MS Sissoko analysed the data and monitored the study. D Coulibaly monitored the study. MA Thera, OK Doumbo, V Lameyre, M Sacko, F Keita, MS Sissoko, OB Traore D Coulibaly and JP Ducret wrote the paper. All authors read and approved the final manuscript.


The authors discuss a study by Philip Bejon et al. that adopted a new approach to balancing the sensitivity and specificity of criteria for defining severe malaria. Plasmodium falciparum parasitemia . A parasite threshold to improve specificity in severe anemia was defined for research purposes in the 1990 WHO guidelines [Finally, while the lengthy descriptions of severe malaria included in current WHO guidelines  are appridelines , but wasies 2,5,1. A paras


Protection against clinical malaria episodes is acquired slowly after frequent exposure to malaria parasites. This is reflected by a decrease with increasing age in both parasite density and incidence of clinical episodes. In many settings of stable malaria transmission, the presence of asymptomatic malaria parasite carriers is common and the definition of clinical malaria remains uncertain.Between February 2002 and April 2003, a country-wide malaria survey was conducted in 24 districts of Mozambique, aiming to characterize the malaria transmission intensities and to estimate the proportion of fever cases attributable to malaria infections in order to establish the malaria case definition. A total of 8,816 children less than ten years of age were selected for the study. Axillary temperature was measured in all participating subjects and finger prick blood collections were taken to prepare thick and thin films for identification of parasite species and determination of parasite density. The proportion of fever cases attributable to malaria infection was estimated using a logistic regression of the fever on a monotonic function of the parasite density and, using bootstrap facilities, bootstrapped estimated confidence intervals, as well as the sensitivity and specificity for different parasite density cut-offs were produced.Plasmodium falciparum was 52.4% . The prevalence of fever (axillary temperature ≥ 37.5°C) was 9.4% . Fever episodes peaked among children below 12 months of life [15.1% ]. The lowest fever prevalence of 5.9%  was recorded amongst children between five and seven years of age. Among 4,098 parasitized children, 498/4,098 (13.02%) had fever. The prevalence of malaria infections associated with fever peaked among children in the less than twelve months age group and thereafter decreased rapidly with increasing age (p < 0.001). High parasite densities were significantly associated with fever (p < 0.04).Overall, the prevalence of The proportion of fever attributed to malaria was 37.8% (95% CI 32.9% – 42.7%). An age-specific pattern was observed with significant variations across different regions in the country. In general, among children less than 12 months of life, the proportion of fever attributed to malaria infection was 43.5% (95% CI 25.8% – 61.2%), in children aged between 12 and 59 months of age was 39.6% (95% CI 30.3% – 48.9%), and among children aged between 5 and 10 years old was 21.5% (95% CI 11.6% – 31.4%).This study confirms that malaria remains a major cause of febrile illness during childhood. It also defines the relation between parasite density and fever and how this varies with age and region. This may help guide case definition for clinical trials of preventive tools, as well as provide definitions that may improve the precision of measurement of the burden of disease. In many malaria endemic regions, children acquire clinical immunity to malaria, and develop anti-parasitic mechanisms during the first few years of life consequent to repetitive exposures . Thus, aet al [The concept of "pyrogenic threshold" has been proposed for defining malaria episode in endemic areas -9. The oet al  proposedet al . In addiIn Mozambique, malaria remains one of the main causes of febrile illness among children. Transmission is perennial with regional variations throughout the country. The incidence of clinical malaria established through weekly active case detection suggests that the risk of clinical malaria is highest between the age of one and three years when children experience an average of more than two episodes per year. Based on a continuous demographic surveillance system and verbal autopsies carried out in Manhiça district, the risk of clinical malaria drops sharply after the age of six .Early diagnosis and prompt treatment of cases, especially in children represent the keystone strategy aiming at reducing the burden of malaria-related morbidity and mortality. However, in most rural areas, diagnosis of clinical malaria is rather presumptive based on fever or history of fever, though a positive blood film is required to confirm the diagnosis in areas where laboratory facilities exist.This paper reports the results of a national malaria survey, carried out in Mozambique, in which the prevalence and the intensity of malaria infections, the establishment of malaria case definition and its relation to age strata across the country were determined.Plasmodium falciparum infections, fever, and malaria parasite infection associated with fever were estimated in each age group for each region and stratum.A detailed description of the methods used in this survey is presented in a companion paper . Brieflyet al [logit(πi) = α + β(xi)τ, and represents a logistic regression model where πI is the probability that observation i with parasite density xi is a fever case. β(xi)t corresponds to model type 3 described by Smith et al and is a monotonic function which is more flexible than the regression on xi or log(xi). The models were fitted using maximum likelihood. To constrain the parameter τ to be positive, the maximum likelihood were estimated for the log(τ). Confidence intervals for the estimated attributable fraction, the τ parameter, the sensitivity and specificity were obtained using the bootstrap methodology [Using the method proposed by Smith et al , the atthodology . AnalysiP. falciparum infections  accounted for 5.7% , but, among febrile children, 72.4% (554/766) were infected. The prevalence of malaria infection associated with fever peaked among children during the first year of life and thereafter decreased sharply with increasing age, and the differences among age groups were statistically significant (p < 0.001).Overall, fever prevalence (axillary temperature ≥ 37.5°C) among children was 9.4% 766/8,816). Table 6. Table P. falciparum parasite densities were significantly associated with fever (p < 0.05)  Table . AccordiThe model define malaria case as any case with at least one parasite, hence sensitivity of parasite density cut-off of one parasite or more will be always 100%. Table After adjusting for region, the fever proportion attributable to malaria was 36.5%, decreasing from the northern to the southern region, the differences were statistically significant (p < 0.05). When adjusted for stratum the attributable fraction of fever was 38.1%, but without significant variations across strata. When adjusted for age group and region the attributable fraction of fever dropped to 36.1% and the differences observed among regions and age groups were statistically significant (p < 0.05). When adjusted for age group, region and stratum there were no differences statistically significant across strata, however, across regions there were differences statistically significant (p < 0.05).The proportion of fever attributable to malaria infection among children less than 12 months of life was 43.5% (95% CI 25.8% – 61.2%)   , representing the proportion of febrile morbidity that would have been removed if malaria infections were eliminated among children in various settings in the study area. On the other hand, the results of this study highlighted the importance of other fever episodes not attributed to malaria infections in the study area.In many malaria endemic areas, the burden of disease, a definition based on fever or reported episode of fever within the previous 48 hours in the presence of any level of parasitaemia, would result in an over-diagnosis of malaria cases. This study was conducted during high malaria transmission season, nevertheless, among febrile children approximately one third did not have malaria parasites. This finding has significant implications on the treatment policy, particularly in rural areas where in the absence of laboratorial diagnosis, all febrile cases would be considered as clinical malaria episodes. Moreover, even among febrile parasitized children, the proportion attributable to malaria infection was less than 40%.The association between malaria infection and body temperature varies significantly among children. Despite, that the definition of clinical malaria have been related to fever episode and presence of parasites in the blood stream, in endemic-malaria areas, manifestations of clinical malaria have a wide spectrum  and the P. falciparum infections were observed. Additionally, the risk of fever among parasitized children was age-dependent, and increased with increasing mean parasite density.In the study area, the majority of parasitized children were asymptomatic carriers, and not all fever episodes were associated with malaria parasites, hence very few fever episodes associated with asexual In the study area, the overall proportion of fever cases attributable to the presence of malaria infection, when adjusted for age showed significant variations. Among younger children, it was 43.5% and decreased with increasing age to reach a low of 21.5% among older children. Therefore, the proportion of fever cases attributed to parasitaemia, the sensitivity and specificity of clinical malaria definition was age-specific. Consequently, among children exposed to malaria infection, the outcome or the risk of developing fever as a clinical manifestation of malaria infection, decreased with increasing age. These findings corroborate with results from other studies carried out in highly endemic areas [Regional variations on the proportion of fever attributable to malaria infection among children have been reported from other community-based surveys in endemic-malaria areas , highligThe case definitions derived from this survey, have shown the relationship between parasite density and fever and how this varies with age and region. This may help guide case definition for clinical trials of preventive tools, as well as provide definitions that may improve the precision of burden of disease assessment.This study confirms that malaria infection remains a major cause of febrile illness during childhood. However, other causes of fever should be considered in case management of febrile illnesses during childhood.The authors declare that they have no competing interests.SM made a substantial contribution on conception and design of the study, coordinated and supervised data collection in all regions, interpretation of data, performed all statistical analysis and wrote the manuscript. JA gave a major contribution on data interpretation and statistical analysis and helped to draft and revised the manuscript. AT helped to draft, gave contribution and critically revised the manuscript. PA gave a major contribution on conception and study design and helped to draft and critically revised the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum (Pf) malaria is only acquired after years of repeated infections and wanes rapidly without ongoing parasite exposure. Antibodies are central to malaria immunity, yet little is known about the B-cell biology that underlies the inefficient acquisition of Pf-specific humoral immunity. This year-long prospective study in Mali of 185 individuals aged 2 to 25 years shows that Pf-specific memory B-cells and antibodies are acquired gradually in a stepwise fashion over years of repeated Pf exposure. Both Pf-specific memory B cells and antibody titers increased after acute malaria and then, after six months of decreased Pf exposure, contracted to a point slightly higher than pre-infection levels. This inefficient, stepwise expansion of both the Pf-specific memory B-cell and long-lived antibody compartments depends on Pf exposure rather than age, based on the comparator response to tetanus vaccination that was efficient and stable. These observations lend new insights into the cellular basis of the delayed acquisition of malaria immunity.Immunity to  Plasmodium falciparum (Pf) is a mosquito-borne parasite that causes over 500 million cases of malaria annually, one million of which result in death, primarily among African children. The development of an effective malaria vaccine would be a critical step toward the control and eventual elimination of this disease. To date, most licensed vaccines are for pathogens that induce long-lived protective antibodies after a single infection. In contrast, immunity to malaria is only acquired after repeated infections. Antibodies play a key role in protection from malaria, yet several studies indicate that antibodies against some Pf proteins are generated inefficiently and lost rapidly. The cells that are responsible for the maintenance of antibodies over the human lifespan are memory B-cells and long-lived plasma cells. To determine how these cells are generated and maintained in response to Pf infection, we conducted a year-long study in an area of Mali that experiences a six-month malaria season. We found memory B-cells and long-lived antibodies specific for the parasite were generated in a gradual, step-wise fashion over years despite intense Pf exposure. This contrasts sharply with the efficient response to tetanus vaccination in the same population. This study lends new insights into the delayed acquisition of malaria immunity. Future studies of the cellular and molecular basis of these observations could open the door to strategies for the development of a highly effective malaria vaccine. Mycobacterium tuberculosis (Mtb), and Plasmodium falciparum malaria Pf infections Pf antigens are inefficiently generated and rapidly lost in the absence of ongoing exposure to the parasite . MBCs recirculate and mediate recall responses after re-exposure to their cognate antigen by rapidly expanding and differentiating into PCs, whereas LLPCs residing in the bone marrow constitutively secrete antibody and provide a critical first line of defense against re-infection.Despite the key role that antibodies play in protection from a variety of infectious diseases, remarkably little is known about the cellular basis of acquiring humoral immunity in response to natural infections in humans. This gap in our knowledge is due in large part to the difficulty in studying natural infections in humans when we cannot predict who within a population will be infected with a given pathogen at a given time. Thus, our current understanding of the acquisition of immunity is largely derived from animal models and studies of humans after vaccination. These studies have established that long-lived, antibody-based immunity requires the generation and maintenance of memory B cells (MBCs) and long-lived plasma cells (LLPCs) - and Pf-specific MBCs and Ab, and also tested three hypotheses: 1) that growth of the MBC compartment depends on immunological experience rather than age, 2) that Pf infection induces non-specific activation of bystander B cells The mechanisms by which antibody responses are maintained over the human life-span remains an open question. In one model, LLPCs survive indefinitely in the bone marrow and independently maintain steady-state antibody levels Pf parasitemia at study enrollment. During the one-year study period there were 380 unscheduled clinic visits, during which 219 cases of malaria were diagnosed, five of which met the WHO criteria for severe malaria Pf asexual parasitemia ≥5000 parasites/µL, and a non-focal physical exam by the study physician. As expected in this region of Mali, all malaria cases were confined to a six-month period that began in July, peaked in October, and ended by January , with age. In Mali, a single TT vaccine is administered to infants less than six months of age and a second TT vaccine is administered to females around 15 years of age to prevent neonatal tetanus. Thus, we measured TT-specific antibody and MBC responses at least 18 months after TT vaccination, a point at which the TT-specific response is likely to be at steady state. In contrast to what was observed for AMA1- and MSP1-specific MBCs, the frequency of TT-specific MBCs among males did not change significantly from age 2 to 25 years , and in a cross-sectional survey at the end of the following dry season (month 12), and compared these frequencies to the pre-malaria season baseline . Malaria episodes were defined as an axillary temperature ≥37.5°C, Pf asexual parasitemia ≥5000 parasites/µL, and a non-focal physical exam by the study physician. Because few adults experienced malaria  to that 14 days after acute malaria . We observed a small, but statistically significant increase in the frequency of TT-specific MBCs from month 0 to convalescence  and naïve B cells  and an increase in resting IgG+ MBCs  and activated IgG+ MBCs . The increase with age of classical MBCs is consistent with the increase in total IgG+ MBCs we observed using the MBC ELISPOT assay  before the malaria season . With inOT assay .Pf exposure is associated with an expanded subset of ‘atypical’ MBCs that express FCRL4 and are hyporesponsive to in vitro stimuli + CD27− CD21− CD20+ CD10− and typically represents <4% of circulating CD19+ B cells in healthy U.S. adults In a subset of 87 individuals from this same study cohort, we previously reported that + 14 days after acute malaria. Within the CD19+ B cell population there were no significant changes in the percent of immature B cells, naïve B cells, or resting MBCs, after acute malaria. Moreover, there was no change in the proportions of resting and atypical MBCs that were IgG+. However, we observed a decrease in the percentage of total atypical MBCs . The decrease in the proportion of atypical MBCs in the peripheral blood suggests that this subpopulation may be trafficking out of the circulation into tissues in response to acute malaria.We investigated the impact of acute malaria on the relative proportion of B cells in each subset in children aged 2–10 years. Compared to the pre-malaria season baseline (month 0), there were no significant changes in the percent of lymphocytes that were CD19Pf asexual parasitemia ≥5000 parasites/µL, and a non-focal exam by the study physician. Because the incidence of malaria was very low in adults during the study period  MBCs in nearly all vaccinees Pf infection disrupts the immune system's ability to generate or maintain MBCs or LLPCs. The differentiation of B cells into long-lived MBCs depends to a great extent on the affinity of their BCRs for antigen. Recently, evidence was presented that affinity maturation of B cells may fail to occur in the absence of adequate Toll-like receptor (TLR) stimulation Pf infection. Although our data do not directly address the role of apoptosis in the gradual acquisition of Pf-specific MBCs, it is worth noting that we found no evidence of Pf-induced ablation of Plasmodium-specific MBCs, as was observed in mice four days after Plasmodium yoelii infection Pf infection also does not appear to be due to a persistent, Pf-induced general immunosuppression as the frequency of TT-specific MBCs increased significantly in most adult females in response to a single TT booster vaccination, an increase that appeared to be maintained for years. In an experimental model of lymphocytic choriomeningitis virus (LCMV) infection, a high antigen-to-B cell ratio disrupted germinal center formation and the establishment of B cell memory Pf infection when the immune system encounters high concentrations of parasite proteins. Indeed, germinal center disruption is observed in mice infected with P. berghei ANKA P. chabaudiPf infection arises from pre-diversified IgM+IgD+CD27+  B cells—analogous to the rapid protective response against highly virulent encapsulated bacteria that do not elicit classical T-dependent responses ex vivo and in vitro assays to rigorously conducted prospective studies of Pf-exposed populations.It is also possible that Pf exposure is associated with a functionally and phenotypically distinct population of FCRL4+ hypo-responsive atypical MBCs Pf exposure is associated with an expansion of FCRL4+ MBCs. The accumulation of atypical MBCs could be linked to the slow acquisition of Pf-specific MBCs, as naïve B cells in response to Pf infection could have a propensity to differentiate into atypical rather than classical MBCs. We also observed that the FCRL4+ MBC population decreased in the peripheral circulation two weeks after acute malaria suggesting that these MBCs are directly involved in the response to Pf infection, possibly trafficking to secondary lymphoid tissues. Although the function of FCRL4+ MBCs is not established, Moir et al. + ‘exhausted MBCs’ contribute to the B cell deficiencies observed in HIV-infected individuals. In contrast, Ehrhardt et al.+ ‘tissue-like MBCs’ in lymphoid tissues associated with epithelium, suggested that these cells may play a protective role during infections. At present, the factors that underlie the expansion of atypical MBCs in this study population are not known. Genetic or environmental factors that are associated with Pf transmission but not accounted for in this study could explain this observation. It will be of interest to understand the origin, antigen-specificity, and function of FCRL4+ MBCs in the context of Pf infection and the potential impact of these MBCs on the ability of children to respond to malaria vaccines.We previously reported that per se may not reliably predict clinical immunity to malaria regardless of antigen specificity. Malaria symptoms only occur during the blood stages of Pf infection and can begin as early as three days after the blood stage infection begins Pf blood stage antigens to differentiate into the antibody-secreting cells that would prevent the onset of malaria symptoms. In contrast, the longer incubation period of other pathogens allows MBCs to differentiate into protective antibody-secreting cells before symptoms develop. For example, follow-up studies of hepatitis B vaccinees have shown that protection can persist despite the decline of hepatitis B-specific antibodies to undetectable levels Pf infection.In multivariate analysis we found no correlation between the frequency of MBCs and levels of Abs specific for AMA1 or MSP1 and malaria risk. This is not necessarily unexpected in light of recent clinical trials that showed that vaccination with either AMA1 or MSP1 did not confer protection Pf-induced polyclonal activation are matched by the rate of loss of senescent TT-specific MBCs. It has also been proposed that non-specific polyclonal stimulation maintains long-lived Ab responses by driving MBCs to differentiate into SLPCs or LLPCs Plasmodium infection generates large amounts of non-specific Ig Pf infection, we did not observe a concomitant increase in TT-specific IgG. This finding is consistent with recent human studies that demonstrate a lack of bystander IgG production after heterologous vaccination or viral infection in vivo upon TLR4 and 9 activation Pf infection, but based on the results of this study we hypothesize that the preponderance of IgG produced in response to malaria is specific for the ∼2400 Pf proteins expressed during the blood-stage of infection in vivo after immunization with an irrelevant antigen Here we also provide evidence concerning the mechanism by which MBCs and LLPCs are maintained. We observed a modest but statistically significant increase in TT-specific MBCs two weeks after acute malaria, in support of the hypothesis that MBCs are renewed by polyclonal or ‘bystander’ activation in vitro suggest that Pf drives polyclonal MBC activation by the cysteine-rich interdomain regions 1α (CIDR1α) of the Pf erythrocyte membrane protein 1 (PfEMP1) Pf-derived TLR agonists It is of general interest to determine which parasite products are responsible for the polyclonal activation of MBCs observed here. Studies Plasmodium infection Pf in longitudinal studies since findings from animal models do not always mirror human biology or pertain to the clinical context Pf infection and to determine the longevity of these cells in the absence of Pf transmission over longer periods of time. Greater insight into the molecular and cellular basis of naturally-acquired malaria immunity could open the door to strategies that ultimately prove useful to the development of a highly effective malaria vaccine.Animal models have provided important insights into the immunobiology of The ethics committee of the Faculty of Medicine, Pharmacy, and Odonto-Stomatology, and the institutional review board at the National Institute of Allergy and Infectious Diseases, National Institutes of Health approved this study (NIAID protocol number 06-I-N147). Written, informed consent was obtained from adult participants and from the parents or guardians of participating children.2) rural village with a population of 1500, located 20 km north of Bamako, the capital of Mali. Pf transmission is seasonal and intense at this site from July through December. The entomological inoculation rate measured in a nearby village was approximately 50–60 infective bites per person per month in October 2000 and fell to near zero during the dry season This study was carried out in Kambila, a small (<1 kmPf. Patients with positive smear results (i.e. any level of parasitemia) were treated with a standard 3-day course of artesunate plus amodiaquine, following the guidelines of the Mali National Malaria Control Program. Anti-malarial drugs were provided exclusively by the study investigators. Children with severe malaria were referred to Kati District Hospital after an initial parenteral dose of quinine. For research purposes, a malaria episode was defined as an axillary temperature ≥37.5°C, Pf asexual parasitemia ≥5000 parasites/µL, and a nonfocal physical examination by the study physician. Severe malaria, as defined by the WHO Pf-specific immune responses and malaria risk: 1) whether or not malaria was experienced, 2) the incidence of malaria, and 3) the time to the first malaria episode. Blood smears were prepared and venous blood samples collected during the two-week enrollment period (month 0), 14 days after the first episode of malaria , and during a two-week period at the end of the six-month dry season (month 12). Hemoglobin was typed from venous blood samples. Stool and urine were examined at enrollment for the presence of helminth infections. Venous blood samples from ten healthy U.S. adult blood bank donors were analyzed as controls. Travel histories for these U.S. adults were not available, but prior exposure to Pf is unlikely.In May 2006, during a two-week period just prior to the malaria season, 225 individuals aged 2–10 years and 18–25 years were enrolled after random selection from an age-stratified census of the entire village population. Enrollment exclusion criteria were hemoglobin <7 g/dL, fever ≥37.5°C, acute systemic illness, use of anti-malarial or immunosuppressive medications in the past 30 days, or pregnancy. All analysis in the present study pertains to an age-stratified subset of individuals (n = 185) randomly selected from those who had complete sets of PBMC samples over the entire study period. From May 2006 through May 2007, participants were instructed to report symptoms of malaria at the village health center, staffed 24 hours per day by a study physician. For individuals with signs or symptoms of malaria, blood smears were examined for the presence of Blood samples (8 ml for children and 16 ml for adults) were drawn by venipuncture into sodium citrate-containing cell preparation tubes  and transported 20 km to the laboratory where they were processed within three hours of collection. Plasma and PBMCs were isolated according to the manufacturer's instructions. Plasma was stored at −80°C. PBMCs were frozen in fetal bovine serum (FBS)  containing 7.5% dimethyl sulfoxide , kept at −80°C for 24 hours, and then stored at −196°C in liquid nitrogen. For each individual, PBMC and plasma samples from all time points were thawed and assayed simultaneously.Pf densities were recorded as the number of asexual parasites/µl of whole blood, based on an average leukocyte count of 7500/µl. Each smear was evaluated separately by two expert microscopists blinded to the clinical status of study participants. Any discrepancies were resolved by a third expert microscopist.Thick blood smears were stained with Giemsa and counted against 300 leukocytes. Hemoglobin was typed by high performance liquid chromatography  as previously described Schistosoma mansoni eggs and other intestinal helminths using the semi-quantitative Kato-Katz method. To detect Schistosoma haematobium eggs, 10 ml of urine were poured over Whatman filter paper. One or two drops of ninhydrine were placed on the filter and left to air dry. After drying, the filter was dampened with tap water and helminths were eggs detected by microscopy.At enrollment, duplicate stool samples were examined for Latitude and longitude coordinates of study subjects' households were measured by a handheld global positioning system receiver  and reported earlier ELISAs were performed by a standardized method as described previously et alet al., unpublished observation). Briefly, PBMCs were thawed and cultured in 24 well plates at 37°C in a 5% CO2 atmosphere for six days in media alone  or media plus a cocktail of polyclonal activators: 2.5 µg/ml of CpG oligonucleotide ODN-2006 , Protein A from Staphylococcus aureus Cowan (SAC) at a 1/10,000 dilution , pokeweed mitogen at a 1/100,000 dilution (Sigma-Aldrich), and IL-10 at 25 ng/ml (BD Biosciences). Cells were washed and distributed on 96-well ELISPOT plates  to detect antibody-secreting cells (ASCs). ELISPOT plates were prepared by coating with either: a 10 µg/ml solution of polyclonal goat antibodies specific for human IgG  to detect all IgG-secreting cells; a 1% solution of bovine serum albumin (BSA) as a non-specific protein control; or 5 µg/ml solutions of either tetanus toxoid (TT), AMA1, or MSP1 to detect antigen-specific ASCs. For AMA1 and MSP1, a 1∶1 mixture of FVO and 3D7 isotypes was used to coat the ELISPOT plates. Plates were blocked by incubation with a solution of 1% BSA. For the detection of antigen-specific ASCs, cells were plated in duplicate in eight serial dilutions beginning with 5×105 cells/well. For detection of total IgG ASCs cells were plated at six serial dilutions beginning at 4×104 cells/well. After a five hour incubation of the cells in the ELISPOT plates, plates were washed four times each in PBS and PBS-Tween 20 0.05% (PBST), and incubated overnight with a 1∶1000 dilution of alkaline phosphatase-conjugated goat antibodies specific for human IgG (Zymed) in PBST/1% FCS. Plates were washed four times each in PBST, PBS, and ddH2O; developed using 100 µl/well BCIP/NBT for 10 minutes; washed thoroughly with ddH2O and dried in the dark. ELISPOTS were quantified using Cellular Technologies LTD plate-reader and results analyzed using Cellspot software. Results are reported as frequencies of MBCs per 106 PBMCs after the six-day culture. The limit of detection of the MBC ELISPOT assay for this analysis was five ASCs per 106 PBMC based on the average number of ASCs on the BSA control. Assay failure was defined as fewer than 1000 IgG+ ASCs per 106 PBMCs after the six-day culture which resulted in the exclusion of 15% of individuals at month 0, 13.2% 14 days after the first malaria episode, and 7.3% at month 12. For individuals with a limited number of PBMCs, priority was given to performing the ELISPOT assay for MSP1, then TT, and then AMA1.Antigen-specific MBCs were quantified by a modified version of the method developed by Crotty + CD21− CD20−), naive B cells (CD19+ CD27−CD10−), immature B cells (CD19+ CD10+), classical MBCs (CD19+ CD27+ CD21+), atypical MBCs (CD19+ CD21− CD27− CD10−) and activated MBCs (CD19+ CD21− CD27+CD20+). FACS analyses were performed on a FACSCalibur flow cytometer (BD Biosciences) using FlowJo software .All phenotypic analyses were performed using mouse mAbs specific for human B cell markers conjugated to fluorophores as previously reported Data were analyzed using STATA  and GraphPad Prism for Windows .The Kruskal-Wallis test was used to compare continuous variables between groups, and the Fisher's exact test was used to compare categorical variables. The Wilcoxon matched-pairs signed-rank test was used to compare measurements of the same parameter at two time points for the same individual. The correlation between different continuous measures was determined by using the Spearman correlation coefficient. The malaria-free probability over the twelve-month study period was estimated by the Kaplan-Meier curve, and the time to the first malaria episode was compared by the log rank test. Cox's proportional hazards model was used to assess the effect of the following factors on the hazard of malaria: age, gender, weight, ethnicity, distance lived from study clinic, self-reported bednet use, hemoglobin type, antigen-specific MBC frequencies and Ab levels. The same list of variables was included in logistic and Poisson regression models to determine their impact on the odds and incidence of malaria episodes, respectively. For all tests, two-tailed p values were considered significant if ≤0.05.Figure S1+ ASCs per 106 PBMCs after the six-day culture. The Spearman's correlation coefficient is given for each plot.Correlative analysis of antibody levels and memory B cell frequencies specific for AMA1, MSP1, and tetanus toxoid. Shown are scatterplots of antibody levels versus memory B cell frequencies specific for (A) AMA1 (n = 64), (B) MSP1 (n = 67) and (C) tetanus toxoid (n = 128). Data are derived from venous blood samples drawn before the malaria season. Only individuals with both antibody and memory B cell data are included. For AMA1 and MSP1 the plots include individuals with antibody levels at or above the limit of detection of the ELISA. Individuals with ‘failed' ELISPOT assays are not included. As described in (3.84 MB TIF)Click here for additional data file.