In Uganda, like in many other countries traditionally viewed as harbouring very high malaria transmission, the norm has been to recommend that febrile episodes are diagnosed as malaria. In this study, the policy implications of such recommendations are revisited.A cross-sectional survey was undertaken at outpatient departments of all health facilities in four Ugandan districts. The routine diagnostic practices were assessed for all patients during exit interviews and a research slide was obtained for later reading. Primary outcome measures were the accuracy of national recommendations and routine malaria diagnosis in comparison with the study definition of malaria (any parasitaemia on expert slide examination in patient with fever) stratified by age and intensity of malaria transmission. Secondary outcome measures were the use, interpretation and accuracy of routine malaria microscopy.1,763 consultations undertaken by 233 health workers at 188 facilities were evaluated. The prevalence of malaria was 24.2% and ranged between 13.9% in patients ≥5 years in medium-to-high transmission areas to 50.5% for children <5 years in very high transmission areas. Overall, the sensitivity and negative predictive value (NPV) of routine malaria diagnosis were high (89.7% and 91.6% respectively) while the specificity and positive predictive value (PPV) were low (35.6% and 30.8% respectively). However, malaria was under-diagnosed in 39.9% of children less than five years of age in the very high transmission area. At 48 facilities with functional microscopy, the use of malaria slide examination was low (34.5%) without significant differences between age groups, or between patients for whom microscopy is recommended or not. 96.2% of patients with a routine positive slide result were treated for malaria but also 47.6% with a negative result.Current recommendations and associated clinical practices result in massive laria over-diagnosis across all age groups and transmission areas in Uganda. Yet, under-diagnosis is also common in children <5 years. The potential benefits of malaria microscopy are not realized. To address malaria misdiagnosis, Uganda's policy shift from presumptive to parasitological diagnosis should encompass introduction of malaria rapid diagnostic tests and substantial strengthening of malaria microscopy. Malaria remains the major public health problem in Uganda with annual estimates of 10 million cases and 43,000 deaths, of which 91% are in children below 5 years of age ,3. GivenNational malaria diagnosis recommendations for health workers are outlined in the recent malaria case-management guidelines and job aids -6, whichThe malaria diagnostic policy in Uganda is currently under revision and a more significant role for parasitological diagnosis is being considered. In support of the policy revision process, this study reports on the accuracy of the current national malaria diagnosis recommendation, the accuracy of routine malaria diagnosis practices, and evaluates the use, interpretation and accuracy of malaria microscopy at facilities where this diagnostic service is available.Between 29 May and 16 August 2007, outpatient malaria case-management was evaluated at all government and private not-for-profit health facilities in four Ugandan districts . The survey methods are described in detail elsewhere [In addition, all patients had, on exit, a finger-prick blood sample taken for malaria microscopy by study laboratory technicians. Thick and thin blood slides were prepared at the facility and stained with 10% Giemsa. At post-survey, two expert microscopists examined independently all study slides at a laboratory based at Malaria Consortium in Kampala. For each study slide 100 high-power magnification fields were examined before the slide was reported as negative. Where results between the two expert microscopists were discordant, a third microscopist re-read the blood slide and the majority decision was accepted as the final result.The primary analysis focused on 1) the accuracy of fever as the national malaria diagnosis recommendation, and 2) the accuracy of routine malaria diagnosis practices. To reflect national guideline recommendations for presumptive malaria diagnosis, the primary analysis was restricted to non-pregnant patients weighing ≥5 kg, without prior use of effective anti-malarials, and presenting to the health facility for an initial, outpatient visit. Fever was defined as axillary temperature of ≥37.5°C and/or the history of fever during the present illness. Routine malaria diagnosis was defined as a patient either diagnosed for malaria or treated with an anti-malarial drug. This adjustment was done since 8% of patients did not have any diagnosis written in their cards but all of them had an anti-malarial prescribed and, therefore, it was obvious that a malaria diagnosis was considered.The secondary analysis evaluated the use, interpretation and accuracy of routine malaria microscopy at health facilities where this diagnostic service was functional during the survey day. The analysis on the use and interpretation of routine malaria microscopy included all patients at these facilities. The clinical process was stratified for patients with and without fever, and subsequently patients with fever were stratified into those for whom the guidelines discourage use of microscopy (i.e. recommend presumptive diagnosis) and those for whom microscopy is recommended . For those patients who had a routine malaria slide performed, further stratification was done by the result of the routine malaria test and the anti-malarial treatment prescribed.The sensitivity, specificity, positive predictive values (PPVs) and negative predictive values (NPVs) of the policy recommendations and current malaria diagnostic practices were calculated, using standard formulae, against the study malaria case definition, i.e. the presence of any parasitaemia in patients with fever after expert slide examination. For those patients who had a routine malaria slide performed, the accuracy of the routine slide result was evaluated using the same accuracy measures against the presence of any parasitaemia on expert slide examination. In all analyses, the precision of proportions  was determined adjusting for the cluster sampling. All analyses were performed across all age groups and separately for patients below and ≥5 years of age. The small sample size precluded meaningful comparisons between individual districts, thus the results are presented from all districts and stratified by the two districts of medium-to-high transmission  versus the two districts of very high transmission  [All data were double-entered into Microsoft Access 2000 by two independent data entry clerks and data files were compared for errors by using a verification programme and referring to original data collection forms. All analyses were performed using STATA, version 9.2 .Ethical approval for this study was provided by the Uganda National Council for Science and Technology (reference number HS 275).In total, 1,763 outpatient consultations undertaken by 233 health workers at 188 health facilities were evaluated. Of 1,763 patients, 278 patients were excluded from the primary analysis evaluating accuracy of malaria diagnosis recommendations and routine malaria diagnosis because they either did not meet the inclusion criterion (n = 274) or had missing results for expert slide examination (n = 4). Therefore, this analysis included 1,485 patients, of which 494 (33.3%) were children below five years of age and 991 (66.7%) were patients five years and older. Of 1,485 patients, 907 were evaluated in the medium-to-high transmission intensity districts (Mubende and Jinja) and 578 in the very high transmission intensity districts (Tororo and Apac). Children below five years of age comprised 288 (31.8%) patients in the medium-to-high and 206 (35.6%) in the very high transmission intensity districts.For the secondary analysis, the routine clinical process was evaluated from 473 consultations undertaken by 70 health workers at 48 health facilities with functional microscopy on the survey day . There were 149 (31.5%) observations for children below five years of age and 324 (68.5%) for patients five years and older. Among all patients 163 routine malaria slides were performed, for which five (3%) expert slide results were not available. Therefore, the accuracy of the routine malaria microscopy was evaluated on 158 slides, of which 48 (30.4%) were for children below five years of age and 110 (69.6%) were for patients ≥5 years of age.Table Conversely, the overall prevalence of fever was high (79.2%) and was significantly higher in children under five years (88.7%) than in patients five years and older (74.5%). The higher prevalence of fever was observed in very high transmission areas (82.5%) compared to medium-to-high transmission areas 77.1%), however the difference was not statistically significant , however, the specificity and PPV of these recommendations were very low (27.4% and 30.5% respectively) across both age groups and transmission settings  and lower sensitivity (89.7% vs 100%)  presented with fever and 131 (27.7%) without fever. Among the patients with fever, there were 57 for whom the national guidelines recommend the use of microscopy, while the remaining 285 febrile patients were cases for whom malaria microscopy is discouraged. Among all patients (473), the overall use of routine malaria microscopy was low  and there were no differences in microscopy use between children below five years of age  and patients five years and older . Patients for whom guidelines recommend the use of microscopy were more frequently routinely tested  than the patients with fever for whom microscopy is discouraged  and patients without fever , however the differences between these categories of patients were not statistically significant.Overall, there were 79 positive and 84 negative slides routinely reported. Nearly all patients with a positive slide result were treated for malaria . Yet, health workers prescribed an anti-malarial treatment for 47.6% (95% CI: 31.3–64.0) of patients with a routinely reported negative slide result. Conversely, health workers respected a negative test result and did not prescribe an anti-malarial for 52.4% (95% CI: 36.0–68.7) of patients. Among patients with fever, 58.1% (95% CI: 37.3–78.8) with a negative test result were treated with an anti-malarial, the proportion being higher in children below five years of age  than in patients five years and older .Table These facility-based data stratified by age and transmission intensity reveal a series of important findings directly relevant for the revision of malaria diagnostic recommendations within the context of deployment of new ACT policies in Uganda.First, the prevalence of malaria parasitaemia among most outpatient groups was lower than initially expected. Indeed, across different transmission settings, which are all traditionally viewed as high malaria transmission intensity areas, only 17–25% of patients above five years of age were parasitaemic. The prevalence rate of 44% was higher in children below five years of age; however, this rate did not exceed 54% even in the areas historically described as having the highest EIRs in the world with between 564 and 1,564 infective bytes per person per year . HoweverSecond, given the low prevalence of malaria (24%), high frequency of fever among outpatients (79%), and health workers' practices largely reflecting recommendations promoting presumptive malaria diagnosis for all febrile patients, the current rates of outpatient malaria over-diagnosis in Uganda are massive, reaching as high as 79% for patients five years and older and remaining high even in the children below five years of age in the study areas of highest malaria transmission (47%). The over-diagnosis rates of routine practices were not much lower than 75% previously reported rates from an area of low and unstable malaria transmission in western Uganda .Yet, it was worrying to observe that despite the minimum overall deviations from national diagnosis recommendations, the current practices in very high transmission areas resulted in a large proportion (49%) of children below five years of age who are not diagnosed although parasitaemic and subsequently not treated for malaria. As observed repeatedly in many studies in the past two decades across Africa -16, any Finally, a shift from the current presumptive policy based on fever to an effective policy based on malaria diagnostics is unlikely to be trivial. The findings at facilities with functional microscopy in study districts suggest that: 1) febrile patients are not sufficiently tested regardless of whether testing is recommended (46%) or discouraged by guidelines (38%), and 2) there is an overwhelming tendency to ignore negative slide results and prescribe anti-malarial drugs (58%). The underuse of malaria microscopy and the prescription of anti-malarials for negative test results revealed in this study mirror well-described patterns of microscopy use and results interpretation reported in past studies in Kenya , Zambia The Ugandan Ministry of Health should be commended for the current revisions of malaria diagnostic policies to promote greater access to parasitological-based diagnosis. An important component of this is the introduction of malaria RDTs to complement microscopy specifically in health facilities without laboratory services. Malaria RDTs are accurate under controlled conditions, easy to use and interpret, provide rapid results, and can be performed with the minimum of training and equipment. Yet, the success of this diagnostic strategy encompassing RDTs will be critically dependent on ensuring high accuracy (in particular sensitivity) of RDTs under field conditions ,26,27, aFinally, prior to the national policy change and scale-up, it would be wise that the implementation package introducing RDTs and strengthening malaria microscopy is piloted at district-level in areas of different malaria endemicity. Such pilots should include an operational research component which should monitor routine health workers' practices and real-world accuracy of both diagnostic tools, and evaluate cost and health benefits of a parasitological-based diagnostic strategy in particular compared with the presumptive treatment strategy in the most vulnerable groups such as children below five years of age. The lessons learned from these pilots should help policy makers to improve upon implementation delivery and take informed decisions if all age groups and transmission settings should be the targets of parasitological-based diagnosis for the national scale-up. Failing to approach the introduction of this new diagnostic tool in a careful and evidence-based manner will mean that no lessons have been learned from the experiences of implementing malaria microscopy.The traditional view of Uganda as a very high malaria endemic area where most of fevers are due to malaria is unlikely to be true. A large majority of outpatients, across all age groups and transmission areas, with the exception of children below five years of age in very high transmission areas, do not have malaria parasitaemia. National recommendations promoting presumptive malaria diagnosis for all patients with fever and health workers practices following these recommendations result in massive over-diagnosis, particularly in patients five years and older. Equally important, in children below five years of age in very high transmission areas, the current practices result in substantial malaria under-diagnosis. The problem of malaria misdiagnosis can only be tackled by using parasitological-based malaria diagnosis, however, the current experiences of malaria microscopy in Uganda suggest that the potential benefits are not realized because of the poor quality of routine reading of slides and irrational clinical practices. There is need for the change of policy from presumptive to parasitological-based diagnosis, however, the modalities of this change and real-world effectiveness of this strategy will be critically dependent on the quality, and coverage, of the implementation process which must be accompanied by a strong operational research component.The authors declare that they have no competing interests.JN and DZ contributed to study design, data analysis and interpretation of results. JNN contributed to data analysis and interpretation of results. JBR and HC contributed to study design, interpretation of results and policy implications of the findings. RWS and JKT contributed to the conception and design of the study, and interpretation of results. All authors contributed to drafting of the manuscript and all read and approved the final manuscript.


Intermittent preventive (or presumptive) treatment of infants (IPTi), the administration of a curative anti-malarial dose to infants whether or not they are known to be infected, is being considered as a new strategy for malaria control. Five of the six trials using sulphadoxine-pyrimethamine (SP) for IPTi showed protective efficacies (PEs) against clinical malaria ranging from 20.1 – 33.3% whilst one, the Ifakara study, showed a protective efficacy of 58.6%.The possible mechanisms that could explain the differences in the reported PE of IPTi were examined by comparing output from a mathematical model to data from the six published IPTi trials.Under stable transmission, the PE of IPTi predicted by the model was comparable with the observed PEs in all but the Ifakara study . When a reduction in the incidence of infection during the study was included in the model, the predicted PE of IPTi increased and extended into the second year of life, as observed in the Ifakara study.A decrease in malaria transmission during the study period may explain part of the difference in observed PEs of IPTi between sites and the extended period of protection into the second year of life observed in the Ifakara study. This finding of continued benefit of interventions in settings of decreasing transmission may explain why rebound of clinical malaria was absent in the large scale trials of insecticide-treated bed nets. Intermittent preventive treatment of infants (IPTi) is the administration of a curative anti-malarial dose to infants, whether or not they are known to be infected, at specified times to prevent malaria . IPTi deA number of explanations for the differences in protective efficacy (PE) of IPTi against clinical malaria between sites has been suggested including the intensity of transmission and consequent malaria incidence, the pattern of antimalarial resistance, the administration of iron and the use of additional control measures, specifically insecticide-treated nets (ITN) . This paP. falciparum (definition of duration of history of fever differed between studies: for Ifakara and Manhica studies it was 24 hours and for remaining studies it was 48 hours). In this paper, all references to the PE of IPTi refers to the PE against episodes of clinical malaria up to 12 months of age based on incidence rates of multiple episodes of clinical malaria, not time to first or only episodes.Data are from IPTi trials conducted in Manhiça (Mozambique), Lambarene (Gabon), Ifakara (Tanzania) and Navrongo, Kumasi and Tamale (Ghana). Detailed descriptions of the study population, methodology and outcome in each study included in this analysis have been published elsewhere -8. A sumThe relationship between the observed PE of IPTi and the following potential determinants of PE were explored: resistance to SP; estimated ITN coverage (% of the study population reporting use of ITN); and malaria transmission intensity . Day-14 parasitological and clinical failure rates were used to define resistance because five out of the six IPTi trials had published this information within two years of conducting the IPTi trial -14. One λ(a), which may vary through time (and hence by age). Once infected and in the asymptomatic state, children return to the susceptible state through one of three routes. First, they may become a clinical case and receive an effective treatment. It is assumed that in clinical trial settings, every case of malaria detected was adequately treated and parasites cleared. Secondly, they may receive antimalarial treatment for asymptomatic parasitaemia eg IPTi. Finally, they may remain asymptomatic and recover naturally. Symptomatic cases of malaria that are not detected by surveillance systems will remain in the asymptomatic state in the model until they die of severe disease or their immune response clears parasites. The model does not incorporate children leaving the asymptomatic pool by death, assuming this will be a very small number because most of the cases would be detected in time to receive effective treatment in a trial setting.An age-structured model Figure  was deveIgnoring mortality from other causes and migration, the model without interventions can be expressed by the following equation:where N is the fixed population size, r(a) is the age-dependent rate of natural clearance of parasitaemia and c(a) is the age-dependent rate of development of clinical disease which is then treated.In endemic areas the risk of developing clinical disease decreases with exposure to infection but rates of parasitaemia remain almost constant in early childhood. The model incorporates functions that mimic the development of immunity so that as children age the rate at which they develop clinical disease decreases and the rate at which they clear parasites increases. For simplicity, the model assumes that both immunity functions are linearly dependent on the expected number of malaria infections at age a:The rate of development of clinical disease is given by the logistic functionϕ is the rate of development of clinical disease in the absence of immunity and α1 and α2 are parameters which determine the number of infections after which full immunity to clinical disease occurs. The rate of natural clearance of parasites is assumed to be linear within the range of interest and hence is given bywhere ω is the mean number of infections after which full parasite immunity is obtained and it was assumed that at full immunity parasites are cleared after a mean of one day.where 1/A generic maternal protective function which acts to reduce the force of infection following birth was incorporated. Maternal protection is complicated and multifaceted , incorpoThe model was numerically evaluated as difference equations in 1-month time-steps using Excel.θ:To compare the model results to the trial data the two interventions were incorporated. Firstly, use of ITNs is included by reducing the force of infection in the group assigned to ITNs by a factor The model only examines the personal protection gained from an ITN and does not examine any other effects, such as effects on transmission.σ) of the population, ie the treatment effect, prophylaxes against new infection (factor σ reducing the force of infection) and reducing the rate of development of clinical disease by the same factor. As the model is defined in monthly time-steps, the parameter σ can be interpreted as the PCR uncorrected day 28 Adequate Clinical and Parasitological Response (ACPR) which measures the proportion that will clear parasitaemia and be protected against new infection 28 days post treatment. For simplicity, it was assumed that the ACPR acts with equal efficacy to clear parasites, to protect against new infection and prevent development of disease.IPT use across all age-groups modelled (two to 24 months) is assumed to act in three ways, clearing parasites in a proportion (1-Equation (6) is applied to the model only for months when IPTi doses are given. For months in those children who used ITNs and receive IPT equations (5) and (6) are combined.For the modelling exercise, the coverage of ITNs (reported ownership) in each trial site was used because the use of ITN at the individual level was not available for all trials. The expected incidence of clinical disease in each trial arm (placebo and IPT) is therefore calculated as a weighted combination of the model predictions with and without ITNs. The protective efficacy of IPT predicted by the model is calculated as 1-relative risk = 1-clinical incidence in IPTi group/clinical incidence in placebo group.λ(a) was assumed to be constant for the baseline scenarios. At a later stage the modelling exercise used scenarios in which the force of infection was decreased in early childhood due to maternal protective factors and separately allowed to increase or decrease linearly as the children aged reflecting changes in transmission over time. The force of infection was initially estimated directly as the mean incidence of clinical malaria in the placebo group. This approximation was based on the observation that the age-specific incidence of clinical malaria is highest in infants, the IPTi target age group and that the age group with the highest incidence should have the lowest immunity. The observed data from the Navrongo study (the only full dataset available to the study team) was compared with the model estimates of clinical disease, which were found to be half of that observed in the study. Thus, the mean incidence of clinical malaria in the placebo group was multiplied by a factor of two to estimate the force of infection. Entomological Inoculation Rates (EIR) were not used as a measure of transmission in the model for two reasons: EIRs measured concurrently with the IPTi trials were not available for most sites and in the studies with EIRs there was no common methodology. Secondly, these large cohort studies had enrolled children from a large geographical area over several years (two to four years). Thus a single measure of EIR would not suffice to represent the whole study area or the whole study period.It is assumed that that on average after 5 infections of malaria an individual is totally protected against clinical disease and that after 50 infections an individual can clear parasites rapidly. A sensitivity analysis for these parameters can be found later in this paper. Parameters that reproduce these patterns are given in Table The proportion of infected children becoming symptomatic and treated in the absence of immunity was assumed to be 90% in one month. This was derived from a study of asymptomatic parasitaemia in 6–59 month old children in a moderate malaria setting in Kampala, Uganda ; in thisFor those children receiving IPT, it is assumed that treatment, prophylaxis, and prevention of developing clinical disease effects of SP will be equally affected by the PCR uncorrected day 28 ACPR of SP. Day 28 PCR uncorrected ACPR is a measure of both the treatment and prophylactic effect combined (it includes both recrudescence's and re-infections) and is more likely to represent the effects of the drugs when used for prevention as opposed to treatment. The sensitivity analysis for how changes in ACPR affect PE is shown in the in the results section. Briefly, as drug resistance increases PE declines. The day 28 ACPR was only available for 2 sites, the sites with the highest and lowest resistances at day 14, namely Ifakara  and TamaFor those children using ITNs, we assume that the protective efficacy of an ITN is 0.5 . The modA sensitivity analysis of how ACPR, ITN coverage and immunity functions affects predicted PE was carried out.Figure The model predicted a similar pattern across the six trials, with a transient decline in incidence among the groups receiving IPTi (with and without ITN coverage) as well as a generally lower incidence among groups with ITNs (with and without IPTi). Using the Navrongo study as an example , Figure The predicted PE of IPTi in the six sites with the observed data are shown in Table rd dose of IPTi given at nine months of age has the dominant effect on the PE. Changes in transmission affect the extent to which rebound or prolonged protection are observed in the months following the IPTi doses independently of maternal immunity. If overall transmission is being reduced, this in turn reduces the probability of infection once the direct protection afforded by IPTi is removed and hence reduces the potential for the rebound effect to be observed and increases the probability of observing prolonged protection.Table The results of the sensitivity analysis of ACPR are shown in Table The high PE of IPTi found in the Ifakara study and a similar preventive trial using amodiaquine in north-eastern Tanzania  triggereThe model shows that PE mainly depends on the level of malaria transmission during the few months which IPTi doses are administered and the length of follow up and transmission intensity when IPTi is not given. To maximise PE IPTi should be given during high malaria transmission and follow up should be short when malaria transmission is low. Supportive evidence for this is demonstrated in the extended analysis of the Navrongo study  and an IThis model also provides a coherent explanation as to why no rebound effect would be observed in situations of decreasing transmission, such as Ifakara or Kenya ,25. The ITNs exert a steady personal protection to the individual sleeping under the net of approximately 50%  as long As with all theoretical studies, the model has some limitations. The model is dependent on some key assumptions regarding the effect of exposure on immunity. First, it is assumed that full clinical immunity was obtained after five infections. This figure was derived from past estimates for severe malaria  but cleaWhat does this study mean for IPTi? This model demonstrates that during a decline in malaria transmission, which Africa is currently experiencing, IPTi can be highly effective and safe. Combining IPTi with ITNs results in greater protection for an individual, further more, if high levels of coverage of ITNs can be attained with a resultant decrease in transmission, then there appears to be synergy between the interventions. However, if transmission subsequently increases a reduction in the efficacy of IPTi  can be expected. In stable conditions, PE does not seem to be greatly affected by levels of transmission, however the higher the level of transmission the more likely a rebound effect is to be seen. The rebound effect is equivalent to delaying of clinical cases of malaria to an older age which may be beneficial as older children appear to develop less severe illness than infants . Indeed,The authors declare no conflict of interest. RG, DC and BG are members of the IPTi Consortium. The views expressed in the paper are those of the authors and not of the IPTi Consortium.RG conceived the idea and developed the model and wrote the paper. AG developed the concept, refined the model and wrote the paper. JD developed the model and wrote the paper. LVS develop the concept and wrote the paper. BG reviewed the early manuscript and wrote the paper. DC developed the concept and model and wrote the paper. All authors read and approved the final version.


In Mali, malaria is the leading cause of death and the primary cause of outpatient visits for children under five. The twin towns of Mopti and Sévaré have historically had high under-five mortality. This paper investigates the changing malaria burden in children under five in these two towns for the years 1998-2006, and the likely contribution of previous interventions aimed at reducing malaria.A retrospective analysis of daily outpatient consultation records from urban community health centres (CSCOMs) located in Mopti and Sévaré for the years 1998-2006 was conducted. Risk factors for a diagnosis of presumptive malaria, using logistic regression and trends in presumptive malaria diagnostic rates, were assessed using multilevel analysis.Between 1998-2006, presumptive malaria accounted for 33.8% of all recorded consultation diagnoses . The monthly presumptive malaria diagnostic rate for children under five decreased by 66% . The multi-level analysis related 37% of this decrease to the distribution of bed net treatment kits initiated in May of 2001. Children of the Fulani (Peuhl) ethnicity had significantly lower odds of a presumptive malaria diagnosis when compared to children of other ethnic groups.Presumptive malaria diagnostic rates have decreased between 1998-2006 among health care-seeking children under five in Mopti and Sévaré. A bed net treatment kit intervention conducted in 2001 is likely to have contributed to this decline. The results corroborate previous findings that suggest that the Fulani ethnicity is protective against malaria. The findings are useful to encourage dialogue around the urban malaria situation in Mali, particularly in the context of achieving the target of reducing malaria morbidity in children younger than five by 50% by 2011 as compared to levels in 2000. Malaria is a major cause of childhood morbidity and mortality in sub-Saharan Africa. In 2006, 86% of the estimated 247 million malaria cases occurred in that region, causing 801,000 deaths (85% among children under age five) . In this paper, monthly outpatient consultations recorded between 1998-2006 in each CSCOM in Mopti and Sévaré were used. Since CSCOMs do not have laboratory facilities and only 0·8% of all presumptive malaria cases were referred to the hospital-based laboratory, these data refer to presumed malaria cases, based on a temperature of 37·5°C or higher and/or a combination of common symptoms of malaria. This diagnostic procedure tends to overestimate the number of confirmed malaria cases -30. HoweAll information was collected between June and August of 2008, recorded in a database, and included age (in months) of each child under five, ethnicity, sex, diagnosis, and CSCOM name Table . QuarterThe proportion of the population served by individual CSCOMs in each year of the study period was calculated by dividing the estimated population in both towns by the estimated catchment population. Since the latter was not estimated by age groups, monthly malaria consultation rates per 1,000 children under five for each CSCOM were calculated utilizing as denominators the product of the annual catchment population by the national estimated proportion of children under five for each year.Finally, information was gathered on malaria control interventions implemented during 1998-2006 through personal communication with the Director of Statistics at the DRS, and through a literature search.th and 75th percentiles. The effect of rainfall on Anopheles density was expected to be lagged in time, although the exact time lag is dependent on temperature and may vary locally [A monthly time series of rainfall from satellite-based estimates provided by the African Data Dissemination Service (ADDS)  was asse locally -37. In tTo accommodate the annual flooding pattern of the inland delta, high-resolution imagery was utilized to calculate the distance from each CSCOM to the nearest body of water in the delta. The distance was measured for non-flood (February-August) and flood (September-January) periods Table . Pan-shaPotential impacts of demographic and environmental variables on malaria diagnoses were investigated with logistic regression models. The response variable indicated if malaria was diagnosed for each child under age five who sought care in the CSCOM. The covariates included in the model were: ethnicity, age, CSCOM, monthly rainfall, sex, and year Table . Age catTrends in malaria consultation rates over time were appraised with a multilevel model for change. Because the data utilized in this paper did not follow individual children over time, the CSCOMs were used as the unit of analysis. For each CSCOM, predictor variables were summarized by month. Individual characteristics were aggregated by month: mean age of all the children; proportion of children who were male; and proportion of children from Bozo and Dogon ethnicities .The multilevel model for change was fitted using a generalized least squares method, and included components at two levels: a level-1 sub-model that described how the monthly malaria consultation rate in each CSCOM changed over time, and a level-2 sub-model that described how these changes varied across CSCOMs. The assumption was that the shape of the hypothesized individual monthly CSCOM malaria consultation rate change trajectory was linear. Assumptions about the error distributions specified univariate normality at level-1 and bivariate normality at level-2. The fit of the model was assessed using the Akaike Information Criterion (AIC) .This study was exempted of human subjects by the Harvard School of Public Health Institutional Review Board (protocol # P16245-101) and by the Ethics Committee of the Faculty of Medicine, Pharmacy, and Odontostomatology, University of Bamako (protocol # 61/FMPOS).The sponsor of the study had no role in the design, data collection, data analysis, data interpretation, or writing of the report. The authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.Monthly consultation records for 51 independent diagnoses were summarized into 14 disease categories using version 10 of the International Classification of Diseases coding. Of the total observations n = 30,198), 283 were incomplete. As there was not a pattern in missing data, all incomplete observations were removed from the analysis. Also, seven of the 606 total monthly records were missing , followed by Bozo (24·5%). The Fulani (Peuhl) ethnic group had the lowest percentage of malaria consultations (6·4%).Univariate tests on malaria interventions indicated that only the distribution of bed net treatment kits was a significant predictor of both the monthly malaria consultation rate (p < 0·001) and the change in the monthly malaria consultation rate over time (p < 0·001). The same procedure was undertaken for the control variables, and those selected for inclusion in the model were: age, sex, ethnicity, annual rainfall, monthly rainfall, lagged monthly rainfall, CSCOM water proximity during the non-flood period, CSCOM water proximity during delta flooding, CSCOM, nurses, and doctors.Results of the logistic regression model are shown in Table Increases in rainfall consistently led to more presumptive malaria diagnoses. Compared to months with <15 mm of rainfall, the odds of a malaria diagnosis increased by 70% in months that had precipitation levels between 15 mm and 90 mm, and by 85% in months that had more than 90 mm of rain. Children aged 18-59 months had 1·29 times the odds of a malaria diagnosis than children age 0-5 months. Finally, the Pearson Chi-Square Goodness-of-Fit test was significant (p < 0·0001).Results of the multilevel model are presented in Table The average monthly malaria consultation rate across all CSCOMs irrespective of time was 4·97, and 13·2% (5·893/(5·893+38·615)) of the total variation in malaria consultation rates was attributable to variation between CSCOMs . Accounting for lagged rainfall, the slope of the monthly malaria consultation rate change trajectory after the bed net treatment kit distribution was 37% lower than prior to the intervention. The AIC suggested that Model D provided a better fit compared to Model C. Standardized residuals fell within +/- 2 standard deviations of their mean, suggesting that the normality assumption was not violated.The results suggest a 66% secular decline in the presumptive malaria consultation rate between 1998-2006 in Mopti and Sévaré, significantly associated with the distribution of bed net treatment kits. In contrast, the bed net social marketing campaign did not reveal significant effects. Substantial variation in the monthly malaria consultation rate was found both between and within CSCOMs, and over time, and corroborated preliminary findings that the Fulani (Peuhl) ethnicity has a protective effect on malaria.The findings are consistent with recent reports of malaria declines observed in Kenya and The Gambia ,7. SimilRegarding ethnicity, children from the Fulani (Peuhl) group had significantly lower odds of a diagnosis of malaria when compared to every other ethnicity. Decreased risk for malaria among the Fulani has been reported in studies in Mali and Burkina Faso, despite few cultural and socioeconomic differences, and similar levels of exposure as measured by the EIR -43. CompThe age of the child is an important consideration in discussions of malaria diagnosis. This study found that children aged 18-59 months were at increased risk  for a diagnosis of malaria as compared to infants (0-5 months). This finding is consistent with a recent study in Kenya that showed that when there is reduced exposure to infection following a decline in malaria transmission, there is a corresponding increase in the mean age of children with malaria . In addiMonthly malaria consultation rates decreased by 37% after the bed net treatment kit distribution. The likely success of this intervention might be explained by the long history of bed net ownership in Mopti and Sévaré, coupled with positive perceptions of the insecticide used to treat them . A 2003 According to the 2006 DHS, 50% of households in Mali have at least one mosquito net, but only one in five is treated . A largeper se can contribute to a reduction in malaria transmission) [This study entails a number of limitations. First, the consultation data is an endogenously selected sample (choice-based sample) as it provides information only on children under age five who sought health care and received a consultation at a CSCOM. It is possible that children who chose not to attend the CSCOM may differ in important ways from those who did. However, the analysis aimed to describe the secular trend in malaria consultation rates only among health-care seeking children. Second, due to the nature of information recorded in the consultation registries, it is impossible to determine whether the same child had multiple records over time. To accommodate this, in the multilevel analysis, the individual unit of observation was the CSCOM and not children. Third, during the rainy season, when malaria transmission is greatest, household revenue is likely to decrease. This could potentially result in less health care seeking and a greater use of traditional medicine . Therefomission) . In addiIn conclusion, with the increasing awareness that infectious diseases, particularly malaria, impose an enormous burden on developing countries, the use of appropriate control interventions is essential. The MoH has set a target year of 2011 to reduce the national level of malaria morbidity by 50% as compared to year 2000 levels, with a particular focus on children under five . ResultsThe authors declare that they have no competing interests.ARW, SD, and MCC participated in the study design. ARW, SD and BT participated in study implementation. ARW collected the data. ARW analyzed the data and interpreted the results under the supervision of MCC. ARW and MCC wrote the paper with input and editing from SD and BT. All authors read and approved the final version of the paper.


Anaemia is caused by many factors in developing countries including malaria. We compared anaemia rates in patients with malaria parasitaemia to that of patients without malaria parasitaemia.A cross-sectional study was carried out from November 2007 to July 2008 in health units in Buea, Cameroon. Adult patients with fever or history of fever were included in the study. Information on socio-demographic variables and other variables was collected using a questionnaire. Malaria parasitaemia status was determined by microscopy using Giemsa stained thick blood smears. Haemoglobin levels were determined by the microhaematocrit technique.The study population consisted of 250 adult patients with a mean age of 29.31 years (SD = 10.63) and 59.44% were females. 25.60% of the patients had malaria parasitaemia while 14.80% had anaemia (haemoglobin < 11 g/dl). Logistic regression revealed that those with malaria parasitaemia had more anaemia compared to those without malaria parasitaemia after adjusting for age, sex, rural residence, socioeconomic status, use of antimalarials, use of insecticide treated nets(ITN) and white blood cell count.In adult patients with fever in this setting, malaria parasitaemia contributes to anaemia and is of public health impact. Our results also provide a baseline prevalence for malaria parasitaemia in febrile adults in health units in this setting. Anaemia is frequent in developing countries and its aetiology is usually multifactorial. The most important factors that contribute to anaemia include parasitic infections, HIV infection, chronic inflammatory disorders, micronutrient deficiencies and genetic disorders -6. The mMalaria related anaemia is associated with many factors which involve increased destruction and reduced production of red blood cells(RBCs) . The relOur objective in this study was to compare anaemia rates in patients with malaria parasitaemia to that of patients without malaria parasitaemia in a population of adult patients with fever or history of fever in Buea, Cameroon.The study was carried out in Buea in the South West of Cameroon. Buea is situated at the foot of Mount Cameroon and has an estimated population of 81 478 inhabitants (2008). The climate of Buea is of equatorial type with temperatures that range from 25-29°C annually. There are two main seasons; the rainy season which starts from June to around October and the dry season which starts from November to May. Malaria is endemic in this region and transmission occurs all year round with an average of 1-100 malaria cases per thousand per year.The subjects for this study were taken from a study on thrombocytopenia and malaria (unpublished data). The study population consisted of patients consulting in 4 health units in Buea. These public health units include a hospital which is the main hospital in the area and 3 health centres. These units were chosen because they serve the majority of the population in the area due to their public nature and their location. Consecutive patients who came to each health unit for consultation were invited to participate in the study. Patients who satisfied all of the following characteristics were included in the study: fever (axillary temperature ≥37.5°C) or history of fever, age 18-65 years, and informed consent provided. Informed consent was obtained from all the participants in the study. Information about the study including possible risks and benefits was provided to the participants. Those who agreed to participate were asked to sign the consent form or provide a thumbprint. The study was approved by The Cameroon National Ethics Committee. A cross - sectional study was conducted from November 2007 to July 2008.The subjects were taken from a study to identify thrombocytopenia as a risk factor for severe malaria in adults, for which the sample size was calculated. The prevalence of severe malaria in adults in the Province in 2006 is 35% (unpublished data). A 15% difference in both groups was considered of public health importance. The estimated sample size with 80% power and 5% significance level was 366.Information on socio-demographic variables was collected through interview and was entered into a structured questionnaire. Other variables like rural residence, employment status, monthly income, marital status, educational status, use of insecticide treated nets (ITN) and use of antimalarials were collected through interview. The monthly income was used as a proxy for socioeconomic status. For all participants, blood samples were collected and sent to the laboratory.The laboratory personnel were not aware of the clinical status of the patients. Thick blood films were prepared for all participants, air-dried, stained with Giemsa , and examined for malaria parasites by light microscopy. Parasites were detected using a binocular Olympus microscope  under oil immersion. Parasites were counted against a minimum of 200 white blood cells for the determination of parasite density. Fifty high power fields (HPF) were scanned to confirm malaria negative slides. Thick blood films were examined for malaria parasites by two experienced microscopists. In case there was discordance in the results, that of the Kenya Medical Research Institute (KEMRI)-certified microcospist was re-examined by a senior scientist for approval. Quality control was ensured by a senior scientist who ensured that the authorised Standard operating procedure for thick blood film microscopy was strictly followed. Furthermore, the senior scientist randomly selected equal numbers of positive and negative slides for confirmation of results of malaria parasitaemia obtained.Packed cell volume values were determined using a microhaematocrit centrifuge  according to the method described by Cheesbrough. BrieflyWhite Blood Cell (WBC), Red Blood Cell (RBC) and platelet counts were determined using a haemocytometer (improved Neubauer counting chamber) as described by Cheesbrough.The Mean Corpuscular Volume (MCV) was calculated from the PCV and the RBC count values using the formula below:Data from the questionnaires was entered using the package EpiData version 3.0 . The data entered was checked for consistency and legal values. Data analysis was done using the package STATA version 8.2 .Baseline characteristics were compared in aparasitaemic and parasitaemic patients using means or proportions. Means were compared using the t-test or analysis of variance (ANOVA). In case of skewed data, the Mann-Whitney test was used. Proportions were compared using the chi-squared test. Quantitative variables were converted to categorical variables by splitting them into standard categories or tertiles. Univariate analysis was done by examining the effect of each measured variable on anaemia using the chi-squared test or the chi-squared test for trend. The T-test was used to compare the geometric means of parasite densities in both the anaemic and non-anaemic patients. The attributable risk fraction (ARF) was calculated by using the formula below:Multivariate analysis was done by entering each measured variable into a Logistic regression model. The variables were entered in a stepwise manner in order of the size of the effect . The models were compared using the likelihood ratio test. A variable was dropped if there was no evidence  that it fitted into the model or if the size of the effect of malaria parasitaemia on anaemia did not change substantially. A test for interaction among the categorical variables was also performed by comparing the model with and without the interaction term. The interaction term was also dropped if there was no evidence that it fitted into the model.The study population consisted of a total of 250 adult patients, 68% 170/250) of the patients had a history of fever while 41.60% (104/250) had measured fever (axillary temperature ≥37.5°C) and 39.2% (98/250) had both history of fever and measured fever. The mean age (± SD) was 29.31 years ± 10.63 and 59.44% of the patients were females. The mean haemoglobin level in the study population was 13.16 g/dl ± 2.21 and the prevalence of anaemia (haemoglobin < 11 g/dl) was 14.80%. The median parasite count was 643 (range = 40 - 275 566) parasites per microlitre of blood and 25.60% of the patients had malaria parasitaemia. The reported use of insecticide treated nets was 16.95%. Patients with malaria parasitaemia were similar to those without malaria parasitaemia in terms of most of the baseline characteristics except for history of fever, temperature at enrolment and platelet count  - Table After adjusting for age, sex, rural residence, socioeconomic status(SES), antimalarial use and WBC count, the effect of parasitaemia on anaemia was more pronounced and this was of borderline significance .After adding the variable ITN use in the model, the effect was even more pronounced and was statistically significant -Table In this study, we sought to investigate the contribution of malaria in the prevalence of anaemia in adult febrile patients attending three principal health institutions in Buea, Cameroon.Our findings indicate that anaemia and malaria parasitaemia are not rare in adult patients consulting in clinics in this setting where malaria is endemic and transmission is perennial. In high transmission areas like this, people usually experience frequent and repeated bites by mosquitoes and they develop immunity by the time they are adults. Adults usually have some level of parasites in them that do not necessarily cause clinical malaria but can lead to anaemia and its related complications. The proportion of individuals with malaria infection found in this population could partly be explained by the low reported use of insecticide treated nets. Our proportions are higher than that of a previous study conducted in a population of Mozambican adults.The relationship between malaria parasitaemia and anaemia is well established in previous studies ,18,20-23The main limitation of this study is confounding. Some potential confounders like helminth infections, micronutrient deficiencies and HIV infection were not measured. The study was conducted over a number of months which means the anaemia rates and even the parasite densities could change over time. However, the malaria transmission in this area is perennial and not seasonal. Human Chorionic Gonadotrophin (HCG) levels indicating the pregnancy status of the female patients was not measured which could affect the levels of haemoglobin. There were about 30% missing values for age which could cause insufficient adjustment for the confounding effect of age. This could have overestimated the effect of parasitaemia on anaemia since those with lower proportion of parasitaemias had missing values for age. The study may have been underpowered which could explain the borderline significance obtained. Microscopy was the only method used for the detection of malaria parasites meaning that some malaria negative patients could have had malaria parasites which could be detected by PCR and this could lead to underestimation of the effect of malaria parasitaemia on anaemia. However, microscopy is the method used in these health facilities for the management of patients and in this study, it was properly conducted using an authorised standard operating procedure, by well trained laboratory scientists.In adult patients in this setting with fever or history of fever, those with malaria parasitaemia are more likely to have anaemia and malaria parasitaemia has a great public health impact on anaemia. Public health interventions on malaria in non-pregnant adults should be geared towards clearing malaria parasites. Our results establish a baseline prevalence of malaria parasitaemia in adult patients (non-pregnant) with fever in this setting which will help clinicians in their differential diagnosis of malaria.The authors declare that they have no competing interests.ENT was involved in the conception and design of the study, analysis and interpretation of the data and drafting of the manuscript. EAA was involved in the analysis and interpretation of the data and drafting of the manuscript. PMN was involved in the analysis and interpretation of the data and drafting of the manuscript. All authors approved the final version of the manuscript.


Negaprion brevirostris fitted with external receivers and transmitters, showed receivers logged interactions between individuals regularly when sharks were within 4 m (∼4 body lengths) of each other, but rarely when at 10 m distance. A field trial lasting 17 days with 5 juvenile lemon sharks implanted with proximity receivers showed one receiver successfully recorded association data, demonstrating this shark associated with 9 other juvenile lemon sharks on 128 occasions. This study describes the use of acoustic underwater proximity receivers to quantify interactions among wild sharks, setting the scene for new advances in understanding the social behaviours of marine animals.Group behaviours are widespread among fish but comparatively little is known about the interactions between free-ranging individuals and how these might change across different spatio-temporal scales. This is largely due to the difficulty of observing wild fish groups directly underwater over long enough time periods to quantify group structure and individual associations. Here we describe the use of a novel technology, an animal-borne acoustic proximity receiver that records close-spatial associations between free-ranging fish by detection of acoustic signals emitted from transmitters on other individuals. Validation trials, held within enclosures in the natural environment, on juvenile lemon sharks  Many animals across a diversity of taxa are recognized to form groups Carcharodon carcharias) used a radio-acoustic positioning system, linked to a base station, to investigate social hunting behaviours 2 providing the resolution to determine inter-individual distances. However, having receivers fixed in location meant that the detectable area was restricted and animals moving out of range could not be included in the analysis. To overcome this problem a very recent study described the use of inter-animal telemetry Carcharhinus galapagensis) were equipped with tags that both transmitted their own code and stored signals (i.e. tag number) from other tagged animals. Field trials determined that the tags were capable of accurately capturing the presence-absence patterns of other tagged sharks. Furthermore, the trials demonstrated that on-shark tags can provide important inter- and intra-specific interaction data among individuals in areas remote from traditional fixed receiver arrays In recent years, researchers have become increasingly reliant on remote devices to address a wide range of science and management questions, in a variety of species, including; marine mammals, turtles, teleosts, chondrichthyans, crustaceans and cephalopods Trichosurus vulpecula) and to examine relationships of contact rate with population density and habitat Meles meles) and dairy cattle in the UK has enabled the identification of high-risk individuals in the transmission of Bovine Tuberculosis (TB) Similar problems have also been identified for terrestrial animals where direct observations might be impractical due to the elusive or nocturnal nature of a species Negaprion brevirostris, testing the detection range and performance of the devices. Finally, we present a demonstration that proximity receivers deployed on free-ranging lemon sharks yield individual interaction data, indicating their potential use in locating shark aggregation sites and determining associative patterns  between sharks within a population. We also discuss other uses for this type of technology in shark research, with an obvious extension to its use being other marine animals, and consider further advances and future experiments that may serve to improve the tag's performance and application.In this study, we investigate whether acoustic telemetry can be used to study grouping behaviours in sharks. We assess a novel acoustic-proximity receiver in a series of controlled experiments using captive juvenile lemon sharks All surgical procedures were conducted in accordance with the animal welfare laws of the country in which they were undertaken.This study was conducted in Bimini, Bahamas, a small chain of islands approximately 85 km east of Miami, Florida, U.S.A . JuvenilThe acoustic receivers used in this study were a prototype system  designed to log the date and time of transmitter-tagged individuals moving within the receiver's detection range . Each ARca. 10. The transmitter will continue this cycle of broadcasting its aural code for 20 seconds and sleeping for 8 seconds until its battery is exhausted (for the IBT tags this is 60 days).The transmitters used in this study can be detected by the ARX receivers, traditional SUR's and manual tracking receivers. Each acoustic transmitter , as per traditional acoustic tracking technology ca. 30 days and is capable of keeping records of 106,000 detections, which equates to just over 2 detections per minute. The ARX's memory can be erased for re-use and the unit itself can be set to a ‘sleep’ mode that saves battery life between trials. It is also important to note that both memory capacity and battery life are dependent on ARX size, and hence, the weight-carrying capacity of the species being studied. In this study, the juvenile lemon sharks used were 0.80–0.90 m in total length, weighing 3.0–3.5 kg in air. The ARX unit weighed 31 g in air (16g in sea water); this is less than 1% of the body weight of these sharks, a percentage that has been shown in other studies not to affect shark behaviour Prior to deployment, the ARX is initiated by placing it on an interface unit that enables an infra-red connection to be established, and that starts an internal clock that counts seconds until stopped. When a transmitter is detected, the time in seconds is logged along with the interval ID (e.g. 910 ms) of the transmitter. This data is then stored in non-volatile memory, within the ARX, and is retrieved through connecting to the interface unit and downloading to a Windows application. The ARX used in this study can differentiate between 50 tags , has a battery life of ca. 300 detections. The percentage of transmitter signals detected by the receiver was then calculated to give an estimate for the measure of efficiency at the two distances.The first preliminary trial that was conducted was a test to check if the unit worked in a stationary, controlled environment. An ARX receiver and transmitter were attached to separate PVC poles and kept at an in-water height of 0.20 m above a flat, sand seabed. Water depth remained between 0.70 and 0.80 m throughout the treatments. Two treatments were completed: (1) A transmitter was stationed 1 m horizontal distance from the ARX receiver, and (2) 10 m horizontal distance. Both treatments lasted 30 minutes. Based on our previous calculation of a theoretical maximum of 10 detections per minute, an expected number of detections for this time period would be N = 5) completed two treatments with each shark fitted with an IBT tag (N = 4). Treatment 1: two sharks were placed together in compartment A or B (one with ARX receiver and one with IBT transmitter); and Treatment 2: individual sharks were placed in separate compartments A and B (one with an ARX receiver and one with a transmitter). Each treatment lasted 10 minutes with sharks given 5 minutes to acclimatise to the compartment and attachment, before trials commenced. An observer recorded the start and end time of each treatment (to the nearest second). The start and end times were compared to detections recorded on each ARX, to determine during which treatments detections were successfully made. During all treatments sharks were observed to swim continuously, indicating that the attachment method did not affect the shark's ability to move around the compartments. When the sharks were together they were observed to interact continuously, performing social behaviours, such as following and circling each other, and so were always <4 m distance from one another. Experiments were performed over a sandy substrate and between 0.70 and 1.00 m water depth. For both of these trials a measure of efficiency (%) was also calculated for the receivers [no. of detections/100 ].A total of 9 juvenile lemon sharks were used in these trials . All sharks were captured using gillnets, immediately measured and transported to a holding pen, see Fifteen juvenile lemon sharks were captured using gill and seine nets from Bone Fish Hole nursery area, Bimini, Bahamas . All werProximity does not necessary imply a social interaction. However, longer durations of proximity are likely to be indicative of interactions. To address this problem in our field data, we used the results from experiment 2 to set a criterion for identifying a social interaction between two sharks in the wild. So for our wild results, if ≥2 detections were recorded in a 60 second period we recorded it as a social interaction lasting 60 seconds. If detections continued to be within 60 seconds of each other the time between the first and last detection were estimated to be the interaction duration, until two detections were separated by a time period of >60 seconds. Single records were also identified on multiple occasions and were included as interactions of <30 seconds. These may be better interpreted as passing encounters rather than actual social interactions, which in this study at least were assumed to have greater longevity.ca. 200 m radius. Previous studies have shown that the average swimming speed of a juvenile lemon shark is about 0.7 m s−1To provide spatial information for the interactions the data retrieved from each seabed-mounted receiver (SUR) was assessed to see if it overlapped by 5 minutes for any detections on the ARX receiver tag. The SURs have a detection range of ca. 4 min−1) indicating a 42% probability of detection. When the distance was extended to 10 m the ARX receiver detected only 3 time intervals in 30 minutes, with a probability of detection at this distance being 1%.This initial trial showed that the ARX receiver worked well at close range distances (1 m), with 127 detections in a 30 minute period , ARX receivers averaged 16.95 detections (±S.D. = 6.63) in 10 min, which is equivalent to a single detection every 40 seconds or a receiver average of 16% probability of detection . For treOne of the 5 ARX receivers deployed on juvenile lemon sharks was recovered with recorded data. The three retrieved receivers that did not work appeared to have malfunctioned due to programming error, such that instead of just ignoring the transmitter paired with the receiver on the same shark, all transmitters were ignored. The ARX receiver that functioned correctly  recorded a total of 315 detections, over a period of 17 days in the Bone Fish Hole nursery area. These detections were from a total of 9 other juvenile lemon sharks, with detections on every day apart from two, and with up to 11 interactions per day of duration ≥60 seconds . Shark 3The principal finding of this study is that miniaturised acoustic technology now exists which operates over appropriate spatio-temporal scales to provide useful remotely-retrieved information about group associations in free-ranging sharks. Quantifying group behaviours in sharks is difficult. This is due to the impractical nature of observing animals that are notoriously elusive, large bodied and fast moving ca. 4 body lengths. The results from this study are therefore really only applicable to marine animals that are similar in body length to the study shark or larger. However, ARX receivers are available with higher or lower detection capabilities, depending on the size of animal that is to be investigated. Further experiments are required in order to test the reliability of such changes to the receiver before deployment on larger or smaller animals. At present, for these units, in order to obtain data the test animal must be recaptured. This means that these receivers are largely applicable to shark species that are small bodied, show high site fidelity and localised movements that will enable recapture and retrieval of the archived data Proximity receivers may not be suitable for all species or types of research questions. In many studies on fishes the distance between individuals is often used to infer group formation; if individuals are within four body lengths of each other then they are deemed to be in a single group Although the field trial did not provide enough data to make any firm conclusions about juvenile lemon shark grouping behaviour in general, it did provide some interesting preliminary results enhancing the claim for the technology's future use. The single ARX receiver tag that functioned correctly made contact with 9 out of 15 juvenile lemon sharks, some on multiple occasions (40 contacts with shark 678) and some continuously for up to 7 minutes (shark 488). In combination with an array of seabed-mounted receivers, the locations of a number of these interactions were also identified. With further replicates, this type of information could be used to determine if sharks are grouping or avoiding particular individuals within the population, based on a number of phenotypic traits such as sex, size and species. In recent years social network analysis has been used as a tool to assess animal social group structure using this type of data in other taxa and so could be applicable here also The formation of lemon shark groups could also be related to abiotic factors such as lunar and tidal cycle, water temperature and depth, as well as biotic factors such as predation risk or prey availability. The ARX receiver tags could be used additionally to monitor much finer scale movement of animals in and out of specific areas, for example caves or refuge sites. It could also prove to be a practical means of quantifying encounter rates between predators and their prey in the marine environment, opening the way for studies of optimal foraging strategies or analysis of predation risk, which have largely eluded detailed study in sharks to date To better understand group living in sharks, previous research has relied primarily on techniques such as captive observations, fishery capture records and position estimates based on triangulation of underwater acoustic receivers


Background. To evaluate the current status of malaria at parturition and its impact on delivery outcome in Nigeria.Methods. A total of 2500 mother-neonate pairs were enrolled at 4 sites over a 12-month period. Maternal and placental blood smears for malaria parasitaemia and haematocrit were determined. Results. Of the 2500 subjects enrolled, 625 were excluded from analysis because of breach in study protocol. The mean age of the remaining 1875 mothers was 29.0 ± 5.1 years. The prevalence of parasitaemia was 17% and 14% in the peripheral blood and placenta of the parturient women, respectively. Peripheral blood parasitaemia was negatively associated with increasing parity (P < .0001). Maternal age <20 years was significantly associated with both peripheral blood and placental parasitaemia. After adjusting for covariates only age <20years was associated with placental parasitaemia. Peripheral blood parasitaemia in the women was associated with anaemia(PCV≤30%) lower mean hematocrit (P < .0001). lower mean birth weight (P < .001)and a higher proportion of low birth weight babies (LBW), (P = .025). Conclusion. In Nigeria, maternal age <20 years was the most important predisposing factor to malaria at parturition. The main impacts on pregnancy outcome were a twofold increase in rate of maternal anaemia and higher prevalence of LBW. Falciparum malaria in pregnancy is an important cause of maternal and perinatal morbidity and mortality in malaria endemic areas. In pregnancy, malaria is more common, more severe, more atypical, and more fatal . PregnanIn Nigeria, several studies focusing on malaria in the peripartum period have been conducted but with small sample sizes and variable findings –15. For With the advent of the Global Fund to fight against Aids, Tuberculosis and Malaria (GFATM), many countries have been able to receive support for scaling up intervention programmes on malaria control including malaria in pregnancy. These activities call for better appraisal of the baseline characteristics of malaria in order to allow for proper impact assessment of the various interventions. Our study was therefore undertaken to evaluate the current status of peripartum malaria and its impact on birth outcomes in Nigeria. This is to serve as a baseline for Nigeria haven only recently developed guidelines for malaria prevention in pregnancy using IPT and ITNs .The study was conducted in four geo-political zones of Nigeria, namely North Western (Kaduna), North Central (Ilorin), South Western (Ibadan) and South Eastern zones (Enugu) between April 2003 and March 2004. Malaria is endemic and transmission perennial in all study sites. Detailed description of the study sites have been reported by Falade et al. .The subjects of the study were mothers and their newborn babies delivered at the study centres within the study period. In addition to willingness to provide written informed consent, continuous residence within contiguous areas of study the centre for at least 2 years and delivery of a life infant were additional inclusion criteria. This study was prospective and descriptive with enrolment of every consecutive delivery fulfilling the inclusion criteria. However to allow for a representative spread of the subjects across the recruitment period, enrolment was spread out over a twelve calendar month period from April 2003 to March 2004. Monthly recruitment was performed to ensure that mother-baby pairs were enrolled during both high and low malaria transmission season in order to determine the seasonal trends in the prevalence of malaria. Further details of the methodology of this study is in the report by Falade et al. .Mother and neonate pairs were enrolled from the labour and delivery unit of each study center. Relevant maternal and neonatal demographic and clinical characteristics were recorded in case record forms. Clinical data obtained include details of labour and delivery, anthropometric parameters of the baby and gestational age. In view of the fact that the subjects were recruited at or near parturition, they could not be placed on Intermittent Preventive Therapy (IPT) using Sulfadoxine-pyrimethamine. Specific note was however made of any malaria preventive measures used by the subjects.Plasmodium species on the thick blood film while thin smears were used for specie identification. Parasite density was determined by counting the number of parasites against 200 leukocytes (or 500 WBC if no parasite is found) on the thick blood film and converted to parasites/μL using an assumed total white blood cell count of 8000 μL [At delivery, thick and thin blood smears were prepared from maternal finger pricks, placental aspirates (within 1 hour of delivery) and baby heel pricks (within four hours of delivery) after initial cardiopulmonary stabilization. The placental aspirate has been previously reported to have a good correlation with wedge biopsy of the placenta . Blood sA procedure manual was produced before study initiation to standardize methodology at all study sites. Baseline training for microscopists and investigators was done. A pilot study was also conducted to evaluate the study process and detect potential site specific problems in order to harmonize the study protocol. Prestudy and mid study (within 3 months) quality assurance of the slides were done in a national reference laboratory. Continuous quality control on a monthly basis was also done, by randomly selecting 10 slides for comparison in the institution's main laboratory by a consultant haematologist or the chief technologist.All centres involved in the study have Federal Wide Assurance (FWA) certification. Ethical approval was provided by the Joint University of Ibadan/University College Hospital Ethical Review Board (Ibadan), the University of Ilorin Ethical Review Committee (Ilorin), Ahmadu Bello University Ethical Review Board (Kaduna) and University of Nigeria Teaching Hospital Enugu Institutional Review Committee. In addition, ethics approval was obtained from the Boston University Institutional Review Board. Written informed consent was obtained from each study volunteer or from the study participant's mother or legal guardian (spouse or grandmother) for study volunteers under 18 years of age. Continuous ethical review was carried out for each year the study was active.Data was collected and recorded into precoded case record forms. The completed case record forms were cross-checked for errors and missing variables by the supervisor. Thereafter, the data was entered into the computer by two data entry clerks using EPI-INFO 6d  programmed with appropriate CHECK commands to minimize error during data entry. t-test or the ANOVA where the assumptions were met. Association between categorical variables was tested using the Chi-square test or Fishers' exact test where applicable. For significant associations, the odds ratio (OR) and the 95% confidence interval (CI) were computed to determine the association between a risk factor and particular categorical outcome variable. For all statistical analysis a P-value less than .05 was considered significant.Relationships between continuous independent variables  and the outcome variables were tested using the Student's For the purpose of this study the following definitions were applied.Baby with birth weight less than 2500 g.Any baby delivered before 37 weeks of gestation. The last menstrual period (LMP) was used for assessing the gestational ages. However this was validated with either the first trimester ultrasound assessment of the gestational age or the Ballard chart. Any discrepancies in excess of 2 weeks of the gestational age by LMP resulted in the exclusion of the subject.A total of 2500 subjects were recruited from the four study centres. However due to the problems with buffering conditions for Giemsa stain and resulting deterioration of the blood smears at the Enugu research center, blood smears could not undergo quality assurance procedure. All mother-baby pairs enrolled at that site were excluded from analysis leaving the final sample size analyzed in this report at 1875 mother-baby pairs.P = .026). The proportion of preterm deliveries was 10% and it was not associated with parity.The majority, 1369 (73%), of the women had no previous history of abortion while 18% of the women had experienced at least one episode of abortion prior to the index pregnancy and 2.5% had had between 2-3 previous abortions. The women enrolled in the study were aged between 14–50 years. The distribution of the age, parity and pregnancy outcome is shown in Of the 1875 women, there were a total of 404  positive smears, thus giving a peripartum prevalence of malaria of 21.6%. Those with positive peripheral smears were 319 (17%) while those with positive placental smears were 267 (14.2%). On the basis of the detection of parasites in either peripheral blood or the placenta, 27.2% of primigravidae, 17.9% of secundigravidae, and 19.4% of multigravidae had peripartum malaria parasitaemia. These differences were statistically significant .P = .016; OR = 2.3: 95% CI = 1.2–4.9) and placental parasitaemia .P = .0001 in both instances). In each case these differences were most significant between primiparous and multiparous women (women in their 2nd pregnancy and above)  .t = 3.27; P = .001 for age: t = −0.75; P = .45 for parity).A linear regression of the age of the women and parity against maternal parasitaemia  however showed only the age of the women was significantly associated parasitaemia in the women .The mean (SD) haematocrit of the women with patent parasitaemia was 34% ± 5.0 while that for those without patent parasitaemia was 37% ± 5.4 . When this was stratified by parity there was no significant difference in the mean haematocrit of primigravidae with positive parasitaemia and multigravidae with positive parasitemia Tables  and 4.Peripheral and placental parasitaemia in the women did not affect the mean haematocrit of the babies Tables  and 4. F-stat = 10.7, P = .001. Low birth weight (LBW) occurred in 128/1875 deliveries thus giving a prevalence of 6.8%. Peripheral parasitaemia was also significantly associated with LBW babies .There was discordance with respect to peripheral and placental parasitaemia on the babies' birth weight. The observed significant differences were with respect to peripheral parasitaemia. Peripheral parasitaemia was associated with significantly lower mean birth weight 3077 g ± (523) versus 3175 g ± (477) among babies born to mothers with patent peripheral parasitemia and those without; In contrast, preterm deliveries and Apgar scores at 1 and 5 minutes were not associated with peripheral parasitaemia in the mother.In this study, we found that 21% of mothers had peripheral  and/or placental parasitaemia at parturition. Previous studies from Nigeria reported parasitaemia rates among pregnant women or at parturition ranging from 24.8–80% , 21–25. In this study, parasitaemia at the time of delivery was found to be associated with low parity and maternal age. Several other studies have reported similar associations –29. A muParasitaemia in the mothers was found to be significantly associated with lower maternal hematocrit. This finding is not unexpected as the association of malaria in pregnancy and low hematocrit has been recognized and reported by previous workers , 30. TheThe main effect of maternal parasitaemia on the babies is the reduction in the birth weight. This is consistent with the observations from other malaria endemic countries –35. ConsThe observed impact of malaria on the mother and their newborns add justification for promoting use of malaria preventive measure in pregnancy. The tools for achieving effective malaria control are now available; these include use of ITNs, IPT, and effective treatment. The use of these tools is also being facilitated with the recent production of a number of treatment guidelines and policy documents , 38, 39.In Nigeria, 1 in every 5 women has malaria  parasitaemia at delivery. Maternal age less than 20 years was the most important predisposing factor. Maternal anaemia, reduction in maternal hematocrit, mean birth weight and a higher proportion of LBW babies were the major deleterious outcomes of malaria in the peripartum period. The findings of this large multicentre study which recorded a prevalence of 21.6% for patent parasitaemia (mother and/or placenta) underscores the need for a focused and concerted effort to address the control of malaria during pregnancy in Nigeria.


HIV-1 infection affects malaria humeral immunity during pregnancy, but data for non-pregnant adults are lacking. This study reports the impact of HIV-1 infection and other variables on the level of malaria humeral immunity in adults with clinical malaria and whether humeral immune suppression was a risk factor for treatment failure.Sera of 224 HIV-1 infected and 115 uninfected adults were compared for IgG to merozoite antigens AMA-1 and MSP2 (3D7 and FC27 types) determined by ELISA, and for IgG to the Variant Surface Antigens (VSA) of three different parasite line E8B, A4 and HCD6 determined by flow cytometry.P = 0.02) and associated with low CD4 count AMA-1 IgG (P = 0.003). Low IgG to all three merozoite antigens was associated with less anemia (P = 0.03). High parasite load was associated with low MSP2 IgG 3D7 and FC27 types (P = 0.02 and P = 0.08). Antibody levels to VSA did not differ between HIV-1 infected and uninfected adults. However, low VSA IgGs were associated with high parasite load (P ≤ 0.002 for each parasite line) and with treatment failure (P ≤ 0.04 for each parasite line).Compared to HIV-1 uninfected adults, AMA-1 IgG was lower in HIV-1 infected (HIV-1 affects humeral responses to AMA-1, but seems to marginally or not affect humeral responses to other merozoite antigens and VSAs. The latter were important for controlling parasite density and predict treatment outcome. Plasmodium falciparum[Humeral responses are of critical importance to blood stage immunity to alciparum. Malariaalciparum. Well-knalciparum. These aalciparum,5 and aralciparum. Variantalciparum, but arealciparum-10.P. falciparum malaria infection, disease and treatment failure [P. falciparum. Differences in anti-malarial antibody levels have been shown in HIV-1 infected pregnant adults [Co-infection with HIV-1 and malaria is common in Africa. HIV-1 i failure -15. Howet adults -18 and at adults , but datThis study assessed the anti-malarial humeral immunity in HIV-1 infected individuals and its association with anti-malarial treatment failureP. falciparum malaria.This study was conducted in Ndola, Zambia, an area of meso- to hyperendemic malaria, betweenP. falciparum malaria infection and pregnancy (if applicable) and were included if they had a parasite density of at least 1,000 parasites/μL. Additional exclusion criteria were: severe malaria; documented intake of SP or AL two weeks prior recruitment; other cause of fever; evidence of underlying chronic diseases ; pregnancy; history of allergy to study drug or other sulfa drugs; non-resident in the study area.The study and the results have been reported elsewhere . Briefly®) and test for HIV-1 infection, CD4 count and viral load (if HIV-1 infected). Residual plasma was separated, stored and transported at -70°C to be assayed by ELISA and FACS for malarial antibody quantification. Patients were followed up until 45 day post-treatment when CD4 count was reassessed, as a proxy of underlying immune suppression. The study was approved by the ethical and scientific committees of the Institute of Tropical Medicine, Antwerp, Belgium, The Antwerp University, the Tropical Disease Research Centre, Ndola, Zambia and Melbourne Health Research Directorate, Australia.Clinical history, body temperature and physical findings were recorded. Venous blood (5 ml) was used to prepare blood films, impregnated filter papers (Schleicher & Schuell) for molecular analysis, measurement of hemoglobin . Parasitaemia/μl was computed using the actual WBC counts. Internal quality control was organized as recommended by WHO[® Counter, Partec, Germany)[® instrument  was used to validate the Cyflow data and served as a quality control. Based on previous research, a CD4 count of 300/μl at study entry and 450/μL at 45 days follow up was used as a threshold between low and high counts[All laboratory technicians were blinded to clinical data. Thin blood films were fixed with methanol and thin and thick blood films were stained with 10% Giemsa. Parasite densities were determined by microscopy as the number of asexual ed by WHO. HIV-1 t Germany). A FACSCgh counts. In ordevar genes, adheres to CD36 and ICAM-1. Of these var genes, A4var  were cultured as described[7/mL with test serum  in microtitre plates and incubated at room temperature for 30 min, washed as before, and incubated for 30 min with rabbit anti-human IgG and (after further washing as before) with Alexafluor® 488-conjugated donkey anti-rabbit IgG  and ethidium bromide . Washed cells were resuspended in 200 μL PBS+ and analyzed on a Becton Dickinson FACSCalibur flow cytometer with Cell Quest software. IE were gated according to ethidium bromide fluorescence, and 1,000 cells positive for ethidium bromide were collected. For each serum sample, the geometric mean Alexafluor 488 fluorescence intensity (MFI) generated by the gated population of IE was recorded. Control serum samples from malaria naïve adults were included in each assay. A positive control pool of hyper-immune serum against VSAs of E8B, A4 and HCD6 was made from the serum of 11 Malawian adults and run in duplicate in each assay. Samples with readings below the mean of the unexposed controls were assigned half the lowest value of that particular VSA variable in order to permit log-transformation; samples given a positive reading were assigned relative values by use of the formula (sample reading minus negative-control reading divided by positive control reading minus negative-control reading) × 100. Samples giving a reading above positive control were further diluted and values were assigned following the same formula and recalculated to the original dilution.Several parasite lines  were used for assessing malarial humeral immunity status. Parasite line E8B, expressing a mix of to ICAM-1) is a mibody BC6  and was described. For floAntibody levels to merozoite antigens were measured by ELISA. Recombinant 3D7 or FC27 MSP2 or AMA-1 proteins  were used to coat microtitre plates overnight at 4°C. Plates were blocked for 1 h with PBS/5% skim milk powder (Blotto) and washed five times with phosphate-buffered saline containing 0.05% Tween (PBS/Tween). Samples diluted 1 in 1,000 in Blotto were added to plates  and incubated for 2 h. After washing, horseradish-peroxidase-conjugated sheep antibody to human IgG  was added and the plates were incubated for 2 h then washed three times in PBS/Tween and twice in deionized water. Peroxidase substrate was added and color was developed, plates were read at 415 nm on a Biorad plate reader version 5.2, and OD results were expressed relative to positive and negative controls as for VSA antibodies. Results were also classified as positive  or negative. To estimate the "breadth" of immunity a binary variable "low responders" was created. Low responders were defined as having antibody levels below the geometric mean on all three antigens.Treatment outcomes were defined according to the most recent WHO classification and are described in detailed elsewhere,21. For t test, non-normally distributed variables by Mann-Whitney's rank sum test (two groups). Differences in proportions were assessed by χ2 analysis. Linear regression modeling was used to assess associations between humeral immunity and other factors. For the linear regression modeling, all non-normally distributed variables were log transformed and factors with a P-value < 0.10 were retained, as well as factors modifying the regression coefficient of the main determinant by more than 10%. All reported P-values are two-sided. Logistic regression modeling was used to assess if humeral immunity was a risk factor for treatment failure. P-values < 0.05 were considered significant.Clinical data were double entered and organized in Epi-info . All data were exported to STATA version 10.0  for analysis. Normally distributed variables were compared by Student's P < 0.001), comprised more women (P < 0.001), had lower CD4 count on day 0 (P < 0.001) and had lower hemoglobin levels (P < 0.001). No differences were found for mean WBC count (P = 0.26), parasite load (P = 0.92) and body weight (P = 0.95).Between October 2004 and June 2005, 339 adult patients were enrolled, 115 (33.9%) of them HIV-1 infected (Table P = 0.02) than HIV-1 negative adults, but this difference in antibody level was not observed for either MSP2 serotypes (P = 0.68 and P = 0.14)   and high parasite density  (Data not shown). After adjustment for CD4 count and age, low responders remained associated with HIV-1 infection (adjusted odds ratio (AOR):2.40; P = 0.01), parasite load  and had less anemia   but not with IgG to the MSP2 serotypes  (Table P = 0.02) and the MSP2 FC27 serotype . No associations between antibody levels with the separate merozoite antigens and CD4 count on day 45 were found in the HIV-1 infected adults. .P = 0.22; A4:P = 0.39; HCD6:P = 0.89)  and weight  were associated with low response to all VSAs.HIV-1 infected individuals and uninfected individuals had similar MFI for all three parasite lines expressing different VSAs . In subjects with a WBC count ≥ 4,900/μL, the parasite density was inversely associated with all VSA antibody levels  (Table P = 0.05) and low WBC count  were associated with a low response to all three VSAs.P = 0.02) though there was an interaction between initial parasitaemia and CD4 count at day 45 .Antibody levels to VSA of E8B and A4 were inversely related to CD4 count at day 45  patients had an adequate parasitological and clinical response at day 45. PCR genotyping identified 16 patients with new infections (6.0%) and 30 patients with recrudescence (11.2%).P = 0.02; P = 0.02 and P = 0.04 respectively) (Table P = 0.04), anemia  and SP treatment . Despite the low power, we observed trends showing SP increasing the risk of new infections , and good response to all VSAs decreasing the risk of new infections .No difference in antibody levels to merozoite antigens or VSA was found between patients successfully treated for malaria and those with recurrent parasitaemia. However, after PCR correction, high antibody levels to the VSAs of E8B, A4, and HCD6 protected for recrudescence HIV-1 infection increases the risks of malaria infection and disease, and (among immune-compromised patients) decreases responses to anti-malarial therapy -14. GiveHIV-1 infection was associated with low antibody levels to AMA-1 and, among HIV-1 infected adults, low AMA-1 antibody levels were associated with low CD4 count. And HIV-1 infected adults were twice as likely as HIV-1 negative adults to have low antibody levels to all three merozoite antigens combined.Reduced concentrations of antibody levels to AMA-1 have also been demonstrated in HIV-1 infected pregnant Malawian women. In thatP. falciparum infection induces a short-lived depression in circulating T cell numbers [Acute  numbers ,27. As H numbers . Limited numbers . Because numbers , malaria numbers  and, und numbers -32. To bSomewhat unexpectedly, within the HIV-1 infected population, low responders were less anemic. The recruited patients came from a community where malaria transmission is relatively homogeneous, so differences in exposure and concomitant development of malarial anemia seem unlikely. Second, the major contributor to the hemolytic anemia is the accelerated destruction of uninfected RBC, probably linked to immune mechanisms. TherefoIn the whole study population, antibody levels to both MSP2 types were negatively associated with parasite density indicating their direct involvement in parasite control whereas AMA-1 antibody levels were not. The results found for MSP2 IgG confirm studies that have shown associations between antibody levels to blood stage antigens and decreased prospective risk of clinical malaria,32,34-36Adult study participants should already have immunity to severe malaria and their antibody repertoire to VSAs may be extensive. It may,Although VSA antibody levels were not directly associated with HIV-1 infection, they appear to have important roles in controlling malaria parasitaemia in this cohort. Among HIV-1 infected adults with a WBC count ≥ 4,900/μL, antibody levels to VSA were inversely related to parasitaemia. Among all patients, for those with a CD4 count >450/μL on day 45 there was a negative association between parasite load and antibody levels to VSAs of parasite lines E8B and A4. Antibody to VSA operates by preventing sequestration of IE in the vasculature (to avoid splenic clearance), or by opsonization of IE by cytophilic antibodies. FurtherAntibody levels to VSA were similar in patients successfully treated for malaria and the patients who experienced recurrent infection, but high levels of antibody levels to VSAs were associated with decreased risk of recrudescence, suggesting that these antibodies may complement the therapeutic response to the anti-malarial drug. Indeed,This study provides evidence that HIV-1 affects humeral responses to AMA-1. Although levels of antibody to the other merozoite antigens and VSA were not or little affected by HIV-1 infection, they appeared to play an important role in controlling parasite density, and the inability to control malaria parasite density is one of the features of HIV-1 immune suppression. Furthermore, recrudescence was associated with low VSA antibody titers to all three parasite lines tested. Specific research in HIV-1 and malaria co-infected adults, with pre-established malaria-immunity, might offer additional insights into malaria immunity.The authors declare that they have no competing interests.J-PVG produced the final dataset, contributed to the analysis plan, data interpretation, wrote the paper and takes responsibility for the integrity of the data and the accuracy of the data analysis. EVE did the analysis and wrote the paper. FY performed the ELISA and FACS analysis. WK and MM organized the collection of data, supervised the study trial and contributed to the data interpretation and writing the paper. UD'A contributed to the writing of the paper. SR supervised the laboratory activities related humeral immunity, contributed to the data interpretation and writing of the paper.