High prevalence of malaria and hepatitis B has been reported among pregnant women in Ghana. In endemic areas, the diagnoses of malaria and hepatitis B among pregnant women on antenatal visits are done using histidine-rich protein 2 (HRP2) and hepatitis B surface antigen (HBsAg) rapid diagnostic tests (RDTs), respectively, which are, however, reported to give some false positive results. Also, socio-economic determinants have been drawn from these RDTs results which may have questionable implications. Thus, this study was aimed at evaluating the prevalence of malaria and hepatitis B by comparing RDTs with polymerase chain reaction (PCR) outcomes, and relating the PCR prevalence with socio-economic status among pregnant women in Northern Ghana.Plasmodium falciparum and hepatitis B virus (HBV) using HRP2 and HBsAg RDTs, and confirming the infections with PCR. Socio-economic and obstetric information were collected using a pre-tested questionnaire, and associations with the infections were determined using Pearson’s chi-square and multinomial logistic regression analyses at a significance level of p<0.05.We screened 2071 pregnant women on their first antenatal visit for P. falciparum mono-infection, 7.9%/7.5% for HBV mono-infection, and 1.9%/1.7% for P. falciparum/HBV co-infection. No statistical difference in prevalence rates were observed between the RDTs and PCRs . Compared with PCRs, the sensitivity/specificity of the RDTs was 97.5%/99.1% and 97.9%/99.4% for HRP2 and HBsAg respectively. Socio-economic status was observed not to influence HBV mono-infection among the pregnant women . However, pregnant women with formal education were at a lower risk for P. falciparum mono-infection  and P. falciparum/HBV co-infection . Also those with good financial status were also at a lower risk for P. falciparum mono-infection .The prevalence of the infections by RDTs/PCR was: 14.1%/13.4% for Our data has shown that, the RDTs are comparable to PCR and can give a representative picture of the prevalence of malaria and hepatitis B in endemic countries. Also, our results support the facts that improving socio-economic status is paramount in eliminating malaria in endemic settings. However, socio-economic status did not influence the prevalence of HBV mono-infection among pregnant women in Northern Ghana. Malaria and hepatitis B virus (HBV) infections continue to be a major public health threats world-wide and among pregnant women. In 2016, global reports of new malaria-cases and malaria-related deaths were 216 million and 445000, respectively –5. The pThe reported prevalence rates of malaria and hepatitis B in Ghana are based on the use of histidine-rich protein 2 (HRP2) and hepatitis B surface antigen (HBsAg) rapid diagnostic tests (RDTs), respectively, which are thought to have sensitivity and specificity drawbacks. In a hospital based study where PCR was used as a gold standard, the sensitivity/specificity of HRP2 RDTs was 56.4/90.0% . On the Factors including gravidity, young maternal age, use of treated bed nets, and use of antimalarial chemotherapy and prophylaxis have been implicated to influence the prevalence of malaria among pregnant women , 18. On This was a cross-sectional study conducted from October 2016 to February 2017among pregnant women on their first antenatal care (ANC) visit at six different health facilities  in the Northern Region of Ghana .The pregInformed consent was obtained from each study participant after an explanation of the purpose, benefits, and risks of the study was provided. The ethics committee of the Tamale Teaching Hospital approved the study .The sample size was calculated based on the national prevalence of malaria  and thatmalaria)+ Screening size based on hepatitis B prevalence (nHBV)Sample size (n) = Screening size based on malaria prevalence /0.0152] + [(1.962 x 0.131 x 0.869)/0.0152] = 2258n = [, and was assigned as good if they earned ≥GH¢ 8.00/day, or poor if they earned <GH¢ 8.00/day. Obstetric history which included gestational age  and gravidity (primigravidae or multigravidae), and participants’ age were obtained from patients’ antenatal records books.Socio-economic and obstetric information of each study participant was obtained using a structured questionnaire. Training was given to six (6) qualified enumerators on the questionnaire, which was then pre-tested. The Questionnaire enumerators were research assistants at the University for Development Studies, and Health personnel at the Zonal Public Health Laboratory, Tamale Teaching Hospital, Ghana. The interviews were administered face-to-face with each pregnant woman. The socio-economic data collected included marital, educational, economic and financial statuses. Educational status was assessed as having any form of formal education  or not. Economic status was assessed from the occupation of the participants as being involved in economic activity  or not . Financial status was assessed based on the minimum daily wage of GH¢ 8.00 as stipulated by the Government of Ghana in January 2016  were stored under room temperature. A week after the sampling period, the DBS were transported by the investigator (Nsoh Godwin Anabire) to the Infectious Disease Laboratory (IDRL) at the West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana, for molecular detection of plasmodium species and HBV. At the IDRL, the DBS were stored at room temperature for a week prior to the molecular detections of the pathogens by the investigator. The RDT cassette, CareStart HRP-2  was used in diagnosing malaria. Commercially available hepatitis B surface antigen (HbsAg) test strips  were used for serological testing for HBV. For both tests, the testing, results reading and interpretations were done with strict adherence to the manufacturer’s protocol. As a quality assurance procedure, two RDTs from each lot containing 50 strips and 25 cassettes were confirmed by testing with known negative and positive samples before such lots were used in the testing process.The chelex extraction method was used to extract DNA from the DBS. Briefly, DBS containing 75 μL of blood were excised into 1.5 mL eppendorf tubes and soaked overnight with 1 mL of 0.5% saponin in 1X phosphate buffered saline (PBS) solution. The eppendorf tubes with the contents were centrifuged at 14000 rpm for 2 mins, saponin suctioned, and the contents of the eppendorf tubes were washed with 1 mLof 1X PBS and suctioned. The washing step was repeated until no haem or red colour was seen on the filter papers. A volume of 50 μL of 20% chelex and 100 μL nuclease free water were added to the tubes, and incubated in a boiling water bath for 20 mins. The tubes were centrifuged at 14000 rpm for 2 mins and 100 μL of the resulting supernatant, which contained DNA, was pipetted into a 96 well plate and stored at -20°C until used.P. falciparum,P. vivax, P. malariae, and P. ovale. For both rounds of PCR, a total reaction volume of 20 μL was used which contained 10X PCR buffer, 25 mM MgCl2,10 μM primers, 10 μM deoxynucleotides, 1 U Taq DNA polymerase , nuclease free water and DNA sample. Cycling conditions of initial denaturation step of 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 Sec, annealing at 58°C for 1 minute, elongation at 68°C for 1.5 minutes, and final elongation at 68°C for 5 minutes were used in both assays. For the detection of HBV, forward and reverse primers for a previously described PCR assay that amplifies the S-gene of HBV genome were used (27). A total reaction volume of 10 μL consisting of10μMprimers, PerfeCTa syber green SuperMix, Low ROX (Quanta biosciences), nuclease free water and DNA sample, was used. The cycling conditions were: denaturation at 95°C for 15 minutes, followed by 40 cycles that included: denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds and elongation at 68°C for 30 seconds. For both plasmodium species and HBV detections, the PCR products were visualized in 2% agarose gels stained with 1.5 μL of ethidium bromide.Plasmodium species were detected using a nested PCR method previously described , 26. TheRedmond, USA), and proofread to ensure there were no errors. The entered data were then exported into SPSS Version 20  and Graphpad Prism 6  for statistical analyses. All comparisons with p-value < 0.05 (two-tailed) were considered statistically significant. Age, a continuous variable, was normally distributed, and therefore was described as mean with standard deviation, and compared across the four groups of pregnant women by one-way analysis of variance (ANOVA). Categorical variables  were presented as frequency and percentages, and compared by Pearson’s chi-square test. Association between socio-economic factors and infection status  was analyzed by the multinomial regression analysis, with the un-infected as the reference group. Infection status was the dependent variable while age, gestation, gravidity and the SES were the predictor variables.Data were entered into Microsoft Excel 2016  pregnant women responded to the questionnaires (95.9% response rate). The remaining 85 pregnant refused to respond to questionnaire due to personal , religious  or no given reasons .n  = 2071) was 27.8±5.8 years old. Majority were married , while the unmarried were either single  or divorced .With regards to gravidity, 78.1% (n  = 1617) were multigravidae, while 21.9% (n  = 454) were primigravidae. As at the time of antenatal visit, 62.8% (n  = 1300) were in the second trimester while 26.7% (n  = 552) and 10.6% (n  = 219) were in their first and third trimesters, respectively. SES was evaluated from the respondents’ educational, economic and financial statuses. Most respondents  had no formal education, that notwithstanding, 62.1% (n  = 1234) were involved in economic activities. Of those, 44.3% (n  = 547) were farmers, 31.6% (n  = 390) were traders, 10.4% (n  = 128) were hairdresser, 8.7% (107) were seamstress and 5.0% (n  = 62) were teachers. Respondents who were not involved in any economic activities were either house wives  or students . Also, more than half of the respondents  were in good financial standing.The average age of the pregnant women , 7.7% for HBV mono-infection (n = 159), 1.9% for P. falciparum/HBV co-infection (n = 39). However, using PCR, the prevalence rates were: 13.4% for P. falciparum mono-infection (n = 278), 7.5% for HBV mono-infection (n = 155) and 1.7% for P. falciparum/HBV co-infection (n = 36). No statistical difference in prevalence rates were observed between the RDTs and PCRs  of pregnant women , however, the proportions in gestational age were significantly different  and good financial standings  differed significantly across the groups . Lastly, the data was checked to ensure there were no outliers. The multinomial analyses showed that age did not influence infection type among the pregnant women  and P. falciparum/HBV co-infection  of <2.000 were included in the multinomial regression model  was associated with lower risk of  malaria . However malaria –41. TherThe study was not without some limitations. Firstly, the performance of only one brand of HRP2 and HBsAg RDTs was evaluated for reporting the prevalence of the infections, thus, the findings may not be reflective of other commercially available RDTs. Also, only serum samples were tested for the presence of HBsAg, prompting the need to assess the performance of the HBsAg RDTs from whole blood samples. In addition, the study may not account for the impact of HIV-seropositivity on performance of the HBsAg RDTs, however, it is reported that specificity of HBsAg RDTs remained high in studies that compared performance between HIV-positive and HIV-negative persons .Our data has shown that, the RDTs are comparable to PCR and can give a representative picture of the prevalence of malaria and hepatitis B in endemic countries. Also, our results support the facts that improving SES is paramount in eliminating malaria in endemic settings. However, SES did not influence the prevalence of HBV mono-infection among pregnant women in northern Ghana.S1 FileA Cross-Sectional Study .(PDF)Click here for additional data file.S1 FigReceiver operator curve of: (A) HRP2 RDTs compared with PCR for the detection of P. falciparum, and (B) HBsAg RDTs compared with PCR for the detection of HBV.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


To investigate whether children with concurrent wasting and stunting require therapeutic feeding and to better understand whether multiple diagnostic criteria are needed to identify children with a high risk of death and in need of treatment.Community-based cohort study, following 5751 children through time. Each child was visited up to four times at 6-month intervals. Anthropometric measurements were taken at each visit. Survival was monitored using a demographic surveillance system operating in the study villages.Niakhar, a rural area of the Fatick region of central Senegal.Children aged 6–59 months living in thirty villages in the study area.Z-score (WAZ) and mid-upper arm circumference (MUAC) were independently associated with near-term mortality. The lowest WAZ threshold that, in combination with MUAC, detected all deaths associated with severe wasting or concurrent wasting and stunting was WAZ <−2·8. Performance for detecting deaths was best when only WAZ and MUAC were used. Additional criteria did not improve performance. Risk ratios for near-term death in children identified using WAZ and MUAC suggest that children identified by WAZ <−2·8 but with MUAC≥115 mm may require lower-intensity treatment than children identified using MUAC <115 mm.Weight-for-age A combination of MUAC and WAZ detected all near-term deaths associated with severe anthropometric deficits including concurrent wasting and stunting. Therapeutic feeding programmes may achieve higher impact if WAZ and MUAC admission criteria are used. Wasting and stunting are implicated in the deaths of almost 2 million children annually and account for over 15 % of all disability-adjusted life years lost in young children. These figures are likely to underestimate burden because the number of wasted children present in a population  found from unadjusted cross-sectional prevalence estimates will miss many incident cases of an acute condition.Wasting and stunting are common conditions in children. It is estimated that 52 million children are wasted, 16 million of whom are severely wasted, and 155 million children are stunted1. Was(–). Wasting and stunting are often present in the same populations and there is evidence that the two conditions share underlying and immediate causal factors. Investigation into whether there is a direct causal relationship between wasting and stunting is ongoing and a number of gaps in the evidence base have been identified. There has also been recent recognition that children may be both stunted and wasted at the same time(–). National estimates of the prevalence of concurrent wasting and stunting in children, calculated for eighty-four countries, range between 0 and 8 %, exceeding 5 % in nine countries. The factors leading to this state of ‘concurrence’ are poorly understood but the available evidence indicates that considerable excess mortality is experienced by these children. The level of excess mortality is not explained by the severity of their wasting and stunting alone. This suggests a multiplicative effect on mortality of having both deficits. The magnitude of the mortality risk associated with concurrent wasting and stunting is similar to that associated with severe acute malnutrition (SAM). This raises the question of whether these children should be included in therapeutic feeding programmes which historically have treated only children with SAM as currently defined by the WHO and UNICEF using three independent diagnostic criteria:1.mid-upper arm circumference (MUAC) below 115 mm;2.Z-score (WHZ) below −3·0; andweight-for-height 3.the presence of bilateral nutritional oedema.Wasting and stunting in children tend to be addressed as separate issues in programmes, policy and research. Recent reviews have called this separation into question–8. W.The presence of one or more of these criteria is enough to warrant admission into a therapeutic feeding programme20..To investigate whether all children with concurrent wasting and stunting (WaSt) require therapeutic feeding and to better understand whether multiple diagnostic criteria are needed to identify children with a high risk of death and in need of treatment, an analysis examining which anthropometric indices are independently associated with near-term mortality is likely to prove a useful approach. The data for this type of analysis should come from a community-based cohort study assessing the risk of death in a representative sample of untreated children. Studies collecting data for such an analysis are no longer possible. The development of community-based management of acute malnutrition (CMAM) programmes capable of delivering effective SAM treatment at high levels of coverage have made it unethical to leave high-risk children untreated. The aim of the present paper is to describe the results of an analysis examining the ability of different anthropometric case definitions to identify children at high risk of near-term death. The analysis was performed using an existing data set from a historical community-based cohort study that pre-dates the development of CMAM programme models. An earlier analysis of the same data set showed strong relationships between a variety of nutritional indicators and mortality, with the highest population-attributable fractions associated with MUAC and WAZ21.. An open cohort of 5751 children, comprising all children under 5 years of age living in thirty villages in the study area, was followed through time. Each child was visited up to four times at 6-month intervals in May and November of 1983 and 1984. Anthropometric measurements were taken at each visit. All measurements were taken by the research staff after careful training and standardisation. Weight was measured to the nearest 10 g using beam scales . Length and height were measured to the nearest 1 mm using ‘Harpenden’ length/height measurement boards . MUAC was measured to the nearest 1 mm using non-elastic (fibreglass) tapes. Survival was monitored for all children using a demographic surveillance system operating in the study villages that was established in 1962. The ages of children at each visit were calculated using the dates of birth taken from this demographic surveillance system and the dates of measurement by research staff. Approval for the original study was obtained from the International Health Department, the Scientific Commission and the Director General of ORSTOM/IRD (the French Institute for Scientific Research Overseas/Research Institute for Development) in France and from the Ministry of Health, Department of Statistics, Census Bureau and ORANA (Office de Recherches sur l’Alimentation et la Nutrition Africaines) in Senegal. Fieldwork was supported by ORSTOM/IRD, ORANA and the European Union’s DG-XII directorate (grant number TDR-36). Participation in the study was voluntary. Oral informed consent was obtained from principal caregivers. The analysis presented here did not involve new data collection and was done using anonymised records; therefore, it did not require further ethical approval.The analysis presented here is based on a data set collected in 1983 and 1984 in Niakhar, a rural area of the Fatick region of central Senegal. These data have been described in detail elsewhere22. AnZ-scores  were calculated using the WHO growth standards. The outcome of interest was death within 183 d (i.e. 6 months) of anthropometric assessment. The analysis was intended to investigate the association between anthropometry and near-term mortality to inform the selection of case-finding and admission criteria for therapeutic feeding programmes. These programmes treat SAM in children aged 6–59 months with the same standard protocol using ready-to-use therapeutic foods. These children represent the vast majority of children currently treated in such programmes. Therefore, the current analysis used data only for children aged 6–59 months at the time of anthropometric assessment (n 5144). The units of analysis used in the analyses are the individual 6-month follow-up episodes (12 638 episodes with 304 deaths).Height-for-age, weight-for-age and weight-for-height standards25. ThData management and data analysis were performed using purpose-written R language (version 3.5.1) scripts managed using the R Analytic-Flow scientific workflow system (version 3.1.8).t test and the common language effect size (probability of superiority) statistic, which estimates the probability that a value drawn at random from the survived group will be greater than a value drawn at random from the died group. The null (i.e. no difference) value for the common language effect size statistic is 0·5. Multiple logistic regression was used to distinguish between real and apparent (i.e. due to confounding) associations. All variables with significant associations in the bivariate analyses were entered into the model. Non-significant variables were removed using backwards stepwise elimination with the least significant variable (P >0·05) removed at each step.Bivariate associations between different anthropometric variables  and death within 6 months of measurement were examined by estimating mean values for died and survived follow-up episodes using Student’s statistic26, wh1.Severely stunted: HAZ < −3·0.2.Severely underweight: WAZ < −3·0.3.Severely wasted: WHZ < −3·0.4.Severely low MUAC: MUAC < 115 mm.Bivariate associations between anthropometric case definitions and death within 6 months of measurement were examined by calculating risk ratios from two-by-two tables. Standard case definitions for severe undernutrition were used;5.WaSt: WHZ < −2·0 and HAZ < −2·0.An additional anthropometric case definition for being concurrently wasted and stunted (WaSt) was also used;Multiple logistic regression with death within 6 months of measurement as the outcome, as described above, was used to distinguish between real and apparent (i.e. due to confounding) associations.. WHZ < −3·0 and MUAC < 115 mm case definitions were used because these are commonly used as admission criteria for programmes treating SAM. An appropriate threshold for a WAZ case definition was found by a directed search for the lowest WAZ threshold that, in combination with MUAC, detected all deaths associated with the WHZ and WaSt case definitions. A second analysis used MUAC < 125 mm instead of MUAC < 115 mm. This threshold is used in programmes integrating treatment of both severe and moderate acute malnutrition. Sensitivity, specificity and Youden’s index:. The maximum value of Youden’s index occurs for case definitions that optimise differentiating ability when equal weight is given to sensitivity and specificity. Risk ratios for death within 6 months of measurement in the populations selected using different combinations of MUAC and WAZ case definitions identified by the Venn diagram analysis were estimated.The possibility of identifying all, or nearly all, children with severe anthropometric deficits who are likely to die within 6 months of measurement using a combination of anthropometric case definitions was explored using four-dimensional Venn diagrams with sets defined by WAZ, WHZ, MUAC and WaSt case definitions27. WHThe effect of changing case definitions on program caseloads was investigated using simple ‘what-if?’ simulations. The model used was:. Prevalence was modelled using the 25th, 50th and 75th percentiles of prevalence estimates from 2426 nutritional anthropometry surveys from fifty-one countries from 1992 to 2015. Coverage for programmes admitting using MUAC was modelled using the 25th, 50th and 75th percentiles of coverage estimates from 227 coverage assessments of CMAM programmes from twenty-nine countries from 2009 to 2017 recorded in a database provided by the Coverage Monitoring Network. Recent CMAM programme coverage estimates for cases meeting the WHZ < −3 admission criteria were unavailable. Work on coverage undertaken in pilots of the CMAM programme model found coverage of cases with WHZ < −3 to be consistently low (i.e. ≤20 %). It was assumed that coverage for WHZ cases would be one-third that of coverage for MUAC cases. It was assumed that cases with WAZ < −2·8 would be identified by growth monitoring or growth monitoring and promotion (GM/GMP) programmes. A literature review was undertaken and found useable coverage data for twenty-three GM/GMP programmes of differing scope and design from fourteen countries(–). GM/GMP coverage was defined as a population coverage of more than three measurements in the previous 6 months. It was assumed that coverage in lower socio-economic groups  would be 80 % of reported coverage. It was also assumed that the interface between GM/GMP programmes and CMAM programmes would be imperfect, with 80 % of cases identified by the GM/GMP programmes being admitted into CMAM programmes. Coverage of children with WAZ < −2·8 was modelled using the 25th, 50th and 75th percentiles of the resulting estimates.The population term was fixed at 17 000 assuming a service delivery unit  with a total population of 100 000 with 17 % of the total population aged between 6 and 59 months. Prevalence and coverage terms were modelled using fuzzy triangular numbers and fuzzy arithmetic with 95 % CI for ratio estimates calculated for a triangular distribution,31. The optimum WAZ threshold  was WAZ < −2·8. Risk ratios for death within 6 months of measurement in the populations selected using combinations of MUAC and WAZ case definitions identified by the Venn diagram analysis – i.e. MUAC < 115 mm; MUAC < 125 mm and MUAC ≥ 115 mm; WAZ < −2·8 and MUAC ≥ 115 mm; WAZ < −2·8 and MUAC ≥ 125 mm – are presented in .The analysis presented here found that MUAC and WAZ were independently associated with death within 6 months of measurement. A previous multivariate (i.e. logistic regression) analysis of community-based cohort data from Bangladesh also reported that WHZ did not help identify high-risk children when MUAC was already in the model40.A combination of MUAC and WAZ was able to detect all near-term deaths associated with severe anthropometric deficits including children who were simultaneously wasted and stunted (WaSt). This suggests that the MUAC admission criterion in therapeutic feeding programmes should be retained and that consideration should be given to replacing the current WHZ admission criterion with a WAZ admission criterion.. This was based on the understanding that it reflected recent severe weight loss. MUAC was introduced a decade later because it is easy to use in the community and is better at identifying children with a high risk of death than WHZ. Using WHZ requires measurement of length or height. This is time-consuming, requires the presence of at least two health workers and requires length/height boards which are not available in many health facilities. Increasing the MUAC cut-off to 120 mm is likely to be more effective for identifying high-risk children than using the current combination of MUAC and WHZ. If two anthropometric criteria are to be used to define severe undernutrition, it is not clear that WHZ in addition to MUAC is a good choice. Community-based cohort studies have repeatedly shown that WHZ is the least useful anthropometric criterion for identifying high-risk children and that WAZ performs significantly and consistently better at this task(–). Recent analyses show that low WHZ is associated with a high risk of death only when it is also associated with stunting defined by low HAZ. This is concurrent wasting and stunting (WaSt). Recent work on the descriptive epidemiology of multiple anthropometric deficits using data from almost 1·8 million children living in fifty-one countries reported that all children who were concurrently wasted and stunted were also underweight, the maximum possible WAZ for children with WaSt was below −2·35 when the WHO growth standards are used, and cases of WaSt can be detected with excellent sensitivity and specificity using WAZ. This, together with the difficulty of providing good spatial and temporal coverage of the height measurements required for calculating HAZ and WHZ, means that WAZ may be used as a practicable indicator of WaSt. WAZ is easier to measure than WHZ because it does not require height or length to be measured. It does, however, lack the simplicity and low cost of MUAC. Any anthropometric indicator that includes an age component, such as WAZ, requires that age be ascertained accurately. This is unlikely to be problem in GM/GMP programmes. Lack of simplicity, relatively high cost and the need to ascertain age accurately are likely to impair the utility of WAZ for detecting cases in the community. Depending on the thresholds used, MUAC has higher attributable risks than WAZ. This means that WAZ may be of most value when it is already being measured in (e.g.) GM/GMP programmes. Linkages between GM/GMP programmes and therapeutic feeding programmes could be forged and children with MUAC < 115 mm or WAZ < −2·8 referred to therapeutic feeding programmes from GM/GMP programmes  than cases identified by MUAC. A similar approach is used in programmes integrating treatment of both severe and moderate acute malnutrition28. Exty risks  suggeststy risks  suggests(–). An extensive literature review found no risk factor for wasting that was not also associated with stunting. Being wasted is a risk factor for being stunted and vice versa. The analysis presented here avoids the distinction between acute and chronic malnutrition and, following earlier work on admission criteria for CMAM programmes, focuses on near-term mortality risk associated with one or more anthropometric deficits. Programmes admitting using MUAC and WAZ, both of which are associated with wasting and stunting, would treat children with anthropometric deficits associated with near-term mortality rather than just ‘acute malnutrition’.Current programme designs tend to focus on either acute malnutrition (wasting) or chronic malnutrition (stunting). The distinction between acute and chronic malnutrition appears arbitrary–11. 1.Repeating the current analysis using data from community-based cohort studies of untreated children from different contexts. This could further inform the selection of a usefully sensitive and specific  case defining WAZ threshold.2.Ascertaining the intensity and duration of treatment required to treat children identified using WAZ as well as appropriate discharge criteria.3.Operational research on linking GM/GMP and therapeutic feeding programmes.Further work is required before the findings of the work reported here can be applied. This includes:The last two issues may be best addressed by small-scale  field studies. Such studies would also allow additional key programme planning data such as cost-effectiveness  and caseloads to be collected.. The caseload simulations are crude, and results should be treated as broadly indicative of the magnitude and direction of the effect of changing case definitions on programme caseloads.The analysis reported here uses data from a single study in a single location. Repeating the current analysis using data from community-based cohort studies of untreated children from different contexts would provide more compelling evidence. Nine additional cohort studies of similar design to the Niakhar study from Bangladesh, Ghana, Guinea Bissau, India, Indonesia, Nepal, Peru, Philippines and Sudan with archived data have been identified and efforts are underway to secure access to these dataTherapeutic feeding programmes concerned with lowering the risk of near-term mortality may achieve higher impact if a combination of MUAC and WAZ admission criteria are used. More work is required before this can be considered as a general recommendation.


P. falciparum infection in United Kingdom adult Phase IIa sporozoite challenge studies (approximately 20–25% sterile protection with similar numbers showing clear delay in time to patency), and greater point efficacy in a trial in Kenyan adults.Heterologous prime boost immunization with chimpanzee adenovirus 63 (ChAd63) and Modified Vaccinia Virus Ankara (MVA) vectored vaccines is a strategy previously shown to provide substantial protective efficacy against We conducted the first Phase IIb clinical trial assessing the safety, immunogenicity and efficacy of ChAd63 MVA ME-TRAP in 700 healthy malaria exposed children aged 5–17 months in a highly endemic malaria transmission area of Burkina Faso.6 PBMC (95% CI 290–387)) many fold lower than in previous trials. No significant efficacy was observed against clinical malaria during the follow up period, with efficacy against the primary endpoint estimate by proportional analysis being 13.8% (95%CI -42.4 to 47.9) at sixth month post MVA ME-TRAP and 3.1%  by Cox regression.ChAd63 MVA ME-TRAP was shown to be safe and immunogenic but induced only moderate T cell responses (median 326 SFU/10This study has confirmed ChAd63 MVA ME-TRAP is a safe and immunogenic vaccine regimen in children and infants with prior exposure to malaria. But no significant protective efficacy was observed in this very highly malaria-endemic setting.NCT01635647.ClinicalTrials.gov Pactr.org PACTR201208000404131. Malaria is the preeminent tropical infectious disease globally, with a devastating effect on human health and society. In 2016, an estimated 216 million cases of malaria occurred worldwide (95% CI 196–263 millions) with an estimated 445 000 deaths [The most advanced malaria vaccine, which received a positive opinion under article 58 from the European Medicine Agency (EMA), RTS,S/AS01, targets antibodies against the circumsporozoite protein (CS), which is expressed by the sporozoite at the pre-erythrocytic stage. A Phase III trial with RTS,S/AS01E showed modest efficacy of 28·3% (95% CI 23·3–32·9%) against clinical malaria in children 5–17 months old who received three doses and of 36·3% (31·8–40·5%) in those given a fourth dose, during 48 months of follow-up. A much lower efficacy was observed in young infants [P. falciparum infection in UK adult Phase IIa sporozoite challenge studies, and 67% efficacy against PCR-determined malaria infection in a trial in Kenyan adults [+ T cell correlate identified in UK adult vaccines [The only other subunit malaria vaccination approach that has demonstrated repeatable partial efficacy in humans involves the use of virally vectored vaccines containing a recombinant genetic insert, encoding the antigen against which the immune response is directed –10. The n adults . T cell vaccines . In a sivaccines  lower T vaccines –14.Based on these promising findings, we undertook a safety, immunogenicity and efficacy trial of the two dose ChAd63 MVA /ME-TRAP malaria vaccination regime in malaria exposed older infants and young children aged between 5 and 17 months in an area of hyperendemic, seasonal and stable malaria transmission in South-Western Burkina Faso.P. falciparum is responsible for more than 90% of all clinical malaria cases [Anopheles gambiae, An. arabiensis and An. funestus. Entomological inoculation rates (EIR) vary from 55 to 400 infected bites/person/year (our unpublished data).The study was conducted in the Banfora trial site, which is located about 400 km from Ouagadougou, the capital city of Burkina Faso. The “Unité de Recherche Clinique de Banfora” (URC-B) research unit is located within the complex of the regional hospital. From recent surveys, the bed net coverage was 80%. There was no implementation of indoor residual spraying or intermittent preventive treatment (IPT) in infants or children in the area. To date in this area there is no evidence of decline in malaria incidence that has been recently reported from other parts of sub-Saharan Africa. In the study area, malaria is very highly endemic. Transmission occurs throughout the year, with a peak during the rainy season (from June to October). ia cases ,16. The The trial enrolled infants and children aged from 5 to 17 months at the time of first vaccination and whose parents or legally accepted guardians / representatives were permanently resident in the study area. They were drawn from six community clinic’s catchment areas of Banfora health district. There was no selection on the basis of pre-existing neutralizing antibodies (NAb) against ChAd63 vector prior to enrolment: antibody levels to this vector are low in Burkina Faso, as described elsewhere [P. falciparum strain T9/96 [P. falciparum pre-erythrocytic antigens fused to the TRAP protein. These epitopes are from six P. falciparum target antigens and are included with the aim to broaden the immune responses in vaccinated volunteers.The recombinant vectors and their generation have been described previously . The antin T9/96 . “ME” is10 vp. The MVA ME-TRAP was manufactured by IDT Biologika GmbH (IDT), Germany. MVA ME-TRAP was supplied as a sterile 0.55ml liquid in 2.0 mL transparent glass injection vials. The dose of MVA ME-TRAP used was 1 × 108 pfu.The ChAd63 ME-TRAP and MVA ME-TRAP were both manufactured under Good Manufacturing Practice conditions. The ChAd63 ME-TRAP was manufactured by the Clinical Biomanufacturing Facility (CBF), Churchill Hospital, Oxford. ChAd63 ME-TRAP is supplied as a sterile 0.5–1.0 ml liquid in 2.0ml glass vials. The dose of ChAd63 ME-TRAP used in this study was 5 x 10The Imovax Rabies Vaccine produced by Sanofi Pasteur SA is a sterile, stable, freeze-dried suspension of rabies virus prepared from strain PM-1503-3M obtained from the Wistar Institute, Philadelphia, PA. The vaccine is supplied as a single dose vial to be administered intramuscularly. The rabies vaccine was chosen as the most potentially beneficial active control vaccine for the study participants.This study was a Phase IIb, double blind, randomized controlled trial. The primary objective was to assess the protective efficacy against clinical malaria of ChAd63 ME-TRAP / MVA ME-TRAP prime-boost immunization, in 5–17 months old infants and children living in a malaria-endemic area, for 6 months after the last vaccination.The trial, participants were randomized 1:1 to receive the candidate malaria vaccine (ChAd63 ME-TRAP and MVA ME-TRAP) or control vaccination with two doses of a rabies vaccine (Imovax Sanofi Pasteur).10vp and 1 x 108pfu, respectively, both to the anterolateral thigh.The prime-boost regimen was given with an eight-week interval between doses. ChAd63 ME-TRAP was followed eight weeks later by MVA ME-TRAP. Both vaccinations were by intramuscular route at doses of 5 x 10ChAd63 ME-TRAP and MVA ME-TRAP are non-replicating genetically modified organisms. Although previous work has failed to identify any evidence of dissemination of these replication-incompetent vectors, to minimize any possibility of dissemination of the vectors into the environment, the inoculation site for all vaccinations (including the rabies vaccine to maintain the blinding) were covered with a dressing after immunization to absorb any vector that may leak out through the needle track. The dressing was removed from the injection site after 30 minutes. Each volunteer was monitored for one hour (or longer if necessary) after each vaccination.Monitoring of solicited adverse events was performed for seven days after each vaccination. Unsolicited adverse events were recorded until one month post each vaccination. Serious adverse events and malaria episodes were monitored throughout the study duration.Trained field workers  under the supervision of the study clinicians visited daily each enrolled child from days 1 to 3 after each vaccination. If necessary, the child continued to be seen by the field worker on subsequent days for follow-up of any adverse event. If any severe (grade 3) event was observed, the volunteer was brought to the vaccination center for examination by a study physician.P. falciparum parasite density. The study participants with clinical conditions requiring hospitalization were referred to the Pediatric ward of the regional hospital of Banfora for admission and further clinical and laboratory investigations. The clinical management was performed in accordance with local standard of care and national guidelines.When a study participant was unwell at any time during the study, the parents were advised to report to the trial site clinic or the nearest health facility for follow up where the study medical staff were available 24 hours a day to identify study participants through the personal identification card and to ensure standardized documentation and appropriate medical management. A duplicate blood film was obtained if the volunteer had symptoms or signs compatible with malaria . A malaria rapid diagnosis test was performed for prompt management of the child while the duplicate blood film was read later to quantify the P. falciparum TRAP. Fresh PBMC were used in all ELISPOT assays using a previously described protocol [The kinetics and magnitude of the T cell response to ME-TRAP were assessed over time by ex-vivo IFNγ ELISPOT assays performed on blood samples taken at days 0, 63, and 243. Ex-vivo IFNγ ELISPOT assays were performed with an 18–20 hour stimulation of peripheral blood mononuclear cells (PBMC) with peptides pools containing up to 10 peptides per pool, including peptides representing the T9/96 and 3D7 strains of P falciparum asexual stages, Giemsa-stained blood slides were read following standard quality-controlled procedures.To establish parasite presence and density of P. falciparum, P. malariae, or P. ovale), and counts for P. falciparum were made for both sexual and asexual parasites. The parasite presence and density were determined independently by two readers for the same slide; if readings were judged to be discordant, a third independent read was organized. The parasite density (parasites/μL) was calculated as the geometric mean of the two positive readings .The presence of parasites on capillary blood samples was assessed by 100 x bright field microscopic examination, assuming 8000 leukocytes/μl of blood. The count was made by species  was 88%.P. falciparum malaria was calculated by the Kaplan-Meier estimate to determine the proportions free by vaccination group (i.e. proportion among vaccinees (Pv) and proportion among controls (Pc)). Vaccine efficacy was determined as 100 x (1- Pv/Pc)%. Confidence intervals were calculated on a log scale.The proportion of children remaining free of any episode of P. falciparum malaria, the incidence of episodes (episodes/person years at risk) for each group were calculated. Kaplan-Meier curves for both groups are shown. The distribution of the survival time was compared with the Wilcoxon test (if efficacy appears to vary with time) or the Log-rank test (if it does not). Vaccine efficacy was assessed using Cox regression models for the first episode. Vaccine efficacy is defined as 1- R where R is the hazard ratio of the malaria vaccine group versus the control group (with 95% CI).For analysis of first or only episodes of The Kaplan-Meier plots were examined for evidence contradicting the assumption of proportionality of hazards. A test based on the Schoenfeld residuals was performed for proportionality of the hazard.Secondary analyses have examined multiple episodes, using the robust clustering method by individual, with examination of proportionality of hazards and calculation of efficacy as described above.Subgroup analysis was conducted by age strata (i.e. 5–12 months and 12–17 months of age). Variation in efficacy by age was tested by an interaction between age and vaccine efficacy, using these categories and using age as a continuous variable.Variation in efficacy according to immunogenicity was assessed, using ELISPOT outcomes, with log transformation to ensure normal distributions, and analyzing only the vaccinees to test for variation in risk using ELISPOT data as a continuous explanatory variable.Analyses were done with Stata version STATA software .The clinical trial protocol and associated documents were reviewed and approved by CNRFP institutional bioethics committee , the Ministry of Health Ethical Committee for Biomedical Research  and Oxford Tropical Research Ethics Committee (OXTREC) . Regulatory approval was given in Burkina Faso by the National Regulatory authority . All study participants’ parents or legally acceptable representatives gave documented informed consent before any study procedures were performed. The trial was conducted according to the principles of the declaration of Helsinki and International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. An independent data safety monitoring board (DSMB) and local safety monitors provided safety oversight and GCP compliance was independently monitored by an external organization .th March 2013 and 14 June 2013. 884 children were screened to enroll 700, from whom 351 received ME-TRAP vaccines and 349 received rabies vaccine. The mean age of study participants was 10.6 months (range 5 to 17.9 months). 334 children received 2 doses of ME-TRAP vaccine and have follow up data, 336 received 2 doses of rabies vaccine and have follow up data and are included in the according to protocol (ATP) analysis. 42 children did not complete the follow up among them 20 consent withdrawals (none related to adverse event), 10 migrations/lost to follow up, and 12 for other reasons such as absence at the time of end of study visit and deaths (4 participants). The study profile is summarized in Recruitment took place between 18The local and systemic reactogenicity profiles are summarized in . Except Solicited systemic symptoms were loss of appetite, irritability, drowsiness and fever (both measured and reported) . ME-TRAPOn final analysis the most common Serious Adverse Events (SAEs) were pneumonia (12 cases in ME-TRAP vaccinees and 10 in rabies vaccine group), non-severe malaria (8 episodes in ME-TRAP group against 9 in rabies vaccine group), gastroenteritis (4 in ME-TRAP group and 3 in rabies vaccine group) and malnutrition (6 in ME-TRAP group and 2 in rabies vaccine group). In total 2 cases of sepsis of unknown causes were recorded during the trial; all in the rabies vaccine group.There were 4 deaths recorded, one in the ME-TRAP vaccine recipients and three in the rabies vaccine recipients. The death in the ME-TRAP vaccinee was a case of sudden death that occurred 80 days after the MVA ME-TRAP vaccination at home while the male study participant was under self-medication for illness with traditional remedies. The three deaths in the rabies recipients were cases of severe malaria, sepsis in a context of acute moderate malnutrition, and cranial trauma. None of the deaths were temporally associated with the study vaccination. All the other serious adverse events recorded were not unexpected events given the study population and were not closely related to vaccination in timing. None were judged likely to be linked to vaccination by the investigators and the DSMB.Hematological values outside the normal range were infrequent. There was no statistically significant difference in the median values for any parameter between the two groups post immunization.Biochemistry values outside the normal range were also infrequent. One-month post MVA ME-TRAP vaccination, the median values for creatinine were statistically higher in the malaria vaccine recipients than in control vaccine recipients . The same trends towards higher values were observed 6 months later for creatinine  and bilirubin . In total 21 comparisons of biochemical and hematological parameters were made .ex vivo T cell responses rose from a median of 16 SFC /106 PBMC before vaccination (95% CI 14–18) to 326 SFC/106 PBMC (95% CI 290–387) after the MVA vaccination Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Plasmodium falciparum infection.Malaria burden remains high in the sub-Saharan region where helminths are prevalent and where children are often infected with both types of parasites. Although the effect of helminths on malaria infection is evident, the impact of these co-infections is not clearly elucidated yet and the scarce findings are conflicting. In this study, we investigated the effect of schistosomiasis, considering soil-transmitted helminths (STH), on prevalence and incidence of S. haematobium eggs and, STH eggs, respectively. P. falciparum carriage was assessed at inclusion, and incidence of malaria and time to the first malaria event were recorded in correlation with Schistosoma carriage status. Stratified multivariate analysis using generalized linear model was used to assess the risk of plasmodium infection considering interaction with STH, and survival analysis to assess time to malaria.This longitudinal survey was conducted in school-age children living in two rural communities in the vicinity of Lambaréné, Gabon. Thick blood smear light microscopy, urine filtration and the Kato-Katz technique were performed to detect malaria parasites, S. haematobium, P. falciparum infections and co-infection with both parasites, respectively. Our results showed that schistosomiasis in children tends to increase the risk of plasmodium infection but a combined effect with Trichuris trichiura or hookworm infection clearly increase the risk (aOR = 3.9 [95%CI: 1.7–9.2]). The incidence of malaria over time was 0.51[95%CI: 0.45–0.57] per person-year and was higher in the Schistosoma-infected group compared to the non-infected group , with a significant delay of time-to first-malaria event only in children aged from 6 to 10-years-old infected with Schistosoma haematobium.The overall prevalence on subject enrolment was 30%, 23% and 9% for P. falciparum infection in schistosomiasis-positive children, and when infected, that schistosomiasis enhances susceptibility to developing malaria in young children but not in older children.Our results suggest that STH enhance the risk for  Trichuris trichiura or hookworm associated with schistosomiasis increase the risk of Plasmodium falciparum infection; whilst schistosomiasis is independently associated with a malaria increase in young children, but not in the older children. Our finding is an additional evidence that optimizing helminth control mainly in children contributes to overcoming malaria in areas endemic for both parasitic infections where children are those who bear the highest burden of these infections.Despite the progress made in the last decade, malaria remains a serious public health issue in sub-Saharan region, where it overlaps with helminths infections. The interactions between both are manifold and complex, requiring further investigation. We report here that  Trichuris trichiura infection was associated with increased malaria prevalence, while an increased helminth burden was associated with increased Plasmodium falciparum or Plasmodium vivax parasitemia  vs 18% [95%CI: 14%-22%], respectively, for P. falciparum and 45% [95%CI: 40%-51%] vs 19% [95%CI: 15%-23%], respectively, for S. haematobium. The prevalence of both infections was similar for age and sex groups.The prevalence of P. falciparum parasite carrier status. Indeed, 168 (78%) of SI children came from Zilé-PK villages while 253(69%) of SU children came from Bindo and Makouké villages (p<0.001). Additionally, prevalence of P. falciparum parasite carriage was significantly higher in the SI group compared to the SU group .Among the participants followed-up for malaria incidence assessment, 216 (37%) were found positive for Schistosoma infection during the study period, including 176 cases on inclusion and additionally 40 new cases during follow-up and assigned to SI group. The 368 (63%) others participants who remained negative during the whole study course were thus assigned to the SU group . As showSchistosoma infection (p = 0.002) and location (p<0.001) were associated with P. falciparum infection. Children infected with S. haematobium had a 1.8 [95%CI: 1.2–2.5] times odds of being co-infected with P. falciparum parasites compared to their non-infected counterpart. After adjustment  while a trend of association with schistosomiasis infection was found (p = 0.06).At crude analysis as given in justment , P. falcTrichuris or hookworm infections on P. falciparum and S. haematobium infections association. As presented in T. trichiura and hookworm infections, there is no effect of S. haematobium on P. falciparum parasite carriage; while among those infected with either hookworm or T. trichiura or a combination, children co-infected with S. haematobium had a 3.1  time odds of being infected with P. falciparum. This finding remained statistical significant when adjusted for age, sex, ascariasis and location . Age, sex and Ascaris infection were forced in the final model of the GLM analysis.In the following analysis, we found effect modification of P. falciparum malaria incidence assessment, 210 (36%) participants had developed a total of 318 new cases of malaria . Assessing the delay until development of malaria according to schistosomiasis status, we found that SI group participants had a 1.6 (Cox model: p = 0.0004) times increased risk of early development of malaria as compared to their counterparts of the SU group. This association remains significant when adjusted for location, age and sex . Stratifying for age yielded a significant delay in time-to-first-malaria episode in the SU group (median time not reach) compared to the SI group, where the median time was 51 weeks (p = 0.00003) for 6–10 year old children. Children with schistosomiasis had a 2.1 (Mantel-Cox test: p = 0.00004) times increased risk of developing malaria compared to children without schistosomiasis. On the contrary, there was no difference in terms of delay in time-to-first malaria episode between the two study groups (p = 0.41) in children aged 11–16 years.The time-to-first malaria episode was assessed for the first twelve months of follow-up of each participant and those who did not develop malaria before the end of that time were censored. Our results show that in the SI group, among the 101 (46%) participants developed malaria, the median time to first malaria episode was 52 weeks. For their counterparts of SU group where 109 (30%) participants developed malaria, the median time to first malaria episode was not reached at the end of 52 weeks of follow-up. As presented in P. falciparum and S. haematobium parasites, comparing to up to 15% of pregnant women co-infected in Cameroon [S. mansoni in a co-endemic area of North-Western Tanzania [S. haematobium. This finding is not surprising since STH is commonly reported to be prevalent in rural areas [S. haematobium is high [P. falciparum association.In area where schistosomiasis is endemic, the question of its effect on malaria outcome is a growing concern. Our study area is endemic for both infections , and ourCameroon  or 23% oTanzania . In our Tanzania ,13,14,24al areas . Therefo is high . Since tP. falciparum carriage and Schistosoma infection. In univariate analysis, Adedoja et al. found that children infected with S. heamatobium have equal chances of being infected with P. falciparum as children with no worm infection [Trichuris and Hookworm infections on the association between S. haematobium and P. falciparum. As well, location was identified as confounding factor. Some authors have previously shown that T. trichiura [P. falciparum parasites. Our analysis was stratified for those two STH infections and adjusted for age and sex which could affect malaria infection [P. falciparum. But, in combination with trichuriasis or hookworm infection, schistosomiasis clearly increases the risk of being infected with P. falciparum. This result shows that S. haematobium alone does not predispose to P. falciparum infection in children instead of combined effect. We hypothesize that the cumulative effects of Schistosoma, Trichuris and Hookworm infections on P. falciparum parasite carriage acts at the immunological level. A potential immuno-modulation effect of a poly-parasitism not measured in our study could explain the combined effect of helminths we have observed. Indeed, there is evidence that Schistosoma infection can modulate the immune system in response to P. falciparum [T. trichiura can exert an influence on the immune response, and for instance negatively affect the antibody response to malaria vaccine candidate in children [Everything else equal, we found a trend of association between risk of nfection , while Anfection . Conflicrichiura  and hookrichiura ,46 indivnfection , and forlciparum ,11. Therchildren . HoweverS. haematobium compared to the uninfected, suggesting that schistosomiasis infection increases the risk of developing malaria. Children infected with S. haematobium infection had 1.4 times the risk of developing malaria than uninfected.The overall incidence of malaria was 0.51 per person-year. This incidence was higher among people infected with S. haematobium developed malaria earlier than those without S. haematobium infection. This finding could support the possible effect of age on the immune responses of malaria in co-infected subjects. However, since the finding is based on statistical significance, biological assessment is suitable for confirmation.Regarding time-to-first malaria infection, we found that malaria occurred earlier in participants infected with Schistosoma than those uninfected even after adjustment for age, sex and location. The main symptom we have considered to define malaria was fever, which is one of the results of some endogenous pyrogen molecules activities, notably pro-inflammatory cytokine TNF-α during the infection. Some authors reported that during malaria infection, the production of pro-inflammatory cytokines as well as of anti-inflammatory cytokines can be affected by co-infection with schistosomiasis infection in an age-dependent manner ,48. In oS. mansoni eggs loads [P. falciparum infection or co-infection with S. haematobium in a Malian study cohort [S. haematobium-infected on malaria instead of becoming infected at time of malaria, which could affect our conclusion compared to the studies mentioned above. On the other hand, it has been shown that STH can affect susceptibility to malaria infection by acting at the immunological level [We can retain that exposure to schistosomiasis enhances incidence of, and susceptibility to develop malaria in our study population. This finding corroborates with previous reports like the one by Sokhna and collaborators who reported an increased in susceptibility to developing malaria in co-infected children, even though it was only in children excreting high gs loads ; supporty cohort . In the al level ,50. Not P. falciparum infection based on prevalence of P. falciparum parasite carriage and malaria incidence. We therefore grouped our study population in relation to the schistosomiasis status. If it was easy at baseline to discriminate children infected or non-infected with S. haematobium, the problem we faced during the follow-up phase was to appropriately group our population in accordance with schistosomiasis status. Subjects infected at baseline or during the follow-up phase were treated systematically. However, they were considered as Schistosoma-infected for the whole follow-up phase and those who were not found positive throughout the survey were considered as non-infected. This approach was sustained by the fact that schistosomiasis is known as a chronic infection and, in areas where schistosomiasis is prevalent, the risk factors as playing habits, swimming, taking baths, washing clothes, distance from river are usually constant [P. falciparum parasite carriage at baseline and on malaria infection during the follow-up study phase.We have assessed the effect of schistosomiasis on clinical and parasitological aspects of constant ,52 and tconstant . Thus, wP. falciparum parasites [P. falciparum parasites as it is the clinical gold standard. However, this may lead to potential misclassification of the participants regarding P. falciparum status at baseline. We think that if prevalence of P. falciparum carriage could be underestimated, this potential misclassification of participants would have been equally distributed in both groups and would not therefore affect the trend of our results.An earlier conducted study in the same population showed that PCR has a better sensitivity than microscopy for the detection of arasites . We haveS. haematobium prevalence is high, a high prevalence of P. falciparum carriage was also found. Indeed, the difference in prevalence observed in favour of the PK area for S. haematobium infection was also observed for P. falciparum. This observation suggest a correlation of factors affecting both infections as either a consequence of the presence of same environmental risk factors. Another explanation could be indeed the effect of S. haematobium infection on P. falciparum infection, as demonstrated above. However, this need to be more investigated.This study confirms that the transmission of schistosomiasis is not evenly distributed in the vicinity of Lambaréné. Schistosomiasis infection is present in many villages but the prevalence varies significantly from one point to another. For example, we found a moderate prevalence for Bindo and Makouké villages where 19% of our study participants were found to be positive when compared to Zilé-PK villages, where 45% of our study participants were found to be positive. This corroborates with a previous pilot study conducted in the same population in 2012. The earlier-indicated prevalences of 15% and 43% for Bindo and Zilé-PK villages, respectively , suggestS. haematobium infection alone does not increase the risk of being infected with P. falciparum parasite but when associated with STH particularly with T. trichiura and hookworm, the risk does increase. On the other hand, in people exposed to schistosomiasis infection, risk and susceptibility of developing a malaria event increase in an age-dependent manner. Our results suggest that Schistosoma and probably STH co-infections in general cumulatively impact on malaria outcome in school-age children and therefore need to be accounted for when designing malaria control programs. Thus, in areas of co-endemicity and in support of higher efficiency, STH and schistosomiasis control should be considered as an additional tool of malaria control.In summary, this study demonstrates that S1 Checklist(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Plasmodium falciparum strains that infect people causing diverse malaria symptoms. This study was carried out to explore the effect of mixed-strain infections and the extent to which some specific P. falciparum variants are associated with particular malaria symptoms. P. falciparum isolates collected during pharmacovigilance study in Nanoro, Burkina Faso were used to determine allelic variation in two polymorphic antigens of the merozoite surface (msp1 and msp2). Overall, parasite density did not increase with additional strains, suggesting the existence of within-host competition. Parasite density was influenced by msp1 allelic families with highest parasitaemia observed in MAD20 allelic family. However, when in mixed infections with allelic family K1, MAD20 could not grow to the same levels as it would alone, suggesting competitive suppression in these mixed infections. Host age was associated with parasite density. Overall, older patients exhibited lower parasite densities than younger patients, but this effect varied with the genetic composition of the isolates for the msp1 gene. There was no effect of msp1 and msp2 allelic family variation on body temperature. Haemoglobin level was influenced by msp2 family with patients harboring the FC27 allele showing lower haemoglobin level than mono-infected individuals by the 3D7 allele. This study provides evidence that P. falciparum genetic diversity influenced the severity of particular malaria symptoms and supports the existence of within-host competition in genetically diverse P. falciparum.There is a large genetic diversity of  Among Plasmodium species infecting humans, Plasmodium falciparum is the most prevalent and the most dangerous, responsible for over 90% of deaths due to malaria2. This species is also the most affected by resistance to antimalarial drugs, which constitutes a major challenge in the fight against malaria. Moreover, there is great genetic polymorphism within P. falciparum species and people living in endemic area are frequently and simultaneously infected by several Plasmodium strains3. This genetic diversity of the parasite is one of the main factors responsible for the slow acquisition  of immunity against malaria. Thus, individuals must meet the entire diversity of circulating parasite populations before they develop an effective anti- malarial immunity3. This genetic polymorphism of the parasite constitutes also a challenge for the development of an effective anti- malarial vaccine4. At the same time, there is a large variability of the clinical manifestations of P. falciparum malaria5. There are no specific symptoms for P. falciparum malaria infection which limits the accuracy of clinical diagnosis. Clinical diagnosis constitutes to date a common practice in poor resource settings due to frequent stock out of rapid diagnostic tests (RDTs)5. However, it is well established that the risk for an individual to fall clinically ill with malaria results from a complex interaction between the parasite, environment and human host7. Considering the factors related to the parasite, the existence of variation in strain virulence has been suggested by several authors9. In animal models, strains of malaria parasite have shown significant differences in virulence and pathogenicity11. However the use of these experimental infection methods to directly estimate the level of strain virulence has always been limited in human malaria for obvious ethical reasons. Nevertheless, in P. falciparum some level of virulence can be assessed indirectly by correlating clinical manifestations of the disease with the biological characteristics of the circulating parasites15. Accordingly, associations between some P. falciparum msp-variants and the occurrence of clinical malaria, severe anemia and cerebral malaria were previously reported20.Malaria is the commonest parasitic disease worldwide leading to about 445000 deaths annually22. When different clones share the same environment (the host and its immune response) and use the same resource (the host red blood cells), the intrinsic growth rate of each clone can be limited by the presence of other clones; that is, within-host competition occurs21. Most evidence for the existence of such within-host competition comes from controlled coinfections experiments in the mouse malaria model, P. chabaudi31. In human malaria, several correlational epidemiological studies support the existence of within-host competition, such that densities of individual parasite genotypes are suppressed when other genotype are present33. Therefore, within-host competition could affect host health through effects on total parasite densities or through the modification of the densities of more or less virulent clones21. Accordingly, previous studies found that within-host genetic diversity was associated with disease severity . In addition, the rising clone multiplicities of Plasmodium falciparum infections increased the likelihood of clinical malaria episodes as well as the severity of the disease39.Besides associations between specific allelic variant of the parasite and particular disease symptoms, the presence of multiple competing clones within a host can also affect disease severitymsp1 and msp2) seem to be the most appropriate to distinguish parasite populations42. This study was carried out using these polymorphic markers to explore: (i) whether some specific family strains of P. falciparum were mostly associated with the occurrence of particular malaria symptoms such as high body temperature, severe anemia, and high parasite densities; (ii) whether within-host competition occurred ; and (iii) whether mixed infections were associated to lower or higher disease severity.Although many polymorphic antigens have been described in several stages of the parasite life cycle, merozoite surface protein 1 and 2  located in the central part of Burkina Faso (West Africa). Data collection was performed at two peripheral health facilities (Nanoro and Nazoanga). The area is characterized by an alternation of two distinct seasons: a rainy season occurring from June/July to October/November in which malaria transmission is highest, followed by a long dry season from November to May. P. falciparum isolates were obtained from patients enrolled in a larger pharmacovigilance trial aimed at assessing the effectiveness of Artesunate-Amodiaquine versus Artemether-Lumefantrine in Nanoro, Burkina Faso. Details of methodology and results of this clinical trial are published elsewhere45. The present study reports on screened patients including: (i) all those who were included for assessment of the effectiveness of the two drugs  and (ii) those classified as severe malaria (n = 52) and were excluded during the effectiveness trial i.e. those with hyper-parasitaemia [Parasite density (DP) ≥ 200000 asexual forms/µL] and severe anaemia [Haemoglobin level (Hb) ≤ 5 g/dl]. Patients were physically examined before enrolment. History of the disease and different symptoms were reported on the case record form (CRF). Blood samples were taken for microscopic examination, haemoglobin (Hb) measurement, and spotted onto Whatman filter papers which were air dried and stored in plastic bags with silica gel for later PCR analysis. Two markers of P. falciparum genetic polymorphism were used to differentiate recrudescence and new infection: merozoite surface proteins 1 and 2 (msp1 and msp2). The present report used data and samples collected on Day 0 (before enrolment) to seek correlation between the presence of msp1 and msp2 allelic families and parasite virulence. Three surrogates of parasite virulence and pathogenicity were used: (i) hyperthermia (high body temperature), (ii) anemia (low haemoglobin level) - two host traits corresponding to objectively measurable malaria signs - and (iii) parasitaemia (parasite density), an important parasite trait.Axillary temperature was determined before enrolment using an electronic clinical thermometer. The tip of the thermometer was inserted under the armpit for both children and adult and numeric value of axillary temperature was recorded.+ was used for photometric measurement of Hb in the field. Undiluted blood was drawn into the cavity of the microcuvette which was inserted into the analyzer and Hb concentration was obtained in g/dL immediately.Screening for anemia was performed by the measurement of Hb level. A HemoCue® 301Parasite density was determined by counting the number of asexual parasites per 200 white blood cells, and calculated per micro liter of blood by assuming the white blood cells at 8,000 per µL. A smear was declared negative when the examination of 100 thick-film fields did not reveal the presence of asexual parasites.P. falciparum DNA was extracted from dried blood spots using the Qiagen DNA extraction Kit  method and 80 µL of DNA template was obtained.Molecular analysis was performed at the laboratory of Centre Muraz in Bobo-Dioulasso, Burkina Faso. msp1 and msp2 allelic families were genotyped in a nested PCR approach as previously described46. Briefly, an initial amplification of the outer regions of the block 2 of msp1 and block 3 of msp2 was performed. The second round used the first PCR product as DNA template with the specific primers of each of the allelic family of the two genes. After DNA amplification, 5 µL of PCR product were used for visualization of DNA bands in ethidium bromide-stained 2.5% agarose gels for 2 hours at 100 V. Culture strains spotted on filter paper were used as positive controls: 3D7 for msp1-K1 and msp2–3D7, D10 for msp1-MAD20 and msp2-FC27 and RO33 for msp1-RO33 allelic family. No DNA template obtained from extraction of a sterile filter paper served as negative control.MOI) was defined as a mean number of parasite genotypes by clinical isolate. Within-host competition was assessed by considering density as a valuable measure of parasite interaction within an infected human host.A sample was considered as belonging to a given allelic family if there was an occurrence of at least one band after DNA amplification using the family specific primers. The prevalence of each allelic family was estimated by calculating the percentage of fragments assigned to this family among the overall number of fragments detected for that locus. Multiplicity of infection  was used to investigate the effect of msp1  and msp2  allelic families, host age, and first order interactions on parasite density. For msp1 allelic family, an additional GLM with a negative binomial distribution was used to explore the effect of multiple infections  on parasite density. Separate GLMs with Gaussian distributions were used to investigate the effects of msp1 (7 levels) and msp2 (3 levels) allelic families, host age, parasite density and first order interactions on axillary temperature and Hb level, respectively (models were checked for homogeneity of variance by using Fligner-Killeen tests)48. For msp1 allelic family, additional gaussian GLMs were used to explore the effect of multiple infections (2 levels: mono versus mixed infection) on axillary temperature and Hb level, respectively. Model simplification used stepwise removal of terms, followed by likelihood ratio tests (LRT). Term removals that significantly reduced explanatory power (p < 0.05) were retained in the minimal adequate model48.All statistical analyses were performed in R (version 3.0.1)This is part of a large study entitled pharmacovigilance for artemisinine based combination treatments in Africa. The ethical approval for the study was provided by the institutional Ethics committee of Centre Muraz, Bobo-Dioulasso, Burkina Faso; the National Ethic committee of Burkina Faso and the WHO-TDR Ethics Review Committee. The study was conducted in accordance with laws and regulations in Burkina Faso and international principles of Good Clinical Practices (GCP). Before enrolment a written informed consent was obtained from patients (adults) or parents/guardians (children). For illiterates, in addition to the thumb impression of the participant/parent/guardian, a signature of an impartial witness was obtained.P. falciparum. Out of them, 748 patients had parasitaemia ≥2000 asexual forms per microliter of blood. All available Day 0 blood spots were systematically considered for msp1 and msp2 genotyping (n = 727). Unsuccessful PCR concerned one sample in both msp1 and msp2 genes. Sex ratio male/female was 1.10 with a median age of 4 (Q25 = 2 - Q75 = 6) years.During the study period (September 2010–October 2012), 1480 patients were screened, 1010 patients were diagnosed positive for malaria and 980 patients were mono-infected by msp1 and msp2 genes yielded 1720 and 1660 different fragments respectively. The multiplicity of infection (MOI) was 2.3 for msp1 and 2.2 for msp2. Table msp1 and msp2 allelic families in each of the two study sites.Overall, 2 tests, all five P-values > 0.05). The commonest msp1 allelic family was the K1 type with a prevalence of 51.2% (881/1720). The most prevalent msp2 allelic family was the 3D7 type with a prevalence of 57.8% (960/1660). No msp1 or msp2 allelic family was overrepresented in uncomplicated or severe malaria groups  of 35814.81 (±3.58). Parasite density was significantly influenced by the msp1 allelic family with highest parasitemia observed for MAD20, followed by RO33 and K1  displayed similar parasite density as isolates with monoclonal infections  did not induce an increase of body temperature compared to isolates with monoclonal infections , and there were no significant interactions.The mean (±se) axillary temperature was 38.4 (±0.03) °C. There was no significant effect of Msp1 allelic diversity had no noticeable effect on Hb levels  . Finally, we found a strong positive relationship between Hb levels and host age  Hb level was 9.6 (8–10.8) g/dL. 49. In this study, the extent to which some specific P. falciparum variants are associated with particular malaria symptoms was explored. Merozoite surface antigen variations are among others, more likely to be involved in malaria symptomatology by participating in erythrocyte invasion and immune evasion49. Accordingly, we found some significant associations between specific clones of P. falciparum and the severity of particular malaria symptoms. In particular, the allelic family msp1-MAD20 was associated with highest parasitaemia. In addition, patients harboring msp2-FC27 allelic family showed the lowest Hb level in both single and mixed infections. In effect, Soulama et al. reported previously that msp2-FC27 complexity was associated with severe anaemia during malaria infection16. Both high parasitaemia and low Hb level have been known to increase disease severity and for this reason, hyper-parasitaemia and severe anaemia are included among criteria defining severe malaria. This finding would mean that the carriage of msp1-MAD20 allelic or msp2-FC27 family is associated with increased susceptibility to severe malaria. Several other studies mentioned msp1-MAD20 and msp2- FC27 allelic families in increasing risk of developing symptomatic malaria or disease severity providing an evidence of association between particular parasite strains and malaria disease outcomes51. Body temperature was influenced neither by the msp1 and msp2 allelic family variation nor by the multiplicity of distinct parasite strains. In contrast, there was a positive relationship between body temperature and parasite density. This could be explained by cell necrosis due to high level of parasitaemia with elevation of produced cytokines such as tumor necrosis factor (TNF) that mediate malaria fever53. However, a previous study reported a positive association of co-infections by msp1-RO33 + K1 with fever14.Severity and malaria disease outcomes stem from a complex interaction between human host, parasite and environment. Understanding of this interaction is crucial to inform disease case management and treatment decision. Evidence of differences in parasite virulence between different strains has been provided in animal modelsmsp1 and msp2) in this study are under immune pressure and this could affect the extent of parasite diversity54. However, no msp1 or msp2 allele was overrepresented in uncomplicated or severe malaria groups in the univariate analysis. This is probably due to the lack of extremely severe cases such as cerebral malaria in this study, which considered only high parasitaemia and low Hb level as severity criteria.Our findings regarding fever and anaemia could be influenced by other pathologies that could cause the same symptoms and this constitutes a limitation of our study. Furthermore, the polymorphic markers  impaired the immune response in this relatively young age group as previously demonstrated57.Except for patients harboring the triple infection K1 + MAD20 + RO33, parasite density decreased with age likely due to the acquisition of natural immunity to malaria in high malaria transmission settings such as NanoroP. falciparum within human host leading to attenuation of the growth of parasites belonging to the MAD20 allelic family. As this allelic family seems to be more virulent through its association with hyper-parasitaemia, the high prevalence of mixed-infections could be beneficial in terms of suppressing this virulent strain family. Factors underlying within-host competition are still under assessment but evidence of this phenomenon in human malaria parasites was previously proposed58. As age-density relationship was similar in all individual allelic families, underlying a similar relationship with host immune response, we postulate that the observed competition could be mediated by red blood cells resources59.Our findings suggest a competition among co-infecting genetically diverse msp1 and the two allelic families of msp2 were represented without significant difference between the two study sites (Nanoro and Nazoanga)44. This was likely due to the proximity between the two sites (around 15 km) with possible important population flow from one to the other. Indeed, Nanoro was one of the RTS,S/AS01 vaccine trial sites and this genetic diversity of circulating parasite clones raises the issue of clone responsiveness i.e. whether different strains observed in our dataset could have responded differently to the vaccine60. In bacterial infections such as meningitis, clone differences constituted for a long time one cause of vaccine failure so that parasite clonal diversity must be taken into account in implementing malaria vaccine in order to avoid this phenomenon61.Finally, a large diversity of circulating parasite strains was observed in Nanoro area with frequent occurrence of multiple infections. All the three different allelic families of P. falciparum genetic diversity on the severity of particular malaria symptoms as measured by parasite density, Hb level and body temperature. While allelic family msp1 and msp2 had no significant effect on temperature, we found that parasite density was influenced by the msp1 allelic family, with highest parasitemia observed for MAD20, followed by RO33 and K1. In addition, clinical isolates containing multiple clones of P. falciparum at the msp1 or the msp2 genes (i.e. multiple infections) displayed similar parasite density as isolates with monoclonal infections, suggesting the existence of within-host competition. For MAD20, parasite densities significantly decreased in mixed infections with K1, suggesting that in those mixed infections, MAD20 cannot grow to the same levels as it would alone, and hence pointing to the existence of competitive suppression. Finally, there was a significant effect of msp2 allelic diversity on Hb level with patients harboring parasite genotype msp2–3D7 in mono-infection showing higher level of Hb than mixed-infected individuals (3D7_FC27).This study explored the effect of Supplementary information


Asymptomatic carriage of the malaria parasites, likewise its misdiagnosis, especially false negatives, due to the use of substandard rapid diagnosis tests (RDTs) has been shown to hinder the progress of the fight against malaria. Plasmodium falciparum-specific protein and histidine-rich protein 2 (PfHRP2) CareStart™ RDT against standard microscopy in the detection of malaria infection among 345 children (1–15 yrs) from two (2) basic schools in Tamale Metropolis.  The study assessed the prevalence of asymptomatic malaria as well as the performance of P. falciparum infections found to be 55.6% and 93.8%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of CareStart™ RDT were found to be 19.23% and 98.45%, respectively. There was an evidence showing a significant relation between CareStart™ RDT and microscopy in determining malaria infection .  From the microscopy (considered as gold standard), prevalence of malaria among the asymptomatic children was found to be 2.6%, with sensitivity and specificity of CareStart™ RDT in detecting PfHRP2 CareStart™ RDT in an asymptomatic population at instances of low parasitaemia. Prevalence of asymptomatic malaria among children was found to be 2.6%. The study reported low sensitivity and PPV for  Malaria continues to remain a global burden and a threat to world health despite increasing awareness and public heath efforts aimed at improving vector control and therapeutics and diagnostics, which in recent times have reduced the incidence of the infection worldwide –3. In 20Following the recent global trend, indications are that malaria in Ghana is on the decline, but the 2017 malaria report from the WHO indicated, however, an upsurge of cases in 2016. In most outpatient departments of the country, it is noted as a major cause of morbidity (40%) and mortality especially among children of school-going age as well as among children under 5 yrs  . MalariaPlasmodium infection found in nonfebrile individuals with no apparent symptoms of malaria, has been brought to the spotlight in recent times as a new challenge to the prevention and control of malaria in sub-Saharan Africa. Individuals with asymptomatic infection carry very low parasite densities for extended periods and usually go untreated. As such, they contribute to malaria transmission especially in areas with seasonal transmission by serving as Plasmodium reservoirs [Asymptomatic malaria, thus servoirs –12. IdenAccurate diagnosis and effective and appropriate treatment of malaria play a key role in the fight against the disease. This has been well recognized in Ghana's National Malaria Control Strategic Plan for 2014–2020, as one of its objectives is to provide appropriate diagnosis to all suspected malaria cases and also prompt an effective treatment regime to 100% of confirmed malaria cases in accordance with treatment guidelines by 2020 . In healThis study therefore sought to determine the prevalence of asymptomatic malaria among children in Tamale in the northern part of Ghana with seasonal transmission whilst assessing the performance of a commonly used malaria RDT in the country for its diagnoses.2 [The study was carried out in two (2) basic schools located in Tamale Metropolis, one of the 26 districts in the northern region of Ghana. The Metropolis has >2000 inhabitants (>36% below 15 yrs.) and a total land area of 646.9 km2 . General2 , 19, TamA school-based descriptive cross-sectional study was employed, targeting children from the ages of 1–15 yrs. Participants were selected from two basic schools with the number of participants per school determined on the basis of their willingness and availability. These two schools were randomly selected from a pool of schools in the Tamale Metropolis. A total of 345 children participated.All children aged 1–15 yrs registered with each participating school were eligible for the study, but only those who were willing and had parent/guardian consent were included. For children under 10 yrs, only those whose parents/guardian consented and also accompanied them to the registration/sample collection point were included, and for those aged between 10 and 15, an endorsed consent form from parent/guardian was enough. However, children who received antimalarial treatment within the past two weeks before the study were excluded.Ethical clearance was obtained from the University for Development Studies School of School of Medicine and Health Sciences and School of Allied Health Sciences Joint Institutional Review Board. Approval was also obtained from the respective school authorities. The study, detailing purpose, benefits, and sample collection procedures, was explained to staff as well as parents in the local language (Dagbani) at a meeting, and informed consent obtained from parents before the study was initiated.Details of each study subject such as age, sex, and place of residence were collected by oral interview of parent/guardian or the participant (>10 yrs), and a unique code for each participant was generated to label the RDT kit and microscope slide. Blood samples from finger pricks were used accordingly for both RDT and microscopy. All those involved in the blood testing process had prior clinical laboratory experience and in addition were given a week's training by an experienced and qualified laboratory technologist.Plasmodium species in participant samples. The RDT was based on lateral flow immunochromatography in cassette format which can detect Plasmodium falciparum-specific protein and histidine-rich protein 2 (HRP 2). The test was carried out on the field and read within 20 min according to the manufacturer's instructions.The rapid malaria test, PfHRP2 CareStart™ , was used to detect Plasmodium asexual forms or gametocytes were found after counting 500 WBCs. The thin films of each specimen were subsequently examined for species identification only in those specimens in which malaria parasites were identified in the thick film. For quality control purposes, a second experienced microscopist randomly selected 5% of the slides for re-examination.Thick and thin blood smears were prepared on either ends of the same slide, air dried in the field, and transported to the Public Health Reference Laboratory at the Tamale Teaching Hospital. The slides were stained with 10% (v/v) Giemsa for 10 min and screened for the presence of plasmodial infections. The slides were read by a trained experienced laboratory technologist who was blinded to the RDT results. Parasitaemia was determined from thick blood films by counting the number of parasites per 200 white blood cells (WBCs). A slide was classified as negative if no Data were entered and analysed using the Statistical Package for Social Sciences (SPSS) version 25. Data were analysed for descriptive statistics, while results of both methods were compared using Cohen's kappa coefficient and the chi-square test. All statistical analyses were done at 95% level of significance. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using the formulae presented below.A total of 345 children participated in the study, of which only 31 (9.0%) were under 5 yrs and the majority  were from 5–10 yrs. Female participants were more  than male . The ovePlasmodium infection was therefore 7.5% and 2.6% per RDT and microscopy, respectively  with a range of 16–80 parasites/μL was recorded. Participants who tested positive either by RDT or microscopy were apparently healthy with no signs of malaria.Using RDT, a total of 23 malaria cases were detected, while microscopy detected 9. The prevalence of ectively . All PlaPlasmodium infection was higher among children <5 yrs in both RDT (12.9%) and microscopy (6.5%). This, however, makes up only 15.38% and 22.22% of the total positive cases recorded by RDT and microscopy, respectively. From the results of the RDT, most asymptomatic infections were found among males (10.5%) while microscopy results indicated a higher prevalence of asymptomatic infection among females (4.3%) (p=0.142) or gender (p=0.126). However, the prevalence by microscopy showed a gender dependency towards females (p=0.014) with a moderate observed power of 0.7  and a rather meagre effect size (Eta squared = 0.02).The prevalence of asymptomatic s (4.3%) . PrevalePlasmodium infection was detected by RDT in 7.5% (26/345) of children. On the other hand, microscopy confirmed Plasmodium infection in only 19.2% (5/26) and the rest thus, were considered false positives. There were also 1.3% (4/319) false negatives. Overall, 98.7% of all RDT negative cases were confirmed to be negative by microscopy. There was no evidence of association between parasitaemia and RDT results (p=0.178). The effectiveness of the RDT in diagnosing asymptomatic malaria as presented by AUC (area under curve) of a receiver operating characteristic (ROC) curve is 0.74.Plasmodium reservoirs are essential to eliminating the source of disease transmission, a crucial strategy in eradication of malaria. In Ghana, where the majority of rural and semiurban setting rely heavily on RDT due to the inability to perform microscopy, low-cost, highly sensitive, and highly specific screening tools for malaria are required.According to the global malaria elimination program, Ghana like many other African countries is in the malaria control phase. Although several years of policy development and control interventions have led to a general decline in malaria-specific mortality, much still remains to be done if the country is to achieve malaria elimination. Detection of asymptomatic malaria and treatment of p=0.126). Microscopy on the other hand did not detect any Plasmodium infection among males, but, however, showed a 4.3% parasite prevalence (lower than that by RDT) among females. Though there was a statistically significant association between gender and malaria in asymptomatic individuals, it is tempting to speculate that the higher prevalence observed in females according to microscopy might be a contributing factor since 20% more female than male participated in the study. Meanwhile, we were careful to avoid any bias in the sample collection; thus, study subjects were randomly recruited. Moreover, though the study demonstrated a modest statistical power, gender had a rather low effect size in relation to malaria status by microscopy. Evaluation of gender as a biological factor in asymptomatic malaria is, however, beyond the scope of this present study and therefore recommended for further studies. Nevertheless, in Myanmar, Huang et al. [Plasmodium infection was observed to be rather prevalent among females [Using microscopy in the current study, the overall prevalence of malaria was lower compared to RDT. This is consistent with recent studies by Sumari et al.  and Huang et al.  and Zaw g et al.  separateg et al.  found as females . RegardlPlasmodium carriage. In this study, however, contrary to a report by Touré et al. [Plasmodium parasites which could be contributing significantly to the disease transmission. As reported by Rovira-Vallbona et al. [P. falciparum infections have been suggested to account for as much as approximately 30% of human-mosquito transmissions in Burkina Faso. Furthermore, unlike other studies also conducted in Ghana [Plasmodium species was observed in this study.Age is deemed as one of the most important factors that correlates with protective immunity in malaria endemic areas. With young children being the most vulnerable, it has been thought of that adults and older children who might have had several episodes of malaria and have acquired some form of immunity are more likely to carry asymptomatic infections . In facté et al. , individé et al. , 24. Buta et al. , asymptoin Ghana , 32, onlμL minimum threshold as set by WHO) current batch of RDTs are not very useful and therefore the need for ultrasensitive RDT especially in areas where even microscopy is a challenge and PCR is simply impossible. In fact, with regards to the general performance of the PfHRP2 CareStart™ RDT, WHO in its recent round of product testing for malaria RDTs reported a 92% panel detection score (PDS) and 0.0% false-positive rate for the RDT at a minimum parasite concentration of 200 parasites/μL and PDS of 100% at higher density (2000 parasites/μl) [Considering microscopy as the gold standard, the RDT generally demonstrated a moderate sensitivity far below that recommended by the WHO while on the other hand exhibiting a good specificity (>90% cf 95%) comparable with the WHO standard . The senites/μl) .k = 0.259; p < 0.001) was found. Stratifying the results by age and gender did not make the agreement between the two tests any different. Other studies, however, have reported a moderate to high agreement between the two test methods [χ2 = 30.579, df = 1, p < 0.05). Assessing the effectiveness of the RDT to diagnose malaria in asymptomatic individuals, the receiver operating characteristic (ROC) curve showed RDT to be a good test method when compared to microscopy with area under the curve (AUC) = 0.74, similar to that reported by Osei-Yeboah et al. [n HRP2 expression, as well as pfhrp2 and/or pfhrp3 gene deletion since in recent studies these deletions were observed in other parts of Ghana [μL.Using Cohen's kappa coefficient to compare the agreement between RDT and microscopy, a fair but highly significant agreement , mass screening and treatment (MSAT), focal screening and treatment (FSAT), and voluntary screening and testing (VSAT), even though such programs have public health and ethical concerns, they present currently a viable way of targeting and eliminating asymptomatic infections [Although microscopy has been recommended by WHO as a gold standard in the diagnosis of symptomatic patients, many studies have, however, reported that low parasite densities especially among asymptomatic carriers are easily missed by this method due to inherent limitations such as expertise in microscopy reading, slide preparation method, staining technique, and detection limits , 27, 40.fections . NonetheP. falciparum are further limitations in this study.Though convenient and affordable and remain the first-line diagnostic tool for malaria in Ghana and many other developing countries, microscopy is less sensitive in diagnosing asymptomatic infections. The use of microscopy as the gold standard is therefore a limitation to evaluating the performance of RDT and ultimately this current study. In addition, the use of a relatively small sample size, the scanty positive cases recorded in this study, and the fact that the RDT used only detects Plasmodium parasite reservoirs among children <16 yrs. Also, though the PfHRP2 CareStart™ has a good specificity and negative predictive value, it however, has a poor sensitivity and positive predictive value and therefore its use among asymptomatic individuals may result in missing true positive malaria cases. Efforts should therefore be made by all stakeholders in the fight against malaria to eliminate these reservoirs by improving upon the performance of current RDTs as well as investing in low-cost, highly sensitive, and specific screening tools for malaria.There could be a substantial amount of