Dihydroartemisinin-piperaquine administered at monthly intervals, but not that dosed once a school term, is a remarkably effective measure for the prevention of incidence of malaria, prevalence of parasitemia, and prevalence of anemia in schoolchildren living in a high-transmission setting. Background. Intermittent preventive treatment (IPT) in schoolchildren offers a promising option for malaria control. However, the optimal drug and dosing regimens for IPT remain to be determined.Methods. We conducted a randomized, double-blind, placebo-controlled trial in 740 schoolchildren aged 6–14 years living in a setting of high malaria transmission in Uganda. Enrolled children were randomized to dihydroartemisinin-piperaquine (DP) given once a month (IPTm), DP given once a school term , or placebo and followed for 12 months. The primary outcome was the incidence of malaria over 12 months. Secondary outcomes included parasite prevalence and anemia over 12 months. Analyses were conducted on an intention-to-treat basis.Results. In the placebo arm, the incidence of malaria was 0.34 episodes per person-year and the prevalence of parasitemia and anemia was 38% and 20%, respectively. IPTm reduced the incidence of malaria by 96% , the prevalence of asymptomatic parasitemia by 94% , and the prevalence of anemia by 40% . IPTst had no significant effect on the incidence of symptomatic malaria or the prevalence of anemia, but reduced the prevalence of asymptomatic parasitemia by 54% .Conclusions. Monthly IPT with DP offered remarkable protection against clinical malaria, parasitemia, and anemia in schoolchildren living in a high-malaria-transmission setting.Clinical Trials Registration.  NCT01231880. Children of school age 5–14 years) benefit relatively little from current malaria control interventions, due to a focus on younger children and pregnant women 4 years b. Yet, inOne intervention under investigation is intermittent preventive treatment (IPT), the administration of curative doses of antimalarials at predefined intervals regardless of infection status. IPT targeted at schoolchildren has resulted in reduced rates of malaria, asymptomatic parasitemia, and anemia, as well as improved cognitive function –13. HowePlasmodium falciparum, with an estimated entomological inoculation rate of 562 infective bites per person per year [This randomized, double-blind, placebo-controlled trial was conducted between February 2011 and February 2012 in Mulanda Primary School, Tororo District, in eastern Uganda. Tororo District is characterized by high-intensity year-round malaria transmission, principally of per year . In the i) known allergy or adverse reaction to artemisinin-based regimens; (ii) history of menarche; (iii) fever (axillary temperature ≥37.5°C) or history of fever in the previous 24 hours; (iv) evidence of severe malaria or danger signs; or (v) ongoing antimalarial treatment. Recruitment was done once and closed after baseline assessments.Mulanda subcounty has 8 primary schools. Mulanda Primary School was selected due to its large population (1320 students) and close proximity (approximately 500 m) to the main public health facility, Mulanda Health Centre IV. Meetings were held to explain the nature and purpose of the trial to teachers and parents or guardians, and written informed consent was obtained. All children with consent from their parent/guardian were screened for eligibility. Children were excluded if they had  was administered.At enrollment, a standardized questionnaire was administered to children fulfilling selection criteria to obtain data on sociodemographics and bed-net ownership and use. A standardized assessment of symptoms and a focused physical examination including measurement of weight and temperature were conducted. A finger-prick blood sample was obtained to assess for A randomization list was computer-generated using fixed blocks of 12 by an individual not involved in patient care. Participants were randomized using previously prepared, consecutively numbered, opaque sealed envelopes and assigned study numbers sequentially. A nurse not involved in care allocated the study group after opening the correspondingly numbered envelope. All other study personnel were blinded to study group assignments, and children were not informed of their regimen, but the color and taste of study medication and placebo were dissimilar. Study drugs were dosed as DP  once daily for 3 consecutive days each month (IPTm), or at the beginning of each school term . DP was dosed using weight-based guidelines targeting a total dose of 6.4 mg/kg dihydroartemisinin and 51.2 mg/kg piperaquine, given with glucose biscuits. Placebo was administered to the placebo and IPTst groups to simulate the IPTm dosing schedule. All treatment was administered at school by the study nurses and was directly observed. After drug administration, children were observed for 30 minutes, and treatment was readministered if vomiting occurred.Plasmodium infection. At the start of each school term, standardized history and physical examination and assessments for hemoglobin were performed prior to treatment. Children absent for any scheduled visits were followed up at home. School attendance was assessed daily by roll call by teachers, with absent children followed up at home by study personnel to establish reasons for absenteeism. Children who were absent due to illness were taken to the study clinic for diagnosis, management, and documentation. Adverse events were assessed and graded according to standardized criteria. Children with illnesses other than malaria received standard of care or were referred to Mulanda Health Centre. Medications with antimalarial activity were avoided whenever possible.Finger-prick blood smears were performed monthly to assess for Monitoring for malaria was by active case detection. Sick children were identified by teachers and sent to the study clinic and absent children were visited at home and taken to the study clinic if ill. The clinic was open 7 days a week. Children with documented fever (axillary temperature ≥37.5°C) or history of fever in the previous 24 hours had a blood sample for thick and thin smears. Malaria was defined as fever (documented or by history) with any quantity of malaria parasites on the blood smear. Episodes of uncomplicated malaria were treated with artemether-lumefantrine, the recommended first-line treatment in Uganda, following standard dosing guidelines and under supervision of the study nurse. Treatment failures within 14 days of prior therapy were treated with oral quinine 10 mg/kg every 8 hours for 7 days. Episodes of complicated malaria  were referred to Mulanda Health Centre IV for intravenous quinine.Blood smears were labeled, air dried, and stained with 2% Giemsa for 30 minutes. Parasite densities were determined from thick blood smears by counting asexual parasites per 200 white blood cells (or per 500 white blood cells if the count was <10 parasites per 200 cells), assuming a white blood cell count of 8000 cells/mL. A smear was considered negative if no parasites were seen in 100 high-powered fields. Gametocytemia was determined using the same methodology. Thin smears were viewed for species identification. Two independent and experienced microscopists read the slides, with a third microscopist resolving discrepancies. Stool examination was by the Kato-Katz technique to assess for helminth infections. Hemoglobin concentration was assessed using a portable hemoglobinometer  and estimated to 0.1 g/dL. Follow-up for malaria, anemia, and parasitemia were conducted for 12 months after enrollment. Participants were withdrawn for change of school, withdrawal of informed consent, or noncompliance with study procedures.The study was designed to test the hypothesis that IPTm would reduce the incidence of malaria compared with placebo by 33% or greater. We assumed that the incidence of malaria would be 0.57 episodes per person year in the placebo arm , and thuP value ≤.05 was considered statistically significant. The primary outcome was the incidence of malaria, defined as the number of incident episodes per time at risk. Time at risk was calculated from the day following the initiation of study drugs to the last day of observation, minus 14 days after each episode of malaria, a standard approach. Malaria within 1 month of IPTst was defined as any episode in March, June, or September, within 2 months as any episode in April, July, or October, within 3 months as any episode in May, August, or November, and within 4 months as any episode in December.Data were double-entered and cross-checked in a bespoke Microsoft Access database . Consistency checks were performed and all discrepancies and queries verified against original paper forms. Statistical analyses were done using Stata version 12.0 software . All analyses used an intention-to-treat approach. Incidence outcomes were compared using a negative binomial regression model, and prevalence outcomes were compared using generalized estimating equations with adjustment for repeated measures in the same study participant. Measures of association were expressed as incidence rate ratios, prevalence ratios, and mean differences. A Secondary outcomes included prevalence of parasitemia, defined as the number of positive smears divided by the total number of smears over 1 year. Parasitemia after IPTst was defined as described above for malaria. Anemia was defined as <11.5 g/dL for children aged 6–11 years and <12.0 g/dL for those aged 12–14 years .2 tests.An adverse event was defined as any untoward medical occurrence, irrespective of its suspected relationship to study medications. All events were graded by severity and relationship to study treatment . The risThe sponsors had no role in study design, data collection, data analysis, data interpretation, or writing the report. The corresponding author had full access to study data and final responsibility for submission of the manuscript. Ethical approval was obtained from the Uganda National Council for Science and Technology and the Makerere University School of Medicine Research and Ethics Committee.Between January and February 2011, 826 children were screened for eligibility, and 740 (89.6%) met inclusion criteria and were randomized to study arms . Compared with the placebo arm, the overall incidence of malaria (Table P < .0001) and 45%  protective efficacies within 1 and 2 months of IPTst administration, respectively. Restricting the case definition of malaria to fever and >2000 parasites/µL of blood yielded similar results and comparisons  and 54% protective efficacy with IPTst  over 1 year of follow-up. Similar to what was observed with malarial incidence, the prevalence of parasitemia in the IPTst group increased with increasing duration after treatment. The prevalence of gametocytemia was significantly lower in the IPTm and IPTst groups compared with the placebo group  and IPTst (18%) arms compared with the placebo (38%) arm, resulting in a 94% protective efficacy with IPTm . In contrast, the mean increase in hemoglobin in children in the IPTm group was significantly greater than that in the placebo group . The overall prevalence of anemia was significantly lower in the IPTm arm (0.12) but not the IPTst arm (0.17), compared with the placebo arm (0.20), resulting in a 40% protective efficacy for IPTm   and in infants (IPTi) living in areas with moderate to high malaria transmission, whereas seasonal malaria chemoprevention (SMC) with monthly AQ + SP is recommended in children aged <5 years living in areas with highly seasonal malaria transmission in the Sahel subregion of Africa . Althougpfmdr1 mutations associated with decreased sensitivity to other aminoquinolines. The impact of chemoprevention with DP on the selection of drug-resistant parasites is unknown. Surveillance of parasites emerging under DP-selective pressure is indicated, and analysis of samples from our trial is planned. Fourth, these findings may not be generalizable to medium- and low-transmission settings. Fifth, the lack of similarity between the placebo and DP may have unblinded study participants, but our outcome measures included clinical and laboratory evaluations by study personnel blinded to the participants' study arms. Considering our results in light of these concerns, further study of the feasibility, safety, and cost-effectiveness of IPT targeted at schoolchildren with DP in different transmission settings will be warranted, but nonetheless, implementation of this highly efficacious intervention in the near future is worthy of strong consideration.Our results suggest that monthly administration of DP will have major benefits for schoolchildren living in areas with high malaria endemicity. However, some concerns regarding this approach need to be addressed. First, the feasibility of monthly administration of a 3-day treatment regimen may be questioned, but it is noteworthy that schools offer relatively simple settings for administration of directly observed therapies. Second, prevention of malaria through chemoprevention may impede the development of immunity, leading to rebound morbidity after completion of the intervention, although rebound after chemoprevention has generally either not been seen, or been modest , and it


Plasmodium falciparum (Pf) incorporate active infection detection strategies to target the subclinical transmission reservoir. Currently available immunochromatographic lateral flow tests lack the sensitivity required for Pf malaria elimination; the limit of detection (LOD) of these rapid diagnostic tests (RDTs) is above that which is required to identify all transmissible infections. Malaria RDTs with a significantly improved LOD would enable more effective elimination interventions while retaining the critical advantages of low cost, ease of use, and rural deployment capability. Histidinerich protein 2 (HRP2) is a high-priority target analyte for identifying individuals at risk of transmitting Pf. HRP2 protein is secreted by Pf parasites in relatively high concentrations, is widely conserved among Pf strains, and persists in human plasma for up to four weeks. These characteristics allow HRP2 antigen detection even in the absence of circulating parasites during cyclical sequestration. However, the highly polymorphic nature of HRP2 makes optimization of RDTs challenging. Current HRP2-detecting RDT reagents primarily target Type 2 (AHHAHHAAD) and Type 7 (AHHAAD) HRP2 motifs, and independent antibody discovery confirms the most common selection of a C1-13/PTL-3 antibody conjugate pair yields the best RDT performance for the majority of parasite strains. In scenarios where malaria transmission rapidly decreases, exclusive use of a diagnostic whose sensitivity varies with the number of HRP2 epitope repeats can result in single axis evolutionary pressure, increasing selection for less-detectable parasite strains. To evaluate the feasibility of developing an HRP2-based RDT that is highly effective in mass testing and treatment programs, we have assessed the technical characteristics of HRP2 reagents. Our aim is to facilitate the development of improved malaria HRP2 RDTs by: (1) incorporating an understanding of HRP2 structure-function relationships and their impact on epitope-antibody interactions and adaptation, (2) correlating HRP2 concentration levels (in pg/mL) with traditional malaria diagnostic metrics (parasites/mL) for comparative evaluation of tests, and (3) predicting optimal capture reagents to improve and sustain HRP2 RDT performance. Based on our ongoing analysis, we present a product development strategy that combines capture reagent and platform innovations that can be used to make highly sensitive Pf malaria RDTs. In addition, we present criteria for the rational selection of HRP2 protein and parasite culture controls to support the product lifecycle from development through commercialization and scale-up.Malaria programs aimed at eliminating


Plasmodium falciparum HRP2, a poly-histidine antigen in urine of febrile patients for malaria diagnosis.Accurate rapid diagnosis is one of the important steps in the effort to reduce morbidity and mortality of malaria. Blood-specific malaria rapid diagnostic tests (RDTs) are currently in use but other body fluid specific diagnostic test kits are being developed. The aim of the present study was to evaluate the performance characteristics of a one-step Urine Malaria Test™ (UMT) dipstick in detecting This was an observational study in which a urine-based malaria test kit was used in malaria diagnosis in a normal field setting. Two hundred and three individuals who presented with fever (≥37.5°C) at seven outpatient clinics in Enugu State during periods of high and low transmission seasons in Southeastern Nigeria were enrolled. Matched samples of urine and blood of consecutively enrolled subjects were tested with UMT and blood smear microscopy.With the blood smear microscopy as standard, the disease prevalence was 41.2% and sensitivity for the UMT was 83.75% . The UMT had an LLD of 120 parasites/μl but the sensitivity at parasite density less than ≤200 parasites/μl was 50% and 89.71% at density ≥201 parasites/μl with specificity of 83.48%. The positive and negative predictive values were 77.91% and 88.07%, respectively.The UMT showed moderate level of sensitivity compared with blood smear microscopy. The test kit requires further improvement on its sensitivity in order to be deployable for field use in malaria endemic regions. Plasmodium species, which is transmitted to humans by the bite of the female Anopheles mosquito. It is estimated that malaria causes about 207 million clinical episodes and about 627,000 deaths annually, mostly in sub-Saharan Africa [Malaria causes significant morbidity and mortality especially among children in sub-Saharan Africa. The disease is caused by the n Africa . One of n Africa . Until rn Africa , 5, but Plasmodium parasite in blood remains the gold standard for the diagnosis of malaria [However, in recent times, with relatively high cost and increasing incidence of resistance to anti-malarial drugs, it has become necessary to confirm cases of malaria using laboratory methods before treatment. Thick smear microscopy based on the demonstration of the presence of  malaria .However, this method is highly operator-dependent and requires initial and ongoing training to maintain high quality testing; such quality assurance practices have often been difficult to implement in resource poor and malaria endemic countries. In addition, there has been reported over-diagnosis of malaria using microscopy in health facilities , 7, withPlasmodium species to detect the parasite antigen in blood of infected patients. Most of these kits use antigens or enzymes derived from parasites that have infected humans. The commonly used one is the histidine rich protein 2 (HRP2), which is produced by Plasmodium during its asexual forms and early gametocytic stages in infected individuals. It is a water soluble substance abundantly found in the parasite cytoplasm and serum of infected individuals. Histidine rich protein 2 is secreted early during infection by the Plasmodium parasite and persists after treatment. Significant levels of HRP2 antibodies in some infected individuals reduce the sensitivity of the protein dependent test kits [These kits use immune-chromatographic materials impregnated with monoclonal antibodies against est kits . Sensitiest kits . Over tiest kits . In ordeest kits , Howeverest kits .Plasmodium falciparum specific HRP2, a poly-histidine protein, or fragment present in the urine of febrile patients.HRP2 have been found in several body fluids such as urine, saliva, blood . These fThe present study was performed to assess the diagnostic accuracy of the UMTin malaria diagnosis in comparison with benchmarked gold standard, blood smear microscopy.This was an observational cross-sectional diagnostic test study in which participants with fever at presentation were enrolled consecutively at different health facilities in both urban and rural communities of Enugu State. Institutional ethical approval was obtained from the University of Nigeria Teaching Hospital Health Research Ethics Committee prior to the start of the study. Individuals who met inclusion criteria were properly consented before enrollment.Enugu State has a population of 3.6 million inhabitants and located in the rain forest region of southeast Nigeria. The region is endemic for malaria and with year-round transmission of the disease. The highest transmission is in the rainy season from the month of April to October.The inclusion criteria were: fever of ≥37.5°C and non-use of anti-malarial drugs in the preceding one week. Adults and children with history of haematuria were excluded from the study . Demographic information such as age, gender, symptoms of disease as well as physical examinations were also obtained from each participant. Subsequently the matched capillary blood drawn from finger/heel pricks and urine samples voided into a universal bottle were respectively stained in accordance with WHO standard microscopy technique  and immeThe technology relies on the fact that clinical malaria commonly results in elevated levels of specific proteins (Histidine Rich Protein-2) or its protein fragments in patient’s urine against which cognate recombinant monoclonal antibody reagents were developed. It is a qualitative assay consisting of a nitrocellulose membrane strip containing relevant antibody reagent and controls that are each immobilized at the specific individual site on the membrane. When the immunochromatographic dipstick is dipped in urine, specific malaria parasite protein present in the urine migrate and interacts with immobilized cognate monoclonal antibody resulting in dark-colored strips on the dipstick. The UMT strips were individually packaged in a sealed Mylar foil pouch with a desiccant, and stored at room temperature for the entire period of the study.To perform the urine test, the UMT strip was dipped in 200 μl of urine for two minutes to allow the sample to wick and saturate the strip. The strip was then removed, placed on its foil pouch packaging and incubated for 20 minutes. The results were reported as negative, positive, or un-interpretable: if two visible lines appeared on the strip (even if very faint) the test was positive; if only the control line appeared, the test was negative. Tests results reported as un-interpretable, i.e., failure to observe a control line or the presence of a darkly stained background that obscured the test lines, were repeated to resolve the discrepant event.The capillary blood samples drawn from finger/heel pricks of the subjects were used to perform thick smear microscopy. Preparation of these blood samples (three thick blood smear slides per participant) for microscopy were in accordance with WHO standard microscopy technique , and reaP. falciparum. The same technique was employed for establishing the parasite density on each of the subsequent blood film examinations.However, if 500+ parasites have been counted without having reached 200 leucocytes, the count was stopped after completing the reading of the last field, and the parasitemia was calculated using the above formula. Also 100 fields of the second thick film were examined to identify mixed infections, which were confirmed on the thin film in case of any doubt. A blood film was assumed negative when the examination of 100 thick film fields did not show the presence of asexual forms of Plasmodium; (ii) on the species of Plasmodium, when one was present; and (iii) on the calculated level of parasitaemia within a 10% error margin [To yield a final microscopic interpretation, the two study microscopists independently agreed on three criteria: (i) on the presence or absence of asexual stages of r margin , 18.All clinical and laboratory data were entered into the corresponding logbooks and transcribed into Excel worksheet  and analysed with Statistical Package for Social Science (SPSS) version 20. . Descriptive statistics were used to ascertain the frequency distribution, mean, median and standard deviation of the subject characteristics; age, temperature, clinical symptoms. The outcome variables: the parasite densities and the UMT results were expressed in proportions (percentages), categorized for determination of sensitivity, specificity and predictive values (positive and negative). Chi square tests were used to test for the significance of the UMT results and other subjects’ derived categorical variables. P value of 0.05 was set for statistical significance.From a total of 638 individuals who were screened, 203 who met the inclusion criteria were enrolled from all the health facilities between June and December 2012, spanning periods of high and low malaria transmission in the study area. Data from 203 subjects were analysed; three children who could not void urine and five that had incomplete information were excluded giving final number of 195 subjects. Sixty four percent of the participants were females and 90.3% were children (aged less than 18 years old). The median age was three years with the range of six months to 60 years  out of the 195 participants had positive malaria Eight-six (44.1%) of 195 were positive with the UMT test. Parasite rates were 41.03% and 3.0% among children (<18 years) and adults, respectively. Overall, the UMT in comparison with the malaria microscopy showed sensitivity of 83.75%  and specificity of 83.48% (95% CI: 75.41% to 89.75%), Table Among the different age groups, false positive results were less frequent among the adults than the children , while the false negative results were less among the children than adults . Overall, the positive predictive (PPV) and the negative predictive values (NPV) for the UMT were 77.91% and 88.07%, respectively.et al. using ParaSight®-F to test urine samples of malaria patients obtained sensitivity and specificity of 81% and 26%, respectively [et al.[The UMT prototype has shown a higher level of sensitivity than previously tested blood specific RDT kits in urine specimens , 13. Genectively , 19 it hectively , 21. Gen [et al. noted th [et al.. They su [et al.. At high [et al., 21 in bP. falciparum a factor which is useful in absolute sensitivity tests against the clinical episodes that was used in this study. However, in areas of low malaria endemicity, this level of false positives may create drug wastage, which the current malaria control efforts seek to reduce. False positive results may be attributed to the ability of all histidine-rich protein 2 (HRP2) antigen malaria test kits to detect the parasite antigen even after malaria illness. The presence of rheumatoid factor and schistosomiasis in a patient may also lead to false positivity, and will need to be further evaluated [The degree of false positive results especially among children may suggest that the UMT is able to maintain acceptable level of false positive results particularly in areas of high malaria transmission , 23. Alsvaluated –26. ThesThe UMT specificity remained constant at significant levels for the different parasite densities although this may cause significant morbidity and mortality in areas of high endemicity. The false negative results with UMT are comparable to those of blood-specific malaria RDTs , 12, 21.Some of the limitations observed with the use of the urine malaria test kit were the high color degradation of the test line within an hour after testing. This is a disadvantage, which makes it difficult to crosscheck the results in cases of doubt. The delay in provision of some subjects, particularly among children, may be a delay factor in the promptness of testing and treatment. The sample size may also may be a factor in the resultant accuracy result, which may probably be answered by the completed larger clinical trials.The Urine Malaria Test kit studied has shown moderate concordance with blood smear microscopy and higher levels of sensitivity than blood-based test kits used for malaria diagnosis with urine. The UMT used for the study is still undergoing further development and evaluation, with opportunity for further improvement particularly in the area of test stability and sensitivity at lower parasite levels before the kit can be fully deployed for general use. Optimization of the detected areas of deficiency will enhance the usefulness of the non-blood based kits such as the UMT as a reliable and easy to use alternative tool in the diagnosis of malaria in the areas where the competence for blood microscopy is not reliable, and safety concerns are expressed.


Treatment of the sub-clinical reservoir of malaria, which may maintain transmission, could be an important component of elimination strategies. The reliable detection of asymptomatic infections with low levels of parasitaemia requires high-volume quantitative polymerase chain reaction (uPCR), which is impractical to conduct on a large scale. It is unknown to what extent sub-clinical parasitaemias originate from recent or older clinical episodes. This study explored the association between clinical history of malaria and subsequent sub-clinical parasitaemia.Plasmodium species. To identify previous episodes of malaria, case records were collected from village malaria workers and local health facilities and linked to study participants.In June 2013 a cross-sectional survey was conducted in three villages in Pailin, western Cambodia. Demographic and epidemiological data and blood samples were collected. Blood was tested for malaria by high-volume qPCR. Positive samples were analysed by nested PCR to determine the Plasmodium vivax was associated with sub-clinical P. vivax parasitaemia in the survey (p < 0.001), but this association was not seen with Plasmodium falciparum (p = 0.253); only three participants had both P. falciparum parasites in the survey and a clinical history of P. falciparum.Among 1343 participants, 40/122 (32.8 %) with a history of clinical malaria were parasitaemic during the cross-sectional survey, compared to 172/1221 (14.1 %) without this history (p < 0.001). Among the 212 parasitaemic participants in the survey, 40 (18.9 %) had a history of clinical malaria, compared to 87 out of 1131 (7.7 %) parasite-negative participants; p < 0.001, adjusted OR 3.3 . A history of P. vivax with artemisinin-based combination therapy without primaquine often resulted in recurrent episodes. Targeting individuals with a history of clinical malaria will be insufficient to eliminate the sub-clinical reservoir as they constitute a minority of parasitaemias.A clinical episode of vivax malaria was associated with subsequent sub-clinical parasitaemia. Treatment of  Plasmodium falciparum was reported   also self-reported having had malaria prior to the survey; 77.7 % (418/538) of those who self-reported having had malaria did not appear in the available malaria case records. Among these 418 participants who reported malaria (but for whom no case record was found), there was a statistically significant association with sub-clinical parasitaemia during the survey [OR 1.5  p = 0.023, Table P. vivax parasites among previously treated cases, which suggests that treatment of P. vivax without primaquine often fails to result in radical cure. There was no clear association between the case records of P. falciparum and subsequent sub-clinical P. falciparum parasitaemia in the survey. Only three participants had both a case record of P. falciparum and P. falciparum parasitaemia in the survey, and all had been infected within the previous 6 months.In Pailin, sub-clinical parasitaemia with any malaria species was three times more common among those with a history of malaria than those without. This may be explained chiefly by the persistence or relapse of P. falciparum parasitaemias in this population. The persistence of sub-clinical P. vivax parasitaemia among previously treated cases highlights again the critical importance of 8-aminoquinolines for the complete cure. 8-aminoquinolines are currently not used widely in Cambodia due to concerns about haemolytic adverse effects among people with glucose-6-phosphate dehydrogenase deficiency.The relationship between malaria incidence and the sub-clinical prevalence of parasitaemia is currently poorly defined. There were 55 malaria cases of all species recorded among residents of the study villages in 2012 . The parasite prevalence during the uPCR survey in June 2013 was 15.8 %, suggesting an approximately fourfold ratio. This information may be of relevance to elimination efforts as it could inform areas to target, but will require confirmation from other studies in other geographic areas. The origin of the large number of sub-clinical parasitaemias among those with no recorded or self-reported history of malaria remains unknown, as does the duration and potential infectiousness of sub-clinical Prior to June 2011 VMW treatment records were not systematically collected. This missing information implies that this study may underestimate the association between treatment and sub-clinical parasitaemia. There is considerable seasonal migration in Pailin and case records of residents who received malaria treatment outside of the study area were not collected. Records could not be obtained from the private sector, where malaria treatment was common practice until it was banned in 2012, and frequently involved the use of ineffective anti-malarial drugs . The souP. vivax with artemisinin-based combination therapy without primaquine often resulted in recurrent episodes. The at least threefold risk of sub-clinical parasitaemia among those with a recorded case history of malaria suggests an easy-to-identify, high-risk population. Other high-risk groups, such as family members of malaria cases or forest workers, have been identified by previous studies [Clinical episodes of vivax malaria were associated with subsequent sub-clinical parasitaemia. Treatment of  studies . None of studies . Therefo


Extensive studies investigating the role of host genetic factors during malaria associate glucose-6-phosphate dehydrogenase deficiency with relative protection. G6PD deficiency had been reported to associate with anti-malarial drug induced with haemolytic anaemia.G6PD normal (type B), heterozygous (type A+) and deficient (type A−) genotypes were determined by direct sequencing of the common African mutations G202A and A376G. Furthermore, multivariate analyses were executed to associate possible contributions of G6PD deficiency with baseline haemoglobin levels, parasitaemia and with severe malarial anaemia.A total of 301 Gabonese, Ghanaian, and Kenyan children aged 6–120 months with severe malaria recruited in a multicentre trial on artesunate were included in this sub-study. G6PD variants. The overall prevalence of G6PD deficiency was 13 % , 14 % (40/278) children were female heterozygous while 73 % (202/278) were G6PD normal [67 % (88/132) females and 78 % (114/146) males] individuals. Multivariate regression revealed a significant association of moderately and severely deficient G6PD genotypes with haemoglobin levels according to the baseline data , but not with severe malarial anaemia (p = 0.66). No association of G6PD genotypes with baseline parasitaemia.Two hundred and seventy-eight children  were successfully genotyped for +) and severely (type A−) G6PD deficiency showed significant association with lower haemoglobin concentrations at baseline in African children with severe malaria without leading to severe malarial anaemia. In addition, there was no association of G6PD variant types with parasite densities on admission.In this study, moderately (type A Plasmodium falciparum and anti-relapse treatment against Plasmodium vivax has been recommended for many years by the World Health Organization (WHO) . In Table G6PD for males and females by study site are shown. Between the study centres, there was a significant difference (p < 0.0001) in the prevalence of A− G6PD deficiency among males. Among females, the frequency of A− G6PD deficiency was lower compared to males. The prevalence was 4 % in Lambaréné (Gabon), 5 % in Kumasi (Ghana) and 0 % in Kisumu (Kenya).Overall, 202 (73 %) children were classified as G6PD normal , while 40 (14 %) were female heterozygous . Furthermore, a comparison between children G6PD normal and heterozygous (p < 0.0001), and between individuals G6PD normal and G6PD deficient (p = 0.009) showed a significant difference in adjusted mean baseline haemoglobin. The G6PD mutant genotypes were associated with a 2.7 g/dL and 1 g/dL decrease in haemoglobin levels in heterozygous and deficient children, respectively. The multivariate regression analysis adjusted for age, gender, centre, weight, haemoglobin levels and temperature did not result in any association (p = 0.29) of the G6PD genotypes on adjusted mean baseline parasite densities. In addition, there was no difference in adjusted log mean parasite densities between G6PD normal and heterozygous individuals (p = 0.18), and between G6PD normal and deficient children (p = 0.07).To investigate the association of p = 0.66) of the haemoglobin levels between SMA children with different G6PD genotypes. However, SMA occurred more frequently among G6PD normal 15/26 (58 %) compared to G6PD heterozygous 8/26 (31 %) and G6PD deficient 3/26 (12 %) children.Following the WHO guidelines  which deOnly 13 patients (5 %) had cerebral malaria. Nine of them were G6PD normal and four were female G6PD heterozygous.Glucose-6-phosphate dehydrogenase deficiency has raised in frequencies in malaria-endemic settings as a consequence of the evolutionary pressure exerted by malaria on the human genome . A plethG6PD genotypes among African children from three geographically countries presenting with severe malaria and participating in the SMAC clinical trial on different artesunate treatment regimens. While meanwhile many G6PD variants have been described [+) and 202A severe G6PD deficiency type A−) in Africa [The main objective of this study was to assess the distribution of escribed , 11, then Africa .versus 14 % reported earlier among males and 7 vs 2 % among females [The prevalence of severe G6PD deficiency as determined genetically was higher compared to previous findings from Gabon with 17 % in the present study  females . In Ghan females . However females . Regardl females .G6PD genotypes and haemoglobin levels according to the baseline data was observed. In fact, compared to children with the normal G6PD genotype (haemoglobin median: 9.3 g/dL), G6PD heterozygous (haemoglobin median: 6.6 g/dL) and deficient (haemoglobin median: 8.3 g/dL) children had 2.7 and 1 g/dL lower haemoglobin concentrations, respectively. The results contradict previous findings, which did not observe any association between G6PD genotypes and haemoglobin levels [G6DP deficient individuals, the association was not significant. An explanation could be the different designs of that and the present study. Here, patients with severe malaria were included, which implies high parasitaemia and haemoglobin concentrations <5 g/dL, whereas in the above cited study, the authors excluded patients with haemoglobin concentrations ≤7 g/L [A significant association of moderately and severely deficient n levels –32. Whils ≤7 g/L  and recrs ≤7 g/L  or who wG6PD genotypes. This may suggest that G6PD deficiency is not associated with this complication. However, this is in fact surprising especially as a significant difference of baseline haemoglobin values between G6PD normal and deficient individuals was observed. Moreover, in a large case–control study in Kenyan individuals, a significant increased risk to severe malarial anaemia associated with lower haemoglobin levels in G6PD deficient children with severe malaria at the time of hospital admission was found [In the case of SMA, there was no difference of haemoglobin levels among children with different as found . The likG6PD genotypes. Other studies have indicated a significantly lower parasitaemia associated with female G6PD heterozygous individuals, suggesting a protective advantage by this genotype [Although not significant, parasite densities were lower in G6PD deficient children than in G6PD heterozygous, compared to G6PD normal individuals. However, this trend is in agreement with previous works , 35. Congenotype –39.P. falciparum parasites in G6PD deficient red blood cells [P. falciparum 6-phosphogluconolactonase mRNA in parasites from G6PD deficient individuals [In comparison to individuals with uncomplicated malaria, this study population was hyperparasitemic according to the criteria of severe malaria provided by WHO with lower parasitaemia in G6PD deficient individuals. However, either in uncomplicated or in severe/complicated malaria, G6PD deficiency was always associated with lower parasitaemia , 33. Altod cells  and slowod cells , 42, morod cells , and lowividuals  both uncG6PD mutations 202G > A and 376A > G, although other variants such as A542T, G680T or T968C have also been reported to contribute to G6PD deficiency, albeit at far lower frequencies. The associations observed between G6PD genotypes and the baseline clinical parameters at admission could in fact also be the result of different circumstances. First, only patients with severe malaria were included, which could explain the findings, especially as the G6PD deficient allele 202A appears to confer protection against cerebral malaria and increases the risk of severe malarial anaemia [Several limitations apply to this study. Although some of the observations are in agreement with previous findings, the study group is rather small. The focus was only on the  anaemia , 17. Sec anaemia  cannot bG6PD genotypes studied to lower haemoglobin levels was observed, suggesting a possible contributions of G6PD deficiency to the reduced production of erythrocytes in affected individuals. This was, however, not related to severe malarial anaemia experienced by some children. There was no evidence of a significant association between lower parasitaemia observed in G6PD deficient individuals compared to normal ones.G6PD deficiency is more common among children from Gabon and Ghana than in Kenya. A significant association of the


Mass screening and treatment campaigns have had limited success in curbing malaria transmission, possibly as a consequence of the high prevalence of subpatent infections that are missed using the currently available field based diagnostic tools. It has been shown that subpatent malaria infections are more likely to occur in households where patent infections are identified suggesting that it may be possible to employ a more focal approach to treatment campaigns using sentinel cases as markers for the presence of a subpatent reservoir. However, it is not known which definition of sentinel case to rely on for initiation of treatment would provide optimal coverage of the parasite reservoir.In a low endemic and highly heterogeneous malaria transmission setting in the western Kenyan highlands we conducted a focal screening and treatment campaign as part of a larger cluster randomized trial. All consenting individuals under 15 years old and febrile adults residing within all households within the defined clusters were tested for malaria with a rapid diagnostic test (RDT). Temperature and blood spots on filter paper were collected from all household members (N = 2083), regardless of RDT result. The parasite reservoir was determined using nested polymerase chain reaction and gametocyte nucleic acid sequence-based amplification; multiple definitions of a sentinel case were assessed to determine which identified the largest proportion of parasitized individuals in the total population.We observed significant household clustering of parasite carriage with a 23.5% (95% CI 21.6-25.3%) increased probability of being positive for malaria by PCR if another member of the same household was also parasitaemic. Sentinel populations of RDT-positive i) febrile individuals, ii) children <5 years of age, iii) children <15 years of age and iv) children <15 years of age and all febrile individuals have variable levels of coverage. However, even with the best approach (iv), only 36.0% to 80.0% of parasitized households corresponding to 46.6% to 90.1% of parasitized individuals were identified. Successful coverage was related to background parasitaemia and was improved considerably if compounds surrounding sentinel cases were included in the intervention.Despite strong clustering at the household level, targeting compounds with parasite positive sentinel cases does not achieve complete coverage of the parasitaemic reservoir. Even in the most optimistic scenario, 20% of households or 10% of the parasitaemic reservoir will go undetected when relying on conventional malaria diagnostics in sentinel populations.


Plasmodium falciparum infection or the risk of developing febrile malaria once P. falciparum infection is established. Here we collected stool samples from a cohort composed of 195 Malian children and adults just prior to an intense P. falciparum transmission season. We assayed these samples using massively parallel sequencing of the 16S ribosomal RNA gene to identify the composition of the gut bacterial communities in these individuals. During the ensuing 6-month P. falciparum transmission season we examined the relationship between the stool microbiota composition of individuals in this cohort and their prospective risk of both P. falciparum infection and febrile malaria.In humans it is unknown if the composition of the gut microbiota alters the risk of P. falciparum infection; however, no relationship was observed between microbiota composition and the risk of developing febrile malaria once P. falciparum infection was established.Consistent with prior studies, stool microbial diversity in the present cohort increased with age, although the overall microbiota profile was distinct from cohorts in other regions of Africa, Asia and North America. Age-adjusted Cox regression analysis revealed a significant association between microbiota composition and the prospective risk of P. falciparum infection in malaria-endemic areas, potentially as an adjunct to partially effective malaria vaccines.These findings underscore the diversity of gut microbiota across geographic regions, and suggest that strategic modulation of gut microbiota composition could decrease the risk of The online version of this article (doi:10.1186/s12864-015-1819-3) contains supplementary material, which is available to authorized users. The human microbiome represents the trillions of microbes that naturally inhabit the human body, the vast majority of which reside within the gastrointestinal tract . Major aSystematic studies aimed at understanding the complex interactions between the host and microbiota have demonstrated that the composition of the gut microbiota varies with age –7, race/Plasmodium falciparum malaria, which is transmitted by Anopheles mosquitos, imposes the greatest disease burden worldwide. The World Health Organization (WHO) estimates that 3.3 billion people are at risk of malaria in 97 countries. In 2013, there were approximately 198 million cases of malaria and an estimated 584,000 deaths, 90 % of which occurred in Africa, with children under 5 years accounting for 78 % of all deaths [Among parasitic pathogens, l deaths .P. falciparum infection seems to occur rarely [P. falciparum infection and the disease it causes is complex and only partially understood [P. falciparum infection can vary greatly between and within individuals [Escherichia coli O86:B7 in the gut—are cytotoxic to α-gal-expressing Plasmodium sporozoites, and thus protect mice from mosquito-transmitted Plasmodium infection [P. falciparum infection in Malian children and adults residing in an area of intense malaria transmission [P. falciparum infection in humans. In addition, Li et al. [Plasmodium berghei resulted in altered metabolite profiles that they attributed to a disturbance of the gut microbial community caused by the parasite. However, it is currently unknown in humans if the composition of the gut microbiota modulates the risk of P. falciparum infection or the risk of developing malarial disease once P. falciparum blood-stage infection is established.In areas of intense malaria transmission, immunity to life-threatening malaria is typically acquired by early childhood, whereas older children remain susceptible to repeated bouts of febrile malaria through adolescence, eventually acquiring near-complete immunity to the disease caused by blood-stage malaria parasites by early adulthood , 19. Howr rarely . The natderstood . Indeed,ividuals . Althougividuals , 24 cleanfection . In the smission , suggesti et al.  noted thP. falciparum and the gut microbiota, we analyzed the bacterial communities in stool samples collected from a cohort of 195 Malian children and adults just prior to an intense malaria transmission season. Community composition was determined by sequencing the 16S ribosomal RNA (rRNA) gene using high-throughput next generation sequencing technology. In a prospective cohort study we examined the relationship between the gut microbiota composition of these individuals and their subsequent risk of P. falciparum infection and febrile malaria. We also compared the microbiota composition of this cohort with published studies conducted in other regions. Consistent with prior studies, we found that gut microbial diversity in this cohort increased with age, although the overall microbiota profile was distinct from cohorts in other regions of Africa, Asia and North America. Age-adjusted Cox regression analysis revealed a significant association between the microbiota composition before the malaria season and the prospective risk of P. falciparum infection during the ensuing malaria season, while no relationship was observed between microbiota composition and the risk of developing febrile malaria once P. falciparum infection was established.To investigate the potential interplay between P. falciparum blood-stage parasites by PCR at enrollment when stool was also collected. The prevalence of intestinal helminths at enrollment was near zero, consistent with the mass-drug treatment program in this region of Mali, whereas the prevalence of Schistosoma haematobium in the urine was 7.4 %, mostly among adolescents and adults. The prevalence of sickle cell trait (HbAS) was 8.5 %. Detailed demographic and clinical data stratified by age are shown in Table From May 11 – May 31, 2011 (end of 6-month dry season) we enrolled 695 healthy individuals in this cohort study. Stool was collected from all subjects at the time of enrollment. A subset of 200 individuals was randomly selected in an age-stratified manner for stool microbiota analysis . The taxonomic assignments were used to generate a sample-taxa count matrix. In the combined dataset, Bifidobacterium, Streptococcus, and family Ruminococcaceae unclassified were the three most abundant taxonomic groups, accounting for 13.7 %, 12.9 %, and 12.4 % of sequences, respectively.Sequence data were successfully generated from 195 out of the 200 stool samples. After processing the raw sequence data, the combined dataset consisted of 1,331,167 sequences. However, not all sequences could be confidently assigned taxonomy to the genus level; therefore we denote these sequences by appending the tag Operational Taxonomic Units (OTUs) were identified in these data by clustering sequences at 97 % sequence identity, and were used to compute microbial community diversity  in these samples. Subsequently, the relationship between diversity and age was assessed using a linear model. This analysis revealed that diversity increased with age in this cohort Fig. ; a similn = 133) and DC2 (n = 62) were significantly different in terms of age  and taxonomic profiles , but had similar gender distributions. Specifically, the average ages of individuals in DC1 and DC2 were 10.3 years and 1.3 years, respectively. The most abundant taxa in the older DC1 group were family Ruminococcaceae unclassified (8.7 %), family Lachnospiraceae unclassified (8.5 %), and Faecalibacterium (7.4 %), while the most abundant taxa in the younger DC2 group were Bifidobacterium (17.1 %) and Streptococcus (11.6 %)  modeling framework  to identterium 7. %, whileMaterial and Methods. The four datasets were analyzed based on taxonomies of their constituent samples. DMM modeling identified 9 Dirichlet components (CC1 – CC9)  compared to CC5 . The HMP samples were predominantly split across CC4 and CC9, both of which were dominated by Bacteroides, but at different abundance levels . The MSD cohort was mostly split across CC1, CC3, and CC8, all of which were dominated by Prevotella to varying degrees (8 % in CC1 and 70 % in CC3) and family Enterobacteriaceae unclassified (9.6 % in CC8). CC7 consisted of Malawi samples and was also dominated by Prevotella (21 %).Next, we compared the gut microbiota composition of the Mali cohort with data from the Malawi, HMP, and MSD microbiome studies described in onents CC – CC9  or uninfected (n = 140). An NMDS ordination of the samples along with their P. falciparum infection status at the end of the dry season is shown in Fig. Bifidobacterium (3.6 %). Together, these findings suggest that age may be a stronger predictor of gut microbiota composition than P. falciparum infection status at the end of the 6-month dry season .In a cross-sectional analysis at the end of the 6-month dry season, a period of negligible malaria transmission , we firscted n = 40. An NMcted n = 40. An NMcted n = 40. An NMP. falciparum blood-stage parasites during the ensuing 6-month malaria season. For this analysis we only included subjects who were not infected with P. falciparum (by PCR) before the malaria season and who had completed study follow-up through the end of the ensuing 6-month malaria season (112 subjects). DMM modeling of the microbiota data obtained from these P. falciparum uninfected subjects identified two Dirichlet components FM1 (38 subjects) and FM2 (74 subjects). These two sample groups had distinct taxonomic profiles  and Streptococcus (10 %) were the most abundant taxa in FM1 while family Ruminococcaceae unclassified (9.3 %), and family Lachnospiraceae unclassified (9.1 %) were the most abundant taxa in FM2. In addition to these taxa, other taxa including Faecalibacterium, Blautia, order Clostridiales unclassified, family Prevotellaceae unclassified, and Escherichia/Shigella were differentially abundant across the two sample groups . The average age of subjects in FM1 and FM2 was 1.4 years and 9.1 years, respectively. During the ensuing malaria season, incident (new) P. falciparum blood-stage infections in these subjects were detected through bi-weekly PCR analysis of fingerprick blood samples. We compared the time to first P. falciparum infection in the FM1 versus FM2 sample groups (irrespective of whether the infection caused symptoms) by generating Kaplan-Meier plots and performing log-rank analysis .Next, we examined the relationship between the stool microbiota composition present before the malaria season and the prospective risk of becoming infected with nts FM1 3 subjectsassified .3 %, andion Fig.  for FM1 P. falciparum blood-stage parasites during the ensuing malaria season. Again, for this analysis we only included subjects who were PCR negative for P. falciparum infection before the malaria season and then became infected with P. falciparum during the ensuing malaria season, as detected through bi-weekly PCR of fingerprick blood samples. Malaria immune subjects (n = 49) were defined as those whose P. falciparum infections never progressed to febrile, symptomatic malaria despite weekly active clinical surveillance; whereas malaria susceptible subjects (n = 63) were defined as those whose P. falciparum infection progressed to a febrile malaria episode . On the basis of the DMM modeling of the microbiota data described above, the same FM1 and FM2 sample groups had similar proportions of malaria immune and susceptible subjects , suggesting that gut microbiota composition is not associated with the risk of febrile malaria once a P. falciparum blood-stage infection is established. On the basis of the same Dirichlet sample groups FM1 and FM2, we also generated Kaplan-Meier plots and performed log-rank analysis for time to first febrile malaria episode  we founP. falciparum infection after accounting for age and other potential confounders. The gut microbiota of subjects at lower risk of P. falciparum infection (FM1) contained a significantly higher proportion of Bifidobacterium and Streptococcus compared to subjects at higher risk of P. falciparum infection (FM2). This finding raises the possibility that modulation of the microbiota with pre- and/or pro-biotic nutritional supplementation that promotes the growth of commensal organisms like Bifidobacterium, particularly in the neonatal period [Bifidobacterium [Using a DMM framework we identified two subject groups with distinct microbiota profiles (FM1 and FM2) that exhibited statistically significant differences in the prospective risk of l period , 33, mayacterium –36. CleaEnterobacteriaceae Escherichia/Shigella compared to subjects at higher risk of P. falciparum infection (FM2)  - an intriguing finding in light of a recent mouse study in which anti-α-gal antibodies induced by the presence of α-gal-expressing Escherichia coli in the gut, were cytotoxic to α-gal-expressing Plasmodium sporozoites in the skin, thus protecting mice from Plasmodium infection [Enterobacteriaceae express α-gal [Enterobacteriaceae are associated with the risk of P. falciparum infection. Moreover, additional studies are needed in various malaria-endemic areas to determine if the findings here can be replicated, since the association we observed between microbiota composition and P. falciparum infection risk may have occurred by chance or may be confounded by other factors not measured in this study.Also of potential interest, the gut microbiota of FM1 subjects contained a significantly higher proportion of the nfection . Becausess α-gal  we are uP. falciparum blood-stage infections were established, possibly due to a lack of statistical power. However, we observed clear changes in the microbiota composition over the age range during which clinical immunity to febrile malaria is slowly acquired in areas of intense malaria transmission [Importantly, we did not observe a relationship between the gut microbiota composition and the prospective risk of developing febrile malaria once smission , suggestP. falciparum infection risk suggests that strategic modulation of gut microbiota composition could decrease P. falciparum infection risk in malaria-endemic areas, potentially as an adjunct to partially effective malaria vaccines.The findings of this study underscore the diversity of the gut microbiota with age and across geographic regions. In addition, the preliminary finding of an association between gut microbiota composition and The Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Sciences, Technique and Technology of Bamako, the Institutional Review Board (IRB) of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and of the J. Craig Venter Institute (JCVI) approved this study. Written, informed consent was obtained from adult subjects and from the parents or guardians of participating children.P. falciparum malaria occurs during the rainy season in this region of Mali from June through December. In May 2011, we enrolled 695 healthy individuals aged 3 months to 25 years in a cohort study to investigate mechanisms of naturally acquired immunity to malaria using systems biology approaches. A detailed description of the cohort has been published elsewhere [This cohort study was conducted in Mali in the rural village of Kalifabougou which has a population of 4,394 and is situated ~40 km northwest of the capital Bamako. Bambara is the predominant ethnic group and ~90 % of residents engage in subsistence farming. Kalifabougou is in the savanna ecoclimatic zone where annual rainfall is 800–1,200 mm/year. Intense transmission of lsewhere . As partlsewhere  all resiPlasmodium parasitemia detected by light microscopy were treated according to the National Malaria Control Program guidelines in Mali. Thick blood smears were stained with Giemsa and counted against 300 leukocytes. Parasite densities were recorded as the number of asexual parasites/μl of blood based on a mean leukocyte count of 7500 cells/μl. Each smear was read in a blinded manner by two certified microscopists. The research definition of clinical malaria was parasitemia of ≥2500 parasites/μL, an axillary temperature of ≥37.5 °C within 24 h, and no other cause of fever discernible by physical exam. To detect asymptomatic Plasmodium infections during the 7-month study period, blood was collected by fingerprick onto filter paper at bi-weekly scheduled visits. At the end of the study period, stored filter papers were analyzed by PCR chronologically from enrollment onwards until the first P. falciparum infection of the malaria season was detected for each individual. A detailed description of the PCR protocol was published elsewhere [After enrollment, individuals were followed during the ensuing malaria season for 7 months. Symptomatic malaria episodes were detected prospectively by self-referral and through weekly active clinical surveillance. All individuals with signs and symptoms of malaria and any level of lsewhere .S. haematobium eggs were quantified by microscopy after filtration of fresh urine samples with Nytrel filters (Vestergaard Frandsen). Schistosoma mansoni and other geohelminth eggs were detected by microscopy of duplicate fresh fecal thick smears using the Kato-Katz method [Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis) was performed as previously described [Stool and urine samples were collected at enrollment. z method . Aliquotescribed . IndividThe sub-cohort that is the primary focus of this study was comprised of 200 subjects—106 females and 94 males ranging in age from 3 months to 25 years. Stool samples were collected at study enrollment in May 2011 just prior to the six-month malaria season. Stool samples were stored in −80 °C freezers in Mali and shipped to the U.S. on dry ice for analysis. Microbial DNA for 16S rDNA gene sequencing was isolated from stool samples using enzymatic lysis followed by phenol-chloroform isoamyl alcohol extraction and ethanol precipitation. Standard Roche protocols were used for sample preparation.The 16S rRNA gene amplicons were quantified and pooled in equal mass ratios such that amplicons derived from samples to be analyzed could be multiplexed in a single Roche-454 sequencing run. Established 454 FLX sequencing methods (Roche) that were developed for the HMP, including universal PCR primers with unique barcode identifiers, were used to amplify and sequence the hypervariable regions (V1-V3) of the bacterial 16S rRNA gene .http://metagenomics.anl.gov/). The Malawi cohort was part of accession “mgp401” (http://metagenomics.anl.gov/linkin.cgi?project=401) [http://www.hmpdacc.org/). The subjects in the HMP cohort ranged in age from 18 to 40 years and 73 were females and 67 males. Data for the MSD study were downloaded from SRA at NCBI and were available under the bioproject accession PRJNA234437. In the MSD study sequence data was generated (using 454 FLX sequencing) from the V1-V2 region of the 16S rRNA gene from DNA extracted from stool samples. Healthy control samples in the MSD study were identified from the full data set using the available study meta-data and only these  were included in the comparison here. The healthy control samples in the MSD study were collected from individuals residing in Bangladesh (117), The Gambia (134), Kenya (140) and Mali (102). These individuals were all under 5 years of age (ranging from 1 to 58 months) and no gender information was available from the study metadata.16S rRNA gene sequence data generated from stool samples collected as part of three other studies were included in the analysis component of this study for comparative purposes only. These additional studies included a cohort in Malawi , the HMPect=401) . This sthttp://www.hmpdacc.org/HM16STR/). OTUs at 97 % sequence identity were computed for the Malian cohort in order to assess sub-genus diversity and structure. The comparisons of the present Malian cohort to the other three datasets were carried out only at the genus level.For the 454 data, the raw flowgram files (Standard Flowgram Format) were deconvolved using the sample barcode information, and the barcodes and primers were trimmed subsequently. All processing of the 454 and Illumina sequences from the various studies were carried out using mothur v1.28 [For the Malian cohort, from the initial 200 samples, 195 samples were successfully sequenced and each sample yielded more than 1,000 16S rRNA sequences after barcode and primer trimming. Together, the samples resulted in 1,500,243 sequences that were used for downstream analysis. Reads were aligned against a reference template, trimmed (keeping only > =100 bp sequences), and checked for chimeras. The resulting output consisted of 1,331,167 sequences. These were assigned taxonomy down to the genus level using mothur's implementation of the RDP classifier (using a bootstrap confidence threshold of 80 %). For OTU analysis, this sequence set was processed further by alignment column trimming, in which the required length of trimmed sequences was > =200 bp. This yielded 968,362 total sequences. These were clustered at 97 % sequence identity to generate OTUs.et al. [K, the number of components, and subsequently, the K with the best model fit using the Laplace approximation to the negative log model evidence was chosen as the optimal value for that dataset. The Dirichlet component means were calculated from the Maximum Posterior Estimates (MPE) of the model hyperparameters [The taxonomic assignments of the 16S sequences were used to generate sample-taxa count matrices. These matrices were analyzed using a DMM modeling framework  to identet al. . The prorameters .Plasmodium, Kaplan-Meier curves were also used to estimate the probability of remaining free of blood-stage P. falciparum infection. Individuals who were infected with Plasmodium malariae were censored at the time of infection. Log-rank analysis was used to test the significance of differences in time to first febrile malaria episode and time to first P. falciparum infection between DMM clusters. A Cox proportional hazards model was used to estimate the risk of P. falciparum infection between DMM clusters and included the following potentially confounding variables: age (per one year increase), HbAS, gender, mild anemia at enrollment, S. haematobium infection, presence of splenomegaly at enrollment (Hackett score >1) and distance from home to river . This combination of variables met the assumptions of proportional hazards. Of these variables, HbAS has been shown to affect the risk of P. falciparum infection [P. falciparum infection risk (unpublished data). Statistical significance was defined as a 2-tailed P value of <0.05. We performed all analyses in R version 3.1.1 (http://www.R-project.org).For continuous variables we compared the differences between group medians with the Kruskal-Wallis test Table ; for binnfection , 47, andThe data sets supporting the results of this article are included within the article ). The sequence data for the Malian cohort are available at NCBI’s Sequence Read Archive under BioProject PRJNA285808. These data are also available at MG-RAST under Project 4793.


Background. Malaria and typhoid fever are major public health problems in tropical and subtropical countries. People in endemic areas are at risk of contracting both infections concurrently. Objectives. The study was aimed at determining the prevalence and associated risk factors of malaria, typhoid, and their coinfection among febrile patients. Methods. A cross-sectional study was conducted on 200 febrile patients suspected for malaria and/or typhoid fever from April to May, 2013, at Ayinba Health Center, Northwest Ethiopia. Blood samples were collected for blood culture, Widal test, and blood film preparation. Data were analyzed using SPSS version 20 statistical software. Results. The prevalence of malaria was 36.5% (n = 73). Among these 32 (43.8%), 30 (41.1%) and 11 (15.1%) were positive for P. falciparum, P. vivax, and mixed infections, respectively. The seroprevalence of typhoid fever was 38 (19%), but 1 (0.5%) with blood culture. Malaria typhoid fever coinfection was 13 (6.5%). 2–5-year-old children and poor hand washing habit were significantly associated with malaria and typhoid infection, respectively (P < 0.05). Conclusions. The prevalence of malaria and typhoid fever was found high. Further studies should be done on the other determinants of malaria and typhoid fever coinfection in different seasons and different study areas. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Approximately half of the world population is at risk of malaria. Most of malaria cases and deaths occur in sub-Saharan Africa. According to the World malaria report 2011, there were about 216 million cases of malaria and an estimated 655,000 deaths in 2010 [Malaria is one of the febrile illnesses and the most common fatal disease in the world caused by one or more species of plasmodium. These are P. falciparum and P. vivax are the dominant species of malaria in Ethiopia, with 60% and 40% relative frequencies, respectively. Plasmodium falciparum is a predominant species in endemic areas and causes complicated disease and death in the country [Malaria is the most communicable disease in Ethiopia and it accounts for about 30% of the overall disability adjusted life years lost. Approximately 68% 54.2 million) of the total population of 78 million lives in malaria risk areas. million  Salmonella enterica serotype typhi (S. typhi) and Salmonella enterica serotype paratyphi  are species that cause typhoid fever. S. typhi is the most common serotype of salmonella that causes typhoid fever [Typhoid fever (enteric fever) is a systemic prolonged febrile illness caused by certain Salmonella serotypes.id fever –6. The eid fever . Poor diid fever , 5.Malaria and typhoid fever are a major public health problem in tropical and subtropical countries caused by very different organisms, protozoa and Gram negative bacilli, respectively, and transmitted via different mechanisms –8. Peopl P. vivax and P. falciparum) and covers an area of 1,270 Km2. It is 27 km away from ancient city Gondar. All febrile patients (age ranged 2–80 years) suspected for malaria and/or typhoid fever who had not taken antimalarial drug and/or antibiotics within 2 weeks were included. Patients with underlying diseases were excluded from the study.The study was conducted from April 2013 to May 2013 at Ayinba Health Center, Dembia district, Northwest Ethiopia. The altitude of the district ranges between 1,750 and 2,100 m above sea level. It has a population of more than 300,000 and the majority of its population depends on subsistence agriculture. The district is malarious  and flagellar (H) antigens kits of S. typhi ). Antibody titration was performed for slide reactive samples. Antibody titer of ≥1 : 80 against O and H antigen of S. typhi was taken as a cut of value based on the manufacturer instruction. The blood smear was read at the health center by laboratory technicians and the result was reported. All blood films were reread by experienced microscopist at the University of Gondar Hospital laboratory who was blinded to initial results. Discrepancies occurred in the result by the two readers were solved by using the third experienced microscopist.Data on the sociodemographic and clinical characteristics of the study participants were collected using a pretested structured questionnaire by interview. After interview, 10 mL blood sample was collected from adult patients by experienced laboratory technologist. Then, 7 mL blood was inoculated immediately to 45 mL Brain Heart Infusion broth. Similarly, 3-4 mL blood was collected from children and 1.5–2 mL blood was inoculated to 9 mL broth to isolateP value <0.05 was considered statistically significant.The data was cleaned, edited, checked for completeness, entered to Epi Info version 3.5.3, and exported to SPSS version 20 for analysis. Chi-square and odds ratio (OR) by logistic regression were calculated to determine associated factors. Ethical clearance was obtained from University of Gondar, School of Biomedical and Laboratory Sciences research and ethical committee. Permission was obtained from Dembia Woreda Health Office and Ayinba Health Center. Written informed consent was obtained from each of the volunteer study subjects or guardian of children. Positive results were given for nurses working in the health center for treatment according to the national treatment guideline.Sociodemographic Data. A total of 200 febrile patients suspected for malaria and/or typhoid fever were included in this study. About 60% of the study participants were males. The mean age was 24.24 ± 13.4 years and majority of the patients (41.5%) were within the age range of 12–25 years, and most of the patients were farmers (71.5%), rural residents (89%), and illiterate (61.5%) ( (61.5%) .Prevalence of Malaria. Malaria was the most prevalent disease in the study area. From the total 200 febrile patients 73 (36.5%) were malaria positive. Of them, 32 (43.8%) were positive for P. falciparum, 30 (41.1%) were positive for P. vivax, and the remaining 11 (15.1%) were positive for both P. falciparum and P. vivax. The positivity rates of P. falciparum and P. vivax were 51.2% and 48.8%, respectively  patients had antibody titers of ≥1 : 80 for both O and H antigens; of them, 7 (18.4%) had ≥ 1 : 320 titers. Different gram negative organisms were grown on the blood culture but there was only one growth of S. typhi  were with P. vivax, and 2 (15.4%) were with mixed infection. The prevalence of coinfection using blood culture was 1 (0.5%).The titration result showed that the prevalence of coinfection was 13 (6.5%). Of them 8 (61.5%) were coinfected withP = 0.04). The chi square analyses showed that this age group was significantly associated with malaria (P = 0.04). There was no significant association of malaria and typhoid fever with sex, residence, occupation, and educational background (P > 0.05) (P > 0.05)  . Clinica > 0.05) .P = 0.01, OR = 2.893, 95% CI = 1.245–6.72)  .The result of this study is comparable with the reports from Akoko State, Nigeria, 37.6%  and Imo  P. falciparum and P. vivax were almost similar . But according to the Federal Ministry of Health report the relative frequency of P. falciparum and P. vivax was 60% and 40%, respectively [ P. falciparum than P. vivax. In case of P. vivax the dormant stage of the parasite can relapse at any time and relatively maintain its prevalence in the community.The positivity rates ofP = 0.337). While other studies showed in Sierra Leone, females (53.4%) are more affected than males (46.6%) [P = 0.04). This might be due to low immune response against malaria infection, inappropriate use of bed nets, and in appropriate use of antimalarial drugs in case of children.In this study the prevalence of malaria was higher in males 64.4% than females 35.6% but there was no statistically significant association ( (46.6%) . This miThe prevalence of the typhoid fever using Widal titration test was comparable with the study in Ebonyi, 21.2% , and lesP = 0.582); however, other previous studies in Sokoto, Nigeria, showed that the frequency of typhoid fever was 29.4% among males and 22.9% among females [The frequency of typhoid fever was greater in females (22.5%) than males (16.7%), but not statistically significant .Because of the high prevalence of typhoid fever and malaria in the tropics, coinfections are common . The higMalaria was the most prevalent disease among febrile patients in the study area. There was a substantial result discrepancy among Widal test and blood culture for the diagnosis of typhoid fever. Poor hand washing habit was significantly associated with typhoid fever. Further studies should be done on the other potential risk factors of malaria and typhoid fever coinfection in different seasons and different study areas. The community should be encouraged to use latrine to reduce the burden of high prevalence of typhoid fever infection in the area. The continued development of better diagnostic tools for both malaria and typhoid fever is still crucial.


Although malaria is declining in many countries in Africa, malaria and anaemia remain frequent in children. This study was conducted to assess the relationship between malaria parasitaemia, intestinal worms, and anaemia, in children <5 years living in low transmission area in Senegal. A survey was carried out in 30 villages in the central part of Senegal. A two-level random cluster sampling technique was used to select study participant. Children <5 years were enrolled after informed consent. For each child, blood thick and smear tests were performed, haemoglobin concentration was measured with HemoCue, and stool samples were collected and examined using the Ritchie technique. A total of 736 children were recruited. Malaria parasite prevalence was 1.5% (0.7–2.6); anaemia was found in 53.4% (48.2–58.9), while intestinal parasites and stunting represented 26.2% (22.6–30.2) and 22% (18.6–25.5), respectively. In a logistic regression analysis, anaemia was significantly associated with malaria parasitaemia (aOR= 6.3 (1.5–53.5)) and stunting (aOR = 2 (1.2–3.1)); no association was found between intestinal parasites and anaemia. Malaria and anaemia remain closely associated even when malaria is declining. Scaling up antimalarial interventions may contribute to eliminate malaria and reduce the occurrence of anaemia among children. Malaria is a major public health problem in African region. According to the World Health Organization (WHO), in 2010, there were an estimated 149 to 274 million cases and 537,000 to 907,000 deaths worldwide [Anaemia also remains a major public health problem, not least in malaria endemic areas, where it is primarily seen in young children in areas with stable malaria, but also in adults in malaria unstable areas , 5. ReceIn tropical regions, there are multiple aetiologies to anaemia and intestinal parasites as well as malnutrition can play a major role in anaemia occurrence , particuThis study was aiming (i) to assess the prevalence of malaria parasitaemia, intestinal parasites (IP), anaemia, and malnutrition among children <5 years and (ii) to explore the relationship between malaria, intestinal parasites, malnutrition, and anaemia.The study was conducted prior to the implementation of an impact study evaluating the impact on malaria burden of a community-based malaria control strategy including effective case management (using RDT and ACT treatment) and IPTc at Lamarame in Senegal, where a demographic surveillance system recently has been established.The study was conducted at the Lamarame health post located in the Keur Socé, a rural community, at 17 Km from the city of Kaolack in central Senegal, and 200 Km from Dakar. The Lamarame health post is in the Ndoffane health district. It covers 34 villages located within a radius of 8 Km from the post, with a total population of about 10,000 inhabitants.Konate et al. unpublished data). Many programs aiming to reduce child health problems are being promoted in this area. These programs included two yearly vitamin A supplementation, and deworming with Mebendazole, in December and June of each year for children aged 1 to 5 years.Malaria is endemic in this area with a seasonal pattern of transmission peaking during the rainy season (July–November); entomological inoculation rates range from 9 to 12 infective bites/human/year , power at 90% (beta 10%), and being multiplied by 2 for an design effect.A code was given to every child after parents' informed consent. Each eligible child was examined by a physician prior to a biological assessment which included blood and stool samples. The mother was interviewed directly concerning the child's symptoms as well as well as sociodemographic characteristics using a standard questionnaire. Data obtained from physical examination and parents interview were assigned on a case report form (CRF).Z-score was used to denote underweight, while height for age Z-score was used as an indicator of stunting. The Z-scores were calculated based on the median values of the National Centre for Health Statistics (NCHS) reference population, United States.Children were weighed by a trained assistant, without their shoes, using Seca scales; height was measured to the nearest 0.1 cm using a calibrated scale and a sliding head piece. Weight-for-age μL using the following formula: numbered parasites ×8000/200 assuming a white blood cell count of 8000 cells per μL. Absence of malaria parasite in 200 high power ocular fields of the thick film was considered as negative.Blood samples were collected using finger prick blood. The first drop was used to prepare thick and thin smears for the diagnosis of malaria. Thick and thin smears were stained with Giemsa and read by a laboratory technician. Malaria was defined as any asexual parasitemia detected on a thick or thin blood smear. Parasitemia was numbered and expressed by number of trophozoites/The second drop of finger prick blood was drawn into a microcuvette for Hb determination (g/dL) using HemoCue machine (HemoCue Hb 301). Moderate and severe anaemia were defined as hb concentration below 11 g/dL and 8 g/dL, respectively.Fresh stools samples were collected into wide mouth screw-cap clean containers. Fecal samples were examined for the detection of intestinal parasite using the Ritchie technique. Intestinal parasite was recorded positive by the presence of helminthes and/or protozoans in the faces.Z-scores for weight-for-age (underweight) and height-for-age (stunting) were derived using Epinut Anthropometry. Children who had Z-scores below −2 standard deviation (SD) of the NCHS reference population median were considered to be malnourished.Data were entered in Excel software and analysed using STATA 10 software. To assess the nutritional status, data were transferred into Epi Info 3.04 d. The For descriptive data, percentage was used to assess the prevalence of each outcome. Proportions were compared using chi-square test or the Fisher exact test ; significance level of the different tests was 0.05 two-sided. A stepwise logistic regression was done for the determination of risk factors possibly associated with anaemia and under-nutrition. The validity of the final models was tested using the Hosmer-Lemeshow goodness of fit test.Seven hundred and thirty-six children  aged 6 months–5 years with a mean age of 2.5 ± 1.3 years participated in this study .Plasmodium falciparum was 1.5%. Moderate anaemia and severe anaemia were found in 53.4% and 12.5% of the children, respectively. A total of 193 (26.2%) children were found with at least one intestinal parasite. Parasites species were represented by Giardia intestinalis 15.6%, Entamoeba coli 10.9, Hymenolepis nana 1.9%, Ascaris lumbricoïdes 0.4%, Enterobius vermicularis 0.4%, and Trichuris trichiura 0.1%. Children with two intestinal parasites species represented 3,01% (22 children); one child  was coinfected with 3 intestinal parasites species. The overall prevalence of stunting and underweight was 22% and 16.3%, respectively ) (Anaemia (Hb < 11 g/dL) was significantly associated with age range from 12 to 23 months (aOR = 3.7 (1.7–7.9)), malaria parasite (aOR = 6.3 (1.5–53.5)), and birth-order higher than 3 (aOR = 1.6 (1.1–3.5)). Stunting was also significantly associated with anaemia in children under 5 years of age (aOR = 2 (1.2–3.1)). There was no statistically significant association between isolated intestinal parasites, such as 8–1.58)) .Giardia intestinalis, Entamoeba coli, Hymenolepis nana, Enterobius vermicularis, and Ascaris lumbricoïdes ), severe anaemia (OR 3.5 (1.9–6.4)), residence zone (OR 3.5 CI 95% (1.6–8.1)), and birth order >3 (OR 2.9 (1.3–6.6)). There was no statistically significant association between malaria and stunting (OR 1.2 (0.3–0.5)) as well as isolated intestinal parasites such ricoïdes .Factors associated with underweight were represented by moderate anaemia (aOR 1.7 (1.1–2.8)), severe anaemia (aOR 4.4 (2.3–8.4)), and age range from 24 to 35 months (aOR 8.9 (1.2–70.6)) .Malaria, anaemia, intestinal parasitic infections, and malnutrition remain serious public health problems especially in developing countries , 10. We P. falciparum infections (1.5%), while a high level of anaemia was observed. Despite the low prevalence of malaria parasitoemia, anaemia was more frequent in children with P. falciparum infections (OR 6.3). It, thus, appears that malaria is still closely associated with anaemia in this area, even in the context of the decline of malaria prevalence. A survey conducted in January 2009 (one year before) in a neighbouring area  showed a higher prevalence of malaria (7.2%) [National Malaria Control program annual report 2009). Scaling up antimalarial strategies such as early case detection and treatment, intermittent preventive treatment as well as long-lasting insecticide treated nets may even contribute to malaria elimination in this area and significantly reduce anaemia as a public health problem in rural Senegalese areas with similar disease patterns. Children with a birth order greater than three had an increased risk of developing anaemia. In other studies, high family size was associated with anaemia and undernutrition of children [Our study conducted in a malaria endemic area showed an unexpectedly low prevalence of a (7.2%) . These rchildren .Giardia intestinalis was the most frequent parasite (15.6%). The observed prevalence of Giardia in this study is consistent with those described in other countries [Giardia intestinalis on growth and health of children has been shown by several studies [Giardia intestinalis and anaemia, as well as malnutrition, this parasite is known for its ability to induce diarrhoea [Lamarame health post report-unpublished data). This can explain the low level of prevalence of helminthic infections. While ensuring an effective action against helminthic infections, mass administration of mebendazole does not act against protozoan infections. Albendazole can have an effective action against both protozoan and helminthic infections and can be a suitable molecule in mass deworming programs, particularly in areas where Giardia is frequent. Thus, replacing mebendazole by albendazole during a mass deworming programme could further reduce the frequency of helminthic infections as well as protozoan infections and, consequently, the occurrence of diarrheal disease and malabsorption syndrome.The study showed a high prevalence of intestinal parasite carriage (26.2%); ountries , 14. Theparasite .6%. The iarrhoea  and malaiarrhoea . Mass deMalnutrition remains frequent in children under 5 years with a prevalence of 22% of stunting and 16.3% of underweight. In 2005, the fourth demographic and population health survey found an average rate of stunting in rural areas of 20.6%, and the prevalence of underweight was 21.6% . Our datStudy Limitations. This study, conducted in a rural Senegalese area, has provided useful data for the control of some frequent tropical diseases. However, sociocultural and behavioural factors which may have a relationship with the occurrence of anaemia and/or malnutrition have not been evaluated in this study, in particular the intake of macro- and micronutrients and the patterns of weaning and supplementary feeding.Anaemia, intestinal parasites, and malnutrition are still frequent in the area of Lamarame, while malaria is declining in this part of Senegal. Despite the decrease of malaria in this area, malaria is still a contributing factor to anaemia in this area, due to the close association between malaria and anaemia. The combination of several antimalarial interventions may contribute to eliminating malaria in this region and reducing the occurrence of anaemia among children.