Fast and reliable diagnosis of malaria requires confirmation of the presence of malaria parasites in the blood of patients with fever or history suggestive of malaria; hence a prompt and accurate diagnosis of malaria is the key to effective disease management. Confirmation of malaria infection requires the availability of a rapid, sensitive, and specific testing at an affordable cost. We compared two recent methods  and 114 (43.4%), respectively. Compared to GM reference standard, the sensitivities of the PT and BN RDT were 100% (95% CI: 96.6-100) and 97.2% (95% CI: 92.0-99.4), respectively, specificities were 97.4% (95% CI: 93.6-99.3) and 93.6% (95% CI: 88.5-96.9), respectively. There was a strong agreement (kappa) between the applied test methods . The average turnaround time per tests was 17 minutes.In 107 (40.7%) of 263 study participants, Plasmodium cannot be identified.In this study two rapid malaria tests, PT and BN RDT, demonstrated a good quality of their performance compared to conventional GM. Both methods require little training, have short turnaround times, are applicable as well as affordable and can therefore be considered as alternative diagnostic tools in malaria endemic areas. The species of  Half of the world's population is at risk of malaria, with an estimated 243 million cases worldwide out of which 85.6% cases occur in Africa . The Wor® (PT)  and the recently established Binax Now® Malaria Rapid Diagnostic Test (BN RDT)  in malaria diagnosis among children from an endemic area using Giemsa stain microscopy as the reference standard. In a first report we compared PT with GM in a separate collective of patients using a real time PCR assay as reference standard [Another major contributing factor is that the laboratory diagnosis of malaria has up to now relied nearly exclusively on light microscopy which is a valuable technique when performed correctly but unreliable and wasteful when poorly executed. A better utilization of microscopy and the development of alternative diagnostic techniques could substantially improve malaria control . This ststandard . In this2 with an estimated population of about 142,000 (projection from 2000 Population Census). Over 40% of the population is under 15 years and over 50% are under 20 years of age. The microscopically confirmed incidence of malaria in children under five who reported at the under-five clinic of the Agogo hospital was 265 per thousand in 2008 .The study was conducted at the Agogo Presbyterian Hospital. It is located in the rural Asante Akim North District of the Ashanti Region of Ghana, West Africa and is the principal hospital of the district. The district is located in the eastern part of Ashanti Region and covers a land area of 1,160 kmBetween June and December 2008 (i.e. from the beginning of the rainy season to the early dry season), 263 cases were enrolled into the study. Samples were obtained from children attending the pediatric clinic of the hospital for the first time with fever or a history suggestive of malaria but without previous antimalarial treatment within two weeks of attending the hospital. Patients diagnosed with malaria were treated with Artesunate - Amodiaquine or Artemether - Lumefantrine as second line treatment representing the Ministry of Health/National Malaria Control Programme approved Artemisinin-based combination therapy for the treatment of uncomplicated malaria in Ghana.Ethical approval for the study was obtained from the Committee on Human Research, Publication and Ethics (CHRPE) of the School of Medical Sciences, KNUST-Kumasi. After information and appropriate explanations parents or legal guardians of all participants willing to participate in the study had to give their consent by appending their signature or thumbprint to the informed consent form before any study related procedures were done. Internationally accepted Good Clinical and Good Laboratory practices were applied in all procedures.A maximum of 0.5 ml capillary blood was taken from each child into an EDTA tube labelled with the patient's barcode and pathology number. These samples were examined immediately using the three methods . All three tests were done immediately and examined by well-trained and competent laboratory staff. For GM, the test was examined independently by two expert microscopists in two different rooms with each not knowing the result of the other. In the case where the two microscopic results were discordant, a third expert reader was employed by an independent slide coordinator. The result of the third reader was then considered as the final result. Written results were communicated immediately to the clinicians.Thick and thin blood films were prepared using standardized blood volumes of 10 μl and 2 μl, respectively. They were air-dried (thin film was fixed in absolute methanol), heat fixed and both stained with 10% Giemsa working solution for 12 minutes. A malaria blood film was considered negative after 100 high power fields (HPF) has been examined and no parasite observed. If parasites were observed, asexual malaria parasites were counted against 200 white blood cells (WBCs) on the thick film but all parasites in a final HPF were counted even if a count of 200 WBCs had been exceeded. The parasite count per microliter of blood was obtained by using the formula: (Parasite count/200WBC) × Absolute WBC count . If para®  is a microscope that has two light sources: in addition to the option of normal light microscopy, a fluorescence light detection through incident UV light can be applied. It uses readily-prepared and ready to-use test slides labelled with an unspecific DNA-binding fluorescent dye (4'-6-Diamidino-2- phenylindole (DAPI); emission 443 nm) that detects intraerythrocytic Plasmodium DNA [The Partec CyScopedium DNA . 5 μl ofP. falciparum specific histidine rich protein 2 (HRP-2) and a panmalarial aldolase common to all four human pathogenic species in venous and capillary blood specimens [et al. [The BN RDT  is an immunochromatographic membrane assay that uses monoclonal antibodies to detect pecimens . This te [et al. . 15 μl o [et al.   for analysis. Sensitivities, specificities, positive predictive and negative predictive values were determined for the various tests and compared with one another. A p-value less than 0.05 was considered significant in the comparisons. Kappa (k) values expressed the agreement beyond chance [Data were double-entered into a predesigned electronic database using Epi info version 6.04 dfr  and cleaned regularly. Data was exported to d chance  and wereFrom June to December 2008 a total of 263 children  up to 16 years, admitted with fever or a history suggestive of malaria were included in the study with a mean ± SD age of 7.3 years ±3.4 years (range: 3-16 years).Plasmodia in 107 blood films (40.7%) with no discordant readings between the two independent microscopists. Out of these positive films 105 slides (98.1%) were positive with mono-infection of P. falciparum (Pf), of which 2 slides showed Pf gametocytes in addition to Pf trophozoites, 1 case (0.95%) each was positive with mono-infection of P. malariae (Pm) and P. ovale (Po), respectively. 156 films (59.3%) were negative of which 2 films (1.3%) were positive for Pf gametocytes only. The geometric mean parasite count was 631.8 parasites/μl (range: 50.0 - 8320.0 parasites/μl) with a turnaround time of 25 minutes per test.Overall, GM expert microscopy Table  detected® revealed 111 slides to be positive for malaria and 107 slides had concordant results with GM. Thus 4 results were false positive with a mean misdiagnosed parasite count of 32.3 parasites/μl, range: 22.0-43.0 parasites/μl. The geometric mean parasite count was 447.2 parasites/μl (range: 40.0 - 4530.0 parasites/μl) and significantly lower than that obtained from the GM (p < 0.001). The turnaround time per test was 5 minutes.The Partec Rapid Malaria TestP. falciparum mono-infection whilst 52 tests showed all three test lines  and 97.2% (95% CI: 92.0-99.4) respectively, and the specificities were 97.4% 95% CI: 93.6-99.3) and 93.6% (95% CI: 88.5-95.7) respectively  and thus this might be inadequate to confirm or refute this point. The BN RDT however, has been shown to have a very good sensitivity (100%) for the detection of pure P. falciparum infection [P. falciparum accounts for almost 99% of malaria infections in the study area  and, therefore, the low sensitivity of the BN RDT for non-P. falciparum malaria may not be a serious cause for concern in this area and most parts of sub-Saharan Africa where malaria infection is predominantly caused by P. falciparum [Plasmodium aldolase [P. falciparum and ≤ 100 parasites/μl for P. vivax [Studies conducted earlier on BN RDT showed a low sensitivity (61.5%) for the detection of pure fections . We founnfection . P. falclciparum . A slighlciparum . Howeveraldolase . The detaldolase ,23. EvenP. vivax . False nP. falciparum infections and has a higher false positive rate due to the persistence of the antigen target HRP 2 [Overall the test is simple to perform, rapid (< 15 minutes), easy to interpret, requires less training, needs no laboratory setup and thus it is applicable for field conditions ,9,21. Thet HRP 2 ,9,26.Plasmodium-infected erythrocytes. The bright shiny dots within infected erythrocytes under UV light is extremely characteristic for malaria and has a very high PPV in areas mainly endemic with P. falciparum [Plasmodia and the differentiation of the species. Compared to GM, PT exhibited four false-positive results which have not been further investigated. PT could also be more sensitive than GM in detecting low number of parasites [® when compared only with conventional Giemsa stained microscopy [Plasmodia species as the disadvantage of PT. However, the characteristic fluorescence of infected erythrocytes has a very high PPV in areas mainly endemic with P. falciparum.PT uses a fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) which detects intracellular double stranded DNA which is present within croscopy  as well croscopy  confirmecroscopy . This stThus we value PT analog to GM thick film as a useful screening method where positive and doubtful results need to be completed by GM thin film examination for confirmation and accurate identification of species later. This strategy takes also into account that Giemsa staining and microscopy is a very elaborate and challenging procedure when performed accurately in a high quality level.® employs fluorescence in which red blood cells are not completely lysed, therefore, they may lie on each other or overlap with each other thereby preventing parasites in red blood cells that may be lying beneath other cells from being identified and counted.Parasitaemia is used to guide treatment, thus underestimation could have significant ramifications for malaria patients. Parasite counts obtained from PT were significantly lower than those obtained from GM. It is unlikely that parasites are hidden during Giemsa stain microscopy, since adequate preparation of the slide ensures visibility through all planes of focus. The lysis of the red blood cells during the staining process reveals the parasites and ease the detection . The ParThe World Health Organization Sexually Transmitted Diseases Diagnostics Initiative (SDI) has developed the ASSURED criteria as a benchmark to decide if tests address disease control needs: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users . Even thThe accepted level of sensitivity for a rapid diagnostic test in diagnosing malaria is a sensitivity of 95% . CompareBoth methods require little training and the BN RDT can withstand temperatures up to 45°C , making There is the need therefore to expand malaria diagnostic services as part of a greater framework of health system strengthening within resource-limited settings. Increasingly, countries and implementing partners have identified that limited diagnostic capacity represents a major barrier to implementation and sustainability of prevention, treatment and care programs for malaria . The PT In this study we demonstrated comparable and acceptable performance and operational characteristics of PT, BN RDT and conventional GM with very good agreements with each other. PT and BN RDT require little training only, have very short turnaround times, are applicable under field conditions as well as affordable even in rural areas. Therefore both methods can be considered as alternative diagnostic tools in malaria endemic areas in addition to or instead of GM.falciparum malaria. Since this is the predominant species causing most mortality and complications in Ghana and sub-Saharan Africa, it is relevant and useful. It is expected that the CyScope® will show similar results for other malaria species especially P. vivax, but this could not be ascertained by this study. Further studies are needed to determine its effectiveness in diagnosing other Plasmodium species.• This study has found the PT to be a reliable diagnostic tool that is sensitive and specific in diagnosing • Based on the ASSURED criteria and the need to expand malaria diagnostic services as part of a greater framework of health system strengthening within resource-limited settings , the PT • In Africa over 70% of malaria cases do not present initially to health facilities but diagnosed and managed at home with traditional remedies or drugs bought from local shops . PatientPlasmodium species.• The PT is not able to differentiate between The authors declare that they have no competing interests.BN designed the study protocol, analyzed the data and headed the writing of the protocol. JM, NB, ET, YAS and FH planned and initiated the study and contributed to the writing of the manuscript. SEKA and LI carried out the tests and also contributed to the writing of the manuscript. SBN contributed to the analysis of the results and writing of the manuscript. All authors have read and approved the final manuscript.The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of any of their affiliated Research Institutions.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/143/prepub


For Cell-Dyn analysers, abnormal depolarization events mainly in the lobularity/granularity and other scatter-plots, and various reticulocyte abnormalities have shown overall sensitivities and specificities of 49% to 97% and 61% to 100%, respectively. For the Coulter analysers, a 'malaria factor' using the monocyte and lymphocyte size standard deviations obtained by impedance detection has shown overall sensitivities and specificities of 82% to 98% and 72% to 94%, respectively. For the XE-2100, abnormal patterns in the DIFF, WBC/BASO, and RET-EXT scatter-plots, and pseudoeosinophilia and other abnormal haematological variables have been described, and multivariate diagnostic models have been designed with overall sensitivities and specificities of 86% to 97% and 81% to 98%, respectively. The accuracy for malaria diagnosis may vary according to species, parasite load, immunity and clinical context where the method is applied. Future developments in new haematology analysers such as considerably simplified, robust and inexpensive devices for malaria detection fitted with an automatically generated alert could improve the detection capacity of these instruments and potentially expand their clinical utility in malaria diagnosis.For more than a decade, flow cytometry-based automated haematology analysers have been studied for malaria diagnosis. Although current haematology analysers are not specifically designed to detect malaria-related abnormalities, most studies have found sensitivities that comply with WHO malaria-diagnostic guidelines, i.e. ≥ 95% in samples with > 100 parasites/μl. Establishing a correct and early malaria diagnosis is a prerequisite for an adequate treatment and to minimizing adverse outcomes. Expert light microscopy remains the 'gold standard' for malaria diagnosis in most clinical settings. However, it requires an explicit request from clinicians and has variable accuracy. Malaria diagnosis with flow cytometry-based haematology analysers could become an important adjuvant diagnostic tool in the routine laboratory work-up of febrile patients in or returning from malaria-endemic regions. Haematology analysers so far studied for malaria diagnosis are the Cell-Dyn For over a century microscopy has been the standard method for routine malaria diagnosis , allowinPlasmodium vivax relapses .,53. P. vick film , or agaiick film   . Furtherida spp. .P. vivax samples also showed salient abnormalities in the WBC/BASO and RET-EXT scatter-plots  counting areas, ≥1 linear or clustered gray events (pixels) had a sensitivity and specificity of 77% and 76%, respectively, for P. vivax  and P. falciparum (n = 30) where developed and evaluated for accuracy against 63 samples from febrile patients, and partially validated against samples from a composite group of 161 febrile patients and healthy individuals  models for each species. One used XE-2100 built-in variables (N-OD1), and the other one used built-in variables and new scatter-plot variables defined by the authors (N-OD2)  Figure  [51]. Th) Figure .Pv and N-OD1Pf models include the increase in the mean number of events detected in the LYMPH-Y (y axis) channel , 'number of granulocyte-coded DIFF abnormalities' ), with ≥4 points being diagnostic; N-OD: non observer-dependent malaria diagnostic model based on logistic regression where the independent variables are obtained from the data gathered from each blood analysis and the outcome or dependent variable is the predicted probability (PP) for malaria that is dichotomized to positive or negative for malaria based on the optimal PP obtained by ROC analysis; N-OD1: N-OD model that only uses raw numerical variables given by the information processing unit of the Sysmex as independent variables; N-OD1Pv and N-OD1Pf are the models for P. vivax and P. falciparum, respectively; N-OD2: N-OD diagnostic model that uses raw numerical variables given by the information processing unit of the Sysmex as well as pixel counts in the WBC/BASO(III) counting area as independent variables; N-OD2Pv and N-OD2Pf are the models for P. vivax and P. falciparum, respectively; PCR: polymerase chain reaction; PLT-O: optic platelet count; PP: predicted probability for malaria obtained from the logistic regression model; QBC: quantitative buffy coat. RBC: red blood cell(s); RDT: Rapid Diagnostic Tests ; RDW-SD: The RBC histogram peak height is assumed to be 100%, the distribution width at the 20% frequency level (y axis of histogram) is RDW-SD (distance between intersections of histogram and a line extended from the 20% frequency), expressed in femtoliters; ROC: receiver operator curve; RT-PCR: real-time polymerase chain reaction; SD: standard deviation; SFL: 90° side-fluorescence detection by the Sysmex XE-2100; SSC: 90° side-scatter detection by the Sysmex XE-2100; U-ODPv: univariate observer-dependent diagnostic model for P. vivax which consists of the recognition of a general pattern of abnormalities on several of the Sysmex XE-2100 scatter-plots; VCS: Volume Conductance-Scatter; WBC: white blood cell(s). WHO: World Health Organization; The following acronyms are product-specific and defined as follows : ΔDIFF/WBC: Ratio between the WBC count in the DIFF channel over the total WBC count multiplied by 103; DIFF: SSC/SFL WBC separation scatter-plot (Sysmex XE-2100); EOS-I: frontal/depolarized side-scatter WBC separation plot (Cell-Dyn 4000); LYMPH-Y: mean number of events detected in the LYMPH-Y (y axis) channel; NEU-EOS: side-scatter/depolarized side-scatter WBC separation plot (Cell-Dyn 4000); RET-EXT: SFL/FSC 'extended reticulocyte' scatter-plot that shows erythrocytes and platelets with varying concentration of nucleic acids (Sysmex XE-2100); WBC/BASO: SSC/FSC WBC separation scatter-plot (Sysmex XE-2100).ACT: Artemisinin Combination Therapy; CBC: complete blood count; CD: Cell-Dyn; DNA: deoxyribonucleic acid; FSC: 0° frontal-scatter detection by the Sysmex XE-2100; HIV: Human Immunodeficiency Virus; HRP2: GCZ, TH and MPG: All authors declare that they have no competing financial or any other interest in relation to their work.GCZ conceived the paper, took the lead in conception and design, and led the drafting of the paper. TH and MPG contributed significantly to the conception and design, and to the writing of the paper. All authors have read and approved the final version of the paper.Expanded Table 1 that includes reference diagnostic tests used, blinding status, and observations for each study.Click here for file


Plasmodium knowlesi malaria causes severe disease in up to 10% of cases in Malaysian Borneo and has a mortality rate of 1 - 2%. However, laboratory markers with the ability to identify patients at risk of developing complications have not yet been assessed as they have for other species of Plasmodium.P. knowlesi malaria were studied. Standardized pigment-containing neutrophil (PCN) count, parasite density and platelet counts were determined and analysed by logistic regression and receiver operating characteristic (ROC) analysis.A case control study was undertaken in two hospitals in Sarikei and Sibu, Malaysian Borneo. One hundred and ten patients with uncomplicated (n = 93) and severe (n = 17) The PCN count was strongly associated with risk of disease severity. Patients with high parasite density  or with thrombocytopaenia  were also more likely to develop complications (odds ratio (OR) = 9.93 and OR = 5.27, respectively). The PCN count yielded the highest area under the ROC curve (AUC) estimate among all markers of severity . However, the difference between all parameter AUC estimates was not statistically significant .P. knowlesi malaria and having a parasite count ≥35,000/μl or ≥1% or a platelet count ≤45,000/μl can be regarded at risk of developing complications and should be managed according to current WHO guidelines for the treatment of severe malaria.Counting PCN is labour-intensive and not superior in predicting severity over parasitaemia and platelet counts. Parasite and platelet counts are simpler tests with an acceptable degree of precision. Any adult patient diagnosed with  Plasmodium knowlesi was thought to be a rare event until an unexpected high incidence of cases was revealed in the Kapit division of Sarawak, Malaysian Borneo in 2004[P. knowlesi from other locations in Sarawak and Sabah, Malaysian Borneo[Plasmodium falciparum infections[P. knowlesi malaria.Human infection with  in 2004. Subsequn Borneo-7. A clin Borneo. Typicalfections. In contP. knowlesi malaria patients at risk of severe disease have not been properly assessed. However, higher peripheral blood parasitemia and lower platelet counts are proposed as markers of severity but precise cut-offs are not available[P. falciparum malaria[P. falciparum malaria circulating pigment-containing WBCs are significantly higher than in uncomplicated cases or healthy controls and are thought to give a measure of the extent and duration of infection[Despite a relatively high rate of complications, markers for identifying vailable. The malvailable. As the vailable. The num malaria,12. In snfection-15 but anfection.P. knowlesi infections was investigated here. Cut-offs predictive of the risk of developing severe disease were computed and the relative merit of each as diagnostic tool discussed.The association between parasitaemia, platelets and pigment-containing neutrophils (PCN) and risk of severe disease in P. knowlesi infection, did not suffer from any significant comorbidity, were not pregnant and had not taken antimalarial treatment within the previous 28 days. At admission, routine haematological testing was performed by using fully automatic Nihon Kohden analyzer  in Sibu hospital and a Sysmex KX-21 (Japan) or Nihon Kohden analyzer  in Sarikei hospital. Patients with uncomplicated P. knowlesi malaria were treated promptly as described elsewhere[Plasmodium species was confirmed by nested polymerase chain reaction (PCR) assays at the Malaria Research Centre, Universiti Malaysia Sarawak, Kuching, as described previously[P. knowlesi were included in the study. Blood samples, blood films and clinical data were collected following written informed consent. The consent forms and the study protocol have been approved by the Malaysian Ministry of Health’s Medical Research and Ethics Committee. All cases with severe disease were retrospectively identified and included (n = 17). Controls were randomly chosen from the pool of patients with uncomplicated malaria (n = 93).This case–control study was conducted in two hospitals in Sarikei and Sibu, Malaysian Borneo, which have a population of mostly Iban and Chinese ethnicity. Adult patients ≥15 years) admitted between July 2007 and January 2010 were included if they had single-species lsewhere. Patientlsewhere. Plasmod5 years aP. knowlesi malaria. Moreover, defining a specific cut-off for hyperparasitaemia in P. knowlesi malaria was an objective of this study. Uncomplicated cases were defined on the basis of symptoms leading to treatment-seeking behavior in the absence of criteria for severity based on organ involvement. Classification was based on clinical and laboratory parameters gained at hospital admission.Classification of severe malaria cases was based on criteria of the World Health Organization (WHO),19. HypeThin blood films were stained with 10% Giemsa Gurr`s improved R66  for 25 minutes after fixation with methanol  for 1 min. Peripheral parasite density was determined from thin blood films by counting the number of asexual forms in eight consecutive microscopic fields, assuming 250 red blood cells per field in an area with regular monolayer and 1,000-fold magnification. Results are given in percent infected red blood cells (IRBC). Standard parasite counts of thick blood films were quantified as follows: number of asexual forms per 500 WBCs x actual WBCs/μl/500. If parasitaemia was high, asexual forms per 200 WBCs were counted. Differential counts were conducted manually on thin blood films for each patient.For a reliable identification of cell morphology, PCN were detected exclusively on thin blood films by counting 100 neutrophils using an Olympus BX53 microscope with 1000-fold magnification  when data were normally distributed. Hypothesis testing was performed with an unpaired t-test. For skewed data, medians and interquartile ranges were presented. When transformation could achieve normal distribution of values a t-test was subsequently used. Otherwise the Wilcoxon rank sum test was employed. D'Agostino's K-squared test was used to check for normality.Linear regression analysis was performed on numerical outcomes. Heteroskedasticity was tested by using the Breusch-Pagan and Cook-Weisberg tests and when positive robust standard errors were calculated. Odds ratios (OR) were calculated for categorical variables by using logistic regression. For testing the overall association of categorical variables and risk of severity, a likelihood ratio test was performed. Possible interaction between variables within multivariate logistic regression models was also checked by the use of the likelihood ratio test. Variables were only included into multivariate logistic regression when they were unlikely to lie on the same causal pathway towards the outcome of interest. A possible application of severity markers as diagnostic tools was determined by receiver-operating characteristic (ROC) analyses. For every possible predictor, the area under the ROC curve (AUC) was computed and compared to other predictors by using the Wald test and following an algorithm suggested by DeLong, DeLong and Clarke-Pearson. Since iP < 0.05 (two-sided) was regarded as statistically significant. All analyses were made by using Stata version 11.0 .Interclass correlation coefficient was used to assess intra-observer reliability in reading 10% of the thin films by the same microscopist for a second time. A vs 36 PCN/μl, p <0.0001). Ten per cent of the films were re-examined and an interclass correlation coefficient of 0.7883 for the PCN count was determined. Thick and thin film parasite counts were also significantly different . One hundred and seven patients (97.3%) had thrombocytopaenia . However, the maximum platelet count in the study cohort was 167,000 platelets/μl. That indicates a generally low platelet count in all of the patients. Platelet counts were lower in severe P. knowlesi malaria compared with uncomplicated disease .A total number of 110 patients were included in the study. According to the WHO criteria for severe falciparum malaria,19, 17 pA detailed crude risk assessment of potential laboratory severity markers was performed by using logistic regression on different categorical variables Table. The PCN2 = 0.75 and 0.65 respectively) of the total variation in circulating pigment-containing neutrophils (Table2 = 0.07 and 0.05 respectively) or amount of circulating pigment-containing neutrophils (R2 = 0.04) and is thus unlikely to lie on the same causal pathway towards the outcome. To address these issues, three different multivariate models were performed. The results are shown in TableLinear regression was performed on the potential severity markers to investigate associations between them. The results indicate that peripheral parasitaemia measured on thick and thin blood films accounts for 75% and 65% , parasite/μl (thick blood film) and platelet counts as potential laboratory markers of severity as identified during risk assessment Figure and5. TP. knowlesi malaria. Such prevalence was reported from a prospective survey in Sarawak, Malaysia[P. knowlesi malaria. For example, any patient having a parasite count ≥35,000/μl or ≥1% parasitaemia can be regarded to be at high risk of severe disease. Multivariate risk assessment of severity markers when using the recommended cut-offs is shown in TableP. falciparum malaria. Having a platelet count ≤45,000/μl is independently associated with a higher risk of severity .Suitable cut-offs for practical use were evaluated for thick and thin film parasite counts and for platelet counts Table. CandidaMalaysia. A minimP. knowlesi malaria causes severe disease in approximately one of 10 patients and can be potentially fatal[P. knowlesi malaria. Pigment-containing monocytes are also a marker of severe P. falciparum malaria[Since ly fatal, the sea malaria. HoweverP. knowlesi malaria. In any case, the observed differences in the AUC estimates are rather small and cannot justify a recommendation of the PCN counts as a routine test at the moment. Flow cytometry can achieve a fast and effective detection of pigment-containing WBCs[A strong association between PCN count and risk of disease severity was observed. The method involved counting 100 neutrophils, took 15–20 minutes and requires skilled personal to identify the correct cell morphology. It is both labour-intensive and time consuming in the routine malaria diagnostic setting. Therefore, the PCN count should have a superior prognostic ability over parasitaemia and platelet counts if it is to be recommended for routine use. Even though the AUC estimate for PCN was the highest among all parameter estimates, PCN counts did not have a higher diagnostic precision when compared with parasite and platelet counts. The lack of statistical significance might be explained by the low sample size. On the other hand, linear regression analysis revealed that the total variation in the amount of pigment-containing neutrophils can be explained by the changes in peripheral parasitaemia to an extent of up to 75%, leaving only a minor proportion in the total variation that might represent sequestration and would give additional prognostic information in ing WBCs but remaP. knowlesi malaria[In contrast, the parasite count on thick and thin blood films is a simpler method and is usually available in resource-poor settings. The peripheral blood parasitaemia on thick blood films has been shown to be a potential marker of severity in  malaria. The autP. knowlesi malaria. A positive predictive value of about 33% as calculated for the recommended cut-off of 35,000 parasites/μl means that two of three patients with a positive test indicating a severe disease or a high risk of developing complications would not suffer from severe malaria at the time of measurement. Managing them according to the WHO guidelines for severe malaria might appear as an overtreatment and potential waste of resources in an often resource-limited setting. However, when using the recommended cut-offs in multivariate logistic regression analysis any tested severity marker is independently associated with high risk of severity (OR 5.27 – 9.93). Therefore, having a positive test result in accord with the recommended cut-offs categorizes a patient to be at high risk of severity regardless of the actual clinical status. Management for severe malaria should consecutively be accomplished to prevent a potential development of severe disease in patients within this high-risk group even if they do not suffer from complications at the time of measurement. Consequently, the results of the study presented here indicate that any adult patient diagnosed with P. knowlesi malaria and a parasite count ≥35,000/μl or ≥1% or a platelet count ≤45,000/μl should be regarded at risk and should subsequently be treated as for severe malaria according to current WHO guidelines.Assessing cut-offs with a more predictive than reflective capability was based on an appropriate relationship between positive and negative predictive values. Recommendation of a specific cut-off required a negative predictive value of at least 95% in the study. This would mean that one in 20 patients with a test result indicating non-severe disease or a low risk of developing complications would have severe disease at the time of measurement. None of the severity markers could reach high positive predictive values in the study, assuming a prevalence of 10% severe cases among hospitalized patients with P. knowlesi and Plasmodium malariae may be an issue. Plasmodium knowlesi is often confused with P. malariae when making the diagnosis by light microscopy[Plasmodium malariae infection causes a median parasitaemia of 8,875 parasites/μl with maximum of about 50,000 parasites/μl[P. knowlesi may be wrongly diagnosed in the presence of a P. malariae infection. In such cases, a parasite count ≥35,000/μl alone may lead to incorrect risk estimation although coupled with low platelet counts may be more specific. In Sarawak and Sabah, P. malariae infection is rare[P. knowlesi would override misdiagnosing P. malariae in a small number of cases.Using these cut-offs in areas with prevalence of both croscopy. Plasmodsites/μl, but rar is rare,24 and tP. knowlesi malaria occur with a prevalence of 10% among hospitalized patients. The prevalence and characteristics of P. knowlesi malaria should be measured within the framework of prospective studies in all locations in Southeast Asia where P. knowlesi malaria occurs[P. knowlesi infections. However, additional simple diagnostic tools to distinguish P. knowlesi from P. malariae infections in settings with limited resources are urgently needed to identify patients at risk of severe P. knowlesi malaria.One major limitation of this study is the assumption that severe cases of a occurs-6 and whP. knowlesi malaria. Counting PCN is not superior in predicting severity but labour-intensive and can therefore not be recommended for routine diagnosis of P. knowlesi infections.Based on the results obtained, parasite and platelet counts are precise tests for indentifying patients at risk of severe The authors declare that they have no competing interests.MW, AA, AS, ITW and LCW collected the blood samples and patients’ data. MW, AA and AS carried out the laboratory work. MW participated in the design of the study and analysed the data. JCS, BS and SK conceived the study and participated in its coordination. MW and JCS wrote the manuscript. All authors read, commented on and approved the final manuscript.


In many areas of the world, including Ethiopia, malaria and helminths are co-endemic, therefore, co-infections are common. However, little is known how concurrent infections affect the epidemiology and/or pathogenesis of each other. Therefore, this study was conducted to assess the effects of intestinal helminth infections on the epidemiology and clinical patterns of malaria in southern Ethiopia where both infections are prevalent.A cross-sectional study was conducted in 2006 at Wondo Genet Health Center and Bussa Clinic, southern Ethiopia. Consecutive blood film positive malaria patients (N=230) and malaria negative asymptomatic individuals (N=233) were recruited. Malaria parasite detection and quantification was diagnosed using Giemsa-stained thick and thin blood films, respectively. Helminths were detected using direct microscopy and formol-ether concentration techniques. Coarse quantification of helminths ova was made using Kato Katz method.Trichuris trichiura infection was associated with increased malaria prevalence while increased worm burden of helminths as expressed by egg intensity was associated with increased malaria parasitaemia which could be a potential factor for development of severe malarial infection with the course of the disease. Majority (77%) of the subjects had multiple helminths infection. T. trichiura, Ascaris lumbricoides, Schistosoma mansoni, and hookworm infestation accounted for 64.5, 57.7 %, 28.4%, and 12.2% of the infections, respectively.The over all magnitude of intestinal parasitic infection was high irrespective of malaria infection . Populations in malaria-endemic areas of southern Ethiopia are multi-parasitized with up to four helminths. Mass deworming may be a simple practical approach in endemic areas in reducing the risk of severe malarial attack particularly for those at high risk of both infections. Plasmodium falciparum and Plasmodium vivax are the major parasites accounting for about 70% and 30% of infections, respectively, during peak transmission periods [Half the world’s population live in malaria-endemic areas, with an estimated 500 million clinical cases and over one million deaths annually . Similar periods .Ascaris lumbricoides, Trichuris trichiura, hookworm and Schistosoma mansoni are widely spread with variable prevalences [A. lumbricoides and T. trichiura, respectively [P. falciparum malaria was reported [Soil-transmitted helminths (STH), on the other hand, are a group of common parasites that infect more than a billion people worldwide . In Ethivalences -12. In mvalences ,14. The ectively . In anotreported .However, how concurrent infections affect the epidemiology and/or the pathogenesis of each other remains controversial -19 raginThis cross-sectional study was conducted at Wondo Genet Health Center and Busssa Clinic, Sidama Administrative Zone, South Ethiopia in a peak transmission season from November to December 2006. These areas are malaria endemic with high prevalence of helminthiasis. All patients presenting with signs and symptoms of malaria during the study period were included (N=230) and asymptomatic individuals with negative result for malaria (N=233). A pretested, structured questionnaire was used to collect socio-demographic and relevant clinical data of the participants.Both thick and thin blood films were made in a single slide and were stained with Giemsa staining solution for detection and quantification of malaria parasites. To detect malaria infections, 200 fields  were examined. The parasite density was expressed per μl of blood assuming 8,000 leucocytes per μl of blood. In brief, a thick fill was selected where the white blood cells were evenly distributed. Using the oil immersion objective, 200 white blood cells were counted systematically, by counting at the same time the number of parasites  in each field covered. Then the number of parasite per μl of blood was calculated by multiplying the number of parasite  counted against 200 leucocytes and 8,000 leucocytes and dividing the product by 200 . After cMalaria patients and their counter part controls were requested to deliver stool specimen. Those who volunteered to deliver the specimen were given a clean, dry, leak-proof plastic. Stool samples were then examined by direct stool examination method using normal saline. The portion of stool was processed by using the Kato Katz technique; about 3 g stool was preserved by 10% formalin for formol-ether sedimentation techniques. Coarse quantification of eggs was obtained by counting the number of eggs on a smear of 41.7 mg faeces, and a quantitative variable scoring  was created for each helminths following the standard procedure used by World Health Organization (WHO) ,35. Packt test and ANOVA. Two-tailed P values was determined and considered significant when found <0.05. Logistic regression analysis was also used to determined association between variables.The data were analysed using to SPSS version 13 statistical packages software. Intestinal parasite densities were transformed to log for analysis and geometric mean was used. Baseline characteristics and malaria parasites densities of groups with and without intestinal parasitic infections was compared by Student’s This study was conducted after institutional  and regional  ethical clearance was obtained and after written and/or verbal informed consent was obtained from the study participants. Participants who were found to be positive for any of the parasites were linked to the health institutions for treatment.A total of 230 patients with malaria infection and 233 malaria negative individuals were included. The mean  age of the subjects was not similar in both groups [17.8 (±14.15) versus 19 + 12.23. The majority (34.5%) of the participants were under nine years of age, and 54% of the subjects were males.T. trichiura, A. lumbricoides, S. mansoni, and hookworm with prevalence of 64.5% (227/352), 57.7% (203/352), 28.4% (100/352) and 12.2% (43/352), respectively. Amongst of those with helminthiasis, 77% (271/352) were having multiple helminths infection. Infection with two, three or four different species of helminths was observed in 37.5% (132/352), 22% (77/352) and 3.4% (12/352) of the participants, respectively. Out of the 271 participants with multiple infections, the frequent combinations of helminths were A. lumbricoides-T. trichiura in 28.8% (78/271); A. lumbricoides-T. trichiura-S. mansoni in 18.5% (50/271), and A. lumbricoides-T. trichiura-hookworm in 8% (22/271) of the patients. In 4% of the patients (11/271) four different helminths: A. lumbricoides, T. trichiura, S. mansoni and hookworm were observed. The prevalence of helminthic infection tends to reach its peak in the age group 10 to 19 years old followed by children of less than nine years old , 46 (20.5%) and six (2.7%) developed light, moderate and heavy infection, respectively. Similarly, out of the 203 participants infected with A. lumbricoides, 92 (45.3%), 96 (47.3%) and 15 (7.4%) developed light, moderate and heavy infection, respectively. Out of the 100 participants with S. mansoni, 51 (51%), 24 (24%) and 25 (25%) developed light, moderate and heavy infection, respectively. The intensity of intestinal worms expressed as geometric mean among the study participants for A. lumbricoides, T. trichiura and S. mansoni was 4,691, 486 and 112, respectively.The overall magnitude of intestinal parasitoses in both groups was 76.9% 352/463). The most frequent intestinal helminths diagnosed were ), 28.4% 00/352 an/463. TheP. falciparum. Plasmodium vivax accounted for 35.2% (81/230) of the cases. The rest, 6.5% (15/230) were mixed infections of P. falciparum and P. vivax , 42.6% (98/230), 22.6% (52/230), and 10% (23/230), respectively. It was only T. trichiura infection that was significantly associated with malaria co-infection (P=0.002) . Similarly, patients with heavy trichuriasis infection seemed to be at higher risk of severe malaria infections compared with light trichuriasis. However, this did not reach a statistically significant level (P=0.066). Both moderate and heavy infections of S. mansoni were also associated with severe clinical malaria infection though the difference was not statistically significant (P=0.067). Worm burden (expressed in helminths egg intensity) was associated with higher mean level of parasitaemia (number of parasite per μl blood) (P=0.032).The rate of helminths co-infection among the malaria patients was 67% 154/230). The magnitude of 4/230. ThP. falciparum (58.3%) while P. vivax and mixed infections accounted for 35.2% and 6.5% of the infections, respectively. The persistently high prevalence of malaria urges a need for evaluation of the malaria control programme in the study area. In Ethiopia, A. lumbricoides, T. trichiura, hookworm, and S. mansoni infections are widespread with variable prevalence that may reach up to 90% in some places among the general population [T. trichiura, A. lumbricoides, S. mansoni and hookworm, with a prevalence of 64.5%, 57.7%, 28.4% and 12.2%, respectively. The high prevalence of helminthiasis in general and the predominance of specific parasites in the area were in agreement with previous reports in the region [Approximately 68% of the total population of Ethiopia lives in areas at risk of malaria . Accordipulation . Likewise region .Schistosoma mansoni was not associated with increased malaria infection in the current study. This, however, contradicts the report from Senegal where the incidence rate of malaria attacks was significantly higher among S. mansoni-infected individuals [A. lumbricoides has protective effect from malaria infection [A. lumbricoides is a risk factor for malaria infection. In line with this, although it did not reach to a statistical significance level, there was a high A. lumbricoides-malaria co-infection rate in the current study. However, it is difficult to strongly conclude the epidemiological interaction of helminths and malaria infection with such cross-sectional study. Hence, it would be useful to check the incidence of malaria in a longitudinal study among cohorts of people having/not having different helminthiasis with and without treatment. Conducting further studies on the co-epidemiology of malaria and helminths in Ethiopia could be of paramount importance to a potential public health value of synergistic opportunities for control.In many tropical areas of the world, malaria and STH co-exist with significant co-infection rates ,14. Thisividuals . On the nfection . This imet al. [Plasmodium density [T. trichiura and A. lumbricoides infections had lower Plasmodium densities than those with moderate or heavy infection. Contrary to this, heavy S. mansoni infection was associated with lower Plasmodium densities [The current study however, tried to determine the effect of helminths co-infection on the clinical manifestation of the malaria cases with only three cases presenting with sign and symptoms of severe malaria. The absence of severe malaria cases in highly ‘wormy’ malaria patients in the current study was, however, consistent with a study conducted by Nacher et al.  where heet al. ,39. Stud density ,18. In lensities . HoweverThe majority of malaria patients in the current study were found to be multi-parasitized with up to four helminths. A strong association between worm burden as expressed by helminths egg intensity and malaria parasite density was observed which could be a dose-dependent risk factor for severe malaria attack with the course of the infection. Mass deworming may be simple practical approaches in endemic areas in reducing the risk of severe malarial attack particularly for those at high risk of both infections.The authors declare that they have no competing interests.AM, ML, BE, TS, AK and DE conceived, designed and proposed the research idea. AM was involved in data collection. AM, ML, BE, DN, TS, AK and DE were involved in data entry, clearance, analysis and interpretation of the findings. AM, BM and YB were responsible for drafting the manuscript. All authors were involved in reviewing the manuscript and gave approval for publication.


Genetic factors play a key role in determining resistance/susceptibility to infectious disease. Susceptibility of the human host to malaria infection has been reported to be influenced by genetic factors, which could be confounders if not taken into account in the assessment of the efficacy of interventions against malaria. This study aimed to assess the relationship between haemoglobin genotypes and malaria in children under five years in a site being characterized for future malaria vaccine trials.Plasmodium falciparum parasitaemia >2,500 parasites/μl and axillary temperature ≥37.5°C or reported fever over the previous 24 hours.The study population consisted of 452 children living in four rural villages. Hb genotype was determined at enrolment. Clinical malaria incidence was evaluated over a one-year period using combined active and passive surveillance. Prevalence of infection was evaluated via bi-annual cross-sectional surveys. At each follow-up visit, children received a brief clinical examination and thick and thin blood films were prepared for malaria diagnosis. A clinical malaria was defined as Frequencies of Hb genotypes were 73.2% AA; 15.0% AC; 8.2% AS; 2.2% CC; 1.1% CS and 0.2% SS. Prevalence of infection at enrolment ranged from 61.9%-54.1% among AA, AC and AS children. After one year follow-up, clinical malaria incidence (95% CI) (episodes per person-year) was 1.9 (1.7-2.0) in AA, 1.6 (1.4-2.1) in AC, and 1.7 (1.4-2.0) in AS children. AC genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 1–2 years [rate ratio (95% CI) 0.66 (0.42-1.05)] and 2–3 years [rate ratio (95% CI) 0.37 (0.18-0.75)]; an association of opposite direction was however apparent among children aged 3–4 years. AS genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 2–3 years [rate ratio (95% CI) 0.63 (0.40-1.01)].In this cohort of children, AC or AS genotype was associated with lower risk of clinical malaria relative to AA genotype only among children aged one to three years. It would be advisable for clinical studies of malaria in endemic regions to consider haemoglobin gene differences as a potentially important confounder, particularly among younger children. Malaria remains one of the most important causes of morbidity and mortality in endemic areas, primarily affecting children under five years of age . The higPlasmodium falciparum is an important adaptive trait of human populations living in endemic areas  and 2–3 years   = 0.63 ; p = <0.001. Among the full cohort, risk associated with AC genotype varied significantly among children younger versus older than three years after adjustment for health district (p for interaction < 0.001).Time to first malaria episode did not significantly differ by AA, AC or AS genotype among the full cohort p = 0.21)  and other variant surface antigens[Abnormal Hb genotype in this cohort was further associated with lower incidence of clinical malaria episodes and delayed first episode among children aged one to three years. These effects were more subtle in infants and older children. Several studies have identified a protective effect of abnormal Hb against clinical malaria ,35. Alth antigens.Alternative findings among cohorts may in part reflect variation in age distribution and associated development of acquired immunity, malaria endemicity, and administration of anti-malarial drugs. Estimates of malaria burden in the current study are likely underestimates, due to prompt treatment of malaria episodes identified by active surveillance. This study is further limited by the small number of children with abnormal genotypes and restriction of follow-up to one year, preventing stronger inferences related to changing risk associated with alternative genotypes throughout early childhood.In this cohort of children under five years of age, AC or AS Hb genotypes was associated with lower risk of clinical malaria relative to normal genotype among children aged one to three years. This age-specific association may suggest influences of HbC and HbS genotypes in the development of naturally acquired immunity in early childhood. Evaluations of anti-malarial interventions in endemic regions should consider Hb genotype as a potentially important confounder, particularly among young children.AA: Wild type; SS: Sickle cell; AS: Sickle trait; AC: Heterozygous for haemoglobin C; CC: Homozygote for haemoglobin C; SC: Haemoglobins S and C; Non-AA: Abnormal haemoglobin ; PCR: Polymerase chain reaction.The authors declare that they have no competing interests.ECB designed the study, collected data and coordinated the study, performed statistical analysis and wrote the first draft of the manuscript. SBS, AT, IN, AO and ATK coordinated and participated in the design of the study, participated in the statistical analysis and procedures and the drafting of the manuscript. EBC, IS, SS and AD participated in the laboratory work and data interpretation. JBY, OE and ECB carried out the study. NW, MS and TJTD contributed to analysis and interpretation and to contribute to writing the paper. All the authors read and approved the final version.


Intermittent preventive treatment of malaria in infants (IPTi) consists of the administration of a treatment dose of sulphadoxine-pyrimethamine (SP) at the time of routine vaccinations. The use of routine Health Management and Information Services (HMIS) data to investigate the effect of IPTi on malaria, anaemia, and all-cause attendance in children aged 2-11 months presenting to 11 health centres in southern Tanzania is described.Clinical diagnosis of malaria was confirmed with a positive blood slide reading from a quality assurance laboratory. Anaemia was defined using two thresholds . Incidence rates between IPTi and non-implementing health centres were calculated using Poisson regression, and all statistical testing was based on the t test due to the clustered nature of the data.Seventy two per cent of infants presenting in intervention areas received at least one dose of IPTi- 22% received all three. During March 2006 - April 2007, the incidence of all cause attendance was two attendances per person, per year (pppy), including 0.2 episodes pppy of malaria, 0.7 episodes of mild and 0.13 episodes of severe anaemia. Point estimates for the effect of IPTi on malaria varied between 18% and 52%, depending on the scope of the analysis, although adjustment for clustering rendered these not statistically significant.The point estimate of the effect of IPTi on malaria is consistent with that from a large pooled analysis of randomized control trials. As such, it is plausible that the difference seen in health centre data is due to IPTi, even thought the effect did not reach statistical significance. Findings draw attention to the challenges of robust inference of effects of interventions based on routine health centre data. Analysis of routine health information can reassure that interventions are being made available and having desired effects, but unanticipated effects should trigger data collection from representative samples of the target population. Plasmodium falciparum-caused malaria to mortality and morbidity of African infants and young children remains substantial [The contribution of stantial . Malariastantial . The monstantial ,3.Intermittent preventive treatment of malaria in infants (IPTi) consists of the administration of a treatment dose of sulphadoxine-pyrimethamine (SP) at the time of routine vaccinations in the first year of life . In a poIn this paper, data from a consolidated HMIS in 11 sentinel health centres in rural southern Tanzania are used to report (i) the uptake of IPTi and coverage of corresponding EPI vaccines, (ii) the incidence of malaria, anaemia, and all-cause attendance at the health centre, and (iii) the effectiveness of IPTi on the incidence of these outcomes in health centre attendees aged 2-11 months.The area has been previously described, but briefly malaria is endemic, with transmission occurring throughout the year, and is the most common primary cause of health centre or hospital admission among those aged under five years ,22. The This study, termed the IPTi Morbidity Monitoring study, took place in the context of a large cluster randomized trial that was designed to determine the community effectiveness of IPTi with SP on malaria and anaemia. IPTi using SP was introduced to 12 of 24 randomly selected divisions, located within five districts, and two regions (Lindi and Mtwara) of rural southern Tanzania  and vaccination records were captured electronically by study-trained local school leavers, using Personal Digital Assistants (PDAs)  , and recIn addition to routine HMIS data, blood slide readings for malaria parasites and haemoglobin data were also collected. Malaria parasite blood slides were prepared using Giemsa staining, and parasite counts per 200 white blood cells (WBCs) were calculated ,27, whilData from PDAs were exported using Pendragon Forms software to Microsoft Access for data management, and subsequently to Stata 10.0 for statistical analysis . Analysis was done according to a pre-agreed analytical plan, incorporating both 'intention to treat' and 'per protocol' analyses. In 'intention to treat' analyses, all children attending a sentinel health centre located in divisions randomized to receive IPTi were compared with those attending sentinel health centres located in IPTi comparison divisions. For the 'per protocol' analyses, children attending health centres in intervention areas who had received IPTi were compared with those attending health centres in comparison areas who had received the corresponding EPI vaccines.st April 2006), and ended at the earliest of either becoming 12 months of age, or the end date of the study (31st March 2007). In order to avoid double-counting the same malaria episode as two separate episodes, 28 days follow up was removed after each malaria diagnosis in analyses of multiple episodes of malaria. Incidence rates were calculated using Poisson regression, and incidence rates of multiple episodes of malaria or all-cause health centre attendance were adjusted for within-child clustering using random effects Poisson regression modelling. The protective efficacy of IPTi was defined as (1-RR) × 100, where RR is the rate ratio. All statistical testing was based on the t test, using a summary measure of the data from each health centre, to take into consideration the clustered nature of the health centre data [Time at risk for malaria, anaemia, and OPD incidence rate calculations began at the latest of the following: date of registration in the consolidated HMIS, date of becoming two months old, or start date of the study  was similar in intervention and comparison areas , 55% for DPT3 (children aged 3-11 months), and 42% for measles (children aged 9-11 months). Similar proportions of children from both areas received DPT2; while more children from the intervention areas received DPT3 and measles vaccines , suggest that the 11 divisions included in the IPTi Morbidity Monitoring study were comparable on variables for which information was available  that IPTi with SP has no impact on malaria, anaemia or all-cause attendance at the health centre in children aged 2-11 months in this setting, (ii) there was insufficient statistical power to detect an effect of IPTi with SP, and linked to this, (iii) that the background rates of malaria and malaria-related anaemia were too low, due possibly to other control measures including net use, to detect an impact of IPTi with the existing study design.Studies, including a pooled analysis of six large RCTs of IPTi using SP , have reAll health centres with functional microscopes in our project area were included in this evaluation (n = 11). However, this provided only 32% power to detect a 30% difference between IPTi intervention and comparison areas. An additional 19 centres  would be required to achieve 80% power [In the 11 divisions included in the Morbidity Monitoring study, there was a 17% fall in malaria parasitaemia between 2004 and 2006 in children aged 2-11 months of age living in IPTi comparison areas, as measured by the representative household surveys. Using the same source data, it was shown that in children of this age group in IPTi comparison areas there was also a 0.5 g/dL increase in mean haemoglobin, and a 39% increase in the reported use of nets by children on the night before the survey. These background changes may also have contributed to the current study lacking sufficient power to detect an effect of IPTi with SP. Longer periods of observation may enable the discernment of trends, and a difference between these trends in intervention and comparison areas. These observations also draw attention to the need to monitor contextual factors which may impact on the evaluation.This study used HMIS data to evaluate the effectiveness of IPTi with SP on health centre attendees. Findings may, therefore, not be representative of children, and their morbidity burden, in the wider community .The quality of data in HMIS remains a critical issue. Monthly supervision visits to all study sites were carried out by the study co-coordinator (WM), data manager (KS), and laboratory assistant. Visits were an opportunity to discuss issues of data completeness and quality with health centre staff. The basis for these discussions was an automatically-, PDA-generated monthly report. These compared numbers of OPD attendances and EPI vaccinations to those recorded during the previous month; highlighted whether all OPD attendances had corresponding laboratory records; and listed the top ten diagnoses made at the centre in the past month, which previously had been prepared manually. Use of PDAs enhanced data capture, ease of retrieval and summarising of the information. However, despite monthly supervision, issues of data completeness across HMIS registers, and the quality of microscopy from the on-site health centre laboratories remained.For example, on average 41% of blood slide results from the health centre laboratory were missing a corresponding record in the OPD register, and this showed variability between the different health centres. This study used blood slide results from the Morbidity Monitoring quality assurance laboratory, along with clinical diagnosis from HMIS, to classify malaria outcome. Health centre microscopy results had an average sensitivity of 89.6% and specificity of 77.3%, using the QA lab as gold standard. This again showed variation between health centres. There was no suggestion that data completeness or quality of microscopy was consistently better or worse in IPTi intervention or comparison areas.The use of HMIS data for monitoring and evaluation should undergo quality control checks, be analysed with due regard to potential selection biases , clustering of the data, and statistical power considerations. The data need to be interpreted bearing in mind the contextual situation and consistency of results. Where unexpected findings are documented, well-designed, well-implemented, representative household surveys may be needed to answer the question of intervention effectiveness at the community level, also capturing data from those populations who do not attend routine health services.This study used routine data from Health Management Information Systems in 11 sentinel health centres in rural southern Tanzania to evaluate the effectiveness of a new malaria control strategy, IPTi with SP. The process highlights the challenges of using routine health centre data to monitor and evaluate the effectiveness of a malaria control programme in health centre attendees. Findings suggest that unexpected results should be investigated by drawing representative samples of the target population to enable robust conclusions to be drawn.The authors declare that they have no competing interests.The paper was drafted by BW, WM, JAS and DS. BW carried out the statistical analyses, with support from JAS. DS, JAS, HM, PA and MT participated in the set up and management of the Morbidity Monitoring study. WM, KS and MC were responsible for data collection. All authors commented on drafts of the paper and approved the final version.


Cotrimoxazole (CTX) has been used for half a century. It is inexpensive hence the reason for its almost universal availability and wide clinical spectrum of use. In the last decade, CTX was used for prophylaxis of opportunistic infections in HIV infected people. It also had an impact on the malaria risk in this specific group.We performed a systematic review to explore the efficacy and safety of CTX used for P.falciparum malaria treatment and prophylaxis.CTX is safe and efficacious against malaria. Up to 75% of the safety concerns relate to skin reactions and this increases in HIV/AIDs patients. In different study areas, in HIV negative individuals, CTX used as malaria treatment cleared 56%–97% of the malaria infections, reduced fever and improved anaemia. CTX prophylaxis reduces the incidence of clinical malaria in HIV-1 infected individuals from 46%–97%. In HIV negative non pregnant participants, CTX prophylaxis had 39.5%–99.5% protective efficacy against clinical malaria. The lowest figures were observed in zones of high sulfadoxine-pyrimethamine resistance. There were no data reported on CTX prophylaxis in HIV negative pregnant women.CTX is safe and still efficacious for the treatment of P.falciparum malaria in non-pregnant adults and children irrespective of HIV status and antifolate resistance profiles. There is need to explore its effect in pregnant women, irrespective of HIV status. CTX prophylaxis in HIV infected individuals protects against malaria and CTX may have a role for malaria prophylaxis in specific HIV negative target groups. Worldwide, malaria is one of the most important causes of morbidity and mortality, with children under five years of age and pregnant women being the most severely affected groups. Artemisinin-based combination therapy (ACT) is currently the mainstay of malaria treatment in both children and adults, while in pregnancy it can be used only in the second and third trimester. HIV infection, through immune suppression, affects the acquisition and persistence of immune response to malaria. P falciparum infections. Anopheles gambiae sensu stricto (ss) mosquitoes CTX is a drug combination consisting of trimethoprim and sulfamethoxazole. Trimethoprim ) belongs to a group of compounds with antibacterial and antimalarial activity. It is an inhibitor of dihydrofolate reductase and has been shown to act as a sulfonamide potentiator. CTX is well known as an antibacterial drug but less as an antimalarial. Nevertheless, considering the reports on the impact of CTX on malaria, both in HIV-infected and uninfected individuals, we reviewed the available evidence on safety and efficacy of CTX as an antimalarial for both preventive and curative use.This systematic review follows PRISMA guidelines. We included all electronically available, peer reviewed articles. We included studies in English as well as abstracts for which a full study was not available such as conference deliberations as long as sufficient data for inclusion were provided. All eligible studies irrespective of sample size were included (see attached flow chart). A protocol for systematic reviews was not used to for this particular review.The studies included comprise sample populations who were administered CTX for treatment or prophylaxis or any other drug administered to compare efficacy and safety with that of CTX. The review also includes data from in-vitro studies that involve CTX. The patients involved include children, adults and pregnant women, HIV/AIDs infected and uninfected individuals.th, 2012. The same terms were used to search other databases such as the ClinicalTrials.gov and the WHO International Clinical trials Registry Platform (ICTRP). The articles identified were downloaded and reviewed. Studies included were prospective and most of them were done in West Africa, East Africa and a few in Southern Africa. Articles selected were stratified according to the target group  and HIV infected population.A literature search was performed to identify publications reporting on safety and efficacy of Cotrimoxazole for malaria treatment or prophylaxis. The search terms included: “Cotrimoxazole prophylaxis and malaria”, “Cotrimoxazole malaria treatment”, “Cotrimoxazole malaria and HIV”, “Cotrimoxazole malaria drug resistance”, “Cotrimoxazole, malaria in pregnancy”, “trimethoprim-sulfamethoxazole and malaria prophylaxis”, “trimethoprim-sulfamethoxazole and sulfadoxine–pyrimethamine”. The search covered the period from 1962 to June 2012. The PubMed was last accessed on June 8P. falciparum malaria, both in children and adults and was generally safe as no adverse effects were reported from the studies reviewed  and body temperature ≥38°C 2 days after the commencement of treatment  in HIV-infected pregnant women taking CTX and HIV–uninfected women taking SP-IPTp The dihydrofolate reductase (dhfr)/dihydropteroate synthetase (dhps) quintuple mutant is known to be associated with SP treatment failure. It is however unclear whether the quintuple mutant affects the protective efficacy of CTX against malaria. Considering the possible cross resistance between CTX and SP, E.coli and therefore may accelerate the development of CTX resistance among respiratory and diarrhoeal pathogens, especially in areas with low CTX resistance at baseline. In Uganda, CTX prophylaxis in HIV-infected individuals did not increase the occurrence of SP-resistant malaria episodes among HIV-uninfected individuals living in the same household. Instead, the latter had a lower incidence of malaria and infections with SP resistant parasites. In-vitro susceptibility testing against sulfadoxine, sulfamethoxazole, pyrimethamine and trimethoprim on P. falciparum isolates from Tanzania and from Thailand revealed incomplete cross-resistance between pyrimethamine and trimethoprim. The cross resistance between CTX and SP did not appear to be absolute at the time of testing Haemophilus influenzae and dysentery-causing bacteria. Cotrimoxazole is often used to treat pneumoccal infections. There were concerns related to the use of CTX and SP regarding increased resistance in pathogens other than pneumococci, such as Artemisinin Combination Therapies (ACTs) are very efficacious and are currently the mainstay for malaria treatment. However, for prophylaxis the options are limited. Despite the high and increasing drug resistance, only SP is at present eligible to be used for IPTp and IPTi. The wealth of information available on the use of CTX in HIV infected people confirms that CTX is effective against malaria infection and in preventing clinical malaria. Further, despite its long term use, CTX is not associated with a higher prevalence of mutations related to antifolate resistance and works synergistically with ITNs in preventing malaria. Therefore, CTX can be successfully used to prevent HIV/AIDS-related opportunistic infections and malaria morbidity, an important feature in sub-Saharan Africa where these two diseases co-exist and health services operate under constrained budgets. Besides being relatively safe CTX is also inexpensive, almost universally available and has a wide clinical spectrum of activity spanning from bacteria, fungi and protozoan infections The safety concerns for CTX is related to its impact in malnourished individuals in whom it may precipitate folate deficiency and pancytopaenia. The fact that in pregnant women CTX prophylaxis showed a similar prevalence of placental malaria in HIV infected women as IPT-SP in HIV uninfected women suggests that daily CTX can similarly decrease the risk of placental malaria. In HIV-infected pregnant women, CTX use was associated with decreased malaria infection, maternal anaemia and increased haemoglobin concentration, a finding consistent with the beneficial effects on birth outcomes. Therefore, CTX may have similar beneficial effects in other groups though there is currently no data available on its use as a malaria preventive measure in HIV uninfected pregnant women or children. The studies reporting on CTX prophylaxis in HIV infected pregnant women were cross-sectional studies and there could be residual confounding from unmeasured factors. Further, participants were only enrolled in the third trimester of pregnancy A limitation of this review is that most information available comes from East and Western Africa. Almost all cited publications are published in English and relevant literature in other languages may have been overlooked. Nevertheless, this review identified the need of determining the benefits of using CTX in HIV-uninfected risk groups, such as children and pregnant women. The constraints related to the use of CTX may relate to the fact that it should be taken daily and issues related to its acceptability, safety, adherence and potential selection of resistant strains including antibiotic resistance.CTX has been extensively used for half a century as an antibiotic worldwide and in malaria endemic areas. CTX antimalarial effect, although scientifically proven, has been ignored. Its long term use in HIV infected children and adults, has proved that CTX is still effective for malaria prevention and treatment. This has been confirmed in a few studies in non-HIV infected population, mostly secondary analyses of HIV studies. More information is required for pregnant women, irrespective of HIV infection for whom no information is available. There is need for more randomized controlled trials to evaluate further the efficacy of CTX as antimalarial, since most of the available data are derived from descriptive studies.Research on CTX safety, adherence and acceptability is still necessary if its role for malaria treatment and prophylaxis in groups other than HIV-infected individuals is to be established.


Regulatory T cells (Tregs) suppress host immune responses and participate in immune homeostasis. In co-infection, secondary parasite infections may disrupt the immunologic responses induced by a pre-existing parasitic infection. We previously demonstrated that schistosomiasis-positive (SP) Malian children, aged 4–8 years, are protected against the acquisition of malaria compared to matched schistosomiasis-negative (SN) children.in vitro studies using PBMC acquired from children with and without S. haematobium infection followed longitudinally for the acquisition of malaria. Levels of Tregs were lower in children with dual infections compared to children with malaria alone  but were similar months later, during a period with negligible malaria transmission. The increased levels of Tregs in SN subjects were associated with suppressed serum Th1 cytokine levels, as well as elevated parasitemia compared to co-infected counterparts.To determine if Tregs contribute to this protection, we performed immunologic and Treg depletion These results suggest that lower levels of Tregs in helminth-infected children correlate with altered circulating cytokine and parasitologic results which may play a partial role in mediating protection against falciparum malaria. Co-parasitic infections such as schistosomiasis and malaria are common in endemic areas. We and others have shown S. haematobium infection to modestly protect against uncomplicated malaria in West African children S. haematobium may be important in facilitating protection from malaria.Regulatory T cells (Tregs) are a subset of CD4+ and thymus-derived, while inducible Tregs develop in the periphery from conventional CD4+ T cells after exposure to regulatory cytokines and other stimuli Tregs are characterized by high expression of CD25 S. haematobium infections. Chronic egg-laying schistosomes exert a persistent stimulatory effect on the host immune system, due in large part to egg antigens S. mansoniS. haematobium-positive Zimbabwean children however a comparison group of children without helminth infection was not included +CD25+ T cells reduce egg-induced liver injury and enhance survival via an IL-10 pathway Little is known about the Treg response to human In vivo depletion of natural CD4+CD25+ T cells prior to infectious challenge with a lethal dose of Plasmodium spp. results in enhanced mouse survival and eradication of parasitemia +CD25+ Treg cells restores the Th1 cytokine balance allowing the murine host to control and eradicate the parasitic infection +CD4+CD25hi T cells and FOXP3 expression In malaria, Tregs may facilitate parasitemia and abrogate host clearance of infection. S. mansoni and falciparum malaria S. haematobium correlates with protection against clinical falciparum malaria in an age-specific manner in Malian children, the role of Tregs as a potential immunologic factor contributing to this phenomenon was of great interest. The complexities of conducting immunologic analysis on young children in remote areas of Africa are well known and, largely responsible for the paucity of immunologic data. In spite of these difficulties, we provide evidence that the presence of S. haematobium in Malian children results in lower Treg levels at the time of a malaria infection compared to children without helminthic infection.Limited data are available regarding Tregs in co-parasitic infected humans. Paradoxically, reduced levels of Treg memory cells were found to be present in Kenyan children co-infected with P. falciparum malaria. The entomologic inoculation rate is 20–60 infected bites per month during peak transmission and, children experience a mean of 1.54 symptomatic malaria episodes per season S. haematobium and mansoni are endemic to the area S. haematobium prevalence of 25% in children aged 4–14 years and ∼50% in adults S. haematobium (SP), were age, gender and residence-matched to a child without schistosomiasis (SN) prior to malaria transmission. Children were followed weekly over the malaria transmission season (25 weeks) and at a dry season follow-up appointment (∼9 months after enrollment at a time when standing water pools had dried and schistosoma transmission had ceased). The primary endpoint of the clinical trial was time to first clinical malaria infection. Clinic personnel were available 24 hours-a-day throughout study duration to detect, examine and treat symptomatic malaria episodes. A clinical episode of malaria was defined as P. falciparum parasitemia and axillary temperature ≥37.5°C on active surveillance, or parasitemia and symptoms leading to treatment-seeking behavior in the absence of other clear cause on passive surveillance. All children were pre-treated with albendazole to eliminate concomitant helminth infections and study samples were drawn at the time of their first clinical malaria episode  and again at the final dry season appointment. Children were optimally treated for schistosomiasis with praziquantel at the final appointment.Bandiagara  is located in Mali, West Africa and has intense seasonal transmission (July-December) of The trial was conducted in compliance with the Declaration of Helsinki. Study protocols were reviewed and approved by the University of Bamako's Institutional Review Board (IRB) as well as the University of Maryland IRB. Village permission to conduct research was obtained from village chiefs, government officials and traditional healers prior to study initiation. Individual written informed consent was obtained from the parent or legal guardian of each child prior to screening and enrollment. All children displaying gross hematuria or symptoms of genitourinary pathology were treated with praziquantel (40 mg/kg) therapy and discharged from the study.6 cellsand the presence of at least 15.0×10 for each child at each time point.Patient whole blood (5–10 mL) was collected at the study clinic into sterile eppendorf and EDTA tubes on admission, prior to institution of anti-malarial therapy, and immediately refrigerated. Sera was processed as previously described 2 and washed. A portion of cells (2.0×106) was removed to serve as negative (media) and positive (stimulation with 10 µg/ml Staphylococcus enterotoxin B (SEB); Sigma, St. Louis, MO) controls and to measure the Treg population prior to stimulation. The remaining PBMC were split into two aliquots consisting of 3.0–4.8×106 cells each and either mock-depleted or depleted of CD25hi cells using Dynabead pan-mouse IgG or CD25 magnetic beads, respectively  at a bead to PBMC ratio of 5∶1. Depleted (mock and CD25hi) PBMC (3.0×106) were stimulated with media, malaria antigen pool  42) S. haematobium antigen pools (soluble egg antigen (SEA) and soluble worm antigen protein (SWAP). Antigen stimulation for the assay was optimized at 5 µg/ml/antigen. The malaria antigens chosen represent two vaccine candidates being tested at the same field site in Mali while the S. haematobium antigens are commonly used in schistosoma research. The 3D7 stain of malaria is well documented at the Malian site and strain specific protection against an AMA1 vaccine has been established Thawed PBMC were rested overnight at 37°C, 5% CO+) were excluded from analysis. Doublets/aggregates were subtracted from analysis and gate placement determined with the aid of Fluorescence Minus One (FMO) controls. Cytokine secretion from macrophages/monocytes (CD14+) was analyzed separately. Specimens were included in the analysis if (1) the cell viability was >75% after thawing and (2) cells were shown to be functionally active as determined by the production of IFN-γ by at least 0.2% CD3+ cells after stimulation with SEB. A response was considered specific if the differential in the number of positive events between experimental  and negative control (media) cultures was significantly increased by χ2 analysis. CD69 has been shown to be upregulated on activated effector T cells but not Tregs +CD19−Vivid−CD4+CD25hiCD69−FOXP3+hi within the stimulated PBMC population. A positive response to Treg cell depletion was defined as (1) The net percentage of cytokine producing cells was ≥0.05% in CD25hi depleted cells as compared to mock depleted PBMC after subtraction of media control values; (2) the number of positive events between the CD25hi depleted and mock depleted cultures was significantly increased by χ2 analysis; and (3) the number of positive events in the stimulant pool compared to the media control in the CD25hi depleted cultures was significantly increased by χ2 analysis.PBMC were stained with fluorochrome-labeled monoclonal mouse anti-human antibodies against surface antigens , CD4-allophycocyanin (APC)-Cy7 , CD8-Alexa700 , CD25-phycoeryhtrin (PE)-Cy7 , CD19 /Vivid-Pacific Blue, and CD14-biotin , followed by fixation/permeabilization by using cytofix/cytoperm solution (eBiosciences) and intracellular staining with monoclonal antibodies to IFN-γ-APC , IL-2-PE , FOXP3-Alexa488 , and CD69-PE-Cy5 . In selected experiments monoclonal antibodies to IL-10-APC  and TGF-β-PE  were also used. Cells were then resuspended in 1% formaldehyde fixation buffer and analyzed using a MoFlo flow cytometer/cell sorter system (Beckman Coulter). PBMC from healthy subjects were used as internal controls in the experiments. A total of 100,000–800,000 events  in the forward and side scatter (FS/SS) lymphocyte gate were collected per sample. List-mode data files were analyzed using WinList 6.0 3D . An amine reactive dye  was used as a dead cell discriminator and B lymphocytes  and fluorescence detection by flow cytometry following the manufacturer's recommendations with modifications, as previously described 2 analysis, using Mantel-Haenszel or Fisher Exact (two-tailed) as appropriate, was performed for categorical data. Pearson product-moment correlation coefficient was calculated utilizing GraphPad Prism 5. A significance level of P<0.05 was considered statistically significant.Statistical analysis was performed on GraphPad Prism 5  and demographic and immunologic data were stratified and evaluated by age group (age 4–8 and 9–14 years). Student t-test (two-tailed) or Mann-Whitney U test were used to compare continuous data and χS. haematobium offered modest but significant clinical protection against P. falciparum malaria in an age-specific fashion (children aged 4–8 years) to children exposed to both parasitic infections 6 PBMC/time point>15.0×10  were thawed and examined. Each experiment consisted of a U.S. malaria-naïve adult control, an SP child and an age-matched SN child. Evaluative data was available from 26 SP children  and 8 who did not acquire malaria  during the transmission season) and 23 SN children, all of who developed malaria  . Very few SN children remained clinically malaria-free throughout the malaria transmission season and did not have adequate cells to pursue the immunologic analyses described. Samples were excluded if the viability or thawed quantity of PBMC was insufficient (n = 3 SN children). SP Mal children had a statistically longer time to first clinical malaria infection, reduced geometric mean parasite density at the time of malaria infection, and a trend towards reduced overall amount of malaria infections detected compared to age-matched SN Mal children (We previously found that underlying infection with children . There w+CD19−Vivid−CD4+CD25hiCD69−FOXP3+ cells (Tregs) were significantly higher in SN Mal children at the time of their malaria infection as compared to matched SP Mal children (1.37 (range 0.28–4.79) vs. 0.49 (0.12–1.28), P = 0.004; age 4–14 years)  and dry season) were examined via multiparameter flow cytometry . The mea4 years) . The U.S4 years) . The per+CD19−CD4+CD8− T cells with minimal levels (<0.03%) observed from both Tregs (CD3+CD19−CD4+CD8−CD69−CD25hiFOXP3+) and monocytes/macrophages (CD3−CD19−CD14+) (data not shown). Significant cytokine expression was measured after malaria antigen stimulation of PBMC in both SP children and SN children  but not in SN children . Demonst+CD4+CD25hiFOXP3+ (Tregs) after antigen stimulation and, although the numbers were low, it did establish that detectable cytokine could be measured in these cells  and 6 SN Mal children with malaria, during the wet and dry season follow-up. Cytokine expression was examined in cells gated on CD3se cells . Measurehi cells) on suppression of CD4+ T cell cytokine expression, equal aliquots of PBMC  were mock-depleted or depleted of CD25hi cells and then stimulated with malaria or schistosoma antigen pools (n = 32). Cytokine expression was then measured after depletion of CD25hi cells (compared to the mock-depleted populations) to determine if cytokine production was increased. The effectiveness of depletion was systematically evaluated by flow cytometry following treatment. Depletion resulted in removal of 90–95% of detectable CD25hi. The results are summarized and a representative experiment is shown in In order to evaluate the effect of Tregs . Similarly, increased cytokine expression (29–50%) was noted in PBMC from the dry season time-point when the children had recovered from active malaria infection. The PBMC from children with enhanced IL-2 production after CD25hi depletion, with a few exceptions, demonstrated enhanced IFN-γ production in response to depletion. Likewise, PBMC from children with increased cytokine production after CD25hi depletion during the wet season tended to demonstrate the same findings at the dry season time point. The net percentage of cytokine increases were measured and there were no significant differences between any one group or season. No responses were observed in the SP no Mal children during the malaria transmission season (n = 3) or in the U.S. adult malaria-naïve controls.After PBMC from wet season samples were depleted of CD25hi depletion of PBMC collected from SP Mal children and stimulated with schistosoma antigen (Increased cytokine expression (compared to mock-depleted PBMC) was also measured after CD25 antigen . Howeverhi depletion (responders). No difference was noted between responders and non-responders. However, the mean percentage of Tregs during the malaria transmission season in SN Mal responders was significantly higher than in SP Mal responders (1.19% (range 0.28–4.79) vs. 0.35% (range 0.12–0.68), P = 0.004 Mann-Whitney U test), which mirrored the findings reported for the groups at large. The mean percentage of Tregs during the wet season for SP Mal responders was the same as in the dry season (0.35% compared to 0.41% (range 0.28–0.63) in the same individuals. Conversely, nearly every SN Mal child responder had higher Tregs percentage at the time of malaria, as compared to the dry season (1.19 (0.28–4.79) vs. 0.60% (range 0.2–0.98), P = 0.07).We also examined the Treg percentages in those individuals with enhanced cytokine production after CD25S. haematobium or between samples evaluated during the transmission season and again at a dry season time point. No differences were noted in SI between mock-depleted PBMC and anti-CD25hi-depleted PBMC after stimulation with malaria antigen (data not shown).Of the 26 SP and 23 SN samples, results were available from 20 SP (12 aged 4–8 years and 8 aged 9–14 years) and 17 SN (11 aged 4–8 years and 7 aged 9–14 years). 2 analysis) (hi PBMC did not result in enhanced proliferative responses at either time point (data not shown).A significant proportion of PBMC collected from SP children during the wet season proliferated after schistosoma antigen stimulation compared to SN children  . Schistonalysis) . This waCirculating cytokine values were available from prior studies S. haematobium infections and measure antigen-specific cytokine production in response to malaria and schistosoma antigen stimulation of PBMC acquired from Malian children. While the limited data published to date suggests that Tregs are elevated in acute malaria, the effect of S. haematobium is unknown. Our studies in Malian children provide strong evidence that the presence of Tregs at the time of an acute falciparum malaria infection is paradoxically reduced in the presence of an underlying schistosoma infection compared to age-matched children without helminth infection. Indirect evidence supports this paradoxical finding in that children with higher percentages of Treg  have 1) reduced circulating Th1 (IFN-γ and IL-2) serum cytokine levels suggesting effector T cell suppression; and 2) elevated parasitemia levels compared to their matched co-infected  counterparts. We did not see marked differences in Th2 cytokines  which have been shown to be produced by inducible Tregs and limit malaria pathogenicity S. haematobium. While a recent study reports measuring Treg in children with S. haematobium, a comparison group of children without schistosomiasis was not included S. haematobium infection may not stimulate marked Treg elevation. The Treg differences measured between SP Mal and SN Mal children disappear after the malaria infection had been treated.Very little is known regarding the activity of Tregs in individual human parasitic infections or the immunologic perturbations that one parasitic infection has upon the immune responsiveness to another infection in the host. We report on the presence of Tregs in both malaria and + T cells hi-depleted PBMC acquired from SP Mal children at the time of a malaria infection, however this was also noted in SN Mal children, as well as in a proportion of samples acquired during the dry season follow up period when the children did not have active clinical malaria infection. This finding suggests that Tregs play a partial role in suppressing the immune response to antigens in a non-specific manner. It is possible that Tregs are stimulated to increase in response to malaria infection and that schistosoma co-infection exerts a down-regulatory effect on the generation of Tregs rather than affecting Treg function per se. In this case, the removal of any measurable amount of Treg followed by cell stimulation at any time point might result in a detectable increase in cytokine expression. It would be important to confirm this hypothesis by longitudinally following a cohort of children prior to the development of the malaria infection and after disease resolution. However, in this trial, we were limited to collecting small blood volumes in ill children and requiring that specimens meet high cell numbers and viability threshold at two time points. The complicated logistics of obtaining PBMC from the large cohort of children at enrollment precluded the collection of samples from being drawn prior to the malaria transmission season.A signature role of T regulatory cells is to suppress CD4Plasmodium spp. to escape from host immune defenses resulting in higher parasitemias but ultimately protecting against pathogenic responses such as cerebral malaria hi-depletion did not lead to increased proliferative responses to malaria antigen in our assays The effect that a reduction in measurable Tregs in co-infected children has on clinical malaria remains speculative. In malaria, Treg-mediated suppression of Th1 responses may aid There are a few issues in the present study, which deserve further investigation. We have previously detected sub-patent, asymptomatic infection during the dry season in children at this site (unpublished data) but we did not examine dry season samples for low-level parasitemia as an explanation for why Tregs may continue to exert suppressive effects on effector responses. Moreover, we are limited to observations seen to only two malaria antigens tested. MSP-1 and AMA-1 are being explored as vaccine candidates in our population and we have extensive experience with the immunologic responses to these proteins but they represent a fraction of the malaria protein repertoire. Parasitized RBCs have been found, subsequent to our analysis, to elicit two populations of Tregs expressing different amounts of FOXP3 and may have provided additional interesting results + T cells, is heterogenous in Tregs +CD25hi population by removing from analyses CD69+ cells (a marker of cell activation) +CD25hi cells. Given that the percentage of FOXP3+ expression within CD25hi cells can vary widely (as low as 10% in some estimates) +FOXP3+ cells have variable levels of CD25 expression hi cells which may result in Tregs remaining after depletion and fail to eliminate Tregs that are FOXP3+ alone (16–35% by some estimates). Thus, results may be underestimated. This may partially explain why Treg results vary widely in previous studies. In controlled experiments involving experimental malaria in naïve adults, TGF-β production correlated with parasitemia and Tregs elevation; however this finding was only noted in 50% of volunteers The definition of what constitutes a Treg cell is a matter of some debate. While CD25 has been thought to be a definitive marker of Tregs In summary, our data provides evidence of a reduction in Tregs during acute falciparum malaria infections in schistsomiasis-infected children. Children with falciparum malaria alone had higher Tregs percentages along with reduced Th1 circulating cytokine and higher parasitemias compared to age-matched children with dual malaria and schistosoma infections. These Th1 cytokines may act to control parasitemia in the short-run while contributing to malaria pathogenicity over time. Whether the reduction of Tregs in the co-infected children mediates protection against acute falciparum malaria remains unclear. Future studies are needed to further dissect the complexities of Treg heterogeneity and to determine if the levels of Tregs in schistosoma-infected children at the beginning of the malaria transmission season contribute to malaria protection over time.Table S1Cytokine Assay Results. Results of enhanced cytokine expression after antigenic stimulation of PBMC acquired in the malaria transmission (wet) and dry seasons. The numerator represents the number of children with significant enhancement of TGF-β or IL-10 expression after pooled malaria (AMA1 and MSP1) or schistosoma antigen (SEA and SWAP) stimulation. The denominator represent the number of children examined. The net percentage of cytokine expressed in CD3+CD4+CD8− T cells, as measured by multiparameter flow cytometry, is also depicted in those experiments with significant increases. Cells were acquired from age-matched children 4–14 years old with or without S. haematobium infection. Also depicted are results from SP children who did not acquire malaria  during the wet season.(DOC)Click here for additional data file.Table S2Serologic Cytokine Levels. Serologic cytokine levels expressed in pg/ml during the first clinical malaria episode of the transmission season between Malian children aged 4–14 years with  or without S. haematobium .(DOC)Click here for additional data file.