The case fatality ratio (CFR) of Ebola virus disease (EVD) can vary over time and space for reasons that are not fully understood. This makes it difficult to define the baseline CFRs needed to evaluate treatments in the absence of randomized controls. Here, we investigate whether viremia in EVD patients may be used to evaluate baseline EVD CFRs.V, from 21% (95% CI 16%–27%) for low viremia (V < 104.4 copies/ml) to 53% (95% CI 44%–61%) for intermediate viremia (104.4 ≤ V < 105.2 copies/ml) and 81% (95% CI 75%–87%) for high viremia (V ≥ 105.2 copies/ml). Compared to adults (15–44 y old [y.o.]), the CFR was larger in young children (0–4 y.o.)  and older adults (≥45 y.o.)  but lower in children (5–14 y.o.) . An order of magnitude increase in mean viremia in cases after July 2014 compared to those before coincided with a 14% increase in the CFR. Our findings come from a large hospital-based study in Conakry and may not be generalizable to settings with different case profiles, such as with individuals who never sought care.We analyzed the laboratory and epidemiological records of patients with EVD confirmed by reverse transcription PCR hospitalized in the Conakry area, Guinea, between 1 March 2014 and 28 February 2015. We used viremia and other variables to model the CFR. Data for 699 EVD patients were analyzed. In the week following symptom onset, mean viremia remained stable, and the CFR increased with viremia, Viremia in EVD patients was a strong predictor of death that partly explained variations in CFR in the study population. This study provides baseline CFRs by viremia group, which allow appropriate adjustment when estimating efficacy in treatment studies. In randomized controlled trials, stratifying analysis on viremia groups could reduce sample size requirements by 25%. We hypothesize that monitoring the viremia of hospitalized patients may inform the ability of surveillance systems to detect EVD patients from the different severity strata. In a retrospective cohort study, Simon Cauchemez and colleagues find viral load can predict case fatality ratios among patients with Ebola Virus. During the current outbreak of Ebola virus disease (EVD) in West Africa, which started in December 2013, there have been more than 28,000 confirmed, probable, and suspected cases of EVD and more than 11,000 deaths from the disease. Ebola virus is transmitted to people from wild animals and spreads in human populations through direct contact with the bodily fluids  or organs of infected people or through contact with bedding and other materials contaminated with bodily fluids. The symptoms of EVD, which start 2–21 days after infection, include fever, headache, vomiting, diarrhea, and internal and external bleeding. Infected individuals are not infectious until they develop symptoms but remain infectious as long as their bodily fluids contain virus. There is no proven treatment or vaccine for EVD, although several treatments are now being assessed in people following promising laboratory studies. Supportive care—given under strict isolation conditions to prevent the spread of the disease to other patients or to healthcare workers—improves survival.Ideally, the efficacy of a potential treatment for any disease is assessed in a randomized controlled trial, a study that compares outcomes among people chosen at random to receive the treatment with outcomes among people given a placebo (dummy treatment). However, because EVD is frequently fatal, randomized controlled trials of potential treatments are considered unethical. Instead, studies evaluating treatments for EVD usually compare the case fatality ratio  among treated patients with a baseline CFR estimated from historical data. But the CFR of EVD varies markedly over time and space for poorly understood reasons . Thus, the CFR in the treatment group could differ from the baseline CFR for reasons that are independent of the treatment. To find a way around this problem, in this retrospective cohort study, the researchers investigate whether there is a relationship between viremia (the amount of virus in the blood) and the CFR among patients with EVD.The researchers used laboratory and epidemiological data  to investigate the relationship between viremia and CFR among 699 patients with confirmed EVD hospitalized in the Conakry area of Guinea between March 2014 and February 2015. In the week following symptom onset, mean (average) viremia remained stable, and the CFR among the patients increased with the level of viremia. Thus, the CFRs for patients with low, intermediate, and high viremia (defined by the number of virus copies per milliliter of blood) were 21%, 53%, and 81%, respectively. Compared to adults aged 15–44 years, young children (aged less than five years) and older adults had a higher CFR, but children aged 5–14 years had a lower CFR. Notably, the CFR in the study population was 14% higher after July 2014 than in the months of March–July 2014, an increase that coincided with a ten-fold increase in the average level of viremia in the population.These findings suggest that viremia is a strong predictor of death that can partly explain variations in the CFR of EVD. Because these findings are based on data collected from hospitalized patients, they may not be generalizable to other settings. Importantly, however, these findings provide estimates of CFR by viremia group that can now be used to adjust risk when undertaking clinical evaluations of EVD-specific treatments. That is, by allowing for differing levels of viremia, it will be possible to assess the efficacy of treatments for EVD more accurately in nonrandomized clinical trials. Moreover, the researchers calculate that stratification of patients by viremia group could reduce the sample size needed in any randomized trials that are undertaken  by 25%. Finally, the researchers suggest that monitoring viremia among patients hospitalized for EVD might provide information about the ability of different surveillance systems to detect patients with different levels of disease severity (probability of death).http://dx.doi.org/10.1371/journal.pmed.1001908.This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at EVD, information about potential EVD therapies, and regular updates on the current EVD epidemic; a summary of the discussion of a WHO Ethics Working Group Meeting on the ethical issues related trials of EVD treatments is available; the WHO website also provides information about efforts to control Ebola in the field and personal stories from people who have survived EVDThe World Health Organization (WHO) provides information about EVDThe UK National Health Service Choices website provides detailed information on http://www.cdc.gov/vhf/ebola/EVDThe US Centers for Disease Control and Prevention also provides information about  An epidemic of Ebola virus disease (EVD) of unprecedented magnitude has been ongoing in West Africa since December 2013 [Currently, most treatments used in Ebola treatment centers (ETCs) rely on supportive care, but several experimental therapies are being assessed for EVD following promising in vitro and limited in vivo findings –5. The mOf course, an important limitation of using historical controls is that, in the absence of randomization, the CFR in the treatment group could differ from the baseline CFR for reasons independent of treatment. Indeed, the CFR of Ebola has varied historically, over time and space ,12,13. FHere, we investigate the relationship between patients’ viremia  and their probability of death, and we assess how this relationship may be used to explain temporal trends in reported CFRs and inform different treatment study designs.We analyzed the laboratory results of a large number of EVD patients from Conakry and surrounding prefectures . These pWe did not seek institutional review board approval for data collection in this study because data were collected as part of routine case management under an emergency response mandate from the government of Guinea. As part of routine practice, patients orally agreed to be tested for Ebola virus infection.d from symptom onset to sample collection: (i) if 3 ≤ d ≤ 10 d, the negative result is definitive; (ii) if d ≤ 2 d, a second sample is collected 3 d after the first sample, and a new RT-PCR test performed, with the results of this second test being final; (iii) if d > 10 d, cases are confirmed using a serological test [Ct value obtained for each sample tested. We had access to the laboratory dataset, where all the laboratory results were recorded, as available on 3 March 2015.In the IPD-LFHP laboratory in Conakry, diagnosis of EVD was performed using real-time RT-PCR ,15. The cal test ,15. In tThe epidemiological line list has already been described in detail elsewhere . In shorOur analysis was restricted to EVD patients from the epidemiological line list who (i) had EVD confirmed by an RT-PCR test performed by the IPD-LFHP laboratory in Conakry, (ii) resided in Conakry or in the surrounding prefectures of Boffa, Coyah, Dubreka, Forecariah, Fria, Kindia, or Telimele (in which diagnoses were mostly performed by the IPD-LFHP laboratory), (iii) had symptom onset between 1 March 2014 and 28 February 2015, and (iv) were hospitalized. Patients were excluded from the analysis if (i) sample collection was done after the day of death or more than 30 d after symptom onset or (ii) one of the following variables was missing or unclear: age, prefecture of residence, date of symptom onset, outcome, date of hospitalization, date of death if died, or date of sample collection. In a sensitivity analysis, we explored the robustness of our findings when different eligibility criteria were used  as a function of age, gender, and time from symptom onset to sample collection.We calculated mean viremia  and selected the best threshold values v1 and v2 (v1 < v2) at maximum likelihood  as a function of viremia We then modeled outcome in all patients in a multivariable logistic regression that included viremia  and the following other predictors of death: age in four classes  and time from symptom onset to sample collection in three classes .Using this model, it was possible to investigate whether the observed temporal variations in CFR could be explained by changes in the distribution of viremia over time. For each case, we computed the probability of death according to viremia, patient age, and time from symptom onset to sample collection. We then averaged these probabilities by month and compared these with the observed monthly CFRs. We explored the consistency of our results when only a subset of cases was used to inform the model. We estimated monthly CFRs using a model that was trained on cases that were hospitalized between 1 March 2014 and 30 September 2014 only and compared the results to when the whole dataset was used.Ct [Finally, as with all assays, the measurement of viremia  is not without uncertainty. It has been estimated that the standard deviation of the assay is around 0.48 Ct . To explWe compared the number of patients to include in an RCT for a new treatment against Ebola depending on whether viremia was/was not used for stratification of the analysis. We used the Cochran-Mantel-Haenszel test for the stratified analysis and the Chi-squared test for the unstratified analysis. We computed sample sizes using the viremia-level groups identified in this study, with their corresponding CFRs. Sample size formulas are reported in Section 7 of n = 71), sample collection after death (n = 11), or long delays from symptom onset to sample collection/death (n = 6) (n = 332) were female  confirmed and probable cases that were hospitalized, 855 (97%) were confirmed, of which 787 (89%) were confirmed by RT-PCR by the IPD-LFHP laboratory and had a viremia measurement available . Eighty- (n = 6) . Our fine female . Mean tie female .4.45 (95% CI 104.32–104.57), and it generally declined sharply among patients still alive after day 7. Mean viremia was comparable in males and females (p = 0.95). It was significantly higher in young children  than in other age groups  ( = 0.02) .10V)2 as a single explanatory variable (see Section 3 of 10V)2 was 1.12 (95% CI 1.10–1.14). This simple model provided an excellent fit to the data (V < 104.4) to 53%  in those with intermediate viremia (104.4 ≤ V < 105.2) and 81%  in those with high viremia (V ≥ 105.2) (n = 243), 24% , and 31% , respectively. Our results were found to be robust to the level of measurement error expected from the viral load assay  patients whose samples were collected within 7 d of symptom onset, a time period when viremia appeared to be stable . Among tthe data . The pro≥ 105.2) . The pro10V)2, age, and time from symptom onset to sample collection using data from all 699 EVD patients. We found that the OR for a unit increase in (log10V)2 was unchanged . We also found that late sample collection (>7 d)  was associated with a significantly increased probability of death compared to those tested <4 d after symptom onset . This rise coincided with an order of magnitude increase in mean viremia (p < 0.01). Our multivariable logistic regression model performed well in predicting individual outcomes  in March–July 2014 to 49% (95% CI 45%–53%) after August 2014   will be the most efficient for testing the efficacy of a new treatment. With the mix of viremia levels across patients observed here  and corresponding CFRs, the total number of patients to include in an RCT would be reduced by 25% with a stratified comparison relative to an unstratified comparison  than that of typical probable and confirmed EVD cases. EVD cases that died before they reached the hospital or before a sample could be collected or that were not hospitalized were indeed excluded from our study. To put our estimates in the wider context of the ongoing epidemic, we provide estimates of CFRs for other groups. From the line list, we estimate that the CFR for all probable and confirmed cases with symptom onset between 26 December 2013 and 3 March 2015 in Guinea was 70% (95% CI 68%–72%), consistent with previously reported figures . HoweverThe increase in mean viremia and CFR in the study population in the months after July 2014 compared to those before is an interesting observation that requires careful consideration. A number of mechanisms could explain this trend. A first possibility is that disease severity increased during this epidemic. This could have occurred, for example, if the virus evolved to become more virulent. However, such rapid adaptive evolution is not supported by the analysis of the currently available genetic sequences from the outbreak ,20, althThis study has some limitations. Our analysis was conducted on a retrospective cohort using data collected as part of routine case management, with no active follow-up of patients who were discharged. We considered individuals discharged from hospital as having recovered; however, it is possible that some died at a later date. This would result in increases in the CFRs reported here, although any differences are likely to be minor. Further, we used data only on hospitalized cases. Our results may not be generalizable to community cases, as the distribution of viremia may be different in these individuals, for example if they represent cases that died before they could seek care or that had only minor symptoms. The route of infection, which is unknown in our cases, may be linked with differential mortality risk . If suchIn summary, in a very large and consecutive sample of patients with confirmed EVD, we have shown that viremia is a strong predictor of death that may in part explain previously observed heterogeneity in CFR estimates. Viremia may also provide an important mechanism for risk adjustment among patients in studies aiming to estimate associations of treatment with outcome, and a mechanism to stratify patients into different risk groups within clinical trials.S1 STROBE Checklist(DOC)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S1 DatasetColumn 1 gives the number of EVD patients with a specific profile, where the profile is given by the month of hospitalization (column 2), outcome (column 3), age (column 4), and viremia group (column 5).(CSV)Click here for additional data file.


Our data may contribute to a better understanding on the persistence of HbE among ethnic groups and its association with malaria.Hemoglobin E (HbE) is one of the most common hemoglobin variants caused by a mutation in the β-globin gene, and found at high frequencies in various Southeast Asian groups. We surveyed HbE prevalence among 8 ethnic groups residing in 5 villages selected for their high period malaria endemicity, and 5 for low endemicity in northern Thailand, in order to uncover factors which may affect genetic persistence of HbE in these groups. We found the overall HbE prevalence 6.7%, with differing frequencies from 0% in the Pwo Karen, the Lawa, and the Skaw Karen to 24% in the Mon. All HbE genes were heterozygous (AE). Differences in HbE prevalence among the studied ethnic groups indirectly documents that ancestries and evolutionary forces, such as drift and admixture, are the important factors in the persistence of HbE distribution in northern Thailand. Furthermore, the presence of HbE in groups of northern Thailand had no effect on the  The hemoglobin E (HbE) variant is caused by a genetic mutation at the codon 26 of the β-globin gene, changing an amino acid GAG (glutamine) in the normal hemoglobin A molecule (A) to AAG (lysine) . HbE carPlasmodium, remains one of the most life-threatening disease in tropical countries [P. falciparum is the most lethal species causing malaria-related deaths. The association of various hemoglobinopathies with protection from malaria was systematically reviewed in a meta-analysis of 62 studies, revealing that most do protect against severe malaria symptoms [Malaria, a parasitic disease caused by protozoa in the Genus ountries . Among fsymptoms , with vasymptoms . Most ofin vitro markers of protection against invasion of red blood cells (RBCs) by P. falciparum, such as the proportion of red blood cells infected in vitro, and the quantity of hemozoin, a product of hemoglobin digestion by the parasite. The data obtained may be helpful in understanding genetics protections against malaria.Various parts of northern Thailand, including the malaria-endemic area along the Salween River, which forms the frontier between Myanmar and northwestern Thailand, and the lowland Chiang Mai-Lamphun basin, have been occupied for hundreds or even thousands of years by several ethnic groups , some ofThe study was approved by the Human Experimentation Committee, of the Research Institute for Health Sciences, Chiang Mai University, Thailand. Among a total of 18 recognized ethnic groups in northern Thailand , we sampHbE, α- and β-thalassemia traits were identified using cation exchange high performance liquid chromatography, using methods described elsewhere ,15. The P. falciparum laboratory 3D7 strain was maintained in continuous culture under standard conditions [The nditions . RBCs frnditions , washed 7 cells was added into 10x108 RBCs (1% parasitemia) and cultured for 96 hours. The percentages of infected RBCs on blood smears were determined by the average count of two human microscopists.Mature schizonts were obtained by gradient centrifugation over 60% Percoll  . Isolate3, and incubating at room temperature for 20 minutes, 100 μl of 2.5% SDS in 1M NaHCO3 was added and then incubated for 30 minutes and absorbance measured at 405 nm and 750 nm on a Spectra MR plate reader .After 96 hours of incubation, formation of hemozoin was determined by methods described elsewhere ,22. Briep-values were calculated by using the R statistical programming language version 3.2 . Analysis of variance (ANOVA) was tested by STATISTICA .Correlations between each pair of parameters and Eight of the selected villages were in the Salween River area in northern and southern Mae Hong Son province, while two were in the Chiang Mai-Lamphun basin in both Chiang Mai and Lamphun provinces. Although the target sample size was 30 subjects per village, in Jong Kham (Shan), Huay Pu Kaeng (Padong Karen), Um Da Neur (Skaw Karen), Nong Du (Mon) and Luang Nuer (Lue) villages there were insufficient number of unrelated persons to satisfy this eligibility criterion. Thus, a total of 269 samples from 114 male and 155 female volunteers were obtained from the 10 villages.HbE was detected in 6.7% (18/269) of the subjects . The HbEp <0.05) was detected between α-thalassemia and malaria prevalence, and G6PD deficiency and β-thalassemia prevalence  had the highest , while the Dong Sa Ngat (Pwo Karen) had the lowest  prevalence of hemoglobinopathies. There was no statistical correlation between the HbE and malaria prevalence for each ethnic population. A slightly significant correlation  and the Austro-Asiatic  linguistic families, while the Sino-Tibetan showed low HbE prevalence . The Sino-Tibetan population also showed low frequency of G6PD deficiency  and β-thalassemia . α-thalassemia was the most prevalent hemoglobin disorder observed for all linguistic families, ranged from 13.6% (14/103) in the Sino-Tibetan to 22% (12/55) in the Austro-Asiatic .P. falciparum, the mean percentages (± SD) of infected RBCs in HbE carrier group (7.3 ± 1.12) was not statistically different from controls who were the same ethnic group with no hemoglobin disorder (7.4 ± 1.33)  or normal RBCs (O.D. 0.492 ± 0.097) .After exclusion of G6PD deficiency and thalassemia, 7 HbE carriers  were willing to participate in the invasion assay study. After 96 hours of incubation RBCs and  ± 1.33) . There w± 0.097) . When coOur survey of HbE prevalence among ethnic groups residing in malaria-endemic area of northern Thailand found 6.7% of HbE carrier, which is slightly lower than a previous observation in the Northern Thai people . The preth to 13th century, then fallen under the Tai’s invasion [The Nong Du (Mon) of Lamphun province exhibited the highest frequency of HbE among our study populations. The Mon was an indigenous ethnic group who established their civilization along the Ping River of northern Thailand during the 8invasion . This waDuring the prehistoric and proto-historic periods, the Mon ethnic group and their Austro-Asiatic speaking relatives occupied the broad areas where are now Cambodia, Northeast/Central Thailand, and Southern Myanmar . SeveralAlthough, many findings have pointed out the importance of HbE against malaria infection, there was no significant correlation between HbE prevalence and malaria incidence reported in ethnic groups of northern Thailand. However, this result should be interpreted with caution as there were a few limitations in our study. First, although most of our selected populations are settled in their present localities possibly since hundreds of years ago, members of the Sino-Tibetan linguistic family, i.e. the Dong Sa Ngat (Pwo Karen) and the Huay Pu Keang (Padong Karen), are still wandering around the border between Thailand and Myanmar . Their recent subdivision populations or founder effect, which is a shift in allelic frequency due to the creation of new and isolated populations, may have more impact on the frequency of HbE than malaria endemicity. The relative lower HbE, G6PD deficiency, and thalassemia frequencies in the Sino-Tibetan than the Tai-Kadai or Austro-Asiatic was corresponding to this founder scenario. Secondly, owing to the fact that most of the ethnic people of northern Thailand tend to practice endogamous marriage, collection of unrelated samples was limited and small sample size from each village was analyzed. Thus, due to the recent demography and small sampling number, further investigations are needed to explore the relationship between HbE prevalence and malaria incidence of ethnic population in northern Thailand.Taking into consideration of sufficient sample numbers by grouping study populations according to their linguistic origins, some speculations for the HbE and malaria relationship could be evaluated. The high HbE prevalence in the Austro-Asiatic family is in confronting with the malaria prevalence which is consistent with the Haldane’s hypothesis of heterozygous advantage selection against malaria . Though P. falciparum in vitro but no inhibitory effect was found. This was consistent with the observations in P. falciparum patients which reported no association between HbE and malaria [in vitro study in different Thai populations which reported inhibitory effect of HbE in vitro [P. falciparum strains used for invasion assay. In addition, the ability to proliferate in both normal and hemoglobinopathic RBCs of different strains of P. falciparum can differ substantially [P. falciparum field isolates, therefore bias due to strain specificity could not be ruled out. It is also worth to take note that due to limitation of light microscope which provides 2-dimentional images of infected red blood cells, we did not enumerate the number of merozoites released from an individual schizont as it could underestimate merozoite number. Further investigation on larger ethnic populations, invasion of P. falciparum field isolates, and enumeration of merozoite number will be of great interest and need to be addressed in the future.Analysis of various hemoglobinopathies indicates that hemoglobin mutations confer protection against severe malaria, but differ in the degree of protection against uncomplicated malaria and asymptomatic parasitemia . Most ob malaria ,29. Howein vitro . The disantially . HoweverHemozoin or malaria pigment is a product from the detoxification process used by malaria parasites to convert toxic free heme, which is an outcome of hemoglobin digestion, into an insoluble crystalline form . It is iP. falciparum growth and hemozoin formation.In conclusion, we reported the overall HbE prevalence of 6.7% among ethnic groups residing in northern Thailand, varying among groups. Ancestry, genetic drift, and admixture have impact on the HbE distribution. Despite of small sample numbers, our data suggest that HbE does not have an effect on


In spite of increasing reports of dengue and chikungunya activity in Tanzania, limited research has been done to document the general epidemiology of dengue and chikungunya in the country. This study aimed at determining the sero-prevalence and prevalence of acute infections of dengue and chikungunya virus among participants presenting with malaria-like symptoms  in three communities with distinct ecologies of north-eastern Tanzania.Cross sectional studies were conducted among 1100 participants (aged 2–70 years) presenting with malaria-like symptoms at health facilities at Bondo dispensary , Hai hospital  and TPC hospital (Lower Moshi). Participants who were malaria negative using rapid diagnostic tests (mRDT) were screened for sero-positivity towards dengue and chikungunya Immunoglobulin G and M (IgG and IgM) using ELISA-based kits. Participants with specific symptoms defined as probable dengue and/or chikungunya by WHO  were further screened for acute dengue and chikungunya infections by PCR.n = 1003) were malaria negative by mRDT. Out of these, few of the participants (<5 %) were dengue IgM or IgG positive. A total of 381 participants had fever out of which 8.7 % (33/381) met the defined criteria for probable dengue, though none (0 %) was confirmed to be acute cases. Chikungunya IgM positives among febrile participants were 12.9 % (49/381) while IgG positives were at 3.7 % (14/381). A total of 74.2 % (283/381) participants met the defined criteria for probable chikungunya and 4.2 % (11/263) were confirmed by PCR to be acute chikungunya cases. Further analyses revealed that headache and joint pain were significantly associated with chikungunya IgM seropositivity.Out of a total of 1100 participants recruited, 91.2 %  contains supplementary material, which is available to authorized users. Dengue and chikungunya are major global health concerns due to their continued spread and intensifying epidemic activities throughout most of the tropical and subtropical regions of the world. Regular outbreaks of dengue have been reported throughout most of the tropical and sub-tropical regions of the world for several decades –3 while Aedes aegypti mosquitoes, furthermore chikungunya has increased its geographical range by its ability to infect Aedes albopictus [These viruses are transmitted between humans by the bite of infected bopictus , 6. Mostbopictus , but canbopictus , 9.Tanzania, as many other African countries, is experiencing a shift in disease transmission patterns with regards to febrile infectious diseases. While the number of cases of malaria are decreasing in several countries of sub-Saharan Africa –13, the Little is known about the general epidemiology of dengue and chikungunya in Africa and Tanzania in particular. Thus, the objective of this study was to determine the sero-prevalence and as well, prevalence of acute infections of dengue and chikungunya virus among participants presenting with malaria-like symptoms  in three communities with distinct ecologies of North-eastern Tanzania.(900–1666 m) and lowland zone (900 m) above sea level. In Hai malaria transmission is classified as moderately high in lower altitude, moderate in mid-altitude and non-endemic in highland areas [We conducted cross sectional studies at three sites in north-eastern Tanzania; Bondo dispensary , Hai hospital  and TPC hospital (Lower Moshi) in May 2013, November 2013 and May 2014, respectively. These sites were selected based on variation in precipitation pattern and varying altitude between the sites resulting in difference in malaria transmission, and as well, evidence of overdiagnosis of malaria . Since mnd areas . Lower Mnd areas , 33.Community sensitization to participate in the study was done through community leaders. Inhabitants aged 2 to 70 years with specific symptoms  were encouraged to voluntarily go for testing at the health facilities at Bondo Dispensary Hai Hospital or TPC hospital. Individuals presenting at the health facilities were informed of the study and on voluntary participation. A written informed consent was obtained from all participants and from parents or guardians for children under 18 years of age who agreed to participate in the study. A questionnaire was used to collect demographic and clinical data such as age, gender and history of illness for each of the participants. All participants were screened for malaria by rapid diagnostic tests  and malaria positive patients were treated with antimalarial according to national guidelines. In addition, auxiliary temperature measurement was done to detect febrile cases (>37.5 0C) and a blood sample of 0.5-1 mL was taken from all consenting participants. A subject number was assigned to each participant and used to label blood samples. All samples collected in this study were not linked to personal identifier.Serum was separated from whole blood by centrifugation and stored at −20 °C. Anti-dengue IgM and IgG were detected using a direct enzyme linked immunosorbent assay (ELISA) kit  while anti-chikungunya IgM and IgG were analysed using Indirect Elisa kits . All assays were performed according to the manufacturers’ procedures and all serum samples were diluted 1 into 100 with sample diluent provided with the kits. The optical density (OD) was measured at 450 nm and the units of antibody concentration and cut-off values calculated as described by the manufacturers. Briefly, for the Anti-dengue IgM/IgG and IgM anti-chikungunya ELISAs the diagnostic cut-off value was calculated as the average OD of negative controls + 0.300. For the IgG chikungunya ELISA the threshold for positivity was based on the OD cut-off value of the cut-of control + 10 %.Only serum samples from participants with specific symptoms  who met World Health Organization (WHO) criteria , 34 for Dengue PCR was conducted as previously described  with res2, 1.8 μL of 10 μM dNTP, 0.5 μL of 20 μM of each primers, 0.3 μL of Go-Tag Flex DNA polymerase, and 10.3 μL of nuclease free water . The PCR programme consisted of an initial denaturation at 95 °C for 2 min and 40 cycles of 30 s denaturation at 95 °C, 30 s annealing at 55 °C, 1 min elongation at 72 °C and final extension at 72 °C for 5 min. The amplified gene products were identified by their molecular weights analyzed by electrophoresis on a 1.5 % agarose gel stained with ethidium bromide and visualized under UV light.The primers Table  used forDengue primers and probeswereadopted from Pongsir et al.,  for deteDengue and chikungunya cases were classified according to WHO , 34. In χ2) was used to compare categorical data while Fisher’s exact test was used in cases when expected counts were less than 5. Association of chikungunya seropositivity and symptoms was analyses by logistic regression. A P-value < 0.05 was considered statistically significant. Descriptive statistics are presented as medians and ranges for continuous variables after checking for normality and as proportions for categorical data.Data were compiled and analyzed using SPSS 20.0 software . Chi-square , of which 61.3 % (674) were female and 61.6 % (678) were adults (≥15 years) , TPC hospital  and Bondo Dispensary , 1100 participants were recruited , followed by TPC hospital at 52.3 % (23/44) and Bondo dispensary at 22.8 % (61/267). Among adults, the prevalence of fever were 53.0 % (115/217) at TPC hospital, followed by Bondo dispensary at 40.4 % (95/235) and lastly, in Hai hospital at 24.5 % (46/188).Among all febrile participants, the total prevalence of dengue IgM positives was 1.3 % (5/381) all of which were from participants residing nearby TPC hospital (3.6 % (5/138)  and IgG sero-prevalence was low with difference between children 3.0 % (11/363) and adults 5.9 (38/640), . The total number of chikungunya IgM positives among febrile participants were 12.9 % (49/381), and there were no difference observed between children 12.0 % (14/49) and adults 13.3 % (34/49) . No apparent differences between the sites were observed; 14.7 % (23/156) in Bondo dispensary, 13.8 % (12/87) in Hai hospital and 10.1 % (14/138) in TPC hospital , .Generally, we observed high IgM sero-prevalence among all participants while no difference between children 14.0 % 51/363) and adults 13.1 % (84/640), . Furthermore, the number of IgM positives was significantly different between the sites; 12.1 % (42/346) in Bondo dispensary, 20.3 % (31/153) in Hai hospital and 10.6 % (13/123) in TPC hospital . Likewise; the observed total chikungunya IgG positives was lower than the IgM positives, at 5.6 % (35/622) though with a significant difference between the sites; 3.2 % (11/346) in Bondo dispensary, 6.5 % (10/153) in Hai hospital and 11.4 % (14/123) in TPC hospital , , though this was not significantly different  and 74.2 % (283/381) cases met the WHO criteria for probable dengue and chikungunya respectively. None of the cases were positive for dengue while 4.2 % (11/263) were confirmed acute chikungunya cases by PCR. Ten of the acute chikungunya cases were from Bondo and a single case came from Hai. Two of these cases were children (2 to 14 years).p < 0.01 and OR = 1.80 (1.25–2.59), p < 0.01 respectively. However when we combined headache and joint pain we did not observe any association, OR = 0.84 (0.55–1.26); p = 0.4. Joint pain were marginally associated with chikungunya IgG seropositivity OR = 0.51 (0.26–0.9), p = 0.05, Tables In uni-variate analysis results, headache and joint pain were significant predictors of chikungunya IgM seropositivity with OR = 3.03 2.06–4.4), .4, p < 0 1.80 1.2–2.59, p Diagnosis of infectious diseases in resource poor settings is a challenge. In malaria endemic areas most febrile illnesses have been and are treated as malaria cases due to lack of diagnostic capacity to properly rule-out malaria as a cause of fever and as well, identify alternative fever-causing pathogens . As for This study was conducted in three sites in northern Tanzania, each community have different characteristics in terms of varying altitude and thus, malaria transmission that may had identified acute cases of dengue and chikungunya infections and variations in prevalence of serological markers of dengue and chikungunya between the sites. Accordingly, the three sites differed in malaria prevalence with TPC hospital having higher malaria prevalence as compared to Bondo dispensary and Hai hospital as based on mRDT diagnosis.We observed few dengue IgM positive cases among febrile participants in TPC hospital in Moshi (3.6 %) and there were no confirmed acute dengue cases by PCR at any sites. Evidence shows that viral RNA can be detected in patients from 0–7 days following the onset of symptoms, which corresponds roughly to the duration of fever while IgM is detected in 3 to 5 or more days after the onset of illness/symptoms in the majority of infected individuals . TherefoOnly 1.3 % of the participants were IgG positive to dengue infection. This is lower than the previously reported (10.7 %) in Moshi by Hertz et al. . This caConversely, we identified a high sero-prevalence of chikungunya IgM (12.9 %) among febrile participants out of which 12.0 % were among febrile children. This is the first report on chikungunya IgM positivity in the study sites. The proportion of chikungunya IgM seropositivity was comparable between children and adults thus indicating similar exposure among the two age groups. The 12 % seropositivity observed in children ismore than twice as high as previously reported by Chipwaza et al.  in Kilosn = 10, 2.0 %). This is somewhat lower than the previous reported in Kilimanjaro region of 7.9 % [We identified an overall prevalence of 4.2 % of acute chikungunya infections among study participants; with Bondo dispensary having the highest number of cases  and the probability of positive diagnosis of chikungunya IgM. These results are in line with other studies, that reported headache, nausea, vomiting, myalgia, and rash were associated with chikungunya infection , 46. We We did observe quite a low number of mRDT positives in the 3 sites; preliminary data suggest that, as expected, a fraction of the mRDT negatives are plasmodium positive when using a sensitive PCR method (data not shown), and therefore the low parasitaemi as are having no impact on fever/symptoms observed in participants, nevertheless after excluding all malaria positive, we found that 38.0 % of the remaining participants reported fever. It is known that in many malaria endemic areas where clear diagnosis cannot be established, all fevers may still be treated as malaria . TherefoThis is the first seroprevalence report of dengue and chikungunya among participants presenting with malaria-like symptoms in three communities with distinct ecologies of in north-eastern Tanzania. Chikungunya virus appears to be actively circulating in the population, while dengue circulates in low level. Among malaria-like symptoms, headache was associated with chikungunya IgM seropositivity.Ethical approval for this study was obtained from the Kilimanjaro Christian Medical University College Research and Ethics Review Committee (CRERC) with a certificate number 658. A written informed consent was obtained from all participants and from parents or guardians for children under 18 years of age who agreed to participate in the study.


Plasmodium falciparum extracts for assays to evaluate the overall anti-blood-stage immune response in humans may be beneficial to malaria pre-elimination programmes. However, there is no consensus on which strain is appropriate for routine analyses. This study aimed to compare the responses of malaria IgG antibodies in serum collections from Dielmo and Ndiop to crude extracts of merozoites and schizonts of local and reference strains of P. falciparum.The standardization of the type of crude P. falciparum reference Palo Alto strain (PA). A total of 218 sera collected in 2000 from inhabitants of the villages of Dielmo and Ndiop were used: 142 from Dielmo and 76 from Ndiop. The biological collection was used to evaluate by ELISA the prevalence of IgG antibodies against crude merozoite and schizont extracts. The genetics of the local and reference strains were compared.Malaria antibodies were evaluated using serological tests for exposure to three local strains  and the p < 0.01). Ig G responses were highest to schizont and merozoite extracts from the field strain of P. falciparum 0703 adapted to in vitro culture. Extracts of P. falciparum strain 0703 isolated from a subject in Dielmo was the most widely recognized , although the responses were high for merozoites from PA [85.3 % (186/218)] the reference strain, and the two strains isolated from subjects living in Dakar: F15 [90.4 % (197/218)] and F16 [72.5 % (158/218)].There was genetic divergence between strains 0703, F15, F16 and PA. IgG responses against local and reference strains correlated well (0.6 to 0.8; For serological studies, the local strain provided the most complete picture of exposure to transmission and malaria prevalence in the local context. However, for the standardization of this method by different laboratories, the reference strain appeared to perform sufficiently well to be used for the evaluation of malaria control programmes. Plasmodium falciparum blood stages [P. falciparum strain reflected the changes of malaria profiles due to various interventions [P. falciparum antigen to be used in standard assays for the evaluation of the overall anti-blood-stage immune response in humans.Programmes of pre-elimination of malaria have been underway in Senegal since 2010, and the burden of malaria has decreased substantially . The chad stages , 12. Antventions . HoweverP. falciparum blood stages in malaria-exposed individuals.This work aimed to compare merozoite and schizont extracts from local and reference strains to identify the most suitable extract for routine analysis of antibody responses to Since 1990, the Dielmo project was established between the Institut Pasteur of Dakar, the Institute for Research and Development, the Senegal Ministry of Health and the University of Dakar. This project was initially approved by the Ministry of Health of Senegal and the assembled village population. The project aim was to study the natural history of malaria and more specifically the determinants of protection and biological markers associated to protection/exposure through longitudinal follow up of the inhabitants of the villages of Dielmo and Ndiop . For thiWritten informed consent from individuals enrolled in the project and parents or guardians of children who provided blood samples was obtained. The consent to participate to the project was regularly renewed. Approval was obtained from the National Ethics Committee for Health Research of Senegal and ad-hoc committees of the Ministry of Health, the Pasteur Institutes (Dakar and Paris), and Institute for Research and Development did audits regularly.This study is based on the samples stored in the biobank and is in line with the objectives of the Dielmo project .This comparative analysis study was conducted using sera collected in 2000 from inhabitants of the villages of Ndiop and Dielmo. A total of 220 sera from individuals less than 20 years old were used: 76 from Ndiop and 141 from Dielmo. In 2000, Ndiop and Dielmo had differences in malaria endemicity and contrasting contexts of transmission: in Ndiop, transmission was seasonal with an entomological inoculation rate of 80 infectious bites/person/year, whereas in Dielmo the transmission was continuous with 482 infectious bites/person/year . Blood s+ erythrocytes in RPMI containing 0.5 % Albumax using the candle jar method [P. falciparum isolated from clinical or asymptomatic malaria-infected Senegalese patients and adapted for in vitro culture as described elsewhere [in vitro culture thereafter [in vitro culture. The Palo Alto Uganda reference strain FCR3  had been isolated from a Ugandan patient [P. falciparum were prepared as previously described [, washed three times in sterile phosphate buffered saline (PBS), counted, and frozen in liquid nitrogen.Four parasite strains  were used in this study and continuously cultured on Or method . Three flsewhere , 18. Theereafter . F15 and patient  and had escribed , 18. Briescribed , 21. In P. falciparum adapted in continuous culture  and from the PA reference strain using a QiAmp ® DNA Blood MiniKit  according to the manufacturer’s recommendations. Nested PCR genotyping was performed for the variable block 3 region of msp-2, considered to be the most informative genetic marker for assessment of P. falciparum genetic diversity [msp-2 (block 3) region was followed by individual nested PCR reactions using family-specific primers for msp-2 (FC27 and 3D7/IC), using previously described standard protocols [et al. [msp-2 products were separated by electrophoresis on 1.5 % agarose gel and the fragments were visualized under UV transillumination with ethidium-bromide. The sizes were estimated by comparison with a 100 bp  molecular weight marker.Genomic DNA was extracted from wild field Senegalese isolates of iversity . The inirotocols . The PCR [et al. , 25. The2O2 (100 μl) and the reaction was stopped by addition of 4 N H2SO4 (50 μl/well). Between each incubation phase, the ELISA plates were washed extensively with PBS-0.05 % Tween. For each assay, plasma samples were tested in duplicates on the same ELISA plate. The optical density (OD) at 450 nm was read in a BIO-RAD Microplate Reader (iMark) [ELISA Maxisorp plates  and a dose effect assay using positive control sera were used to determine the optimal dilution of the extracts to use in the assay. The optimal dilution found for each extract was used for coating the plates. Plates were coated with 100 μl of water-soluble extract for all crude extracts. Non-infected red blood cell extract was prepared in the same conditions, incubated overnight at 4 °C and washed. Then, 200 μl of a blocking buffer (2 % BSA in PBS with 0.05 % Tween) was added to each well and the plates were incubated for 1 h at 37 °C. Plasma samples were diluted at 1/200 in a dilution buffer (1 % BSA in PBS with 0.05 % Tween) and 100 μl of diluted serum was distributed into each well. Negative and positive controls were included on each plate: positive controls were from Dielmo/Ndiop hyperimmune individuals and negative controls were from European individuals. Polyclonal goat anti-human IgG conjugated to peroxidase at a dilution of 1/6,000 in the dilution buffer (1 % BSA in PBS-Tween 0.05 %) was then added. Bound peroxidase was detected with ortho-toluidine /H (iMark) . The fin (iMark) , 26.R software was used for statistical analysis. Non parametric tests  and the Spearman test were used to study non-parametric quantitative variables. Fisher’s exact test was used for qualitative variables. Differences were considered statistically significant for p < 0.05 [p < 0.05 .msp2 locus  was determined by assessing length polymorphism within the rho values of 0.62, 0.6, and 0.56 respectively for PA, F15 and F16; p < 0.01) . The OD ratios obtained with IgG against PA, F15 and F16 merozoites was also significantly correlated with OD ratios obtained for 0703 merozoites  . The response against meroPA was significantly higher than those against meroF15 and meroF16 (p < 0.01).Mean antibody responses, expressed as OD ratios were 4.44 for meroPA, 3.63 for meroF15, 2.79 for meroF16 and 4.64 for mero0703 , but there was no difference for strains F15 (p = 0.06) and 0703 (p = 0.26). The mean level of the antibody response against 0703 was higher than those of the other strains although the difference with meroPA was not significant.Antibody responses against schizont extracts and merozoite extracts were determined and compared Fig. . The meavs.75.2 % for schizont extract but not for strains F16 (72.5 % vs. 71.1 %), F15 (90.4 % vs 77.5 %) and 0703 (91.3 % vs. 81.2 %) (Table The percentages of positive responders (OD ratio > 2) to merozoite and schizont crude extracts were compared Fig. . There wP. falciparum blood stages in individuals exposed to malaria.The study reported here involved an analysis, by an Enzyme Linked ImmunoSorbent Assay approach, of IgG responses to extracts of merozoites and schizonts from local and reference strains; the aim was to identify the most suitable extract for routine analysis of the levels of antibodies to P. falciparum strains. The mps2 genotype was used here to compare the parasite strains included in the study. Various genotypes were identified, based on PCR fragment size, revealing genetic diversity of the parasite strains included [Samples from immune individuals from Dielmo and Ndiop were tested for their reactions to crude extract of four included .P. falciparum antibody responses in malaria endemic areas. The correlation between local and the reference strain means that laboratories could use reference strains for routine analysis; this is an advantage because adapting and maintaining wild parasite strains in continuous in vitro culture is complicated, and often unsuccessful [The IgG responses obtained with the four strains were strongly and positively correlated. The strong and positive correlation between strains PA, F15, F16 and 0703 for IgG antibody levels observed indicates that any of these crude extracts could be used to determine total anti-ccessful .P. falciparum isolates to continuous in vitro cultivation [P. falciparum extract (merozoites or schizonts) to use for serological studies. There is a need for a standard assay, in particular for inter-laboratory comparisons of immune responses against strains from different origins.Immunoassays using crude extracts from local strains as antigens are useful for evaluating immunity but, often, require the adaptation of tivation , 19. To The high IgG values observed in response to schizonts and merozoites of strain 0703, the local strain isolated in Dielmo and Ndiop, are consistent with the study population’s contact with this local strain, and the higher prevalence of positive responders against merozoites and schizonts confirmed the good recognition for this strain. These observations are in agreement with previous studies . A high Although there was a significantly higher signal detected for IgG responses to 0703 than to other strains, notably F15, the frequencies of responders were less different: 81.2 % (177/218) vs 77.5 % (169/218) and 91.3 % (199/218) vs 90.4 % (197/218) for schizont and merozoite extracts, respectively. Therefore, it is not clear whether the high signal reflects a better IgG reactivity to a geographically-matched isolate or an immune response strongly elicited by more frequent inoculation. But whatever the origin of this higher signal the fact that frequencies of responders are less different between strains leads to the conclusion that for standardization using the parameter of frequency of responder as an indicator of malaria dynamics could be a better choice. This point raises the important issue of the cutting-off value to adopt for defining positivity. In this study the cutting-off chosen was values of OD ratio (mean sera OD/mean controls OD) > 2. This is an arbitrary value but, in the authors’ opinion, this value is high enough to exclude very low titers which could be difficult to interpret. Another method of defining positivity could be to adopt for positivity values > mean OD of controls + three standard deviations. This leads to higher number of positive responders as in these assays the standard deviations of controls are very low.In high to moderate transmission areas as those studied here very low cut-off values would not be suitable for following malaria transmission dynamics as it could be not enough sensitive. But in areas where malaria transmission is very low and where elimination is envisaged, lower cut-off than ratio > 2 could probably be though.The greater recognition of strain 0703 suggests that the use of a local strain may allow optimal determination of total anti-falciparum Ab responses in an area. These observations are also relevant to issues about the malaria indicators used in serological studies and some of the incoherencies related to the crude extract at different scales in dings for the level and prevalence of antibodies revealed no difference between merozoites and schizonts of the local strain . TherefoThe findings reported herein indicate that, for the analysis of serological data, distinctions should be made between local and reference strains. The use of a reference strain should help with obtaining homogeneous and comparable data from different laboratories in different study areas and be useful for monitoring. However, local strains may be valuable for detecting low level infections in local context, although further work is needed to confirm this possibility.


Malaria still infects many Malawian children, and it is a cause of death in some of them. Regulatory T cells (Tregs) help in negating immune-related pathology, it but can also favor multiplication of malaria parasites. The question remains whether children recovering from uncomplicated malaria (UCM) have higher Tregs and interleukin (IL)-10 levels in convalescence.We recruited children between the ages of 6 and 60 months presenting with acute UCM in Blantyre (low transmission area) and Chikwawa (high transmission area). We observed the children after 1 month and 3 months and analyzed their blood samples for parasitemia, lymphocyte subsets, and levels of the cytokines interferon (IFN)-γ, IL-10, and transforming growth factor (TGF)-β. Blood samples from age-matched controls were also analyzed for the same parameters.P < .0001) higher in convalescence compared with acute disease, whereas in Blantyre, these were as low as in healthy controls both during acute disease and in convalescence. Blantyre had a higher percentage of parasiteamic children (15% versus 12%) in convalescence compared with Chikwawa, but none of these developed symptomatic malaria during the study duration. Concentrations of TGF-β were higher at time points for the study participants and in controls from Blantyre compared with those recruited in Chikwawa.Compared with controls, acute UCM was associated with mild lymphopenia, splenomegaly, and high levels of IFN-γ, tumor necrosis factor-α, and IL-10, which normalized in convalescence. In Chikwawa, Treg counts were significantly (The high transmission area was associated with high Tregs counts and IL-10 concentrations in convalescence, which could have an effect on parasite clearance. We recommend that children recovering from UCM, especially those from high transmission area, should sleep under insecticide-treated nets, be screened for parasitemia, and a provision of antimalarial prophylaxis should be considered. Plasmodium falciparum malaria causing more deaths [Malaria affects between 300 and 600 million people annually and contributes towards more than 1 million deaths worldwide, with e deaths . Of the P falciparum malaria infection develops into severe disease are not clearly known, current evidence suggests that some interplay between the parasite and host's immune response has a role [Although factors that determine whether s a role –6. Succes a role , 8.If the proinflammatory response is left uncontrolled, instead of just achieving the parasite clearance and controlling the parasite replication , it can Plasmodium yoelii [Plasmodium berghei-infected mice [Regulatory T cells (Tregs), a subset of T cells, have been implicated in playing a role in the severity of malaria and also in the clearance of parasites in blood-stage infection in both mouse models and in human malaria , 15. Depm yoelii . Both inted mice .P falciparum malaria infection coinciding with increased parasite growth rate [Studies in humans have shown that Treg levels increase during acute wth rate , which iwth rate .A longitudinal study conducted in Kenya found an association between the percentage of Tregs and risk of subsequent clinical malaria upon reinfection . A separWe therefore conducted the current study to compare and contrast Tregs and cytokine levels in children presenting with UCM at a location with a high (Chikwawa) and a location with a low (Blantyre) malaria transmission rate.We conducted a prospective cohort study at Ndirande Health Centre (NHC) in Blantyre  betweenA 5 mL venous blood sample was collected from each participant at recruitment and during follow-up. Participants presenting with UCM were treated with coformulated tablets of 20 mg of artemether and 120 mg of lumefantrine , per Malawi Government guidelines as first-line treatment of UCM, before blood sample collection. Ethical approval for the study was obtained from College of Medicine Research and Ethics Committee, and written informed consent was obtained from the parent or guardian of every participant.Human immunodeficiency virus testing was performed using 2 rapid test kits; Determine  and Unigold . Where discordant results were obtained, polymerase chain reaction was used to confirm the results. Thick and thin blood smears on slides were prepared by standard methodology. Total white blood cell (WBC) counts, percentage, and absolute counts of lymphocytes were determined using an HmX hematological analyzer (Coulter).+ Tregs as a subset that expressed CD4+CD25hiFoxP3+CD127low.Immunophenotyping of blood samples by flow cytometry and lymphocyte subset identification was performed as reported elsewhere . A totalp-Nitrophenyl phosphate (Sigma-Aldrich) per well, and optical density was measured after 30 minutes of incubation using BioTek reader ELx800 (BioTek Instruments) at 405 nm. Concentrations of serum cytokines were determined using plotted standard curve.High protein binding 96-well enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immuno) were coated with monoclonal antibodies for IFN-γ, TNF-α, IL-10, and transforming growth factor (TGF)-β: 1-D1 K, TNF3/4, 9D7, and MT593 , respectively, at 2 µg/mL in PBS (Sigma-Aldrich) and adding 100 µL per well. Coated ELISA plates were incubated overnight at 4°C and washed twice with PBS adding 200 µL per well. Plates were blocked with PBS with 0.05% Tween 20 containing 0.1% bovine serum albumin  (incubation buffer) and incubated at room temperature for 1 hour. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20. A total of 100 µL of test serum and standards were added to appropriate wells, diluted in incubation buffer, and incubated at room temperature for 2 hours. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20 and appropriate biotinylated mAbs for detection of serum IFN-γ, TNF-α, IL-10, and TGF-β: 7-B6-1-biotin, mAb TNF5-biotin, mAb 12G8-biotin, mAb MT517-biotin (MABTECH AB), respectively, at 1 µg/mL were added at 100 µL per well and incubated at room temperature for 1 hour. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20 and 100 µL Streptavidin-ALP diluted 1:1000 (MABTECH AB) in incubation buffer added per well and incubated at room temperature for 1 hour. Enzyme-linked immunosorbent assay plates were washed 5 times with PBS containing 0.05% Tween 20 and 100 µL SIGMAFAST Spleen size grading was performed using a protocol described previously . In brieU test was performed between groups to detect statistically significant differences. A P value of < .05 was considered to show a statistically significant difference. Regression analysis was used to assess linear associations between the concentration of IL-10 and the percentage of Tregs at the 2 sites, with the Spearman's correlation value (r) of 1 indicating a strong correlation and anything lower showing a weaker correlation.Statistical analysis was performed using GraphPad Prism. A Mann-Whitney In Blantyre, 33 children presenting with UCM were recruited with a median age of 34.5 months (5.7–60). Twenty-seven of these children were observed at 1 month convalescence, and 26 children were successfully observed again after 3 months (Table P < .005). Both of these parameters normalized during convalescence  . Although a similar trend was observed in Blantyre, the decrease in percentage Tregs during convalescence was not significant.When lymphocytes were presented as a percentage of total WBCs Figure A and B ae Figure C and D.  P = .034  in convalescence compared with the cell counts in acute infection  and absolute counts (P = .0210) of Tregs compared with the controls in Chikwawa  . Levels of TNF-α  .Serum from children presenting with acute malaria at both sites had significantly higher levels of IL-10 compared with that of controls  Figure , and theP = .0017) and TGF-β (P = .0014) compared with participants in Chikwawa . Healthy controls in Blantyre had significantly higher concentrations of IFN-γ (P = .0448) and TGF-β (P = .0005) compared with healthy controls in Chikwawa , suggesting that Tregs are an important producer of IL-10.There was significant linear correlation between the IL-10 levels and the percentage of Tregs Figure  during t+ T cells, we found that they were significantly higher in acute UCM compared with levels in healthy controls only at Chikwawa site. These then normalized in convalescence with the difference being significant only during the second follow up. Although a similar trend was observed in Blantyre, the differences were not significant between any stages.We found that children recruited with acute UCM presented with lymphopenia and splenomegaly and higher than normal levels of IFN-γ, TNF-α, and IL-10, which normalized 1 month into convalescence. We also found that 15% of the children recruited with acute UCM were parasitemic during convalescence, but none of these participants developed any further form of symptomatic malaria. When Tregs were presented as percentages of the CD4P < .0001) higher in convalescence compared with acute disease , and even more at the 3-month follow-up stage, none of these participants developed any symptomatic malaria during the 3-month period of convalescence.The observed lower IL-10 levels in healthy controls compared with acute disease is consistent with results found in Malian children , adding We did not expect to find any child having parasitemia during convalescence at both sites because we anticipated that the antimalarial treatment would clear all parasites from the infected children. Although a majority of the parents and guardians of the study participants reported to be using insecticide-treated nets during convalescence period, we did not determine whether this parasitemia resulted from new infection or was due to the children failing to completely clear the initial parasitemia. These results need to be replicated in another study, nevertheless such parasitaemia prevalence post treatment might justify administering an additional antimalarial prophylaxis in the affected individuals.Further studies need to be done to establish the role of Tregs in the transition from UCM to the more severe clinical forms of malaria, namely CM and SMA. In addition, by having larger sample sizes and observing the study participants for as long as 1 year or even 2 years, researchers can provide a better understanding of Tregs perturbations at times when the children are purely parasitemic and asymptomatic, when they develop UCM, and when they transition from UCM to the life-threatening CM. In addition, with malnutrition known to affect cytokine production and T-cell differentiation , subsequ


Plasmodium ovale is rarely described in Senegal. A case of clinical malaria due to P. ovale wallikeri in West Central of Senegal is reported.Plasmodium falciparum HRP-2 RDT was negative, he was treated with antibiotics. However, owing to persisting symptoms, he was referred to the emergency unit of the Youssou Mbargane Diop Hospital, Dakar, Senegal. Clinical examination found impaired general condition. All other physical examinations were normal. Laboratory tests showed anaemia (haemoglobin 11.4 g/dl), severe thrombocytopaenia (platelets 30 × 109/mm3), leukopenia (3650/mm3), lymphocytopenia (650/mm3). Renal function was normal as indicated by creatininaemia and uraemia  and liver enzymes were slightly elevated . Blood smear evaluations in Parasitology Laboratory of Aristide Le Dantec Hospital showed malaria parasites of the species P. ovale with a 0.08 % parasitaemia. Molecular confirmation was done by real time PCR targeting the 18S rRNA gene. The P. ovale infection was further analysed to species level targeting the potra gene and was identified as P. ovale wallikeri. According to the hospital’s malaria treatment guidelines for severe malaria, treatment consisted of intravenous quinine at hour 0 (start of treatment) and 24 h after initial treatment, followed by artemether–lumefantrine 24 h later. A negative microscopy was noted on day 3 post-treatment and the patient reported no further symptoms.A 34-year-old male baker in Dakar, with no significant previous medical history, was admitted to a health clinic with fever and vomiting. Fever had been lasting for 4 days with peaks every 48 h. As monospecific Malaria due to non-falciparum species is probably underestimated in Senegal. RDTs specific to non-falciparum species and/or pan specific RDTs should be included as tools of diagnosis to fight against malaria in Senegal. In addition, a field-deployable molecular tool such as the loop-mediated isothermal amplification can be considered as an additional useful tool to detect low malaria parasite infections and for speciation. In addition, national malaria control policies should consider other non-falciparum species in treatment guidelines, including the provision of primaquine for the treatment of relapsing parasites. Plasmodium ovale is endemic in tropical Africa, malaria due to P. ovale is rarely described in Senegal [Plasmodium falciparum [P. ovale infections were described in the south of Senegal in the context of the longitudinal study at Dielmo village located 280 km South East of Dakar [P. ovale was determined, among symptomatic and asymptomatic villagers of all groups age, to be 5.5 % either as a mono-infection or as mixed infection with P. falciparum [P. ovale infections were rarely reported [P. ovale in Kedougou only as mixed infections with P. falciparum at 11.5 % of prevalence [Despite the fact that  Senegal . The moslciparum . In the of Dakar . During lciparum . Concertreported . Recentlevalence .Plasmodium ovale consists of two distinct species: P. ovale curtisi (classic type) and P. ovale wallikeri (variant type) [P. ovale tryptophan-rich antigen (potra) allows for the distinction between the two P. ovale species. Sequence and size variations were noted between the tryptophan-rich antigen genes from P. ovale curtisi (poctra) and P. ovale wallikeri (powtra) [poctra; 245 bp for powtra). In addition, Tanomsing et al. developed a semi-nested PCR protocol which efficiently discriminate P. ovale wallikeri and P. ovale curtisi and found that the 299 bp fragment was overlapping between the two subspecies [P. ovale wallikeri.nt type) . The twont type) . The rec(powtra) . This wa(powtra) , where tbspecies  and the P. ovale wallikeri is described in a patient admitted to the emergency unit service of the Youssou Mbargane Diop Hospital of Rufisque, a department of the peripheral region of Dakar in Senegal.A case of malaria due to P. falciparum). The test was negative for P. falciparum malaria and the patient was treated by three intravenous (IV) injection of cefotaxim  at 100 mg per kg per day to cover potential bacterial infection. Owing to persisting symptoms 4 days later, he was referred to the emergency unit of the Youssou Mbargane Diop Hospital, Dakar, Senegal.A 34-year-old male baker living in Diamniadio (Dakar region) sought a consultation at a health centre near his home for fever, headache, vomiting and diarrhoea. Fever had lasted for 4 days with peaks every 48 h. The patient was suspected of malaria and was tested using an HRP-2 based RDT (detecting only 9/mm3), leukopaenia (3650/mm3) and lymphocytopaenia (650/mm3). Kidney function was normal as indicated by creatininaemia and uraemia . Transaminases were slightly elevated.The patient had no particular previous medical history. He was born in Thies and moved to Touba at the age 12 and finally at age 23 to Diamniadio, where he has lived since. The patient claimed that he had never been out of these three areas, which are all in West Central of Senegal. Physical examination found impaired general condition but the patient was conscious. Abdomen was tender. All other physical examinations were normal. Another HRP-2 based RDT was performed and was also negative. Other laboratory tests showed anaemia (haemoglobin 11.4 g/dl), thrombocytopaenia . Some infected RBCs were oval shaped and some were fimbriated. However, Schüffner’s dots were not observed likely due to staining defect Fig. . All malP. ovale, infection  [P. ovale species as P. ovale wallikeri. This is most likely the first time P. ovale species determination have been undertaken when describing P. ovale infections in Dakar, Senegal.In Senegal, patterns  and secothogenic . The pre (potra)  were useP. ovale infection was likely acquired locally. Since Diamniadio is a crossroads town located at the town exit of Dakar where many different human populations from neighbouring regions and countries intersect there are two possible likely sources of the P. ovale parasites: (1) from an existing P. ovale reservoir in or near Diamniadio, or (2) that the source of the P. ovale infection came from another region or neighbouring countries. In the 1990s, in a study in Dielmo, exposure to P. ovale was reported across all ages and perennial transmission of P. falciparum was also noted [P. falciparum cross immunity could protect against non-falciparum species [The patient in this study denied having travelled outside Diamniadio suggesting that the so noted , therefoadio, or  that thePlasmodium ovale infections present unique challenges because of the reported long latency period and the fact that two distinct species are known to exist [P. falciparum is the most prevalent parasite detected when patients are suspected of malaria. Because of this, the national malaria control programme provides all health clinics of the country with HRP-2 based RDT, which are specific to P. falciparum. Therefore, it is possible that many cases of non-falciparum malaria infections are just not detected. This case report highlights the urgent need to develop standardized protocols capable of detecting all Plasmodium parasites in Senegal in order to achieve the malaria elimination agenda. In addition, although the existence of P. ovale hypnozoite remain controversial [P. ovale and other non-falciparum malaria parasites in Senegal and in other parts of Africa as previously suggested [This present findings highlight the need to consider non-falciparum malaria infections in clinical settings both for the appropriate treatment of the infection and in order to further understand the epidemiology of malaria in the country. to exist . In factoversial , they pouggested .Plasmodium species [P. falciparum, ACT and quinine are the recommended and registered anti-malarials. The patient discussed in this case report was treated using IV quinine followed by oral ACT, which according to the current Senegalese treatment guidelines, is still the treatment of choice for severe malaria in adult patients [P. falciparum and non-falciparum infections after ACT [P. ovale infected patients. Currently, the national malaria control programme plans to evaluate primaquine in the north of the country in an effort to end malaria in this region [P. falciparum was considered resistant to chloroquine, this drug was withdrawn and national malaria control programme did not define treatment guideline for non-falciparum malaria explaining how neglected was the non-falciparum species in Senegal [The current World Health Organization treatment guidelines recommend intravenous (IV) artesunate for the treatment of severe malaria due to all  species . In Senepatients . ACT haspatients . Howeverfter ACT . Importas region . Also, c Senegal .P. ovale exists in West Central of Senegal and the prevalence is likely to be underestimated due to the diagnostic tools in place.In summary, this case report demonstrates that P. ovale malaria is not as benign as previously thought and that it is important to consider other non-falciparum malaria infections for appropriate patient care and for the success of the malaria control programme.This case also highlights the fact that


Background: Imported malaria is a major threat to neighboring malaria-eliminating countries such as P.R. China and is difficult to monitor. A molecular survey of febrile patients with a history of traveling abroad along the Myanmar-China endemic border areas from January 2008 to August 2012 was carried out. The rates of infection with species of Plasmodium and compliance of microscopy diagnosis with nested PCR (Polymerase Chain Reaction) results were calculated.Results:Plasmodium genus-specific nested PCR confirmed that 384 cases were positive. Further species-specific nested PCR showed that the rate of Plasmodium vivax infection was 55% (213/384); that of Plasmodium falciparum was 21% (81/384) and 17% (67/384) of cases were co-infection cases of P. vivax and P. falciparum; the remaining 6% (23/384) of cases were caused by other species, such as Plasmodium ovale, P. malaria, P. knowlesi or mixed infections of Plasmodium. In total there was 13% (50/384) false microscopy diagnosis including 6% (22/384) error in species diagnosis and 7% (28/384) undiagnosed cases in co-infection or low parasitemia malaria cases.Conclusions: This study indicates that there are considerable numbers of malaria cases in the China-Myanmar endemic border areas that remain undiagnosed or misdiagnosed by microscopy, especially in low-level and/or complex co-infection cases. It is urgent to develop accurate rapid diagnostic tests and apply PCR confirmation for efficient surveillance. Plasmodium spp. infection by examining Giemsa-stained blood smears under the microscope. Although effective and inexpensive, the method is laborious and time-consuming and its sensitivity drops with the decrease in parasitemia during the disease elimination stage [Plasmodium, i.e. Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi [Plasmodium spp. [Malaria is a major infectious disease in the Greater Mekong Subregion (GMS) countries, namely P.R. China, Laos, Myanmar, and Vietnam. In the GMS, Myanmar accounts for approximately one-fifth of the region’s population, more than half of the malaria cases and approximately three-quarters of malaria deaths. “Border malaria” is extremely difficult to monitor; frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries . Yunnan on stage . The Polon stage , 26. A vknowlesi , 22, 5, ium spp. .A molecular survey was carried out of samples collected randomly in the malaria-endemic area along the border line of southern China, in Yunnan province from January 2008 to August 2012. The present study aims to evaluate PCR amplification in comparison with light microscopy examination.The research proposal was approved by the ethics committee at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, in particular regarding collecting blood samples from local residents or migrant workers by finger prick. Written informed consent was obtained from all adult participants and from the parents, or legal guardians of children. Any participant found to be parasitologically positive received anti-malaria treatment, except for those with contraindications.P. vivax infection cases, 96 P. falciparum cases and 63 cases of vivax and falciparum co-infection were referred to as the Microscopy-Positive group (MP group). The remaining 189 samples were the Microscopy-Negative group (MN group). Parasite density per 200 leukocytes was counted, then calculated as the number of parasites per microliter by assuming a leukocyte count of 7000.The population of the Tengchong village is served by a general hospital, a polyclinic at the village’s Center for Disease Control and Prevention (CDC), and 15 government health clinics. A total of 562 blood specimens were collected from febrile patients in China-Myanmar endemic border areas at Tengchong Hospital, the polyclinic of the village’s CDC and the government health clinics. All these patients in Tengchong village come from different countries and most of  these febrile patients had a history of being abroad in Myanmar during the last year. Finger-prick blood samples were collected and the Giemsa-stained blood smears were examined by microscope from January 2008 to August 2012. Thick blood smears were examined under 1000× magnification by microscopists with extensive experience in identification of malaria parasites. The thick film was considered positive when malaria parasites were present and negative if no parasites were seen after 500 leukocytes were counted. In total, 373 samples including 214 DNA was extracted from the blood spots using the QIAamp DNA Mini Kit (QIAGEN China (Shanghai) Co., Ltd.) according to the manufacturer’s instructions and the method of Foley et al. . Approxi2, 100 mM KCl, 20 mM Tris-HCl, pH 8.3. DNA amplification was carried out under the following conditions: 94 °C for 5 min, 30 cycles of , followed by a final extension at 72 °C for 5 min. One microliter of the first PCR product was used in the second amplification. Conditions and concentrations used for the second amplification were identical to those used for the first, except that rPLU3/rPLU4 were used as primers  and analyzed by MEGA version 5.1 (http://mega.software.informer.com/5.1b/)Nested PCR amplification was performed according to Snounou et al.  using a  primers  and amplarasites . KnowlesPlasmodium genus-specific nested PCR confirmed that 384 cases were positive . So, the nested PCR confirmed all of the MP group malaria cases  and diagnosed 13 malaria cases in the MN group  which may have been ignored because of the low parasitemia conditions. Further species-specific nested PCR identified that among the 384 malaria cases, the P. vivax infection rate was 55.5% (213/384), that of P. falciparum was 21.1% (81/384) and 17.4% (67/384) of cases were co-infections of P. vivax and P. falciparum, with 6.0% (23/384) of cases caused by other infections, including five cases of P. ovale and three of P. malariae; and various cases of mixed infections: two cases of P. knowlesi and P. falciparum, three cases of P. vivax, P. falciparum and P. ovale, two cases of P. vivax and P. ovale, two cases of P. falciparum and P. ovale, two cases of P. falciparum and P. malariae, one case of P. vivax, P. falciparum and P. ovale, one case of P. vivax, P. malariae and P. ovale, one case of P. vivax, P. falciparum, P. malariae and P. ovale, and one case of P. malariae and P. ovale. In total there was 13.0% (50/384) error in microscopy diagnosis including 5.7% (22/384) error in species diagnosis and 7.3% (28/384) undiagnosed cases in co-infection or low parasitemia malaria cases , while the remaining 189 samples were diagnosed as negative (MN group). ates see .Figure 1The long border of 4061 km in southern Yunnan, directly adjacent to the northwest of Laos, northeast of Myanmar and north of Vietnam, is considered the most prevalent area of malaria in P.R. China . DespitePlasmodium species, a technique appropriate for routine clinical diagnosis. However, its sensitivity is limited and diagnostic accuracy depends greatly on the particular expertise of laboratory staff. The detection limit of malaria parasites by microscopy is 20–30 parasites/μL of blood [Low-level infection appears to be common across malaria-endemic areas, often as complex mixed infections. A number of studies demonstrated that PCR is more reliable than microscopy in detection of malaria in low parasitemic areas , 20, 21.of blood . Frequenof blood , 14.P. ovale, rarely seen in P.R. China, was distributed widely in Myanmar [P. ovale, and P. malariae in East Asia than previously reported when using alternative diagnostic methods, such as acridine orange staining and PCR-based methods. The rate of P. knowlesi co-infections with other malaria parasites was reported to be about 30% in southern Myanmar near Yunnan Province of China [P. knowlesi co-infection rate detected in this study was much lower than that reported in southern Myanmar, which may be because all the field samples we collected were mainly in the villages adjacent to northeastern Myanmar, so only two knowlesi malaria cases were detected. To establish long-term cooperation in prevention and control of important infectious diseases in cross-border areas, a proposal for malaria prevention and control in border areas of Yunnan has been suggested. A more sensitive and specific diagnosis method is needed to obtain a rapid-response emergency mechanism for the important infectious diseases [It is reported that  Myanmar ; here, w Myanmar  reportedof China . The P. diseases .P. falciparum malaria has already been reported [Plasmodium loss of malarial susceptibility to artemisinin in Thailand suggested that the resistance gene also exists in P. vivax-endemic areas. The latest research work in northern Angola has demonstrated that because P. malariae and P. ovale are sympatric with P. falciparum across the continent and are frequently present as co-infections, populations of P. falciparum previously dominated by chloroquine (CQ)-resistant genotypes are now under different drug selection pressures. In short, it is essential that the treatment of malaria patients depends on the correct diagnosis of malaria species, particularly with the spread of parasitic resistance to antimalarial drugs [Artemisinin resistance in reported , 19. Uhlreported  reportedal drugs , 11. ThuBabesia microti, is emerging in China-Myanmar border areas in Yunnan province, P.R. China [Babesia in malaria-endemic regions is possible and sometimes babesiosis has been diagnosed as malaria due to the similarities of symptoms and morphology. Laboratories in babesiosis-endemic areas need broad access to modern diagnostic methods for a more rapid and reliable microbiological diagnosis in cases of suspected cases of malaria and/or human babesiosis. We should also pay attention to P. ovale, P. knowlesi and P. malariae as well as Babesia, for there may be co-infection with the other two dominant Plasmodium species.Our latest study showed that human babesiosis caused by another intraerythrocytic protozoan, R. China . So, theThis study indicates that there are considerable numbers of malaria cases in the China-Myanmar endemic border areas that remain undiagnosed or misdiagnosed by microscopy, especially in low-level and/or complex co-infection cases. It is urgent to develop accurate, rapid diagnostic tests suitable for these intraerythrocytic protozoans besides the two dominant plasmodium species, and apply PCR confirmation for accurate surveillance.


Under trial conditions insecticide-treated nets have been shown to provide significant clinical and mortality protection under a range of malaria transmission intensity conditions. There are, however, few operational impact data, notably in very intense transmission conditions. This study, reports on malaria infection among Kenyan schoolchildren living in areas of intense malaria transmission and their reported use of insecticide-treated bed nets.5188 children in 54 schools were randomly sampled from seven counties surrounding Lake Victoria between May and June 2014. A questionnaire was administered to schoolchildren in classes 2–6 on the use of a long-lasting, insecticide-treated net (LLIN) the night before the survey and provided a single blood sample for a rapid diagnostic test for malaria infection. Analysis of the impact of insecticide-treated net use on malaria prevalence was undertaken using a multivariable, mixed effects, logistic regression at 95 % confidence interval (CI), taking into account hierarchical nature of the data and results adjusted for school clusters.The overall prevalence of malaria infection was 48.7 %, two-thirds (67.9 %) of the children reported using LLIN, 91.3 % of the children reported that their households own at least one LLIN and the household LLIN coverage was 2.5 persons per one LLIN. The prevalence of infection showed variation across the counties, with prevalence being highest in Busia (66.9 %) and Homabay (51.8 %) counties, and lowest in Migori County (29.6 %). Generally, malaria parasite prevalence differed between age groups and gender with the highest prevalence occurring in children below 7 years (50.6 %) and males (52.2 %). Adjusting for county and school, there was a significant reduction in odds of malaria infection among the schoolchildren who reported LLIN use the previous night by 14 % .Malaria transmission continues to be high around Lake Victoria. Despite evidence of increasing pyrethroid resistance and the likely overall efficacy of LLIN distributed several years prior to the survey, LLIN continue to provide protection against infection among school-aged children.The online version of this article (doi:10.1186/s12936-015-1031-6) contains supplementary material, which is available to authorized users. Malaria continues to pose a significant public health threat across Africa. Achievements in increasing coverage of efficacious interventions have changed the landscape of transmission and disease burdens in some areas but this has not been universal between or within countries –3. This Understanding the intensity of parasite transmission in a community is a fundamental prerequisite to the design of malaria control . How traThis study reports on a survey of malaria infection among Kenyan schoolchildren living in areas of intense malaria transmission and the relationship between infection in this age group and their reported use of insecticide-treated bed nets (ITN).Plasmodium falciparum. By 2009, this region of Kenya continued to represent the majority of Kenya’s hyper-holo-endemic transmission [circa three million LLIN in October 2014.The densely populated counties in Western Kenya, which are close to Lake Victoria, have historically supported the most intense, perennial transmission of smission , changessmission , and supsmission . In addismission . Confronsmission  and procP. falciparum infection prevalence of 24 % (range 2–66 %) [Seven counties were selected for the purposes of the current investigation, Bungoma, Busia, Homa Bay, Kakamega, Kisumu, Migori, and Vihiga, all within 50 km of Lake Victoria. These counties have historically been high-transmission districts and form part of the priority county selection for increased control investment by the Kenyan Government. However, Siaya County, nested within the seven counties, was not included in this study since it currently forms part of intensive, community-based malaria investigations . Surveys 2–66 %) , 17.P. falciparum infection prevalence based upon the county predicted infection prevalence in 2009 [circa 100 children per school. In schools where the desired sample could not be achieved because of low enrolment, all the students in classes 2–6 were recruited.Schools were randomly sampled from each county to be able to provide adequate precision  for county level estimates of  in 2009 , the num in 2009 , 19. At Pf device, Orchid Biomedical Systems, [P. falciparum. All children with a malaria-positive RDT were treated with artemether-lumefantrine according to the national malaria treatment guidelines and written advice on subsequent doses provided to the child and class teacher [Trained interviewers asked each participating child about details related to their age, LLIN use the night before the survey, absenteeism from school in the previous 2 weeks, and any illness on the day of the survey. In addition, children were asked to report on the numbers of LLINs in their household. Each child was asked to provide a finger-prick blood sample for a rapid diagnostic test (RDT)  to deteInformation was captured electronically during the interviews using Open Data Kit for android-based smartphones  that stoAll field staff were trained in survey procedures prior to the survey including mock surveys. The teams used in this study had participated in school based health surveys for many years and all were familiar with protocols for sampling and data collection using mobile phone applications. The RDT tests were read according to the manufacturer’s instructions by trained laboratory technologists.The study protocol received ethical approval from the KEMRI and National Ethics Review Committee (number 2801). Additional approval was provided by the appropriate county-level health and education authorities, who were briefed about the survey. At school level, parental consent was based on passive, opt-out consent rather than written opt-in consent owing to the low risk and routine nature of the study procedures . IndividPlasmodium falciparum infection was defined as a positive RDT result and reported use of a net was assumed to be an LLIN as these have replaced untreated nets since 2006 [nce 2006 , 17, 23.nce 2006 . ProportP. falciparum and 67.9 % (95 % CI 65.7–74.2) reported having slept under an LLIN the night before the survey. Prevalence varied markedly by school (0–2 %) and by county, the county-level infection prevalence was highest in Busia (66.9 %) and Homa Bay (51.8 %), and lowest in Migori (29.6 %) and Bungoma (35 %) counties. Similarly, the reported use of LLINs varied across the counties with the lowest reported use of LLIN being in Kakamega County (51.9 %), and the highest LLIN use by schoolchildren being in Kisumu County (82 %)  and 50.3 % of the children were female. Overall, 48.7 % (95 % CI 45.3–57.3) of the children were positive for P. falciparum infection were investigated using univariable and multivariable mixed effects logistic regression reporting OR at 95 % CI and taking into account two-level hierarchy of the data (county and school). The results from univariable analysis are provided as supplementary information  and considerably higher than previous reports of 33 % LLIN use among schoolchildren in the lakeside region in 2009–2010 . This inIt should be noted that the survey occurred before the large mass distribution campaign of LLIN in this area in October 2014. It is not possible to gauge how bio-effective the LLINs were as it was not recorded when the nets were issued; the last mass distribution was in 2011. It is possible that many of the nets reported as being used by the schoolchildren might have been at the end of their useful life . In addiP. falciparum infection was significantly reduced among children reported to have slept under treated nets [This study’s findings are contrary to previous studies of the impact of treated nets on infection prevalence in this area of Kenya. During one of the pivotal, randomized control trials of ITNs in the 1990s, undertaken in Western Kenya, two cross-sectional surveys among adolescent girls attending 28 schools showed no impact of treated nets on peripheral malaria infection . Analysited nets .An interesting observation from this study was that males had a higher significant risk of parasitaemia compared with female counterparts. These sex differences in malaria infection were significant in all counties except Kisumu, Homabay and Bungoma. This observation is consistent with findings from other studies , specifiSchool surveys provide a rapid means of establishing the use of interventions in the community and the likely impact of these interventions on the risks of malaria infection. They however suffer from the inability to examine the interventions in use in the children’s households and depend on school children’s responses only. In addition, children absent from school on the survey day are not included and these sampling biases might influence estimates of protective effects of ITN. Although such bias is expected to be higher in areas of lower transmission intensity compared to the area selected during the present study.P. falciparum infection in the lakeside zone with overall significant reduction in the adjusted odds of malaria risk of infection. The results from this study therefore have implications for Kenya’s malaria control programme, which focuses on persons with symptomatic P. falciparum infection [This study found a significant association between LLIN use and nfection . Future One major limitation of the present study was the use of a few selected pupils in a few sampled schools and therefore this might not be representative to county population. This study relied on the affirmative answers of the pupils on the use of nets. Finally, this being a cross-sectional study by nature seasonal fluctuations were not reproduced or captured.