Overnight stays in farming huts are known to pose a risk of malaria infection. However, studies reporting the risk were conducted in the settings of poor net coverage. This study sought to assess whether an overnight stay in a farming hut is associated with an increased risk of malaria infection if insecticide-treated bed nets (ITNs) are properly used.A pair of cross-sectional surveys was carried out in the Lamarm district of Sekong province, Laos, in March (dry season) and August (rainy season) in 2008. Questionnaire-based interviews and blood examinations were conducted with farmers and their household members from three randomly selected villages in March  and August . Logistic regression analysis, adjusted for potential confounding factors, was used to assess the association between malaria infection status and frequency of overnight stays for the two weeks prior to the study in both the seasons.In March, 13.7% of participants reported staying overnight in a farming hut at least once in the previous two weeks. The percentage increased to 74.6% in August. Not only adults but also young children stayed overnight as often as adults. The use of an ITN the preceding night was common both in farming huts , and in main residences . Logistic regression analysis showed no statistical association between malaria infection status and frequency of overnight stays in farming huts in either study period. However, people sharing one family type net with five people or more were significantly more likely to have malaria than those sharing a net with up to two people in the dry season.This study showed that staying overnight in farming huts was not associated with an increased risk of malaria infection in the setting where ITNs were widely used in farming huts. It suggests that malaria infection during overnight stays in farming huts might be preventable if ITNs are properly used in rural Laos. Laos is located in Southeast Asia, where malaria transmission is intensive in and on the fringes of forests . With 70During the rice farming season, farmers and their family members often move from their village to a farm, where they stay in a temporary shelter (farming hut). Likewise, in Thailand, movements entailing overnight stays on farms occur throughout the year for rice cultivation, and peaked between June and September . A studyThese seasonal movements in relation to agricultural activities have been identified as risk factors associated with malaria infection ,7-11. InTwo environmental factors for higher risk of malaria infection have been identified among those who stayed overnight in farm huts. First, malaria transmission is more intense in the farm huts than in the village settlements. Second, farm huts are poorly constructed with fewer barriers against mosquitoes . In addiRecently, the Global Fund to Fight AIDS, Tuberculosis and Malaria and other global efforts rapidly increased the coverage of ITNs in many malaria endemic countries. In Laos, ITN coverage reached 77.5% of the target population in 2003 . FurtherAlthough the coverage has been increased in many countries, few studies have documented the association between malaria infection risk and overnight stay in a farming hut in a setting of high ITN coverage. Most of the previous studies about overnight stay provided little information on whether ITNs or any nets were used among their target populations ,7-9, andThis study was carried out in the Lamarm district of Sekong province, in the southern region of Laos in 2008. The annual incidence of malaria in 2003 was 15.4 cases per 1,000 people in the province, the highest in the country . The raiSekong province consists of four districts. With 27,000 people and 66 villages, Lamarm is the most populous district. This district was selected because access to villages in rainy seasons is known to be very difficult in other two districts. The remaining one district contained only a small number of villages. Governmental healthcare facilities in the Lamarm district include one provincial hospital and four health centres that serve rural communities.In the district, the National Malaria Control Programme has been annually distributing conventional bed nets since 2000. During distribution, bed nets are sold in communities at a subsidized price ; insecticide treatment services are provided for free. Since 2009 long-lasting ITNs have been distributed instead of conventional nets with insecticide treatment. Indoor residual spraying has not been adopted. The use of personal spray, mosquito coil, and repellent appeared to be rare.For the target village selection, two-stage cluster random sampling was used. First, one out of the four health centres was randomly selected, and then three of the ten villages were randomly selected from the catchment area of the health centre.From the three selected villages , a total of 143 households  were registered in the village census. An attempt was made to include all of the households in a survey. Data were collected from 134 households in the March survey, and 135 in the August survey. In both the surveys, seven households that were not engaged in farming were excluded. In total, data of 127 households  with 891 people  and 128 households  with 919 people  were analysed in the March survey and in the August survey, respectively. Of the 127 households surveyed in March, 104 with 761 people were included in both surveys.Plasmodium falciparum, as measured by rapid diagnosis test . Malaria infection was defined by positive test result with or without clinical symptoms. The independent variable was the frequency of overnight stays in farming huts in the two weeks prior to each survey, as measured by a question with possible response options such as 0 day, 1-4 day, and 5 days or more. Because the respondents of an interview pre-test had difficulty in answering the number of exact days where they slept in huts, the question was asked in the categorical manner. Other variables included socio-demographic variables , socio-economic variables , bed net-related variables , presence of hung ITN in main residence, and presence of hung ITN in hut), rice farming-related variables (type of rice farming and distance to farming hut), and a cluster-related variable (village). These variables were measured by conducting interviews with household members and observing the bed nets.The outcome in this study was the infection status of In this study, ITN was defined as a conventional net, which was treated with insecticide in the past twelve months. The possibility that households possessed a long-lasting ITN was not taken into account, not only because the National Malaria Control Programme had not provided long-lasting ITNs in the study site, but also because they were rarely available at retailers. The use of ITN was defined as the sleeping under an ITN during the previous night.® is a rapid diagnostic test kit which detects Plasmodium falciparum-specific protein (histidine-rich protein-2) in human blood. In Laos, this test tools are widely used in two settings: one is at rural health centres where microscopy examinations are often unavailable and another is by village health volunteers in communities [Paracheck Pfmunities . A reviemunities . Parachemunities .With the exception of four variables , measurements were based on questions drawn from the Multiple Indicator Cluster Survey Questionnaire 3 developed by the United Nations Children's Fund. Surveyors also confirmed the presence and type of bed nets left in each household's main residence. To increase the validity of the measurements, interview procedures were pre-tested with surveyors at 25 households in a village. Questions that respondents had difficulty in answering were modified.A pair of cross-sectional surveys was conducted in March (during the dry season) and August (during the rainy season), 2008. Each survey consisted of interviews with household members with particular focus on heads and blood examinations aiming at all household members.Five health workers from local health offices were recruited and trained as surveyors. They made household visits and conducted interviews with household heads. The surveyors invited a household head to be the main interviewee. In cases where the head could not participate in the interview, the next responsible person (e.g. a spouse) was chosen. Other household members were encouraged to help the household head to correctly respond to the interview questions.P. falciparum in the villages were conducted on the same days as the interviews. All villagers were invited to assemble at a mobile laboratory that was set up at a designated place including a village health volunteer's house and a primary school. Blood samples were collected from those who gathered and consented to their participation. For child participants, their guardians consented to their participation. Two laboratory technicians from the Center of Malariology, Parasitology, and Entomology, collected a finger prick blood sample from the participants for testing by rapid diagnosis test. Collected blood was immediately used for testing. Results of Paracheck assay were read at 15 minutes from the starting of testing and interpreted by both the technicians. In case that Paracheck turned out to be invalid or technicians did not reach an agreement on interpretation, a blood sample was collected for re-testing. Laotian medical doctors from the same institute treated all participants who tested positive by giving Coartem® or artesunate suppository according to the National Malaria Treatment Guideline. Except one field surveyor, the same members worked for the data collection both in the March and August surveys.Blood examinations of villagers to determine the prevalence of A sample size of 516 people was needed to detect a statistically significant difference in malaria prevalence between people who stay overnight in farming huts and those do not, with 80% power at 95% significance level. It was hypothesized that 90% of villagers would be farmers or a farmer's family members and that half of them would have stayed overnight in a farming hut in the two weeks prior to the survey. It was also hypothesized that 70% of them would participate in a blood examination on the basis of the study conducted in the district , and theP-value of < 0.05 was accepted as statistically significant.Bivariate and multivariate analyses were done to examine the association between the outcome variable and both independent and confounding variables. For the analysis, each confounding variable was categorized as follows: age ; number of household members ; educational attainment ; possession of asset ; number of people sharing the same family type net ; distance to farming hut (< 3 km or ≥3 km); and type of rice farming . Odds ratios (OR) and 95% confidence intervals (CI) of the outcome for each variable were estimated using logistic regression analysis. The multivariate model was adjusted for all the variables other than the independent variable (frequency of overnight stays). Statistical analysis was performed using SPSS 17.0 . A This study was approved by the National Ethics Committee for Health Research, Ministry of Health, Lao People's Democratic Republic (No. 044/NECHR). The participants knew that their participation would be voluntary and that all data obtained would be confidential. Verbal and written consent was obtained from all the participants before conducting the survey.The mean number of household members was 7.0 891/127) in the March survey, and 7.2 (919/128) in the August survey  in the March survey and 80.2% (138/172) in the August survey. These percentages were higher than any other groups: for children aged 5-14 years the percentages were 7.7% (21/273) in the March survey and 75.1% (208/277) in the August survey; for adults there were 14.8% (68/459) in the March survey and 72.1% (339/470) in the August survey.Use of ITN was common both in farming huts and main residence. In the March survey, 66.3% (53/80) of those who stayed overnight in farming huts used an ITN, and 85.8% (680/793) of those who stayed overnight in their main residence used one. In the August survey, 95.2% (555/583) of those who stayed overnight in farming huts used an ITN, and 92.5% (309/334) who stayed overnight in their main residence used one.People commonly shared one net with multiple people. In the March survey, the most commonly reported number of people sharing a net was three to four , followed by one to two  and five or more . Likewise, in the August survey the most commonly reported number of people sharing was three to four , followed by one to two  and five or more .For blood examination, 73.8% (658/891) and 69.4% (638/919) participated in the March and August surveys, respectively. The prevalence of falciparum malaria was 15.3% (101/658) in the March survey and 22.9% (146/638) in the August survey. Of the people who participated in the blood examination in March, 64  had self-reported fever that occurred in the past one week and the remaining 594  had no fever. Of the people who participated in the blood examination in August, 60  had fever and the remaining 578  had no fever. Of the 101 people who were tested positive in March, 18 were also tested positive, 59 negative, and 24 not participating in August.In total, there were 528 nets in the March survey and 560 in the August survey Table . In the Logistic regression analysis for the March survey showed no association between malaria infection status and frequency of overnight stays in huts even after adjustment for the confounding variables . However, it is unlikely that many of them resulted from false-positives; studies that assessed the performance of Paracheck among asymptomatic population in Asian settings showed that sensitivity, specificity, positive predictive value, and negative predictive value were 92.3%, 97.2%, 83.7%, and 98.8% in Thailand , and 94.4%, 89.0%, 70.3%, and 98.3% in India , respectively ,29.This study asked people's behaviors regarding overnight stay in farming huts in the past two weeks. The use of the two weeks duration is reasonable to minimize the potential recall bias associated with self-report, but may not be enough to assess an association between sleeping in farming hut and malaria infection, particularly when the extended period of persistence of histidine-rich protein-2 is considered. However, it is likely that the study population who frequently slept in farming huts for the past two weeks were more likely to choose to sleep in farming huts around the survey period, compared to those who never slept in farming huts.In this study there were five major limitations. First, nearly 30% of the people did not participate in blood examinations. Logistic regression analysis was conducted using data from those who participated. Thus, results are not free from selection bias. Those who did not participate in blood examinations were more likely to sleep in huts for five days or more compared to those who participated in March (10.7% vs. 5.9%) and in August (52.1% vs. 48.7%). It suggests that one reason for non-participation might be due to people working in the farms and that the association between malaria infection and frequency of overnight stays might be underestimated. In a future study, performing blood examinations at the time of interview is recommended to avoid potential loss due to non-response. Second, because any entomological surveys were not conducted, it is unclear how intensive transmissions were in permanent residences and farming huts. Third, because the data collection was limited to only one health zone in the district, the samples of this study might not reflect the entire population of the district. However, as other health zones have the similar ecological setting and the same ethnic group (Lao Thueng), differences in malaria epidemiology and people's behaviors seem to be small. Fourth, the association might be somewhat underestimated in case sleeping in farming huts was associated with low-level parasitaemia. Finally, although the estimated minimum sample size was multiplied by 1.5, the increase might be insufficient to fully address the design effect of the sampling strategies used in this study. The estimated sample size should have been multiplied with consideration to design effect related to cluster surveys.This study showed that staying overnight in farming huts was not associated with an increased risk of malaria infection in the setting where ITNs were widely used in farming huts. It suggests that malaria infection during overnight stays in farming huts might be preventable if ITNs are properly used in rural Laos.The authors declare that they have no competing interests.DN was the principal investigator and was responsible for the entire process. SL, JK, KB, VH, and SP contributed to the development of the study design and coordinated the field work. JK, SK, JY, and MJ contributed to data analysis and to review of the manuscript. All authors read and approved the final manuscript.Logistic regression analysis which examined the associations of variables with malaria infection status in March survey.Click here for fileLogistic regression analysis which examined the associations of variables with malaria infection status in August survey.Click here for file


Plasmodium falciparum, Plasmodium berghei, and Plasmodium yoelii.Michael Delves and colleagues compare the activity of 50 current and experimental antimalarials against liver, sexual blood, and mosquito stages of selected human and nonhuman parasite species, including  Malaria remains a disease of devastating global impact, killing more than 800,000 people every year—the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance.Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony.Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle–wide analyses of drugs for other pathogens with complex life cycles.Please see later in the article for the Editors' Summary Plasmodium parasite, which is transmitted to people through the bites of infected mosquitoes. According to latest global estimates, about 250 million people are infected with malaria every year with roughly 800,000 deaths—most occurring among young children living in Africa. Malaria also causes severe morbidity in children, such as anemia, low birth weight, and neurological problems, which compromise the health and development of millions of children living in malaria endemic areas. In addition to strategies that scale up and roll out the prevention of malaria, such as country-wide programs to provide insecticide-treating bednets, in the goal to eradicate malaria, the global health community has refocused efforts on the treatment of malaria, including finding new compounds that target different stages of the parasite life cycle as it passes from vector to host and back.Malaria is a life-threatening disease caused by the The interruption of malaria transmission worldwide is one of the greatest challenges for the global health community. In January 2011, this journal published a series on The Malaria Eradication Research Agenda , which described a set of research and development priorities, identified key knowledge gaps and the necessary tools needed, and introduced a draft research and development agenda for the worldwide eradication of malaria.Most currently available antimalarial drugs primarily target the disease-causing parasites' stages in the human blood system. But to eradicate malaria, new drugs that block transmission of the parasite between the human host and the mosquito vector, and eliminate the various stages of the parasite during its cycle in the human body, are needed. In this laboratory study, the researchers compared the profiles of all available and experimental antimalarials and analyzed each drug for activity against each specific stage in the malaria parasite's life cycle to provide a reference set of methods and data, that might serve as a benchmark to help guide the malaria research community in assessing the potential of newly discovered antimalarials. Furthermore, this analysis could provide insights into which chemical drug classes might provide transmission-blocking capabilities—an essential component of malaria eradication.Plasmodium species encompassing every major cellular strategy of the malarial life cycle including drug resistant parasite strains. In their comparative analysis, the researchers undertook a chemical profiling approach to identify the drugs that block transmission from the host to the mosquito vector and additionally suppress transmission from the mosquito to the human host.The researchers used novel laboratory techniques under standardized conditions to develop a series of novel assays to analyze the activities of 50 antimalarial compounds (current drugs and those under development) against three P. falciparum and P. vivax isolates show a very good correlation, suggesting that most of the pathways inhibited by antimalarials in P. falciparum may also be valid targets in P. vivax. The researchers also have shown that approved drugs, such as pyronaridine and atovaquone, can target liver and sexual stages in addition to asexual blood stages. Furthermore, the researchers found promising results for new compounds currently in clinical trials, such as the endoperoxide OZ439, a stable synthetic molecule currently being studied in a phase IIa clinical trial, which seemed to be a strong inhibitor of gametocyte maturation and gamete formation. The new 8-aminoquinoline, NPC-1161B, also inhibited sporogony.The researchers highlighted some encouraging results; for example, the potencies of some antimalarials against the asexual blood stage of cultivated The results of this analysis provide a valuable guide to help researchers decide which drugs and compounds show most promise as potential future antimalarial drugs for blocking the transmission of malaria. This study could also help researchers make decisions about which molecules could be best combined to provide the next generation of drugs that will succeed artemisinin compound therapy and support the eradication of malaria. Furthermore, this comprehensive approach to drug discovery could be applied to test drugs against other pathogens with complex life cycles.http://dx.doi.org/10.1371/journal.pmed.1001169.Please access these Web sites via the online version of this summary at malERA a research agenda for malaria eradication sponsored collection, published by PLoS in January 2011, comprises 12 Review articles that discuss agendas in malaria research and developmentThe  Anopheles mosquito, Plasmodium sporozoites reach the mammalian liver, where they invade hepatocytes and either lie dormant or develop over several days, eventually forming the schizonts that are the prelude to a blood stage infection P. vivax and P. ovale. Because only 100 or so sporozoites may be introduced by a bite, there are likely to be many orders of magnitude fewer parasites at this stage than in an active blood stage infection, reducing the possibility of resistance arising. A second bottleneck occurs during sexual development. At each round of schizogony ∼1% of merozoites differentiate into gametocytes Malaria remains one of the most widespread infectious diseases of our time. The latest estimates reveal that ∼250 million people are infected with malaria across the globe, of whom ∼800,000 die every year Given that it would be highly desirable for candidate drugs to have activity against hepatic and sexual forms of the malarial parasite, it is surprising that few clinical trials, to date, have examined whether gametocyte carriage can be reduced following drug treatment. The only drugs found to be effective at reducing gametocyte carriage include artemisinin Here we report the development of a series of novel assays against liver, sexual blood, and mosquito stages of the malaria parasite, using both drug-susceptible and drug-resistant parasite strains. We applied these assays to the current portfolio of schizonticidal compounds, consisting of 50 anti-infectives currently in use or under development..All work involving laboratory animals for the host-to-mosquito transmission studies was performed in accordance with the European Union (EU) regulations “EU Directive 86/609/EEC” and within the regulations of the United Kingdom Animals (Scientific Procedures) Act 1986, sanctioned by UK Home Office Licence PLL70/6347 awarded in January 2008. Protocol design and implementation was guided by the principle of the three Rs  and are of mild-to-moderate severity. Protocols are regularly reviewed and revised following approval by the Imperial College Ethics Review Committee.P. berghei parasites constitutively expressing GFP (PbGFPcon) P. falciparum NF54 strain parasites were maintained in culture as described previously 3H]-hypoxanthine incorporation assay P. falciparum obtained from MR4. Results were expressed as the concentration resulting in 50% inhibition (IC50).In vitro antimalarial activity was measured using the –hypoxanthine incorporation assay.(TIF)Click here for additional data file.Table S3A comparison of antimalarial exposure in human blood against predicted early vector-stage potency. The IC50 values of selected antimalarials in the P. falciparum exflagellation assay was estimated using additional data generated by screening at 1 µM (*) and compared to drug Cmax values obtained from the literature. The in vitro assay contains both blood and serum. Most of the cells in culture (∼97%) are uninfected RBCs. The medium that the assay is set up in is derived from RPMI and contains 10% human serum.(TIF)Click here for additional data file.Table S4A comparison of the reported asexual blood stage potencies of selected antimalarials against P. falciparum (field and laboratory isolates) and P. vivax (field isolates).(TIF)Click here for additional data file.


Plasmodium falciparum-infected child develops uncomplicated or severe malaria. TLR2 has been identified as a receptor for P. falciparum-derived glycosylphosphatidylinositol (GPI), and polymorphisms within the TLR2 gene may affect disease pathogenesis. There are two common polymorphisms in the 5' un-translated region (UTR) of TLR2, a 22 base pair deletion in the first unstranslated exon (Δ22), and a GT dinucleotide repeat in the second intron (GTn).In malaria endemic areas, host genetics influence whether a These polymorphisms were examined in a Ugandan case control study on children with either cerebral malaria or uncomplicated malaria. Serum cytokine levels were analysed by ELISA, according to genotype and disease status. In vitro TLR2 expression was measured according to genotype.p = 0.04), and long GT repeat alleles were associated with elevated TNF (p = 0.007).Both Δ22 and GTn polymorphisms were highly frequent, but only Δ22 heterozygosity was associated with protection from cerebral malaria . In vitro, heterozygosity for Δ22 was associated with reduced pam3cys inducible TLR2 expression in human monocyte derived macrophages. In uncomplicated malaria patients, Δ22 homozygosity was associated with elevated serum IL-6 (Reduced inducible TLR2 expression may lead to attenuated pro-inflammatory responses, a potential mechanism of protection from cerebral malaria present in individuals heterozygous for the TLR2 Δ22 polymorphism. Plasmodium falciparum infection that occurs much less commonly than uncomplicated malaria. Genetic factors influence infection outcome, and polymorphisms in genes encoding proteins involved in disease pathogenesis are strong candidates for disease association studies. As a receptor for parasite derived GPI, TLR2 is likely involved in disease pathogenesis [TLR2 Δ22) in the first un-translated exon was highly polymorphic in Kenyans and in a Japanese population but was not associated with a disease phenotype [n) is present within the second intron, approximately 100 bp upstream of the translational start site. Repeats of varying length have been associated with susceptibility to tuberculosis, reversal reactions in leprosy and colorectal cancer [n repeats are associated with reduced TLR2 reporter activity and TLR2 surface expression in vitro [Plasmodium falciparum derived GPI activate TLR2 to induce TNF, IL-12, IL-6 and nitric oxide in murine bone marrow derived macrophages [In some holoendemic countries, individuals are bitten by malaria-infected mosquitoes 60-300 times a year ,2. The mogenesis . Reporteogenesis ,6. A 22 henotype ,7. A GT l cancer -10. Thesin vitro . Alteredin vitro . Reportsin vitro ,12. Plasrophages -15. Simirophages -19. In trophages . Althougrophages  these cyrophages .n, in the pathogenesis of cerebral malaria was evaluated. Genomic DNA and serum were collected from Ugandan children participating in a case control study examining children with cerebral malaria and uncomplicated malaria. This data indicates heterozygosity for Δ22 polymorphism was protective , but there was no disease association with the GTn polymorphism. In malaria naïve donors, Δ22 heterozygosity was associated with reduced pam3cys induced TLR2 stimulation in human monocyte derived macrophages. In Ugandan children involved in the case control study, none of these polymorphisms was associated with serum cytokines in the cerebral malaria group. In the uncomplicated malaria group, homozygosity for the Δ22 insertion allele was associated with elevated IL-6 (p = 0.04), and at least one long GT repeat allele was associated with elevated serum TNF levels (p = 0.007). This data suggests that the TLR2 insertion deletion polymorphisms may be involved in cerebral malaria pathogenesis through alteration of induced TLR2 expression and down-regulation of specific pro-inflammatory cytokines.In the current study, the role of TLR2 insertion/deletion polymorphisms, Δ22 and GTP. falciparum-smear positive, and no other signs of encephalopathy. Lumbar puncture was performed to rule out meningitis or encephalitis. Children with UM were enrolled at the acute-care clinic or a malaria outpatient clinic that is sponsored by the University of California San Francisco (UCSF). Children were enrolled as UM patients if they exhibited the signs and symptoms of malaria , were P. falciparum-smear positive, had no malaria-related complications , no other sign of acute illness, and were not hospitalized for their malarial illness. Details of study enrolment have been reported previously [Children aged three-12 years were recruited for a study assessing complications of cerebral malaria, which was conducted at Mulago Hospital in Kampala, Uganda . Eighty-eviously . GeneticP. falciparum was determined by light microscopy of thin and thick blood smear with two independent readings. A third independent reading was performed if necessary to resolve any discrepancies between the initial two readings.Exclusion criteria for children in all three groups was (1) a history of meningitis or encephalitis, or any other brain disorder (including CM); (2) a history of developmental delay; (3) prior admission to the hospital for malnutrition; (4) a history of chronic illness. Ethical approval for these studies was granted by the institutional review boards for human studies at Makerere University Faculty of Medicine, University Hospitals of Cleveland, Case Western Reserve University, Indiana Wesleyan University, and the University of Minnesota. Upon admission, study participants' blood was collected onto Whatman filter paper . Genomic DNA was extracted from dried blood spots using the QIAamp 96 spin blood kit . Presence of 2, 125nM primers, and 0.8 units Taq polymerase in a reaction volume of 25μl. PCR primers and amplification conditions have been described previously [TLR2 Δ22 genotypes were assigned based on size discrimination of PCR products on a 4% agarose gel. TLR2 GTn genotypes were assigned based on size discrimination of PCR products run on a 6% polyacrylamide gel as described elsewhere [Genomic DNA was extracted from blood using the QIAamp 96 spin blood kit . PCR was performed using a master mix consisting of 1× PCR buffer, 125μM dNTPs, 2.5 mM MgCllsewhere .PCR amplification products from local donors were purified using the QIAquick PCR purification kit . Purified PCR products were sent to MWG Biotech, High Point, NC for sequencing. Sequences were analysed using the Sequencher software .Serum levels of IFNγ, TNF, IL-1β, IL-10, and IL6 in children with uncomplicated malaria and cerebral malaria were compared according to TLR2 genotype. Serum samples for cytokine measurement were obtained at the time of admission (CM) or outpatient enrollment (UM). Cytokine testing was done using cytometric bead array (CBA) technology using the Bioplex-Luminex system (Austin TX), or ELISA as previously described .T-test using SPSS for windows (version 13.0). Because of errors due to multiple comparisons, Bonferroni correction assuming two individuals tests was applied. At a false positive rate of 5%, the adjusted significance threshold becomes p < .025.Allele and genotype frequencies were calculated for the different polymorphisms, and the Hardy-Weinberg exact test  was performed for each population using Arlequin version 3.01. HaplotyVenous blood was collected from 18 malaria naïve North American volunteers. Peripheral blood mononuclear cells were isolated by Ficoll-Paque separation  and stained with TLR2-FITC (clone TL2.1) and CD14-APC . Cells were read on an LSRII flow cytometer . Monocytes were distinguished by scatter characteristics and CD14 positivity. Median fluorescence intensity of isotype controls were subtracted from each sample to determine relative TLR2 fluorescence. Ethical approval for these studies was granted by the institutional review board for University Hospitals of Cleveland, Case Western Reserve University.6 cells/ml and stimulated with Pam3Cys (200 ng/ml) for 24 h. Human monocytes were isolated by immunomagnetic purification using the Miltenyi monocyte isolation kit II (miltneyi) to obtain unlabelled monocytes which were 80-95% pure. Purified monocytes were cultured for six days in RPMI supplemented with 2 mM L-glut, 20 mM HEPES, pen/strep, and 10% autologous serum to allow differentiation into monocyte derived macrophages. Cells were washed three times with PBS, and adherent cells were removed with cell dissociation buffer (vendor) for flow cytometry.Peripheral blood mononuclear cells (PBMC) were cultured in RPMI 10% human serum AB, 2 mM L-glut, 20 mM HEPES, and pen/strep. PBMC were plated out at 1 × 10Plasmodium falciparum strain 3D7 parasites were cultured and the parasite life cycle was synchronized as described [9 cells/ml. Parasite cultures were passed through Miltenyi LD columns (Miltenyi), allowing uninfected RBCs and rings to pass through the column. Miltenyi columns were washed two times 1 ml Macs buffer, and late stage parasites were eluted with 3 ml Macs buffer. Trophs/schizonts were washed three times and counted. Schizonts were lysed by sonication . Following sonication, schizonts were brought up to the appropriate concentration in RPMI. Schizonts or intact trophozoites were added to macrophage cultures at a ratio of 10:1.escribed . Trophoz6 monocytes were isolated for qPCR. Total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). 100 ng of total RNA was reverse transcribed into cDNA using commercial reagents (Invitrogen) and non-specific Oligo-DT primers. Quantitative PCR was carried out using the following gene specific primers; TLR2 - forward 5'-ATTGTGCCCATTGCTCTTTC-3', reverse, 5'-CTTCCTTGGAGAGGCTGATG-3'; GAPDH - forward 5'-AAGATCATCAGCAATGCCTCCTGC-3', reverse 5'-ATGGACTGTGGTCATGAGTCCTTC-3'. Samples were analysed on a 7300 Real Time PCR System (Applied Biosystems), and normalized using the comparative threshold method [Approximately 1 × 10d method ,26. HEK TLR2 insertion-deletion polymorphisms. The Δ22 polymorphism was highly polymorphic in this study, but there was no significant difference in allele frequency between the two groups . The GTn polymorphism was highly polymorphic but similarly distributed between the two groups, as demonstrated in Figure A total of 65 children with cerebral malaria and 52 children with uncomplicated malaria were genotyped for TLR2 Δ22 and GTn genotype frequencies are displayed in Table p = .005 OR 0.34, 95%CI 0.16-0.73), and this was below the adjusted significance threshold of p < 0.025. Additionally, in the uncomplicated malaria group there was a slight deviation from Hardy Weinberg Equilibrium (p = 0.065), indicating an excess of heterozygotes in this group. Consistent with how the GTn repeats have been analysed previously [n repeat polymorphism were present in the groups studied here, and these genotypes were similarly distributed between cerebral malaria patients and controls with uncomplicated malaria .eviously ,27, the n polymorphisms. TLR2 expression was highly variable on monocytes from local donors ex vivo, and was not associated with Δ22 genotype in this study , but not on macrophages from heterozygous donors (p = 0.20). P. falciparum strain 3D7 parasites were synchronously cultured to trophozoite stage and added to human monocytes derived macrophages and incubated for 24 h. Neither intact, infected RBCs or uninfected RBCs up-regulated TLR2 expression on human monocyte derived macrophages , indicating that the odds of cerebral malaria are 66%  lower in heterozygotes compared to either homozygote. Due to the low frequency of Δ22 homozygotes, and the small sample sizes, the dominant genotype model  is also a possibility, and future studies with larger samples sizes need to be conducted to more precisely define the role of this polymorphism in susceptibility to cerebral malaria. Future studies should investigate whether this polymorphism is protective against other forms of severe malaria, such as severe malarial anaemia. Individual co-infections may have influenced the frequencies of TLR2 genotypes observed here. The Δ22 polymorphism was associated with enhanced susceptibility to acute adenolymphangitis events in lymphatic filariasis-infected Papua New Guineans . Lymphatic filariasis is one of many parasitic diseases transmitted in Uganda [This is the first study to show an association between severe malaria and a n Uganda  and the TLR2 gene, Δ22 is located in the first untranslated exon of TLR2, approximately 60 bp downstream of an NF-kB site critical for chromatin remodelling and transcription factor binding [n repeat is located approximately 100 bp upstream of the transcriptional start site, potentially affecting inclusion of the single coding exon in the transcript. Because of their location within the 5' UTR and proximity to critical cis-elements within the TLR2 gene, it is possible that Δ22 and GTn may affect TLR2 expression. Although short and long GT repeats were too uncommon in local donors to detect a difference in TLR2 expression, there was reduced TLR2 expression in response to pam3cys in human monocyte derived macrophages. There was a similar, although insignificant hypo-responsiveness in human monocytes from Δ22 heterozygotes (data not shown). The focus was on macrophages in this study to avoid donor specific effects on monocyte differentiation and subsequent TLR2 expression. There was no significant TLR2 up-regulation in response to either intact parasitized RBCs or schizont lysate. This does not rule out the possibility that P. falciparum may directly up-regulate TLR2, but it was not detected in this study, possibly because there was insufficient GPI activating TLR2 in either of the parasite preparations to up-regulate TLR2 expression in a manner similar to pam3cys induction. Future studies should examine whether purified parasite derived GPI up-regulate TLR2. None of the North American donors homozygous for Δ22, and therefore the effect of this genotype on either constitutive or inducible TLR2 expression could not be determined.Within the  binding . The GTnn genotypes. There was no significant association between the TLR2 polymorphisms examined here and serum cytokines in the cerebral malaria group. In the uncomplicated malaria group Δ22 homozygosity was associated with elevated IL-6 production (p = 0.04), and at least one long GTn allele was associated with elevated serum TNF (p = 0.007). In these children, neither IL-6 nor TNF were correlated with severe disease [n genotypes may elucidate the relationship between TLR2 genotype and infection outcome. Examining serum cytokines is non-specific, and many cells contribute to serum cytokine levels. Future studies should examine PBMCs from donors with different TLR2 genotypes and measure cytokine responses upon stimulation with intact and lysed infected RBCs. One potentially confounding variable in the current case control study would be the presence of a MAL SNP Ser180Leu. This molecule is a downstream adaptor in the TLR2 signalling pathway, and this polymorphism is associated with protection from malaria and reduced signalling in vitro [The reduced pam3cys inducible TLR2 expression on macrophages from Δ22 heterozygous donors suggests that this polymorphism may attenuate TLR2 induced signalling cascades and downstream cytokine production. To test this, serum cytokine responses were examined in the Ugandan children involved in the case control study according toΔ22 and GT disease , though in vitro . The Ugain vitro . The SNPCollectively, this data suggest that heterozygosity for a 22 bp deletion within the first un-translated exon of TLR2 is associated with protection from cerebral malaria, and that this protection may be due to a dampening of the pro-inflammatory response. Pam3Cys inducible TLR2 expression was significantly reduced in macrophages from heterozygous donors. Reduced surface expression may attenuate TLR2 mediated responses. Although there were no significant differences in serum cytokines according to TLR2 genotypes in children with cerebral malaria, future studies examining responses to pam3cys and parasitized RBCs in donors with different TLR2 genotypes will help identify cell responsible for cytokine production.RBC: red blood cell; TLR: Toll-Like Receptor; GPI: glycosylphosphatidylinositol; CM: cerebral malaria; UM: uncomplicated malaria; UTR: unstranslated region; OR: odds ratio; SNP: single nucleotide polymorphism.The authors declare that they have no competing interests.JG performed genotyping, in vitro work, statistical analysis, and drafted manuscript. JXK and CCJ conceived and designed the study. PAZ was involved in data analysis. NSA conducted ELISAs. ROO was involved in study sample collection. All authors read and approved the final manuscript.Table S1. Cytokine association tests according to TLR2 genotype in Uganda children with cerebral malaria or uncomplicated malaria.Click here for file


Increasing international travel and migration is producing changes in trends in infectious diseases, especially in children from many European cities. The objective of this study was to describe the epidemiology and determine the trends of imported malaria in patients under 20 years old in the city of Barcelona, Spain, during an 18-year period.odds ratio (OR) with a 95% confidence interval (CI) and Chi-square for a linear trend (p < 0.05).The study included malaria cases that were laboratory confirmed and reported to the malaria register at the Public Health Agency of Barcelona from 1990 to 2008, residing in Barcelona and less than 20 years old. Patients were classified as natives (born in Spain) or immigrants. Differences in the distribution of demographic, clinical characteristics, and incidence per 100,000 person-year evolution were analysed. Natives and immigrants were compared by logistic regression by calculating the Plasmodium falciparum, and 108 (62.1%) were visiting friends and relatives (VFR) as the reason for travel. Among the immigrants, 99 (67.8%) were from Equatorial Guinea. Immigrant cases more frequently travelled to Africa than natives (p = 0.02). The factors associated with imported malaria among immigrant residents was travelling for VFR (OR: 6.2 CI 1.9-20.2) and age 15-19 (OR: 3.7 CI 1-13.3). The incidence increased from 1990 to 1999 (p < 0.001) and decreased from 2000 to 2008 (p = 0.01), although the global linear trend was not statistically significant (p = 0.41). The fatality rate was 0.5%.Of the total 174 cases, 143 (82.1%) were immigrants, 100 (57.5%) were female, 121 (69.5%) Plasmodium falciparum was most frequently detected. The trend analysis of the entire study period did not show a statistically significant decline. It is recommended to be aware of malaria, especially among children of immigrants who travel to their parent's home country for VFR. Better access to pre travel advice should be provided.The majority of cases of malaria in population less than 20 years in Barcelona were immigrants, travelling to Africa for VFR and  Plasmodium falciparum and immigration from sub-Saharan Africa are characteristics of the majority of imported malaria cases in Spain [The study of imported malaria has become of more interest in recent years than ever in before. The concern within the scientific community reflects a problem that is secondary to increasing international travel to endemic regions and recent immigration from low-income countries ,2. Identin Spain -9, as wein Spain -17.Imported malaria characteristics and incidence evolution for vulnerable populations such as children are still not well understood in spite of a disproportionally high number of cases ,11. For Affected children in European countries and cities are typically resident immigrants of non-endemic countries who travel to their country of birth or their parent's birthplace to visit friends and relatives (VFR) ,19-21. RPopulation-based studies, which would show incidence rate evolution and define the problem, are scarce. The objective of this study was to describe the epidemiological characteristics of imported malaria cases among children and young adults under 20 years of age and to examine the incidence evolution in a European city during a long period of time. Identification of the patient profile will allow for a connection between specialized healthcare centres and public health measures to prevent imported malaria in children and young adults.A population-based observational study design was implemented to examine confirmed imported malaria cases in children less than 20 years of age, residents of the city of Barcelona, and reported to the malaria registry of the Public Health Agency of Barcelona (Spain) Epidemiology Service between January 1, 1990 and December 31, 2008. Relapses (cases with new symptoms and/or parasitaemia who have not travelled again) were excluded. Reporting imported malaria cases to public health is mandatory in Spain. In Barcelona a laboratory surveillance system exists but cases are reported by the clinicians. Epidemiological surveys are completed by public health nurses.Imported malaria was defined as acquired in an endemic area and compliant with diagnostic criteria of microscopic observation of parasites in a thick peripheral blood smear or genomic detection by amplification techniques in a non-endemic country [immigrant was defined as born in an endemic area and resident of the city of Barcelona. VFR immigrant was defined as having travelled to their country of origin to visit friends and relatives, and a recently arrived immigrant was defined as having arrived in Barcelona with malaria before residency. Immigrant children born in Barcelona were considered natives [ country . An immi natives .Cases and species were confirmed by microscopic observation of parasites in a thick and thin blood film and by PCR as previously described .The following variables from the epidemiology survey for each case were systematically reviewed: socio-demographic characteristics , epidemiological characteristics , reason for travel , diagnostic variables (species and technique), chemoprophylaxis, treatment, and date of symptom onset, diagnosis, hospitalization admittance and discharge. Laboratory registries and clinical histories were reviewed at the tropical disease reference centre of Barcelona for additional information.2 test was used to compare qualitative variables. ANOVA and corresponding non-parametric tests were used to compare quantitative variables. Incidence rates were calculated per 100,000 person-years according to the total population average for a five-year period (data was obtained from the city census) [2 and adjusted linear trend test.A descriptive analysis of the variables was performed. Quantitative variables were described using medians and interquartile ranges (IR) according to their no normal distribution. The x census) . Recentlodds ratio (OR) with a 95% confidence interval (CI). Variables from the bivariate model with a p-value less than 0.05 and those of epidemiological interest were included in the multivariate model. Fatality rates were calculated according to the total number of deaths and total cases per 100. Statistical package SPSS 18 and Epi Info 6 were used for the statistical analysis.Differences between immigrant and native cases on bivariate and multivariate levels were compared using a logistic regression model and P. falciparum .The 174 cases reported during the study period were included. The median age was 9.6 years (IR: 5-15 years), one hundred cases (57.5%) were female and 143 (82.1%) were immigrants. The majority of cases had travelled to Africa , of which 53.4% travelled to Equatorial Guinea (EG) and 8% to Cameron. Of the 125 cases who were prescribed chemoprophylaxis, almost all did not take it correctly . The most common reason for travel was VFR  and the most commonly detected species was Imported malaria incidence increased from 1.65 cases per 100,000 persons under 20 years old in 1990 to four cases per 100,000 in 1997, at which point it declined to reach 1.6 cases per 100,000 in 2008. The linear trend analysis showed a significant increase from 1990 to 2000 (p < 0.001) and a decrease from 2001 to 2008 (p = 0.01). However, analysis of the entire study period did not show a statistically significant decline (p = 0.41) . Excluding recently arriving immigrants with malaria, no difference in sex, district of residence, country of travel or detected species between resident immigrants and natives were found. Similarly, no difference was seen in hospitalization or chemoprophylaxis completion between the two groups  had a previous episode of malaria in their life, 97.2% did not correctly complete chemoprophylaxis and 27 cases (11.1%) required hospitalization. Six cases (6.5%) were hospitalized between 1990 and 1999, and 21 cases (26.3%) were hospitalized between 2000 and 2008 (p < 0.001). During the 18-year study period, one death due to malaria was reported .P. falciparum and were treated without need of hospitalization. Age 15-19 years and VFR were associated factors to resident immigrants compared to natives. This study also found a decline in imported malaria incidence in recent years, which was not statistically significant.Imported malaria in young adults of Barcelona predominantly affected immigrants over five years of age who travelled to Africa (most frequently EG) to visit friends and relatives, did not complete chemoprophylaxis, were infected with Plasmodium falciparum accounted for 78% of all malaria cases, 95% of which were acquired in sub-Saharan Africa [As described in adult populations of similar settings in Spain and other European countries ,16,22-25n Africa . A high n Africa ,25,28. Tet al described in a recent study; VFR children were less likely than adults to receive pre travel medical advice [Many immigrants that are established residents in Spain visit their home country  with thel advice . Like thl advice ,14.P. falciparum infection, and incomplete chemoprophylaxis use, are similar to those described in other cosmopolitan cities of the world [Plasmodium falciparum is the most frequently identified species in children [The association between imported malaria and VFR in patients under 20 years old has been reported in various European cities and countries ,18,30 anhe world ,22. Plaschildren ,30,31 nochildren ,32-36, ochildren ,15,17,26children ,38.The infrequent use of chemoprophylaxis is a concern on a global scale. Although anti-malarial drug resistances are emerging in endemic countries, chemoprophylaxis is still effective and selection of a good regimen, along with barrier and repellent precautions, is key to decreasing the risk of acquiring malaria . The ratThe increase in incidence from 1990 to 1999 can be attributed to the high in international travel to endemic regions and infrequent use of chemoprophylaxis. The later decrease in incidence between 2000 and 2008 could be due to decreased incidence in endemic countries by the use of artemisinins and mosquito control programmes. Better travel advice, higher quality of travel health information in hospitals and primary care facilities and the correct use of chemoprophylaxis could also had a role in the decrease of incidence. There are many other factors that could also affect the incidence calculations such as migration flow, the census development and case reporting. Nonetheless, according to the linear trend analysis, the decline over the entire study period was not significant. Malaria incidence among children had not been previously described but one study from The Netherlands reported decreasing incidence among adults . The incStudies that have assessed imported malaria among children have showed the important aspects of the clinical epidemiological patterns in various non-endemic countries ,40. One The fatality rate was similar to previous studies performed in industrialized cities, except in Italy, Japan, Great Britain and Sweden, in which no deaths were reported ,26. The Challenges for health systems in developed countries in the control of imported malaria include the improvement of information dissemination of preventative measures, the correct use of chemoprophylaxis when necessary and rapid diagnosis of clinical cases -45. To rP. falciparum was the most frequently detected species. It is recommended to be aware of infectious diseases, such as malaria, especially in children and young adults who travel to their parent's home country to visit friends and relatives. Furthermore, worldwide prevention programmes targeting these vulnerable populations should be enhanced by providing better access to travel advice to prevent the disease.The majority of cases of malaria in population less than 20 years in Barcelona were immigrants, travelling to Africa to VFR and The authors declare that they have no competing interests.JPM, PG, MGV and JC designed the study, collected the data, analysed and prepared the first draft. All authors put forward different ideas, contributed to the interpretation of the data, early drafts and agreed the final draft.


The effectiveness of anti-vectorial malaria protective measures in travellers and expatriates is hampered by incorrect compliance. The objective of the present study was to identify the determinants of compliance with anti-vectorial protective measures (AVPMs) in this population that is particularly at risk because of their lack of immunity.Compliance with wearing long clothing, sleeping under insecticide-impregnated bed nets (IIBNs) and using insect repellent was estimated and analysed by questionnaires administered to 2,205 French military travellers from 20 groups before and after short-term missions (approximately four months) in six tropical African countries . For each AVPM, the association of "correct compliance" with individual and collective variables was investigated using random-effect mixed logistic regression models to take into account the clustered design of the study.i.e., before midnight), the type of stay (field operation compared to training), the absence of medical history of malaria, the absence of previous travel in malaria-endemic areas and the absence of tobacco consumption.The correct compliance rates were 48.6%, 50.6% and 18.5% for wearing long clothing, sleeping under bed nets and using repellents, respectively. Depending on the AVPM, correct compliance was significantly associated with the following factors: country, older than 24 years of age, management responsibilities, the perception of a personal malaria risk greater than that of other travellers, the occurrence of life events, early bedtime (There was no competition between compliance with the different AVPMs or between compliance with any AVPM and malaria chemoprophylaxis.Interventions aimed at improving compliance with AVPMs should target young people without management responsibilities who are scheduled for non-operational activities in countries with high risk of clinical malaria. Weak associations between compliance and history of clinical malaria or variables that pertain to threat perception suggest that cognition-based interventions referencing a "bad experience" with clinical malaria could have only a slight impact on the improvement of compliance. Further studies should focus on the cognitive and behavioural predictors of compliance with AVPMs. Each year, approximately 25 to 30 million travellers from temperate countries visit malaria-endemic areas, and about 30,000 of them suffer from travel-associated clinical malaria . TravellPlasmodium falciparum and Plasmodium vivax may be resistant to most drugs used in chemoprophylaxis, AVPMs should be considered as the first line prevention against malaria among non-immune travellers. Regardless of the preventive measures employed, effectiveness depends on their appropriate usage, including user's compliance [Because mpliance -8. The dmpliance ,9-13. Inmpliance ,7,11. Pompliance  and a leNon-immune travellers sent to malaria-endemic areas for professional purposes, such as building sites, opencast working, tree-felling, plantations, humanitarian operations within governmental or non-governmental organizations (NGOs), or military operations, can contract malaria, and their risk increases if they work in field conditions -17. To ii.e., 4-5 months) professional travellers in African malaria-endemic areas.The aim of the present study was to identify the determinants of compliance with AVPMs among short-term  in one of the following African countries: the Ivory Coast (n = 8 groups), Gabon (n = 4 groups), Chad (n = 3 groups), the Central African Republic (n = 2 groups), Senegal (n = 2 groups) and Djibouti (n = 1 group). Individuals were required to take malaria chemoprophylaxis and apply AVPMs.All members of these groups were eligible for the study and were invited to participate. Two self-administered questionnaires were filled out by each individual and validated during a face-to-face interview with a member of the research team. The first survey was administered no earlier than 15 days before their departure, the second being completed no later than 15 days after returning home. People who were absent on the dates of the surveys or who left their group between the first survey and their departure for Africa or during their stay in the field were excluded from the analysis.i.e., when individuals were outside the IIBN). For each of the above three AVPMs, the level of compliance was assessed as either "never", "seldom", "often" or "always". People spent their night time in one of the following locations and activities: in the base without professional activity , in the base with professional activity , outside the base with professional activity  and outside the base without professional activity . For each of the three dependant variables, a global score was calculated by weighting the compliance with AVPMs in each situation by the time spent in each of these locations and activities.Dependent variables included the level of compliance with the following AVPMs: using IIBNs while sleeping, using insect repellents and wearing long clothing at night  was defined as a global score reflecting responses such as "always", while "incorrect" compliance was defined as one of the three other possible responses: "often", "seldom" or "never". In the case of insect repellent, the prevalence of a global score suggesting an "always" response was unexpectedly low, which could have hampered calculations. The "correct" compliance for this AVPM was therefore defined as a global score reflecting an "always" or "often" response. For each individual, independent variables that could be potential determinants of compliance were recorded upon either departure or return.Mission-specific variables included the dates of departure and return, country and type of stay, either for field operations  or training, which occurred mainly in urban conditions . Demographic variables included age, gender, management responsibilities and previous travel overseas to other malaria-endemic areas. Behavioural variables included tobacco consumption, wake and sleep time during the stay and usual morningness or eveningness determined by the Horne and Ösberg Morningness-Eveningness Questionnaire ,19. MornCognitive variables concerned subjective items including perceived risk of malaria during the stay, perceived frequency of mosquito bites and perceived individual attractiveness for mosquitoes. The perceived risk of malaria during the stay was associated with three conditions: perceived individual likelihood of acquiring clinical malaria, severity of the disease if malaria were personally contracted and a dangerous situation of any possible nature.i.e., positive thin blood smear, positive quantitative buffy coat malaria diagnosis system test, or histine rich protein 2 rapid diagnostic test).Variables relative to medical history or medical events during the African stay included presence of clinical malaria, clinical dengue fever, allergy to insect bites and usual cutaneous response to insect bites. Cases of clinical malaria were recorded by the military weekly disease epidemiological surveillance system and defined as clinical attacks occurring during the stay or within 16 weeks after returning to France with biological confirmation of plasmodial infection .The variable pertaining to chemoprophylaxis was the declared compliance. It was defined as "correct" if no dose was reported missed for the entire stay on the return questionnaire. Otherwise, it was considered "incorrect" (http://www.EpiData.dk) and checked for consistency before analysis using SAS version 9.1.3  and STATA 9.0 . Subjects were considered in the statistical analysis only if they completed both questionnaires. Missing values affected less than 1% of the participants and were replaced using a single imputation method. When a question was not asked to one or more groups, the responses were coded as missing data for those groups.Data were recorded using EpiDATA, version 3.0 . The logistic model was also adjusted using a generalized estimating equations (GEE) approach. Random-effect and GEE regression models allowed for the estimation of group-specific and population-averaged effects, respectively [The confidence intervals for "correct" compliances were estimated by taking into account the clustered design of the study using the "svy" commands under STATA 9.0. Compliance with AVPM was analysed as a dependent variable according to individual and group characteristics using a random-effect mixed logistic regression model with the GLIMMIX procedure under SAS. The model was designed to take into account the intra-group correlations that could exist due to the sampling design . Informed consent was obtained from each participant at the beginning of the study after a thorough explanation of the study's purpose.Of the 3,051 eligible individuals from the 20 groups surveyed, 846 were excluded because they did not complete both questionnaires, leaving a working sample of 2,205 subjects. The most common reason for not completing the questionnaires was the absence of the subject at the time of the survey , which was independent of the purpose of the study and the dependent variables. Some individuals who had planned to go to Africa and completed the first questionnaire did not travel and were replaced by others who had not completed the first survey. Some individuals returned to France before the rest of their group or were transferred to another city and were not present at the time of the second survey.The descriptive characteristics of the groups are summarized in Table The "correct" compliance rates were 48.6% : 46.5 - 50.7%; ranging from 2.6% to 88.2%), 50.6%  and 18.5%  for wearing long clothing at night, using IIBN while sleeping and using insect repellents, respectively. Gender, rainy season, mosquito bite burden, perceived mosquito attractiveness compared with other people and perception of the severity of malaria were not associated with compliance with any of the AVPMs in bivariate analyses.Of the responses corresponding to incorrect compliance, the compliance with wearing long clothing at night was scored as "often" in 497 individuals (22.5%), "seldom" in 407 individuals (18.5%) and "never" in 230 individuals (10.4%).In the final mixed multivariate logistic regression model, "correct" compliance was significantly associated with the country of the stay , age over 24, management responsibilities, occurrence of any familial event, medical history of malaria and "correct" compliance with the use of IIBN and chemoprophylaxis , "seldom" in 385 individuals (17.6%) and "never" in 478 individuals (21.7%).In the final mixed multivariate logistic regression model, "correct" compliance with the use of IIBNs was significantly associated with the country of the stay , absence of tobacco consumption, bedtime before midnight and "correct" compliance with both wearing long clothing at night and chemoprophylaxis Table . "CorrecOf the responses corresponding to "incorrect" compliance, the compliance with the use of insect repellents was scored as "seldom" and "never" in 695 individuals (31.5%) and 1103 individuals (50%), respectively. "Correct" compliance was scored as "always" and "often" in 328 individuals (14.9%) and 79 individuals (5.6%), respectively.In the final mixed multivariate logistic regression model, "correct" compliance with use of insect repellents was significantly associated with age over 24, absence of previous overseas travel in malaria-endemic areas, perception of personal malaria risk greater than that of other individuals and "correct" compliance with wearing long clothing at night .In endemic areas, malaria is a major health burden for employees of international companies, including expatriates and other non-immune workers. During the construction of the 1,000 km-pipeline from Chad oilfields to the coast of Cameroon, up to 18.5% of workers suffered from malaria 12 months after the start of construction . MalariaP. falciparum to most anti-malarial drugs makes the appropriate use of AVPMs even more important. Few studies have assessed compliance with AVPMs. Compliance has mostly been studied as a secondary outcome in the context of assessment of compliance with chemoprophylaxis [Resistance of phylaxis ,9,11,22.i.e., wearing long clothing and using IIBNs) may be higher than the pressure to comply with non-visible measures, such as the use of repellents.The prevalences of "correct" compliance with wearing long clothing and the use of IIBNs were more than 2.5 times higher than the prevalence of "correct" compliance with the use of insect repellents. The need for repeated applications of repellents could explain this difference from other AVPMs. Moreover, the social and staff pressures to be compliant with visible measures . These findings are in agreement with previous reports [Some studies have shown no association  or even  reports ,17,23 thIn a French cohort of 899 travellers , age oveBedtime after midnight was associated with "incorrect" compliance with the use of IIBNs and long clothing. In other studies, eveningness, a concept closely related to bedtime after midnight, has been associated with "incorrect" compliance with chemoprophylaxis . Eveningi.e., the two aspects of individual representation of health behaviours in the "Health Belief Model") did not appear to have the same relationship with compliance with chemoprophylaxis. In accordance with the "Health Belief Model", these results suggest that even if the perceived probability of a threat and its perceived severity are the first two dimensions generating perception of risk and motivation for action, they are insufficient to predict any health behaviour. Perceived barriers appear to be the most powerful predictor of health action [The perception of personal malaria risk and severity comprises the concept of threat perception, one of the two aspects of health behaviours in the "Health Belief Model" . In a prh action .Medical history of malaria was associated with "correct" compliance with wearing long clothing and with "incorrect" compliance with the other AVPMs and chemoprophylaxis . These fParticipating in field operations was associated with "correct" compliance with the three AVPMs. The same finding applied for chemoprophylaxis , suggesti.e., from 7.5 and 9.0 clinical malaria cases/100 person-years in the Ivory Coast and Central African Republic, respectively) than in the other countries  during the 2005-2006 period, according to the French Forces epidemiological surveillance. Individually driven compliance and staff pressure to ensure "correct" compliance could be modulated according to the environment and the perceived or documented level of malaria risk. More favourable living conditions, such as sleeping in permanent structures or air-conditioned rooms during training or less intrusive missions, could have made individuals feel safer towards mosquito bites, thus impairing their compliance with AVPMs. Because a country was the theatre of only one type of stay (either for field operations or training), it was not statistically feasible to segregate the two variables.The prevalence of "correct" compliance with wearing long clothing and using IIBNs was higher in countries where the incidence rate of clinical malaria was known to be higher (The group effect remained significant in all three final multivariate models. Furthermore, the rate of compliance was heterogeneous between groups. These results suggest that individual behaviours were probably not independent within a group and that significant collective behaviour determinants were not identified in the present study.Although this study was performed among French military personnel travelling for a four-month period in tropical Africa, the present results could be directly extrapolated to other aforementioned groups of non-immune workers staying in malaria-endemic areas. Extrapolation to individual travellers staying for a shorter duration is less straightforward due to the supposed absence of collective factors. However, data about compliance with AVPMs are scarce among such populations ,7,11. ThThe authors declare that they have no competing interests.ES participated in the data analysis and drafted the manuscript. NR, VM and LO participated in the data management and in the data analysis. EOP, GT, FP, RM, BP and SB participated in the data collection. LO, EOP, GT, FP, RM, AB, CTT and CR participated in the design of the study. CR conceived the study and coordinated the data collection, the data management, the data analysis and the redaction of the manuscript. All authors read and approved the final manuscript.


Change of Kenyan treatment policy for uncomplicated malaria from sulphadoxine-pyrimethamine to artemether-lumefantrine (AL) was accompanied by revised recommendations promoting presumptive malaria diagnosis in young children and, wherever possible, parasitological diagnosis and adherence to test results in older children and adults. Three years after the policy implementation, health workers' adherence to malaria diagnosis and treatment recommendations was evaluated.A national cross-sectional, cluster sample survey was undertaken at public health facilities. Data were collected using quality-of-care assessment methods. Analysis was restricted to facilities with AL in stock. Main outcomes were diagnosis and treatment practices for febrile outpatients stratified by age, availability of diagnostics, use of malaria diagnostic tests, and test result.The analysis included 1,096 febrile patients (567 aged <5 years and 529 aged ≥5 years) at 88 facilities with malaria diagnostics, and 880 febrile patients (407 aged <5 years and 473 aged ≥5 years) at 71 facilities without malaria diagnostic capacity. At all facilities, 19.8% of young children and 28.7% of patients aged ≥5 years were tested, while at facilities with diagnostics, 33.5% and 53.7% were respectively tested in each age group. Overall, AL was prescribed for 63.6% of children aged <5 years and for 65.0% of patients aged ≥5 years, while amodiaquine or sulphadoxine-pyrimethamine monotherapies were prescribed for only 2.0% of children and 3.9% of older children and adults. In children aged <5 years, AL was prescribed for 74.7% of test positive, 40.4% of test negative and 60.7% of patients without test performed. In patients aged ≥5 years, AL was prescribed for 86.7% of test positive, 32.8% of test negative and 58.0% of patients without test performed. At least one anti-malarial treatment was prescribed for 56.6% of children and 50.4% of patients aged ≥5 years with a negative test result.Overall, malaria testing rates were low and, despite different age-specific recommendations, only moderate differences in testing rates between the two age groups were observed at facilities with available diagnostics. In both age groups, AL use prevailed, and prior ineffective anti-malarial treatments were nearly non-existent. The large majority of test positive patients were treated with recommended AL; however, anti-malarial treatments for test negative patients were widespread, with AL being the dominant choice. Recent change of diagnostic policy to universal testing in Kenya is an opportunity to improve upon the quality of malaria case management. This will be, however, dependent upon the delivery of a comprehensive case management package including large scale deployment of diagnostics, good quality of training, post-training follow-up, structured supervisory visits, and more intense monitoring. Health workers' adherence to diagnostic and treatment guidelines is one of the critical aspects determining effective implementation of malaria case management policies . In lineBy the end of 2006, the new treatment policy was implemented countrywide . The finBetween 2007 and 2009, the Kenyan Division of Malaria Control reinforced health workers' case management practices to adhere to case management guidelines. In this paper, malaria diagnosis and treatment practices observed during the national malaria case management survey undertaken in 2010 are reported, approximately three years after the new AL policy was implemented countrywide.th January and 12th February 2010. From the universe of 6,094 public facilities in Kenya the following categories of facilities were excluded from the sampling frame: 1) facilities from Nairobi province due to absence of malaria transmission and requiring special studies to evaluate malaria case management, 2) tertiary hospitals since they serve mainly as referral facilities, and 3) facilities run by other than Ministry of Health (MoH) and Local Authorities (LA) because they provide services to special patient groups such as military or prisoners. In total, 861 facilities were excluded and therefore our sampling frame consisted of 5,233 health facilities. For the purpose of sampling, facilities belonging to the faith-based and non-governmental organizations were classified into one category. Similarly, MOH and LA owned facilities were grouped into government category as well as smaller facilities such as dispensaries and health centres which also represented one category. Therefore, in each of seven provinces, four strata based on the facility type  and ownership (government versus faith based/non-government) were formed. Finally, from each of the 28 strata, a simple, random sample proportional to the number of facilities in a stratum was drawn. A cluster was defined as all outpatient encounters between health workers and patients occurring on a survey day.A cross-sectional, cluster sample, health facility survey was undertaken in public facilities between 18Data at each facility were collected over a single survey day using a range of quality-of-care methods including health facility assessments, health worker interviews, and exit interviews with caretakers and patients. Firstly, all patients presenting to the outpatient departments underwent rapid screening when they were ready to leave the facility. Non-referred and non-pregnant patients weighing 5 kg and above and presenting for an initial outpatient visit with fever underwent a detailed interview. Of relevance for this report, the information about patients' age, weight, temperature, main complaints, routine malaria diagnostics requested, results reported and medications prescribed was collected during the exit interview and from the patient-held cards. Secondly, each facility was assessed to determine the survey day availability of anti-malarial drugs, antibiotics, malaria RDTs and functional malaria microscopy service. Finally, at the end of the working day, all health workers who saw recruited patients on the survey day were interviewed to collect information on their demographics and exposure to malaria case management interventions. Informed written consent was obtained for all participants.The study definitions reflected national guidelines for management of uncomplicated malaria, which were valid at the time of the study . In summTherefore, to reflect criteria for testing and AL treatment, all analyses were restricted to febrile, non-pregnant patients weighing 5 kg and above, presenting for an initial outpatient visit without being referred or admitted for hospitalization. Patients aged five years and older presenting with another obvious cause of fever, as well as the children less than five years of age meeting the same criteria in low risk areas, were excluded from the analysis. Another obvious cause of fever was defined as febrile patients presenting concomitantly with runny nose, sore throat, oral thrush, wounds, urinary problem, skin problem or abscesses. Fever was defined as axillary temperature of ≥37.5°C or a history of fever during the present illness.To ensure comparable evaluation of anti-malarial treatment practices based on different age-specific recommendations for malaria diagnosis, the focus of the analysis was observations from health facilities where AL was in stock during the survey, stratified by the availability of diagnostics and patients' age (under five and over five years of age). At facilities with malaria diagnostics, anti-malarial treatment practices were further stratified by the use of malaria diagnostic tests and the patient's test result. Combined microscopy and RDT results are presented since the small number of patients with malaria RDTs precluded a meaningful analysis stratified by type of diagnostics.Data entry and management was undertaken using Access , through customized data entry screens with in-built range and consistency checks. All forms were entered twice by independent data entry clerks and data files were compared for errors using a verification programme and referring to original questionnaires. All analyses were performed using STATA, version 11. The precision of proportions  was determined adjusting for the cluster sampling at the health facility level.Ethical approval for the survey was provided by the Kenyatta National Hospital/University of Nairobi-Ethics & Research Committee (reference number KNH-ERC/A/383).The survey was undertaken at 174 public health facilities, of which the majority (70.1%) were government facilities, followed by faith-based (25.9%) and non-governmental organization facilities (1.2%). Most facilities were dispensaries (70.1%), while health centres and hospitals accounted for 18.4% and 11.5% of facilities, respectively. Parasitological diagnosis of malaria was available on the survey day at 96 (55.2%) facilities, more commonly using malaria microscopy  than RDTs . Five health facilities had both diagnostic capacities. AL was available in 94.3% facilities, while SP tablets, quinine injections and quinine tablets were respectively in stock at 88.5%, 77.6% and 69.0% of facilities. Non-recommended AQ was available in only 23.6% of facilities.Malaria diagnosis and treatment practices were analysed for 1,976 febrile patients who met the inclusion criteria , at 159 facilities where AL was in stock on the survey day. At five facilities with AL in stock, no febrile patients meeting the inclusion criteria were seen during the survey days. Of these 1,976 febrile patients, 1,096 (567 aged <5 years and 529 aged ≥5 years) were seen at 88 facilities with malaria diagnostic support. The remaining 880 patients (407 aged <5 years and 473 aged ≥5 years) were seen at 71 facilities without malaria diagnostic capacity.Table Table Among test positive children, anti-malarial treatment was prescribed universally and 74.7% (95% CI: 61.0-88.5) of children were treated with AL, 18.7% (95% CI: 6.5-30.9) with AL+QN and 5.5% (95% CI: 0.8-10.2) with quinine alone. Notably, none of the test positive children were treated with ineffective AQ or SP monotherapies  and older children and adults (29%). While the absence of malaria diagnostics in nearly half of the facilities due to partial deployment of RDTs provides an explanation for low testing rates in older children and adults, the testing practices observed at facilities with available diagnostics are worth noting. At these facilities, despite the increase in testing rates compared with all facilities, the testing rates for febrile older children and adults did not exceed 54%, and at variance with presumptive recommendations for children under 5 years of age, 34% of these children were still tested. The testing patterns found in this study concur with reports from studies undertaken during the early AL implementation in Kenya, which suggested underuse of testing in older children and adults and important presence of testing in children under five years of age [The in-service malaria case management training for health workers - the key implementation activity reinforcing age-specific recommendations for malaria testing between 2007 and 2009 - deserves special attention. The issues related to the quality of in-service training and unclear or incorrect case management messages communicated to health workers during the early AL implementation phase in 2006 were previously reported . DespiteNevertheless, in 2010, Kenya has changed diagnostic policy and case management guidelines to unambiguously recommend parasitological testing of all febrile patients regardless of the age category . TherefoThe findings of this analysis, three years after AL was delivered to health facilities in Kenya, have shown that AL treatment practices have prevailed over other anti-malarial treatments in both age groups (64% in young children and 65% in older children and adults). Despite non-adherent practices that are still present, it is important to note that the use of ineffective monotherapies such as SP or AQ was nearly non-existent. However, the use of the non-recommended combination of AL and quinine was particularly common among test positive children under five years of age 18%). These findings reveal a major shift in treatment practices compared with reports during the early AL implementation, which were characterized by low AL use in both age groups, with prevailing practice of prescribing non-recommended AQ, SP and their combinations %. These .There are several possible explanations for these patterns. Firstly, despite the change of policies and deployment of AL to peripheral facilities, the discontinuation of old treatment practices and the translation of new policies into recommended clinical practice is a relatively slow process. The slow changes in treatment practices observed during the early and late implementation process in Zambia  concur wAnti-malarial treatment practices for test negative patients deserve special attention since failure to adhere to test negative results severely compromises the cost-benefit of testing-based malaria case management strategies . These fThere are several possible explanations for these patterns in this study. While in young children, the disregard of test negative patients could be explained by the promotion of presumptive treatment (but without testing) during the study period, the magnitude of practices in discordance with implemented guidelines is worrying in older children and adults. Reduced availability of non-recommended AQ in public health facilities has not resulted in more rational use of anti-malarial drugs for test negative patients but alongside more established AL policy in switch of practices from AQ to AL. However, non-adherence to test-negative results is not unique to Kenya, and larger scale evaluations have often reported similar results across Africa under the microscopy-based ,24,25 anOverall, malaria testing rates were low and despite different recommendations for malaria diagnosis for patients under and over five years of age, only moderate differences in testing rates between the two age groups were observed at facilities where diagnostics were available. More positively, in both age groups, AL treatment practices have prevailed over other anti-malarial treatments and the use of ineffective monotherapies was nearly non-existent. Furthermore, the large majority of test positive patients were treated with recommended AL, while in the smaller subset of non-adherent treatments the use of AL+QN was observed. However, the use of anti-malarials for test negative patients was widespread, with AL being the dominant treatment choice. Further improvements in malaria case management are urgently required and they will be dependent upon the delivery of a comprehensive case management package including deployment of RDTs, good quality training, post-training follow-up, structured supervisory visits, and more intense monitoring. The recent change of diagnostic policy abandoning presumptive treatment and recommending universal parasitological diagnosis across all age groups should be an opportunity to strengthen the quality of malaria case management.The authors declare that they have no competing interests.Both authors contributed to the study design, data analysis, interpretation of the results, policy implications of the findings and drafting and finalization of the manuscript.