Intermittent preventive treatment of malaria in children (IPTc) is a promising strategy for malaria control. A study conducted in Mali in 2008 showed that administration of three courses of IPTc with sulphadoxine-pyrimethamine (SP) and amodiaquine (AQ) at monthly intervals reduced clinical malaria, severe malaria and malaria infection by >80% in children under 5 years of age. Here we report the results of a follow-on study undertaken to establish whether children who had received IPTc would be at increased risk of malaria during the subsequent malaria transmission season.Morbidity from malaria and the prevalence of malaria parasitaemia and anaemia were measured in children who had previously received IPTc with SP and AQ using similar surveillance methods to those employed during the previous intervention period.1396 of 1508 children (93%) who had previously received IPTc and 1406 of 1508 children (93%) who had previously received placebos were followed up during the high malaria transmission season of the year following the intervention. Incidence rates of clinical malaria during the post-intervention transmission season (July –November 2009) were 1.87 (95% CI 1.76 –1.99) and 1.73  episodes per child year in the previous intervention and placebo groups respectively; incidence rate ratio (IRR) 1.09 (95% CI 0.99 –1.21) (P = 0.08). The prevalence of malaria infection was similar in the two groups, 7.4% versus 7.5%, prevalence ratio (PR) of 0.99 (95% CI 0.73–1.33) (P = 0.95). At the end of post-intervention malaria transmission season, the prevalence of anaemia, defined as a haemoglobin concentration<11g/dL, was similar in the two groups  (P = 0.84).IPTc with SP+AQ was not associated with an increase in incidence of malaria episodes, prevalence of malaria infection or anaemia in the subsequent malaria transmission season.NCT00738946ClinicalTrials.gov  Malaria is one of the most important infectious diseases in the world and 40% of the world's population is at risk. Despite increasing use of current control strategies such as rapid diagnosis and treatment of clinical cases, use of insecticide impregnated materials and indoor residual spraying with insecticides, malaria remains an important cause of morbidity and mortality, particularly in sub-Saharan Africa. More than 90% of the deaths due to malaria occur in this region and 88% of these deaths occur in children aged less than 5 years of age Intermittent preventive treatment of malaria (IPT), defined as the administration of a curative dose of an anti-malarial drug or drug combination at predefined time intervals to an at risk population regardless of whether or not they are known to be infected, is a promising new strategy for malaria control in areas where the infection still causes substantial morbidity and mortality The sample size of these initial studies was relatively small, with fewer than 500 subjects per treatment arm, and they were conducted in areas where the utilisation of insecticide treated bednets (ITNs) was low. Therefore, two larger studies of IPTc (about 1500 subjects per arm) were conducted in Mali and Burkina Faso in 2008 A concern over the use of IPTc is that it may impair the acquisition of naturally acquired immunity leading to an increase in the incidence of malaria in the period after treatment is stopped, as was seen when chemoprophylaxis was given to African infants or children The protocol of the trial and its amendment , and CONDetails of the study sites, the overall design of the trial and the surveillance procedures used have been published previously The study was conducted in two villages, Djoliba and Siby, and in the small town of Ouelessebougou in the district of Kati in the savannah region of Mali. Djoliba and Siby are located 40 and 30 km south west of the capital city Bamako respectively and Ouelessebougou is located 80 km south of Bamako. Each locality has a community health centre. A research team composed of physicians and medical residents was established in each of the three community health centres to follow up and provide health care to the study participants.www.vestergaard-frandsen.com) was provided at enrollment to all the participants. Parents or guardians were instructed on how to use the net and the importance of using the net regularly was emphasized. Participants were followed to the end of the 2008 malaria transmission season (intervention period) to assess the safety and efficacy of the intervention and to the end of the 2009 transmission season to look for any rebound effect from the intervention given during the previous year.The study was an individually randomised, placebo controlled trial. A total of 3017 children aged 3–59 months were randomized in a 1: 1 ratio to receive IPT with sulphadoxine-pyrimethamine (SP) plus amodiaquine (AQ) (n = 1509) or their placebos (n = 1508) on three occasions at monthly intervals during the peak period of malaria transmission starting in August 2008. A long lasting insecticide treated net (LLIN)  the incidence of clinical malaria during the whole post-intervention period (December 2008 to November 2009), (ii) the incidence of severe malaria defined according to the WHO criteria P. falciparum parasitaemia during the high transmission season and at the end of the malaria transmission season, (iv) the prevalence of mild, moderate or severe anaemia defined as a haemoglobin (Hb) concentration<11 g/dL, <8 g/dL and <5 g/dL respectively at the end of the malaria transmission season and (v) the incidence of hospital admission defined as a stay of at least 24 hours in hospital for treatment during the follow-up period.The primary endpoint for the follow-up study was the incidence of clinical malaria during the follow-up malaria transmission season (July to November 2009). Clinical malaria was defined as the presence of fever (axillary temperature ≥37.5°C) or a history of fever in the past 24 hours and the presence of http://www.indepth-network.org/index.php?option=com_ content&task  = view&id =  96&Itemid = 184).Passive surveillance for clinical malaria continued after the end of the intervention period  to the end of the subsequent transmission season. Parents were encouraged to bring their child to a study health centre, where medical staff were available 24 hours a day and 7 days a week, if their child became unwell. A finger prick blood sample was obtained from any study child with fever (an axillary temperature of 37.5°C or higher) or a history of fever within the previous 24 hours for preparation of a blood film, measurement of Hb concentration and for a rapid diagnostic test (RDT) for malaria OPTIMAL IT® . Children who had a positive RDT for malaria were treated immediately with artemether/lumefantrine (AL). Severe cases were admitted to the health centre or referred to the paediatric ward of the Gabriel Touré Hospital in Bamako. Causes of death were assessed within a month of death using a modified version of the INDEPTH post-mortem questionnaire . During these home visits, the use of a LLIN was assessed by asking if the child had slept under an LLIN the previous night and the presence of the net was checked by field staff. In addition, the axillary temperature of each child was taken and a blood film obtained regardless of whether or not the child had fever to measure the prevalence of malaria infection.At the end of the 2009 malaria transmission season, a cross-sectional survey of all the children enrolled into the study was carried out. A clinical examination was performed, thick and thin blood films were prepared and haemoglobin concentration was measured.Thick blood films were air dried, stained with Giemsa and examined for malaria parasites by two well-trained technicians. One hundred high power fields were examined before a film was declared negative. Parasite density was determined by counting the number of parasites present per white blood cell (WBC) on a thick smear and assuming a WBC count of 8,000 per µl. In the case of a discrepancy (positive/negative or a difference in parasite density greater than 30%), a third reading was done. The median parasite density of two or three readings was used. Haemoglobin concentrations were measured using a haemoglobin analyzer  on blood obtained by finger prick.Data were collected on standardized forms, double-entered and verified using MS Access and then exported to Stata  for additional cleaning and analysis. A data analysis plan was written and submitted to the DSMB prior to analysis. An intention-to-treat analysis was performed. The intervention period was defined as the period from the first dose of IPT administration to the end of November 2008 (42 days after the third dose of IPT). The post-intervention period of high malaria transmission was defined as the period from July to November 2009 and the whole post intervention period was defined as the period from December 2008 to November 2009, which included the low malaria transmission season (dry season from December 2008 to June 2009).Incidence rates of clinical malaria, severe malaria and hospital admissions were calculated by dividing the number of episodes by the total child days at risk. Children were not considered at risk for 21 days after treatment for an episode of malaria and these days were not included in the calculation of the child days at risk. The incidence rates in the two treatment groups were compared using Cox regression to estimate the incidence rate ratio (IRR), with adjustment for age, gender and locality, and using a robust standard error to allow for the lack of independence among repeated episodes in the same child. Time to first episode of clinical malaria in the two arms was examined using Kaplan-Meier plots and compared using the log rank test. The prevalence of malaria infection was compared between the previous intervention and control groups using a generalized linear model, and prevalence ratios (PRs) were adjusted for age, sex and village.http://www.who.int/childgrowth/software/en/) was used to define wasting, stunting and being underweight and to code these variables as binary using z-scores with a score of<−2 as a cut-off Anthropometric data collected at the end of the 2009 malaria transmission season were analysed as previously described http://www.pharmaclin.com).The study protocol was reviewed and approved by the Ethical Committee of the Faculty of Medicine, Pharmacy and Dentistry, University of Bamako, Mali and by the Ethics Committee of the London School of Hygiene and Tropical Medicine. Individual, written, informed consent was obtained from a parent or guardian of each child prior to screening and enrolment. A Data and Safety Monitoring Board (DSMB) was established and monitored the trial with the support of a local medical safety monitor. GCP monitoring of the trial was performed by PharmaClin  and 34 were lost to the control arm (3 had died and 31 migrated). The proportion of children who completed the whole study was 89.8% (1355/1509) in the intervention arm and 90.9% (1372/1508) in the control arm (P = 0.29). The age and sex distributions of the children available for follow-up were similar in the two arms  initially randomised to receive IPTc were available for follow-up on July 1udy area . One thotwo arms . The meaThe incidence of clinical malaria in the two study groups during the high malaria transmission period of the post intervention year is presented in Kaplan-Meier survival estimates of the proportion of children who remained free of malaria during the high malaria transmission period of the post intervention year were similar in both groups .There was not strong evidence of an increase in the incidence of malaria post-intervention (data not shown) when a malaria episode was defined as fever or history of fever and positive asexual parasitaemia of any density  (P = 0.13).The incidence of severe malaria in children in the two study groups during the post- intervention period is presented in During the high malaria transmission season of the post intervention period, eight hospital admissions were recorded in children who had previously received IPTc compared to seven in children who had received placebos. The incidence rates of hospital admission did not differ between the two groups of children  (P = 0.77). This observation was not altered when the low malaria transmission (dry) season post intervention was included  (P = 0.21) .During the high malaria transmission season of the post intervention year, five deaths were recorded amongst children who had previously received IPTc compared with four deaths among those who had previously received placebos.From July to November 2009, home visits were made to 1352 children who had previously received IPTc and to 1348 children who had previously received placebos to measure the prevalence of malaria infection; 172 and 168 children in the two groups were not found and microscopy results were available for 1179 and 1180 children respectively. The number of children who carried a malaria infection was 87 (7.4%) in children from the former intervention arm and 89 (7.5%) in children from the former control arm  (P = 0.95). The geometric mean parasite densities in positive films were also similar in the former intervention  and control groups  (P = 0.96), the ratio between the two treatment arms was 1.01 (95%CI 0.48–2.12).At the end of post intervention malaria transmission season, 1355 children from the former intervention group and 1372 children from the former control group were studied . MicroscDuring the 2009 end of malaria transmission season cross-sectional survey, haemoglobin concentrations were measured in 1350 children who had previously received IPTc and in 1360 children who had previously received placebos . Mean HbZ scores were calculated for 1051 children from the former intervention arm and for 1057 children from the former control arm . The proThis study assessed whether, in a context of a high usage of ITNs, children who had received intermittent preventive treatment with SP +AQ during one malaria transmission season were at increased risk of malaria morbidity during the subsequent malaria transmission season as consequence of lack of exposure to malaria parasites and consequent impairment of immunity. The study showed a small increase in clinical episodes of malaria during the post intervention high malaria transmission season in children who had previously received IPTc, a rate ratio of 1.09, with a confidence interval from 0.99 to 1.2. This is consistent with the results obtained in previous smaller studies undertaken in Mali, Senegal and Ghana that found no evidence of an important increase in malaria morbidity after administration of IPT in children for a single transmission season ITN usage in children enrolled in these three previous studies was low (<5%) while in our study more than 96% of the children used an ITN. A meta-analysis of the three previous studies found a similar increase  in malaria morbidity In a parallel study conducted in Burkina Faso, with a similar design and a similar large sample size, a 12% increase in malaria morbidity was seen in the post intervention period, [Unpublished]. The confidence interval was narrower in that study, reflecting the larger number of malaria episodes.In line with previous studies No evidence was found for an increase in malaria parasite prevalence or parasite density in subjects who had previously received IPTc during the post-intervention period in line with the results of previous studies At the end of the post intervention malaria transmission season, the prevalence of anemia, moderate anemia and nutritional indicators  were similar among the subjects who had previously received IPTc and those who had not. Similar results were found in the parallel study in Burkina Faso [Unpublished]. However, Kweku et al In summary, the results of this study are internally consistent, and consistent with those of other similar studies in different settings with different levels of malaria transmission, in showing that administration of an effective IPTc regimen for one malaria transmission season may result in a small increase in the incidence of clinical episodes of malaria during the subsequent malaria transmission season but no increase in the prevalence of malaria parasitaemia, anaemia or malnutrition. It is possible that the risk of rebound malaria may be influenced by the level of transmission but currently there are too few data to determine this. No increase in severe malaria has been seen in this or any other study but the numbers of subjects studied so far have been too small to exclude a small effect. The possibility of a small rebound effect must be set against the major benefit seen during the period in which the intervention was given, with an 80% reduction in the incidence of severe malaria and a probable, substantial reduction in all cause mortality A limitation of this and previous studies that have assessed the post-intervention consequences of IPTc is that the interventions have been given during a single malaria transmission season whilst, if IPTc is to be deployed as a malaria control tool, it is envisaged that children would receive seasonal IPTc for several years and it is possible that this would have a bigger impact on the acquisition of naturally acquired immunity than seen in this study. In a previous study of seasonal chemoprophylaxis undertaken in an area of the Gambia with a pattern of malaria similar to that of Mali, the risk of a rebound in clinical malaria increased with each year that a child had received seasonal prophylaxis only becoming substantial when a child had received prophylaxis for several seasons following birth Protocol S1Trial Protocol(DOC)Click here for additional data file.Checklist S1CONSORT checklist(DOC)Click here for additional data file.


An epidemiological and entomological study was carried out in Balaghat district, Madhya Pradesh, India to understand the dynamics of forest malaria transmission in a difficult and hard to reach area where indoor residual spray and insecticide treated nets were used for vector control.This community based cross-sectional study was undertaken from January 2010 to December 2012 in Baihar and Birsa Community Health Centres of district Balaghat for screening malaria cases. Entomological surveillance included indoor resting collections, pyrethrum spray catches and light trap catches. Anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite protein.Plasmodium falciparum infection accounted for >80% of all infections. P. vivax 16.5%, P. malariae 0.75% and remaining were mixed infections of P. falciparum, P. vivax and P. malariae. More than, 30% infections were found in infants under 6 months of age. Overall, an increasing trend in malaria positivity was observed from 2010 to 2012 . Twenty five Anopheles culicifacies  were positive for circumsporozoite protein of P. falciparum (44%) and P. vivax (56%). Additionally, 2 An. fluviatilis, were found positive for P. falciparum and 1 for P. vivax (sibling species S and T). An. fluviatilis sibling species T was found as vector in forest villages for the first time in India.These results showed that the study villages are experiencing almost perennial malaria transmission inspite of indoor residual spray and insecticide treated nets. Therefore, there is a need for new indoor residual insecticides which has longer residual life or complete coverage of population with long lasting insecticide treated nets or both indoor residual spray and long lasting bed nets for effective vector control. There is a need to undertake a well designed case control study to evaluate the efficacy of these interventions. Malaria is a global issue and India contributes substantially to global malaria incidence Plasmodium species and the effect of antimalarial measures. Therefore, the objective of this study was to understand the dynamics of forest malaria transmission by conducting an epidemiological and entomological study in a difficult and hard to reach area where IRS, ITNs, RDTs and ACT were used for malaria control.Factors influencing malaria in India are highly diverse and vary greatly from the epidemiological setting of any other country. The presence of various malarial parasites and vector species, climatic diversity favoring growth and proliferation of the parasite and vector as well as a highly susceptible human population have resulted in high malaria transmission in forested areas of central India (Madhya Pradesh). Indoor residual spraying (IRS) with synthetic pyrethroid and distribution of ACT were in use since 2008. Insecticide treated nets (ITNs) were introduced in this state since 2010. Despite these new tools, a very large number of malaria cases with high spleen rate were reported from district Balaghat, Madhya Pradesh Balaghat district is a region of deep valleys, hills and hillocks with thick dense forest . There are several perennial streams and their tributaries which provide numerous permanent breeding sites for mosquitoes. The villages are located near streams and most of the villages are unapproachable during rains (July - September).This study was undertaken in 18 villages of Birsa (Population 11536) and 12 villages of Baihar (Population 6218) community health centres (CHCs) which are almost approachable throughout the year and on the border of Rajnandgaon and Kawardha districts of Chhattisgarh state . These vBefore undertaking this investigation, data from previous years of the district were obtained from District Malaria Officer (DMO), Balaghat along with details of insecticide used for spray, population covered, types of antimalarial used and their numbers and RDTs used etc , 2. The Monthly cross sectional fever surveys were carried out door-to-door in study villages from January 2010 to December 2012. Finger-pricked blood smears were collected from all fever cases and cases with 14 days history of fever as per the guidelines of National Vector Borne Disease Control Programme (NVBDCP). The blood smears thus collected were stained with the JSB stain P. falciparum cases were given ACT [Artesunate Combination Therapy (Artesunate + Sulfadoxine Pyrimethamine)] and Primaquine (PQ) and all P. vivax cases were given Chloroquine (CQ) and PQ as per NVBDCP guidelines Spleen examination was done in children between 2–9 years with or without fever by Hackett’s method Overall 105 patients (aged 1–59 years) were enrolled with ACT orally over a three-day period. Clinical observations were recorded daily for the first eight days (0–7 days) and during follow- up following standard protocol Indoor resting Anopheles mosquitoes per man hour (MHD) were sampled once in a month in the early morning (0600 hrs) for 15 minutes each by a team of two insect collectors with flashlights and mouth aspirators in four villages (two from each CHC) as per standard techniques An. culicifacies using DDT (4%), malathion (5%) and deltamethrine (0.05%) test papers following standard techniques et al. (1987) P. falciparum and P. vivax. PCR and D3 domain of 28S rDNA gene sequencing was used to identify all specimens of the An. culicifacies and An. fluviatilis species complex as described earlier Insecticide susceptibility tests were carried out on field collected The data were double key-entered in MS Access 2003  based data entry screen. Age was recoded in categorical variable  before analysis. 2×2 contingency table analyses were used to describe the differences in case prevalence by age groups. Data analysis was done using SPSS 17 for Windows .P. falciparum.The slide positivity rate (SPR) is the proportion of examined thick films found parasite positive and the related slide falciparum rate (SFR) and slide vivax rate (SVR) were calculated. Gametocyte carriers were recorded only for Informed written consent was obtained from each adult and from guardian of the children enrolled as per ethical guidelines of Indian Council of Medical Research, New Delhi, India. Purpose, potential risk and benefits of the study has been communicated in local language (‘Hindi’) and consent was documented, even for those who were unable to read and write by taking thumb impression in the presence of a witness specifically from the same community. Generally school teachers, forest guards and other influential members of their community are very helpful in explaining benefit of the study and obtaining consent. The study was approved by the institutional review board of Regional Medical Research Centre for Tribals, Jabalpur, India (IRB00006471).P. malariae was found only in 2012.Of the total 5683 males and 5939 females screened for malaria, 1881 males (33%) and 1868 females (31.5%) were positive for malaria . P. falciparum and 18 for P. vivax. However, SFR was highest in relatively older children > 4–8 years  compared with adults (P<0.001). P. falciparum proportion was higher in all age groups as compared to P. vivax (P<0.001). The mean gametocyte rate was 14.4% and monitoring of gametocyte prevalence revealed that young children less than 5 years have significantly more gametocytes as compared to relatively older children (>5 years and above) .The SPR was lowest in adults (aged >14 yrs) when compared with other age groups  (P< 0.0001). The SVR was highest in infants < = 1 year)  when compared with adults (P<0.001) . FurtherP. vivax prevalence was highest in summer . Spleen rate was 48.5% in 2010 which increased to 58.3% in 2012 . The average spleen rate in children was 43.8% (2269/5182) and the average enlarged spleen was 1.99 (95% CI 1.94–2.04). Year wise analysis revealed  and lowest in summer 18.7% . The gamrevealed  that SPROver all therapeutic efficacy study showed 100% adequate clinical and parasitological response and follow up of patients up to 28 days did not show clinical symptoms and malaria parasites.An. culicifacies Giles, An. subpictus Grassi, An. fluviatilis James, An. annularsis Van Der Wulp were the most abundant species in indoor resting collection (MHD)  with a peak in monsoon months. The relative abundance of An. culicifacies was high throughout the year (39%) with a peak in post monsoon season (October - November). An. fluviatilis was collected mainly in winter season (December - January) and was almost absent during the dry hot months of April to June. The trend of anopheline prevalence was more or less similar in MHD  . An. vagr in MHD  and PSC r in MHD  except io indoor .An. culicifacies assayed, 25 were positive for CSP of which, 14 were reactive for two polymorphs of P. vivax (7 VK 210 and 7 VK 247) and remaining for P. falciparum. The positive An. culicifacies were found during most part of the year except in January, February and March. They were caught from HD, Cattle shed (CS), PSC and LT (indoor/outdoor) and represented sibling species C, D and E (An. culicifacies (species C) was found positive from outdoor collection (LT). Additionally out of 67 An. fluviatilis, two were reactive for P. falciparum and represented sibling species T (An. fluviatilis (sibling species S) was positive for CSP of P. vivax (VK 247). D and E  . In this study >50% of the cases were reported in children less than 8 year and 31% cases were reported in older children between 8 to 14 years while only 18% cases above 14 years of age. The number of malaria cases and age group indicated that the risk for the age group under 8 years was 3.4 times greater than in adults suggesting a considerable degree of immunity in the adults.P. falciparum or P. vivax were less than 6 months of age. A review of age pattern of malaria revealed that as transmission increased, there was a shift of malaria towards younger age groups, regardless of seasonality P. falciparum (80%) followed by P. vivax. It is interesting to point out that both P. vivax polymorphs, Pv 210 and Pv 247 were present almost equally while in North East India, VK 247 was predominant polymorphs P. vivax overlap P. malariae infections were recorded only in 2012 P. malariae is sustained at low infection rates among the sparse and mobile human population P. falciparum and P. vivax malaria throughout the year with high sporozoite rate estimated for P. falciparum and P. vivax indicates that the vector control measures were inadequate and not effective in reducing adult vector density. Moreover, the environmental and climatological situations permit the continual breeding of vectors in permanent breeding sites. The sporozoite rate of An. culicifacies found in this study explains that An. culicifacies plays a major role in malaria transmission and An. fluviatilis played a role in enhancing P. falciparum transmission period along with An. culicifacies well beyond the wetter months. Moreover, for the first time An. fluviatilis sibling species T was found playing a major role in malaria transmission in forest villages along with species S in India.More than 30% infants found infected with P. falciparum, P. vivax, P. malariae and mixed infection of P. vivax, P. falciparum and P. malariae suggest that vectors had a long life expectancy. Further, the patterns of infections are also determined by the occurrence and the ability of the vector species to be infected by different parasite species simultaneously An. culicifacies was also found positive in outdoor catches. The outdoor infectivity of An. culicifacies is of significance from a malaria control stand point, because the vector may avoid contact even with an effective insecticide sprayed inside the houses. Further research is required about the extra domiciliary transmission as the villagers frequently spent the night in the open, presumably providing a source of infection to the anophelines prevalent outdoors. During the survey, people also told that these bednets were never re-impregnated. Therefore, providing LLINs in such communities can remedy the short coming associated with ITNs. The World Health Organization also recommended the use of LLINs as these nets are designed to maintain their biological efficacy against vector mosquitoes for three years, obviating the need for regular insecticide treatment The high infection rates with Tremendous progress has been made in some countries in reducing malaria related morbidity and many countries are striving for elimination of malaria ITNs and IRS which are so effective in other parts of world


Severe malaria remains a major cause of death and morbidity amongst adults in the Asiatic tropics.Plasmodium falciparum malaria and prospectively recruited to malaria studies in western Thailand between 1986 and 2002, was performed to assess the factors associated with a fatal outcome. Different severity scores and classifications for defining severe malaria were compared and, using multiple logistic regression, simple models for predicting mortality developed.A retrospective analysis of the clinical and laboratory data of 988 adult patients, hospitalized with The proportion of patients fulfilling the WHO 2000 definition of severe malaria was 78.1%, and their mortality was 10%. Mortality in patients given parenteral artesunate or artemether  was lower than in those given parenteral quinine  (P < 0.001). Models using parameter sets based on WHO 1990, 2000 and Adapted AQ criteria plus blood smear parasite-stage assessment gave the best mortality prediction. A malaria prognostic index (MPI), derived from the dataset using five clinical or laboratory variables gave similar prognostic accuracy.The mortality of severe malaria in adults has fallen and the switch from quinine to artesunate has probably been an important contributor. Prognostic indices based on WHO 2000 definitions, and other simpler indices based on fewer variables, provide clinically useful predictions of outcome in Asian adults with severe malaria. Plasmodium falciparum malaria still kills ~0.7 million people each year. The majority of those who die are children in sub-Saharan Africa, but ~43,000 patients still die each year in the Asia-Pacific [-Pacific . Admissi-Pacific -6. For A-Pacific   has produced three guidelines which include definitions of severe malaria  score was developed , based oThe clinical and laboratory features of adults with falciparum malaria recruited prospectively to hospital-based studies on the western border of Thailand between 1986–2002, conducted by the Mahidol University-Oxford Tropical Medicine Research Unit and colleagues, were analysed. The specificity and sensitivity of the different severe malaria definitions and scoring systems were examined and simple models to identify adults at risk of death built.P. falciparum present on peripheral blood slides, admitted to hospital with malaria and then recruited to clinical research studies on the western border of Thailand between 1986 and 2002 were analysed. Patients were recruited at hospitals in Kanchanaburi , Sangklaburi , and Mae Sot  and described in a series of papers [Data from 988 adult (≥15 years) patients with asexual forms of f papers . All patAs patients were recruited to a variety of studies, clinical and laboratory evaluations varied. All patients had a full history and examination performed and haematocrit and parasitaemia determined. Thick and thin blood films were stained immediately with Field’s stain and parasites counted and staged ,8. AdmisThe anti-malarial treatment regimens used in clinical studies in western Thailand changed over the 18 years as new anti-malarials were introduced and were:1. Non-artemisinin-based parenteral treatment:Intravenous quinine dihydrochloride with or without a 20 mg salt/kg or 7 mg/kg loading dose followed by 10 mg/kg every 8 h followed by oral quinine salt 10 mg/kg every 8 h, combined, when the patient was able to take oral medication, with oral quinine alone or with oral tetracycline, doxycycline, mefloquine ) or single dose primaquine to give a total treatment course of 7 days.2. Non-artemisinin-based oral treatment:Oral quinine sulphate 10 mg salt/kg every 8 h alone or with oral tetracycline, doxycycline, mefloquine , single dose primaquine or proguanil to give a total treatment course of 7 days.Or oral mefloquine  at 15 mg base/kg on the first day with or without 10 mg/kg on the next dayOr oral halofantrine  with tetracycline plus or minus oral quinine3. Artemisinin-based parenteral treatment:Intravenous artesunate ; 2.4 mg/kg stat, 1.2 mg/kg at 12 h followed by 1.2 mg/kg every 24 h) or intramuscular artemether (3.2 mg/kg stat followed by 1.6 mg/kg every 24 h) for 7 days alone or with oral doxycycline, tetracycline or mefloquine  with or without primaquine. Sixty-nine patients were treated with iv artesunate combined with iv quinine followed by the above oral regime .4. Artemisinin-based oral treatment:Oral artesunate or artemether (4 mg/kg for 3 days or 2 mg/kg for 7 days) alone or with oral doxycycline, tetracycline or mefloquine  with or without primaquine. Eighteen patients received oral dihydroartemisinin (4 mg/kg) in replacement for one artesunate daily dose .Supportive treatment was in accordance with guidelines ,27. FaciSix main models have been used to define severe malaria in adults, those published by WHO and the AQ, MSA and CAM scores -23 Addi. The accStatistical analyses were performed using Stata . All univariate comparisons between survivors and patients who died were performed using logistic regression and adjusted for study site. Ten sets of clinical and laboratory assay variables Tables  were useVariables selected into any final logistic regression model, based on published criteria (see above), were used to define the malaria prognostic index (MPI). Since for all logistic models (above), inclusion of study site as an independent variable did not improve the model nor change the co-efficients for other covariates significantly, it was not included in the development of the MPI. Each variable was categorized into four groups using rounded quartiles or commonly used cut offs (as for GCS). Univariate logistic models (with sets of corresponding binary variables) were fitted and categories with similar (P > 0.05) odds ratios (OR) were grouped together. Co-efficients of the final multivariate model were rounded to the nearest integer and used to calculate for each patient a linear combination of variables  – the MPI. The MPI was calculated for each patient and the ROC analysis used to evaluate its prognostic utility.The predictive value of the MPI was further evaluated using cross validation  on a subOf 988 hospitalized patients enrolled Tables , 7 and 8Mortality did not significantly differ between the three sites . Artemisinin derivatives were given to 7% of patients in Kanchanaburi, 77% in Sangklaburi and 83% in Mae Sot (P < 0.001), reflecting temporal changes in treatment policy and study protocols. Patients in Kanchanaburi had received prior malarial treatment more frequently than at other sites and women there were more often pregnant. Patients in Mae Sot presented with lower coma scores (69% with coma score of 15 compared to >80% at the other sites and 20% with coma score ≤11 compared to 9-10% in the other sites), higher parasitaemia, higher proportions of trophozoites on admission film and a greater likelihood of a palpable liver or spleen (Tables Forty-one admission variables were significantly associated (P < 0.05) with death on bivariate analysis Tables  and 8. V4/μL or a parasitaemia of >5 x 105/μL [Staging of parasite development on peripheral blood smears provides prognostic information additional to the parasite count itself; the median percentage of ring stages amongst those who survived was 98% and amongst those who died 48% (P = 0.0001). The percentage of the modal parasite stage was significantly lower amongst those who died than in those who survived (P = 0.0002), suggesting that lower circulating parasite stage synchronicity was associated with death. Using the cut offs of Silamut and White of pigmented stages >10x 105/μL , the senThe median (range) coma recovery time and time to death were 24 (1–188) h (N = 46) and 44 (2–641) h (N = 76) respectively Table . Artemisversus 34% (15/44) (P = 0.547). Mortality was higher in patients with renal impairment; 82% (18/22) in those with a serum creatinine >264 μmol/L compared with 28% (23/83) in those with lower levels (P < 0.001).Of 111 patients with cerebral malaria (GCS < 11), 97 had evaluable data. Of these, 68 (70%) had plasma lactate >4 mmol/L and/or serum bicarbonate <15 mmol/L and/ or serum creatinine >264 μmol/L, and 36 (53%) of these died compared with three (10%) among 29 cerebral malaria patients without these abnormalities (P = 0.001). Among cerebral malaria patients, mortality did not significantly differ between those with and without jaundice : 42% (23/55) All final models were adjusted for treatment with ACT and study site. No interactions between covariates and treatment were significant in any of the models and study site was not significant (P > 0.05). Tables Considering the WHO- and AQ-based models, the WHO 1990, WHO 2000, Adapted AQ and Adapted AQ + Pigmented stages, variable sets gave the best predictive power and the areas under the ROC curves (AUROCCs) were not significantly different Tables , and 4. The BCAM and RCAM scores could be calculated for 662 and 873 patients, respectively, and mortality rose with increasing score for both Table . Of 498 For the MSA score  Additio using adConsidering all ten models, those based on the MSA score and the Adapted AQ with pigmented parasites had the best predictive power, but AUROCCs for models based on WHO criteria were not significantly lower. Simple rules for classification of severity as a MPI Table , gave seThe prognosis of severe falciparum malaria has improved markedly since the introduction of parenteral artesunate ,32. ThisModels using variable sets based on WHO 2000 and Adapted AQ with pigmented stages gave the best prediction of mortality, and were comparable to the results based on the MSA score using a smaller sample. Very similar results were also obtained with the MPI based on the most commonly selected variables, GCS, plasma lactate, parasitaemia, serum bilirubin and percentage of pigmented parasites. This will need to be evaluated in independent series of adults with falciparum malaria in similar settings of unstable malaria transmission. This score suffers from the disadvantage, unlike the RCAM score, that determination of 4/5 variables  requires skilled technicians and equipment/consumables and quality assurance that are seldom available where severe malaria is common.There are at least four limitations of this analysis: specifically, not all patients admitted to the study hospitals were recruited, recruitment criteria varied, there are missing values, and a variety of different doctors reviewed the patients with consequent variability in the nature of both clinical assessment and inpatient management. However, any such differences are likely to result in false negative, rather than false positive associations. The MSA score differs from the others scores discussed, as it is not strictly an admission predictive prognostic score, including mechanical ventilation during hospitalization. Some potential prognostic factors such as haemoglobinuria and abnormal bleeding were too infrequent to allow reliable conclusions to be drawn. Serum bilirubin and plasma lactate were not measured in the South East Asian Quinine Artesunate Malaria Trial (SEAQUAMAT) and therefore the MPI could not be calculated for this dataset .This study differs from that of Hanson , which oThe wide variation in mortality estimates in this dataset among those with severe malaria (10-18%) and indeed the wide range of estimates of severe disease (6–60%), depending on which definition is used, illustrates the importance of definitions in comparisons between studies, in meta-analyses, and in understanding the host and geographical variability in the presentation and outcome of severe malaria. Various terms, such as ‘uncomplicated’ and ‘mild’, are used to refer to malaria that is not severe. Severe disease is also equated with ‘complicated’ malaria. To avoid confusion, terminology should be standardized with ‘severe’ malaria referring to malaria with clinical and/or laboratory features suggesting a clinically significant risk of death (e.g. >5%) despite anti-malarial treatment, and ‘uncomplicated’ as those without such prognostic features and that the terms ‘complicated’ and ‘mild’ are not used.The present study suggests that, if laboratory tests are available, the history of the illness and the physical examination, apart from GCS and respiratory rate, are relatively unimportant in assessing prognosis in a population of malaria patients. Unlike in other series , increasIn this series, the mortality of cerebral malaria was increased three-fold if concurrent acidosis and/or renal failure were present and these factors are crucial in predicting death. The ease of identifying patients with cerebral malaria may have made study of other complications less of a focus and these data suggest that more research on the pathophysiology and treatment of acidosis and renal dysfunction would provide information that would improve management and outcome ,14,32. TThe authors declare that they have no competing interest.PN and KS analysed the data and wrote the first draft of the manuscript. PN, AD, KS, WC, SK, TMED, YS, BA, SP, RR, JH, ND, NW looked after the patients and revised the manuscript. All authors read and approved the final manuscript.Severe malaria definitions.Click here for fileWorld Health Organization (1990) criteria for severe malaria and outcome.Click here for fileWorld Health Organization (2000) criteria for severe malaria and outcome.Click here for fileAdapted ‘AQ’ criteria, from Hien et al.[en et al.,20, and Click here for file


Trypanosoma brucei parasite subspecies. We therefore examined the effect of Trypanosoma brucei gambiense sleeping sickness on the specificity of malaria RDTs.In endemic settings, diagnosis of malaria increasingly relies on the use of rapid diagnostic tests (RDTs) instead of microscopic examinations. False positivity of such RDTs is poorly documented, although it may be particularly relevant in infections for which the differential diagnosis includes malaria, such as sleeping sickness, a fatal but treatable disease caused by Plasmodium.Blood samples of 117 sleeping sickness patients and 117 matched non-sleeping sickness controls were prospectively collected in the Democratic Republic of the Congo. Reference malaria diagnosis was based on microscopy corrected by a four primer real-time PCR. Ten commonly used rapid diagnostic tests for malaria were evaluated including three two-band tests and seven three-band tests, based on the detection of Pf-HRP-2, Pf-pLDH and/or pan-pLDH antigens of Pf-pLDH test lines were not affected.Specificity of RDTs for diagnosis of malaria in controls was between 97.5 and 100% and was between 11.3 and 98.8% in sleeping sickness patients. For seven out of 10 RDTs, specificity was significantly lower in sleeping sickness patients compared to controls. Decreased specificity of malaria RDTs in sleeping sickness was mainly caused by false positivity of the pan-pLDH test lines, but also occurred frequently for the HRP-2 test lines.The Specificity of some malaria RDTs in sleeping sickness is surprisingly low, and constitutes a considerable risk for misdiagnosis or delayed diagnosis of sleeping sickness.


This study aimed to investigate baseline data on malaria before the evaluation of new vector control strategies in an area of pyrethroid-resistance of vectors. The burden of malaria was estimated in terms of infection  and of clinical episodes.Between December 2007 and December 2008 in the health district of Ouidah - Kpomassè - Tori Bossito (southern Benin), a descriptive epidemiological survey of malaria was conducted. From 28 selected villages, seven were randomized from which a total of 440 children aged 0 to 5 years were randomly selected. Clinical and parasitological information was obtained by active case detection of malaria episodes carried out during eight periods of six consecutive days scheduled at six weekly intervals and by cross-sectional surveys of asymptomatic infection. Entomological information was also collected. The ownership, the use and the correct use of long-lasting insecticide-treated nets (LLINs) were checked over weekly-survey by unannounced visits at home in the late evening.P. falciparum asexual forms per μL of blood (95%CI 504-680). Pyrogenic parasite cut-off was estimated 2,000 P. falciparum asexual blood forms per μL. The clinical incidence of malaria was 1.5 episodes per child per year (95%CI 1.2-1.9). Parasitological and clinical variables did not vary with season. Anopheles gambiae s.l. was the principal vector closely followed by Anopheles funestus. Entomological inoculation rate was 5.3 (95%CI 1.1-25.9) infective bites per human per year. Frequency of the L1014F kdr (West) allele was around 50%. Annual prevalence rate of Plasmodium falciparum asymptomatic infection was 21.8% (95%CI 19.1-24.4) and increased according to age. Mean rates of ownership and use of LLINs were 92% and 70% respectively. The only correct use of LLINs (63%) conferred 26% individual protection against only infection (OR = 0.74 (95%IC 0.62-0.87), p = 0.005).Mean parasite density in asymptomatic children was 586 The health district of Ouidah-Kpomassè-Tori Bossito is a mesoendemic area with a moderate level of pyrethroid-resistance of vectors. The used LLINs rate was high and only the correct use of LLINs was found to reduce malaria infection without influencing malaria morbidity. Anopheles gambiae was resistant to pyrethroids, have reported that significant reduction in the efficacy of pyrethroids was applied either in treated nets or IRS [Despite considerable worldwide efforts made in recent years to control malaria , the diss or IRS ,12. In os or IRS ,14. In trd General Census of the Population and the Environment (RGPH3) of February 2002. The population is rural and lives on agriculture with scattered settlement. The main ethnic group is Aïzo. The climate is essentially sub-equatorial, with two dry seasons (a long dry season from December to March and a short dry season in August and September), and two rainy seasons (a long rainy season from April to July and a short rainy season from October to November). The average annual rainfall is around 1,200 mm, of which 700-800 mm and 400-500 mm rain down respectively in the first and in the second rainy season. The hottest months (31°C) are February to April and the coldest months (27°C) are July to September. Less than 30% of children are present at the health centre when sick. They are mostly treated by traditional medication [This epidemiological study was carried out in the Ouidah-Kpomassè-Tori Bossito health district in southern Benin Figure , from Dedication . A recendication ,17. Maladication .Twenty eight villages were chosen according to the following criteria: having between 250-500 inhabitants, distance between any villages greater than two kilometers and the absence of a local health centre. From these, seven villages were randomly selected. Geographical, demographical and environmental characteristics are described in Table Active case detection (ACD) for malaria episodes was carried out during eight periods of six consecutive days at six weeks intervals throughout the year. Each day a nurse assisted by a local village helper trained for the study, visited the households in the sample. A physician supervised the field work. The presence or absence and state of health of each child were recorded daily on a specially prepared form (one form per household). The nurse examined and recorded data on every case of sickness detected at home. A thick blood film was taken from every sick child. Children were treated according to the clinical diagnosis made by the nurse. When malaria was suspected, the patient was treated with artemether-lumefantrine for three days according to the recommendations of WHO and NMCP ,20. CrosAn. gambiae complex and Anopheles funestus group were stored in individual tubes with silicagel and preserved at -20°C for P. falciparum circumsporozoite index estimation and molecular identification.Data were collected two weeks before each clinical monitoring. Adult mosquitoes were caught using Human Landing Catches (HLC) technique . In the ® 2.0) which were distributed in October 2007 were checked over weekly-survey. The visits of the nurse were unannounced and took place in the late evening around 9.00 PM when children were expected to be asleep [The ownership, the use and the correct use of LLINs  were determined according to the method of Favia [P. falciparum circumsporozoite protein (CSP) [kdr allele [After scoring field-collected Anopheles mosquitoes and identifying the species of each specimen by Polymerase Chain Reaction (PCR) , the preof Favia . Infectiin (CSP) . The metr allele .P. falciparum asexual blood forms, density of P. falciparum asexual blood forms in parasite positive blood thick films and prevalence of P. falciparum gametocytes. A generalized estimating equation (GEE) approach, which can be used with normal distributions and discrete data was used for statistical analysis of repeated measures. To take into account the interdependence of observations made on the same person, an exchangeable correlation structure was used in which the correlation between these observations made on one person at different times was assumed to be the same. The prevalence of asymptomatic malaria infections was analyzed as a binomial response by using a logistic regression model. The parasite density was log transformed for a normally distributed response and analyzed with a link function by using a linear regression model.Demographic, parasitological, clinical and entomological data were double entered independently in the Access 2003 database. Parasitological and clinical data were analyzed using the svy command (STATA 11.0). For each person only one blood sample per monitoring period was considered for analysis. When a pathological condition was detected, the blood sample taken during the clinical episode was retained for analysis. Parasitological data were analyzed separately in terms of prevalence of versus asymptomatic state) as the dependent variable, and parasite density as the independent variable. In this type of model, a random intercept variable is allowed to vary with subjects, and this random subject-specific intercept allows the interdependence of the observations made on the same person to be taken into account. For each pathological period, the probability that it was caused by malaria was estimated by the Attributable Fraction (AF) calculated from the odds ratios associated with the estimated parasite density in the logistic model [The association between the parasite density and the occurrence of clinical episodes was tested using a Poisson regression model, taking clinical status  were analyzed according to demographic , environmental (season and villages) and sanitary  variables. The Chi2 test was used to compare the rate of ownership, use and correct use of LLINs. An optimum pyrogenic parasite density cut-off was calculated using the estimated AFs with a logistic model. The sensitivity and the specificity were similarly determined [The three dependent variables  was expressed as the number of anopheles bites per human per night. The sporozoite index was calculated as the proportion of mosquitoes found to be positive for CSP. The entomological inoculation rate (EIR) was calculated as the product of the HBR and the sporozoite index and expressed as the number of infected bites per human per year.A total of 440 children in seven villages were parasitologically and clinically monitored during 18,262 person-days from which 402 (2.2%) were missing for the following reasons: 366 not found and 36 refusals. Ten children died during the study. The mean age of the children at inclusion was 2.1 years. The female/male ratio was 1:1. Each child in the survey was visited on an average of 42 days out of the 48. A total of 3,074 thick blood films were taken, comprising 2,838 in asymptomatic children and 236 in sick children, with an average of seven per child.Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale were present alone or mixed . In the multivariate random-effects logistic regression model, age of children, season, village and correct use of LLINs, but not the ownership and the use of LLINs were significantly associated with the prevalence of infection (Table versus 7.8% (CI95% 5.2-10.5)). The prevalence of infection was higher during the dry season (24.7% (CI95% 21.6-27.8) than during the rainy season (18.6% (CI95% 15.7-21.5)). The prevalence of infection was higher in Satré, Wanho, Kindjitopka and Hèkandji than in Dokanmè, Aidjèdo and Guézohoué.ed Table . The annon Table . The corP. falciparum asexual forms per μL of blood (95%CI 504-680). According to multivariate random-effects linear regression model, increased parasite density was associated with some villages (Dokanmè and Satré) but not with age of children, season, ownership and use of LLINs  and differed significantly between the dry (3.8% (95% CI 2.9-4.8)) and the rainy season (2.2% (95%CI 1.4-3.0)), p = 0.008.The mean parasite density in positive asymptomatic children was 586  95%CI 50-680. AccP. falciparum alone, three individuals with P. malariae alone, two individuals with P. ovale alone and three individuals with mixed infection. The P. malariae single infections showed densities of 480, 2,360 and 200 parasites/μL and P. ovale single infections showed densities of 4,800 and 9,800 parasites/μL. Three mixed infections with P. falciparum and P. ovale showed combined densities of 3,760 Pf + 720 Po, 960 Pf + 400 Po and 280 Pf + 120 Po   and 67 An. funestus  were caught in the seven villages. The number of CSP positive An. gambiae s.l. and An. funestus were nine and four respectively. The aggressiveness of culicidae and malaria vectors (An. gambiae s.l. and An. funestus) were 5,541  and 74 (95%CI 17-318) bites per human per year. The annual EIR was 5.3 (95%CI 1.1-25.9) infected bites per human per year. The 1014F kdr allele was present in both molecular M and S forms. The frequency of this mutation was respectively 0.47 (95%CI 0.37-0.57) and 0.61 (95%CI 0.43-0.80) in the M and S forms.Overall 13,602 mosquitoes including 115 The LLINs ownership rate reached 91.8%  and remained high through the year. Use was significantly higher during the rainy season than the dry season 73%  and 67%  respectively, p = 0.0001. It significantly decreased to 31%  in the middle of dry season. Correct use was also the highest during the rainy season ) compared to the dry season ), p < 0.0001, Figure .This prospective longitudinal study allowed the epidemiology of malaria in the health district of Ouidah-Kpomassè-Tori Bossito after the nation-wide distribution of LLINs to children in October 2007 to be characterized. Previous studies have described the epidemiology of malaria in Benin, by focusing mainly on malaria transmission  and on cAn. gambiae to pyrethroids, the L1014F kdr allele reached 50%, in accordance with previous studies carried out in southern Benin [The entomological findings showed that the health district of Ouidah-Kpomassè-Tori Bossito is a mesoendemic area with a mean annual EIR of 5.3 infected bites 95%CI 1.1-25.9). This EIR was found in conjunction with the annual prevalence rate of 21.8% (95%CI 19.1-24.4) observed in young asymptomatic children ,49,50. Irn Benin . The annrn Benin . Here, wrn Benin ,53. The .1-25.9. P. falciparum asexual blood forms per μL. The use of AF to define the pyrogenic parasite cut-off allows the best trade-off between sensitivity and specificity level [P. falciparum parasitaemia is dependent on the season and age, which affects the malaria-AF of pathological episodes and thus the malaria case definition according to pyrogenic parasite density cut-off [falciparum asexual blood forms per μL was close to the value of 1,000 found in mesoendemic area on children under 3 years [The calculated AF of pathological episodes to malaria helped to determine the optimum parasite pyrogenic cut-off at 2,000 ty level . In stab cut-off ,55. In t 3 years  and to t 3 years .P. falciparum high-endemic area, the pyrogenic cut-off of parasitaemia in persons of a given age is similar for all Plasmodium species [P. malariae could have been responsible for one malaria clinical case with 2,360 parasites/μL and P. ovale for two cases with a parasitaemia of  respectively.In the health district of Ouidah-Kpomassè-Tori Bossito, one pathological episode out of three was attributed to malaria. To avoid a maximum of missed cases the malaria case definition took into account signs evoking malaria or history of fever during the 48 hours preceding the first day of ACD as advised Mcguinness . Mean an species . Given tIn 2001 before the national distribution of LLINs, in south of Benin, 4.3% of household owned a treated net (ITN) and 2.4% of children under five years old slept under ITNs . In 2006In conclusion, the health district of Ouidah-Kpomassè-Tori Bossito is a mesoendemic area characterized by a moderate level of pyrethroid resistance of vectors and a high heterogeneity of malaria infection between villages. Malaria infection and disease did not vary through the year. The used LLINs rate was high and only the correct use of LLINs was found to reduce malaria infection without influencing malaria morbidity.The authors declare that they have no competing interests.The design of the study was conceived by FC and M-CH. Data were collected in the communities by GBD, AD and A-SBB. M-CH, AD and VC carried out laboratory analysis. CR, M-CH, GBD, AD and VC participated in the data analysis. The results were discussed by GBD, M-CH, AD, VC, CR, FC, MA, DK-G and AM. GBD and M-CH draft the manuscript. All authors read and approved the final manuscript.


Iron deficiency is highly prevalent in pre-school children in developing countries and an important health problem in sub-Saharan Africa. A debate exists on the possible protective effect of iron deficiency against malaria and other infections; yet consensus is lacking due to limited data. Recent studies have focused on the risks of iron supplementation but the effect of an individual's iron status on malaria risk remains unclear. Studies of iron status in areas with a high burden of infections often are exposed to bias. The aim of this study was to assess the predictive value of baseline iron status for malaria risk explicitly taking potential biases into account.We prospectively assessed the relationship between baseline iron deficiency (serum ferritin <30 µg/L) and malaria risk in a cohort of 727 Malawian preschool children during a year of follow-up. Data were analyzed using marginal structural Cox regression models and confounders were selected using causal graph theory. Sensitivity of results to bias resulting from misclassification of iron status by concurrent inflammation and to bias from unmeasured confounding were assessed using modern causal inference methods.The overall incidence of malaria parasitemia and clinical malaria was 1.9 (95% CI 1.8–2.0) and 0.7 (95% CI 0.6–0.8) events per person-year, respectively. Children with iron deficiency at baseline had a lower incidence of malaria parasitemia and clinical malaria during a year of follow-up; adjusted hazard ratio's 0.55 (95%-CI:0.41–0.74) and 0.49 (95%-CI:0.33–0.73), respectively. Our results suggest that iron deficiency protects against malaria parasitemia and clinical malaria in young children. Therefore the clinical importance of treating iron deficiency in a pre-school child should be weighed carefully against potential harms. In malaria endemic areas treatment of iron deficiency in children requires sustained prevention of malaria. Childhood iron deficiency is prevalent globally with highest estimates in children in developing countries. In sub-Saharan Africa, 33–66% of children are iron deficient, most of whom are under the age of five years In recent years concerns have focused mainly on the possible harmful effects of treating iron deficiency rather than on the effects of iron status itself Severe Anemia Study (SevAna study), a large case-control study investigating the etiology, pathophysiology and outcome of severe anemia in southern Malawi This is a secondary analysis of follow-up data from a cohort of pre-school children recruited as the control group for the SevAna study was approved by the Ethics Committees of the College of Medicine, Malawi . Prior to enrolment in the study, written informed consent was obtained from the parent or guardian of all study participants.The SevAna study recruited 381 cases and 757 controls between July 2002 and July 2004  or history of fever . Severe malaria was defined as a positive blood slide with either severe anemia (hemoglobin <5.0 g/dl) or coma Plasmodium falciparum asexual parasites per 200 white cells was counted and expressed as the number per microliter of blood. Malaria parasitemia was defined as the presence of one or more Pl. falciparum asexual parasites Hemoglobin (Hb) concentrations were measured by HemoCue B-Hemoglobin analyzer  to judge eligibility-criteria. Hb measured by Coulter counter analyzer  was used for statistical analyses. Moderate and severe anemia were defined as an Hb of <11.0 g/dL and <5.0 g/dL, respectively. Plasma levels of C-reactive protein (CRP) and serum ferritin were analyzed on Modular P800 and Monular Analytic E170 systems . Iron deficiency was defined as serum ferritin <30 µg/L The incidence of clinical malaria and parasitemia was calculated for a follow-up period of one year. Multiple events per child were registered. A child was considered not to be at risk for incident malaria for the 28 days following malaria treatment (SP), as the prophylactic effect of malaria treatment was assumed Directed Acyclic Graph theory, to model the causal relationship between iron status, malaria risk and any potential (measured and unmeasured) confounders  were identified from The main predictor, iron deficiency, was defined as serum ferritin <30 µg/L. Possible confounders considered for inclusion were: sex, age, area of residence, season, socio economic status, genetic predisposition of malaria, cellular immunity, humoral immunity, cumulative malaria exposure, malaria immunity, history of other infections than malaria, HIV infection, hookworm infection, nutritional status and zinc deficiency . We usedDue to inadequate sample volume in 29% of the children serum ferritin was not available. The missing data were assumed to be missing at random; basic characteristics between children with and without available ferritin assessments were comparable . BecauseSchoenfeld residuals against time and tested using cox.zph procedure in R To compare the incidence of malaria during follow-up between iron deficient and iron replete individuals, a marginal structural Cox proportional hazards model was used to adjust for confounding. Marginal structural models are a new class of statistical models to estimate causal effects We assessed to which extent the outcomes could have been biased because of possible misclassification of the iron deficiency indicator (serum ferritin), by reanalyzing the data using a more sensitive and a more specific definition of iron deficiency Robins et al.(2×ID - 1), with ID = 1 or 0 as indicator of iron deficiency and α the degree of unmeasured confounding or, in other words, the average difference in malaria risk at baseline on a ratio-scale and conditional on the covariate distribution.In addition, to evaluate how large the average difference in susceptibility to malaria at baseline between the iron deficient and iron-replete group would need to have been to partly or fully explain our observed differences in malaria incidence, we used a method for sensitivity analysis for unmeasured confounding developed by Follow-up data were available for 96.0% of all study participants (727/757). Baseline characteristics are presented in During the follow-up year the overall incidence of malaria parasitemia and clinical malaria was 1.9 (95% CI 1.8–2.0) and 0.7 (95% CI 0.6–0.8), respectively. This incidence was higher in the rural areas and in the children less than 24 months of age . There wThe marginal structural Cox model showed that iron deficiency was an independent predictor of malaria parasitemia and clinical malaria; HR (95%CI) 0.55 (0.41–0.74) and 0.49 (0.33–0.73) respectively . The effAnalyses assessing sensitivity to misclassification of iron status are presented in In this study, children with iron deficiency at baseline (serum ferritin <30 µg/L) had a lower incidence of malaria parasitemia and clinical malaria during a follow up period of one year. This finding suggests that iron deficiency protects against malaria parasitemia and clinical malaria in pre-school children living in malaria endemic areas. The results were consistent across study sites with varying transmission intensity and across age-groups despite changing susceptibility with age. In addition, the difference in risk between baseline iron status groups persisted throughout the follow-up period.et al also showed an effect on all cause and malaria associated mortality Our findings are in line with results of the few other studies assessing influence of iron status on malaria risk As with any observational study, the results of this study are subject to bias. Therefore, we explicitly evaluated the potential impact that misclassification and confounding bias could have had on our results. Firstly, because serum ferritin levels are influenced by concurrent inflammation it may not adequately reflect true iron status. As a consequence, misclassification of baseline iron status may have occurred and thus resulted in biased effect measures. We evaluated whether use of a more sensitive and a more specific definition, taking concurrent inflammation into account, would substantially change the results. As expected, use of a more specific definition slightly increased the magnitude of effect and the use of a more sensitive definition led to a slightly weaker effect. Importantly, this did not change our conclusions, suggesting that the findings are robust to misclassification bias.Plasmodium in the red cells In the directed acyclic graph (DAG) in In the interpretation of our analyses we assume that iron status measured at baseline remained relatively stable throughout the year of follow-up. Unfortunately, iron status measurements were not repeated after baseline, so we are not able to check this assumption. It is possible that children changed from the iron deficient group to the iron replete group and vice versa. However, because our study population was closely followed and adequately treated for infections it is more likely that iron deficient children became iron replete than the other way around. The resulting misclassification would have resulted in an underestimation of the effect.For the underlying mechanism of the protective effect of iron deficiency on susceptibility to malaria infection, several hypotheses have been raised. It is suggested that iron deficiency alters the membrane structure of parasitized erythrocytes which facilitates their elimination by phagocytic cells, yet further research is needed to further support this hypothesis We cannot fully exclude that the observed effect was the result of increased susceptibility in the iron replete group resulting from the month of iron supplementation, rather than a protective effect of iron deficiency. In 2006 a large supplementation trial in Zanzibar was stopped prematurely after increased morbidity and mortality was observed in iron replete children receiving iron and folic acid In conclusion, iron deficiency seems to protect against malaria parasitemia and clinical malaria. However, because we lack direct measures of baseline immunity to malaria our results should be confirmed by well conducted randomized controlled trials or observational birth cohort studies. Our findings support the theory of nutritional adaptation to infectious diseases Figure S1Flow chart study population. This flowchart presents number of children enrolled in the main study, a case control study investigating etiology of severe anemia (I); number of children enrolled in the cohort study (II) and the number of children attending the follow-up visits (III).(PDF)Click here for additional data file.Table S1Baseline characteristics of the study population.(DOC)Click here for additional data file.


Insecticide-treated bed nets (ITNs) are known to be highly effective in reducing malaria morbidity and mortality. However, usage varies among households, and such variations in actual usage may seriously limit the potential impact of nets and cause spatial heterogeneity on malaria transmission. This study examined ITN ownership and underlying factors for among-household variation in use, and malaria transmission in two highland regions of western Kenya.Cross-sectional surveys were conducted on ITN ownership (possession), compliance , and malaria infections in occupants of randomly sampled houses in the dry and the rainy seasons of 2009.Despite ITN ownership reaching more than 71%, compliance was low at 56.3%. The compliance rate was significantly higher during the rainy season compared with the dry season (62% vs. 49.6%). Both malaria parasite prevalence (11.8% vs. 5.1%) and vector densities  were significantly higher during the rainy season than during the dry season. Other important factors affecting the use of ITNs include: a household education level of at least primary school level, significantly high numbers of nuisance mosquitoes, and low indoor temperatures. Malaria prevalence in the rainy season was about 30% lower in ITN users than in non-ITN users, but this percentage was not significantly different during the dry season.In malaria hypo-mesoendemic highland regions of western Kenya, the gap between ITNownership and usage is generally high with greater usage recorded during the high transmission season. Because of the low compliance among those who own ITNs, there is a need to sensitize households on sustained use of ITNs in order to optimize their role as a malaria control tool. Plasmodium falciparum infection prevalence in the community is often over 40% [Insecticide-treated mosquito nets (ITNs) used for protection against mosquito bites have proven to be a practical, highly effective, and cost-effective intervention against malaria . The eviover 40% ,2. Commuover 40% -8. Scaliover 40% -11. Targover 40% -14 is a over 40%  in areasPlasmodium infection [Unlike the lowlands where perennial malaria transmission is attributed to high vector densities throughout the year , transminfection ,19. In anfection , hydrolonfection , climatenfection , land-usnfection , and drunfection . Given tnfection .Although ITN distribution has been massively expanded in most parts of malaria endemic sub-Saharan countries since 2005, there is limited information on community based actual use of nets owned, area specific reasons for non-use, and the possible impact of the variations in use on malaria vector densities and transmission in either Kenyan highlands or other countries where malaria is seasonal and unstable. Most studies of ITN use are intervention trials that attempt to explain why vulnerable groups, such as children under five or pregnant women, are or are not using an ITN ,25, or d2 [The two study regions, Iguhu  in Kakamega district, and Emutete  in Emuhaya district are situated in the highlands of Western Kenya  coordinates of each sampled house were taken and then mapped using a handheld GPS unit. Owners of the selected houses were requested to sign a freely administered informed consent form for participation in the study, and for entomologic, parasitological and questionnaire surveys.A total of 600 houses, 300 houses from each of the two sites were randomly selected within an area of approximately 4 × 4 kmMosquitoes were collected using the Pyrethrum Spray Collection (PSC) method  during tParacheck-Pf® , were used for field identification of infections, but microscopy was used to determine infection prevalence rates. Thin and thick blood smears were taken in the field and the slides were stained with 4% Giemsa for 30 minutes [Occupants from sprayed houses who freely consented to participate in the study were screened for the malaria parasite during both the dry and the rainy season in 2009. Rapid diagnostic tests kits (RDTs),  minutes . Househo minutes . ParticiHouseholds were interviewed about ITN ownership and use as a means of malaria vector control. In general, a household was defined as a unit headed by a male or female with his ⁄ her dependents and spouse, and who share a cooking pot/common eating place and sleep under one roof . An ITN 2-test was used to determine the differences between ITN ownership and actual usage. The Student t-test was used to determine differences in vector densities between houses with at least a functional ITN and those with none in use.Data was collected and entered in Excel spread sheets (Microsoft Corporation) and statistical analysis was performed by the use of STATA SE 9 . The χth 2008 and the Institutional Review Board of the University of California at Irvine. A freely administered informed consent with interpreters was given to residents for participation in the study.Ethical clearance was obtained from the Ethical Review Committee of Kenya Medical Research Institute, "Ecology of African highland malaria (II), SSC No. 1382 (N)" dated May 15During the dry and the rainy seasons, a total of 600 houses per season (300 houses from each site) were surveyed. A total of 1,160 and 1,265 participants of all ages were surveyed in the dry and rainy seasons respectively . In both regions, participants' ITN usage was significantly higher in the rainy than the dry season  and 53.4% vs. 41.8% in Emutete , respectively.Reported and confirmed net ownership and usage among the study participants is summarized in Figure 2 = 13.28, d.f. = 1, P < 0.001) . There was also a significant inter-site difference in ITN usage for different age groups . Determination of ITN coverage was a limiting factor as this would only be ascertained by doing spot checks very early in the morning to confirm participants who had their nets properly hung.Compared with ownership, actual ITN use was considerably lower, regardless of seasonality and inter-site variation Figure . There wFigures An. funestus densities in the two highlands sites, this data was categorized into season for analysis. Unlike in the dry season where there was no difference in the number of indoor An. funestus collected in the houses with or without ITNs, there was significantly high number of An. funestus collected in houses without an ITN compared with those with one during the wet season . Table An. gambiae s.l. density during the two study seasons. During the dry season, we did not detect significant differences in indoor resting female An. gambiae s.l. density between ITN owning houses and houses without ITNs  in houses using ITNs compared with 0.80 f/h/n in houses not using ITNs . An. gambiae s.l. density in Iguhu was 0.10 females/house/night (f/h/n) in houses using ITNs compared with 0.09 f/h/n in houses not using ITNs . However, during the rainy season, indoor resting female An. gambiae s.l. density in houses with functional ITNs were significantly lower, 43% lower, compared to those not using ITNs in both Emutete and Iguhu , whereas those densities were 0.90 f/h/n and 1.56 f/h/n in Emutete .Due to the very low Regardless of the types of nets, 86.3% and 94.3% of the nets in Iguhu and Emutete respectively were in good condition, i.e., not torn. In Iguhu 89.8% of the surveyed nets were LLINs (85.1%) or retreated regular ITNs (4.7%), whereas this number is 86.2% in Emutete (62.4% LLINs), the remainders were untreated nets. Overall, about 20% of the surveyed ITN/LLINs were not considered as good for preventing mosquito biting because they had at least one tear.Education level and knowledge about malaria transmission were some of the significant reasons affecting ownership and usage of ITNs Table . When coMost parts of Kenya have approached or met the RBM household ITN coverage target of at least 60% as reported by previous studies . SimilarEven though ITN ownership was similarly high across seasons, seasonality in use was remarkable during this survey in both regions of study. Net use was considerably higher in the rainy, cooler season of April-May than in the dry, hot season of January-February. During the rainy season, the non-compliance rate was lower compared with the dry season. Similar findings have also been reported in surveys in ITN intervention trials in the lowlands of western Kenya and northern Ghana ,39. As iThe proportion of households possessing mosquito net(s) and the proportion of children less than 5 years of age who slept under a net the preceding night are two of the key RBM bed net ownership indicators used to investigate the strengths and weaknesses of monitoring malaria control . In thisThe protective effect of nets on infection prevalence during the rainy season among sampled participants was consistent across age-groups except for ages under 5 years in Iguhu. Unlike in the rainy season, the nets' protection role was not clearly observed in older children and adults in the dry season. Children under 5 years old in both regions had the largest protection margin with net use. Overall, during the dry season infection prevalence among net users was lower than in non-users by 56.7% in Emutete and by 26.2% in Iguhu among this age group. During this season, the odds of malaria infection among this age group were 2.3 fold and 1.4 fold higher among non-net users in Emutete and Iguhu respectively. While the approach of targeting interventions to protect at-risk individuals is based on solid scientific grounds ,45,46, aThe impacts of ITNs very much depend on their excito-repellent and insecticidal properties ,49. FurtThis study demonstrates that in hypo-meso endemic highlands of western Kenya, despite high mosquito net ownership, actual usage is still remarkably low. To track progress and draw inferences about this principal malaria intervention tool, both ownership and actual use indicators must be taken into account. Regular, region-specific rapid assessments of household possession, use of nets, and respondent's knowledge should complement ongoing distribution, and findings should be incorporated into programme policy. Based on these findings, ITN use in this region could be increased, if IEC messages on malaria transmission were strengthened either through community health workers or net provider programs to encourage participants to use nets they already possess. Since the analyses found that there were disparities in net use across age groups, distribution of free or subsidized nets to the entire population may optimize coverage as well as help increase ITN use.The authors declare that they have no competing interests.HA carried out the field surveys, assembled data, analyzed and drafted the manuscript. GZ performed statistical analysis and revised the manuscript. MCL participated in writing and revised the manuscript, YA and IM participated in study coordination and revised the manuscript. AG and GY designed the study and helped with manuscript preparation. All authors read and approved the final manuscript.


The annual incidence and temporal trend of severe malaria and community-acquired bacteraemia during a four-year period in Muheza, Tanzania was assessed.Data on severely ill febrile children aged 2 months to 14 years from three prospective studies conducted at Muheza District Hospital from 2006 to 2010 was pooled and analysed. On admission, each enrolled child had a thin and thick blood film and at least one rapid diagnostic test for falciparum malaria, as well as a blood culture. The annual incidence of bacteraemia and severe malaria among children coming from Muheza was calculated and their temporal trend was assessed.Haemophilus influenzae and pneumococcal invasive bacterial infections decreased as well but were much fewer in number.Overall, 1, 898 severe falciparum malaria and 684 bacteraemia cases were included. Of these, 1, 356 (71%) and 482 (71%), respectively, were from the referral population of Muheza. The incidence of falciparum malaria and all-cause bacteraemia in Muheza decreased five-fold and three-fold, respectively, from the first to the fourth year of surveillance (p < 0.0001). During this period, the median ages of children from Muheza admitted with severe malaria increased from 1.7 to 2.5 years (p < 0.0001). The reduction in all-cause bacteraemia was mainly driven by the 11-fold decline in the incidence of non-typhoidal salmonellosis. The annual incidences of These results add to the growing evidence of the decline in malaria associated with a decrease in non-typhoidal salmonellosis and possibly other bacteraemias. Malarial prevention and control strategies may provide a greater benefit than the mere reduction of malaria alone. Salmonella (NTS) infections. NTS, mainly Salmonella typhimurium and Salmonella enteritidis, are a leading cause of bacteraemia in African children [Community-acquired bacteraemia causes significant morbidity and mortality among children in sub-Saharan Africa. A recent comprehensive review that included 43, 130 children admitted to African hospitals with a blood culture, found that 3, 527 (8%) had a bloodstream infection . That machildren . In contchildren , invasivchildren ,15,17,21et al [et al [et al [The common occurrence of NTS septicaemia during malaria outbreaks was first reported in British Guiana in the 1920s . Duggan et al  found thl [et al  proposedl [et al ,18,21. Il [et al  found thet al reported lower rates of invasive NTS infection in children associated with reductions in malaria [During the last decade, the intensity of malaria transmission has decreased in parts of sub-Saharan Africa -28. In T malaria . Most re malaria . Using dP. falciparum malaria. In 2007, 7% of antenatal clinic attendees [Haemophilus influenzae type b (Hib) had just started in March 2009 and there are plans to introduce pneumococcal vaccine [The study site consisted of two paediatric in-patient wards of the Teule District Hospital in Muheza, north-eastern Tanzania. Each of the wards has 35 beds and together receives 6, 000 child admissions per year. Teule's referral base is the Muheza district  but the hospital accepts patients from all over the Tanga region. Tanga, one of 26 regions of Tanzania, has an area of 26, 808 square kilometres and is divided into the following districts: Lushoto, Korogwe, Muheza (and Mkinga), Tanga, Pangani, Handeni, and Kilindi Figure . First-lttendees  and 4% ottendees  were HIV vaccine .Malaria diagnostic data and blood culture results from prospective studies of febrile children undertaken at the Teule paediatric wards from 2006 to 2010 were pooled and analysed ,34,35. TP. falciparum malaria using a PfHRP-2 based  and/or pLDH based  kits. From each child, thin and thick blood films were prepared, Giemsa-stained and read by experienced laboratory technicians. At least 100 high-power microscopic fields of the thin film were examined to exclude the diagnosis of malaria. One to 10 ml of blood (depending on body weight) were collected from each child and inoculated into a BactALERT™ Paediatric-fan bottle  and incubated in the BacT/ALERT 3D automated microbial detection system. Blood cultures were processed according to standard methods. The laboratory participated in a bacteriology external quality assurance programme organized by the Network for Surveillance of Pneumococcal Disease in the East African Region http://www.netspear.org and identified unknown samples provided by a nearby laboratory as part of the College of American Pathologists external quality assurance programme. Bacterial identifications were independently confirmed for Streptococcus pneumoniae and Haemophilus influenzae isolates by the KEMRI/Welllcome Trust Centre for Geographic Medicine, Kilifi, Kenya and for some of the Salmonellae isolates by the Queensland Health Forensic and Scientific Services, Brisbane, Australia.The point-of-care and laboratory investigations conducted are described in detail elsewhere ,34,35. EEach study used its standard case record form that documented demographic details including age and residence, clinical information, as well as point-of-care and laboratory results. Data analysis was performed using Stata software (version 11.0). Fever was defined as history of a rise in body temperature as recalled by a care-giver or presence of axillary temperature ≥37.5°C on presentation. Bacteraemia was defined as fever with isolation of pathogenic bacteria from blood culture. One blood culture was performed on admission. For the purpose of this analysis, severe falciparum malaria was defined as fever with a positive RDT or presence of asexual malaria parasites on the blood film and at least one clinical sign of severe malaria as described above. A severe febrile illness was defined as fever with at least one severity sign as described above with the addition of neck stiffness, bulging fontanel and fever of three or more days.To apply consistent criteria for inclusion across the studies, we excluded children without signs of severe illness in the first study from the analysis Figure . To ensuThe three studies (from which the pooled data was obtained) were conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from the parent or guardian of each eligible child prior to his/her participation. The three studies were approved by the Tanzania Medical Research Coordinating Committee. In addition, the first febrile illness study was approved by the London School of Hygiene and Tropical Medicine (UK), the severe malaria drug trial was approved by the Oxford Tropical Research Ethics Committee (UK) and the surveillance study to assess the burden of typhoid fever was approved by the International Vaccine Institute - Institutional Review Board (Republic of Korea).During the study period from June 2006 to May 2010, a total of 6, 836 severely ill febrile children between 2 months and < 15 years old who participated in the study were included in the analysis: 3, 091, 778 and 2, 967 from the first, second and third studies, respectively  were > 2 months to 2 years, 2, 125 (31%) were > 2 years to 5 years and 688 (10%) were > 5 years of age. We ranked the pathogenic bacterial isolates according to frequency, by age group (Table E. coli bacteraemia did not change (p = 0.56) whereas that of typhoid fever increased from 7 cases/100, 000 children/year during the first year to 15 during the fourth year of surveillance (p = 0.002).The children with falciparum malaria and bacteraemia were classified by district Table . Of the As an indicator of hospital utilization the annual number of deliveries from 2002 to 2010 was assessed Figure . There wThere was an impressive decline in the incidence of malaria in the study site, which is consistent with previous reports from other locations -30. FromThe decline of malaria in Muheza occurred in parallel with a reduction in invasive bacterial infections, particularly in non-typhoidal salmonellosis. Although over a shorter time period, our findings are similar to those from The Gambia, where the estimated incidence of NTS infection in Fajara fell from 60 (1979-1984) to 10 (2003-05) cases per 100, 000 person-years. In Basse, also The Gambia, the estimated incidence of NTS infection fell from 105 (1989-1991) to 29 (2008) cases per 100, 000 person-years . In thatE. coli, a gram-negative organism ubiquitous in the bowel, did not decrease. The incidence of Hib and pneumococcal bacteraemia in the study site decreased over the relatively short period of four years. It is unlikely that these reductions were due to immunization, as neither Hib nor pneumococcal vaccines were widely available during the study period. Hospital-based surveillance data from Malawi showed a decline in the number of invasive pneumococcal infections from 2000 to 2009, which paralleled the scale-up of national antiretroviral therapy [That malaria increases the risk for bacteraemia is plausible for several reasons. The hemodynamic or inflammatory processes that occur during severe malaria can predispose to invasive bacterial infection. For example, malarial haemolysis may lead to impaired macrophage and neutrophil function, saturation of iron-binding proteins, and increased iron availability to NTS, a siderophilic organism . Alterna therapy . These ta priori. Prospective studies on community-acquired bloodstream infections in sub-Saharan Africa are scarce [There are several limitations of our study. First, the analysis used pooled data from three studies with slightly different enrolment criteria. To ensure consistent inclusion criteria in our analysis, the non-severely ill children in the first study were excluded from the analysis. The period from June 2007 to February 2008 had the highest percentage of children with severe malaria (55%) as the enrolment criteria during the drug trial was focused on signs of severe malaria and did not include neck stiffness, bulging fontanel and fever of three or more days. This more stringent enrolment criteria likely resulted in the failure to detect some clinically important bacteraemia cases, particularly typhoid fever, as reflected in the lowest incidence during the second year. But the overall trend showed a decrease in bacteraemias from the beginning to the end of surveillance. Second, the analytic plan was not developed e scarce  and the e scarce , as wellThese findings provide additional evidence for the association of malaria with bacterial disease. Strategies that prevent malaria also inhibit the risk of non-typhoidal salmonellosis and other bacteraemias. This has important implications for the impact of malaria control on all-cause child mortality. In Bioko there was a large drop in under-five mortality from 152 to 55/1000 births following malaria control .From 2006 to 2010, a decline in malaria infections in Muheza was associated with a reduction in invasive NTS disease and other bacteraemias. Continued surveillance in Muheza would be important to understand longer-term trends.The authors declare that they have no competing interests.Conception, design and implementation of the studies: GM, BA, ICEH, AMD, LVS, HR, and JD. Laboratory supervision: BA. Data management: AM, DRK. Contributed to the data analysis: GM, BN, AM, DRK and JD. Participated in the writing and revision of the manuscript: GM, BA, BN, ICEH, AMD, RLO, JDC, LVS, HR and JD. All authors read and approved the final manuscript.