Patterns of decreasing malaria transmission intensity make presumptive treatment of malaria an unjustifiable approach in many African settings. The controlled use of anti-malarials after laboratory confirmed diagnosis is preferable in low endemic areas. Diagnosis may be facilitated by malaria rapid diagnostic tests (RDTs). In this study, the impact of a government policy change, comprising the provision of RDTs and advice to restrict anti-malarial treatment to RDT-positive individuals, was assessed by describing diagnostic behaviour and treatment decision-making in febrile outpatients <10 years of age in three hospitals in the Kagera and Mwanza Region in northern Tanzania.Prospective data from Biharamulo and Rubya Designated District Hospital (DDH) were collected before and after policy change, in Sumve DDH no new policy was implemented. Diagnosis of malaria was confirmed by RDT; transmission intensity was evaluated by a serological marker of malaria exposure in hospital attendees.Prior to policy change, there was no evident association between the actual level of transmission intensity and drug-prescribing behaviour. After policy change, there was a substantial decrease in anti-malarial prescription and an increase in prescription of antibiotics. The proportion of parasite-negative individuals who received anti-malarials decreased from 89.1% (244/274) to 38.7% (46/119) in Biharamulo and from 76.9% (190/247) to 10.0% (48/479) in Rubya after policy change.This study shows that an official policy change, where RDTs were provided and healthcare providers were advised to adhere to RDT results in prescribing drugs can be followed by more rational drug-prescribing behaviour. The current findings are promising for improving treatment policy in Tanzanian hospitals. Malaria accounts for more than 800,000 deaths a year worldwide , of whicSince the 1980s, malaria control has incorporated presumptive treatment of fever with anti-malarials, treating all febrile episodes suspected of malaria with a full therapeutic dose of anti-malarials, chloroquine  or sulpMicroscopy is the gold standard method for diagnosis of malaria, but the provision of microscopic facilities may not always result in accurate diagnosis ,8,14,17.Here, the impact of the policy change was assessed by describing diagnostic behaviour and treatment decision-making in febrile outpatients under 10 years of age in three hospitals in the Kagera and Mwanza Region in Tanzania. Transmission intensity in these areas, all nearby Lake Victoria, was assessed by serological markers of malaria exposure.In this study, common practices in fever diagnosis in the outpatient departments were described for three designated district hospitals (DDH) in northern Tanzania: Biharamulo DDH and Rubya DDH, both situated in the Kagera Region, and Sumve DDH, situated in the Mwanza Region. Biharamulo is situated at 1,490 m, Rubya at >1,500 m and Sumve at 1,100 m. The transmission intensity of the catchment areas is suspected to be different based on their distance to Lake Victoria and altitude . HoweverHealthcare at these outpatient departments was largely provided by clinical officers with two to three years' clinical training. Pneumonia, HIV, tuberculosis, gastrointestinal infections, various parasite infections and malaria are common conditions in outpatients according to hospital records. Malaria transmission is related to the long rains in March-April and the less reliable short rains in October -November. The study was approved by the Kilimanjaro Christian Medical Centre Research Ethics Committee. Written informed consent was obtained from parents or care-takers of all participants.Data were prospectively gathered from all patients under the age of 10 years presenting at the outpatient department of Sumve DDH with reported or measured fever in the months September 2009 - January 2010. In Biharamulo DDH and Rubya DDH, identical data were collected in September - October 2009. This is the period before the policy change when microscopy was the only available tool to diagnose malaria. At the end of October 2009, the Kagera Region was the first region in Tanzania where a policy change was implemented by the National Malaria Control Programme (NMCP) of the Ministry of Health and Social Welfare. This policy change comprised the replacement of microscopy as the main diagnostic tool for malaria by RDTs , ICT Diagnostics) with a sensitivity of 82% at parasite densities of 200 parasites/μL, of 97% for parasite densities >2000 parasites/μL and a false positivity rate of ~1% [The same information was collected before and after policy change. The following information was collected in a questionnaire: age, gender, month attending the hospital, resident or no resident of the area in which the hospital was situated, bed net use, use of medication in the previous two weeks, auxiliary temperature, clinical diagnosis made by a clinical officer, request for blood slide, result of blood slide, treatment given in the hospital, and result of RDT. In Biharamulo and Rubya another question 'request for RDT' was added in the second period. Auxiliary temperature was measured with digital thermometers; blood slides were prepared according to hospital routine, stained with 10% Giemsa solution and read by a single experienced microscopist.Plasmodium falciparum. The RDT was not part of hospital routine, but was used to facilitate objective diagnosis for the current study. The RDT was used according to the manufacturer's instructions and is based on the detection of histidine-rich protein II. This RDT has a sensitivity of 75% at parasite densities of 200 parasites/μL, of 100% for parasite densities >2,000 parasites/μL and a false positivity rate of ~7% [Before the policy change, a finger prick blood sample was taken for malaria parasite detection by RDT  in each patient enrolled for an objective assessment of infection with For an objective assessment of exposure to malaria, serum samples were collected using a single finger prick (~20 μL) from all individuals, both adults and children, attending these three hospitals in Northern Tanzania in November 2009. This group included patients, people coming for routine follow-up visits and people accompanying patients. This sampling method has previously been validated to obtain a selection of samples representative of the general population in the hospital catchment areas . Informaet al [Serum was eluted from filter papers as described by Corran et al . Immunoget al ,23 usinget al . To geneet al .Statistical analyses of data were performed using SPSS version 18.0.0 and Stata 9.2 . Categorical variables were compared between groups by the Pearson Chi-square test or Fisher's Exact test, odds ratios (OR) with 95% confidence intervals (95% CI) were calculated. The main outcomes of the analyses were the proportion of individuals treated with anti-malarials despite a negative RDT and the proportion of individuals who did not receive anti-malarials despite a positive RDT. AMA-1 ELISA data were used to generate an age-seroprevalence plot. OD values were expressed as percentage of the positive control . The annual seroconversion rate (SCR), λ was estimated by fitting a simple model of the acquisition and loss of antibodies to the age-specific prevalence of the antibodies using maximum likelihood methods assuming a binomial distribution . If visuFrom September 2009 - February 2010, a total of 1,608 outpatients below 10 years of age presenting with (reported) fever were included. Age, reported bed net use and socio-demographic factors were similar between the three clinics  of patients without a slide taken was treated with anti-malarials before the policy change compared to 27.0% (27/100) of patients without a RDT after the policy change. In Rubya DDH this proportion changed from 67.5% (52/77) to 2.0% (2/98), indicating presumptive treatment was largely abandoned after the policy change.Together with the decrease in anti-malarial prescription, there was an increase in the proportion of individuals who received antibiotics. These were mostly broad-spectrum antibiotics like co-trimoxazole, ciproxin or amoxicillin. In RDT negative patients in Biharamulo DDH 12.8% received antibiotics before and 59.7% after the policy change. In Rubya DDH the number increased from 70.0% to 94.6%.From Biharamulo DDH, Rubya DDH, and Sumve DDH, a total of 298, 320 and 304 serum samples were successfully tested in the AMA-1 ELISA, respectively. The overall seroprevalence of AMA-1 antibodies was 52.0% (158/304), 40.3% (129/320), and 56.7% (169/298); there was a clear increase in seroprevalence with age in all sites .Click here for file


This study was conducted at the Kintampo Municipal Hospital in Ghana to determine whether there was any benefit (or otherwise) in basing the management of cases of suspected malaria solely on laboratory confirmation (microscopy or by RDT) as compared with presumptive diagnosis.Children under five years who reported at the Out-Patient Department of the Hospital with axillary temperature ≥37.5°C or with a 48 hr history of fever were enrolled and had malaria microscopy and RDT performed. The attending clinician was blinded from laboratory results unless a request for these tests had been made earlier. Diagnosis of malaria was based on three main methods: presumptive or microscopy and/or RDT. Cost implication for adopting laboratory diagnosis or not was determined to inform malaria control programmes.In total, 936 children were enrolled in the study. Proportions of malaria diagnosed presumptively, by RDT and microscopy were 73.6% (689/936), 66.0% (618/936) and 43.2% (404/936) respectively. Over 50% (170/318) of the children who were RDT negative and 60% (321/532) who were microscopy negative were treated for malaria when presumptive diagnoses were used. Comparing the methods of diagnoses, the cost of malaria treatment could have been reduced by 24% and 46% in the RDT and microscopy groups respectively; the reduction was greater in the dry season (43% vs. 50%) compared with the wet season (20% vs. 45%) for the RDT and microscopy confirmed cases respectively.Over-diagnosis of malaria was prevalent in Kintampo during the period of the study. Though the use of RDT for diagnosis of malaria might have improved the quality of care for children, it appeared not to have a cost saving effect on the management of children with suspected malaria. Further research may be needed to confirm this. Malaria is a disease with a significant socio-economic impact on countries in the developing world especially in sub-Saharan Africa. Some studies have shown that more than 50% of patients who were microscopy negative for malaria were treated for the disease The ease of operation and portability of malaria rapid diagnostic tests (RDTs) as compared with that of microscopy There have been some opposing views on whether it is probably time for changes in current guidelines recommending that African children with fever should be managed presumptively for malaria. While English et al., (2009) have expressed reservations with any attempt to introduce policy changes seeking to change presumptive treatment of malaria in favour of laboratory confirmed diagnosis and treatment With these varied opinions in mind, we investigated the benefit or otherwise in restricting the use of ACTs to cases of malaria diagnosed by RDT/Microscopy alone compared with those presumptively diagnosed for children less than five years of age reporting to the OPD of the Kintampo Municipal Hospital. We believe that the results from this study will contribute to narrowing the knowledge gaps in the advantages or otherwise of confirmatory diagnostics (microscopy and RDTs) in the management of malaria.This was a cross-sectional study spanning the period from January 2009 to February 2010. It was an all-year round study that allowed for seasonality comparisons.The study was conducted at the Kintampo Municipal Hospital in the Kintampo North Municipality of the Brong-Ahafo Region of Ghana, which has a resident population of about 75,000 people. The municipality is located within the forest-savannah transitional ecological zone of Ghana.Anopheles gambiae and Anopheles funestus with slightly more than a quarter of children under five using bed nets The rainy season in the study area occurs between April and November each year with an average rainfall of 1250 mm per annum and mean monthly temperatures between 18°C and 38°C. The area is holoendemic in terms of malaria transmission with a parasite prevalence of more than 50% among asymptomatic children less than 10 years of age Facilities for malaria microscopy are usually available at the hospital and the private clinics while RDTs are mainly used at the peripheral clinics. The community chemical shops are usually the first point of seeking medical care in the community and malaria diagnosis in the shops is mainly presumptive The municipal hospital is the referral point for the 13 Community–Based Health Planning and Service (CHPS) compounds, three (3) health centres at the sub-district levels and four private clinics in the municipality.Using morbidity data for 2006 from the hospital OPD, a sample size of 845 children was calculated which enabled the study to have 90% power with 95% confidence. The sample size calculation was done using Stata 8.2.P.falciparum specific histidine rich protein-2(Pf.HRP-2) and Pan malaria specific Plasmodium Lactate Dehydrogenase (pLDH). The sensitivity and specificity ascribed to the test are 100% for both malaria positive and malaria negative samples Prior to the start of the study, laboratory staff on the project were trained on the correct use of the Parascreen® RDT Inclusion criteria for the study were children below five years of age reporting to the Out-Patient Department of the Hospital with fever (i.e. axillary temperature ≥37.5°C) or with a history of fever within the previous 48 hours. Children five years of age and above and any child admitted to the hospital with severe disease were excluded from the study.Plasmodium falciparum parasites identified were counted against 200 white blood cells. A smear was declared negative if no parasites were found after examining 100 high power fields. The parasite density was determined from the positive smears. Laboratory results from any of the tests mentioned above were made available to the clinician only upon request. No attempt was made by the study to change the treatment practices at the hospital at that time (which in most cases was presumptive).Project staff who were stationed at the OPD identified potential study participants who were children already seen and managed by a clinician. A finger-prick blood sample (approximately 1 ml) was taken from the child by a trained laboratory technician using sterile procedures for preparation of a blood smear for microscopy examination and an RDT to confirm the presumptive diagnosis made by the clinician. The smears were independently read by two microscopists who were blinded to the results of the RDT as well as the diagnosis (es) made by the clinicians. If there was any discordance between the results of the two readers, a third and most experienced microscopist read the slide the third time, the agreement between the third reader and any of the earlier two was accepted as the final. Any asexual At the time of the study children were treated according to the national IMCI guidelines which included presumptive treatment with artesunate amodiaquine and followed up. However, if a child came back unwell, the laboratory results including the blood slide results were made available to the clinician.All study forms were checked by the study coordinator for completeness and consistency prior to submission for data entry. Data was double entered independently into Microsoft Access database and verified. Consistency and range checks were also done and problems identified were resolved.The cleaned dataset was analyzed using appropriate tests in Stata 11.0. Socio-demographic characteristics of study participants that were categorical variables were summarized into proportions, while quantitative variables such as MUAC were summarized into means together with their standard deviations. The level of agreement between microscopy and RDT was estimated using the Kappa Statistic.The study also sought to determine any differences in the cost of malaria treatment based solely on presumptive diagnosis or by laboratory confirmation. Various cost scenarios were evaluated: one was the total cost of anti-malarial treatment prescribed for subjects for whom a presumptive diagnosis of malaria had been made. This cost covered only the costs of ACTs prescribed and did not include the cost of services provided. Similar costs were calculated for subjects who were diagnosed as positive for malaria by the other two methods of diagnosis – the “Cost of malaria treatment”. Another cost was calculated separately for each group of subjects who had a positive diagnosis of malaria by either of the two laboratory diagnostic methods. This was done for each child in each group by adding the cost of antimalarials prescribed for that child to the cost of the diagnostic method – $1.00 for each RDT and $2.50 for each microscopy done . The unit cost of malaria microscopy used in the analysis was based on the cost of malaria microscopy under the Mutual Health Insurance Scheme in Ghana at the time of the study. In the second cost scenario, the two types of cost were each calculated per subject for the three diagnostic methods. Both cost scenarios were assessed separately for the wet and dry seasons. The cost of antibiotics was not included in the data analysis.The study received ethical clearance from the institutional ethics committee of the Kintampo Health Research Centre (KHRC), Ghana Health Service (GHS). Mothers/caretakers voluntarily signed or thumb- printed an informed consent form after the study was fully explained to them before their children were enrolled in the study. Data was stored in locked cabinets to ensure participant confidentiality, and was only accessible to investigators and permitted members of the study team. Participants were only identified with a unique study code.Nine hundred and forty (940) caregivers were contacted to participate in the study out of which 936 (99.6%) consented to participate and their children were enrolled into the study. All these children had both microscopy and RDT results for comparison with the study clinicians' diagnoses and were used for the data analysis. Children recruited into the study were between 1 month and 59 months of age (mean = 24 months) with 52.7% of them being males. Children between the ages of 24 months and 59 months constituted the largest proportion of study participants. There was not much difference between the proportions of children in the 0–11 month and 12–23 month age groups (24.7% vs. 28.0%). The Z-scores were based on the WHO 2005 Standard population.The proportions of malaria which were diagnosed presumptively, by RDT and by microscopy were 73.6% (689/936), 66.0% (618/936) and 43.2% (404/936) respectively. The data was disaggregated by seasonality . Of the Only one percent (1%) of children diagnosed as non-malaria cases by RDT in the dry season was identified as positive for the disease by microscopy. Almost a quarter (23.8%) of study subjects diagnosed as malaria cases by RDT were not confirmed by microscopy .The sensitivity of the presumptive method in diagnosing malaria in the wet and dry seasons was 93.3% and 80.3% respectively . The RDTIf the diagnosis of malaria had to be confirmed by any of the laboratory methods before ACTs were given, the reduction in over-diagnosis using RDTs would have been 4.1% and 24.2%  in the wet and dry seasons respectively. With microscopy, the reduction would have been 30.5% and 29.8%  respectively. Overall, over-diagnosis of malaria would have been reduced by 7.6%  and 30.5%  using RDT and microscopy respectively.This study showed that the highest frequency of treatment for malaria (73.6%) was recorded by the presumptive method of diagnosis followed by RDT (66%) and microscopy (43.2%). Results from this study are similar to those in Tanzania which showed that over 50% of patients for whom antimalarials were prescribed may not have had the disease The high levels of agreement recorded between RDT and microscopy as diagnostic methods means in transmission areas comparable to ours, one of these diagnostics methods if available, is sufficient as a diagnostic method for malaria. The World Health Organization (WHO) recommends laboratory confirmation (either by microscopy or RDT) of all suspected malaria cases before treatment is commenced and that presumptive treatment should only be considered where such confirmation cannot be done In this study, the RDT had a high sensitivity (97.7%) but a rather low specificity (58.1%) for detecting malaria. In terms of diagnosing malaria there was moderate agreement between RDT and microscopy (Kappa = 0.53) As earlier stated, almost a quarter of study subjects diagnosed as malaria cases by RDT were not confirmed by microscopy . This waWith regards to the cost of treatment in the various groups, overall the cost of treatment (per subject)  did not Notwithstanding the high total cost of malaria treatment in the RDT group, a potential limiting factor for its use, the RDT can still be said to be an effective tool in reducing the over-diagnosis of malaria and the consequent use of ACTs in non-malaria cases, due to its high negative predictive value. The quality of care of such children will therefore be improved One limitation of the study was that since it was a cross-sectional study, there was no follow up of the subjects. It was therefore not possible to ascertain the clinical outcomes of the children who were presumptively treated for malaria  and those who were not treated for malaria even though they had been diagnosed as having the disease by the two laboratory methods.Another possible limitation that since the study was conducted only in children less than five years of age and not in participants across all age groups, the possible cost-saving effect of the RDT could not be determined conclusively.


To the Editor: The rise in international travel to malaria-endemic areas in recent years has been followed by an increase in the number of cases diagnosed in countries where malaria is not endemic . endemic . Tourist endemic ,3. In sp endemic ,5.1 We rP. vivax and P. malariae. In a stool specimen, Entamoeba histolytica, Trichiuris trichura, Endolimax nana, and Blastocystis hominis were observed; stool cultures were negative.A 31-year-old man with no relevant medical history was treated in the internal medicine department of our hospital. He reported a history of poorly defined malaise, night sweats, sleeplessness, tinnitus, and episodic diarrhea with no pathologic products during the previous 6 days. He did not report fever, chills, or headache. Two weeks earlier, he had spent 10 days in Puerto Plata in a tourist resort, without traveling to any other place. He had not received any antimalarial chemoprophylaxis. Physical examination showed no abnormalities. Laboratory values, including levels of sodium, potassium, liver enzymes, creatinine, and coagulation factors, as well as results of hemogram and chest radiograph, were within normal limits. A blood film showed trophozoytes of Treatment was initiated with chloroquine (4 doses) and primaquine for a period of 14 days; metronidazole and paromomycin were administered for the intestinal infestations. Symptoms resolved in 48 hours, and control blood films showed clearance of the parasitemia. Two months after the end of treatment, the patient remained asymptomatic.Plasmodium involved are notable. The Dominican Republic is considered a low-risk area for malaria, although some places in the west, on the Haitian border, are malaria-endemic. In addition, according to available information, autochthonous malaria cases increased after Hurricane George  , Indonesia (12.1%), South America (11.4%), and West Africa (11.4%). Only 0.2% of the cases of P. vivax infection were acquired in the Caribbean, none of them in the Dominican Republic  . Previountinent 1%, Indone<10% of patients do not exhibit fever or chills, and some report only poorly defined complaints or other atypical symptoms. Among these, gastrointestinal symptoms are the most frequently reported (Second, the clinical features were atypical. Malaria usually starts as a febrile syndrome, accompanied by chills, headache, malaise, and arthromyalgia. However, sometimes symptoms are unspecific. In fact reported . In the P. falciparum may be the cause of the disease; these species likely induce more atypical forms of malaria.In summary, clinicians should include malaria in the diagnostic workup of tourists who become ill after traveling to the Dominican Republic. Species other than


Plasmodium falciparum infection, the mechanisms underlying severe disease remain poorly understood. We have previously shown evidence of endothelial activation in Ghanaian children with malaria, indicated by elevated plasma levels of both von Willebrand factor (VWF) and its propeptide. In the current prospective study of children in Malawi with retinopathy confirmed cerebral malaria, we compared these markers with uncomplicated malaria, non malarial febrile illness and controls.In spite of the significant mortality associated with Children with cerebral malaria, mild malaria and controls without malaria were recruited into the study. All comatose patients were examined by direct and indirect ophthalmoscopy. Plasma VWF and propeptide levels were measured by ELISA. Median VWF and propeptide levels were significantly higher in patients with uncomplicated malaria than in children with non-malarial febrile illness of comparable severity, in whom levels were higher than in non-febrile controls. Median concentrations of both markers were higher in cerebral malaria than in uncomplicated malaria, and were similar in patients with and without retinopathy. Levels of both VWF and propeptide fell significantly 48 hours after commencing therapy and were normal one month later.In children with malaria plasma VWF and propeptide levels are markedly elevated in both cerebral and mild paediatric malaria, with levels matching disease severity, and these normalize upon recovery. High levels of both markers also occur in retinopathy-negative ‘cerebral malaria’ cases, many of whom are thought to be suffering from diseases other than malaria, indicating that further studies of these markers will be required to determine their sensitivity and specificity. Plasmodium falciparum transmission. African children bear 90% of the brunt of mortality from falciparum malaria, with high case fatality rates in children with cerebral malaria even with good treatment Falciparum malaria is one of the commonest potentially fatal infections in sub-Saharan Africa. Most deaths from malaria occur in young children living in areas of intense In vivo von Willebrand Factor (VWF) biosynthesis is limited to endothelial cells and megakaryocytes. VWF is produced and released by vascular endothelial cells, and is frequently used as an indicator of endothelial cell activation in vascular disorders such as thrombotic thrombocytopenic purpura (TTP) and sepsis in vivo.P. falciparum infection had significantly increased plasma VWF and propeptide levels, consistent with acute endothelial cell activation P falciparum parasitaemia P. falciparum malaria is associated with circulating ultra large VWF multimers P. falciparum adhesion to platelet-decorated VWF strings P. falciparum-infected erythrocytes (IE) might sequester in vascular beds which do not express appropriate host endothelial receptors. These findings together suggest the possibility that the secretion of VWF by activated endothelial cells may be a mechanism that enhances the severity of P falciparum malaria.In a prospective study of patients from Ghana with different malaria presentations, we showed that patients with severe Severe malaria is commonly misdiagnosed in Africa, because a patient with severe disease of any cause may have a parasitaemia that is coincidental and not responsible for the illness P. falciparum parasitaemia who had severe disease or uncomplicated febrile illness and controls without parasitaemia. We used malarial retinopathy to differentiate patients with definite cerebral malaria from those with encephalopathy accompanied by parasitaemia.In the current prospective study we investigated plasma VWF and propeptide levels in paediatric malaria. We measured mature VWF and propeptide concentrations in plasma samples collected from children with P falciparum parasitaemia presenting with uncomplicated febrile illness or with encephalopathy were recruited for the study. Controls were febrile children without parasitaemia and well children undergoing routine surgery. Children (mean age = 33 months) were recruited for both the control and index groups after their parents or guardians had given written informed consent to their participation in the study. Clinical details were recorded for all cases at the time of recruitment, and all subsequent analyses were carried out blind to these details. All patients with malaria received standard supportive and anti-malarial treatment.The study was carried out at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi. Children with P. falciparum parasitaemia and no other identifiable cause of coma Patients (n = 100) were recruited when admitted with an initial clinical diagnosis of cerebral malaria (CM), defined as a Blantyre Coma Score (BCS) of 2 or less Patients with mild malaria (MM) were children attending the same hospital's Paediatric Accident and Emergency Unit (PAEU). These children were febrile and parasitaemic, had no complications suggestive of severe malaria Children (6–60 months) with a non-malaria febrile illness (NMFI) were recruited from among attenders at the same PAEU. This group comprised children not requiring admission, who had a documented febrile illness (>37.5°C) with no malaria parasites on a single thick blood film, a negative rapid diagnostic test for malarial antigens , and no history of having taken any antimalarial medication in the previous two weeks (n = 32).These were well children with no febrile illness (NFI) admitted for elective surgery (n = 20). A Paracheck malaria rapid antigen diagnostic test was performed on all controls and only those negative were included in the analysis (n = 17).To develop a Malawian standard for VWF and propeptide, and a normal range for a Malawian (adult) population, we recruited 50 healthy Malawian adult blood donors through the Malawi Blood Transfusion Services (MBTS). These donors were negative for both human immunodeficiency virus (HIV) by serology and for malaria by thick blood film microscopy and rapid diagnostic test. Citrated plasma samples were pooled together to provide a local standard.Clinical details were taken using a research proforma at the time of admission, and for children with CM through timed data capture points during ward admission. Retinal examination in CM was performed by one ophthalmologist with extensive previous experience of observing malarial retinopathy. Mydriatic eyedrops were used in all cases, and the retina was examined by both direct and indirect ophthalmoscopy.For the measurements of plasma VWF and VWF propeptide in hospital and emergency room patients and for the MBTS blood donors, venous blood samples were collected in citrated tubes (1∶9 vol/vol), and immediately placed at 4°C until spun. The 30 days post-admission samples were collected in lithium heparin. Samples were centrifuged and plasma was aliquoted and stored at −80°C until analysed batchwise.VWF and VWF propeptide concentrations were measured by enzyme-linked immunosorbent assay (ELISA) as previously described This study was approved by the ethics committees of the College of Medicine, University of Malawi and Liverpool School of Tropical Medicine.Data were analyzed using the GraphPad Prism v4.03 and SPSS (version 18) statistical packages. As all of the continuous variables had very positively skewed distributions, all analyses were conducted using distribution-free statistical tests; results are summarised using medians (ranges) unless otherwise stated. Plasma concentrations of VWF and propeptide were compared across the study sub-groups using the Kruskal-Wallis test with Bonferroni correction for post-hoc multiple pairwise testing. The Friedman test was used to compare median plasma concentrations of VWF and propeptide in children during and after treatment. Comparisons of groups based on malaria retinopathy, illness outcome, HIV status and gender were made using the Mann-Whitney U-test. Correlations between variables were estimated using the Spearman correlation coefficient. Statistical significance was set at the conventional 5% level for all analyses.falciparum malaria are summarized in A total of 100 children with CM were recruited, and 99 underwent ophthalmology examination, with 79 (79.8%) having retinopathy. Of these, 13 children died, and nine were discharged with clinically significant neurological deficits. The remaining children responded to antimalarial drug therapy and made a full recovery. The clinical and laboratory findings in different subsets of children with and without The median plasma concentrations of VWF and propeptide of the NFI controls were 37.1 (9.3–62.1) nM and 3.4 (1.0–30.6) nM, respectively . These vMedian plasma concentrations of both VWF and propeptide at admission were significantly higher in children with both cerebral and mild malaria when compared with the non-malarial control groups (p<0.001 both variables) - and were also significantly higher in children with cerebral malaria compared to those with mild malaria  was studied prospectively to relate plasma VWF and propeptide levels at the time of admission to plasma levels 48 hours and 30 days after the start of treatment. At 48 hours, median plasma VWF and propeptide levels had dropped significantly from 189.9 (101.0–378.4) to 112.9 (59.8–192.0) nM and 19.1 (11.6–31.0) to 12.6 (6.7–22.2) nM, respectively , median VWF and propeptide levels in this subset of children were significantly lower than at admission (p = 0.016 for both variables) or at 48 hours  , with meVWF and propeptide plasma concentrations were compared between cerebral malaria children with retinopathy and those without retinopathy. Median concentrations and ranges of the two proteins were similar in these two groups , died 176.5 (81.1–440.1); p = 0.873. Similarly, median propeptide levels did not differ significantly: survived 28.4 (5.3–107.5), died 26.6 (4.8–66.0); p = 1.000 .The median circulating platelet count differed significantly between the NMFI group and the cerebral and mild malaria groups  and 153.5 (48.5–247.4) nM respectively, and for propeptide were 30.2 (4.8–107.5) and 25.4 (6.4–44.5) nM (p = 0.059 and p = 0.137 respectively).Neither VWF nor propeptide concentration correlated with plasma lactate levels . There was no significant difference in median VWF or propeptide concentration between children with lactic acidosis (plasma lactate >5 nM) (VWF: 224.5 (63.4–378.4); propeptide: 26.4 (4.8–50.7) and those with plasma lactate <5 nM (VWF: 185.0 (40.5–484.3); propeptide: 28.4 (5.3–107.5) (p = 0.340 and p = 0.504 respectively). Median VWF and propeptide levels did not differ between male children ; propeptide: 26.6 (4.8–107.5)) and female children ; propeptide: 29.9 (11.6–77.4)) children (p = 0.418 and p = 0.168 respectively).et al., P. falciparum infection, and that VWF and propeptide levels are markers of endothelial activation in falciparum malaria, at higher levels than seen in other infections, suggesting a specific perturbation in malaria.We studied the patterns of increase in the plasma concentrations of VWF and propeptide in Malawian children with mild and severe malarial disease in order to assess the relationship of these biomarkers with disease severity. Our findings show that VWF and propeptide levels are markedly elevated in patients with retinopathy positive cerebral malaria but less so in mild malaria . In someThe recovery of cerebral malaria patients was associated with a significant decline in both VWF and propeptide levels towards normal values in healthy Malawian controls . Since lA novel aspect of this study is the assessment of plasma VWF and propeptide levels in cerebral malaria cases confirmed on the basis of malarial retinopathy. The presence of malarial retinopathy in histopathological studies of fatal paediatric cerebral malaria has been reported to have a positive predictive value of 95% and a specificity of 90% for the detection of histopathologically confirmed cerebral malaria in parasitaemic patients The observation that plasma VWF and propeptide levels were not significantly different between children who died and those who recovered  suggestsin vitro studies have demonstrated platelet-mediated IE adhesion to TNF-activated brain endothelium by acting as a bridge between the endothelium and IE in vitro), and present in early asymptomatic infection in humans (in vivo) Platelets were significantly lower in patients with cerebral malaria than in those with mild malaria. Platelet changes have long been known in malaria, thrombocytopenia being a usual feature of plasmodial infections. Several independent lines of evidence suggest that platelets play a role in the pathogenesis of cerebral malaria: (1) postmortem studies in African children with cerebral malaria have revealed that in some cases platelets co-localize with sequestered IE at brain microvascular sites, suggesting that platelets can play a role in the pathogenesis of cerebral malaria; (2) In summary, we have confirmed that plasma VWF and propeptide levels are markedly elevated in both cerebral and mild paediatric malaria, discriminate between these two conditions and that in cerebral malaria these normalize upon recovery. It is not possible to say whether VWF and VWF propeptide are more markedly elevated specifically in retinopathy positive cerebral malaria than other central nervous system (CNS) severe illnesses in the presence of malaria infection. Further work on the specificity and sensitivity of VWF and VWF propeptide is therefore required to ascertain their role as prognostic biomarkers for malaria disease severity.


Relative contribution of these infections on anemia in pregnancy is not certain. While measures to protect pregnant women against malaria have been scaling up, interventions against helminthes have received much less attention. In this study, we determine the relative impact of helminthes and malaria on maternal anemia.A prospective observational study was conducted in coastal Kenya among a cohort of pregnant women who were recruited at their first antenatal care (ANC) visit and tested for malaria, hookworm, and other parasitic infections and anemia at enrollment. All women enrolled in the study received presumptive treatment with sulfadoxine-pyrimethamine, iron and multi-vitamins and women diagnosed with helminthic infections were treated with albendazole. Women delivering a live, term birth, were also tested for maternal anemia, fetal anemia and presence of infection at delivery.P. falciparum microscopy-malaria infections. At delivery, 34% of women had moderate/severe anemia and 18% of infants' cord hemoglobin was consistent with fetal anemia. While none of the maternal infections were significantly associated with fetal anemia, moderate/severe maternal anemia was associated with fetal anemia.Of the 706 women studied, at the first ANC visit, 27% had moderate/severe anemia and 71% of women were anemic overall. The infections with highest prevalence were hookworm (24%), urogenital schistosomiasis (17%), trichuria (10%), and malaria (9%). In adjusted and unadjusted analyses, moderate/severe anemia at first ANC visit was associated with the higher intensities of hookworm and More than one quarter of women receiving standard ANC with IPTp for malaria had moderate/severe anemia in pregnancy and high rates of parasitic infection. Thus, addressing the role of co-infections, such as hookworm, as well as under-nutrition, and their contribution to anemia is needed. International guidelines recommend routine prevention and treatments which are safe and effective during pregnancy to reduce hookworm, malaria and other infections among pregnant women living in geographic areas where these infections are prevalent. Despite their effectiveness, programs to address common infections such as hookworm, schistosomiasis and malaria during pregnancy have not been widely adopted. Hookworm, malaria and other infections have been associated with anemia in children, but the studies on the impact of these infections on anemia in pregnancy have not been as clear. This study was undertaken to evaluate the prevalence of parasitic infections among women attending antenatal care which provided the nationally recommended malaria preventive treatment program in coastal Kenya. At the first ANC visit, more than 70% of women were anemic, nearly one-fourth had hookworm and about 10% had malaria. Women with high levels of hookworm or malaria infections were at risk of anemia. Anemia affects nearly 25% of all pregnancies worldwide and more than 40% of those in Sub-Saharan Africa Schistosomiasis haematobium), and almost 43% with malaria , while more than 46% of women were co-infected Although anemia in pregnancy is multi-factorial, poor nutrition and infection are common causes. In Sub-Saharan Africa, soil-transmitted helminthes (STH) including hookworm, urogenital schistosomiasis, and other parasitic infections such as malaria contribute to the high anemia rates in women and young children P. falciparum malaria increases risk for moderate and severe maternal anemia Parasitic infections, including hookworm, may be evaluated by intensity of infection, as measured by the concentration of eggs in the stool Many studies have focused on the effects of a single infectious agent on pregnancy outcome and maternal anemia, although a few studies have attempted to understand the relative effects of multiple agents with conflicting results With preventative treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) as recommended by the World Health Organization (WHO) in 2004 We conducted a secondary analyses of a large, prospective cohort study on fetal immunity conducted in coastal Kenya from 2006–2009. During that period trained study nurses recruited pregnant women between 20 and 32 weeks gestation who were residents of the area for the study at their first antenatal care (ANC) visit at Mswambweni District Hospital, Mswambweni, in Coast Province Kenya. Women who delivered live, term births at the study hospital had follow-up exam completed. At the first ANC visit, blood, stool and urine samples were collected, in addition to maternal anthropometrics, and basic demographic information. All women diagnosed with helminthic infections were treated with albendazole. Women were not treated for urogenital schistosomiasis during pregnancy, but treatment  was delayed until after delivery, per standard care in Kenya during the study period. All women enrolled in the study received IPTp-SP, one month of iron tables (60 mg/day) and folate (400 ug/day), and multi-vitamins for the duration of their pregnancy per Kenyan national guidelines.All women provided written informed consent prior to study enrollment. Institutional review board approval was received by Case Western Reserve University, Kenya Medical Research Institute, and the University of North Carolina at Chapel Hill.P. falciparum malaria was determined by microscopy using the standard Giemsa stain (thick and thin slices) on site and post-study by PCR/Ligase Detection Reaction Fluorescent Microsphere Assay, as previously described Blood was drawn at ANC visits to make both thick and thin blood smears at antenatal care and at delivery for maternal peripheral, cord and placental samples. The remaining blood was processed and stored at the laboratory, under temperature controlled conditions, for subsequent PCR analyses. Ascaris lumbricoides, Trichuris trichuria, Strongyloides stercoralis). Approximately one gram of fresh stool specimen was processed and examined by Richtie's concentration method S. haematobium) and results expressed as number of eggs/mL. Schistosomiasis was also categorized as light (0–<50 eggs/mL) or moderate (≥50 eggs/mL), according to WHO criteria Maternal stool and urine samples were collected at the first ANC visit and at delivery. Study participants brought a morning stool sample to the respective visit and these stool specimens were brought to the laboratory immediately following the collection by the health worker. Stool samples were tested for hookworm infection and other STH (Hemoglobin (Hb) levels were measured at the first ANC visit and at delivery by Coulter counter (Beckman Coulter Inc.). Women were classified as anemic (Hb<11 g/dL) and then categorized as being moderately to severely anemic (Hb<9 g/dL), as the primary outcome, and being mildly to non-anemic (Hb≥9 g/dL) according to the WHO classification of anemia 2. Since pre-pregnancy BMI was unavailable, to assess BMI, low BMI was defined as the lowest 10th percentile for the gestational age at measurement. The overall BMI ranged from 19.5 to 31.4 kg/m2 and the 10th percentile cut-off for GA at measurement ranged from 19.8 to 20.7 kg/m2. Trained study nurses interviewed the women at antenatal care visit to obtain key socio-demographic and basic medical history.Maternal height and weight were taken at the first ANC visit  and body mass index (BMI) calculated as kg/min utero malaria infection on neurodevelopmental outcomes (NCT00314899). Analyses were performed in SAS version 9.3 . Descriptive analyses were performed. Parity, gestational age, maternal age, maternal education, and socio-economic status (as measured by monthly household expenditures) were evaluated as potential confounders, based on previous research a priori cut-off (p<0.15) was defined for variable to be considered significant and retained in the final regression model. Analyses were restricted to those with complete case information.The study was a secondary analysis of data collected as part of a larger study to evaluate the association of Of the 813 women screened at ANC, 706 (88%) consented women had blood and urine samples available for anemia, malaria, and schistosomiasis evaluation, respectively. Of these participants, 544 (71%) provided stool samples at antenatal care for measurement of STHs and 394 had outcomes at delivery available.At enrollment at first ANC, the mean gestational age was 24.5 weeks (SD 3.8 weeks). 516 (71%) were anemic (Hb<11 g/dL) and 190 (27%) had moderate to severe anemia (Hb<9 g/dL). For subsequent analyses, moderate/severe anemia was evaluated as the primary outcome of interest. About 19% of the women were <20 years of age, nearly 86% were married, 21% had no formal education, and about 23% were primagravidas . In unadP. falciparum), and urogenital schistosomiasis (S. haematobium) are summarized in P. falciparum PCR-positive , hookworm , and urogenital schistosomiasis infection  were higher among those <20 years compared to women ≥20 years. Risks for infection did not differ significantly by maternal education levels. Primigravidity was associated with increased risk of P. falciparum PCR-malaria  and urogenital schistosomiasis , but not hookworm infection. About one-fourth (25.7%) of the women reported no use of insecticide-treated bednets (ITNs) prior to enrollment, which was associated with increased risk of malaria infection .The association of demographic characteristics and the prevalence at first ANC of hookworm infection, PCR-positive malaria , S. haematobium (17.1%), and T. trichuria (10.1%) were the most common infections at the first ANC visit. Of women positive for one of these infections, approximately 10% were co-infected, 4% with urogenital schistosomiasis and hookworm, and 2% with either malaria and hookworm or malaria and schistosomiasis, and the remaining with another combination (data not shown). Hookworm intensity ranged from 1 to 1035 eggs/g; thus all were considered ‘light’ according to the WHO criteria (light defined as <1999 eggs/g). To further evaluate whether relative intensity of infection was associated with outcomes, we also classified the highest intensity of infection (≥100 eggs/g) among the cohort as ‘moderate’ infection.Hookworm (23.7%), P. falciparum PCR-positive and microscopy positive . S. haematobium and T. trichuria, although common, were not significantly associated with moderate/severe anemia and few had moderate burden of infection. S. stercoralis and A. lumbricodes were observed in about 1% of women, using a single sample, which may be an under-representation since single-sample fecal assays have low sensitivity, especially for detecting S. stercoralisWe next examined the risk for moderate/severe maternal anemia at ANC associated with these infections, in unadjusted and adjusted analyses . In analFor those women who delivered live, term births at the study hospital, we evaluated the association between infection at the first ANC visit with maternal and fetal anemia at delivery, as well as the association of infections detected at delivery for those women and their fetuses who had stool (n = 210), or urine and blood samples (n = 394) available at delivery. Women whose births were included were comparable whose births were excluded , on socio-demographics, maternal characteristics and infection at ANC  (data not shown).P. falciparum malaria (PCR and microscopy) and schistosomiasis at delivery, none of these infections were significantly associated with maternal or fetal anemia at delivery; however, there were insufficient numbers of high-burden hookworm at delivery to test for this association. Furthermore, hookworm infection at ANC was associated with risk of hookworm infection at delivery and P. falciparum malaria infection was associated with risk of malaria infection at delivery (p<0.0001 for both).At delivery, 34.2% of the women had moderate/severe anemia and 18.4% of the neonates had fetal anemia (cord Hb<12.5 g/dL). Moderate hookworm burden at the first ANC visit was associated with moderate/severe maternal anemia at delivery , but other infections at first ANC visit were not significantly associated with risk of moderate/severe maternal anemia at delivery . Fetal aIn exploratory analyses, we also examined the association of the anemia at ANC with maternal and fetal anemia at delivery among 210 women and their newborns with both measures available. Women with moderate/severe anemia at first ANC visit had increased risk of maternal anemia at delivery . Fetal anemia was also associated with moderate/severe maternal anemia at first ANC visit and moderate/severe maternal anemia at delivery  (data not shown).P. falciparum microscopy-positive malaria infection  remained significantly associated with moderate/severe maternal anemia at ANC, when adjusting for primigravid status and low maternal BMI  and rnal BMI .P. falciparum PCR-malaria had fallen to 9% from previous rates of 40% at delivery reported in a similar region in Kenya, prior to widespread IPTp-SP and ITN's P. falciparum malaria as diagnosed by microscopy and moderate hookworm infections at ANC were associated with moderate/severe anemia at the ANC visit, while urogenital schistosomiasis and trichurisis and light infections were not. No association between PCR-detected malaria infections and anemia was found, probably because malaria detected by PCR included many low-level parasitemias. Thus, our findings are consistent with previous studies showing an association with anemia among populations with higher rather than low-intensity parasitic infection Few studies have examined the burden of helminthic infection and under-nutrition in pregnancy on maternal and fetal anemia in malaria-endemic regions. This is now especially important in geographic areas with a declining incidence of malaria found with widespread use of insecticide-treated bednets and IPTp-SP Socio-demographic factors assessed including age, gravidity, education, socio-economic and marital status, and low BMI were not significantly associated with moderate/severe maternal anemia in this cohort. However, the study was conducted among a relatively homogenous community, and thus these disparities may not have been large enough to be detectable. One limitation was that pre-pregnancy BMI and additional measures of under-nutrition were not available for this cohort and thus a more sophisticated assessment of the relationship of nutritional intake and anemia was not possible. Our findings are also consistent with research suggesting that in the context of low socio-demographic status, even light infections such as hookworm and malaria may be associated with anemia In low-resource areas where hookworm, malaria, and other parasitic infections, in addition to poor nutritional intake, are common, maternal anemia is prevalent and adversely affects the health of both women and their children. In this study, maternal anemia was associated with increased risk of fetal anemia. While we found no significant association of fetal anemia with maternal infection, these results should be interpreted with caution given the restriction to live, term births and the relatively small sample size, reducing our ability to detect differences. While fetal anemia has been less well studied, emerging research suggests that it may also be common in areas with high-burden of infection Effective, safe treatments are available to prevent and treat hookworm and malaria, both of which were associated with maternal anemia in this study. While numerous studies have evaluated preventative treatment for malaria in pregnancy, fewer have assessed antihelminthic treatment in the context of malaria treatment. Of those that have assessed hookworm, the results suggested that benefit may be most pronounced among women with higher burden of hookworm infection In contrast to the Uganda study, in our study, while all women in this study received antenatal care including IPTp-SP for malaria, treatment for hookworm as indicated, and iron/folic acid, most were not enrolled until after 20 weeks gestation. Thus, even with relatively good antenatal care, treatment was not initiated until the second trimester at which time anemia was prevalent in this cohort. Furthermore, unlike the region where this study was conducted and despite the international recommendations, uptake of treatment for hookworm, malaria, and schistosomiasis in ANC is still low in many parts of Africa Checklist S1STROBE checklist.(DOC)Click here for additional data file.


The aim of this study was to determine the incidence and seasonal pattern of malaria in children in South-West Burkina Faso, and to compare, in a randomized trial, characteristics of cases detected by active and passive surveillance. This study also enabled the planning of a malaria vaccine trial.Households with young children, located within 5 kilometers of a health facility, were randomized to one of two malaria surveillance methods. In the first group, children were monitored actively. Each child was visited twice weekly; tympanic temperature was measured, and if the child had a fever or history of fever, a malaria rapid diagnostic test was performed and a blood smear collected. In the second group, children were monitored passively. The child’s parent or caregiver was asked to bring the child to the nearest clinic if he was unwell. Follow up lasted 13 months from September 2009.Incidence of malaria  was 1.18 episodes/child/year in the active cohort and 0.89 in the passive cohort . Malaria cases in the passive cohort were more likely to have high grade fever; but parasite densities were similar in the two groups. Incidence was highly seasonal; when a specific case definition was used, about 60% of cases occurred within the 4 months June-September.Passive case detection required at least a 30%–40% increase in the sample size for vaccine trials, compared to active detection, to achieve the same power. However we did not find any evidence that parasite densities were higher with passive than with active detection. The incidence of malaria is highly seasonal and meets the WHO criteria for Seasonal Malaria Chemoprevention (SMC). At least half of the malaria cases in these children could potentially be prevented if SMC was effectively deployed. Malaria remains a major public health problem in Burkina Faso. Malaria control policies include free distribution of insecticide treated bednets (ITNs), Intermittent Preventive Treatment (IPT) for pregnant women and treatment of malaria cases with artemisinin-based combination therapy (ACT). Seasonal Malaria Chemoprevention (SMC) was adopted as policy in 2012 and a national plan for introducing SMC has been prepared but has yet to be implemented. Coverage of ITNs and access to treatment with ACTs is relatively low Malaria surveillance relies essentially on two methods. Passive cases detection, whereby malaria cases are detected only at the clinic or by a village health worker when the child is brought for treatment, is generally now preferred over active case detection in many malaria intervention studies Factors affecting the proportion of malaria morbidity detected passively include distance from health centre and treatment-seeking behavior et al., 1989, in The Gambia et al. (2010) in Kenya et al. (2013) As the trial has to be substantially larger if passive rather than active detection is used, it is surprising that few studies have compared active and passive detection directly. Snow The present study was conducted in the Cascades region in south-western Burkina Faso within an area selected for a phase 2b efficacy trial of the GMZ2 malaria vaccine candidate. The aim was to determine the incidence of malaria in this area, and to compare, in a randomized trial, characteristics of cases detected by active and passive surveillance; finally, the study aimed also to determine the burden and seasonal pattern of malaria disease in children.Findings served to plan for sample size estimates and surveillance methods for the main GMZ2 malaria vaccine candidate trial.2. The climate is characterized by a rainy season from May to November and dry season from December to March . Malaria is the main cause of morbidity. Malaria transmission is stable throughout the year, but peaks during the rainy season (May through November). Plasmodium falciparum accounts for some 90% of malaria cases. The study area is served by two health facilities staffed by a minimum of three nurses each. Each health facility includes consultation room for sick visits, maternity ward and a pharmacy. These health facilities report to the Banfora health district hospital that is 5 km away.The study was conducted in Banfora Health District in the Cascades region, the south-western administrative area of Burkina Faso. The Cascades regional population in 2008 was estimated at 558,893 inhabitants of which 272,807 were residents in Banfora district It was estimated that about 100 households minimum were required assuming an average of 7 children per household. This would give over 90% power to detect a difference in incidence if the incidence in the passive arm was less by 20% or more than the active arm; and if the incidence rate in the active arm was 1 episode per child per year assuming a coefficient of variation of 0.3 between households.In August 2009, prior to the start of the study, meetings were held in the villages in the study area to explain, in the local languages the objectives of the study and the procedures involved and to answer questions from the residents. In September 2009, eligible households  were randomly allocated to active case detection (ACD) or passive case detection (PCD) surveillance arms. Among the 271 household in the study area, 230 were eligible and therefore invited to participate to the random allocation process. This was done in a public lottery ceremony where heads of households were asked to participate to a traditional stick-drawing game. Sticks were white and red colors ended representing respectively the PCD and the ACD. The colored ends of the sticks were fixed in a box filled with sand so that they could not be seen by the participants. When a stick was drawn, the children in the household were assigned to the corresponding cohort. Neighboring households could be assigned to different surveillance methods . ChildreEach eligible child underwent a brief clinical examination by a physician, medical history was taken, and tympanic temperature measured. A finger-prick blood sample was obtained for measurement of haemoglobin using a hemocue®, , and for making malaria smears. A malaria rapid diagnosis test  was performed for children with fever (axillary Temperature ≥38.0°C) and/or history of fever within the last 24 hours. Artemether-Lumefantrine (AL) was given to those with positive test as per national malaria treatment guidelines. If severe malaria was suspected the child was referred to the nearest health facility.Follow-up of study participants took place from September 2009 until September 2010, a 13 months period. ID cards were provided to caregivers to facilitate recognition of the study participants by project staff. In the PCD cohort, parents/guardians of children were encouraged to take their child to the nearest health centre any time the child became unwell. Trained nurses were based at both health facilities in the study area to identify children in the trial and to ensure that they were seen, properly investigated in a standardized manner and treated appropriately. Local key informants in each village were requested to inform the study team when a child moved out of the study area or if a child died.Parents/guardians of children in the ACD cohort were also encouraged to take their child to the clinic at any time they were sick. In addition, children in the ACD cohort were visited by trained field workers  twice each week. At each visit the field worker administered a standardized questionnaire to the parents/guardians of the child to collect information about any illness since the last visit, symptoms experienced, use of healthcare facilities and any medications.At each home visit in the ACD cohort, and whenever a child from either cohort attended clinic, tympanic temperature was measured using a digital thermometer. If the child had a fever (tympanic temperature ≥38.0°C), or history of recent fever (within the last 24 hours), a RDT  for diagnosis of malaria was used to guide treatment and a thick blood smear was obtained for malaria microscopy. Children with a positive RDT were treated with AL for malaria based on National Malaria Control Program guidelines.Thick and thin blood films were prepared, air-dried, giemsa-stained, and examined using a light microscope fitted with 100X oil immersion lens. The number of parasites and leucocytes were counted to reach 200 leucocytes for positive slides. Slides were declared negative only after 100 high power fields had been read. Each slide was read twice by two independent microscopists. A third reading was performed in case of discrepancy between the two. Discrepancy of reading was defined as following: The ratio of densities from the first two readings is >1.5 or <0.67; or <30 parasites were counted with an absolute difference in the number of parasites >10; or discordance in positive-negative or species. The final result was based on the two most concordant readings. The parasitaemia was estimated on the basis of an assumed white cell count of 8,000/µL.Microscopists competency was evaluated through an external quality control (EQC) program carried out by the College of American Pathology proficiency testing. It includes a set of 20 slides provided to each microscopist for reading thrice a year. Only those with a score of at least 80%, graded as ‘competent’, were involved in the reading of study participants’ slides.In adherence to the Council for International Organizations of Medical Sciences (CIOMS) guidelines, community permission was obtained before the start of the study. Written informed consent was obtained from parents/guardians of children who participated in the study. The study was approved by the National Ethical Committee of Burkina Faso (Deliberation N° 2009-36).Data were reviewed for completeness and consistency then double entered into a Microsoft Access® database. The validated database was transferred to Stata 12.1  for analysis. The primary endpoint was malaria defined as the presence of fever (tympanic temperature ≥38°C) with a parasite density of 5,000/µL or more. Seven other case definitions were also considered see .The incidence rate of malaria was estimated for each case definition, including all malaria episodes that met the definition. All children were included in the denominator without deducting any time at risk after treatment. In the PCD cohort, observations were censored if the child was known to have left the study area or died, in the ACD cohort observations were censored if the child was lost to follow-up or died. In the primary analysis, any episodes occurring within 14 days of a primary episode which met the same case definition, were assumed to be relapses and did not contribute towards incidence. All children who were enrolled and followed up were included in the analyses. Jackknife confidence intervals using the household as the cluster were generated for incidence rates to allow for the lack of independence among multiple events in the same household. The Kaplan-Meier failure function was used to estimate the cumulative risk  by the end of surveillance period, and the Nelson-Aalen cumulative hazard was used to estimate the mean number of episodes per child.Cox regression was used to compare incidence rates in the ACD and PCD cohorts, using the household as the unit of randomization with a robust standard error to allow for the lack of independence among multiple events in the same household. The time scale used for the Cox regression was age. A Poisson regression with robust standard errors was used to examine the effect of surveillance method on non-zero parasite densities. A logistic random effects model that accounted for household-level effects was used to examine the effect of surveillance method on the proportion of cases with non-zero parasite density that exceeded 39.0°C and the proportion with parasite density of more than 20,000/µL.An examination of the effect of season used calendar time categorized into six month bands of December to May and June to November, and interaction between surveillance method and season was examined.th September 2009, 698 children from 230 households were enrolled; 359 from 114 households were assigned to the PCD cohort and 339 from 116 households to the ACD cohort. Subsequently a further six children were enrolled in the PCD cohort upon attendance at a clinic and a further 72 children were enrolled in the ACD cohort after being identified at a household visit, from the randomized households  of follow-up time. Total follow-up was 421 person-years in the ACD cohort and 382 person-years in the PCD cohort.In total ten children were admitted to hospital during the study period. Among them, three were from the ACD cohort and seven from the PCD cohorts. For children in the ACD cohort, the main cause was severe malaria (two cases of severe anemia and 1 case of prostration). In the PCD cohort, causes for admission were severe gastroenteritis (1 case), severe malaria (2 cases with convulsions and 1 case of respiratory distress), moderate anemia (1 case) and respiratory infection (1 case).Six children died in the course of the study. In the ACD cohort, three boys aged one, one boy aged two and one girl aged four died; among them three occurred during the low transmission season (April) and two during the malaria high transmission season (in June and August). One girl aged one year from the PCD cohort died during the malaria transmission season. Cause of deaths is not known .The 411 children in the ACD cohort contributed 40,490 visits, median 101 per child (IQR 99–102). Almost all of these children  attended the health clinic, with a median of 5 clinic visits (IQR 2–7) per child for a total of 1952 clinic visits. Of the 365 children in the PCD cohort, 329 (90%) contributed to 1286 clinic visits for a median of 3 visits (IQR 2–5) per child.Malaria with fever and parasitaemia 5,000/µL or more: In the ACD cohort the number of episodes per child ranged from 0 to 5, with a mean of 1.18. In the PCD cohort, the mean number of episodes per child was 0.89 (range: 0 to 6). Timing of cases is shown in Other case definitions of clinical malaria episodes: A greater number of episodes of malaria were observed by active case detection compared to passive case detection for all case definitions  that fell in the four months June to September 2010 ranged from 39% to 64% depending on the case definition used and the type of surveillance; the higher figure corresponding to the more specific malaria case definitions . For theThe proportion of clinical malaria cases presenting with a high grade fever (≥39.0°C) was higher in the PCD cohort (26%) than the ACD cohort (19%) , this efParasite density varied by season . Mean paROC curves were produced showing sensitivity and specificity of parasite density cut-offs for identifying malaria cases (parasitaemia with tympanic temperature ≥38.0°C or parasiteamia with tympanic temperature ≥38.0°C/History of fever within the last 24 hours), using data collected at the baseline survey  and 8. ISpecificity of Optimal-IT® is summarized in Malaria remains the main cause of severe illness and death among children in Burkina Faso. Our study confirms that incidence remains very high despite recent efforts to scale-up the use of LLINs and access to ACTs, with an average incidence of over 2 episodes of malaria confirmed by RDT per child per year. Incidence was high even among children reported to be using LLINs. Coverage of LLINs was only 50% in the children in this study, we did not measure coverage in other age groups in the population, but it is likely to be lower. The overall effect of LLINs may be greater if higher levels of community coverage can be achieved. Resistance to pyrethroids, known to be common in southern Burkina Faso, has become widespread in the country in recent years Malaria morbidity showed marked seasonal variation, with higher incidence during the rainy months of June-November. The degree of seasonality was underestimated when less specific case definitions were used; using a specific case definition , 62% of annual cases occurred in 4 months of the year. The WHO now recommends that children living in areas of the Sahel sub region with highly seasonal malaria, defined as 60% of cases falling in 4 months of the year benefit from seasonal malaria chemoprevention (SMC) This study was conducted in the context of field site characterization to prepare for a phase IIb efficacy trial of the GMZ2 malaria vaccine candidate Incidence of malaria was substantially lower using passive detection through clinics, even though all households had good access to clinic. For our primary endpoint of documented fever with parasitaemia of 5,000/µL or above, the rate ratio ACD:PCD during the main transmission period was 1.39; so PCD would require a 39% increase in the sample size for a vaccine efficacy trial, compared to ACD.et al. also compared active and passive surveillance in a randomized study where randomization was done by village, but they did not consider differences in clinical characteristics of the cases.Strength of our study is that it was randomized by household, and sample size was relatively large (230 households enrolled while minimum sample size required was 100 households). The location of participants could be a confounder for comparison between surveillance methods given the known heterogeneity in malaria exposure P. falciparum infection is common. The cut-off of parasite density that gives same level of sensitivity and specificity is comparable to what was reported in other part of Burkina Faso Occurrence of clinical malaria is the endpoint most commonly used in the field to measure not only the efficacy of interventions to prevent malaria but also to assess the public health burden of the disease. However it is not always straightforward to clearly define what constitutes an episode of clinical malaria especially in areas of high malaria endemicity where asymptomatic carriage of The specificity of testing by Optimal RDT in our study was lower during the malaria high transmission season. It is possible that in our context with malaria high endemicity, a high proportion of individuals are carrying low parasites density not detected by microscopy despite the experience of microscopists and the quality control using double reading of each individual blood smear.It is likely to be many years before a highly effective malaria vaccine is available. Our study shows that malaria burden in young children remains unacceptably high in South West Burkina Faso despite recent efforts to improve access to insecticide treated bednets and effective treatment with ACTs. Alternative measures are urgently needed. We have shown that malaria incidence is highly seasonal in South West Burkina Faso making this area suitable for SMC. Over 60% of malaria cases each year could potentially be prevented if SMC was provided over 4 months of the year from July to October.File S1Monthly incidence rates of malaria by calendar month, September 2009 through to September 2010. Monthly incidence rates are presented for all eight case definitions of malaria. A breakdown is provided for children in the passive and active case detection cohorts as well as incidence rates for all children under follow-up. Rates are presented in both tabular and graphical format. In addition, the proportion of cases  falling in the four months of June to September 2010 is provided.(XLSX)Click here for additional data file.File S2Incidence rates of malaria by yearly age groups, September 2009 through to September 2010. Yearly incidence rates are presented for all eight case definitions of malaria. A breakdown is provided for children in the passive and active case detection cohorts as well as incidence rates for all children under follow-up. Rates are presented in both tabular formats.(XLSX)Click here for additional data file.


Plasmodium knowlesi is a common cause of human malaria in Malaysian Borneo and threatens the prospect of malaria elimination. However, little is known about the emergence of P. knowlesi, particularly in Sabah. We reviewed Sabah Department of Health records to investigate the trend of each malaria species over time.The simian parasite P. malariae and P. knowlesi were considered as a single group due to microscopic near-identity.Reporting of microscopy-diagnosed malaria cases in Sabah is mandatory. We reviewed all available Department of Health malaria notification records from 1992–2011. Notifications of P. falciparum peaking at 33,153 in 1994 and decreasing 55-fold to 605 in 2011, and P. vivax peaking at 15,857 in 1995 and decreasing 25-fold to 628 in 2011. Notifications of P. malariae/P. knowlesi also demonstrated a peak in the mid-1990s (614 in 1994) before decreasing to ≈100/year in the late 1990s/early 2000s. However, P. malariae/P. knowlesi notifications increased >10-fold between 2004 (n = 59) and 2011 (n = 703). In 1992 P. falciparum, P. vivax and P. malariae/P. knowlesi monoinfections accounted for 70%, 24% and 1% respectively of malaria notifications, compared to 30%, 31% and 35% in 2011. The increase in P. malariae/P. knowlesi notifications occurred state-wide, appearing to have begun in the southwest and progressed north-easterly.From 1992–2011 total malaria notifications decreased dramatically, with P. knowlesi notifications following reduced transmission of the human Plasmodium species, and this trend threatens malaria elimination. Determination of transmission dynamics and risk factors for knowlesi malaria is required to guide measures to control this rising incidence.A significant recent increase has occurred in  Plasmodium knowlesi is a common cause of malaria in Malaysian Borneo; however, little is known about its emergence over time, particularly in Sabah. We reviewed all available Sabah Department of health malaria notification records from 1992–2011, and considered notifications of P. malariae and P. knowlesi as a single group due to their microscopic similarity. We found that malaria notifications in Sabah have decreased dramatically, with P. falciparum and P. vivax notifications peaking at 33,153 and 15,877 respectively during 1994–1995, and falling to 605 and 628 respectively in 2011. Notifications of P. malariae/P. knowlesi fell from a peak of 614 in 1994 to ≈100/year in the late 1990s/early 2000s, however increased >10-fold between 2004 (n = 59) and 2011 (n = 703). In 1992 P. falciparum, P. vivax and P. malariae/P. knowlesi monoinfections accounted for 70%, 24% and 1% respectively of malaria notifications, compared to 30%, 31% and 35% in 2011. The increase in P. malariae/P. knowlesi notifications occurred state-wide, appearing to have begun in the southwest and progressed north-easterly. This significant recent increase in P. knowlesi notifications following reduced transmission of the human Plasmodium species threatens malaria elimination; further research is required to determine transmission dynamics and risk factors for knowlesi malaria.The simian parasite  Plasmodium knowlesi, transmitted by the forest-dwelling Anopheles leucosphyrus group of mosquitoes, is now a common cause of human malaria in the eastern states of Sabah and Sarawak, and presents an increasing threat to malaria elimination Malaria elimination is now a goal of many countries in Southeast Asia and the Western Pacific, and large reductions in malaria prevalence have been achieved P. knowlesi from P. malariae by microscopy. Although the first naturally acquired case of human knowlesi malaria was reported from Peninsular Malaysia in 1965 P. knowlesi is now the most common cause of human malaria in several districts throughout Sabah and Sarawak P. knowlesiDocumentation of the emergence of this species over time is limited by the inability to distinguish P. knowlesi existed in macaques in Southeast Asia more than 100,000 years ago, with infection in humans likely occurring from the time of human arrival in the region P. malariae by microscopy P. malariae in Sarawak when PCR methods are used P. knowlesi. When PCR was performed on the earliest P. malariae slides available, taken in 1996, 35/36 (97%) were positive for P. knowlesi, with only one being positive for P. malariaeP. malariae” accounted for 9% of all malaria notifications in Sarawak, and 20% of cases in the Kapit district Whether this apparent increase in cases however is due to a true emergence of the species or increasing recognition remains uncertain. Evolutionary analyses of sequence data from samples obtained from Sarawak indicate that P. knowlesi infection in humans may have increased only recently. In 2001, only 96/6050 (1.6%) malaria slides referred to the Sabah State Public Health Laboratory were diagnosed as P. malariae monoinfection by microscopy, with the proportion increasing to 59/2741 (2.2%) in 2004 P. malariae” accounted for 621/1872 (33%) of malaria cases reported to the Sabah Department of Health in 2011 .In Sabah, limited available evidence suggests that the situation may differ from that of Sarawak, and that P. knowlesi represents an emerging infection in humans.In this study, we reviewed the Sabah Department of Health records of malaria notifications from 1992–2011, in order to investigate the trend of each malaria species over time, and in particular to determine if The study was approved by the Medical Research Sub-Committee of the Malaysian Ministry of Health and the Menzies School of Health Research, Australia. All data analysed were anonymised.2 and a population of 3.2 million The north-eastern Malaysian state of Sabah has an area of 73,600 kmMalaysia has a long history of malaria control programs dating back to the early 1900s, with an initial focus on environmental management techniques. The launch of the Malaria Eradication Program in 1967, followed by state-wide malaria control programs during the 1970s and 1980s, led to large reductions in malaria prevalence, with cases falling from 240,000 in 1961 to around 50,000/year during the 1980s An. balabacensis and An. donaldiP. knowlesi hosts, the long-tailed and pig-tailed macaques, are found throughout the state.Mosquito vectors in Sabah include P. malariae/P. knowlesi are mostly reported, and hence notified, as P. malariae.In Sabah mandatory reporting of all malaria cases to the Sabah State Health Department is generally done by nursing staff, with species normally reported according to microscopy results. Blood slides with parasites resembling Plasmodium species annually for each district in Sabah from 1992–2011, in addition to the age and sex distribution and seasonal variation of each species from 2007–2011.We reviewed all available malaria notification records held by the Sabah State Health Department. Hard copy summaries of annual malaria notifications by species and by district were available from 1992. From 2007 yearly Excel databases were also available that included limited demographic/epidemiological information for each malaria notification. We therefore recorded the number of notifications of each Plasmodium species. Median ages were compared using Wilcoxon rank-sum test, and proportions were assessed using the Chi-square test. Edwards' test was used to assess seasonality of the Plasmodium species.Data were analysed using Stata statistical software, version 10.0 . Spearman's correlation coefficient was used to analyse the association between annual notification rates of the P. malariae and P. knowlesi were considered as a single group , due to the inability to distinguish these species by microscopy. Mixed-species infections were recorded as a single group, with analysis of these cases limited to annual notification rates.Notifications of P. falciparum notifications peaking at 33,153 in 1994 and decreasing 55-fold to 605 in 2011, while P. vivax notifications peaked at 15,857 in 1995 and decreased 25-fold to 628 in 2011 , before decreasing to around 100/year in the late 1990s and early 2000s. Until 2003, annual notifications of P. malariae/P. knowlesi strongly correlated with those of P. falciparum  while P. malariae/P. knowlesi notifications remained stable from the late 1990s to 2006, and then increased markedly from 2007 , and with P. malariae/P. knowlesi notifications until around 2008 . Since 2008 P. vivax notifications decreased while P. malariae/P. knowlesi notifications increased, although this relationship was not statistically significant.Notifications of P. falciparum and P. vivax peaked at 16.0 and 7.36/1000 people/year respectively during 1994–1995, and decreased to 0.18 and 0.19/1000 people respectively in 2011  and 701/6980 (10%) of malaria notifications respectively, and Tenom from 1998–1999 and Tuaran in 1994 and 1999, where approximately 6% of malaria notifications were P. malariae/P. knowlesi.The 23 districts of Sabah  in generistricts . P. malaP. malariae/P. knowlesi  notifications of P. malariae/P. knowlesi have been fewer, although have been increasing since 2008.Since the early 2000s most districts have experienced an increase in notifications of knowlesi . This inP. malariae/P. knowlesi notifications , followed by Ranau , Keningau , Tenom  and Kota Marudu .In 2011 Kudat district accounted for the highest number of P. malariae/P. knowlesi (31 years) was significantly higher than that of patients with P. vivax or P. falciparum . Males with P. malariae/P. knowlesi demonstrated an approximately normal age distribution, with a mean, median and interquartile range of 33, 30 and 20–45 years respectively  of female cases were aged 15–50 years, with 28% occurring in children <15 years old and 24% occurring in adults >50 years. Among adults (≥15 years) with P. malariae/P. knowlesi, females were significantly older than males .Epidemiological characteristics of notifications according to species were assessed from 2007–2011, when relevant data were recorded for each notification. This time period included 16,011 malaria notifications, although species was not recorded for 373 (2.3%). The overall median age of patients with ectively . In cont50 years . While mP. vivax and P. falciparum the overall median age was lower among females than it was among males . As with P. malariae/P. knowlesi however, adult females with P. vivax were older than adult males .Among patients with P. malariae/P. knowlesi cases as a proportion of all malaria notifications increased from 43/244 (17.6%) in 2007 to 131/263 (49.8%) in 2011 (p<0.0001).The median age of all malaria patients increased progressively from a median of 24 years in 2007 to 27 years in 2011 . The proportion of patients >50 years old also increased, from 244/3191 (7.7%) in 2007, to 364/4135 (8.8%), 345/4009 (8.6%), 244/2644 (9.2%) and 263/2032 (12.9%) in the years 2008, 2009, 2010 and 2011 respectively (p<0.0001). Among patients >50 years old, P. malariae/P. knowlesi were male , and this proportion increased among those aged 15–60 years, of whom 82% were male, compared to 63% outside this age range (p<0.0001).A greater proportion of patients with Plasmodium species, with maximum notifications occurring in July, April and June for P. falciparum (p = 0.0001), P. vivax (p = 0.002) and P. malariae/P. knowlesi (p = 0.0001) respectively  may have been prevalent at low levels for decades, a significant increase in notifications has occurred over the past decade. This increase follows a dramatic reduction in notification rates of P. vivax and P. falciparum. In fact over the past decade, a strong inverse correlation has occurred between notification rates of “P. malariae/P. knowlesi” and P. falciparum.Although P. malariae/P. knowlesi” notifications during the last two decades is actually P. knowlesi, with PCR testing only instituted at the Sabah State Reference Laboratory in 2005 P. falciparum and P. vivax was high, it is possible that a significant number of P. malariae cases also occurred. However recent studies demonstrate that, at least since 2007, PCR-confirmed P. malariae in Sabah is rare. Although eight cases of P. malariae were detected by PCR from 49 “P. malariae” blood films taken from Sabah during 2003–2005 (with six of these from Kudat) P. malariae infections among 1286 patients with PCR-confirmed Plasmodium infection in Sabah from 2007 to 2011 P. malariae infections were identified from 475 patients with PCR-confirmed Plasmodium infections in Kudat from 2009–2011, including 365 with microscopy-diagnosed “P. malariae” P. malariae was detected by nested PCR in only two of 318 (0.6%) microscopy-diagnosed P. malariae cases referred to the Sabah State Public Health Laboratory in 2009 P. malariae/P. knowlesi” notifications since 2007 in the current study are very similar to those described in a previous study in Kudat, in which 345 patients with PCR-confirmed P. knowlesi were analysed P. knowlesi, this strongly suggests that a large majority of “P. malariae/P. knowlesi” notifications, at least since 2007, are indeed P. knowlesi cases.Available evidence does not allow us to determine what proportion of “P. knowlesi in this and previous studies P. knowlesi requires further investigation, but may possibly relate to lower forest exposure among young adult females; this may also account for the finding in this and other studies The reason for the older age group affected by P. knowlesi. Firstly, increased recognition of the species may account for increased reporting by microscopists. Although this possibility cannot be excluded, the previous high prevalence rates of malaria in Sabah ensured that microscopy skill levels were maintained at high levels. It seems unlikely therefore that large numbers of “P. malariae” slides would have been misdiagnosed as P. vivax or P. falciparum. In fact, in a study involving blood films obtained from 243 patients with PCR-confirmed P. knowlesi in Sarawak between 2001–2006, only 4.5% and 6.6% were misdiagnosed by microscopy as P. falciparum and P. vivax respectively P. knowlesi. The consistency of notification trends across districts further supports the overall reliability of the microscopy reports and the State Department records. Furthermore, the number and proportion of all malaria patients aged >50 years increased significantly between 2007 and 2011. Given that this age group is over-represented among patients with knowlesi malaria P. knowlesi cases and cannot be attributed to increased recognition.There are several possible explanations for the emergence of P. knowlesi in Sabah has increased, and that this has occurred as a result of environmental change together with reducing rates of the other human malaria species. The extensive deforestation that has occurred in Sabah has led to encroachment of humans into previously forested areas, resulting in increased interaction with mosquito vectors and simian hosts. Furthermore, the removal of habitat together with malaria control activities may have led to a change in vector behaviour, or a vector shift, as has been seen in the Kinabatangan region where the previously dominant malaria vector An. balabacensis appears to have been displaced by An. donaldiP. knowlesi, although further research regarding P. knowlesi vectors in Sabah is needed.We believe, therefore, that the prevalence of P.knowlesi appears to have increased very recently, long after Sabah's most extensive period of deforestation during the 1970s and early 1980s P. vivax and P. falciparum are likely to have contributed directly to this trend. Possible explanations for this may be derived from examining the relationship between P. falciparum and P. vivax, as in other regions prevalence of P. vivax has increased as rates of P. falciparum decrease P. vivax and P. falciparum have demonstrated lower than expected rates of mixed infections P. falciparum and P. vivax, a phenomenon also demonstrated in early syphilis studies in which P. falciparum was found to suppress P. vivax parasitemia when both species were inoculated simultaneously P. knowlesi and either P. falciparum or P. vivax may explain the malaria trends in Sabah, with density-dependent regulation possibly accounting for previously low rates of symptomatic P. knowlesi. The occurrence of density-dependent regulation may also explain the lack of earlier reports of severe “P. malariae”, similar to reports from other regions that cases of severe vivax malaria increased as the prevalence of P. falciparum reduced Finally, the finding in this study that the prevalence of P. falciparum, antibodies were found to bind to the surface of P.knowlesi merozoites, although erythrocyte invasion was not prevented P. vivax were less susceptible to infection with P. knowlesiP. falciparum or P. vivax may be particularly relevant given that P. knowlesi tends to effect older individuals; frequent exposure to P. falciparum and P. vivax may previously have protected this age group from infection with P. knowlesi.In addition, it is possible that cross-species immunity may play a role in the malaria prevalence patterns observed in Sabah. Although heterologous immunity does not generally occur between human malaria species, it has been argued that a degree of cross-resistance may be more likely to occur between species infecting different hosts P. knowlesi have increased following decreasing prevalence of the other malaria species has implications for malaria control in any country where P. knowlesi is known to occur, which includes nearly every country in Southeast Asia P. knowlesi is now the most common cause of malaria, and based on current trends, is likely to become increasingly dominant and may extend to previously unaffected districts. Furthermore, human-to-human transmission, if not already occurring, may become more likely as prevalence continues to increase. Close monitoring of P. knowlesi in Sabah and elsewhere is therefore essential, including accurate reporting of microscopy-diagnosed “P. malariae” as P. knowlesi, as has been previously recommended P. falciparum and P. vivax, malaria elimination will not be achieved unless control of knowlesi malaria is addressed.The finding that notification rates of