Massive implementation of malaria diagnostics in low-resource countries is regarded as a pivotal strategy in control and elimination efforts. Although malaria rapid diagnostic tests (RDTs) are considered a viable alternative, there are still obstacles to the widespread implementation of this strategy, such as reporting constraints and lack of proper quality assurance of RDT-based programmes at point-of-care (POC).TM, former codename “GenZero”), that assists users at POC to process RDTs. After informed consent, patients were enrolled into the study and blood samples were collected for thick blood smear (TBS) and RDT. The RDT results were interpreted by both visual inspection and Deki Reader device and concordance between visual and device interpretation was measured. Microscopy corrected by real-time polymerase chain reaction (PCR) and microscopy were “gold standard” tests to assess the diagnostic performance.A prospective cohort of patients, seeking routine care for febrile episodes at health centres in malaria-endemic areas of Colombia, was used to assess the diagnostic performance of a device based on smartphone technology  and 98.7% (95% CI 98.5-99.4) for P. falciparum, and 98.0% (95% CI 90.3-98.9) and 97.9% (95% CI 97.1-98.5) for P. vivax. Percentage concordance between visual and device interpretation of RDT was 98.5% and 99.0% for P. vivax and P. falciparum, respectively.The RDT, when compared to TBS, showed high sensitivity and specificity for P. falciparum in both visual and device interpretation, and good overall diagnostic performance for P. vivax. Comparison between PCR as gold standard and visual and device interpretation showed acceptable overall performance for both species.In total, 1,807 patients were enrolled at seven health centres in malaria-endemic areas of Colombia. Thirty-three The diagnostic performance of the Deki Reader was comparable to visual interpretation of RDTs (without significant differences) for both malaria species. Providing standardized automated interpretation of RDTs at POC in remote areas, in addition to almost real-time reporting of cases and enabling quality control would greatly benefit large-scale implementation of RDT-based malaria diagnostic programmes. Plasmodium parasites in blood circulation, is not only critical for appropriate case management [Malaria is transmitted in about 106 countries in tropical and subtropical regions where ~3.3 billion people, half of the world’s population, are at risk of malaria transmission. The World Health Organization (WHO) estimates that over 219 million cases (uncertainty range of 154 to 289 million) and about 660,000 malaria deaths  occur worldwide every year [Plasmodium species on thick blood smear (TBS) has been for decades the standard method for malaria diagnosis despite its field accuracy, errors in species identification and its operator-dependence. Although it is less expensive and easier to handle than other malaria diagnostic methods developed more recently, it is laborious and time consuming, and it requires electricity as well trained personnel, frequently not available in remote malaria-endemic areas. Recently, a number of techniques aimed at reducing the time of processing and some other constraints of TBS have been developed. Some of them include fluorescence stains but still require microscopes, others are based on detection of parasite nucleic acids in human blood circulation but require expensive and complex equipment, and others are based on the use of antibodies that recognize parasite components [Microscopic detection of mponents ,4. The mmponents . Attemptmponents .Malaria diagnostic methods based on the use of monoclonal antibodies recognizing parasite antigens ,4 have bThe reliability of RDT results is pivotal to ensure the safety of withholding anti-malarial treatment in test-negative patients -10. As rThe performance of health professionals using RDTs can be improved through simple redesign of job aids ,8,13,14,The present report describes a field trial in malaria-endemic areas of Colombia of a system that includes mobile, battery-operated devices based on smartphone technology that assists users at POC to process RDTs, performing automated interpretation, reducing notification time to a cloud-based database and allowing malaria case geo-location and remote QA activities. The main objective of the trial was to assess the concordance between automated interpretation of RDTs by the device and by visual inspection by laboratory experts.Plasmodium falciparum as predominant parasite species. Buenaventura is the main port of Colombia on the Pacific coast with a population of ~360,000 habitants, with similar prevalence of both P. falciparum and Plasmodium vivax infections. Monteria is the political capital of the department of Córdoba, with a population of ~300,000 inhabitants and Tierralta, a town located about 100 km south of Monteria, with a population of ~80,000, with predominance of P. vivax infections. Patients were mostly Afro-descendants of both genders, seeking routine care for febrile episodes and were enrolled into the study after signing an informed consent form. Before the conduct of the trial, the protocol was reviewed and approved by the institutional ethics committee .The present study involved a prospective cohort of consecutive patients from August 2011 to January 2012 at seven sites in four towns located in malaria-endemic areas of Colombia  and about three to four blood drops were used for preparation of TBS, RDTs, and a filter-paper blood spot for real-time polymerase chain reaction (PCR). The latter was performed as quality control of microscopy. Technicians working at the reference laboratory were blinded to results of both visual and device interpretation of RDTs performed at the site. Likewise, results of TBS microscopy examination and PCR (when applicable) were blinded to site technician who performed the RDTs. The processing of the RDT was performed according to manufacturer recommendations and guided by a job aid displayed on the screen of the Deki Reader device (former codename “GenZero”). The device also assisted technicians at the sites in keeping track of the incubation time for each RDT processed. Once the incubation period was completed, the study technician interpreted the RDT by visual inspection of the strip and captured the result using the touch-screen of the device. The study technician then immediately inserted the cassette into the device to perform the automated interpretation of the test. The results of the device’s interpretation of the RDT were concealed at all times, and were generated in all cases after the study site technician had entered results of her/his visual interpretation of RDT test results. All results of RDTs interpretation, along with a high-resolution image of the RDT captured by the device, and the patient data collected were encrypted and automatically transmitted to a central cloud database specifically designed for the purpose of the trial. Patient management was performed according to official protocols of the national malaria control programme , based oSymptomatic patients were bled by finger prick and TBS were prepared and stained using the standard 10% Giemsa method . Slides P. falciparum HRP2 protein and the other one specific for P. vivax pLDH enzyme. Interpretation of the results was based first on the presence of the control line, and then on the presence of one or two lines.The malaria RDT used in the current study was SD Bioline Malaria Antigen Pf/Pv , referred here to as RDT. This is a lateral flow immunochromatographic test that contains a membrane strip encased in a flat plastic cassette. The strip is pre-coated with two monoclonal antibodies: one specific for et al.[et al. [P. falciparum and P. vivax, the 20 μl reaction mix contained 2 μl DNA, 1× TaqMan Master Mix , 200 nM forward and reverse primer, 200 nM P. falciparum probe and 100 nM P. vivax probe. The PCR programme consisted of an initial step of 10 min at 95°C followed by 40 cycles of 10 sec at 95°C and finally 60 sec at 60°C. Each experiment included the test sample in duplicate, non-infected DNA as negative control and a positive control with a known parasitaemia for P. vivax and P. falciparum. Parasitaemia was calculated using an average standard curve of pre-determined parasitaemia. The detection limit calculated was one parasite/ml based on the upper limit of the calibration curve at the lowest detected concentration. In addition, PCR was performed in a randomly selected subset of samples, and used in an ad hoc analysis with PCR results as gold standard (n = 274).PCR was adapted from Rougemont et al., and per.[et al. . PCR wasThis is a mobile, ruggedized, battery-operated device Figure , manufacSeven laboratory microbiologists with five years of university education and former training on malaria microscopy were engaged to operate the Deki Reader and to fill up all required documentation for the study. These laboratory personnel were trained in a two-day workshop on all study procedures, the operation of RDTs assembled to a Deki Reader, as well as on the collection of patients’ information, including the informed consent form.P. falciparum and P. vivax) at the study areas was estimated to be 10 to 15%, P. vivax being responsible for ≥75% of the malaria cases. Based on this prevalence, the sample size was estimated to be between 1,800 to 2,000 subjects, under the assumption that it would provide approximately 120 to 225 positive results for P. vivax and 40 to 75 positive results for P. falciparum. Assuming that the sensitivity and specificity of the interpretation of RDT is the same as the human-eye interpretation, this sample size would provide 95% confidence intervals within 5% of the point estimate. Results of RDT were interpreted by both visual examination and by the Deki Reader device.The prevalence of malaria cases  regulations and Good Clinical Practice (GCP) guidelines.There were three outcomes of interest for the purpose of the analysis in the current trial: a) microscopy results: performed on TBS by laboratory technician at reference laboratory and entered manually into the dataset; b) visual interpretation of RDT: performed by laboratory technician at the site following visual inspection of the RDT at the appropriate time, and entered into the Deki Reader device; and, c) device interpretation of RDT: performed by the device, which was automatically collected and transmitted to the database by the device. Data collected at the cloud database were reconciled to data collected in the internal memory card of each Deki Reader device to ensure that all data points were correctly transmitted to the cloud database. Data analysis was performed using JMP Version 8.0.2 and R Version 2.12.1 . The primary analysis was conducted using microscopy corrected by PCR as gold standard to determine diagnostic performance characteristics ,26  were excluded due to lack of compliance with the protocol: 238 (62%) were samples collected in a centre where RDT processing was performed in major violation of RDT manufacturer’s recommendation; 78 (20%) patient samples due to incomplete written informed consent process, and 68 (18%) due to RDT processing by non-study authorized personnel . Positive cases varied among the sites between 1.89 and 5.84% for P. faciparum, and between 1.52 and 10.54% for P. vivax, which is in agreement to the local known epidemiology concerning distribution of Plasmodium species infections [P. falciparum infections was higher in the sites of Tumaco and Buenaventura , while P. vivax infection was higher in Tierralta .The main demographic features of the cohort are described in Table fections . PrevaleOf the 2,191 samples, in 37 (1.7%) no primary or secondary outcomes (regarding results of the interpretation of RDTs), either visual or by the device were found in the dataset, most likely due to software malfunction, and these patients were not included in the analysis. Therefore, the final analysis dataset consisted of 1,770 samples  when a positive result was obtained, 99.1% (95% CI 98.5-99.4) when a negative result was obtained, and an overall percentage agreement of 99.0% (95% CI 98.4-99.4) (Table P. vivax was 98.9% (95% CI 94.0-99.5), when a negative result was obtained; 99.1% (95% CI 97.8-99.0) when a positive result was obtained; and an overall agreement of 98.5% (95% CI 97.9-99.0). There was no statistically significant difference in the sensitivity or in the specificity between the two methods of interpretation for any of the Plasmodium species.The percentage agreement between visual interpretation and Deki Reader interpretation of RDTs for 4) Table . The perP. falciparum or P. vivax results and two showed discordant results for both Plasmodium species. In 28/30 (93%) discrepant interpretations, the visual inspection failed to detect a line, but the device reported a “positive” result. In the remaining two cases, it was the opposite. In ten (33%) of the 30 discrepant results, a closer review of the images of the RDTs revealed the presence of artefacts and bizarre smearing of the test strip that could have caused the software in the device to interpret as presence of a line. The presence of these unusual patterns in the flow of the sample along the diagnostic strip renders the test inconclusive and therefore should have resulted in processing a new RDT. In an additional seven cases, including the two where the visual inspection reported the presence of a line, the discrepancy seems to be originated from clerical mistakes by either registering a negative result when clearly visible lines were present in the RDT, registering a positive result when no lines were visible and no parasites were reported either by microscopy or PCR, or by mistakes in the identification of the tests. In an additional three cases, results of microscopy and/or PCR confirmed the presence of parasites and therefore were true positive cases (device interpretation was correct). In the remaining ten cases, all of which the device interpreted as positive with line intensity values very close to the cut-off value, both microscopy and PCR failed to detect presence of parasites and therefore correspond to false positive results (0.56%) (device interpretation was incorrect).From a total of 30 discrepant test results, discordant results were obtained in 26 RDTs (1.47%) for either Plasmodium by both diagnostic methods (TBS and RDT), 1,525 were negative by both diagnostic tests and 132 had discordant results. Of these discordant samples, 70 (4%) were positive only by RDT, 43 (2%) were positive only by TBS and 19 (1%) had discordant parasite species results. This discrepancies were solved using PCR as a tie-breaker, finding that 24 (34%) of the 70 RDT-positive samples were positive, 39 (90%) of the 43 TBS-positive samples were negative and 74% (14/19) the samples with species discrepancies had PCR results that were in line with RDT results.Of the 1,770 samples tested, 94 were positive for P. falciparum infections, and sensitivity of 72.1%  and a specificity of 96.5% (95% CI 95.6-97.4) for detection of P. vivax infections. The correspondent sensitivity of expert visual interpretation of RDTs was 35.1%  and specificity of 98.4% (95% CI 97.7-98.9) for detection of P. falciparum infections, and sensitivity of 71.8%  and a specificity of 97.4% (95% CI 96.5-98.0) for detection of P. vivax infections  and a specificity of 98.1% (95% CI 97.3-98.7) for detection of P. falciparum infections, and sensitivity of 98.0% (95% CI 90.3-98.9) and a specificity of 97.9% (95% CI 97.1-98.5) for detection of P. vivax infections  and the specificity 99.0% (100% CI 98.0-99.14). Finally, the performance of microscopy was 60.6% (95% CI 44.9-78.5) and 98.0% (95% CI 97.3-98.6) for sensitivity and specificity against P. falciparum; and 75.0% (95% CI 66.0-83.4) and 98.4%  for sensitivity and specificity against P. vivax  and a specificity of 98.7% (95% CI 98.5-99.4) for detection of P. falciparum; and 68.0% (95% CI 46.5-85.0) and 98.8% (95% CI 96.5-99.75) for sensitivity and specificity against P. vivax (Table When compared to PCR results performed in a subset of 274 randomly selected samples the performance of microscopy was 54.6% (95% CI 23.4-83.3) and 98.9% (95% CI 96.7-99.8) for sensitivity and specificity against Early and adequate malaria diagnosis is critical to opportune and effective treatment to prevent clinical complications and death. Among the malaria diagnostic tools currently available, microscopic examination of TBS remains the gold standard for defining malaria infection, not only because of the relative ease and low cost, but also for its ability to provide a quantitative result. Malaria RDTs are particularly useful in remote areas of difficult access and lack of basic infrastructure required in a microscopy post. The interpretation of two test lines contained in the RDT may be challenging from the point of view of personnel with low level of education, training and supervision  located in remote rural areas. Although the SD Bioline Malaria Antigen Pf/Pv RDT imposes this difficulty, it was decided to use this RDT in combination with the Deki Reader due to the known presence of both parasite species in the study areas. A previous study conducted in rural health facilities in Tanzania demonstrated that interpretation of SD Bioline Malaria Ag Pf/Pan RDT by Deki Reader is substantially equivalent to that of visual interpretation as performed by laboratory technicians with vast experience in RDT processing and interpretation , suggestPf/Pv RDTs was at least as good as the visual interpretation by experts, with sensitivity, specificity, positive and negative predictive value similar to visual interpretation of the same RDT , most of which (238 = 10.8%) presented major violation of RDT manufacturer’s recommendation, indicated that although constant monitoring was available, the two-day training may have been insufficient. The PI and study coordinators had permanent access to the database via internet portal especially designed for the study and were able to monitor the progress of the study and to review images of the RDTs which allowed them to redirect training efforts to the areas where images suggested RDT mishandling. Device users in the field were able to perform the data collection using the touch screen and processing and interpretation of RDTs without major problems. Likely, due to software malfunction early in the course of the study, data from 37 samples (1.7%) were not recorded in the device nor transmitted to the database.Although some places in remote endemic regions do not yet have reliable mobile phone connectivity, the device saves all data which can be transmitted later under good connections to mobile networks. The data and images displayed in the portal created for this study allowed to follow up closely the performance of field operators. It has also demonstrated the potential to be greatly beneficial to health programme managers by providing a tool to perform remote QA monitoring of field activities. The ability to assist in POC diagnosis, and simultaneously capture and make immediately available pivotal epidemiological data at source of origin makes this a very attractive tool to enhance epidemiological surveillance, strengthen health care systems, and contribute to global mapping efforts of malaria and other infectious diseases -39. The Although the function of the Deki Reader is to interpret the presence or absence of a test line signal, independent of the analyses that are finally deposited on it, these results could not be extrapolated to other RDTs. Important differences in RDT clinical diagnostic performance have been well documented, and therefore the results presented here are only applicable to SD Bioline Malaria Antigen Pf/Pv RDT. Nevertheless, the performance of the Reader should be independent to the underlying immunochemistry and thus should provide similar performance characteristics for other RDTs using the same immunochromatographic test.The diagnostic performance of the Deki Reader was comparable to visual interpretation of RDTs (concordance rate >98%) for both malaria species. Providing standardized automated interpretation of RDTs at POC in remote areas, in addition to almost real-time reporting of cases and enabling quality control would greatly benefit large-scale implementation of RDT-based malaria diagnostic programmes.The authors declare that they have no competing interests. SF and MC are employees of Fio Corporation.SH, MA-H and SF conceived and designed the study. SH, MA-H, AV and SF wrote the manuscript. MC and JPQ collected and monitored the data, and provided comments on the manuscript. All authors read and approved the final manuscript.


In Tanzania, malaria is the major cause of morbidity and mortality, accounting for about 30% of all hospital admissions and around 15% of all hospital deaths. Severe anaemia and cerebral malaria are the two main causes of death due to malaria in Tanga, Tanzania.This was a prospective observational hospital-based study conducted from October 2004 to September 2005. Consent was sought from study participants or guardians in the wards. Finger prick blood was collected from each individual for thick and thin smears, blood sugar levels and haemoglobin estimations by Haemocue machine after admission.A total of 494 patients were clinically diagnosed and admitted as cases of severe malaria. Majority of them (55.3%) were children below the age of 5 years. Only 285 out of the total 494 (57.7%) patients had positive blood smears for malaria parasites. Adults aged 20 years and above had the highest rate of cases with fever and blood smear negative for malaria parasites. Commonest clinical manifestations of severe malaria were cerebral malaria (47.3%) and severe anaemia (14.6%), particularly in the under-fives. Case fatality was 3.2% and majority of the deaths occurred in the under-fives and adults aged 20 years and above with negative blood smears.Proper laboratory diagnosis is crucial for case management and reliable data collection. The non-specific nature of malaria symptomatologies limits the use of clinical diagnosis and the IMCI strategy. Strengthening of laboratory investigations to guide case management is recommended. Malaria is the leading cause of morbidity and mortality affecting about 300-400 million people worldwide and accounts for about 1-2 million deaths every year. It is also the most prevalent tropical disease especially in sub-Saharan Africa . Accordiet al, unpublished) data collection activities had to be stopped after failure to obtain suitable candidates despite most of the patients having clinical symptoms suggestive of malaria.Due to their low acquired immune status, children below five years of age are the most affected by malaria in high transmission areas, whereas in low transmission areas all age groups are equally affected ,11. AlthApart from these two studies which have shown very low malaria prevalence, there is still inadequate information on the epidemiology of laboratory confirmed malaria and the underlying associated risk factors. It was the objective of this study, therefore, to determine the malaria disease pattern and the proportion of malaria associated deaths in patients admitted at Bombo regional hospital. The study was conducted for a period of one year from October 2004 to September 2005 and involved prospective data collection from severe malaria cases admitted to Bombo hospital.The study was conducted in the city of Tanga, at Bombo regional hospital, north-eastern Tanzania. The city lies at about 5.17°, 5.33°S and 38.17°, 38.33°E along the Indian Ocean seashore. The area receives two seasons of rainfall; short rains during the months of October - December and long rains in March - June, with a humidity of about 100%. Temperature ranges between 27°C and 32°C. Tanga city covers an area of about 600 sq. km and has an estimated human population of 248,696 (2003 estimated at a growth rate of 2.1% per annum). The hospital has a capacity of 500 beds for inpatients among which 60 beds are reserved for paediatric patients. It has a general laboratory for both in-patient and out-patient services.Based on data from Bombo regional hospital for the year 2003, malaria was indicated as the leading cause of hospital admissions in the intensive care unit (ICU), medical and paediatric wards, accounting for 56.8%, 39.0% and 47.5% respectively. Severe anaemia and severe malaria were noted to be the leading cause of deaths in the paediatric ICU with rates of 61.5% and 45.8% respectively. Anaemia was most probably related to malaria, especially in children.This was a prospective observational hospital based study conducted from 2004 to September 2005. Malaria case admissions and outcome were collected and analysed. The data were collected during official working hours and only on five working days of the week.Study aims were explained to Bombo hospital management team in order to make them aware and get fully involved in the study execution. Permission to conduct the study in Bombo regional hospital was sought from the Regional and City administrative authorities. During all these meetings, study aims and activities were elaborated and clarified when necessary. Oral and written consents were sought from the study participants or guardians/caretakers in the wards. Consent for adult patients with cerebral involvement was sought through relatives/caretakers. Patients' data were taken as confidential entities. The study was granted ethical clearance by the Medical Research Co-ordinating Committee of the National Institute for Medical Research.Patients were admitted at the out-patient department (OPD), usually by clinical officers. The study group comprised of all patients admitted with a clinical diagnosis of severe malaria. Both sexes and all ages were eligible for inclusion in the study. Proposed diagnoses by the hospital staff were entered in the morbidity form as they were. Morbidity questionnaire was completed for all malaria admissions in the wards. Clinical reassessment and laboratory investigations were done in the ward by the research team. From each individual, personal data including name, sex, age, ward, village/street, as well as clinical and parasitological examination results were entered in the morbidity form. Clinical notes of every patient were reviewed on discharge to see if there was any re-evaluation and subsequent change of the initial diagnosis. Patients who had a change of diagnosis from malaria to another febrile illness on discharge were excluded from the study. Cerebral involvement was assessed using Blantyre coma score ,17 by inFinger prick blood was collected from each individual for thick and thin smears. For all cases, finger prick blood was also taken for Hb level and blood sugar examinations using Haemocue machine. The blood smears were stained using 10% Giemsa for 30 minutes and examined under the microscope for identification and enumeration of malaria parasites. Malaria parasites were counted against 200 leukocytes. A blood smear was considered negative if no malaria parasites were seen after scanning 200 high power fields. When sexual parasites were present, they were enumerated against 500 leukocytes. Hyperparasitaemia was defined as parasite density ≥ 250,000 rings/μL of blood . AnaemiaInitial assessment was done at the OPD on admission to ascertain the presence of any severe signs which would require special attention. Parenteral/oral paracetamol was prescribed as antipyretic and analgesic whereas parenteral diazepam was prescribed to patients presenting with convulsions. Blood transfusion was provided in the case of severe anaemia (Hb < 5 g/dL) after screening for HIV, grouping and cross-matching of the blood. According to national policy quinine is the drug of choice. The drug is administered by loading and maintenance doses. There is a slight difference between the regimen for adults and for children. The loading dose for adults is 20 mg/kg body weight of quinine hydrochloride salt diluted in 10 ml/kg of 5% dextrose by intravenous infusion. The drip is left to run for over 4 hours. A maintenance dose of quinine follows 8 hours from the beginning of the previous (loading) dose and is given at 10 mg/kg in similar IV drip, again running over 4 hours. The maintenance dose is repeated every 8 hours, calculated from the beginning of the previous infusion, until patient improves to an extent that he can swallow. Then oral quinine (tablets), 10 mg salt/kg, is given 8 hourly intervals to complete a 7 days treatment course. As for children, a loading dose of quinine is administered exactly like in adults (20 mg/kg), running over 4 hours, followed by a maintenance dose (10 mg/kg) 12 hours after the start of the loading dose, given over 2 hours. This maintenance dose is repeated every 12 hours, estimated from the beginning of the previous drip. When the patient improves and can swallow, the quinine dose is given orally, 10 mg/kg 8 hourly to complete a 7 day treatment course.Data collected from this study were handled using Epi-info software and were double entered to minimize errors during data entry process while check files were created to restrict entrance of invalid data. Data analysis was done using STATA version 9. Proportions were compared by chisq test. Logistic regression analysis was used to compare malaria clinical signs and symptoms with blood smear results in patients admitted for severe malaria. P-value < 0.05 was considered significant.A total of 494 patients were clinically diagnosed and admitted as cases of severe malaria during the one year period. Males accounted for 61.5% (304) of the patients. Among the patients admitted to IPD, children aged 1-4 years were majority (34.4%), while children in the age group 5-9 years were the least (13.6%), Table Plasmodium falciparum and P. malariae were the only malaria parasites species detected. P. malariae was only found as mixed infection and it accounted for 6.7% (19) of all infections. Highest smear positive rate was recorded in children under the age of 5 years and thereafter, the prevalence showed a sharp drop that levelled off after the age of 10 years  of the cases had positive blood smears for malaria parasites. for 6.7% 9 of all The highest parasite density of 10734 asexual parasites/μL (95% CI: 3912 - 33307) was recorded in patients aged 10-19 years whilst the lowest parasite density  was seen in adults aged ≥20 years, . The highest rate of fever was recorded in children aged 1-4 years followed by infants and adults aged ≥20 years. Figure  had positive BS for malaria parasites. The prevalence of severe anaemia was highest in children under the age of 5 years and decreased with increasing age.Anaemia was a prominent feature among the patients, with an overall rate of 69.8% (345). Only 30.2% (149/494) of the patients had normal Hb levels (Hb > 11 g/dL). Majority (55.2%) of the cases had mild/moderate anaemia. Severe anaemia was recorded in 14.6% (72) of the patients and a significant proportion of them (p = 0.003). Most of these patients were children below the age of 5 years. Assessment by the Blantyre coma scale revealed that 129 out of total 273 under-fives (47.3%) admitted had signs and symptoms indicative of cerebral malaria but only 89 of the 129 children (69.0%) had positive BS for malaria parasites , Table . Out of (p = 0.006) only in infants (Table p = 0.001) as shown in Table Hyperpyrexia, hyperparasitaemia, hypoglycaemia, convulsions and jaundice were among the accompanying clinical complications of severe malaria observed across all age groups. Among all these complications, hyperpyrexia appeared to be relatively high (19.6%) Table . Again, ts Table  & 3. HypThe majority of patients in the study had been sick for varying lengths before admission, ranging from 1 to 90 days. The longest period of sickness was documented in adults of 20 years and above who had a median duration of sickness of 7 [IQ range: 4-33] days. On the other hand, a high proportion of the adults aged 20 years and above gave a history of being sick for durations exceeding 2 weeks (Table Most of the patients (95.8%) were successfully treated and discharged. However, 5 (1.0%) were transferred to other health facilities for various reasons and 16 (3.2%) of the 494 patients died during the one year period. Most of the deaths occurred in patients from the urban areas of Tanga city (81.2% vs 18.3%). Deaths reported were of infants (12.5%), children aged 1-4 years (43.8%) and adults (37.5%) of over 19 years of age, and all of the deceased adults had negative BS for malaria parasites. Only 6 out of these 16 (37.5%) deaths had positive BS for malaria parasites of whom 2 were infants and 4 were children aged 1-4 years.According to health facility statistics, malaria in Tanzania is the leading cause of morbidity and mortality. Ministry of Health data from 2003 show that around 30 per cent of all hospital admissions and 15 per cent of all hospital deaths in Tanzania are due to malaria . ResultsAn interesting observation was that a considerable proportion of patients with negative blood smear for malaria parasites and without fever was also admitted as severe cases of malaria. The inclusion of afebrile/apyrexic patients and those with BS negative results for malaria parasites as severe malaria shows that malaria is over-reported in this health facility using clinical diagnosis as the principal method of diagnosis. This is likely to occur in most Sub-Saharan Africa and is facilitated through the syndromic approach, popularly termed as integrated management of childhood illness (IMCI) strategy , which iThe high rate of cases with negative BS for malaria parasites found in our study could have been attributed to the inclusion of patients with acute febrile illnesses caused by aetiologies other than malaria. This is similar to what Njama-Meya  reportedet al [et al [The proportion of cases with hypoglycaemia in our study was relatively lower than those reported in other areas of Sub-Sahara Africa. For instance, Taylor et al  in a stul [et al  reportedMoreover, the low sensitivity of laboratory techniques used in diagnosing malaria could have played a part in the increase of BS negative results. Microscopy has some technical drawbacks that make it less sensitive than polymerase chain reaction (PCR) technique in the detection of malaria parasites . It has According to the scores obtained with the Blantyre coma scale, it seems that cerebral malaria is a common manifestation of severe malaria among the under-fives in this area. It was observed that among the sick children assessed, 47.3% had altered consciousness indicative of cerebral involvement, and about one third of these had negative BS for malaria parasites. Considering the fact that a case definition of malaria requires the presence of asexual parasites in the blood,40, it iet al. in preparation). Unfortunately under-trained personnel are normally the first to be consulted even in most regional hospitals in Tanzania. It is true that malaria is the leading cause of hospital attendances and hospital admissions in Tanzania so far and probably in sub-Saharan Africa as a whole. But the findings of this study portray doubt on the reliability of the accuracy of data from health facilities.The considerable proportion of patients observed with signs and symptoms of severe malaria, and others presenting with hyperpyrexia (body temperature ≥ 39.5°C) but having no parasitaemia indicates very serious diagnostic errors and case management problems in our hospitals. In this study, for instance, 38.5% of patients who presented with severe clinical signs and symptoms of disease pertaining to severe malaria and 19.6% of patients who had hyperpyrexia had no parasitaemia. This in part is due to inadequate quality of care resulting from dependence on under-trained personnel for most of the clinical work in most of our health facilities, and absence of or insufficient laboratory diagnostic services, particularly in the primary health care units . The trend showed that where there was an increase in the proportion of BS positive results for malaria parasites, there was a corresponding increase in the proportion of severe anaemia cases, and vice versa. This phenomenon was more marked in children below the age of five years who also happened to be the group with the highest proportion of BS positive for malaria parasites. In many malaria endemic countries anaemia has been reported to be the most common complication of falciparum malaria and one of the commonest causes of deaths in children ,43,44. TIt has been noted that patients reported various duration of illness. Some had been sick for periods as short as one day, whereas a number of them were unwell for almost 3 months. Duration of illness documented in adult patients (≥20 years) was noted to be significantly long among those with negative BS, indicating that some causes other than malaria, like HIV/AIDS, might have been responsible for the sickness in this age group.Low death rates might have resulted from good access to health services for the majority of the patients living within the city . Prompt treatment by hospital staff due in part to over-diagnosis of malaria  played aAlmost half of the patients enrolled in our study had a history of taking antimalarial drugs prior to admission. These patients most likely got their medicines either from low level health facilities before they were referred or they might have obtained the drugs over the counter, which is a common practice in Tanzania. Some studies conducted in the country show that over 71% of patients treat themselves even before consulting a health facility ,53.The high proportion of fever cases in patients with BS negative results for malaria parasites poses the question as to what could be the actual cause of illness. There is a need to find out if malaria still continues to be the leading cause of morbidity and mortality in Tanzania and sub-Saharan Africa as is believed to be, particularly bearing in mind the impact of HIV/AIDS. Use of more specific RDT might also help. This would reduce the problem of over-diagnosis of malaria and bring about rational use of antimalarials and other relatively expensive drugs. We need to revisit the usefulness of IMCI in terms of its impact on disease over-diagnosis and over-prescription of drugs. Improvement of clinical and laboratory diagnosis of malaria is crucial for proper case management and reliable data collection in the changing epidemiological setting. Blood and urine culture tests could be an important component of management of febrile illnesses in general and cases of non malaria febrile illness in particular.The authors declare that they have no competing interests.HAM designed the study, collected data and prepared the manuscript. MLK designed the study, analyzed the data and prepared the manuscript. MML designed the study and prepared the manuscript. BPM and FFT participated in data analysis. SHS, EKM and JAA collected data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/256/prepub


Placental malaria and pre-eclampsia occur frequently in women in tropics and are leading causes of maternal and perinatal morbidities and mortality. Few data exist concerning the interaction between placental malaria and pre-eclampsia.A case control study was conducted in Medani Hospital, which locates in an area of unstable malaria transmission in Central Sudan. Case (N = 143) were women with pre-eclampsia, which was defined as systolic blood presure≥140 mm Hg or diastolic blood pressure ≥ 90 mm Hg and proteinuria. Controls were parturient women (N = 143) without any blood pressure values > 139/89 mm Hg or proteinuria. Obstetrical and medical characteristics were gathered from both groups through structured questionnaires. Placental histopathology examinations for malaria were performed.P = 0.04 of the cases vs. controls, had placental malaria infections. Five (2%), 1 (2%) and 22 (28.0%) vs. 1, 2 and 13 of the placentae showed acute, chronic and past infection on histopathology examination in the two groups respectively, while 115 (80.4%) vs.127 (88.8%) of them showed no infection, P = 0.04. In multivariate analysis, while there were no associations between age, parity, educational level, lack of antenatal care, blood groups and body mass index and pre-eclampsia; family history of hypertension and placental malaria  were significantly associated with pre-eclampsia.Twenty-eight (19.6%) vs. 16 (11.2%); Placental malaria was associated with pre-eclampsia. Further research is needed. Pre-eclampsia, one of the most common medical complications of pregnancy, it affects approximately 10% of all human births . It has Few data exist concerning placental malaria and its association with pre-eclampsia. While some of these studies have reported the association between malaria and pre-eclampsia and malaria and hypertension during pregnancy ,9, otherPlasmodium falciparum is the sole malaria species in the area  in preeclamptic vs. controls women. The coverage of bed nets was low in the two groups, Table During the study period, there were 4,620 deliveries. Of these, 160 had pre-eclampsia. One hundred and forty-three pre-eclamptic women had complete data including placental histology and were included in the final analyses. These data were compared with equal number of controls with complete data, Table P = 0.04 of the cases vs. controls, had placental malaria infections. Five (2%), 1 (2%) and 22 (28.0%) vs. 1, 2 and 13 of the placentae showed acute, chronic and past infection on histopathology examination in the two groups respectively, while 115 (80.4%) vs.127 (88.8%) of them showed no infection, P = 0.04.Maternal and placental blood films for malaria were positive in one of the cases and one of the controls had positive placental blood film. Twenty-eight (19.6%) vs. 16 (11.2%); P = 0.1), educational level  blood groups  and body mass index  and pre-eclampsia, Table There were no associations between the age  and placental malaria  were significantly associated with pre-eclampsia, Table Primigravidae, lack of antenatal care and history of malaria in the index pregnancy were associated with pre-eclampsia in univariate analysis only. In multivariate analysis family history of hypertension  compared with women without pre-eclampsia. This is consistent with the previous observation in Senegal with a 3.3-fold increase in the risk of placental malaria infections within women with diastolic blood pressure greater than or equal to 90 mm Hg compared with controls, where it was less than 90 mm Hg [et al [In the current study women who had placental malaria infections were twice at higher risk for pre-eclampsia (OR = 2.3, 95% CI = 1.0-5.2; 90 mm Hg . The dif90 mm Hg . Likewis90 mm Hg . Interesg [et al ,11.The majority of the placental infections in the current study were past infections.There is a critical phase in early/mid pregnancy when transformation of the spiral arteries should take place, which is critical in pathogenesis of future pre-eclampsia.et al [Massive sequestration of parasites in the placenta leads to placental ischemia and loss of placental integrity, increases production of pro-inflammatory cytokines, and increases endothelial dysfunction ,25. Moreet al  have sugFamily history of hypertension and placental malaria were associated with pre-eclampsia. Larger longitudinal studies are needed.The authors declare that they have no competing interests.IA and MIE coordinated, carried out the study and participated in the statistical analysis and procedures, EME participated in the clinical work and statistical analysis. AM and MS participated in the lab work. All the authors read and approved the final version.


Plasmodium falciparum infection results in high levels of parasite-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, individuals in endemic areas who are repeatedly infected are often asymptomatic and have low levels of iRBCs, even young children. We hypothesized that febrile malaria alters the immune system such that P. falciparum re-exposure results in reduced production of pro-inflammatory cytokines/chemokines and enhanced anti-parasite effector responses compared to responses induced before malaria. To test this hypothesis we used a systems biology approach to analyze PBMCs sampled from healthy children before the six-month malaria season and the same children seven days after treatment of their first febrile malaria episode of the ensuing season. PBMCs were stimulated with iRBC in vitro and various immune parameters were measured. Before the malaria season, children's immune cells responded to iRBCs by producing pro-inflammatory mediators such as IL-1β, IL-6 and IL-8. Following malaria there was a marked shift in the response to iRBCs with the same children's immune cells producing lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines . In addition, molecules involved in phagocytosis and activation of adaptive immunity were upregulated after malaria as compared to before. This shift was accompanied by an increase in P. falciparum-specific CD4+Foxp3− T cells that co-produce IL-10, IFN-γ and TNF; however, after the subsequent six-month dry season, a period of markedly reduced malaria transmission, P. falciparum–inducible IL-10 production remained partially upregulated only in children with persistent asymptomatic infections. These findings suggest that in the face of P. falciparum re-exposure, children acquire exposure-dependent P. falciparum–specific immunoregulatory responses that dampen pathogenic inflammation while enhancing anti-parasite effector mechanisms. These data provide mechanistic insight into the observation that P. falciparum–infected children in endemic areas are often afebrile and tend to control parasite replication.In malaria-naïve individuals,  Malaria remains a major cause of disease and death worldwide. When mosquitoes infect people with malaria parasites for the first time, the parasite rapidly multiplies in the blood and the body responds by producing molecules that cause inflammation and fever, and sometimes the infection progresses to life-threatening disease. However, in regions where people are repeatedly infected with malaria parasites, most infections do not cause fever and parasites often do not multiply uncontrollably. For example, in Mali where this study was conducted, children are infected with malaria parasites ≥100 times/year but only get malaria fever ∼2 times/year and often manage to control parasite numbers in the blood. To understand these observations we collected immune cells from the blood of healthy children before the malaria season and 7 days after malaria fever. We simulated malaria infection at these time points by exposing the immune cells to malaria parasites in a test-tube. We found that re-exposing immune cells to parasites after malaria fever results in reduced expression of molecules that cause fever and enhanced expression of molecules involved in parasite killing. These findings help explain how the immune system prevents fever and controls malaria parasite growth in children who are repeatedly infected with malaria parasites. Plasmodium falciparum parasites rapidly replicate and almost invariably induce fever and other symptoms of malaria P. falciparum infections more commonly cause a mild febrile illness or no symptoms at all, and parasite numbers in the blood are generally kept in check, even in young children P. falciparum-induced inflammation while maintaining control of parasite replication remains elusive In previously unexposed individuals, blood-stage P. falciparum antigens P. falciparum pathogen-associated molecular pattern (PAMP) molecules such as GPI anchors The notion of malaria ‘tolerance’ has long been invoked to explain the common finding of low-level, asymptomatic blood-stage infection in endemic areas P. falciparum infection in humans ‘tolerizes’ immune cells in an analogous manner is unknown. More specifically, there is no direct evidence that febrile malaria in humans induces regulatory responses that limit the production of pro-inflammatory cytokines and chemokines upon re-exposure to P. falciparum parasites relative to responses induced at homeostasis before malaria in the same individual. Also, it is unclear how current views of malaria-induced tolerance/regulatory responses account for the observation that most children in endemic areas are able to restrain P. falciparum-induced inflammation and simultaneously control parasite replication, since generalized suppression of P. falciparum-triggered immune responses predicts that parasite replication would proceed unhindered and cause severe disease.Interestingly, it has long been speculated that parallels exist between malarial tolerance and bacterial endotoxin tolerance  tolerance in murine macrophages to show that the regulation of LPS-triggered inflammation is component-specific, such that pro-inflammatory mediators are transiently silenced or ‘tolerized’ while antimicrobial effectors are primed or enhanced upon re-challenge with LPS A potential solution to this problem is that febrile malaria temporarily alters immune cells such that the host responds to P. falciparum parasites compared to that induced at homeostasis before malaria in the same children. In addition, given the pivotal role of IL-10 in regulating Plasmodium-induced inflammation in murine models P. falciparum-specific IL-10-producing cells. We further asked if P. falciparum-inducible regulatory responses that limit inflammation are maintained in untreated children who harbor chronic asymptomatic infections, and whether these responses can be recalled in children who have not been exposed to P. falciparum for extended periods of time. To address these questions we applied a systems biology approach P. falciparum transmission.Here we tested the hypothesis that febrile malaria alters children's immune cells such that pro-inflammatory mediators are downregulated and anti-parasite effector responses are upregulated upon re-exposure to P. falciparum re-exposure with cells producing lower levels of pro-inflammatory cytokines and chemokines and higher levels of anti-inflammatory cytokines compared to responses induced at homeostasis before malaria. Re-exposure was also associated with enhanced expression of pathways involved in phagocytosis and activation of adaptive immunity. This shift was accompanied by a marked increase in P. falciparum-specific CD4+Foxp3− T cells that co-produce IL-10, IFN-γ and TNF. IL-10 remained partially inducible in untreated children with chronic asymptomatic infections whereas IL-10 was no longer inducible in children whose infections had been cleared by treatment.We found that febrile malaria induces a marked shift in the response to P. falciparum parasites, and 7 days after treatment of their first febrile malaria episode of the ensuing malaria season, when malaria symptoms had resolved. The average age of these children was 8.5 years and 32% were female. At their first febrile malaria episode of the season, children had an axillary temperature of >37.5°C (or their parents reported fever within 24 hours), were infected with P. falciparum  and had no other cause of fever discernible on physical examination. The average incidence of febrile malaria during the six-month malaria season was similar for the 34 children in this study compared to children in this age group in the larger cohort  P. falciparum-specific antibodies that reliably protect from febrile malaria. Individual demographic and clinical data are shown in To obtain a global view of transcriptional changes that persist in children's PBMCs after the clinical resolution of febrile malaria, compared to each child's own healthy baseline, we profiled RNA expression of PBMCs collected from 34 healthy Malian children before the six-month malaria season, when blood smears were negative for P. falciparum-induced fever and inflammation IL1B and TNF as well as the pro-inflammatory chemokines IL8, CCL3 (MIP-1α) and CXCL2 (MIP-2α), were suppressed after the resolution of malaria to lower levels than observed at baseline (P = 2.15e-6), “Fcγ receptor-mediated phagocytosis in macrophages and monocytes” (P = 1.8e-4), “Production of nitric oxide and reactive species in macrophages” (P = 1.09e-7), “Antigen presentation pathway” (P = 0.0463), “T cell receptor signaling” (P = 1.19e-5) and “Interferon signaling” (P = 5.08e-6) (TLR)2 and TLR4 was increased after malaria relative to baseline . Principal components analysis of the microarray data showed segregation of transcription profiles based on time-point , but not age, gender or batch effects . Within-baseline . Conversbaseline . Specifi5.08e-6) . The expbaseline , consist+ or CD8+ T cells, B cells or monocytes after the resolution of febrile malaria relative to the healthy baseline  following in vitro stimulation with P. falciparum-infected red-blood cell (iRBC) lysate. iRBC-inducible gene expression as well as secreted and intracellular cytokine production were examined with each child serving as his or her own healthy baseline control.These data indicate that febrile malaria induces transcriptional changes in PBMCs that persist after the resolution of febrile malaria; namely, we observed that the expression of genes encoding pro-inflammatory mediators is suppressed while the expression of molecules involved in microbial killing and activation of adaptive immunity is enhanced after malaria relative to baseline. On the basis of these findings we hypothesized that re-exposure to P = 3.36e-34). Within this functional category, all differentially expressed pro-inflammatory cytokines and chemokines  were downregulated in response to iRBC stimulation after the resolution of malaria relative to the iRBC-induced response at baseline , “Antigen presentation pathway” (P = 6.87e-5), “T helper cell differentiation” (P = 1.88e-3) and “T cell receptor signaling” (P = 0.035)  . Togetheex vivo and iRBC-stimulated experiments at both time points . We found a positive correlation  for gene expression as detected by microarray and qRT-PCR -PCR to analyze PBMCs from the 17 children who had microarray data from the  qRT-PCR . The qRTeriments . TherefoIL10 in the microarray data. Given the known temporal dissociation between IL-10 transcription and translation P. falciparum-inducible IL-10 production was higher after the resolution of febrile malaria relative to the healthy baseline of the same children before the malaria season , and after the 6-month dry season. Consistent with the kinetics of P. falciparum-inducible secreted IL-10 production described above  represented only a small percentage of IL-10-producing T cells. P. falciparum-inducible IFN-γ- and TNF-producing CD4+ T cells also increased after the resolution of malaria compared to baseline, and like IL-10, reverted to homeostasis after the dry season  and T cell receptor engagement. We magnetically isolated CD4+ T cells that had been collected after the resolution of febrile malaria and found that they failed to produce IL-10 in response to iRBC stimulation in the absence of antigen-presenting cells (+ T cells was abrogated in PBMC cultures in the presence of antibodies that block major histocompatibility complex (MHC) class II molecules , and upregulation of immune mechanisms involved in control of inflammation (e.g. IL-10-producing CD4+ T cells) and parasite clearance   . The maiP. falciparum-inducible inflammation in early life contributes to the rapid acquisition of strain-transcendent immunity to severe malaria which may occur after only one or two symptomatic infections These data shed light on the long-standing and enigmatic clinical notion of ‘premunition’—a partially effective, exposure-dependent immune response that protects against illness and high numbers of parasites in the blood without completely eliminating the infection P. falciparum PAMPs and their respective PRRs have been identified. These include GPI anchors (TLR2>TLR4) in vitro and in animal models show that these PAMPs drive monocytes/macrophages to produce pro-inflammatory cytokines and chemokines such as IL-1β, IL-6, IL-8 and TNF P. falciparum re-exposure relative to that induced at the healthy baseline of the same individuals was unknown. Here we show that the capacity of monocytes/macrophages to produce the canonical pyrogenic cytokines IL-1β and IL-6 is reduced upon re-exposure to P. falciparum parasites relative to that induced at the healthy baseline of the same individuals—a finding we observed at the mRNA level by microarray and qRT-PCR in PBMCs, and at the protein level in isolated monocytes/macrophages.The prospective design of this study, in which each subject served as their own healthy control, provides a rare view of the regulation and functional plasticity of innate and adaptive immune cells in response to a natural infection in humans. In general, innate immune cells such as monocytes/macrophages first detect pathogens through PRRs such as TLRs and NOD-like receptors (NLRs) which recognize highly conserved PAMPs P. falciparum-inducible inflammation remain to be fully elucidated, this study offers insight into the multiple levels at which this regulation might occur. For example, we observed that NFKB1 expression was downregulated after malaria relative to baseline. NFKB1 encodes the p50 component of the canonical p65/p50 NF-κB heterodimeric transcription factor that stimulates the expression of pro-inflammatory cytokines such as IL-1β and IL-6 NLRP3 after malaria relative to baseline. NLRP3 encodes a component of the NALP3 inflammasome which is expressed in myeloid cells and activates caspase-1, thereby promoting the maturation and secretion of IL-1β TLR2 and TRL4 were upregulated after malaria relative to baseline, suggesting that the regulation of P. falciparum-inducible inflammation does not occur at the level of TLR expression. Interestingly, TLR expression is also upregulated in the context of tolerance induced by gram positive bacteria Although the precise molecular mechanisms that regulate IL18BP, IL1R2, CTLA4, BTLA, SAMHD1 and TNFSF10, as well as decreased expression of genes encoding proteins that promote inflammation including PTGS2 and TREM1. Further studies are needed to more clearly elucidate the signaling networks involved in regulating the immune response to P. falciparum infection and to fully understand the relationships between perturbations to these networks and the variability in malaria clinical outcomes.After the resolution of febrile malaria we also observed increased expression of genes encoding proteins that limit the inflammatory response including P. falciparum parasites was associated with downregulated expression of chemokines that recruit macrophages  and chemokine receptors (increased) enhances the sensitivity of monocyte/macrophages to detect decreased concentrations of chemokines.The gene expression microarray data also shed light on the regulation of chemotactic responses Plasmodium-induced inflammation P. falciparum-induced inflammation during a single malaria episode. However, whether febrile malaria conditions the immune system to modify the production of IL-10 and TGF-β upon subsequent exposure to P. falciparum within the same individual remained an open question. Here we show that P. falciparum-inducible IL-10 and TGF-β production/expression is upregulated after the resolution of febrile malaria relative to that which is inducible at the healthy baseline of the same individuals. In addition, we observed that IL-10 blockade in vitro enhanced IL-6 and TNF production in some but not all children, consistent with a role for IL-10 in controlling inflammation in the setting of P. falciparum re-exposure, but also highlighting the complexity of regulatory responses that restrain P. falciparum-induced inflammation.Murine models clearly demonstrate that IL-10 and TGF-β play critical roles in regulating Plasmodium-induced inflammation, we sought to illuminate the identity, function and kinetics of P. falciparum-specific IL-10-producing cells. Previous studies in humans have shown that total FOXP3+ T regulatory cells (Tregs) increase in response to experimental P. falciparum infection  and non-opsonic  phagocytosis, phagolysosome maturation, antigen processing and presentation, and T cell co-stimulation  induces de novo expression of immune-related genes.To our knowledge, no human study has evaluated the genome-wide transcriptional response to a natural infection in which each subject serves as his or her own healthy control. Nearly all individuals at the study site become infected with P. falciparum-specific regulatory responses that limit pathogenic inflammation and enhance anti-parasite effector responses upon P. falciparum re-exposure. These findings offer mechanistic insights into several long-standing clinical observations in malaria including the high incidence of asymptomatic P. falciparum infection in endemic areas Plasmodium infections in humans P. falciparum exposure Plasmodium-induced hetero-tolerance to endotoxin challenge P. falciparum will refine our understanding of the mechanisms underlying the regulation and dysregulation of Plasmodium-induced inflammation and may help define the potential for interventions that safely prevent or mitigate Plasmodium-induced immunopathology without compromising control of parasite replication.In summary, this longitudinal study of Malian children shows that febrile malaria induces exposure-dependent The Ethics Committee of the Faculty of Medicine, Pharmacy, and Dentistry at the University of Sciences, Techniques, and Technologies of Bamako, and the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health approved this study. Written informed consent was obtained from the parents or guardians of participating children.P. falciparum transmission occurs from July through December. The cohort is an age-stratified random sample of the entire village population. A detailed description of the study site and design of the cohort study has been published elsewhere P. falciparum transmission. Individual demographic and clinical data are given in Study subjects were enrolled in an observational cohort study conducted in Kambila, Mali, a rural village of ∼1500 inhabitants where intense seasonal P. falciparum densities were recorded as the number of asexual parasites/µl of whole blood based on an average leukocyte count of 7500/µl. Each smear was evaluated separately by at least two expert microscopists. P. falciparum was detected by PCR from dried blood spots preserved on 903 Protein Saver filter paper (Whatman) as previously described Thick blood smears were stained with Giemsa and counted against 300 leukocytes, and Blood samples (8 ml) were drawn by venipuncture into sodium citrate-containing cell preparation tubes  and transported 20 km to the laboratory where PBMCs were isolated and frozen within three hours according to the manufacturer's instructions. PBMCs were frozen in fetal bovine serum (FBS)  containing 7.5% dimethyl sulfoxide , kept at −80°C for 24 hours, and then stored at −196°C in liquid nitrogen. For each individual, PBMCs from all time points were thawed and assayed at the same time. The trypan blue dye exclusion assay consistently demonstrated >80% viability of PBMCs after thawing.affy package library in the R project for Statistical Computing (R Core Team 2013). Nine outlier chips were identified among the unstimulated samples using quality control plots from Partek Genomics Suite software  and principal components analyses (PCA) computed using R. An empirical Bayes moderated paired T-test was computed using the limma package library in R to obtain false discovery rate (FDR) adjusted p-values and fold changes. Probes were considered statistically significant if their FDR-adjusted P values were <0.05 and their absolute fold change was >1.25. Heatmaps were generated with the gplots package library in R. Log fold change ratios, p-values and false discovery rates from the empirical Bayes T-tests were imported into Ingenuity Pathways Analysis to examine enrichment of pathways and functional groups.RNA extraction, cDNA amplification, synthesis and labeling was performed as previously described CXCL5), interleukin-1 beta (IL1B), interleukin-6 (IL6), interleukin-10 (IL10), toll-like receptor 2 (TLR2), and transforming growth factor, beta 1 (TGFB1). All six probe and primer sets were designed using Primer Express version 3.0  and are listed inex vivo unstimulated’ and ‘in vitro stimulated with iRBC’). Template preparation and q-RT-PCR analysis was performed as described previously Human yeast Sfi1 homolog spindle assembly associated gene (Sfi1) was selected as a reference gene based on its low coefficient of variation (CV) across DNA microarray analysis. Seven mRNAs were analyzed by q-RT-PCR to validate DNA microarray findings: chemokine (C-X-C motif) ligand 5  supplemented with 10% heat-inactivated ORh+ human serum , 7.4% Sodium Bicarbonate  and 25 µg/ml of gentamycin , at 37°C in the presence of a gas mixture containing 5% O2, 5% CO2 and 90% N2. Parasite cultures were shown to be free of mycoplasma and acholeplasma using an ELISA-based Mycoplasma Detection Kit (Roche) which contains polyclonal antibodies specific for M. arginini, M. hyorhinis, A. laidlawii and M. orale. P. falciparum schizont iRBCs were isolated in RPMI 1640 medium supplemented with 0.25% Albumax  and 7.4% Sodium Bicarbonate  using magnetic columns . Control preparations of uninfected red blood cells (uRBC) from the same blood donor were obtained and tested in all experiments. Lysates of P. falciparum-infected and uninfected RBCs were obtained by three freeze-thaw cycles in liquid nitrogen and 37°C water bath.3D7 2 atmosphere. 500,000 PBMCs were stimulated with lysate of infected red blood cells (iRBCs) or uninfected RBCs (uRBCs) in a ratio of 3 RBCs per PBMC for 18 h, with or without 1.25 µg/ml Brefeldin A (BFA) (Sigma-Aldrich) for the last 15 h of stimulation. A 3∶1 ratio of RBC to PBMC was used on the basis of titration experiments (from 5∶1 to 1∶1) and is consistent with previous reports Staphylococcus aureus (SEB) (Sigma-Aldrich) was used as a positive control for cytokine production in supernatants and within cells. Following stimulation, cells were centrifuged and supernatants were recovered and frozen at −80°C for cytokine analysis. Cells stimulated in the presence of BFA were centrifuged, washed and recovered for intracellular staining and flow cytometry analysis.PBMCs were cultured in complete RPMI  in flat-bottom 96 well plates, at 37°C in a 5% COP. falciparum-infected RBCs and cytokines were measured in supernatants.Monocyte/macrophages were isolated from PBMCs of Malian children by negative selection using the MACS Pan Monocyte Cell Negative Isolation kit II (Miltenyi Biotec), an indirect magnetic labeling system for the isolation of untouched monocytes/macrophages. Non-monocyte/macrophage cells were directly depleted by using a cocktail of biotin-conjugated antibodies followed by magnetic removal of labeled cells. Monocyte/macrophage purity was verified by flow cytometry using fluorescently labeled antibodies specific for CD3 PE (UCHT1), CD4 APC (RPA-T4), CD8 APC-Cy7 (SK1), CD14 FITC (M5E), CD16 Pacific blue (3G8) (BD Biosciences), CD19 PerCP-Cy5.5 (SJ25C1) (eBioscience), and 7-Aminoactinomycin D (7-AAD) viability staining (BD Biosciences). FACS analysis was performed on a BD LSR II Table flow cytometer (BD Bioscience) and analyzed using FlowJo software (Tree Star). Purified monocytes/macrophages were then stimulated in a ratio of 30 RBCs per monocyte for 6 h with lysate of PBMCs were washed in PBS with 4% heat-inactivated FCS and cells were incubated for 30 min at 4°C with fluorescently labeled antibodies specific for CD3 PE (UCHT1), CD4 APC (RPA-T4), CD8 APC-Cy7 (SK1), CD14 FITC (M5E) and CD16 Pacific blue (3G8) purchased from BD Biosciences; and CD19 PerCP-Cy5.5 (SJ25C1) purchased from eBioscience. All phenotypic analyses were performed using mouse mAbs specific for human markers conjugated to fluorophores. FACS analyses were performed on a BD LSR II Table flow cytometer (BD Biosciences) and analyzed using FlowJo software .Supernatants were thawed and immediately analyzed with Bio-plex human cytokine assays  as recommended by the manufacturer. The following cytokines were measured: IL-1β, IL-6, IL-8, IL-10 and TNF. Briefly, 25 µL of supernatant was diluted 1∶2 in medium and incubated with anti-cytokine antibody-coupled magnetic beads for 30 min at room temperature shaking at 300 RPM in the dark. Between each step the complexes were washed three times in wash buffer, using a vacuum manifold. The beads were then incubated with a biotinylated detector antibody for 30 min before incubation with streptavidin-phycoerythrin for 30 minutes. Finally, the complexes were resuspended in 125 µL of detection buffer and 100 beads were counted with a Luminex 200 device . Final concentrations were calculated from the mean fluorescence intensity and expressed in pg/mL using standard curves with known concentrations of each cytokine.2 atmosphere. 500,000 PBMCs were stimulated for 18 h with lysate of infected (iRBCs) or uninfected RBCs (uRBCs) in a ratio of 3 RBCs per PBMC in the presence of anti-IL-10  and anti-IL-10R  or in the presence of the respective isotype controls. Following stimulation cells were centrifuged and supernatants were recovered for cytokine analysis.PBMCs were cultured in complete RPMI in flat-bottom 96 well plates, at 37°C in a 5% CO6 PBMCs were sequentially stained for surface and intracellular markers in round-bottom 96-well plates at room temperature. To exclude dead cells, PBMCs were stained for 30 min using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen) followed by a surface staining with PerCP-Cy5.5 anti-human CD27 (M-T271) and APC-H7 anti-human CD45RO (UCHL1) for 20 min. After fixing and permeabilizing the cells according to the manufacturer's protocol using the FoxP3 Staining Buffer Set (eBioscience), the cells were stained with BD Horizon V500 anti-human CD3 (UCHT1), PerCP anti-human CD4 (SK3), Alexa Fluor 700 anti-human IFNγ (B27), FITC anti-human TNF (MAb11), APC anti-human IL-10 (JES3-19F1), PE-Cy7 anti-human CD25 (BC96) and PE anti-human FoxP3 (236A/E7) for 30 min. Fluorescently labeled antibodies against TNF, CD25 and FoxP3 were purchased from eBioscience, the remaining antibodies were purchased form BD Biosciences. Cells were acquired using a BD LSR II Table flow cytometer (BD) and analyzed using FlowJo software (Tree Star) and SPICE software After stimulation a total of 1×10+ T cells were isolated from PBMCs of Malian children by negative selection using the MACS CD4+ T Cell Negative Isolation kit II (Miltenyi Biotec), an indirect magnetic labeling system for the isolation of untouched CD4+ T helper cells. Non-CD4+ T cells were directly depleted by using a cocktail of biotin-conjugated antibodies against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR g/d and Glycophorin A and anti-Biotin microbeads, followed by magnetic removal of labeled cells. CD4+ T cells purity was verified by flow cytometry, using fluorescently labeled antibodies specific for CD3 (PE) and CD4 (APC) (BD Bioscience) and a BD LSR II Table flow cytometer  and analyzed using FlowJo software . Purified CD4+ T cells were then incubated either in the presence or absence of non-CD4+ cells and stimulated for 18 h with lysate of infected (iRBCs) or uninfected RBCs (uRBCs), for cytokine analysis of supernatants.CD42 atmosphere. 500,000 PBMCs were stimulated for 18 h with lysate of infected (iRBCs) or uninfected RBCs (uRBCs) in a ratio of 3 RBCs per PBMC, in the presence of anti-human leukocyte antigen HLA-DQ , anti-HLA-DP  and anti-HLA-DR , or in the presence of respective isotype controls. Following stimulation, cells were centrifuged and supernatants were recovered for cytokine analysis.PBMCs were cultured in complete RPMI in flat-bottom 96 well plates, at 37°C in a 5% COP value of ≤.05. Statistical tests were computed using R version 2.13.2 (http://www.R-project.org), GraphPad Prism version 5.0d (http://www.graphpad.com/scientific-software/prism/) or JMP 10.0 (www.jmp.com).Continuous data were compared using the paired or unpaired Student's T-test, paired Wilcoxon rank sum test or permutation tests of mean paired differences as appropriate. Bonferroni adjustments were applied to correct for multiple comparisons when appropriate. A linear mixed model for repeated measures ANOVA with Tukey HSD post hoc tests was also used to compare continuous variables. Pearson correlation coefficients and linear regressions with 95% confidence bands were used to examine the correlation between continuous variables. Fisher's exact test was used for contingency table analyses. The statistical test used is specified in the figure legends. Statistical significance was defined as a 2-tailed Figure S1P. falciparum iRBC stimulated PBMC microarray experiments. The graphical elements are as described in (B).(A) Principal components analysis of the microarray data showed that transcription profiles of the unstimulated PBMCs segregated on the basis of time-point , but not age, gender or batch effects. The samples of the nine individuals that did not pass the microarray quality assessment are indicated in gray. (B) Ingenuity Pathway Analysis (IPA) summary showing canonical pathways that remained affected after the resolution of febrile malaria relative to the healthy pre-malaria baseline in the unstimulated PBMC microarray experiments. The graphs show the BH adjusted p values (yellow line) of the enrichment of canonical pathways. The bars indicate the percentage of genes in a given pathway that are differentially expressed with the total number of genes in each pathway shown on the right Y-axis. The red and green portions of the bars indicate the percentage of genes within each pathway that were upregulated or downregulated, respectively. (C) Ratio of monocyte percentage  vs the ratio of the expression level of monocyte-derived mediators of the inflammatory response . Each point represents an individual subject. (D) Heat map showing RMA-normalized log2 ratios  of genes identified as myeloid-specific  (rows) in unstimulated PBMCs for each child (columns). Statistically significant differentially expressed genes are indicated with an asterisk (n = 50 paired samples). (E) IPA summary showing canonical pathways that remained affected after the resolution of febrile malaria relative to the healthy pre-malaria baseline in the (TIF)Click here for additional data file.Figure S2+ before and after monocyte/macrophage enrichment, shown in pink. (B) Percentage of live monocyte/macrophages of total PBMCs before (PBMCs) and after (M0) monocyte/macrophage enrichment in PBMCs collected at healthy baseline (HB) and 14 days after the first malaria episode of the season (d14) . P values determined by ANOVA with Sidak's multiple comparisons test.(A) Flow cytometry gating strategy to detect monocyte/macrophage enrichment. FACS plots of PBMCs of a representative Malian child. Within the total PBMC gate the monocyte population is defined by live CD14(TIF)Click here for additional data file.Table S1Demographic and clinical data of study subjects and assays in which PBMC samples were used.(PDF)Click here for additional data file.Table S2Ex vivo differentially expressed genes from before to after malaria. Transcripts are significant if FDR-adjusted p-value<0.05 and absolute fold change >1.25. Transcript ID is the Affymetrix accession number.(PDF)Click here for additional data file.Table S3in vitro P. falciparum stimulation from before to after malaria. Transcripts are significant if DR-adjusted p-value<0.05 and absolute fold change >1.25. Transcript ID is the Affymetrix accession number.Differentially expressed genes in response to (PDF)Click here for additional data file.Table S4P. falciparum in vitro stimulation.Microarray Expression and q-RT-PCR values of selected genes from 18 individuals at healthy baseline and day 7 after the first malaria episode with and without (PDF)Click here for additional data file.Table S5Sequences of primers and probes used for q-RT-PCR validation.(PDF)Click here for additional data file.


WHO now recommends test-based management of malaria across all transmission settings. The accuracy of rapid diagnostic test (RDT) and the outcome of treatment based on the result of tests will influence acceptability of and adherence to the new guidelines.We conducted a study at the Kintampo hospital in rural Ghana to evaluate the performance of CareStart, a HRP-2 based RDT, using microscopy as reference. We applied IMCI treatment guidelines, restricted ACT to RDT-positive children and followed-up both RDT-positive  and RDT-negative  cases over 28 days.436 children were enrolled in the RDT evaluation and 391 (children with haemoglobin >8.0 gm/dl) were followed-up to assess treatment outcomes. Mean age was 25.4 months (s.d. 14.6). Sensitivity and specificity of the RDT were 100.0% and 73.0% respectively. Over the follow-up period, 32 (18.5%) RDT-negative children converted to positive, with 7 (4.0%) of them presenting with fever. More children in the non-malaria group made unscheduled visits than children in the malaria group (13.3% versus 7.7%) On all scheduled follow-up visits, proportion of children having a temperature higher than that recorded on day 0 was higher in the non-malaria group compared to the malaria group. Reports of unfavourable treatment outcomes by caregivers were higher among the non-malaria group than the malaria group.The RDT had good sensitivity and specificity. However a minority of children who will not receive ACT based on RDT results may develop clinical malaria within a short period in high transmission settings. This could undermine caregivers' and health workers' confidence in the new guidelines. Improving the quality of management of non-malarial febrile illnesses should be a priority in the era of test-based management of malaria.NCT00832754ClinicalTrials.gov  IMCI guidelines promoted the presumptive treatment of malaria for all febrile illnesses in children in high transmission settings. The use of RDT would more clearly delineate non-malaria febrile illnesses and it is argued that this will lead to improved management of such cases As the incidence of malaria declines, increasingly more children will present to health facilities with low levels of parasitaemia. At low levels of parasitaemia, the probability of a false-negative result is higher and this could cause delay in the initiation of treatment. On the other hand, in high transmission settings a positive RDT result does not necessarily rule out bacterial or viral co-infections, as symptomatic malaria parasitaemia is not uncommon. In the era of presumptive treatment, nearly all children who presented with febrile illness received an antimalarial. In addition to clearing parasites in children with malaria, this “opportunistic presumptive treatment” probably prevented new malaria infections through post treatment prophylaxis in all children. It is hypothesised that restricting ACTs to RDT-positive cases would lead to increased frequency of malaria in children since a large number of children would be denied this inadvertent prophylactic benefit and make them prone to malaria sooner and more frequently than children who receive antimalarials. The over-emphasis on malaria over the years has been at the expense of attention to non-malarial febrile illnesses. Very little is known about the aetiology and epidemiology of non-malaria fevers in sub-Saharan Africa. p.f are being considered for roll out nationally. Studies in Africa show considerable variability in sensitivity and specificity of HRP 2 antigen detection tests. p.f. for the diagnosis of malaria in Ghana, and the treatment outcome in both RDT positive  and RDT negative  cases. We evaluated CareStart p.f. because it was the RDT that the NMCP was considering deploying in the country at the time of initiating this study.In Ghana, the management of fever in under-five children has for many years been based on guidelines in the Integrated Management of Childhood Illnesses (IMCI). The protocol for this study is available as supporting information; see The study was conducted in Kintampo North municipality of Ghana which lies within the forest-savannah transition belt and has malaria transmission all year round. The entomological inoculation rate was estimated at 269 infective bites per person per year. Peak incidence of malaria (8.6 per child per year) occurs in children aged 12–35 months. TM, perform rapid test for malaria using CareStartTM, and prepare thin and thick blood smears for microscopy. Filter paper samples were taken for PCR analysis. Children were excluded if they had any of the IMCI danger signs or other signs of severe disease. Children with Hb<8 gm/dl were also excluded as a precautionary measure given uncertainty about the accuracy of the RDT and the practice of restricting ACT to RDT-positive cases at the time of initiating the study. Children were also ineligible if they were enrolled in other clinical studies. Children were enrolled as and when they presented at the clinic and were found to be eligible. This occurred within normal OPD working hours (0800–1400 hrs), Mondays to Fridays.Caregivers residing within 15 km of the hospital who presented with children aged 3–60 months, with complaints that included fever, either on presentation (temperature>37.5°C) or within the previous 48 hours, were requested to take part in the study. After individual informed consent had been obtained, 50 µg of capillary blood was taken from all children to estimate haemoglobin using HemocueAll the children were assessed by a study physician using a standard morbidity assessment questionnaire that ensured comprehensive IMCI-based case management. After clinical assessment, 5 mls of venous blood was taken under aseptic conditions for blood culture. Malaria was diagnosed and treated with an ACT only when RDT was positive. Other medications were prescribed according to identified co-morbidities. RDT-negative cases were managed on the basis of the conditions that were diagnosed using IMCI case management guidelines.The first doses of all prescribed medications were administered under direct observation by a study nurse. Caregivers were educated on the correct procedure for administering the remaining medications at home. The follow-up schedule and the procedures were explained to caregivers prior to leaving the facility. All children (irrespective of RDT results) were followed-up in the clinic on days 1, 2, 3, 7, 14 and 28 post-treatment. Caregivers who failed to show up by mid-day of a scheduled appointment were followed-up to their places of residence. The reason for non-attendance was ascertained and assistance was offered to enable attendance at the clinic within 24 hours. Caregivers were reimbursed for their transportation costs. On each follow-up visit, a standard morbidity questionnaire was administered by a trained research assistant. Caregivers were asked to indicate whether they considered the condition of the child to be “improving”, “unchanged”, “worsening” or with “new illness”. On days 1, 7, 14, 28 post-treatment and on all unscheduled visits, blood sample was taken for RDT and blood smear microscopy. Whenever a caregiver reported “unchanged”, “worsening” or “new illness”, the child was further assessed by the study clinician.Notwithstanding the scheduled appointments, caregivers were advised to bring their children to the hospital at any time that the condition of the child did not improve or when new symptoms developed (unscheduled visits).The primary end point for estimating the sample size was the sensitivity of RDTs to diagnose malaria. We assumed that the RDTs should have at least 95% sensitivity to be useful. To estimate a sensitivity of 95% with a 95% confidence limit of +/− 3%, 203 true positive cases of malaria were required. Our second assumption was that the proportion of participants clearing fever by day 3 post-treatment would be 95% in the RDT positive cohort and 85% in the RDT negative cohort. The loss to follow up by day 3 would be <5%. To have 80% power to detect this difference between the two cohorts with 95% significance, 170  participants were needed in each cohort.All blood smear microscopy was performed at the clinical laboratory of the Kintampo Health Research Centre using standard staining and examination techniques. Parasites were counted against 200 white blood cells and slides declared negative only after 100 fields had been examined. Two microscopists independently read slides while discordant slides were read by a third microscopist. The agreement between two of the microscopists was taken as the final result. All microscopists were blinded to the results of RDTs. Due to logistic constraints, PCR analyses were limited to cases where there were conversions from RDT negative to positive during follow-up. All the RDTs used in the study were stored between 21°C and 35°C, consistent with the recommendations of the manufacturer. Blood culture was done using BACTEC blood culture system, with subculture of positive vials and gram staining for identification.The clinical laboratory at KHRC participates in the external quality assessments schemes provided by the College of American Pathologist, UK National External Quality Assessment Scheme (UK-NEQAS) and the National Institute for Communicable Diseases in South Africa.All data were double-entered in a database and verified/cleaned using FoxPro version 6. Analyses were performed using STATA version 10. The results of microscopy were used as reference to assess the accuracy and reliability of RDT results. Treatment outcomes were assessed on the basis of (1) changes in RDT result (from that obtained on day 0) within the 28-day follow-up period and the correlation with clinical outcome, (2) frequency of unscheduled visits, (3) caregiver perception of clinical outcome, (4) occurrence of fever (axillary temp>37.5°C) and (5) the reduction (from baseline) in haemoglobin level. Caregiver perception of clinical outcome was assessed on the basis of the condition of the child as reported by the caregiver during follow-up visits. Caregiver perception was considered favourable when improvement in the condition of the child relative to the time of initial presentation was reported and unfavourable when the condition was reported to be “unchanged”, “worsened” or with “new illness”. The treatment outcomes were compared between the two groups. However, given that the natural history and treatment outcomes of malaria and non-malaria fevers are likely to be inherently different, the relative risks of various outcomes were not calculated.Written informed consent was obtained from all caregivers before enrolment. The protocol for the study was approved by the Institutional Ethics Committee of the Kintampo Health Research Centre, the Ethics Review Committees of the Ghana Health Service and the London School of Hygiene and Tropical Medicine. The study was conducted in accordance with the principles of Good Clinical Practice. The study is a component of the trial registered in the clinical trials registry under NCT00832754.A total of 1224 children aged 3 months −59 months were screened and 436 children were enrolled  The reasThe mean age was 25.4 months (S.D. = 14.6) and only 44.3% of the children had fever (axillary Temp>37.5°C) at the time of enrolment. The children who had a RDT positive result were slightly older, more likely to have axillary temperature>37.5°C (P<0.01) and anaemic compared to children who had a RDT negative result (P<0.01). No child in the RDT negative group had malaria parasitaemia. The geometric mean parasite density was 6861/ul . Staphylococcus aureus (24), Non-typhoid Salmonella (8), E.coli (1), S. pneumonia (1), Acinetobacter sp (1), Proteus sp (1) and Pseudomonas sp (1). Although a slightly higher proportion of RDT-negative children had a positive culture than RDT-positive children , the difference was not statistically significant (Blood culture was positive for 37 (9.5%) out of 391 children, and the isolates included nificant .Acute respiratory infection (ARI) (63.4%), malaria (55.5%), diarrhoea (16.9%) and skin infections (14.1%) were the leading diagnosis established among the 391 children at enrolment. About a third (28.6%) of them had both ARI and malaria. A higher proportion of RDT-negative children had ARI at enrolment than in RDT-positive children . There were no statistically significant differences in the proportion of children presenting with diarrhoea or skin diseases between the RDT-negative and RDT-positive children . UnspeciA higher proportion of RDT-negative children were given antibiotics than RDT-positive children . Children diagnosed with ARI (95.2% vs 42.7%: p<0.01), skin diseases  or diarrhoea (80.3% vs 75.1%: p = 0.37) were more likely to be prescribed an antibiotic. However, those diagnosed with either malaria (61.8 % vs 97.3%: p<0.01), or unspecified acute febrile illnesses (57.1% vs 76.7%: p = 0.09) were less likely to be prescribed an antibiotic.The sensitivity of CareSart for the diagnosis of malaria on day zero was 100.0%. Specificity was however 73.0% (95% C.I. 67%–78%) while positive and negative predictive values were 75.7% (95% C.I. 70%–81%) and 100% respectively. The false positive error rate at day zero was 27% (95% C.I. 19%–30%). Over the 28-day follow-up period, the rate of RDT conversion from positive to negative was markedly slower than blood smear parasite clearance. As a result, RDT false positive error rates were over 50% by day 7 and 14, and 43.5% on day 28 post-treatment . HoweverOver the 28-day follow-up period, 32 (18.5%) children who tested RDT negative on day 0, converted to RDT positive. The conversions were 6 (3.5%), 7 (4.0%), 8 (5.2%) and 11 (10.4%) on days 1, 7, 14 and 28 respectively. All 32 were microscopy and PCR negative on day 0. In 7 (4.0%) of these children, conversion to RDT positive was associated with fever (axillary temperature>37.5C), giving a malaria incidence of 0.53 per person year. In 5 of them, the children were presented on unscheduled visits, while in the remaining 2, the caregivers reported “new illness” on their day 28 visit. In all 7 cases, conversion to RDT positive was confirmed by positive microscopy and they were diagnosed with uncomplicated malaria. No case of severe malaria or death was recorded.Forty (10.2%) children made an unscheduled visit during the 28-day follow up period as a result of caregiver's perception of adverse outcome. A higher proportion of children with non-malaria illnesses made unscheduled visits on account of febrile illness than children diagnosed with malaria (13.3% vs 7.8%). The sensitivity, specificity and positive predictive value of RDT for the diagnosis of malaria on these unscheduled visits were 100%, 65.5% and 47.3% respectively. Diagnoses other than malaria made at these unscheduled visits included ARI (47.5%) and diarrhoea (10.0%). In 6 (15.0%) cases, both malaria and acute respiratory infections were diagnosed.No caregiver reported a worsening of the condition of the child compared to the status at enrolment. However, the proportion of caregivers who reported the emergence of “new illness” or “unchanged” condition increased from 5.1%, (day 1) to 14.7% (day 28). On all scheduled follow-up days (except on day 14), the proportion of caregivers reporting less favourable treatment outcomes was higher in the non-malaria group compared to the malaria group .The proportions of children having fever were 6.1%, 2.3%, 1.8%, 1.5%, 2.6% and 4.6% on days 1,2,3,7, 14 and 28 post-treatment respectively. On day 3, a higher proportion of children in the non-malaria group had fever than children in the malaria group (2.89% versus 1.82%). On all scheduled follow-up days, a higher proportion of children in the non-malaria group had temperature above values recorded on day 0 .The proportions of children with haemoglobin lower than that recorded at enrolment were 61.1%, 52.1%, 39.9% and 32.7% on day 1, 7, 14 and 28 post-treatment respectively. The proportion of children having a reduction in Hb level on day 1 post treatment compared to day 0 was higher in the malaria group than in the non-malaria group (46.8% vs 72.4%). In contrast, the proportion of children having lower Hb levels at day 28 post-treatment compared to day 0 Hb levels was higher in the non-malaria group than the malaria group (44.8% versus 23.4%) . On averThis study was primarily powered to test the accuracy of the RDT and the treatment outcomes were secondary end-points. The comparative analyses of treatment outcomes were exploratory and should be interpreted with caution. The findings will require further investigation in appropriately-powered studies. Notwithstanding these limitations however, a pattern of comparatively unfavourable outcomes in children in the non-malaria group emerges that raises concern about their recovery when managed using current guidelines, and possibly the safety of restricting ACT to RDT positive cases in high malaria transmission setting. First, the RDT negative to positive conversion rate of 18.5% over a 28–day period appears high when compared to 0.8% found in a cohort of children (average age of 5years) in a study in a region of low malaria transmission in Uganda. If previous IMCI guidelines  had been applied in these cohorts of children however, nearly all of them would have been given an antimalarial. For those who did not have malaria (RDT negatives), such antimalarial would have offered some prophylactic benefit that would have conferred some protection from malaria for possibly 4–12 weeks depending on the half-life of the partner antimalarial in the ACT that is given. On the other hand unnecessary ACT use would have exposed the partner drug to selective mutation of falciparum resistance strains. In the Ugandan and Tanzanian studies The decision to deploy test-based management of malaria therefore needs to be carefully evaluated, within the context of local levels of malaria transmission. In high-transmission settings the risk of malaria and delayed treatment could outweigh the advantages of targeted treatment. Health worker perceptions that children who are denied ACT tend to return to the clinic sooner, would undermine the confidence with which they adhere to the revised test-based treatment guidelines.In Tanzania, a study among severely-ill children (2 months – 13 years) that used different brands of HRP-2 RDT found sensitivities levels of between 96.4% and 98.2%. The over-emphasis on malaria has been at the expense of diagnosis and effective treatment of other causes of childhood fevers in malaria high transmission settings. While a comparison on the basis of giving  or denying ACT  has been presented, it is important to acknowledge that the two groups of children represent different pathologies. For this reason, it has been suggested that statistical testing of observed differences between the two groups is not appropriate, and observed trends in the malaria cohort cannot be used as a comparator against which to assess trends in the non-malaria cohort. S. aureus were the major organisms isolated as in the Kenyan and Tanzanian studies. This contrast to the findings of some other studies in the same countries where bacteraemia rates among outpatients were much lower and other organisms other than non-typhoid Salmonella and S. aureus dominated. The 9.5% rate of positive blood culture among out-patient children in this study is lower than rates of between 21.3% and 26% among hospitalised patients in two studies in Kumasi, Ghana. Although the RDT used in this study showed a perfect sensitivity of 100%, in about 1 out of 4 cases, the positive results on day 0 were false positives. Misleading interpretation of such positive RDT results have the potential to lead to the withholding of life-saving antibiotics. www.actconsortium.org ) that are specifically-designed to address the question of safety in restricting ACT to RDT-positive cases in under-five children in high and low transmission settings are currently underway. The findings of these studies will further clarify the issues raised in this study.Two studies funded by the ACT Consortium Click here for additional data file.