Trypanosoma brucei gambiense.In endemic settings, diagnosis of malaria increasingly relies on the use of rapid diagnostic tests (RDTs). False positivity of such RDTs is poorly documented, although it is especially relevant in those infections that resemble malaria, such as human African trypanosomiasis (HAT). We therefore examined specificity of malaria RDT products among patients infected with Pf-pLDH and/or pan-pLDH antigens. Rheumatoid factor was determined in PCR negative subjects. Specificity of the 10 malaria RDT products varied between 79.5 and 100% in HAT-negative controls and between 11.3 and 98.8% in HAT patients. For seven RDT products, specificity was significantly lower in HAT patients compared to controls. False positive reactions in HAT were mainly observed for pan-pLDH test lines (specificities between 13.8 and 97.5%), but also occurred frequently for the HRP-2 test line (specificities between 67.9 and 98.8%). The Pf-pLDH test line was not affected by false-positive lines in HAT patients (specificities between 97.5 and 100%). False positivity was not associated to rheumatoid factor, detected in 7.6% of controls and 1.2% of HAT patients.Blood samples of 117 HAT patients and 117 matched non-HAT controls were prospectively collected in the Democratic Republic of the Congo. Reference malaria diagnosis was based on real-time PCR. Ten commonly used malaria RDT products were assessed including three two-band and seven three-band products, targeting HRP-2, Specificity of some malaria RDT products in HAT was surprisingly low, and constitutes a risk for misdiagnosis of a fatal but treatable infection. Our results show the importance to assess RDT specificity in non-targeted infections when evaluating diagnostic tests. Rapid diagnostic tests (RDT) for malaria are currently rolled-out as the backbone of parasite-based diagnosis, and their diagnostic accuracy is sufficiently high to substitute microscopy. One decade ago, attention has been given to occurrence of limited false positivity in a number of malaria RDTs, but false positivity of RDTs has remained poorly documented since then. In the last years, the number of available RDT products has dramatically increased and test performance has improved. False positivity may therefore not be perceived as a problem anymore. In this manuscript, we demonstrate that specificities of malaria rapid diagnostic tests detecting parasite antigens are seriously affected by human African trypanosomiasis (sleeping sickness), with values down to 11%. Malaria constitutes the main differential diagnosis of human African trypanosomiasis, and the false-positive results for malaria RDTs increase the risk of misdiagnosis or delayed diagnosis of human African trypanosomiasis which is a fatal but treatable infection. Plasmodium in thick blood films, which is labour-intensive, time-consuming and requires technical skills. Malaria rapid diagnostic tests (RDT) offer an attractive alternative for microscopy. They detect parasite antigens in blood through an antibody-antigen reaction, made visible by a red line on a nitrocellulose strip. Test formats include two- and three-band tests. Two band RDT products consist of a control line and a test line, three-band tests have a control line and 2 test lines, of which at least one usually targets a Plasmodium falciparum (Pf) specific antigen. The main target antigens are histidine rich protein 2 (HRP-2) specific for Pf and Plasmodium lactate dehydrogenase (pLDH), either detecting all human infective species (pan-pLDH) or specific for Pf (Pf-pLDH). Accuracies of RDTs are now such that they can substitute microscopy: average sensitivity and specificity have been estimated 95% for HRP-2 test lines, and respectively 93 and 99% for pLDH detection tests Pf infection Traditional diagnosis of malaria relies on microscopic detection of Trypanosoma brucei (T.b.) gambiense and rhodesiense, which are transmitted by tsetse flies. Association of HAT with a strong polyclonal B-cell activation and with rheumatoid factor-like anti-immunoglobulin antibodies has been reported T.b. gambiense infection has been shown to decrease the specificity of antibody detection tests for HIV diagnosis Occurrence of false positivity in RDTs may be particularly relevant for infections prevailing in malaria endemic regions, for which the differential diagnosis includes malaria. An example of such an infection is human African trypanosomiasis (HAT) Before enrolment into the study, participants gave written informed consent. Parents or guardians provided consent on behalf of all child participants. Ethical clearance for the study was obtained from the institutional review board of ITM and the ethical committees of the University Hospital in Antwerp, Belgium (study registration number B30020108363) and of the Ministry of Health of the Democratic Republic of the Congo (DR Congo).HAT patients and paired non-HAT endemic controls were prospectively included in DR Congo, Bandundu Province between July and December 2010. Malaria transmission in most parts of DR Congo, including in Bandundu Province, is high (>1 case per 1000 population) and DR Congo is considered as suffering from the highest malaria burden in Africa Plasmodium falciparum (Pf), Plasmodium vivax (Pv), Plasmodium malariae (Pm) and Plasmodium ovale (Po) as previously described Plasmodium species was included for each test run. If Pf-PCR, Pv-PCR, Pm-PCR or Po-PCR was positive, malaria-PCR was considered positive, if negative for all species, malaria-PCR was considered negative. Standard microscopy was performed on thick blood films by an expert microscopist. Parasite density was assessed by counting asexual parasites against 200 white blood cells (WBC) in thick blood films and converted into parasites/µl using the standard of 8000 WBC/µl DNA was extracted from EDTA blood mixed with GE buffer using the Maxwell 16 DNA Purification Kit . A four primer real-time PCR was performed for detecting single and mixed infections of Pf-pLDH were added. The following two-band tests were used: 1° Paracheck Pf ; 2° ICT Malaria Pf Cassette Test ; 3° Advantage Pan Malaria Card . The three-band tests were: 4° Malaria Antigen Pf (HRP-2/pLDH) ; 5° SD malaria Ag Pf/Pan (Standard Diagnostics Inc); 6° SD Malaria Antigen Pf (Standard Diagnostics Inc.); 7° ICT Malaria Combo Cassette Test (ICT Diagnostics); 8° Carestart Malaria HRP2/pLDH (Pf/Pan) Combo Test ; 9° Carestart Malaria pLDH (Pf/pan) (Acces Bio); 10° First Response Malaria Ag (pLDH/HRP2) Combo Rapid Diagnostic Test . Tests were performed according to the instructions of the manufacturers, using thawed EDTA blood. Three experienced readers who were blinded to each other's and to other results, except for the HAT status, read the RDT results, at daylight and within the prescribed delay. After reading, RDT results were immediately photographed. Test line intensities were scored as negative (no line visible), faint (barely visible), weak , medium , strong (stronger than the control line). Invalid tests (no control line) were repeated. Based on the three scores, a consensus line intensity score was established. If two out of three readers had scored the line intensity the same, this was taken as the consensus. If the three readers had scored the intensity differently, the photograph was used for taking the final decision. For analysis of test line results, faint, weak, medium and strong line intensities were considered positive, absence of test lines was recorded as negative. Interpretation of the RDT result was as prescribed by the manufacturer. An RDT was considered negative for malaria when all test lines were negative, and positive when at least 1 test line was positive.In total, ten rapid diagnostic tests for malaria were evaluated. Malaria RDTs in a cassette format were selected based on (i) demonstrated diagnostic accuracy Pf-PCR negatives was measured using the VITROS chemistry Products RF reagent on the VITROS 5600 Integrated system . The procedure consists of an antigen-antibody reaction occurring between rheumatoid factor from the test sample and denatured human IgG adsorbed to latex particles from the test reagent, resulting in agglutination. The agglutination is detected as an absorbance change, with the magnitude of the change being proportional to the quantity of rheumatoid factor. The detection limit was 9 IU/ml, as suggested in the instructions, values ≥12 IU/ml were considered abnormal.The presence and concentration of rheumatoid factor in plasma of all Pf-pLDH lines was assessed respectively as the number of HRP-2 or Pf-pLDH line negatives/number of Pf-PCR negatives; sensitivity was defined as the numbers of HRP-2 or Pf-pLDH line positives/number of Pf-PCR positives. Likewise, specificity of pan-pLDH lines was calculated as the number of pan-pLDH negatives/number of malaria PCR negatives and the sensitivity as the number of pan-pLDH positives/number of malaria PCR positives. Specificity of three-band RDTs for diagnosis of malaria was calculated as the number of RDT negatives/number of malaria PCR negatives or number of RDT negatives/number of Pf-PCR negatives if the RDT consisted only Pf specific test lines. Proportions were calculated with 95% exact confidence intervals (CI). In a primary analysis, differences between proportions were tested for significance using the Fisher exact probability test (STATA 10.0), in order to avoid data loss and as the effect of matching, which was done for another study component, was expected to be small on RDT performance. For comparison of specificity, this primary analysis was followed by a secondary analysis using McNemar chi square, after matching controls and HAT again taking into account the PCR result. For sensitivity, the low number of PCR-matched samples precluded meaningful comparison with the McNemar test. Inter-reader reliabilities were assessed for the test results expressed as positive and negative line readings and kappa values for each pair of readers were calculated.For calculation of specificity and sensitivity, PCR was used as the reference method . Specifip = 0.01). Twenty-two  thick blood films were positive for Plasmodium. Due to low Plasmodium parasite densities, the Plasmodium species could be defined only in 16 thin blood films, and was Pf in 15, Po in one. In total, 79/233 participants were malaria-PCR positive (1/234 sample of blood on GE buffer missing), including all 22 that were thick blood film positive. Species identification as defined by PCR were 87% Pf (69/79), followed by Po , Pm  and Pv .An overview of the study population and reference test results is shown in Pf-pLDH test lines in three malaria RDT products was 97.5–100% (Pf-pLDH test were respectively 21.1–28.9% in controls (n = 38) and 9.7% in HAT patients (n = 31). True positive reactions consisted of faint (4–8/69) and weak lines .Specificity of 7.5–100% . There w7.5–100% . Sensiti 0–5/69, . For nonPf/pan and Carestart Malaria HRP2/pLDH (Pf/Pan) Combo Test, a similar tendency towards lower specificity of HRP-2 in HAT was observed, but the difference lost significance. Line intensity scores are shown in Specificity of HRP-2 test lines in seven malaria RDT products for controls and HAT patients are summarized in Pf-PCR positive controls (n = 38) and HAT patients (n = 31) ranged between 47.4–71.1% and 25.8–74.2% respectively. No significant difference in sensitivity of HRP-2 was observed between controls and HAT patients. The majority of positive lines scored weak (8–23/69), medium (1–4/69) or strong , there was substantial to almost perfect agreement between at least two readers in HRP-2 line intensity .For pan-pLDH tests as well, a significant lower specificity in HAT patients compared to controls was observed with five out of seven RDT products . These dp<0.001) and Carestart Malaria pLDH (Pf/pan) (respectively 0.0% (0–8.0) versus 11.4% (3.2–26.7), p<0.04). The majority of positive line intensities scored only faint (4–29/79) or weak (0–22/79), but some medium (0–2/79) or strong tests line intensities (0–7/79) were observed as well. On all 233 samples tested, there was substantial to almost perfect agreement between at least two readers .Sensitivity of pan-pLDH test lines in malaria-PCR positive controls (n = 44) was 0–63.6%, in HAT patients (n = 35) it was 11.4–77.1%. Sensitivity in controls was significantly lower than in HAT patients for two out of seven RDTs: ICT Malaria Combo Cassette Test (respectively 25.0% (13.2–40.3) versus 77.1% (58.9–89.6), Pf-pLDH or pan-pLDH, specificity was significantly lower in HAT patients compared to controls. These differences were confirmed after taking into account matching. In both tests combining Pf-pLDH with pan-pLDH test lines ), no difference between HAT patients and controls was observed, and specificities were 96.3 to 100%.Specificity of the combined test result in the seven three-band RDTs in controls and HAT patients are summarized in After subtraction of patients who were on anti-malaria drugs prior to sampling, specificities in controls (n = 55) and HAT (n = 50) for the different RDTs ranged between 78.2–100% and 12.0–100% respectively, and remained significantly lower in HAT patients compared to controls for three out of five three-band RDT products incorporating HRP-2 test lines  Combo Test). In Malaria Antigen Pf (HRP-2/pLDH) and First Response Malaria Ag (pLDH/HRP2) Combo Rapid Diagnostic Test, a similar tendency towards lower specificity in HAT was observed, but the difference lost significance.p = 0.06). There was no association between false positivity of any of the HRP-2, pan-pLDH, or Pf-pLDH test lines and the presence and concentration of rheumatoid factor .Proportions of rheumatoid factor positive samples were 7.6% (6/79) among controls (maximum concentration 47 IU/ml) and 1.2% among HAT patients  respectively. This difference was not statistically different . Cross-reactions of specific trypanosome antigens present in blood of HAT patients with the antibodies used in the malaria RDTs also seems unlikely, because of the large variation of proportions of false positive results between the different RDT products.Although malaria constitutes the main differential diagnosis of HAT We withhold from proposing a particular malaria RDT to be used in HAT and malaria co-endemic regions since actually we cannot present relevant data on sensitivity of the RDTs in clinical malaria. Parasite densities in immune patients exhibiting symptoms range from 2,500 in infants to 30,000/µl in adults Our results emphasize the importance, in general, to explore the impact of non-targeted infections when evaluating diagnostic tests.File S1Pf Plasmodium falciparum; Sens sensitivity; Spec specificityCalculation of specificity and sensitivity using PCR as the reference method. HAT Human African trypanosomiasis; C control; (PPTX)Click here for additional data file.File S2Specificities of RDTs for malaria diagnosis in controls and HAT using microscopy as a reference.(DOC)Click here for additional data file.


Plasmodium falciparum, clinical malaria morbidity and higher rate of lactase persistence compared to sympatric tribes. Lactase non-persistence, often called lactose intolerance, is the normal condition where lactase activity in the intestinal wall declines after weaning. Lactase persistence, common in Europe, and in certain African people with traditions of raising cattle, is caused by polymorphisms in the enhancer region approximately 14 kb upstream of the lactase gene.Fulani are a widely spread African ethnic group characterized by lower susceptibility to -13910 polymorphism upstream of the lactase gene was sequenced. DNA samples used in the study originated from 162 Fulani and 79 Dogon individuals from Mali.To evaluate the relationship between malaria and lactase persistence genotypes, a 400 bp region surrounding the main European C/T-13910 was by far the most common polymorphism, with an allele frequency of 37%. Three other single-nucleotide polymorphisms were found with allele frequencies of 3.7%, 1.9% and 0.6% each. The novel DNA polymorphism T/C-13906 was seen in six heterozygous Fulani. Among the Fulani with lactase non-persistence CC genotypes at the C/T-13910 locus, 24% had malaria parasites detectable by microscopy compared to 18% for lactase persistent genotypes (P = 0.29). Pooling the lactase enhancer polymorphisms to a common presumptive genotype gave 28% microscopy positives for non-persistent and 17% for others (P = 0.11).Among 79 Dogon only one heterozygote of the lactase enhancer polymorphism was detected, whereas all others were homozygous for the ancestral C allele. Among the Fulani, the main European polymorphism at locus C/TPlasmodium falciparum parasitaemia in asymptomatic Fulani is more common in individuals with lactase non-persistence genotypes, but this difference is not statistically significant. The potential immunoprotective properties of dietary cow milk as a reason for the partial malaria resistance of Fulani warrant further investigation. During the last 10,000 years protection against malaria and lactase persistence have been two of the strongest selection forces shaping the human genome . ProtectThe Fulani are traditionally a nomadic, pastoralist, trading people, herding cattle, goats and sheep across the vast dry hinterlands of their domain, where they live in sympatry with the local agricultural populations. As lactose tolerance is generally rare among African ethnic groups, the Fulani with around 50% clinical lactose tolerance are an obvious exception among their sympatrics -5.LCT) at 2q21-22 [Lactase non-persistence  is a genetically determined normal trait where down-regulation of lactase activity occurs during childhood . Mutatio 2q21-22 ,7-10.Fulani people have been shown to be more resistant to malaria infection compared to other sympatric tribes in Western Africa . Some diThe hypothesis of this study postulates that the partial malaria resistance could be associated to the dietary habits of the Fulani. Fulani has a tradition of abundant milk consumption and are known to use more milk than sympatric tribes. In the study area the Dogon also use milk, but not as much as the Fulani. To test this hypothesis, the 400 bp region covering the known lactase persistence variants was screened and the association between lactase persistence genotypes and malaria indices in 162 Fulani and 79 Dogon individuals was analysed.P. falciparum. There is a dry season from October to May and a rainy season between June and September, leading to seasonal transmission of malaria, with most cases registered from July until December. Entomological assessment in September 2001 revealed the same entomological inoculation rate (EIR) of 0.38 infective bites/person/night in the two ethnic groups [The field study was carried out in rural villages located in the district of Koro where the Fulani and Dogon live in sympatry. The study villages are located in Sahelian area in Mali, the area is situated halfway between Mopti and the country border to Burkina Faso, approximately 850 kilometre North-East of Bamako, the capital city of Mali. The Malaria Research & Training Center of the University of Bamako has established a permanent community based health centre in the village of Manteau. The area is mesoendemic for malaria and infections are mainly caused by c groups .Blood samples from 162 Fulani and 79 Dogon individuals from an age and gender matched control cohort were collected in a cross-sectional study during the rainy season in 2005 as previously described ,14. The Plasmodium genetic material was assessed from blood samples by polymerase chain reaction (PCR). As markers for repeated or chronic malaria infections, the genotype groups for presence of splenomegaly and anaemia (defined as hemoglobin less than 110 g/L) were compared. The material for this study was a cross-sectional cohort, and thereby the malaria positives cases represent asymptomatic parasitaemia, not clinical disease.Thick blood smears were collected from all patients and stained with 3% Giemsa. Smears were read by microscopists. The number of parasites present per 300 leukocytes was counted and parasite density assuming mean leukocyte count was calculated. Study participants had several indices recorded including age, body temperature, occurrence of spleen enlargement as observed by palpation. Furthermore, haemoglobin levels and presence of DNA was extracted from filter papers using Chelex-100 method ,15. Brie-13910 in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase gene [The DNA fragment covering 400 bp around the original lactase persistence variant C/Tase gene  was amplSamples were purified from excess primers by digestion using 2.5 U of Shrimp Alkaline Phosphatase USB and 5 U of Exonuclease I (New England Biolabs) in 37°C for 60 minutes preceding inactivation of 15 minutes at 80°C. Purified samples were prepared for sequencing using the BigDye 3.1 terminator (Applied Biosystems) as instructed by manufacturer. Sequencing reaction was as follows: initiating step of 96°C for 1 min, 25 cycles of 96°C for 10 sec, 53°C for 5 sec, 60°C for 4 min. Sequence samples were purified with Millipore Multiscreen plates  with Sephadex G-50 Superfine sepharose , electrophoresis with an ABI 3730 DNA Analyzer (Applied Biosystems) and base calling using the Sequence Analysis 5.2 software (Applied Biosystems). The results were analysed by Sequencher 4.1.4 software .P. falciparum parasitaemia of 25% in unexposed (lactase non-persistent). The calculations were done for a significance of 95% and a power of 80%. Statistical analyses were conducted using SPSS 15.0 for Windows (SPSS Inc.) and StatCalc EpiInfo version 6 (CDC). Mann-Whitney U-test was used for comparing parasite densities for different genotypes, which did not follow normal distribution.Each of the malaria variables were grouped into two categories enabling application of chi square test of statistical significance for all variables. Power calculations for the sample size were done assuming a prevalence of -13910 is the main causative mutation for lactase persistence [The enhancer region 14 kb upstream of the lactase gene, where the previously identified functional polymorphisms are located, was sequenced. In populations with European origin, C/Tsistence . In Afri-13910 mutation, which is rare in most African populations, was found with a frequency of 37% in Fulani. Previously it has been detected with a minor allele frequency of 48% in Fulani from Sudan [In the sequenced region, four single nucleotide polymorphisms (SNP) were detected in the Fulani and Dogon under investigation Table . C/T-139-14107 previously reported in Ghana [-13915 is the founder lactase persistence variant among Urban Saudi population [-13906, in the immediate proximity of the main European mutation, was found in six of the Fulani samples.The mutation G/Ain Ghana  had a mipulation , and wasOf the 79 individuals belonging to the Dogon ethnic group, only one individual had a variant in the analysed region. This was a heterozygote of the globally most common mutation C/T at locus -13910.-13910 variant in Fulani was analysed  and in 51% of lactase persistence (CT/TT) .To assess the hypothesised association between lactase persistence and malaria, the occurrence of the main functional C/Ted Table . At thisAmong the subjects with the CC genotype, splenomegaly was seen in 39% (25/65) and among the TC/TT genotypes in 35% (34/97) of the Fulani. Anemia was present in 56% (36/64) of CC individuals and in 58% (56/97) of TC/TT individuals. These differences were not statistically significant (p > 0.05).P. falciparum with microscopy were compared with different genotypes. The mean parasite density for the lactase non-persistance CC-13910 genotypes was 4,755 parasites/μL and for the CT/TT-13910 genotypes 2,584 parasites/μL . The geometric means were 1,290 parasites/μL and 1,360 parasites/μL, respectively.Parasite densities of the Fulani positive for Assuming that all the detected SNPs in the lactase enhancer region would be functional, all patients with at least one polymorphic allele were pooled and then compared against the wild-type homozygotes Table . The difSeveral studies in Burkina Faso have indicated that people belonging to the Fulani tribe are less parasitized, have fewer clinical episodes of malaria, and higher levels of anti-malarial antibodies as compared to the sympatric ethnic groups Mossi and Rimabé, despite similar transmission intensity -21. In MThe reason for malaria resistance in Fulani is still unknown, but suggested mechanisms include a functional deficiency of regulatory T cells  and diff-13910 is the main causative mutation for lactase persistence [-13910 was by far the most common polymorphism with an allele frequency of 37%. This means that 60% of the Fulani carry the lactase persistence genotype. Possibly the above described sympatry with other ethnic groups is responsible for the lower minor allele frequency found in this study than the one described by Enattah et al [The hypothesis of the present study postulates that the increased ability to drink milk in individuals with lactase persistence genotypes may offer protection against malaria infection. In human evolution, lactase activity has been maintained by mutations in the enhancer region of the lactase gene in those populations which herd livestock and will thus benefit substantially from the ability to use dairy products as part of the adults' diet. Several such mutations have enriched over time in isolated livestock herding populations, although smaller than expected differences among these genetically diverse populations have been reported . In popusistence . In Afriah et al . Three oP. falciparum parasitaemia in asymptomatic Fulani is more common in individuals with lactase non-persistence genotypes. However, this finding was not statistically significant as this study did not have power to detect a difference with odds ratio smaller than 2.7. The rarity of the SNPs among Dogon and among other non-pastoralist populations in Africa further supports the hypothesis. Asymptomatic parasitaemia was used as the main phenotype, assessment of the suggested protective effects of lactase persistence on clinical malaria episodes, severe malaria or malaria mortality was not applicable. The novel T/C-13906 polymorphism detected in this study cannot be claimed to be functional without lactose tolerance test or intestinal biopsy on individuals with this variant. However, the location only 4 bp from the main mutation supports the possibility of functionality. In addition, the lack of polymorphisms in the Dogon subjects suggests that the variability in the Fulani lactase enhancer region is caused by ongoing selection for which functionality is a prerequisite.Results show that p-aminobenzoic acid (PABA), as milk is lacking PABA in contrast to normal diets [Plasmodium species can synthesize PABA de novo, but probably not in sufficient amounts to survive without dietary intake by the host [The potential protective role of milk consumption in malaria infections has been debated for more than half a century. Studies in the 1950s indicated that a cow milk diet has a suppressive effect on malaria infections in rats, mice and monkeys . The supal diets . Plasmodthe host .Infants under six months of age have a lower incidence of severe malaria than older children, which mainly has been explained by maternal antibodies acquired through breast-feeding. Deficiency of PABA might be another reason, especially for children exclusively breast fed. Additionally, there might be other still unidentified anti-malarial properties of milk, as it consists of numerous probiotics, innate immunity components , cytokines, chemokines and anti-inflammatory factors .The relationship between nutrition and malaria seems to be complex. In the 1990s it was suggested that lactase non-persistence would have been selected by malaria, like beta-thalassaemia and glucose 6-phosphate dehydrogenase deficiency ,32. ThisThe potential protective role against malaria infection achieved by abundant milk consumption, allowed by lactase persistence genotypes, could have many explanations. First, milk might provide a generally increased nutritional status being rich in energy, proteins and fatty acids. Secondly, milk consists of a large number of immunomodulating components. Third, a diet dominated by dairy products might lead to a relative PABA deficiency, protecting from malaria. Additionally, there might be still unrecognized factors in milk providing protection against malaria and other infections.Plasmodium falciparum parasitaemia in asymptomatic Fulani is more common in individuals with lactase non-persistence genotypes, but this difference is not statistically significant. A novel polymorphism in the LCT enhancer region was discovered with potential functional importance. The lack of LCT enhancer region polymorphisms in the sympatric Dogon suggests positive selection in the Fulani who have access to dietary milk. The potential immunoprotective properties of dietary cow milk as a reason for the partial malaria resistance of Fulani warrant further investigation.The authors declare that they have no competing interests.IL extracted the DNA from blood samples stored in Stockholm University and performed sequencing and analysis of sequence data in University of Helsinki. VH introduced the original idea for study and drafted the manuscript together with IL. IJ supervised laboratory work in University of Helsinki and provided expertise in lactase genetics during writing of the manuscript. SM provided insight to the discussion of immunologic properties of milk and infection biology of malaria. EI and MTB supervised laboratory work in Stockholm University. Furthermore, MTB provided the blood samples while EI participated in the collection of the samples. BM, AD and OKD provided expertise of the research material, described the study population, and collaborated in organizing the field work for collecting the data. All authors read and approved of the final manuscript.


Whether this translates into a clinical benefit in HIV-infected patients residing in malaria-endemic regions is unknown. We studied the incidence of malaria, as defined by blood smear positivity or a positive Plasmodium falciparum histidine-rich protein 2 antigen test, among 444 HIV-infected women initiating antiretroviral treatment (ART) in the OCTANE trial . Participants were randomized to treatment with PI-containing vs. PI-sparing ART, and were followed prospectively for ≥48 weeks; 73% also received cotrimoxazole prophylaxis. PI-containing treatment was not associated with protection against malaria in this study population.HIV protease inhibitors (PIs) show antimalarial activity  The majority of HIV-infected individuals live in malaria-endemic regions, and available evidence suggests that the interaction between HIV and malaria infection is complex, synergistic and bidirectional. Reported effects include increased HIV replication resulting from immune activation in vitro activity against the two most important malaria parasites, Plasmodium falciparumP. vivaxin vivo activity against rodent malaria parasites Plasmodium falciparum-infected erythrocytes HIV protease inhibitors (PIs) have moderate To explore the effect of PI-based ART on HIV-infected people residing in malaria-endemic regions of sub-Saharan Africa we undertook a retrospective study examining the incidence of malaria in a group of women enrolled in AACTG 5208 P. falciparum histidine-rich protein two (HRP2) by antigen-capture immunoassay forms the basis of most rapid diagnostic tests for falciparum malaria. This water-soluble protein is released into the circulation of individuals infected with P. falciparum, and has been shown to persist in the circulation after clinical symptoms of malaria have resolved and parasites have been cleared from the circulation. It has been reported to remain in the blood for 7 days following treatment in 62–99% of patients, for 14 days in 35–98% of patients, and for 28 days in 27–92% Detection of The OCTANE study (AACTG 5208) consisted of two concurrent randomized trials; detailed methods and primary results have been published P. falciparum Histidine-rich protein 2 [HRP2]) was detected in the plasma samples that were collected at study visits. All available samples from each participant at the relevant sites were tested for HRP2 antigenemia using a commercially available ELISA kit . Each plasma sample was tested in duplicate as stated in the manufacturer's instructions with positive and negative controls on every plate. Evaluation of this kit before the study indicated that it was more sensitive than other commercially available plate-based HRP2 ELISA kits and was between 30 and 120 fold more sensitive than malaria rapid diagnostic kits (data not shown).The current study includes data obtained from participants enrolled at the six OCTANE study sites where malaria transmission has been reported to occur . Study subjects were considered to have malaria if they had a positive blood smear, a positive malaria rapid diagnostic test (RDT), or if malaria antigen (http://www.openepi.com). Multivariate analyses were not performed given the small numbers of samples with a confirmed diagnosis of malaria.As malaria was not detected in two of the study sites and some participants changed ART regimen and/or received cotrimoxazole, a drug with known prophylactic activity against malaria in HIV-infected populations HRP2 antigenemia was assessed by examining available plasma samples totaling 3,516 from 444 women. All available samples where a diagnosis of malaria had been made by blood smear, on-site rapid diagnostic test or HRP2 ELISA where also tested for malaria in our laboratory using immunochromatographic rapid diagnostic tests . An additional 113 samples from these 444 women were tested for malaria during the original study (on site blood smear or RDT). As blood smear slides were not retained these diagnoses could not be revalidated. Therefore, laboratory data for malaria were available from a total of 3629 samples from 444 subjects  of the 3629 samples tested  of study subjects were taking daily cotrimoxazole prophylaxis at the time of blood sampling . While tIn this study, we observed a low incidence of malaria as defined by positive malaria smears, RDTs, and antigenemia, and did not find that PI-based ART was associated with a lower incidence of malaria. Fifty three (20%) of the women in the study had at least one episode of laboratory-confirmed malaria over the study interval, 34 (64%) of these women were receiving PI-based ART at the time of malaria diagnosis .Although the results indicate that PI therapy did not exert a protective effect against malaria in this population, a number of circumstances that may have masked a protective effect should be considered. In the first instance, it is possible that the low numbers of malaria diagnosis made in our cohort underpowered the study and prevented the identification of a protective effect. While this study was predicted to have sufficient power to answer our question when it was first designed, there was a significant reduction in the prevalence of malaria in many parts of Africa in the interval between design and study execution The high rate of cotrimoxazole prophylaxis (80%) observed in our study population, an intervention with a protective effect against malaria in HIV-infected populations, may have also impacted our results by reducing the episodes of malaria. While pharmacokinetic interactions are also possible when drugs are co-administered, and are a particular concern with ritonavir, a drug well known to affect the pharmacokinetics of many drugs including antimalarials, an interaction of this drug with cotrimoxazole seems unlikely to be an explanation for any lack or antimalarial effect observed among subjects in the current study. Both groups receiving cotrimoxazole, regardless of their ART treatment regimen had reduced episodes of malaria .A pharmacodynamic interaction may be associated with the inability of PI-based ART to protect the current cohort of women from malaria infection. While the primary mode of action of cotrimoxazole is understood, the antimalarial mode of action of the PIs is still not known. A better understanding of the basis of the antimalarial effect of PIs may enable prediction of clinically significant pharmacodynamic interactions. Both cotrimoxazole and individual PIs may also have off-target effects that alter the activity of the other drug or additional drugs that may be used to treat HIV-infected patients with opportunistic pathogens. These interactions must be understood to ensure the use of the most effective drug treatment regimens. The ability of the PIs to modulate aspects of the immune system, decrease the expression of CD36 and interfere with the phagocytosis of parasitized erythrocytes Indirect antimalarial benefits, that were not examined in this study, including reduced cytoadherence


Plasmodium falciparum infection in childhood. Therefore it is important to understand the pathology underlying the development of CM and SMA, as opposed to uncomplicated malaria (UM). Different host responses to infection are likely to be reflected in plasma proteome-patterns that associate with clinical status and therefore provide indicators of the pathogenesis of these syndromes.Cerebral malaria (CM) and severe malarial anemia (SMA) are the most serious life-threatening clinical syndromes of Plasma and comprehensive clinical data for discovery and validation cohorts were obtained as part of a prospective case-control study of severe childhood malaria at the main tertiary hospital of the city of Ibadan, an urban and densely populated holoendemic malaria area in Nigeria. A total of 946 children participated in this study. Plasma was subjected to high-throughput proteomic profiling. Statistical pattern-recognition methods were used to find proteome-patterns that defined disease groups. Plasma proteome-patterns accurately distinguished children with CM and with SMA from those with UM, and from healthy or severely ill malaria-negative children.We report that an accurate definition of the major childhood malaria syndromes can be achieved using plasma proteome-patterns. Our proteomic data can be exploited to understand the pathogenesis of the different childhood severe malaria syndromes. Plasmodium falciparum has an estimated annual global disease burden of 300 million clinical episodes, leading to one million deathsHuman malaria caused by P. falciparum blood stage parasitemia with coma, severe anemia or respiratory distress Cerebral malaria (CM) and severe malarial anemia (SMA) are the major severe disease syndromes in African children with a high level of mortality in the under-five age group. The current WHO case definitions for severe malaria combine Previous studies have attempted to define malaria syndromes by studying plasma correlates of severity using reductionist approaches with variable successP. falciparum childhood malaria from the uncomplicated cases and also from well or unwell children without malaria.We hypothesized that the plasma proteome during malaria infection reflects the molecules that are modulated as the severe status is established. In the present study we show that distinctive plasma proteome-patterns distinguish the different severe presentations of Parents or guardians of study participants gave informed written consent. This research was approved by the joint ethics committee of the College of Medicine of the University of Ibadan and the University College Hospital Ibadan.All study participants were recruited under the auspices of the Childhood Malaria Research Group (CMRG) at the 600-bed tertiary hospital University College Hospital (UCH) in the city of Ibadan, Nigeria in west sub-Saharan Africa. Ibadan is a densely populated urban setting with a population of 2.5 million inhabitants. Ibadan has a lengthy 8 months rainy season from March to October with malaria transmission and severe disease present all year round (holoendemic).The study site is located in the UCH Ibadan Department of Paediatrics. We screen about 12,000 children attending the hospital (ill and well) for malaria parasites per year. Our studies report 11.3% SMA and 19.7% CM admissions in the parasitized children under five years of age The participants in this study were recruited during 2006 to 2009 as part of a larger prospective case-control study of childhood severe malaria currently ongoing under the auspices of the CMRG. This case-control study was divided into a Discovery Cohort consisting of those patients recruited during 2006 to 2008 and a Validation Cohort made up of those recruited in the 2008 to 2009 period.Malaria parasites were detected and counted by microscopy following Giemsa staining of thick and thin blood films P. falciparum malaria P. falciparum parasitemia with normal cerebrospinal fluid. A Blantyre coma score less than 2 was used to assess coma status. Children with hypoglycemia were excluded from the study. Added to the strict clinical and laboratory definitions of CM, our study patients recover consciousness after effective antimalarial therapy. We excluded from this study those CM patients who died. Our overall mortality rate for CM is of the order of 10%. Severe malarial anemia cases were defined as conscious children with Packed Cell Volume (PCV) less than 16% in the presence of P. falciparum parasitemia. We excluded from this study those SMA patients who died. Our overall mortality rate for SMA is less than 1%. Uncomplicated malaria cases were defined as febrile children with P. falciparum parasitemia who did not require hospital admission. Our study was designed to discover and validate plasma proteome changes in dichotomous cases for which we only included those children with CM and UM with PCV greater than 20% , Severe Malarial Anemia (SMA) and Cerebral Malaria (CM). The malaria-negative children, the controls, are Disease Control (DC) and Community Controls (CC). We followed the WHO criteria for severe than 20% . We exclThe DC group consists of malaria-negative children with infectious diseases such as meningitis, otitis media, diarrhea and upper respiratory tract infections. It also includes mild to moderately anemic children and children admitted for surgery.g, 10 minutes), aliquoted and frozen at −80°C no later than 4 hours following collection.Participants’s clinical data were collected using a malaria-tailored questionnaire designed by the CMRG. A 2.5 ml blood sample was obtained from each participant in an EDTA blood collection tube for subsequent plasma separation. Blood samples were kept on ice and transferred to the central malaria laboratory. Plasma for this study was harvested by centrifugation (1000 Packed cell volume (PCV) was measured using the microhaematocrit method Malaria parasites were detected and counted by microscopy following Giemsa staining of thick and thin blood films m/z) clusters were detected and clustered using ProteinChip® Datamanager Client 4.1 software (BioRad). Mass spectrometer calibration was performed using All-in-1 Peptide and Protein calibrants (Bio-Rad). Reproducibility was determined by measuring the inter-ProteinChip® coefficient of variation (CV) for the quality control spectra, based on all peaks in the spectrum with intensity >1 µA. Overall interchip CV for the quality control sample was 20%, consistent with similar studies.Crude plasma was profiled using Surface Enhanced Laser Desorption/Ionization-Time Of Flight (SELDI-TOF) mass spectrometry. All plasma samples underwent two freeze-thaw cycles prior to analysis. Plasma samples were coded, blinded and randomized before application onto the following solid-phase fractionation surfaces (ProteinChip® arrays Bio-Rad): weak-cation exchange (CM10), strong-anion exchange (Q10) and reverse phase (H50) as previously described Liquid-phase anion-exchange fractionation of plasma samples was carried out using the ProteinChip® Fractionation Kit (Bio-Rad) according to the manufacturer’s instructions with a Biomek 3000 Laboratory Automation Workstation. Six fractions were obtained from each sample eluting at pH 9.0 (f1), pH 7.0 (f2), pH 5.0 (f3), pH 4.0 (f4), pH 3.0 (f5) and organic phase (f6).We selected subsets of the most relevant mass clusters in the discovery cohort groups using the weighted Kernel-based Iterative Estimation of Relevance Algorithm Our multivariate statistical tests included testing against age or sex to ascertain that significant pattern changes in the proteome were not dependent on those variables in the population studied.To visualize the covariance within the mass spectral profiles we used Principal Component Analysis (PCA). PCA encapsulates the covariance within a set of variables by extracting a ranked set of independent factors or principal components. The first 3 components encompass a high proportion (∼95%) of the informational content of a multivariate dataset. We plotted each patient with respect to the first 3 components, in 3-dimensional space, color-coding according to patient group.A total of 946 children participated in this study as part of the discovery and validation case-controlled cohorts. The discovery cohort comprised of 367 malaria-positive children with either Cerebral Malaria (CM), Severe Malarial Anemia (SMA) or Uncomplicated Malaria (UM), and 289 malaria-negative children who were either Disease Controls (DC) or Community Controls (CC) . The valTo compare the proteome-patterns of the study groups, we fractionated plasma samples by three different chromatography procedures on solid-phase surfaces  followed by Time-Of-Flight mass spectrometry. The resulting mass spectra from each of the surfaces contained a series of mass/charge ratio (m/z) peak clusters, each representing a protein of a particular mass. A set of proteins that are present, absent or at a different level in the samples defines a proteome-pattern that may discriminate between two or more of the study groups. To discover such patterns we applied statistical pattern recognition algorithms to the profiles and the selected number of discriminating proteins for each of the pairwise group comparisons is shown in Overall, 22 to 33 proteins composed the discriminatory patterns with accuracies above 90% across the three comparisons  on the selected proteins to visualize the separation of patient groups. The CC group clustered tightly together  indicatiWe simplified further the complexity of the plasma proteome by high-throughput liquid-phase anion-exchange fractionation followed by solid-phase weak-cation exchange fractionation prior to protein mass determination in the spectrometer on a subset of the samples. We assessed the discriminatory accuracy of relevant proteins obtained from each of the six anion-exchange fractions . The redWe carried out an overall analysis of plasma proteome pattern overlap by comparing the discovered sets of proteins that discriminate UM, CM, SMA  and DC  ill children from the malaria-negative well children CC  ratio can be exploited to guide the identification of the set of proteins relevant for discrimination between syndromes. Plasma proteome profiling has been used to define a variety of disease statesOur large cohorts allowed us to statistically validate the pattern-based proteome definitions of the major childhood malaria syndromes. Although the mass spectrometry platform used in our study does not provide direct molecular identification, the chromatographic chemistry used and the mass-to-charge Click here for additional data file.Data S2Liquid-phase fractionation data.(XLS)Click here for additional data file.


P. falciparum malaria, urinary tract infections and bacteremia among under-fives presenting with a febrile illness at the Makongoro Primary Health Centre, North-Western Tanzania.In malaria endemic areas, fever has been used as an entry point for presumptive treatment of malaria. At present, the decrease in malaria transmission in Africa implies an increase in febrile illnesses related to other causes among underfives. Moreover, it is estimated that more than half of the children presenting with fever to public clinics in Africa do not have a malaria infection. Thus, for a better management of all febrile illnesses among under-fives, it becomes relevant to understand the underlying aetiology of the illness. The present study was conducted to determine the relative prevalence and predictors of From February to June 2011, a cross-sectional analytical survey was conducted among febrile children less than five years of age. Demographic and clinical data were collected using a standardized pre-tested questionnaire. Blood and urine culture was done, followed by the identification of isolates using in-house biochemical methods. Susceptibility patterns to commonly used antibiotics were investigated using the disc diffusion method. Giemsa stained thin and thick blood smears were examined for any malaria parasites stages.P. falciparum malaria and bacteremia respectively. In general, 11.5%  of the children had two infections and only one child had all three infections. Predictors of urinary tract infections (UTI) were dysuria  and body temperature (40-41 C) . Predictors of P. falciparum severe malaria were pallor  and convulsion . Escherichia coli were the common gram negative isolates from urine  and blood . Escherichia coli from urine were 100% resistant to ampicillin, 97% resistant to co-trimoxazole, 85% resistant to augmentin and 32.4% resistant to gentamicin; and they were 100%, 91.2% and 73.5% sensitive to meropenem, ciprofloxacin and ceftriaxone respectively.A total of 231 febrile under-fives were enrolled in the study. Of all the children, 20.3% , 9.5%  and 7.4%  had urinary tract infections, Escherichia coli was the common cause of febrile illness in our setting. Improvement of malaria diagnosis and its differential diagnosis from other causes of febrile illnesses may provide effective management of febrile illnesses among children in TanzaniaUrinary tract infection caused by multi drug resistant  P. falciparum prevalence rates in children aged 2-10 years from 37% before the year 2000 to 17% after 2000 has been documented [The etiology of fever in areas where it can be due to multiple causes brings serious challenges in management, especially in areas where presumptive treatment is the norm . For mancumented . In addicumented . AlternaIt is estimated that more than half of the children presenting with fever to public clinics in Africa do not have a malaria infection . Thus, pP. falciparum malaria, urinary tract infections (UTI) and bacteremia among under-fives presenting with febrile illnesses at the Makongoro Health Centre, North-Western Tanzania, thus establishing the differential of fever attributable to either condition. In addition, the study investigated the antimicrobial susceptibility pattern of the isolates from blood and urine samples.At the Makongoro Health Center in Mwanza, Tanzania, presumptive treatment of all fever with antimalarial and antibiotics is a common practice and is supported by the guidelines of the Integrated Management of Childhood Illness (IMCI) programme. IMCI was introduced in 1996 in primary health care facilities ,13 whereThe study was conducted at the Makongoro Health Center, a primary health care facility located in Nyamagana District within Mwanza City, Tanzania. Approximately 17,431 people seek health services at this health facility per year. Over 50% of the patient population seeking services is under-fives.Mwanza City lies on the southern shores of Lake Victoria at an altitude of 1140 m above sea level. The basin is characterized by a tropical and subtropical climate with temperatures varying from 18°C to 20°C during the rainy season and 26°C to 30°C during the dry season. Annual rainfall ranges from 700-1000 mm with long rain seasons occurring from February to May. The Lake Victoria basin is located in the holoendemic area for malaria transmission with intense transmission observed during the long rain season (February-May) and at the end of the long rain season (May-June).A cross-sectional analytical survey was conducted from February to June 2011. During this period, blood and urine cultures were obtained from all under-fives who fulfilled the inclusion criteria. In addition, thin and thick blood smears were obtained for malaria diagnosis.All children aged two months to five years of age presenting with a febrile illness at the health facility were recruited for the study. The inclusion criteria were age between two months to five years, fever (defined as axillary temperature ≥ 37.5°C) and parents/guardians/caregivers willing to give written informed consent for their children to participate in the study. On the other hand, children were excluded if they presented with a history of use of antimalarials or antibiotics for the past seven days.The required sample size for the study was calculated using a formula for a single population proportion. Kish and Leslie formula  was usedBlood culture was performed as previously described in the literature . BrieflyUrethral catheterization methods were used in infants and pre-toilet trained children to collect urine samples ,20. For Antimicrobial susceptibility pattern was determined by disk diffusion method according to CLSI . The folPlasmodium parasite, whereas the thin film was used to identify the Plasmodium species. A slide was considered negative for malaria parasite if no parasites were seen in at least 100 oil-immersion fields on the thick film [A drop of venous blood obtained for culture was used for preparation of thick and thin blood films for malaria parasites examination. The prepared blood slides were fixed in absolute ethanol (only the thick smear) and stained in 10% Giemsa . The slides were examined under a light microscope using objective × 100 under oil-immersion for any malaria parasite stages. The thick film served to confirm the presence or absence of the ick film . Parasitick film .For quality control purposes, all blood smears that were positive for malaria and a 10% sample of slides that were considered negative were subsequently re-examined by a second independent microscopist. In case of any disagreement between the first and second microscopist, the issue was resolved by a third microscopist. Malaria parasitaemia was categorized as follows: 1-500/μL, 501-1000/μL and > 1,000/μL .All patients enrolled in the study were managed according to the Tanzanian guidelines for malaria and bacterial infection. Antibiotic therapy was reassessed according to culture results and sensitivity patterns.Ethical approval was obtained from Weill-Bugando University College of Health Sciences/Bugando Medical Centre, Institution Review Board. Further ethical clearance was obtained from the regional health and administrative authorities of Mwanza region. For participation of the underfives, parents/guardians/caregivers were informed about the study objectives and gave informed written consent prior to inclusion into the study.2). Univariate and multivariate model were developed to determine the independent predictors  of P. falciparum malaria, urinary tract infections and bacteremia. The model reconsidered the significance of each risk factor by stepwise backward logistic regression at a P-value of < 0.2 for inclusion in the final multivariate model. Odds ratios with 95% Confidence Intervals were used to measure the strength of association at statistical significance level of P < 0.05.Data entry was done using Microsoft Access  and analyzed using STATA version 11 . Statistical test between dependent and independent variables was done by using Chi-squared test  were recruited  had severe malaria as per WHO definition [2 = 1.8, P = 0.2). Among the P. falciparum positive children, 4.5% (1/22) had 1-500 parasites/μl, 4.5% (1/22) had between 501-1,000 parasites/μl and 90.9% (20/22) had ≥ 1000 parasites/μl.Parasitological diagnosis of malaria parasites revealed that out of the 231 underfives, only 9.5%  had a blood slide for malaria parasite positive. All the malaria infections were due to finition . The pre2 = 1.3, P = 0.3).The proportion of blood cultures that yielded a clinically significant positive result was 7.4%  among febrile under-five children. The distribution of bacteremia by sex was 9.5% and 5.6% in male and female children respectively. However, the observed difference in the prevalence of bacteremia between sex was not statistically significant  of the positive blood cultures. Among them, the majority were Escherichia coli  and the other isolates were Klebsiella pneumoniae (10%), Salmonella species  and Gram Negative Rods . Of all the positive blood cultures, gram positive were isolated in 41.2%, with Staphylococcus aureus  and coagulase-negative Staphylococci  being the only isolates.versus female: 12.4% versus 26.9%, P < 0.01). Escherichia coli were isolated from the majority of urine samples 34 . Other isolates were K. pneumoniae (21.28%), Proteus species (4.26%) and Pseudomonas spp (2.13%).The overall prevalence of urinary tract infection was 20.3% (47/231) with female under-fives having the highest prevalence  had two infections with the majority having co-infections of P. falciparum malaria and urinary tract infection . Other dual infections observed were between urinary tract infections and bacteremia  and P. falciparum malaria and bacteremia .In general, 37.6%  of all the children had Escherichia coli isolated from blood and urine samples was observed. In addition, Escherichia coli from urine were 32.4% resistant to gentamicin. However, the Escherichia coli and Klebsiella spp isolates from urine were between 74%-100% susceptible to meropenem, ceftriaxone, ciprofloxacin and ceftazidime, while those from blood (E. coli isolates) were 100% sensitive to gentamicin, ciprofloxacin, ceftriaxone and meropenem.A high prevalence of resistance (97%-100%) to ampicillin and co-trimoxazole especially to Staphylococcus aureus isolates were 75% resistant to clindamycin and erythromycin.For gram positive isolates from blood samples, P. falciparum malaria (parasites ≥ 1000 parasites/μl) were strongly associated with young age, body temperature (40 C - 41 C), palmar pallor and convulsion (Table P = 0.025) and convulsion  remained significantly associated with severe malaria.In univariate analysis, P < 0.001), body temperature (40 C-41 C)  and female sex  remained independently associated with urinary tract infections , female sex and abdominal pain were found to be associated with urinary tract infections. On multivariate analysis only; dysuria (OR = 12.51, 95%CI, 4.28-36.57, P. falciparum malaria and bacteremia among febrile under-fives attending a primary health care facility in North-Western Tanzania. The study has shown that the three infections are prevalent among febrile underfives and a number of factors remained independently associated with these infections.The present study documents the frequent occurrence of urinary tract infections, P. falciparum. This species is known to cause almost 95% of all malaria cases in the country [P. falciparum malaria observed in the present study was lower than the 12% recently reported among under-fives in Western Tanzania [P. falciparum-positive blood film [P. falciparum prevalence.In this study, 9.5% of all the study population had malaria positive slides, of which all were infections with  country . The preTanzania . In Gaboood film . The varEscherichia coli has been found to be the commonest organism isolated from urine samples and the isolates have been shown to account for up to 75% of urinary tract infections in all pediatric age groups [Klebsiella spp, Proteus spp and Pseudomonas species [The prevalence of urinary tract infections of 20.3% observed among the under-fives of the present study was considerably higher than 3.3% and 9% reported in developed countries  and lowee groups , followe species ,21,28.The bacteremia prevalence observed here was comparable to other similar studies in Africa whereby the prevalence ranged between 7.1 - 12.2% ,29,30. HHaemophilus influenzae and Streptococcus pneumoniae were not isolated in this study which could partly be explained by the technique used in our study. In the case of Haemophilus influenzae, the use of HIB vaccine among under-fives and seasonal variation could possibly explain the negative result.In contrast to studies conducted in other developing countries , gram-poP. falciparum malaria and urinary tract infections, and least had urinary tract infections and bacteremia. These findings were similar to previous findings in African settings [Co-occurrences of multiple diseases in an African setting are norm rather than exception . In the settings ,34,35.Escherichia coli; this isolate was found to be resistant to commonly used antibiotics like ampicillin, co-trimoxazole, amoxycillin/clavulanate and gentamicin. In addition, more than 20% of these strains were resistant to third generation cephalosporins. A recent similar study within the region has reported a resistance rate of up to 98.4% and 95.3% of Escherichia coli isolated from urine samples among febrile children [Klebsiella pneumoniae as compared to E. coli [. Fortunately, all the organisms causing urinary tract infections and bacteremia observed here remained sensitive to meropenem and about 95% of them are still sensitive to ciprofloxacin. As a matter of fact, meropenem is an expensive injectable beta-lactam antibiotic and is not available in many drug stores in Tanzania. It is therefore less misused. As for ciprofloxacin, it is not commonly used in children below 12 years. As this study recruited patients were from primary health care facility, the high resistance rate of Escherichia coli to ampicillin, co-trimoxazole and augmentin is possibly indicating a problem with these particular isolates within the community. This could be explained by an inappropriate use of these antibiotics in the community, i.e. wrong prescription or poor adherence. Furthermore, the 32.4% and 26.5% resistance rates of Escherichia coli to gentamicin and ceftriaxone is not low for community isolates; this observation calls for a continuous local epidemiologic surveillance to monitor the resistance patterns of these antibiotics within the community.The commonest isolate from urine and blood was children . The stu E. coli . FortunP. falciparum malaria in under-fives [Several factors have been independently associated with severe er-fives ,37. The er-fives ,38. In oer-fives .A number of previous studies have reported various demographic, clinical signs and symptoms associated with urinary tract infections in children ,28,39. FOur study is however subject to some limitations. Using a cross-sectional survey design makes it difficult to clearly show the temporal relationship between urinary tract infections, malaria and bacteremia with other variables. In addition, HIV status was not investigated in the present study.Escherichia coli was identified as the most common cause of fever, which co-existed with malaria and bacteremia. Several independent factors were found to be associated with UTI and malaria. The isolates identified from urine were highly resistant to commonly used antibiotics in our setting. The growing resistance to commonly used antibiotics calls for the necessity of a continuous epidemiologic surveillance in primary health care facilities in Tanzania. In addition, improvement of the diagnosis of UTI and other causes of febrile illnesses will provide more effective measures in the management of febrile illnesses among under-fives in TanzaniaIn conclusion, urinary tract infections caused by multi drug resistant The authors declare that they have no competing interests.BM participated in collecting specimens, collecting clinical data and follow up of the patients, SEM participated in design of the work, performing microbiological procedures, data analysis, interpretation of data and writing of the manuscript, AH participated in recruitment, patients management and manuscript writing, HM participate in the design of the work, data analysis and manuscript writing, DM participated in interpretation of the results and manuscript writing. All authors have read and approved the final manuscript.


Plasmodium species.There are few detailed etiologic studies of severe anemia in children from malaria-endemic areas and none in those countries with holoendemic transmission of multiple P. falciparum infection (19.4 (6.7–62.6)), vitamin A deficiency (13.5 (5.4–37.7)), body mass index-for-age z-score <2.0 (8.4 (2.7–27.0)) and incomplete vaccination (2.94 (1.3–7.2)). P. vivax infection was inversely associated (0.12 (0.02–0.47), reflecting early acquisition of immunity and/or a lack of reticulocytes for parasite invasion. After imputation of missing data, iron deficiency was a weak positive predictor (6.4% of population attributable risk).We examined associates of severe anemia in 143 well-characterized Papua New Guinean (PNG) children aged 0.5–10 years with hemoglobin concentration <50 g/L , malnutrition and genetic predisposition are prevalent, we studied children from Papua New Guinea who were hospitalized with severe anemia and healthy non-anemic community-based controls. Factors associated with severe anemia were parvovirus B19 infection, falciparum malaria, vitamin A deficiency, low body mass index for age and incomplete vaccination. Vivax malaria was inversely associated reflecting early acquisition of immunity or perhaps lack of circulating red blood cells for parasite invasion. Iron deficiency was only a weak positive predictor. These data show that severe anemia is multifactorial in Papua New Guinean children, strongly associated with under-nutrition and certain infections, and potentially preventable through vitamin A supplementation and improved nutrition, completion of vaccination schedules, and prevention or active treatment of malaria. Plasmodium falciparum is the major cause in malaria-endemic areas 12 were significantly associated with severe anemia. Iron deficiency, considered the most common cause of anemia worldwide P. falciparum was only associated with severe anemia in children in urban areas with seasonal transmission but not in surrounding holoendemic rural sites Severe anemia is a common reason for pediatric hospitalization in developing countries P. falciparum have varying effects on the prevalence of severe anemia. There have been inconsistent reports of associations between parvovirus B19 (B19V) and severe anemia P. vivax infections most commonly present as severe anemia in West Papua P. vivax parasitemia appears to attenuate the post-treatment nadir in hemoglobin in Thai adults with co-existent P. falciparum infections Infections other than P. falciparum, P. vivax and B19V, and a high risk of micronutrient deficiencies.Since available data suggest that specific epidemiologic settings will have their own hierarchy of causes underlying severe anemia, we carried out an etiologic study in Papua New Guinean (PNG) children with a high incidence of red cell polymorphisms such as alpha-thalassemia, exposure to infections with P. falciparum and P. vivax with approximately 50 infective bites per child per year The study was performed in Madang Province on the north coast of mainland PNG. Most of the population of 450,000 are subsistence farmers and their families who live on lowland coastal plains. There is hyperendemic transmission of Approval for the study was obtained from the PNG Institute of Medical Research Institutional Review Board and the Medical Research Advisory Committee of the PNG Health Department. Written informed consent for participation was obtained from parent(s)/guardian(s) and, where possible, children gave assent to study procedures.Children with severe anemia (hemoglobin concentration <50 g/L) were identified as part of an observational study of all children aged 0.5–10 years admitted to the pediatric ward of Modilon Hospital, the tertiary referral hospital for Madang Province, between October 2006 and November 2009 A standardized case report form was completed by trained clinical research nurses who detailed each child's demographic details, history of current and/or past illness, examination findings, results of laboratory investigations, treatment and outcome. Vaccination history was identified from the health record book when this was available. It was assumed children without a documented vaccination history were unvaccinated. The expanded programme of immunization in PNG recommends two doses of vitamin A at 6 and 12 months In the children with severe anemia, between 5 and 10 mL of venous blood were drawn if the clinical situation allowed, and 4–6 mL blood were collected from the healthy control children. Initial hemoglobin concentrations for cases and controls were measured using HemoCue . Two skilled microscopists independently examined thick blood smears. Parasite density was calculated per 200 leukocytes using an assumed peripheral blood leukocyte count of 8000/µL. A senior microscopist adjudicated discrepant findings. Additional on-site tests in children with severe anemia comprised i) whole blood glucose  and lactate  assay, ii) a full blood count , and iii) blood culture . Light-protected aliquots of plasma from all children were stored at −70°C prior to routine biochemical testing, B19V IgM and DNA assay, and measurement of serum vitamin A concentrations. Cell pellets were stored at −20°C for red cell folate assay, and host and parasite DNA extraction. Serologic testing for HIV, assays for G6PD enzyme activity and genotyping, and stool microscopy for intestinal parasites, were not performed.Children found to have severe anemia were treated in accordance with PNG treatment guidelines 12. Other than vitamin B12  all biochemistry assays were performed on the COBAS INTEGRA 800 platform  using reagents supplied by the manufacturer. Red cell folate concentrations were measured using a 20 µL red cell pellet . Vitamin A concentrations were measured by high performance liquid chromatography with UV detection (wavelength 325 nm) following protein precipitation and liquid-liquid extraction. An internal standard (retinol acetate) was used to correct for extraction recovery. All biochemical assays were monitored for accuracy and imprecision using appropriate internal quality control procedures as under the Quality Assurance Programme of the Royal College of Pathologists of Australasia and satisfying the requirements of external standards (ISO15089:2003). Vitamin B12, folate, vitamin A deficiencies were considered present if concentrations were <150 pmol/L, <260 pmol/L and <0.7 µmol/L, respectively. Iron deficiency was defined as a ratio of sTFR to the log10 of ferritin >5.6 Plasma was assayed for concentrations of electrolytes, urea and creatinine, albumin and total protein, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), total bilirubin, calcium, phosphate, C-reactive protein (CRP), total cholesterol, triglycerides, creatine kinase, ferritin, soluble transferrin receptor (sTFR), vitamin BPlasma was assayed for B19V IgM by EIA kit  and for viral DNA using two specific oligonucleotide primers 2O according to the supplier's instructions. We performed genotypic tests for red cell polymorphisms that are common in coastal Melanesian populations. These included 3.7-Kb and 4.2-Kb α-globin deletions associated with α+-thalassemia, a 27 bp deletion associated with South Asian Ovalocytosis (SAO), a 3 bp deletion in glycophorin C (GLYC) and genotyping of complement receptor-1 (CR1). The methods for the genotypic assays are detailed elsewhere DNA was extracted from 200 µL venous whole blood collected into EDTA anti-coagulant using QIAamp 96 DNA Blood Mini Kit  and eluted in a final volume of 200 µL dHt-test or Mann-Whitney test for parametric and non-parametric continuous variables, respectively, or the Chi-squared test for dichotomous or nominal data. Associates of severe anemia were assessed using backward stepwise logistic regression analysis. Variables other than age were included based on biologic plausibility and P<0.10 on bivariate regression analysis and the most parsimonious model chosen based on Aikake's Information Criterion (AIC).We used the statistical package R for all analyses Ad hoc methods such as replacing a missing value with a sample mean or median creates potential for bias. An alternative approach is multiple imputation (MI) in which each missing value is imputed a number of times (commonly five) using informative prior knowledge of the distribution  of each variable. MI generates a number of complete datasets that facilitate refinement of logistic regression models through comparisons of AIC and statistical testing In conventional logistic regression, a missing value for a single variable means that other valid data for an individual are lost. One hundred and forty three children with severe anemia were recruited see . In the The demographic and anthropometric features of cases and controls are summarized in 12, folate and vitamin A concentrations were all significantly lower in the severe anemia group and more of these children were deficient in each case. Serum vitamin A concentrations were higher in those children who had received both doses of vitamin A when compared to one or no doses . Serum ferritin concentrations were, consistent with serum CRP concentrations and the clinical features, higher in the severe anemia group.The laboratory features of cases and controls are summarized in Klebsiella spp. was cultured after 5 days of hospitalization and was associated with disseminated ascariasis. A second child with severe anemia had acute bacterial meningitis with Hemophilus influenzae type b isolated from CSF. No other invasive bacterial infections were identified.Due to difficulty with venesection, occasional parenteral concerns about the amount of blood to be taken from an unwell child, and prioritisation of on site and other tests requiring relatively small volumes, there was sufficient blood for culture in only 100 children in the severe anemia group (70%). In one of these children, an isolate identified as a P. falciparum, vitamin A deficiency, wasting and incomplete vaccination were also independently associated with severe anemia. The presence of P. vivax was negatively associated with severe anemia. Although included in the initial backward stepwise logistic regression model, maternal education, mosquito bednet use, red cell folate deficiency, vitamin B12 deficiency and alpha-thalassemia genotype did not prove to be independent associates of severe anemia. Iron deficiency was also not significantly associated with severe anemia. When this variable was forced into the most parsimonious model, there was a trend to significance .The summary of the most parsimonious logistic regression model is shown in P. falciparum infection, P. vivax infection, vitamin A deficiency, wasting, and incomplete vaccination. Additionally, all logistic regression models using imputed data included iron deficiency as an independent associate.Severe anemia was multifactorial in the majority of children. Only three of 112 severely anemic children with complete datasets did not have at least one independent risk factor compared with 34 of 113 in the control group, whilst 88.3% and 15.1% of cases had at least two and four risk factors for severe anemia, respectively. After performing MI, logistic regression modelling was performed on each of the five complete datasets. The ORs for each of the variables in each model and the mean values across all 5 models are presented in P. falciparum, vitamin A deficiency, wasting and incomplete vaccination were calculated using the R package ‘pARccs’ with P. vivax included as a confounder. In this analysis, nutritional deficits accounted for 51.3% of attributable risk, specifically vitamin A deficiency (36.0%), wasting (8.9%) and iron deficiency (6.4%). P. falciparum and B19V accounted for 24.5% and 10.4%, respectively. Incomplete vaccination had a partial attributable risk of 9.8%.The significant independent risk factors accounted for 96.0% of population attributable risk. The partial attributable risks for B19V, P<0.0001). Iron deficiency  and B19V  were less common. The median red cell distribution width (RDW) was lower in children with falciparum malaria . Serum bilirubin and CRP concentrations were also higher in this group. Of the red cell polymorphisms tested, those with a homozygote genotype for the GLYCΔex3bp deletion were under-represented amongst the severely anemic children with falciparum malaria .The demographic, clinical, laboratory and genetic features of the 84 severely anemic children with falciparum malaria (58.7%) and those without are shown in P. vivax. The fourth child died following a prolonged illness accompanied by wasting, lymphadenopathy and hepatosplenomegaly. After failing empiric therapy for tuberculosis, a presumptive clinical diagnosis of lymphoma was made.Four (2.8%) children with severe anemia died. Two were comatose on admission and had cerebral malaria in addition to severe anemia. One had acute renal failure and metabolic acidosis due to P. vivax infection was negatively associated with severe anemia. The presence of iron deficiency was predictive in our children only after imputation of missing data, with a relatively small overall contribution to total attributable risk. Although vitamin B12 and folate deficiency were present in 9.0% and 9.5%, respectively, of our severely anemic children, neither was independently associated with severe anemia, which was also the case for adoption, ethnicity, maternal education, bednet use and all of the red cell polymorphisms assessed. The causes of severe anemia in PNG children can, therefore, be considered as comprising two main categories, namely under-nutrition (vitamin A deficiency and wasting) and infection , which are both associated with a mainly hypoproliferative anemia The present study shows that severe anemia is common and multifactorial in PNG children. Parvovirus B19 infection, falciparum malaria, vitamin A deficiency, wasting and incomplete vaccination were the main etiologic factors, while 12 and folate deficiency and severe anemia in the present study, it is possible that these might act in concert with deficiencies in vitamins B6 and E, riboflavin, zinc and copper to substantially reduce erythropoiesis in our under-nourished children Epidemiologic studies have shown an association between low serum vitamin A and anemia in different populations In contrast to the African case-control study P. falciparum and P. vivax parasitemias and severe anemia. As in most studies conducted in sub-Saharan Africa P. falciparum infection in our PNG children. This reflects the increase in incidence of falciparum malaria during first three years of life in PNG children living in holoendemic areas P. vivax in this epidemiologic situation is rapid, such that children have almost complete clinical immunity by the age of 5 years P. vivax infections are common in PNG children aged 2–10 years, they are associated with only small decreases in hemoglobin P. vivax is an important cause of severe anemia in very young infants with an excess risk over that associated with P. falciparumWe found opposing associations between P. vivax reflects the fact that this parasite invades young red cells, especially reticulocytes, which are absent or present in low numbers in established severe hypoproliferative anemias P. vivax itself to the hypoproliferative state P. vivax parasitemia detectable by light microscopy, it does not exclude a significant prior contribution. The importance of antecedent parasitemia as an independent determinant of anemia has been highlighted in an African study of falciparum malaria and acute and relapsing vivax malaria might be an even greater cause of a subsequent presentation with severe anemia An alternative explanation for the inverse relationship between severe anemia and Plasmodium species P. falciparum when there is a mixed infection with P. vivaxP. vivax appears to abrogate the nadir hemoglobin resulting from malaria due to P. falciparumP. vivax provides protection against P. falciparum clinical disease P. vivax being suppressed in severe P. falciparum malaria, either directly by P. falciparumCross-sectional and longitudinal surveys in PNG and neighbouring Vanuatu have shown negative correlations between different P. falciparum also had a lower median RDW, a measure that has been associated with erythropoietic activity where reticulocyte data are unavailable P. falciparumGLYCΔex3bp deletion in the severely anemic P. falciparum group, but this association has not be observed consistently in other studies from PNG The children with severe anemia and falciparum malaria were more likely to be vitamin A deficient than the severely anemic aparasitemic children, but it is unclear whether this represents a cause or consequence of malaria infection Our data confirm and extend the body of evidence implicating B19V as a cause of severe anemia in PNG. Although it had the highest OR (75.8) for an individual, it accounted for only 10% of the population attributable risk. In a previous study of severe anemia in PNG children, B19V and the effects of malaria were the only etiologic factors considered Our study had limitations. Due to socio-cultural barriers to testing, HIV serology was not performed. Despite being a risk factor for severe anemia in African children P. falciparum infection are the most important contributors. Vitamin A supplementation has been shown to reduce malaria infection The present study confirms that severe anemia in PNG children is multi-factorial and suggests that vitamin A deficiency and Checklist S1STROBE checklist of items that should be included in reports of cross-sectional case-control observational studies with detailed referencing of requirements to the text of the paper.(DOC)Click here for additional data file.


There is a paucity of data on malaria among hospitalized children in malaria endemic areas. We determined the prevalence, presentation and treatment outcomes of malaria and anemia among children in two hospitals in Rakai, Uganda.Children under five years hospitalized in Kalisizo hospital or Bikira health center in Rakai district, Uganda between May 2011 and May 2012 were enrolled and followed-up until discharge, death or referral. Data were collected on social-demographic characteristics, current and past illnesses and clinical signs and symptoms. Blood smears, hemoglobin (Hgb) levels and HIV testing were performed from finger/heel prick blood. The associations between malaria infection and other factors were estimated using log-binomial regression to estimate adjusted prevalence risk ratios (aPRR) and 95% confidence intervals (CIs), controlling for clustering at health facilities.2471 children were enrolled. The most common medical presentations were fever (96.2%), cough (61.7%), vomiting (44.2%), diarrhea (20.8%), and seizures (16.0%). The prevalence of malaria parasitemia was 54.6%. Children with malaria were more likely to present with a history of fever  and seizures . Confirmed malaria was significantly lower among girls than boys , HIV infected children (aPRR 0.60 CI 0.52–0.71), and children with diarrhea . The overall prevalence of anemia (Hgb<10 g/dl) was 56.3% and severe anemia (Hgb<6 g/dL) was 17.8%. Among children with severe anemia 76.8% had malaria parasitemia, of whom 93.1% received blood transfusion. Malaria associated mortality was 0.6%.There was a high prevalence of malaria parasitemia and anemia among inpatient children under five years. Malaria prevention is a priority in this population. Plasmodium falciparum, the predominate species, accounts for 99% of all malaria infections in Uganda There are 300–500 million annual clinical cases and nearly one million deaths from malaria in children globally, 90% of which occur in sub-Saharan Africa Plasmodium falciparum resistance to artemisinins and mosquito resistance to pyrethroids in ITNs have been reported and potentially threaten malaria control Scale up of interventions to prevent and/or treat malaria such as use of artemisinin-based combination therapy (ACT), distribution of insecticide treated bed nets (ITNs), intermittent preventive treatment of pregnant women and indoor residual spraying (IRS) have reduced malaria prevalence in some endemic settings In many endemic settings efforts to evaluate the impact of interventions on malaria infections has mainly focused on community-based surveys The study was approved by Uganda Virus Research Institute-Science and Ethics Committee (UVRI-SEC), the Uganda National Council of Science and Technology (UNCST) and the Indiana University Institutional Review Board. Parents or guardians of children enrolled in the study provided written informed consent for their children before the children enrolled into the study. A written parental/guardian consent was also obtained for children who were tested for HIV.This was a health facility-based observational study designed to estimate the prevalence, presentation and treatment outcomes of confirmed malaria cases in children under five years admitted at two health facilities in Rakai, Southwestern Uganda. Rakai district is on a plateau at an altitude ranging between 2500 and 3000 feet and has two rainy seasons. Malaria is meso- to holo-endemic with year round transmission and increased intensity during the rainy seasons or in communities adjacent to lakes and other mosquito breeding sites Children were eligible for this study if they were under five years of age and admitted to the inpatient wards at the two participating facilities. We sought written parental or guardian consent to enroll children within 24 hours of admission, but some children under five years were not enrolled. An effort was made to only enroll first admissions, but some repeat admissions or referrals between the two facilities may have occurred.In this study, all clinical and laboratory procedures were routine clinical care procedures conducted following Uganda MoH clinical guidelines Two laboratory technicians from each facility received a 2-week intensive training in microscopic examination of blood smears for MPs at the Malaria Diagnostic Center in Kisumu, Kenya The facilities treated malaria according to Uganda MoH clinical guidelines Trained hospital staff collected enrollment and follow-up data using standardized case report forms (CRFs). Information included the child's gender, date of birth, date of admission and location; presenting symptoms and duration; temperature, weight, mid-upper arm circumference, level of consciousness, pulse, respiration rates, treatment (including blood transfusion) and treatment outcomes . MPs results were abstracted from the laboratory request forms and Hgb and HIV tests were recorded directly into the CRFs. CRFs were entered into an electronic database in Visual FoxPro by trained RHSP data entry clerks.Malaria parasitemia (MP), was defined as the presence of any asexual (or other forms) of malaria parasites on a thick blood smear. Febrile illness was defined as an axillary temperature of ≥38°C. Anemia was defined as Hgb concentration <10 g/dl, and categorized as severe (Hgb<6 g/dl), moderate (Hgb 6–7.9 g/dl) and mild (Hgb 8–9.9 g/dl). Severe anemia associated with malaria was defined as Hgb<6 g/dl and a positive smear. According to the Uganda MoH clinical guidelines, blood transfusion is indicated when the Hgb concentration is below 6 g/dl We estimated the prevalence of malaria parasite positivity among children under five years by gender, age, presenting symptoms, mid upper arm circumference (MUAC) and HIV-1 status. The association between risk factors, presenting symptoms and MP was assessed using generalized linear models with binomial log link regression to estimate the prevalence risk ratios (PRR) and 95% confidence intervals (CIs) controlling for age, gender, MUAC, HIV status and adjusting for clustering of children at health facility level. Anemia was excluded from this analysis and analyzed independently. In this setting where malaria is endemic, anemia is more likely to be a consequence rather than a risk factor of malaria. Student's t-tests were used to compare mean ages of children with and without malaria parasites. Treatment outcomes and factors associated with treatment outcomes were estimated using bivariate and multivariable analyses. All statistical analyses were conducted using STATA software package version 12 . P values were double sided and considered significant when <0.05.th, 2011 and May 31st, 2012, of whom, 2507 (67.3%) were enrolled into the study. However, 36 (1.4%) observations were excluded because the children either had duplicate identification numbers, missing age or failed to meet enrollment criteria (i.e. age ≥5 years), leaving an analysis sample of 2471. Greater than 95% of clients who were approached enrolled in the study.A total of 3727 children under five years were hospitalized at the two health facilities between May 18The overall prevalence of confirmed malaria parasitemia was 54.6% . ParasitThere were 77 HIV exposed children of whom 84.4% (n = 65) were confirmed HIV infected. The overall prevalence of HIV infection among hospitalized children under five years was 2.6% of whom 38.5% (n = 25) were confirmed to be under HIV care. HIV infected children were less likely to be hospitalized and diagnosed with malaria than HIV uninfected children (aPRR 0.60 (CI 0.52–0.71). The majority of the HIV infected children (61.5%) were less than 2 years of age.X2 for trend p<0.001).At admission, the mean hemoglobin concentration of the study population was 9.0 g/dl (CI 8.9–9.1). The overall prevalence of anemia was 56.3% . The mean Hgb was significantly lower in children with parasitemia as compared to children without , and the prevalence of confirmed malaria was higher among children with lower Hgb (X2 for trend p = 0.004). Children with either anemia or severe anemia were at no increased risk of death when compared to other children, .Of the 503 (20.4%) children who received blood transfusion, 79.5% had severe anemia. Among the 439 children with severe anemia, 76.8% had parasitemia and 93.1% received a blood transfusion. BHC transfused significantly more children (52.9%) than KH (4.8%); p<0.0001; . The preOverall, treatment outcomes were available for 2439 children. Of these, 2375 (97.4%) were discharged, 25 (1.0%) died and 39 (1.6%) were referred. Children with confirmed malaria had significantly lower case fatality (0.6%) compared to children without malaria (1.6%); p = 0.02. Mortality among enrolled children was 1.0% compared with 1.6% in non-enrolled children; p = 0.051.This study documented a high prevalence of malaria parasitemia and anemia, but a low malaria case fatality among hospitalized children in Rakai district, Southwestern Uganda. Over half of the admissions in children under five years were diagnosed with malaria. Despite scale up of malaria prevention and treatment Nearly all (94.5%) of the inpatient children under five years were presumptively diagnosed with clinical malaria, yet the lab confirmation showed only 56.4% of these children had malaria parasitemia, suggesting substantial over diagnosis based on clinical presentation. This is consistent with other studies HIV infected children had a lower prevalence of malaria than uninfected children, possibly because 38.5% of HIV infected children were on co-trimoxazole prophylaxis which has been shown to reduce liver stages of P. falciparum Although both malaria-associated fatality and all cause case fatality were low in our study population, children who were diagnosed with malaria were less at risk of death than those who actually did not have malaria. This difference in case fatality is consistent with a study in a Ugandan teaching hospital which documented a 1.5 times higher risk of mortality among smear negative children and a more than 3-fold increased risk of mortality amongst children who were presumptively diagnosed with malaria when compared to those with a positive malaria smear Anemia was diagnosed in over half of the children, and was 68.7% in children with MPs consistent with other studies from similar settings Our study had several limitations. Data collection was done by health workers from the health facilities who were inexperienced in research and placed priority on clinical care. However, all staff were trained on the protocol and good clinical practice. The RHSP quality control team audited all CRFs to ensure that any errors were corrected by study nurses  before data entry. Although the study was designed to enroll all children, it was not possible in practice; some parents declined to participate, or their children were too sick to obtain parental consent, while others were admitted late at night and missed investigations such a blood smear, Hgb, etc prior to treatment. The study also failed to enroll some children who died before they had an opportunity to be enrolled, as indicated by the higher but not statistically significant mortality among non-study admissions. A concerted effort was made to only enroll first admissions at each institution, but some repeat admissions or referrals between the two facilities may have occurred.Unlike other retrospective studies using routinely reported clinical records Malaria parasitemia and anemia were the two most common diagnoses among hospitalized children under five years in this rural Ugandan population. Although malaria case fatality was low, malaria prevention and access to early, effective treatment needs to be improved and consistently monitored to reduce the burden of life-threatening malaria and to effectively manage other non-malarial illnesses.Study Approval S1(ZIP)Click here for additional data file.Protocol S1(ZIP)Click here for additional data file.