Plasmodium falciparum and Plasmodium vivax endemic. Such spatial and temporal clustering of malaria only serves to underscore the importance of regularly collecting up-to-date malaria surveillance data to inform decision-making in malaria control. Cross-sectional school-based malaria surveys were conducted across Oromia Regional State to generate up-to-date data for planning malaria control interventions, as well as monitoring and evaluation of operational programme implementation.In Ethiopia, malaria transmission is seasonal and unstable, with both Plasmodium infection; Bayesian geostatistical methods and Kulldorff's spatial scan statistic were employed to investigate spatial heterogeneity.Two hundred primary schools were randomly selected using a stratified and weighted sampling frame; 100 children aged five to 18 years were then randomly chosen within each school. Surveys were carried out in May 2009 and from October to December 2009, to coincide with the peak of malaria transmission in different parts of Oromia. Each child was tested for malaria by expert microscopy, their haemoglobin measured and a simple questionnaire completed. Satellite-derived environmental data were used to assess ecological correlates of Plasmodium infection was found to be 0.56% (95% CI: 0.46-0.67%), with 53% of infections due to P. falciparum and 47% due to P. vivax. Of children surveyed, 17.6% (95% CI: 17.0-18.1%) were anaemic, while 46% reported sleeping under a mosquito net the previous night. Malaria was found at 30 (15%) schools to a maximum elevation of 2,187 metres, with school-level Plasmodium prevalence ranging between 0% and 14.5%. Although environmental variables were only weakly associated with P. falciparum and P. vivax infection, clusters of infection were identified within Oromia.A total 20,899 children from 197 schools provided blood samples, two selected schools were inaccessible and one school refused to participate. The overall prevalence of These findings demonstrate the marked spatial heterogeneity of malaria in Oromia and, in general, Ethiopia, and provide a strong epidemiological basis for planning as well as monitoring and evaluating malaria control in a setting with seasonal and unstable malaria transmission. Following the recent achievements in global malaria control , there iBuilding on the Kenyan experience, this paper presents results from the first, large-scale school survey of malaria in Ethiopia. Malaria transmission in Ethiopia is temporally and spatially dynamic , with trThis study was undertaken throughout Oromia Regional State, the largest of Ethiopia's 11 regional states. Oromia covers approximately one third of the country's landmass  Ethiopia office Table . MalariaA two-stage sampling design was employed, whereby schools (primary sampling unit) were selected using probability proportional to size, then within schools a fixed number of children (secondary sampling units) were randomly selected. Therefore, the number of schools sampled from each ecological zone was proportional to the number of schools in each zone, with the exception of the 'highland occasional epidemic' zone , which was under-sampled as a result of low expected prevalence and a need to maximize the power of the survey in more stable transmission areas. These criteria excluded one administrative zone from the sampling frame , while the remaining 16 administrative zones in Oromia were included in the survey.The sample size was determined using 95% confidence limits, 80% power and assuming design effect of 2. It was consequently estimated that the sample size for each ecological zone should be 3,925 children from 40 schools to detect a prevalence of 1% with 0.5% precision. Sampling 40 schools from each of five ecological zones would give a final sample size of 20,000 children from 200 schools. It was decided to select schools from ecological zones using probability proportional to population size, due to the uneven distribution of primary schools in Oromia between ecological zones  assent obtained from selected children before samples were collected. In each school, 10 boys and 10 girls (plus one reserve boy and one reserve girl) aged between five and 18 years were selected from each of grades 2-6 using computer-generated random number tables. If fewer than 110 children were enrolled in the school or present on the survey day, all children aged five to 18 present in the school were included in the survey; this, in some instances, resulted in a small school sample size. Detailed logistical and ethical considerations for this style of school survey have been presented elsewhere .® Pf-HRP2 Pan-pLDH, Access Bio, USA) to allow immediate diagnosis and treatment. This test was shown to have 85.6% sensitivity and 92.4% specificity for Plasmodium falciparum, and 85.0% sensitivity and 97.2% specificity for Plasmodium vivax in Ethiopia [Finger-prick blood samples were used to prepare thick and thin blood films for microscopy, and haemoglobin concentration was estimated to an accuracy of 1 g/L using a portable haemoglobinometer . In addition, blood spots were collected on filter paper for serological analysis at a later date ,17. ChilEthiopia . The locPlasmodium species was recorded, but quantification of parasite density was not conducted. A second reading was carried out for a proportion of blood films, by highly experienced microscopists at the malaria reference laboratory for Oromia Regional State in Adama. Criteria for a second microscopy reading were: slides positive for Plasmodium spp. at first microscopy reading; individuals with discrepant microscopy and RDT results; severely anaemic individuals (<80 g/L); and a randomly selected 5% of negative slides. Slides with discrepant results between first and second readings were settled by a third, expert microscopist from the Ethiopian Health and Nutrition Research Institute, the national reference laboratory in Addis Ababa  at 5 km2 resolution [2 resolution. The distance to permanent water bodies was extracted from the World Wildlife Fund (WWF) Global 200 Ecoregions database at 5 km2 resolution [2 resolution were extracted from data provided by the Moderate Resolution Imaging Spectroradiometer (MODIS) instrument aboard the Terra (EOS AM) and Aqua (EOS PM) satellites [Elevation was extracted from the shuttle radar topography mission (SRTM) digital elevation model at 1 kmsolution ,21. Landsolution . Estimattellites , for thetellites ,25. EnviMicroscopy results were entered into a Microsoft Excel 2007 spreadsheet . Questionnaire data from school surveys, including RDT results and haemoglobin measurements, were entered into a customized Microsoft Access 2007 database that had been developed to automatically conduct range and consistency checks. Any errors or inconsistencies were corrected with reference to the original paper forms. Survey data were exported from Access and Excel into a combined dataset in STATA 9.0  for cleaning. Point prevalence maps were developed in ArcGIS 9.3.Plasmodium infection, P. falciparum and P. vivax infection individually and anaemia, were presented with binomial 95% confidence intervals (CI) by sex, age group, ecological stratum and survey period. Use of malaria prevention measures, specifically LLINs and IRS, were presented by sex, age group, ecological stratum and survey period with binomial 95% CI, with associations tested using Chi squared test.Individuals aged over 18 years n = 260) or with missing parasitological data (n = 270) were excluded from the school survey analysis. Anaemia was defined according to WHO classifications, adjusted by age and elevation [0 or withP. falciparum or P. vivax infection) and individual covariates were assessed by random effects logistic regression to control for clustering of infection by school. Full multivariable models to describe association between LLIN use or IRS with Plasmodium infection were developed, using zero inflated Poisson (ZIP) models to account for the large proportion of schools with zero prevalence. ZIP models were favoured over standard Poisson models on the basis of the Vuong test [Crude univariate associations between outcomes  were subsequently included in full multivariable models, and non-significant (p < 0.5) covariates excluded sequentially in order of least significance to generate minimal adequate models. Excluded covariates were retested in the minimal model to confirm lack of significance. Bayesian spatial multivariate models were then developed in WinBUGs version 1.4  to explicitly model unexplained spatial correlation between schools. The number of examined and slide-positive individuals for each species at each survey location were modelled as binomial outcomes, including covariates as described above and a geostatistical random effect that modelled spatial correlation using an isotropic, stationary exponential decay function .P. falciparum and P. vivax in Oromia, the existence of spatial clusters of high malaria prevalence were investigated using Kulldorff's spatial scan statistic . A PoisThis study received ethical approval from the national health research ethics review committee of the Ethiopian Science and Technology Ministry (RDHE/2-89/2009). Approval for the study was given by the Oromia Regional Health Bureau and the Oromia Regional Education Bureau.Written consent for the survey was provided by each school director, but parents maintained the right to withdraw their child from the survey. Each child selected for inclusion was required to provide written assent (or thumbprint) after having the procedures explained. Schools where the director refused consent were not included in the school survey, and pupils refusing assent were excluded. Individuals with a positive malaria RDT were treated according to Ethiopian national guidelines . IndividA total of 21,166 children, age five to 18 years , from 197 rural primary schools in Oromia took part in the survey, with a similar number of boys and girls included . A mean 106 children were enrolled from each school (range 43-112). Blood films from 267 children were missing or unreadable. Consequently, these individuals were excluded from analysis, leaving 20,899 children (98.7%).Overall, 46.0% : 45.3-46.7%) of school children reported using a LLIN the previous night. In locations where the school elevation exceeded 2,000 m, 42.0% (95% CI: 39.7-43.6) of children reported using a LLIN; however, this may be in part attributable to kebeles with large altitude ranges still falling within the National Malaria Control Programme (NMCP) objectives to target areas under 2,000 m with LLIN distribution . ReportePlasmodium infection was 0.56% (95% CI: 0.46-0.67%). Of children with Plasmodium infection, 52.1% were infected with P. falciparum, 47.0% with P. vivax and 0.9% with mixed infections. Mixed infections were not analysed separately but included with each of the single species infections. The overall prevalence of P. falciparum was 0.30% (95% CI: 0.23-0.38%) and P. vivax 0.27% (95% CI: 0.20-0.35%). Only 18% of children with P. falciparum reported fever on the day of the survey; however, 72% had had fever in the past month. A greater proportion of P. vivax infections were sub-clinical, with only 7% of children reporting fever on the survey day, but 56% reporting to have felt fever in the past month. In total, 17.6% of children were anaemic, and the mean haemoglobin concentration was found to be 132.8 g/L (95% CI: 132.6-133.0).The overall prevalence of P. falciparum and P. vivax prevalence by school. Thirty schools (15%) were found to have at least one child with Plasmodium spp. infection on the day of the survey , and prevalence by school ranged between 0 and 14.5%. All schools with detectable infection were located at an elevation between 1,183 and 2,187 metres above sea level. The median time taken to walk to school, an indicator of distance the child lives from school, was 30 minutes (range 0-240 minutes). The schools with highest prevalence of infection were found in Jimma and South West Shoa administrative zones . There was strong evidence that the P. falciparum and P. vivax rates differed by ecological zone (both p < 0.001). Prevalence of anaemia varied markedly between schools  and temperature (LST) were seen, but the effect was small. In multivariate ZIP models, LLIN use was no longer found to be associated with either P. falciparum or P. vivax infection. The association between IRS in the household and increased risk of infection, however, remained in multivariate models: IRR of 2.91 for P. falciparum  and IRR of 3.92 for P. vivax .There were no statistical differences in P. falciparum or P. vivax prevalence by school and population density, elevation or land cover class, once residual spatial correlation was accounted for. Parameters associated with the magnitude and timing of seasonal changes in LST and EVI did show some association with P. falciparum and P. vivax infection; although these covariates did not improve model fit  was large for both species. Similarly, for both species the distance at which spatial correlation dropped to below 5% was very large (in excess of 200 km) when compared with other spatial models of malaria infection [Bayesian multi-variable modelling suggested there were no significant associations between it Table . The varnfection . This suP. falciparum and P. vivax infection . A significant cluster was seen for P. vivax infection, which with a radius of 169.0 km, was larger than the P. falciparum infection cluster. This cluster included 4,782 children from 44 schools, with 49 P. vivax infections found compared to an expected number of 12.5. The relative risk of infection in this cluster was 55.1 times higher than outside of the cluster (p = 0.001). Parameters for seasonal changes in EVI and LST showed some association with presence inside the P. falciparum and P. vivax infection clusters, while proximity to water was associated with location inside the P. vivax cluster .SaTScan analysis identified two clusters of high prevalence of n Figure . A smallPlasmodium to that found in the 2007 national MIS survey [P. vivax prevalence to be comparable to that of P. falciparum, and highlighted the marked spatial heterogeneity in infection observed in Oromia and, indeed, Ethiopia. Furthermore, the results are consistent with other cross-sectional study findings [This first application of school-based surveys to inform targeting of malaria interventions in Ethiopia has revealed a comparable prevalence of S survey , but lowS survey . The stufindings , with mafindings .P. falciparum infection identified by crude univariate analysis include history of fever and anaemia. Only fever history was, however, associated with P. vivax infection. These findings indicate that fever in the previous month is predictive of malaria due to both species, but there remained a high proportion of identified infections that were asymptomatic. Cross-sectional surveys from a range of transmission settings have shown that a strong association exists between P. falciparum and reported fever [Plasmodium infections to elicit clinical symptoms, but the current findings dispute this. Since parasite density was not calculated in this study, it is not possible to determine if asymptomatic infections were due to very low parasite density. Asymptomatic infections will contribute to ongoing transmission in a community, but are unlikely to be detected or treated in a context where only individuals feeling unwell access diagnostic services. If Ethiopia is to achieve focal malaria elimination in areas of current low, unstable transmission, alternative strategies must be used to identify and treat asymptomatic infections and halt transmission. In São Tomé and Príncipe, for example, mass screening by means of cross-sectional country-wide surveys, wherein all residents were screened with a RDT and RDT-positive individuals were treated with an ACT, has contributed to recent dramatic reductions in malaria transmission [Risk factors for ed fever . In low smission .Plasmodium infection and resultant anaemia. Overall, these findings indicate that iron supplementation should be considered as a possible school health strategy, targeting boys and girls.Absence of age-dependency for infection in the current findings is consistent with lack of acquired immunity among individuals living in low malaria transmission settings , and finWhile documented scale-up in LLIN distribution and coverage in Ethiopia has been very successful , the preP. falciparum and P. vivax infection in multivariate models. The lack of protective effect of IRS in these findings may be a result of near-universal resistance to DDT ethylene) in Ethiopian anopheline mosquitoes . The most likely explanation for this association between IRS and increased malaria risk is that the NMCP targets IRS to locations of known malaria endemicity; therefore living in a location where IRS is conducted is predictive of being in a malarious area.Somewhat surprisingly, LLIN use was associated with increased odds of malaria in crude univariate analysis. However, multivariate models did not find such association. Previous cross-sectional studies have found that net use is protective against malaria among school-aged children ,43, and Plasmodium infections due to P. vivax than previously described in Ethiopia [P. falciparum and P. vivax in equal proportion overall but P. vivax dominating in the highland epidemic ecological stratum (90%). The variation in species distribution may be a result of increased use of artemisinin-based combination therapy and P. falciparum-detecting RDTs at peripheral health facilities, impacting on transmission of P. falciparum and changing the epidemiology of this parasite in Ethiopia. Alternatively, the findings may simply be due to the highly variable and unstable transmission setting, where increased P. vivax cases may be a result of focal epidemics in highland areas at the time of the survey, or a result of the tendency for P. vivax to cause long-term chronic infections and show less seasonality in transmission than P. falciparum [P. vivax infections. These infections are known to cause morbidity, including anaemia, malnutrition and respiratory distress [P. falciparum-detecting RDTs. Challenges remaining in control of P. vivax include examination of drug-efficacy and potential adjustment of national policy, in light of identified foci of chloroquine resistance [P. vivax infections. Furthermore, similar to other settings, it is likely that as prevalence of P. falciparum in Ethiopia is reduced by effective control interventions, the burden of malaria attributable to P. vivax will increase [The current surveys found a greater proportion of Ethiopia ,12, with stratum 0%. The vdistress ,45, but sistance -49, as wincrease ,51.Plasmodium infection, only a minority of parasitaemic individuals identified in schools reported any fever on the day of the survey. Similar high proportions of asymptomatic Plasmodium infections have been found in other low transmission settings [It is the commonly held belief that in low transmission settings, a high proportion of children with malaria would be symptomatic and therefore absent from school. The present findings, however, indicate that although self-reported fever during the previous month is predictive of settings . AttemptPlasmodium infections [Plasmodium than microscopy [The poor sensitivity of microscopy to detect low-density fections  may havecroscopy ,54, and The current study was unable to create a valid model, based on environmental covariates, to predict malaria endemicity across Oromia, because strong environmental predictors for location of transmission foci were lacking. Risk mapping using similar strategies had previously been successful in Afghanistan, with a comparable prevalence of infection (0.49%) . This inPlasmodium are detected using an enzyme-linked immunosorbent assay, reflecting exposure to infection over a longer time period. This method has been used successfully in other low and unstable transmission settings [Identification of all areas where malaria transmission is ongoing may be possible using an alternative diagnostic method where IgG antibodies to settings ,58,59. Asettings , but thePlasmodium parasite rates among children attending school, there is a need to conduct a rigorous comparison to indicators determined from standard community surveys, such as MISs. This will determine if findings from school-based surveys are representative of all school-aged children in a community or, indeed, the whole community; if representative, school-based surveys could become an alternative survey method to the more costly and labour-intensive community surveys. While it is not expected that there are differences in risk of infection by age in Ethiopia, there is a need to further explore what proportion of school-absenteeism is due to malaria, as well as whether there are differences in malaria risk between enrolled and non-enrolled children. These findings will define the potential role of schools in malaria surveillance, monitoring and control in Ethiopia and other low transmission settings. Envisaged roles of schools in malaria surveillance could be to provide data on coverage of major interventions and parasite prevalence during routine school surveys, and to alert service providers of epidemics using information on school-absenteeism and from active case finding [While the current cross-sectional surveys have provided data regarding the  finding .Results of cross-sectional school surveys in Oromia demonstrated marked spatial heterogeneity in malaria. Although several foci of infection were identified, large areas appear to be non-endemic for malaria. While these findings allow malaria control interventions to be targeted to identified endemic areas, this likely does not reflect the true extent of malaria in Oromia. Research is ongoing to further validate the use of school surveys in identifying transmission foci, as well as to investigate other potential uses of schools in malaria surveillance including monitoring and evaluation of control programme implementation.The authors declare that they have no competing interests.RA coordinated project management, data collection, analysis and developed the draft manuscript. TK, GT and DY were responsible for fieldwork supervision and project coordination and contributed to the final manuscript. RP contributed to data analysis and undertook spatial modelling. SB, RR and JK were responsible for the study design, interpretation and scientific guidance. All authors read and approved the final manuscript. The opinions expressed in this paper are those of the authors and may not reflect the position of their employing organisations nor of their funding sources.Microscopy results quality control flowchart.Click here for filePlasmodium falciparum and P. vivax and potential risk factors among school children in Oromia Regional State, Ethiopia in 2009, adjusting for clustering within schoolsUnivariate analysis for associations between .Click here for file


Plasmodium knowlesi is a simian malaria parasite that is widespread in humans in Malaysian Borneo. However, little is known about the incidence and distribution of this parasite in the Sandakan division, Malaysian Borneo. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of P. knowlesi as well as other Plasmodium species in this division based on a most recent developed hexaplex PCR system (PlasmoNex™).Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene.A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients . Identification of Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections . Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study.A total of 178 samples were positive for P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malaria.The discovery of  Plasmodium, is one of the most deadly parasitic diseases in the world. According to the World Malaria Report 2011, an estimated 3.3 billion people were at risk of malaria in 2010. Of this total, 2.1 billion people were at low risk whereas 1.2 billion people were at high risk  and living mostly in the WHO African (47%) and Southeast Asian regions (37%) [Malaria, a tropical disease caused by infection with single-celled parasites of the genus Anopheles mosquitoes, such as Anopheles balabacensis, Anopheles sundaicus and Anopheles flavirostris[Plasmodium species, which include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. These four Plasmodium species are well-recognized to cause human malaria worldwide [P. falciparum is severe and may be fatal in the absence of prompt recognition of the disease and its complications, while the disease caused by P. vivax, P. ovale and P. malariae is usually milder and rarely fatal.In Sabah, malaria is transmitted by various virostris. Traditiorldwide . InfectiPlasmodium knowlesi, has been recognized as the fifth human malaria species by WHO [Plasmodium knowlesi is a malaria parasite of Old World monkeys, which was formerly known to cause malaria only in macaques [P. knowlesi was generally high in Southeast Asian countries especially Malaysia. Unfortunately, this parasite is life-threatening and has caused mortality in humans [Recently, a simian malaria parasite, s by WHO . Plasmodmacaques . Howevermacaques -11 and Pmacaques ,12-14. Tmacaques -17, Thaimacaques ,19, Myanmacaques ,21, Philmacaques , Indonesmacaques ,24, Vietmacaques  and Cambmacaques , pinpoinn humans ,28.P. knowlesi infection can be challenging for an inexperienced microscopist because the early trophozoite stages of this parasite are morphologically similar to those of P. falciparum[P. knowlesi are indistinguishable from P. malariae[P. knowlesi and P. malariae, naturally acquired P. knowlesi infections are often misdiagnosed via microscopy as P. malariae[The diagnosis of alciparum,29 whils malariae, includi malariae,29.P. knowlesi (later diagnosis done with molecular tools) but were diagnosed initially as P. malariae infections via microscopy detection. Hence, in order to overcome the limitations of microscopy for the detection of malaria, molecular tools such as polymerase chain reaction (PCR) are necessary for accurate identification of P. knowlesi as well as other Plasmodium parasites in order to avoid mortality. This method has proven to be more specific and sensitive than conventional microscopy [This was evident when six out of 14 malaria deaths reported in Sabah during 2010–2011  were actcroscopy . Recognicroscopy .Plasmodium species in these districts of Sabah, Malaysian Borneo based on a most recent developed hexaplex PCR system in order to obtain accurate epidemiological data which serves as an important foundation in the effectiveness of malaria control strategy. The straightforward single-step hexaplex PCR system  targeting five human Plasmodium 18 s small subunit rRNA (ssu rRNA) was developed by Chew et al.[Plasmodium species simultaneously as well as mixed infections in a single step PCR with high sensitivity and specificity. Therefore, this system was the method of choice for the detection of malaria parasites in this study instead of nested PCR because it is rapid and less labour-intensive.However, information on malaria incidence in eastern districts of Sabah is scarce. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of the ew et al.. This sySabah is one of the 13 member states of Malaysia. It is situated at the northern part of Borneo Island and it shares a land border with Sarawak and Indonesia. This state covers an area of 73,620.1 sq km, comprising of 24 districts with a population of 3,120,040 people. Sabah is a famous tourist destination . Currently, there are more than 30 officially recognized ethnic groups in Sabah, the largest indigenous groups being the Kadazan-Dusun people, followed by Bajau and Murut. Chinese is the largest non-indigenous ethnic in Sabah. In this research, samples of the studied subjects were obtained from four districts  within Sandakan division which is the largest division in Sabah. It has approximately 19.4% of Sabah's total population and covers an area of 28,205 sq km. A majority of the ethnic groups in Sandakan division are Chinese, Suluk and Bajau Simunul. Tongod is at the central part of Sabah, whereas Kinabatangan, Beluran and Sandakan are situated in the eastern part of Sabah.P. knowlesi, the long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques as well as the vectors .The major economic contributions for Sandakan division are oil palm plantation, timber and eco-tourism. Most of the indigenous populations in this region reside in communities close to palm oil plantations or dense rain forest. This region is surrounded by forest fringe, contains an array of wildlife habitat, which usually act as a natural habitat for the hosts of A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. Telupid Health Clinic is the only diagnostic laboratory in Sandakan Division outside of Sandakan town. Usually, patients in Sandakan town receive treatment at the Sandakan Hospital. However, this hospital is about 130 km away from Telupid Health Clinic and it takes about 2–3 hours drive to get there. Therefore, samples from Sandakan and neighbouring districts may be sent to Telupid Health Clinic for diagnosis. Blood samples were collected from Blood Film for Malaria Parasites (BFMP)-positive patients who attended the clinic. The patients who participated in the study were all microscopically malaria positive patients before recruitment. Blood samples included in the study were collected from Sandakan and neighbouring districts such as Kinabatangan, Beluran and Tongod. In this study, 3 ml of blood was collected from each BFMP-positive patient that attended the clinic by qualified medical laboratory staff. A small amount of each sample was used for routine haematological profiling in the laboratory, and genomic DNA was extracted from the remaining sample. Basic demographic data of the patients if available, was obtained and recorded. Complete demographic details and haematological profiles were obtained from 85 patients .Patients’ consent was obtained prior to blood sample collection. For all child participants, parents/guardians provided the consent on their behalf. This study was approved by the Ethics Committee reviewer board of University of Malaya Medical Center (UMMC), Malaysia (MEC reference number 709.2).Blood smears were prepared prior to anti-malarial treatment for patients with suspected malaria. The thick and thin blood smears were stained with Giemsa and examined by experienced microscopist at the Telupid Health Clinic, Sabah. All blood smears were examined under the microscope at a magnification of ×1000 with immersion oil. Routine diagnosis reports were prepared by medical laboratory staff at this clinic. Species of parasites were determined based on the morphology of parasites. Parasite developmental stages such as early trophozoite, late and mature trophozoite, schizonts, and gametocytes were determined and recorded.The parasitaemia of infected patients in this study was estimated using a simple modified method, which involved counting of parasites in thin blood films by using the plus system . A code Haematological profiles of patients such as haemoglobin level (Hb), platelet counts and total white blood cell counts (TWBC) were obtained and analysed. All these laboratory procedures were carried out at the Telupid Health Clinic, Sabah. Anaemia was confirmed when the Hb level is <110 g/L for children aged six months to two years, <115 g/L for children five to 11 years, <120 g/L for children 12–14 years, <120 g/L for women and <130 g/L for men . ThromboDNA template for the hexaplex PCR assay was extracted from 200 μl thawed EDTA-blood using the QiAamp DNA Blood Mini Kit , following the manufacturer’s recommendations. Approximately 100 μl of DNA template were obtained from the blood and 1.5 μl of DNA template was used for the hexaplex PCR assay. The extracted DNA was kept in -20°C until further use.Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene [2, 0.2 mM of dNTP mix, Plasmodium primer mixture, 1 U of Taq DNA polymerase and 1.5 μL (~10 ng) of template DNA. Cycling condition of the PCR amplification was as follows: initial denaturation at 95°C for 5 min, 35 amplification cycles at 95°C for 30 sec, 56°C for 30 sec, and 65°C for 40 sec, followed by final extension at 65°C for 10 min in a thermal cycler . The amplified products were visualized on a 3.0% (w/v) agarose gel stained with ethidium bromide.Identification of RNA gene . PCR amps) test as they were not normally distributed. A Pearson’s Chi-square (X2) test was used to test the association between each variable. The level of statistical significance was set at p < 0.05 for each test. For each of the statistically significant variable, odd ratios (ORs) and 95% confidence interval (95% CI) were calculated to explore the association between variables of interest.Statistical analysis was carried out using the SPSS software programme for Windows version 17 . The intensity of malaria infections (parasitaemia) was estimated by a simple modified method which involved counting of parasite numbers in thin blood films by using the plus system and was categorized into four main categories: +, ++, +++ and ++++. For descriptive data, percentage was used to describe the characteristics of the studied population, including the prevalence of anaemia, thrombocytopaenia, leukopaenia and leukocytosis. The distribution of Hb, platelet and TWBC were presented as median and interquartile range (IQR) after being examined for normality using the Shapiro-Wilk test. The correlation between continuous variables was examined using Spearman’s correlation coefficients  were P. vivax, 25.9% (49) were P. malariae, 1.1% (two) were mixed P. vivax/P. malariae and 0.5% (one) was mixed P. falciparum/P. vivax infections  had asexual parasites only, 7.8% (five) had both asexual parasites and gametocytes. For P. falciparum-positive blood films, 60.3% (44/73) had asexual parasites alone, 39.7% (29) had asexual parasites and gametocytes. As for P. malariae-positive blood films, 98.0% (48/49) had asexual parasites alone while only 2.0% (1/49) had both asexual parasites and gametocytes  were Given that the complete demographic details and haematological profiles could only be obtained from 85 patients , the results pertaining to demographic and haematological profiles will be based on 85 and not 189 patients. The ethnicities of patients that participated in this study were Dusun (50.6%), Bugis (18.8%), Filipinos (5.9%), Rung (4.7%) and others . Among the four districts of Sandakan division, Kinabatangan has the highest malaria cases detected  in this study followed by Sandakan , Beluran  and lastly Tongod .With regard to the age groups, there was a total of 19 children and 66 adults. The age range was between two to 60 years old . Malaria was more prevalent among adults (77.6%) compared to children (22.4%). With regard to gender, male patients (84.7%) were almost five-fold more compared to female patients 15.3%) (Table % (Table 9 cells/L (IQR = 5.1-8.8), respectively (Table 9 cells/L). The median Hb value  and TWBC count  among infected children were lower than the Hb value  and TWBC count  of adults. However, the platelet count among infected children  was higher when compared to the adults . With regard to gender, male had higher Hb value  and platelet count  compared to female’s Hb value  and platelet count . However, the TWBC count of female  was slightly higher than male .According to the haematological profiles, the present study showed that the overall median concentration of Hb, platelet count and TWBC for the patients was 128.0 g/l (IQR = 109.0-140.0), 91,000 cells/cmm  and TWBC was 6.6 × 102 = 6.30; p = 0.012) compared to adult. Study also showed that children had higher risk  of getting anaemia compared to the adults. With regard to gender, it was observed that anaemia was significantly higher in female  compared to male. The risk of female suffering anaemia was 1.8 times higher than male .Study showed that an overall of 48.2% (41/85) of the patients had anaemia, 56.5% (48/85) had thrombocytopaenia, 8.2% (7/85) had leukopenia and 7.1% (6/85) had leukocytosis Table . The preHowever, the prevalence of thrombocytopaenia, leukopaenia and leukocytosis were not significantly associated with age and gender. Although the prevalence of thrombocytopaenia in adult (59.1%) was higher compared to children (47.4%), the difference was not statistically significant. Similarly, there was no significant difference between leukopaenia and leukocytosis with age although the prevalence of leukopaenia was higher in children (10.5%) and leukocytosis was higher in adult (7.6%). There was also no significant difference between thrombocytopaenia, leukopaenia and leukocytosis with gender.Plasmodium species by using PlasmoNex™, however, 11 (5.8%) samples were found to be negative. Plasmodium falciparum was identified in 68 samples (38.2%) which was the highest number of species detected by PlasmoNex™ in this study followed by 64 cases (36.0%) of P. vivax and 42 (23.6%) cases of P. knowlesi and two (1.1%) cases of P. malariae. Overall, only two (1.1%) of the samples were found to have mixed-species infections  by PlasmoNex™. For PlasmoNex™ confirmed P. knowlesi infection, there were only 4.8% of the samples (2/42) that had both asexual parasites and gametocytes while 95.2% of the samples (40/42) had only asexual stages.Out of the 189 samples analysed, a total of 178 samples were positive for P. malariae by microscopy examination, only two (1.1%) were found to be P. malariae by PlasmoNex™ . Figure P. knowlesi cases detected in this study. No P. ovale was detected by using PlasmoNex™. Six P. falciparum cases were misdiagnosed as P. malariae by microscopy examination as well as one mixed infection of P. falciparum/P. vivax was misdiagnosed as P. vivax/P. malariae (Table Of the 49 samples which were reported as only two .1% were P. vivax (24.6%) compared to children (8.3%) whereas the infection percentage for both P. falciparum and P. knowlesi patients was almost the same for adult and children. The oldest age range for patients with P. knowlesi infection were slightly higher (51–60 years old) than that of patients with P. falciparum and P. vivax (41–50 years old). The youngest child with PCR-confirmed knowlesi malaria was nine years old. This showed that patients with P. knowlesi infection demonstrated a wide age distribution . Among P. knowlesi infected patients, the percentage of male patients (95.0%) was much higher compared to female patients (5.0%) . The haematological analysis of the present study focused only on single infection. Data showed that the patients within the age group 21–30 years (39.1%) were the most susceptible group to malaria in this study Table . There w. vivax 2.6% compas) test was used to study the relationship between Hb value, platelet count and white blood cell count with parasite density. Overall, there was a significant correlation between parasite density with Hb value and platelet value but not with white blood cell count for all the species. There was a significant inverse correlation between parasite density with Hb value and platelet value. Moderate inverse correlation was noted between increasing parasite density and decreasing Hb value . Likewise, the parasite density also significantly correlated with platelet value. Increasing parasite density correlated with decreasing platelet value  for all the Plasmodium species. As for TWBC, no significant correlation was observed with parasite density.Spearman’s correlation co-efficients . Besides, the overall median concentration of platelet count for knowlesi patients  was the lowest platelet counts compared to P. falciparum (94 000 cell/cmm) and P. vivax  malaria patients , nine (60.0%) and nine 45.0%) patients with .0% patiefalciparum malaria  were found to have the highest cases of severe thrombocytopaenia followed by knowlesi malaria patients  and vivax malaria patients . This showed that P. falciparum and P. knowlesi patients had higher risk of getting severe thrombocytopaenia compared to P. vivax patients. Plasmodium falciparum patients also had the highest number of moderate thrombocytopaenia  followed by P. knowlesi infected patients  and six of them were P. vivax-infected patients   was observed in all malaria species. There were 13 patients who had severe thrombocytopaenia, 27 patients had moderate thrombocytopaenia and 15 had mild thrombocytopaenia whereas 14 patients had no thrombocytopaenia Table . Data shP. knowlesi cases was observed in Sabah between 2004 and 2011 and this trend threatens malaria elimination [P. knowlesi cases and it appeared to have begun in the south-west of Sabah (Interior division), followed by West Coast division and gradually progressed north-easterly (Kudat division). This increase appears to have begun initially in the Interior division and there was a steady increase of P. knowlesi cases from 2000 to 2011 in this region. West Coast division experienced an increase of P. knowlesi cases later, from 2001 to 2009. After that, the tip of Borneo, Kudat division experienced the most noticeable increase in P. knowlesi cases with more than 200 cases for the years 2009 and 2011. Plasmodium knowlesi infection has also been reported in eastern districts of Sabah (Sandakan and Tawau), although the cases reported in these two districts were fewer compared to other districts, it has been increasing since 2008 [P. knowlesi cases were also detected in the districts of Sandakan division. This shows that P. knowlesi is generally prevalent in Sabah. Therefore, the main objective of this study is to investigate the incidence and distribution of P. knowlesi as well as other human Plasmodium species in eastern Sabah.Malaria is one of the most severe parasitic infections and it is also a major public health problem in many countries in Southeast Asia. In the 1980s, the prevalence of malaria drastically decreased and was eliminated in many areas of Peninsular Malaysia due to the efficient malaria control programme. However, malaria is still prevalent in Malaysian Borneo and among the ethnic minority groups . Recentlmination . Since tnce 2008 . FindingP. knowlesi were identified in 42 (23.6%) samples, however, microscopic examination did not show P. knowlesi in any samples examined. Thirty-five infections diagnosed as P. malariae by microscopic examination were found to be P. knowlesi. This finding was similar to the studies carried out by other researchers in Malaysian Borneo [P. malariae infections were actually proven to be P. knowlesi infections when molecular approach was used. However, the transmission dynamics of this infection still remain unknown despite wide distribution of this parasite in Malaysian Borneo. The most prevalent Plasmodium species identified in the study was P. falciparum followed by P. vivax and P. knowlesi. It was reported that P. vivax was the most prevalent human malaria parasites in Malaysia [P. falciparum was found to be the most prevalent species in this study.Hexaplex PCR (or PlasmoNex™) findings of the present study demonstrated that n Borneo ,10,28,35Plasmodium on thin and thick blood films has been considered as the “gold standard” for the diagnosis of Plasmodium parasite infection because it is cost-effective and simple [P. knowlesi is rather challenging due to its morphology, which is indistinguishable from P. falciparum and P. malariae. Underdiagnosis of P. knowlesi by microscopy examination was observed in the present study. Thus, molecular tool, such as PCR, is necessary to overcome the limitations of microscopy for malaria detection especially in generating epidemiology data.Conventional diagnostic technique, microscopic examination of asexual stages of d simple . Howeverd simple . The diaPlasmodium species in this study. Molecular methods based on DNA amplification have been used for malaria diagnosis since 1980s [The recently developed PlasmoNex™ , was usece 1980s -49. Sevece 1980s -52. Besice 1980s ,53-55.Plasmodium species. The fewer PCR reactions reduce the usage of reagents and consumables and are also less labour intensive. Mixed infection detected by single-step hexaplex PCR in this study indicated that this system is robust and capable of detecting at least up to two species level mixed infections.PlasmoNex™ is a single-step hexaplex PCR system , which iP. knowlesi cases had been reported in the Interior division and north-eastern state of Sabah [P. knowlesi in Sabah has increased markedly and studies reported on the discovery of this parasite by using molecular approaches have greatly increased recently. In the early 1990s, there was around 50,000 malaria cases reported in Sabah and a continuous decrease was observed from the year 2001 to 2003. There were 6,050 cases in 2001, and this number fell to 5,096 cases in 2002 and subsequently to 1,770 cases in 2003 [P. falciparum and P. vivax in Sabah had drastically decreased over the past decade [P. falciparum and P. vivax had decreased to around 600 for the year 2011, however, a significant increase of P. knowlesi had occurred following the reduction of P. falciparum and P. vivax in Sabah. It showed that when human malaria cases decreased, P. knowlesi cases were on the rise. It is postulated that environmental changes together with decreasing rates of P. falciparum and P. vivax were likely to have contributed to the rise of P. knowlesi in Sabah [P. knowlesi infection was detected in all four districts of Sandakan divisions with the highest incidence of this parasite in the Kinabatangan region.Recently, of Sabah ,28,35. I in 2003 . Previout decade . The notin Sabah . Human aP. knowlesi in this region is not surprising as this region is surrounded by dense primary and secondary forests which act as suitable habitats for mosquito vectors  [M. fascicularis and pig-tailed macaques, M. nemestrina) which are the natural hosts for P. knowlesi in Sabah. Previously, An. balabacensis was well recognized as the predominant vector of human malaria in Sabah [Anopheles donaldi was reported to have replaced An. balabacensis in the Kinabatangan region in 2005 [An. donaldi mosquitoes occurs between 18:00 and 19:00. This is the period most people are being bitten by this mosquito when the adults are working outside and children are having outdoor activities. Although An. balabacensis was recognized as the vector of human malaria in Sabah, more studies should be carried out to investigate the current vector situation in this region. Perhaps, the situation of vectors in this region has changed due to recent development and mass forest clearance in the area. Nevertheless, communities should be informed to take necessary preventive measures against this parasite in order to prevent outbreaks of P. knowlesi infection.The wide distribution of acensis)  and macain Sabah . However in 2005 . The peaP. knowlesi malaria was diagnosed in male patients more often than in female patients, and only small proportion of knowlesi malaria cases occurred in children, this may be related to the limited outdoor or forest activities by this group of patients. These findings were in concordance with those reported from two studies in Sarawak [knowlesi malaria occurred in children and no clustering of cases was reported. Findings of these two studies suggested that P. knowlesi infection occurs outside people’s home and adults were the high risk group of this infection due to greater forest exposure, and it is postulated that human to human transmission did not occur. However, previous study postulated that the transmission of P. knowlesi may occur from human to human due to the discovery of family clustering cases [P. knowlesi cases was found in the present study.Malaria patients who participated in the study comprised ages from young children to the elderly, however, this infection was more prevalent among adults especially the males. The epidemiological data showed that  Sarawak ,9, whereng cases . NeverthP. falciparum and P. vivax samples compared to P. knowlesi samples. This could infer that infected humans were getting the knowlesi infection from mosquitoes that fed on monkeys and not from humans. Consistent with previous studies [P. knowlesi were older than those with P. falciparum or P. vivax. The possible reason for the older age group being infected by P. knowlesi was discussed in a study done by William and his colleagues [P. knowlesi were detected in this district. Long working hours in the forest may increase the risk of exposure to the mosquito vectors, resulting in successful transmission of P. knowlesi. Despite these observations, epidemiological risk factor of P. knowlesi malaria still remains unclear, hence further investigations are required in order to develop successful control strategies for knowlesi malaria.Microscopic findings showed that there were more gametocytes found in the  studies ,35,57, plleagues . It was P. knowlesi infection are almost similar to the other four human malaria species, however, it is more severe than the disease caused by P. vivax and more virulent because it has a short life-cycle of 24 hours [P. knowlesi[P. falciparum[P. vivax[The symptoms of human 24 hours  which le24 hours ,57,59-61 knowlesi,35,62-64alciparum,66 and PP. falciparum patients suffered severe and moderate thrombocytopaenia followed by P. knowlesi and P. vivax patients. This might explain the role of platelet activation in the pathogenesis of liver-attacking malaria. Platelets are produced by a process called thrombopoiesis, which occurs in the bone marrow by budding off from megakaryocytes. Thrombopoietin is a hormone that is secreted for the regulation of platelet production and megakaryocytes. This hormone is produced by the liver and kidneys. Hyperparasitaemia of P. falciparum and P. knowlesi parasites due to their shorter lifecycle may destruct the production of this hormone in the liver and thus decreased the production of platelet and caused thrombocytopaenia. However, this hypothesis requires further investigation. Many studies have shown that P. knowlesi patients suffered severe thrombocytopaenia [P. knowlesi than in P. falciparum malaria. Besides, findings of the present study are also in line with the study conducted by Patel and his colleagues [P. falciparum malaria were found to have lower platelet counts than patients with P. vivax malaria. Therefore, findings of present study demonstrated that the platelet count of P. knowlesi-infected patients were lower than those in P. falciparum-infected patients and P. vivax-infected patients, which, in turn, were lower than those in the uninfected patients and a platelet count below 75,500 cells/cmm should increase the suspicion of P. knowlesi infection. Nonetheless, it is suggested that in order to verify this hypothesis, more samples need to be included in future. It is believed that thrombocytopaenia is more common in P. falciparum malaria compared to P. vivax malaria. However, recent studies have shown the trend that thrombocytopaenia was found to be equally common in P. vivax malaria compared to P. falciparum[P. vivax malaria. The findings were in accordance with those in Brazil [knowlesi malaria.The present study showed similar results with previous studies whereby thrombocytopaenia occurred in all species of human malaria parasites indicating that thrombocytopaenia is a common feature of malaria and its occurrence should increase the suspicion of malaria. However, there are several other causes of thrombocytopaenia such as leukaemia, hereditary syndromes, HIV-associated thrombocytopaenia and also dengue fever. Thrombocytopaenia can be caused by decreased platelet production or peripheral destruction. It is postulated that malarial antigen caused the lysis of immune complexes, which leads to sequestration of the injured platelet by macrophages in the spleen ,68. Howetopaenia ,35,69,70lleagues  which realciparum-74. A stn Brazil  and Qatan Brazil . AlthougP. knowlesi and P. falciparum infections [P. knowlesi patients indicating that anaemia is common in human malaria infections. However, malarial anaemia appears to be multifactorial, factors such as genetic disorders , haemoglobinopathies, poor dietary intake and socio-economic causes may also contribute to anaemia. It was observed that anaemia is significantly higher in female patients  compared to male patients in the present study. This might also be due to blood loss of female during menstruation. With regard to age, the present study demonstrated that the prevalence of anaemia was significantly higher  in children compared to adults. This is in agreement with other previous studies where malaria-related anaemia is more severe in children rather than in adults [P. knowlesi and Plasmodium cynomolgi. The study reported that anaemia was observed in P. knowlesi-infected monkeys after primary and/or repeated infection. Severe anaemia was notable in macaques after repeated infection by P. knowlesi[P. knowlesi-infected humans or macaques. Malaria-related anaemia is a consequence of hyperparasitaemia and it has been associated with immune-mediated lysis of erythrocytes by schizonts or erythrophagocytosis in the spleen. Findings of the present study showed that both anaemia and thrombocytopaenia are common in malaria-infected patients, including P. knowlesi patients.Anaemia is also another haematological complication that is usually associated with malaria especially in fections ,57,78. Tn adults ,80 due tn adults  carried  knowlesi. This shP. falciparum[P. vivax[P. knowlesi infection was scarce. This showed that more investigations have to be conducted in order to study the correlation of platelet count with parasite density in malaria infection. Low Hb level due to increased parasite density might be explained by increased haemolysis or decreased rate of erythrocyte production due to the invasion of erythrocytes by malaria parasite [There was a significant inverse correlation between parasite density with Hb and platelet value for all the species of malaria in the present study. This was in line with other studies where malaria-infected patients tended to have significantly lower Hb levels and platelet counts ,82. The alciparum,84 but n[P. vivax. Howeverparasite ,86.P. vivax malaria patient was lower than P. falciparum malaria patient, which was in contrast to previous study where WBC in P. falciparum patients were lower than those in P. vivax-infected patients and, in turn, lower than those in healthy patients [White blood cell count (WBC) during malaria are usually characterized as being low to normal ,87, a deP. knowlesi infection, could not be deduced. Nevertheless, despite all these limitations, this study is one of the few comprehensively analysed studies on the occurrence and epidemiology study of malaria in the eastern districts of Sabah.The present study had several limitations. Firstly, haematological profiles such as Hb level, platelet counts and TWBC were only available for 85 patients. The statistical analyses of these parameters were mostly not significant due to the small sample size. Secondly, the baseline demographic data is incomplete and, unfortunately, detailed clinical features of patients could not be retrieved. There were several reasons for the incomplete data in the study. Demographic data and haematological profiles were unable to be retrieved for the samples that were sent from other districts of the division to the clinic. Case notes of all these patients were kept in the respective hospital or clinic therefore the data required were not available. In addition, sometimes, patients with severe malaria were referred to the district hospitals immediately before the full blood profiles were tested in the clinic. There were also some patients with uncomplicated malaria who have refused admission and, therefore, full blood counts were unable to be performed. Thirdly, travel history of patients was not available and therefore the information on where patients acquired malaria infection, especially P. knowlesi infection in Sandakan division was misdiagnosed as P. malariae by microscopic examination. The wide distribution of human P. knowlesi infection indicated that this parasite is generally able to infect humans and there is high potential for knowlesi outbreak to occur given that the full knowledge of the dynamics of transmission between human-monkey-mosquito is not adequately understood. This study also observed that there was no clustering of cases of P. knowlesi suggesting that transmission may occur in forested areas away from people’s homes. In addition, given that microscopic findings showed that gametocytes were only detected in two P. knowlesi patients, indicating that there was less chance of human to human transmission occurring. With regard to haematological analysis, thrombocytopaenia and anaemia was shown to be the most frequent malaria-associated haematological complications. However, larger prospective studies are needed to investigate the relations between parasite species as well as the parasite density to the haematological complications associated with malaria. It is crucial that prospective studies on epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are comprehensive in order to accrue accurate epidemiological data, which serves as an imperative foundation in formulating effective malaria control measures that will subsequently contribute to the successful elimination of malaria in Malaysia by 2020. In order to reduce mortality, it is pertinent for malariae suspected samples to be confirmed by PCR. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malaria. Given the severity of knowlesi malaria and its fatality, confirmed P. knowlesi cases should be reported as such by the doctors treating these cases so that the actual disease burden is known.Present study revealed that most of the The authors declare that they have no competing interests.YALL, IV and KHC conceived and designed the experiments. PCL and VN carried out the laboratory diagnosis. XTG, CHC and NJY carried out the molecular genetic studies. XTG, RN and TCT analysed the data. XTG, YALL, TCT and IV drafted the manuscript. KHC, PCL and YALL contributed the reagents/materials/analysis tools. All authors read and approved the final manuscript.


Plasmodium falciparum malaria, resulting in a substantially worse outcome. Whether a biological relationship exists between malaria infection and IBI susceptibility remains unclear. We, therefore, examined the extent, nature and evidence of this association.Severe malaria remains a major cause of pediatric hospital admission across Africa. Invasive bacterial infection (IBI) is a recognized complication of P. falciparum malaria using the search string ‘ AND .’ Eligiblity criteria also included studies of children hospitalized with malaria or outpatient attendances in sub-Saharan Africa.We conducted a systematic search in August 2012 of three major scientific databases, PubMed, Embase and Africa Wide Information, for articles describing bacterial infection among children with A total of 25 studies across 11 African countries fulfilled our criteria. They comprised twenty cohort analyses, two randomized controlled trials and three prospective epidemiological studies. In the meta-analysis of 7,208 children with severe malaria the mean prevalence of IBI was 6.4%  5.81 to 6.98%). In a further meta-analysis of 20,889 children hospitalised with all-severity malaria and 27,641 children with non-malarial febrile illness the mean prevalence of IBI was 5.58 (95% CI 5.5 to 5.66%) in children with malaria and 7.77% (95% CI 7.72 to 7.83%) in non-malaria illness. Ten studies reported mortality stratified by IBI. Case fatality was higher at 81 of 336, 24.1% (95% CI 18.9 to 29.4) in children with malaria/IBI co-infection compared to 585 of 5,760, 10.2% (95% CI 9.3 to 10.98) with malaria alone. Enteric gram-negative organisms were over-represented in malaria cases, non-typhoidal Salmonellae being the most commonest isolate. There was weak evidence indicating IBI was more common in the severe anemia manifestation of severe malaria.The accumulated evidence suggests that children with recent or acute malaria are at risk of bacterial infection, which results in an increased risk of mortality. Characterising the exact nature of this association is challenging due to the paucity of appropriate severity-matched controls and the heterogeneous data. Further research to define those at greatest risk is necessary to target antimicrobial treatment. Malaria remains a leading cause of childhood morbidity and mortality worldwide, accounting for 7% of deaths in children younger than five years of age . In the Plasmodium falciparum malaria are at risk of invasive bacterial infection (IBI). Blood stream infection, largely secondary to enteric Gram-negative organisms (EGNOs), with a predominance of non-typhoidal salmonella (NTS) species, has been widely reported as a complication of severe malaria. However, it remains uncertain whether malaria infection is a risk factor for invasive bacterial disease since the majority of children in malaria-endemic Africa are frequently infected by P. falciparum throughout childhood and only a minority will develop severe disease. A sub-analysis within a comprehensive systematic review of blood stream infections in Africa indicated that 6.5% of 11,814 malaria infections had concomitant bacteremia [Over time there has been a piecemeal accumulation of data indicating that children with This is important for two reasons. Firstly, concomitant IBI in children hospitalized with evidence of recent or intercurrent malaria, with or without severe manifestations, results in a significantly worse outcome. In one epidemiological study an estimated one third of all SM deaths were attributable to bacteremia . SecondlWe reviewed published observational and epidemiological data in order to establish whether there was evidence of an association between malaria and IBI. We sought to determine the prevalence of co-infection in children with malaria infection, the risk factors for co-infection and the mortality effect in African children, drawing on this knowledge to consider the implications for future treatment guidance.Studies were eligible for inclusion in the review if they were conducted among children with malaria, who had blood cultures taken and were admitted to hospitals or outpatient clinics in sSA. Although we restricted our review to studies reporting bacteremia, we did not exclude studies describing other bacterial co-infection, such as urinary tract infection or meningitis.P. falciparum malaria. Search terms used were ‘’ AND ‘’ in three major scientific databases, PubMed, Embase and Africa Wide Information (AWI). The Internet search was carried out on 13 and 14 August 2012. Searches of PubMed and Embase were limited to humans while AWI was limited to humans and scholarly (peer reviewed) journals. Abstracts and titles from all years were compiled in Endnote (Thomson Reuters) and screened, after the removal of duplicates. Prospective studies were required to recruit children systematically or consecutively and to evaluate all cases. Our review was restricted to articles either written in English or translated into English. Unpublished data were not included. The protocol and review are not registered.We performed a systematic search for articles describing bacterial infection among children with Eligible publications were retrieved in full text if available online or from academic libraries. Both prospective and retrospective case series of children with malaria undergoing blood culture were subdivided according to principal inclusion criteria . Studies principally reporting epidemiological associations were tabled separately. The definitions of SM varied widely. In order to facilitate agreement between the reviewers for study inclusion we used the following criteria for assessment of the quality of studies reporting on severe malaria (SM): 1) criteria for SM well-defined; 2) bacterial species defined or quality assurance verified that contaminants were excluded; 3) mortality data for ‘high risk’ SM cohort at least 7.4%  [For each study we worked out case fractions for malaria IBI co-infection as ‘number of children with malaria AND IBI’ (numerator) divided by ‘number of children with malaria’ (denominator). We used the Q test, as a measure of heterogeneity among studies, calculated as the weighted sum of squared differences between individual study effects and the pooled effect across studies. Iability) . Since tability) .Our search of the published literature yielded 10,200 articles and 7,897 unique articles following removal of duplicates. After reviewing the abstracts 72 full text reports were identified for closer evaluation, which were reduced to 21 studies after applying the exclusion criteria ; however, not all children had blood cultures taken [We excluded 15 studies since they described specific populations not primarily defined by malaria status. Studies alluding to an association between malaria and IBI but with incomplete information or unsuitable study design were tabulated separately , two randomized controlled trials and three epidemiological studies, summarized in Tables Table 2 statistic were 40.1 and 77.5, respectively, indicating substantial heterogeneity and between-study variability; thus, a formal meta-analysis was not performed. Of note, other studies, not included in this table, described other bacterial infections  compneumonia -24.P. falciparum gametocytes and/or pigment on a blood film). However, interpreting data from these studies, which used hospitalized children with non-malarial febrile illness as a comparator group, one must consider the possibility of selection bias. If the threshold for admission in children with confirmed malaria is lower, whereas children with a non-malarial febrile illness require one or more manifestations of severe disease in order to be hospitalized, the groups may no longer be appropriately matched for comparison.The second section of Table Two prospective studies  at three and four time points between 1979 and 2008, the incidence of NTS infections in children was compared to both the proportion of malaria-positive thick blood films in outpatients and the percentage of malaria admissions (Fajara only) . NTS incIn general, African children hospitalized with bacteremia have a poor prognosis . The outOne prospective study of children admitted with clinical features of SM included 182 film-positive children: 23 had bacteremia (12.6%) and 2 of these died (8.7%) compared to 69 slide-negative children where 28 (41%) had bacteremia and of these 11 died (39.2%). Overall, morality was greatest in children younger than 18 months . Other sP <0.001) [P <0.0001) [A retrospective study including 783 Kenyan children with SM showed a three-fold increase in mortality (10.4% to 33.3%) in those with IBI compared to those without IBI ( <0.001) . A simil <0.001) . The ass <0.001) . In Moza<0.0001) . Convers<0.0001) ,39.Staphyloccocus aureus, compared to only 3% due to Streptococcus pneumoniae and, thus, may be not representative due to the possibility of contamination. Were et al. [Only four of the ten studies reporting case series of febrile children with all severity malaria included data on case fatality rates for both malaria and malaria/IBI groups ,35,40,41S. aureus was the major pathogen isolated [S. aureus [Salmonellae Typhi was reported in isolated cases, predominantly in older children [S. typhi.Overall, 16 of the 22 studies in Table isolated ,40,42 an. aureus . Overall. aureus ,38,41,43. aureus . Salmonechildren ,44,45, wComparing the proportion of NTS in malaria versus non-malaria infection we found that two of three SM studies with a comparator non-malaria cohort recorded a higher proportion of NTS bacteremia in the malaria group ,24. Of tS. pneumoniae is the most common cause of IBI in children in Africa [S. aureus featured as a common culture isolate in children with malaria, exceeding 35% of all isolates in five of these studies; however, there were no suggestions of a focus of infection and contamination remains a possibility.Among Gram-positive organisms, n Africa . Howevern Africa ,23,36,47n Africa ,22,24. AOne study has reported the case fatality of malaria complicated by Gram-positive bacteremia to be substantially lower than mortality in malaria complicated by Gram-negative bacteremia  . Case faE. coli being the most common organism  [A number of in-hospital studies have examined clinical manifestations associated with IBI co-morbidity, including age, SM clinical phenotype, malaria endemicity, hyperparasitemia, and underlying illness ,41,43,46ganisms) . IBI co-ganisms) . The eviganisms) . In Westganisms) ,41.The clinical phenotype of SM appears to be an important factor. Nigerian and Gambian children ,43 with While there is enormous epidemiological potential for co-occurrence of malaria infection, malnutrition and HIV in much of sSA, the limited data available do not support such an association. Concomitant IBI was more common in underweight children (Z-score < -2)  and highet al. [Simple clinical criteria proposed by Nadjm et al.  identifiCollated data examining the potential biological relationship between malaria and invasive bacterial infection across a range of studies conducted in sSA is by no means conclusive. For SM the case fraction of children with IBI co-infection was 6.4% (95% CI 5.81 to 6.98%) but with substantial between-study variability, mostly due to differences in study methodology including the case definition of SM. In prospective studies of febrile children admitted to health facilities with all-severity malaria, the case fraction with IBI was lower in children with malaria (5.6%) compared to non-malaria febrile illness (7.8%). For children with all-severity malaria (including recent infection) NTS was more commonly isolated than in non-malarial febrile illness. Added to this, several large studies  provide Teasing apart the association between malaria and IBI has been challenging for several reasons. Foremost, both malarial parasitemia and IBI are common in children presenting with fever in malaria endemic areas. Rainy seasons in the tropics predispose to blood borne infection with enteric pathogens as well as malaria outbreaks ,51. HospThe findings of this review are subject to methodological limitations. Heterogeneity was high between studies. Only a few papers recorded sufficient data to allow standardization of populations. Inclusion of recent malaria infection, using antigen-based rapid diagnostic tests, has shed more light on the prevalence of co-infection and needs further investigation. We were also unable to control for differences in study design and quality of microbiological data. For many studies, the prevalence of IBI co-infecting malaria was not the primary question, making a formal meta-analysis potentially misleading. Unpublished data were not included and, therefore, our findings may have a publication bias towards studies that report high level of concordance. In children with SM the strength of a biological association with IBI is even more difficult to establish due to the paucity of data from an appropriate control population with severe life threatening illness. Our findings do, however, concur with the conclusions of the systematic review and meta-analysis of community acquired bacterial infection indicating co-infection in 6.5% of children with malaria (any type) .A putative clinical association between malaria and IBI was first suggested in the 1920s in Guyana by Giglioli who observed both increased prevalence and virulence of paratyphoid fever during malaria seasons with a co-infection rate of 29% . A similP. falciparum-infected erythrocytes within the endothelial bed of the gut, particularly at the tip of intestinal villi [The propensity for co-infection with EGNOs has led to speculation that the source of the microorganisms is most likely to be from the intestine due to impaired gut barrier function . A recenal villi ,59. The al villi  has alsoal villi  both comal villi . This maal villi . We hypoAlthough current recommendations for managing putative sepsis in children with SM are unclear ,64, the Establishing the relationship, both proof and effect, between malaria and IBI has been challenging. The accumulated evidence suggests that children with recent or acute malaria are at risk of bacterial infection, which results in an increased risk of mortality. Establishing which children with SM are at greatest risk of bacteremia would inform a policy for targeted antibiotic therapy that could substantially reduce malaria-associated mortality while minimizing the risks of excess antibiotic prescribing.EGNO: enteric gram-negative organism; IBI: invasive bacterial infection; NTS: non-typhoidal salmonella; SM: severe malaria; SMA: severe malarial anaemia; sSA: sub Saharan Africa.The authors declare that they have no competing interests.JC did the literature search, review of eligible manuscripts, constructed the first table of data and wrote the first draft of the manuscript. KM conceived the idea for a systematic review; reviewed manuscript selection; advised on design, edited tables, performed the statistical analysis and writing of the manuscript. Both authors read and approved the final manuscript.JC is a Paediatric specialty trainee and Honorary Research Fellow at Queen Mary, University of London and was a visiting research fellow at the KEMRI-Wellcome research programme last year. KM is a Professor in Tropical Paediatric Infectious Diseases at Imperial College, London, based fulltime in East Africa at KEMRI Wellcome Trust Programme.Table S2. Excluded studies referring to malaria and invasive bacterial infection.Summary of excluded studies. Click here for file


Assessment of malaria endemicity at different altitudes and transmission intensities, in the era of dwindling vector densities in the highlands, will provide valuable information for malaria control and surveillance. Measurement of serum anti-malarial antibodies is a useful marker of malaria exposure that indicates long-term transmission potential. We studied the serologic evidence of malaria endemicity at two highland sites along a transmission intensity cline. An improved understanding of the micro-geographic variation in malaria exposure in the highland ecosystems will be relevant in planning effective malaria control.Plasmodium falciparum MSP-119 were measured in an age-stratified cohort  in 795 participants from an uphill and valley bottom residents during low and high malaria transmission seasons. Antibody prevalence and level was compared between different localities. Regression analysis was performed to examine the association between antibody prevalence and parasite prevalence. Age-specific MSP-119 seroprevalence data was fitted to a simple reversible catalytic model to investigate the relationship between parasite exposure and age.Total IgG levels to 19 seroprevalence and density were observed in the valley residents than in the uphill dwellers. Adults (> 15 years) recorded high and stable immune response in spite of changing seasons. Lower responses were observed in children (≤ 15 years), which, fluctuated with changing seasons particularly in the valley residents. In the uphill population, annual seroconversion rate (SCR) was 8.3% and reversion rate was 3.0%, with seroprevalence reaching a plateau of 73.3% by age of 20. Contrary, in the valley bottom population, the annual SCR was 35.8% and the annual seroreversion rate was 3.5%, and seroprevalence in the population had reached 91.2% by age 10.Higher MSP-1The study reveals the micro-geographic variation in malaria endemicity in the highland eco-system; this validates the usefulness of sero-epidemiological tools in assessing malaria endemicity in the era of decreasing sensitivity of conventional tools. Malaria still thrives in the African highlands, in spite of low vector density exposure . The wesPfHRP2 based rapid diagnostic test (RDTs) presents additional challenges at low parasite densities.Assessing variation in malaria endemicty at different altitudes across regions with differing malaria transmission intensities can be achieved directly by determining exposure to malaria-infected mosquitoes, the entomological inoculation rate (EIR) , or indiPlasmodium falciparum has been explored as a marker of human exposure to malaria [Prevalence of antibodies to  malaria ,14,21-24 malaria . Thus th malaria ,14,27. A malaria ,14,27.19 as the most suitable immunological marker for assessing malaria endemicity at varying altitudes along transmission intensity cline.However, the use of inadequate serological markers may underestimate exposure by virtue of their lack of sensitivity. For instance, in the case of circumsporozoite protein (CSP), sporozoites injected by infected mosquitoes have a relatively short life-span in the blood. Some rapidly develop into liver stages and others are taken up by macrophages, processed, and presented to the immune system . The amo42) is further cleaved to produce a 33-kDa fragment (MSP-133) and a 19 kDa C-terminal fragment (MSP-119); with the later remaining attached to the merozoite surface and present on ring forms in newly invaded erythrocytes [19 is thus a recognized target of protective immunity [MSP-1 is secreted as a 195-kDa precursor anchored via glycosylphosphatidylinositol . This isimmunity .This study was therefore conducted to investigate serologic evidence of malaria exposure at a highland site along a malaria transmission intensity cline to characterize differences in malaria endemicity. The current study reports an improved understanding of the micro-geographic variation on malaria endemicity in the complex highland eco-system; and potentially identifies vulnerable groups in the event of hyper-transmission. This study confirms that sero-epidemiology provides valuable information for planning effective malaria control strategies and surveillance systems.Anopheles gambiae sensu stricto and A. funestus, [The study areas included two highland sites located in Iguhu and Lidambiza villages , and Sigalagala and Museno villages , all in Kakamega county, western Kenya. The terrain of the study area is typical of the highlands and consists of hills and river valleys. The hillside is mostly dotted with maize fields and subsistence crops. Six different seasonal streams flow within the valleys in the study villages and empty into the main Yala River, which runs through the area from east to west. The inhabitants live in houses of stick frames with mud walls and thatch or corrugated metal roofs. The area experiences two rainy seasons, and averages 2,000 mm rainfall per year. The long rainy season typically occurs between April and May, with an average monthly rainfall of 150-260 mm, while the short rainy season typically occurs between September and October, with an average monthly rainfall of 165 mm. The main dry season occurs from December to March . The meaunestus, . Most mounestus, ,37.A serological cross-sectional survey was carried out during the dry season in February-March 2009, and again in the rainy season in June-July of the same year, corresponding to the low and high transmission seasons, respectively. To obtain informed consent, residents in individual, randomly selected households were approached, the study goals and procedures were explained and potential participants were invited to enroll in the study. All willing individuals signed consent forms or provided a thumbprint in the presence of witnesses. Parental consent was obtained for children and all participants were transported to the Iguhu district government hospital. Individuals of all age groups from the study sites were eligible to participate except infants of less than 6 months of age. Demographic data including age and gender were taken, and, as altitude was a critical feature of the study, participants were also required to confirm their village of residence. Subsequently, clinicians and nurses obtained venipuncture blood (~ 3 ml). Sera was isolated and transported to the Kenya Medical Research Institute (KEMRI) Kisumu laboratories for storage in freezers at -80°C until further processed. Thick and thin blood smears were prepared according to standard protocols described elsewhere , to detePfMSP-119 FVO recombinant protein has been described elsewhere [PfMSP-119 FVO specific antigen were measured in serum by indirect ELISA [SPECTRAMAX 340pc  and the serial dilutions were used to fit a four-parameter curve using SoftMax Pro v4.1 (Molecular Devices). Results were expressed in titer values, the titer endpoint being defined in this study as the calculated serum dilution yielding an optical density of 1.0. A serum pool obtained from 30 naïve US donors never exposed to malaria was used to define an assay cutoff; the mean titer +3 SD represented the cutoff value for all negative responses to the PfMSP-119 antigen. The resultant titer value obtained as cutoff was 63.3; any titer above this value was considered as positive. A pool of malaria positive individuals from hyper-endemic areas in Kenya was used for positive controls.The expression and purification of the lsewhere ,40. Totact ELISA . Briefly19 seroprevalence is the number of positive responders out of the total number of participants tested; this is simply referred to in the whole text as seroprevalence. Differences in the proportion of seroprevalence of MSP-119 between age-stratified, uphill and valley residents were compared by the χ2 test with p < 0.05 considered statistically significant. The Mann-Whitney test was used to test if medians of seroprevalence were different between localities. Multinomial logistic regression was used to examine the association between MSP-119 seroprevalence and parasite prevalence adjusting for age in the valley and uphill population. Linear regression was used to examine the trend of parasite prevalence and age at different localitiesMSP-119 seroprevalence data to a simple reversible catalytic model using the maximum-likelihood method that assumes a binomial error distribution;We fitted age specific MSP-1t Pis the proportion of individuals aged t that is seropositive, constant λ is the annual rate of seroconversion and ρ is the annual rate of reversion to seronegative. This was done to investigate the relationship between force of parasite exposure and age [PfMSP-119 antibody titer, the data were analyzed and graphed using GraphPad Prism software . The age-seroprevalence model was fitted using JMP 9.0 .where  and age . For PfMThe distribution of the participant's population in terms of age and locality are shown in Table 12 = 0.52, p = 0.02). A similar trend was observed in the valley bottom residents where parasite prevalence generally declined with increasing age, however, this was marginally significant  and  respectively. In these two groups we observed parasite prevalence as high as a 2-fold increase in the valley residents compared with the uphill residents. However in the adult group we observed no significant differences in parasite prevalence between uphill and valley residents .In Figure In Table 2 = 33.78, df = 1, p < 0.001, rainy season χ2 = 69.96, df = 1, p < 0.001). However, when the two sites were combined there was no significant inter-seasonal variation in seroprevalence in < 5 and ≥ 15 years age groups . In contrast, the seasonal variation in seroprevalence was significant in children within the 5-14 years age group .Furthermore, highly significant differences were found when seroprevalence was compared between the uphill and valley residents in both seasons (dry season χP < 0.0001),  and in the valley population . Adjusting for age, the multinomial logistic regression used to assess the association between MSP-119 seroprevalence and parasite prevalence, revealed that seropositive individuals were more likely to have been exposed to parasites compared to seronegative individuals , and total IgG titers, together with malaria infection prevalence, we have observed considerable variation in human exposure to malaria in western Kenya. Seroprevalence of residents at the valley bottom was almost two fold higher than that of the uphill residents. Comparison of the median total IgG titers in the study population revealed a13-fold difference between the uphill and valley bottom residents. Similar trends have been observed in entomological and parasitological studies, which have consistently found higher transmission intensity, vector abundance, and parasite prevalence at the valley bottom, in comparison to the top of the hill [Using age-specific MSP-1the hill ,17,38. Tthe hill ,10,17,38Immunity to malaria is a function of exposure challenge; it develops gradually as a consequence of experiencing multiple parasite exposures or persistent infection for years. Consequently, seroprevalence reflects cumulative exposure and, thus, is less affected by seasonality or unstable transmission due to the longer duration of the specific antibody response ,21,41. IAge-specific seroprevalence has been used to estimate seroconversion rates (SCR) as a measure of malaria transmission intensity. Earlier studies in neighboring Tanzania have shown that these estimates are tightly correlated with EIR measurement ,14,27. AIn very low transmission settings, where parasite prevalence and EIR are insensitive, serological measures offer a way of accurately assessing endemicity and identifying focal areas of transmission supporting the potential for elimination . HistoriWhen we compared age-specific IgG titers from the two sites, there was a highly significant difference in the IgG titers between the valley bottom and the uphill residents, indicating a considerable variation in malaria endemicity within the highland area. The observed seasonal variation in the IgG titres in the 5-14 year group suggests that the level of exposure may not result in the development of stable humoral responses by the age 14. If this is solely due to seasonal exposure to malaria parasites, then it may represent a vulnerable group in the valley bottom area that may pose a public health problem in an event of hyper malaria transmission. As individuals remain seropositive for several years, the level of antibody response (IgG titers) can reflect fluctuations in recent exposure. It is known  that antThe observed parasite prevalence in the current study is lower compared to earlier studies conducted in the same area ,40. Muny19 responses that are highly varied within the same age group and altitude transects. Other studies have observed similar variation in exposure, susceptibility and even disease patterns at the individual level [An. gambiae salivary gland protein (gSG6-P1) previously validated as a marker of mosquito bite exposure [We observed a spectrum of MSP-1al level ,49. Thisal level , MSP-1 pal level  or antigal level . Intrinsal level , and proal level ,22 may bexposure .19, a leading vaccine candidate was used as a marker of immune response and a proxy of parasite exposure between the valley bottom and uphill residents. We observed higher immune response to the Plasmodium falciparum antigen in the valley residents than in the uphill dwellers indicating a possible higher exposure to infections. Odds ratios indicated seropositive individuals were more likely to be parasite positive than seronegatives. High and stable humoral immune responses were observed in older residents (> 15 years) at our highland site in spite of changing seasons. However, in children (≤ 15 years) we observed lower responses that also fluctuated with changing seasons. If the MSP-119 immune responses reflect functional immunity, something that was not directly determined in this study and is currently uncertain, uphill populations, particularly children ≤ 15 years of age, may be at a higher risk of (severe) clinical disease. This hypothesis requires further longitudinal studies.MSP-1The authors declare that they have no competing interests.KB carried out the field survey, the serological experiments, and the serological analysis and wrote the first draft of the manuscript. GY, YA and AG developed the study protocol, YA led the field operations, data and laboratory specimen collection, and GZ supported field team operations and data analysis. JW, JL, EA and AS coordinated laboratory procedures and serological testing, EA provided the plate antigen and participated in the study design and implementation, GY, AG, DJP and JMO conceived the study, participated in its design and implementation and supervised fieldwork, laboratory investigations, data analysis and manuscript development. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/50/prepub


Plasmodium falciparum malaria, but the incidence of severe and complicated vivax malaria tends to be increasing. We report two cases of severe Plasmodium vivax malaria from Malang, a nonendemic area in Indonesia. Patients exhibited anaemia, thrombocytopenia, jaundice, renal disturbance, and melena. Microscopic peripheral blood examination and amplification of parasite 18s rRNA by polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum. All patients responded well to antimalarial drugs.Most of the complications of malaria such as anaemia, thrombocytopenia, jaundice, and renal failure are commonly found in  Plasmodium falciparum is the most dangerous, since it can cause more severe manifestations and multisystem organ failure. Previously, Plasmodium vivax was thought as a benign parasite causing nonsevere and uncomplicated malaria, but recent reports about life-threatening complications from endemic regions such as Ethiopia [Plasmodium vivax seems to cause complications of severe malaria, including cerebral malaria [Several hepatic and renal complications are associated with complicated and severe falciparum malaria. Of the five human pathogenic species, Ethiopia , India [Ethiopia –5, BraziEthiopia , and PapEthiopia , 8 impli malaria –5, renal malaria  liver dy malaria , 5, thro malaria , and sev3, platelet count of 42000/mm3, glucose of 89 mg/dL, urea of 18.8 mmol/L, and serum creatinine of 0.56 mg/dL. The liver function tests showed an AST of 24 IU/L, ALT of 24 IU/L, total bilirubine of 2.59 mg/dL, direct bilirubine of 1.3 mg/dL, and indirect bilirubine of 1.25 mg/dL. Serum electrolyte analysis showed low potassium (2.94 mmol/L) and normal sodium levels (136 mmol/L). Blood smear showed Plasmodium vivax (parasitaemia 0.6%) as well as PCR result  of 8 mg/dL, leucocyte count of 4890/mmR result . No seizR result .3, platelet count of 54000/mm3, glucose of 68 mg/dL, urea of 77.7 mmol/L, serum creatinine of 1.46 mg/dL, AST of 354 IU/L, ALT of 88 IU/L, albumin of 1.88 g/L, total bilirubine of 4.61 mg/dL, direct bilirubine of 3.27 mg/dL, and indirect bilirubine of 1.34 mg/dL. Serum electrolyte analysis showed normal values. No seizures or cerebral symptoms were observed. Based on PCR results, the patient was positive for Plasmodium vivax confirming the microscopic diagnosis (parasitaemia 0.3%)  . The patia 0.3%) . There wMalang is known to be a nonendemic area of malaria in Indonesia, but some of the imported cases of malaria admitted to the Dr. Saiful Anwar Hospital showed severe clinical and laboratory complications. All patients in this study were admitted to the emergency unit. Clinical and laboratory examinations including Giemsa thin smear and polymerase chain reaction (PCR) were targeted against the 18S rRNA gene of the parasite were conducted to confirm the diagnosis .vivax infection. Females are at greater risk of hospitalization with Plasmodium vivax malaria than males and in one large analysis were more likely to present with anaemia. Pregnant women with Plasmodium vivax infection have a 2-fold higher risk of moderate anaemia than uninfected [Plasmodium species is the red blood cell. Plasmodium vivax has a very strong predilection for young red blood cells that have emerged from the bone marrow within the last 14 days. Despite reaching lower densities than P. falciparum, P. vivax causes similar absolute reduction in red cell mass because it results in proportionately greater removal of uninfected red blood cells [Both cases had moderate anaemia with haemoglobin levels <10 mg/dL . Anaemia is a common and frequently severe consequence of infected , 12. In infected . The priod cells .Mild hypoglycaemia occurred in the second case (WHO criterion for severe hypoglycaemia is <40 mg/dL). As the patient was pregnant, hypoglycaemia could also be due to pregnancy . Malaria3. Severe thrombocytopenia is common in isolated falciparum and mixed falciparum/vivax malaria but is very rare in isolated Plasmodium vivax infection. The mechanism of thrombocytopenia in malaria is uncertain. In vivax infection, it may be due to direct lysis of the platelets, reduced platelet survival, and IgG mediated destruction [The first case had a platelet count less than 50000/mmtruction , 16.The incidence of leucopenia in this study was consistent with other reports. The leukocyte count in malaria is from low to normal due to the localisation of leukocytes away from peripheral circulation to spleen and other marginal pools rather than actual depletion or stasis. This is a transient finding like thrombocytopenia and normalises after antimalarial therapy .Plasmodium vivax malaria based on WHO criteria . Malaria-associated renal and hepatic dysfunctions are severe complications that are increasingly becoming a problem of great concern in malaria endemic countries. According to the World Health Organization, serum creatinine levels above 4.77 mg/dL are an indication of acute renal failure. Serum creatinine level in the second case showed only acute renal disturbance. The mechanisms proposed for acute renal disturbance in Plasmodium vivax infection are heavy parasitaemia, acute tubular necrosis, volume depletion, intravascular haemolysis, renal ischemia, and microvascular sequestration [The current study documented the severe manifestations of stration .The increase of serum level of hepatic enzymes, transaminases (AST and ALT), and alkaline phosphatase is the markers of liver damage . The secPlasmodium vivax infection have also been reported by Lakhar et al., (1996) who explained that DIC is a major cause of bleeding [μL. Acute bleeding due to thrombocythopenia is unlikely as long as platelet function is still effective.Gastrointestinal bleeding in the form of melena occurred in the second case. The bleeding can occur for many reasons, one of which is the DIC. Cases of melena in bleeding . PlateleVivax malaria has been long considered to have a benign course. It is known for multiple relapses, but the typical complications seen with falciparum malaria are not found with vivax monoinfection. However, in the past few years severe and complicated clinical manifestations of vivax malaria are frequently reported, sometimes even fatalities [Plasmodium vivax malaria can also cause severe complications such as anaemia, thrombocythopenia, melena, acute renal disturbance, hepatic dysfunction, and jaundice. Pregnancy in Plasmodium vivax infection is clearly associated with serious complication as mentioned in this case and reported previously [Plasmodium vivax malaria, and emerging drug resistance, emphasis must be on strict preventive measures to lower the burden of this disease [talities , 23. In eviously . Rapid d disease .