In malaria-endemic areas, residents of modern houses have less malaria than those living in traditional houses. This study will determine if modern housing provides incremental protection against clinical malaria over the current best practice of long-lasting insecticidal nets (LLINs) and prompt treatment in The Gambia, determine the incremental cost-effectiveness of the interventions, and analyze the housing market in The Gambia.Plasmodium falciparum infection, parasite density, and the prevalence of anemia. Exposure to malaria parasites will be assessed using light traps, followed by detection of Anopheles gambiae species and sporozoite infection. Ancillary economic and social science studies will undertake a cost-effectiveness analysis and use qualitative and participatory methods to explore the acceptability of the housing modifications and to design strategies for scaling-up housing interventions.A two-armed, household, cluster-randomized, controlled study will be conducted to assess whether improved housing and LLINs combine to provide better protection against clinical malaria in children than LLINs alone in The Gambia. The unit of randomization will be the household, defined as a house and its occupants. A total of 800 households will be enrolled and will receive LLINs, and 400 will receive improved housing before clinical follow-up. One child aged 6 months to 13 years will be enrolled from each household and followed for clinical malaria using active case detection to estimate malaria incidence for two malaria transmission seasons. Episodes of clinical malaria will be the primary endpoint. Study children will be surveyed at the end of each transmission season to estimate the prevalence of The study is the first of its kind to measure the efficacy of housing on reducing clinical malaria, assess the incremental cost-effectiveness of improved housing, and identify mechanisms for scaling up housing interventions. Trial findings will help inform policy makers on improved housing for malaria control in sub-Saharan Africa.ISRCTN02622179. Registered on 23 September 2014.ISRCTN Registry, The online version of this article (doi:10.1186/s13063-016-1400-7) contains supplementary material, which is available to authorized users. Metal-roofed houses more frequently have closed eaves, so installing metal-roofs and closing the eaves should reduce the entry of malaria vectors. Because screening the doors and windows also reduces mosquito house entry [The hypothesis supporting housing as being protective against malaria in sub-Saharan Africa is based on the observation that 79–100 % of malaria transmission occurs indoors at night ; so prevse entry , we hypoThe long-term effectiveness and potential impact of housing as a malaria intervention will depend on its cost-effectiveness, acceptability to householders, and the feasibility of implementation at scale. Therefore, alongside the trial, we will undertake a cost-effectiveness analysis and use qualitative and participatory methods to explore the acceptability of the housing modifications and to design strategies for scaling up housing interventions. The incremental costs and benefits associated with house improvements, as well as the demand and supply of construction material and skill, are invaluable data to inform policymakers in their decision to adopt the interventions as policy. However, very limited evidence exists on the cost-effectiveness of improved housing on health, and the little information available is focused on interventions implemented in high-income countries . AnalysiWe view improved housing not as a stand-alone-intervention but as one that should be incorporated with established and effective vector-control interventions such as LLINs. Thus, our goal is to determine whether there is any additional benefit in improving rural homes over the current best practice of using LLINs alone for protecting children against clinical malaria, to determine the incremental cost-effectiveness of the interventions, and analyze the housing market in The Gambia.The primary clinical objective its to assess whether improved housing  and LLINs provide added protection against clinical malaria in children with the LLINs alone over two malaria transmission seasons of follow-up.To determine whether improved housing reduces the rate of parasite infection, parasite density, and anemia in childrenTo assess whether improved housing is associated with a rise in respiratory infectionsThe secondary clinical objectives include the following:The primary entomological objective is to assess whether improved housing and LLINs reduce vector density inside houses when compared with the LLINs alone.The primary economic and social science objective is to assess the incremental costs, benefits, and cost-effectiveness of these interventions.To explore the acceptability of the house modifications to the residentsTo analyze the housing market in The Gambia for the identification of economic barriers to scale-upThe secondary economic and social science objectives are as follows:2 and is bisected by the river into the north and south banks. The population of the region was 182,586 in 2003, the most recent census, with most living in small discrete rural villages in houses made with mud or cement walls and thatched or metal roofs. The study’s field station is based at Basse Santa Su town , which is the only sizeable urban area in the region.The study is situated in the far east of The Gambia, in the Upper River Region (URR), an area of open Sudan savanna. The climate consists of a single rainy season from May to October followed by a long dry season. This defines the highly seasonal malaria transmission, with most malaria episodes experienced during or immediately following the rainy season. This region covers an area of 1995 kmRural villages located on the north and south bank of the Gambia River will be selected. We will discuss the study with village leaders, including women in each potential study village, and proceed if given approval. Community sensitization will be followed by a survey to identify suitable houses.A total of 800 traditional thatched-roofed houses constructed with mud walls, open eaves, and without ceilings or screening, with two children aged 6 months to 13 years, will be selected. Consent will be sought from the house owners (and residents) for them to join the study by field assistants . After explaining the purpose of the study to the household heads, only children whose parents have given written, informed consent for their child to be included in the study will be enrolled .The control arm, representing traditional houses, will be left with thatched roofs and open eaves (n = 400) until the end of the study. A metal-louvered screened door will be installed in the front of the house, and a wooden-screened door, at the back [Enrolled houses will all have thatched roofs and mud walls in good condition, and the occupants, provided with a sufficient number of LLINs  to cover all sleeping places. At the time of donation, we will follow national guidelines to encourage their correct use, as this is the current best practice. In the intervention arm, represented by modern housing, we propose to modify existing rectangular-plan and circular-plan thatched roof houses, so they will have metal roofs, closed eaves , and scrthe back . HousehoThe baseline clinical survey of all study children will take place in May/June 2016 and will determine malaria infection, splenomegaly, and anemia. Clinical follow-up will start in June 2016, and cover both rainy seasons (June to December 2016 and 2017). Incidence of clinical malaria will be determined by ACD during twice weekly house visits from June to November each year [No treatment will be administered by the study personnel. However, any patient diagnosed with malaria or suspected to have pneumonia or other illnesses will be referred to the local health system for treatment according to the Gambian National Guidelines.The main morbidity outcome will be the incidence of clinical disease assessed by ACD; data from this will provide the primary endpoint of the study. The axillary temperature will be taken twice weekly and if ≥ 37.5 °C, a rapid diagnostic test  for malaria will be made, and the breathing rate will be checked. The presence of a cough and either a raised age-specific respiratory rate or chest indrawing will also be assessed in children deemed to be unwell by the parent or guardian. Serious adverse events (SAE) will be recorded during both transmission seasons. Sick children will receive referral notes to the nearest health post/facility, and treatment will be documented at the next biweekly visit.Plasmodium parasites. Blood films will be stained in the field by project staff, transported to the laboratory, and read independently by two microscopists blinded to the identity of the child. Any discrepancy will be resolved by a third senior microscopist. Children with sickness or disease will be referred for treatment.During the cross-sectional surveys, clinical surveys will be done of all study children, who will be examined for general health and the presence of an enlarged spleen. A finger prick sample will be taken to measure the blood hemoglobin immediately using a spectrophotometer  and to prepare thick smears for later detection and quantification of An. gambiae s.l. and other species, recorded. The presence of sporozoites in An. gambiae s.l. will be identified using an enzyme-linked immunosorbent assay [An. gambiae s.l. females, typed to species by PCR [Indoor mosquito collections made using Centers for Disease Control (CDC) light traps will be used to estimate the potential exposure to malaria vectors and will be carried out once a month for 6 months from June to December in 2016 and 2017 in the bedrooms of the study children. Mosquitoes will be identified by microscopy, and the numbers of nt assay , and An.s by PCR .Data on incremental costs of house improvements and their sources will be collected alongside the interventions. Incremental benefits due to malaria cases averted will be estimated based on malaria treatment cost data collected from selected health facilities. The acceptability of the intervention will be captured during the trial using (1) observations and informal conversations during the house modification process, (2) photo-voice  , and (3)No potential pharmacological intervention or treatment by project staff is proposed in this study. However, the possibility remains that our modified housing may increase the risk of acute respiratory infections. We will therefore assess the study children for respiratory disease at each study visit, i.e., twice weekly during the transmission season. Since respiratory infections are common in children in this area , we seleParticipants are free to withdraw from the study at any time without giving a reason. In the unlikely event that homeowners require their house to be returned to the pre-intervention state, we will remove the items we have added and replace the metal roof with a thatched one. Withdrawals may also occur if a study subject has any clinically significant AE, laboratory abnormality, intercurrent illness, or other medical condition or situation that occurs such that continued participation in the study would not be in the best interest of the participant. No replacements will be made during the surveillance period in either year, but in May 2016, children who have withdrawn or are no longer in the study for any reason will be replaced by a child of a similar age from the same house. If a household withdraws consent, no further follow up will be made in that household. If the house was participating in the entomology collections, it will be replaced by a neighboring house of the same type, if possible.P. falciparum parasites detected by microscopy.The primary clinical endpoint will be the incidence of clinical malaria, which is determined by active case detection (ACD) and defined as a body (axillary) temperature of ≥ 37.5 °C, together with the presence of P. falciparum parasite rates, (2) prevalence of splenomegaly, (3) prevalence of anemia, and (4) an incidence of respiratory infection, measured as a cough and either a raised age-specific respiratory rate or chest indrawing.The secondary clinical endpoints will be (1) An. gambiae s.l./light trap/night.The primary entomological endpoint will be the mean number of female The secondary entomological endpoint will be the estimated entomological inoculation rate (EIR) in each study arm .The primary economic and social sciences endpoints will be the mean and median costs of house improvement interventions/house improved and the mean and median costs of treating one case of childhood malaria.The secondary economic and social sciences endpoints will be the percentage of home owners who agreed to house modifications in the intervention arm, key observations and discussions on house modifications with villagers and builders in the trial, trends in the types and quantities of housing materials imported/exported, the construction skills developed during at least the previous 5 years in the country, and a road map for scaling up of the housing modifications.p value of less than 5 % for the difference between the control and the intervention arm was the estimated power. This shows that when following one child per house for 2 years, five houses in 80 villages are required in each study arm  to detect a 35 % reduction in malaria cases with more than 80 % power at the 5 % significance level . In order to demonstrate a 50 % reduction in indoor-entering mosquitoes  associated with housing interventions, with 80 % power at the 5 % level of significance, we would require 47 houses in each arm of the study respectively. Since the number of mosquitoes rise and fall during the rainy season, we propose to sample from each house six times each year. To allow for loss to follow-up during the study period, we propose to conduct entomological monitoring in 60 houses in each arm of the study each year.We postulate that metal-roofed houses, closed eaves, and screening on the doors will reduce indoor collections of malaria vectors by at least 50 %. Based on a study using light traps in the proposed study area in 2011, we expect that the mean number of female The demographic data will be recorded by fieldworkers, and the clinical data by study nurses on standardized data forms. Each child in the cohort will be identified by a unique identification number. All forms and datasets will identify participants by these numbers, and names will not be entered. The study will follow the MRC Unit The Gambia’s specific policy to maintain anonymity of study participants. Personal identifiers will be removed from the transcripts of interviews, and discussions with participants will be identified only through a study identification number. Transcribe and translated transcripts will be entered into NVivo for management and analysis. Participants in the photo-voice exercise will be specifically requested and trained not to take photographs containing images of identifiable people, and any photographs containing such images will not be entered into the NVivo database. Thus all data will be anonymized at data entry, including those participating in the subsidiary studies (economic and perceptions of the housing intervention). Databases will be password protected and accessible only to authorized personnel. All documents will be securely stored in locked filing cabinets and accessible only to authorized personnel.The PI will maintain appropriate medical and research records for this study in compliance with the principles of good clinical practice and regulatory and institutional requirements for the protection of confidentiality of participants. The study team members will have access to records. The authorized representatives of the sponsor, the ethics committee(s), or regulatory bodies may inspect all documents and records required to be maintained by the investigator, including but not limited to medical records  for the participants in this study. The clinical study site will permit access to such records. The results of the study will be made publicly available. Blood slides and mosquito DNA will be stored and made available for future studies.A per-protocol and an intention-to-treat analysis will be conducted.Outcome 1—malaria morbidity. Protective efficacy against clinical malaria will be determined by comparing incidence rates of clinical malaria between arms. After any treatment for malaria, the child will not be considered at risk for 4 weeks, and this period will be censored. History of travel away from the household will be captured by twice-weekly surveys during the malaria season, and the time at risk will be censored for such periods. An initial unadjusted analysis will be based on comparisons of the incidence rates between the two arms. Formal analysis will use a mixed effects Poisson  model to test the difference in incidence rate between the two arms, allowing for the repeated measurements within house and within village and the effect of year. With multiple control and intervention houses within each village, the intervention * village interaction can also be quantified. Possible confounders such as age of child, gender, ethnicity, and rainfall will be tested using the mixed effects model.A comparison of the time to the first episode of clinical malaria and repeat episodes of clinical malaria will be examined using a survival analysis approach. Initial analyses will be based on Kaplan-Meier curves with further adjustment for confounders performed using a Cox regression model.Outcome 2—malaria transmission. Differences in malaria transmission experienced in the two groups will be made by comparing the mean number of mosquitoes (as a proxy for transmission) caught indoors in houses between the intervention groups. Generalized estimating equations will be used to estimate differences in the numbers of indoor-resting mosquitoes, adjusting for repeated measures within houses and possible covariates.Outcome 3—incremental cost-effectiveness ratio. The incremental cost-effectiveness ratio of averting a clinical case of malaria by home improvement interventions will be calculated through probabilistic decision tree analysis. The health impact will be also expressed in terms of disability-adjusted life years.Outcome 4—housing market supply. Data on the types and quantities of construction material produced and imported during at least the previous 5 years, as well as of construction skills developed within the country, will be analyzed against the resources used within the trial. Time-trend analysis will be also performed to generate future predictions of the quantities potentially available in the next few years, while controlling for confounding factors.Given the nature of the intervention, it is impossible to conduct this study in a fully blinded manner, but those parts of the data collection that can be blinded will be. Observer bias will be reduced where feasible. Blood films will be read by microscopists blinded to the identity and intervention status of the subjects. Mosquito collection bias will be reduced by using standard light traps that do not rely on the ability of the fieldworker to collect specimens. Trap catches will not be examined and analyzed by those who collected them but by different technicians who will not know the trap location. Datasets will only be unblinded after all those critical for the listed primary and secondary endpoints have been locked.https://www.mrc.ac.uk/documents/pdf/good-clinical-practice-in-clinical-trials/). In particular, the SSC will concentrate on the progress of the trial, adherence to the protocol, patient safety, and the consideration of new information, and the SSC will formally report to the sponsor (Durham University). The Data Monitoring Committee will determine if additional interim analyses of trial data should be undertaken, and assess any additional safety issues that may arise during the study. They will ensure the safety, rights, and well-being of the study participants and will report to the SCC at regular intervals. The roles and responsibilities of the steering committees are described in Additional file An SSC will provide overall supervision of the trial and ensure that the trial is conducted to the standards set out in the Medical Research Council’s Guidelines for Good Clinical Practice  and the School of Biological and Biomedical Sciences Ethics Committee, Durham University, on 1 December 2014 (ref: SBBS/EC/1401/RooPfs 12 09 14). Approval for any important protocol modifications will be sought from both ethics committees.Pfs study is novel because we are not using chemicals as an intervention. This is important because the major threat to global malaria control today is from strains of vectors that are resistant to the insecticides used for treating bed nets or spraying indoors [Most malaria-control interventions are based on the use of chemicals. While drugs and vaccines are used to protect people from the disease, insecticides are used to protect people from potentially infective mosquito bites. The Roo indoors . Since vThe quality of housing is improving across many parts of sub-Saharan Africa. Despite the global economic recession, Africa’s economy is the fastest growing of any continent , and the economies of sub-Saharan Africa are anticipated to grow at a rate of 6 % per annum over the next decade. Such rapid development is leading to marked changes in the housing stock with the traditional thatched houses disappearing in many areas. With a rapidly growing population, the need for further housing is pressing, and this provides an opportunity for new housing construction that will protect the occupants from vector-borne diseases such as malaria.A recent systematic review suggests that improved housing could reduce the amount of malaria infection and disease by half . Our preThe potential risk exists to study children that screening houses from mosquitoes will reduce the air circulation in the house, making it hotter, and perhaps increasing the risk of respiratory disease. We think this is unlikely but will record respiratory disease in the study cohort. It should be appreciated that all study children will be have access to LLIN at the start of the study, and that the nets limit ventilation sharply . Indeed,Study subjects will benefit from this study by having a desired commodity: a metal-roofed and screened house. Children will also have a health check at the surveys. However, a number of limitations exist for the use of improved housing to control malaria. First, we do not know whether the housing interventions will be acceptable to local people. Second, we do not know how durable the interventions will be. Third, this intervention is most likely to be introduced outside the health sector, so innovative new pathways to scale up will need to be developed. Finally, we are unaware of the costs of this intervention but recognize that they are likely to be greater than conventional vector-control strategies. All of these issues will be addressed in ancillary studies being carried out in parallel with the randomized controlled study. Furthermore, improved housing is not an “evolution proof” intervention. If vectors adapted to entering houses are prevented from doing so, it is possible that vectors will be selected for biting outdoors, possibly early in the evening, when they are most likely to feed. Such changes in behavior have been seen when LLINs or IRS have been used on a large scale . This em17 July 2015, version 2.0.The trial is currently recruiting.PfPR, Plasmodium falciparum parasite rates; PI, principal investigator; RDT, rapid diagnostic test; SSA, sub-Saharan Africa; SAE, serious adverse event; WHO, World Health Organization; WHOPES, WHO Pesticide Evaluation SchemeACD, active case detection; AE, adverse event; CDC, Centers for Disease Control and Prevention; CRF, case report form; CV, coefficient of variation; EIR, entomological inoculation rate; GCP, good clinical practice; Hb, hemoglobin; ICH, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use; ID, identification number; LLIN, long-lasting impregnated nets (permethrin-treated); PCR, polymerase chain reaction;


Plasmodium infection carriers represent a major threatto malaria control worldwide as they are silent natural reservoirs and do not seekmedical care. There are no standard criteria forasymptomaticPlasmodium infection; therefore, its diagnosis relieson the presence of the parasite during a specific period of symptomless infection.The antiparasitic immune response can result in reducedPlasmodiumsp. load with control of disease manifestations, which leads to asymptomaticinfection. Both the innate and adaptive immune responses seem to play major roles inasymptomatic Plasmodiuminfection; T regulatory cell activity(through the production of interleukin-10 and transforming growth factor-β) andB-cells (with a broad antibody response) both play prominent roles. Furthermore,molecules involved in the haem detoxification pathway (such as haptoglobin and haemeoxygenase-1) and iron metabolism have emerged in recent years as potential biomarkers and thus are helping to unravelthe immune response underlying asymptomatic Plasmodium infection.The acquisition of large data sets and the use of robust statistical tools, includingnetwork analysis, associated with well-designed malaria studies will likely helpelucidate the immune mechanisms responsible for asymptomatic infection.Asymptomatic  It is estimated that two-three billion people are at risk of contracting malaria, andnearly one million people die from this disease each year . The spePlasmodium sp. touninfected persons ,individuals with API harbour the parasite as evidenced by positive parasitaemia. However,these individuals do not develop any symptoms during a defined period of time . API is  persons , (ii)as persons , and against ite load . The immite load . In contite load .Immunity to malaria does not necessarily prevent infection; however, it does limit parasitedensity and symptoms . APIindPlasmodium infection as well as new approaches to understand APIimmunity.The immune system seems to play a major role in malaria outcomes, and our object herein isto uncover the partial protective immune response to infection in API to unravel themechanisms of disease resistance. Here, we review both innate and adaptive immune responsesto Plasmodium sp. clones; multiclonal infections may bea marker of immunity and confer protection against malaria by inducing a broader immuneresponse and tolerance to infection  have an increased HBV viraemia yet a decreased malaria parasitaemia, reduced levels of cerebral chemokines, increased levels of nitricoxide, and higher IgG seroreactivity to P. falciparum antigens , haemoglobin C (HbC), andα-thalassaemia, have been associated with protection from malaria . API chiantigens . Other hantigens , southeaantigens , glucoseantigens , and bloantigens  are assoP.falciparum antigen-specific IgGs was extracellular and indicated a key rolefor CD32/FcγRII; however, the production of ROS in response to whole merozoites wasalmost completely within the cell, suggesting that the underlying mechanism wasphagocytosis activity is correlated with acquired disease resistance to malaria in endemic regions. In thisocytosis . The innocytosis . Further malaria . The indentation . In a sientation . Cells o+ and CD8+T-cells are important formalarial immunity in humans as well as in mouse models  in recognising and binding diverse antigens through a repertoire of cellsurface receptors . Ithas e models . In a clmic area . CD4+ceed IL-10 . In thisnfection . Thus, infection .+CD25+FOXP3+) appear tomediate their effects by direct cell contact or by induction of the regulatory cytokinesIL-10 or transforming growth factor (TGF)-β  than didpatients with uncomplicated and severe malaria, suggesting a role for Treg reduction inmalaria protection (STAT6 and IL10RA), which may influence Treg cellsand malaria protection (T regulatory (Treg) cells (CD4 (TGF)-β . Treg ceoduction . Neverthotection .Intriguotection . Congoleotection .P. falciparum are short-lived and fail to boost uponre-infection. Thus, immunological memory is a challenge in many vaccine trials ,P. falciparum CLAG 9 , and malaria-infected erythrocyte variant surface antigens, includingP. falciparumerythrocyte membrane protein 1, P.falciparum merozoite surface protein 1 3D7 (MSP142), P.falciparum VarO rosetting variant, andP. falciparumerythrocyte binding-like and reticulocyte binding-like proteins , alanine aminotransferase (ALT), indirectbilirubin, and serum creatinine as well as higher levels of Hb than do individuals withmild or severe symptomatic P. vivax malaria  and which may explainthe immunological control of clinical disease. However, parasite burden control mayinvolve a more complex host response in addition to the moderation of TNF levels  areassociated with a decreased risk of mild malaria compared to asymptomatic infection byreducing plasma levels of IL-6 and TNF  and TGF-β production through superoxide dismutase(SOD)-1-dependent mechanism and prevents the release of haem. The complex Hp-Hb is recognised by CD163 on themacrophage and hepatocyte surfaces in the spleen and liver, respectively , andexposure to CO may reduce severe complications  and markers of tissue damage  and augmented levels of regulatory cytokines (TGF-βand IL-10) when compared with those of the symptomatic groups ,artificial neural, Bayesian, and network analysis methods are some tools that can beused to characterise a molecular signature of resistance or susceptibility to malaria. Many stnfection . Using aoutcomes . Networkmalaria). Furtherolerance . Othersolerance . In thisP. vivax infection is an active and acquired state, and itcan control parasitaemia and limit organ dysfunction by an as yet poorly understoodimmune mechanism. Asymptomatic individuals carrying the parasite are natural reservoirsrepresenting a challenge for malaria eradication, primarily in low and moderate-endemiccountries. The use of mass drug administration or mass screening and treatment schemesis controversial  but is not associated with immunity and inflammatory tolerance.Asymptomatic oversial . Overall


Plasmodium and a subspecies of Plasmodium using filter paper in malaria endemic province, Sanliurfa, in Turkey, compare the results of nested PCR (nPCR) with microscopy for the diagnosis of malaria and present the epidemiological data of malaria.The objective of this study to detect Plasmodium gene with genus and specific primers. The results of the microscopy were compared to the nPCR results.This study was carried out in malaria-endemic Sanliurfa between 2008 and 2011. Finger prick blood samples, thick and thin Giemsa-stained blood smears, were collected from 153 malaria-suspected farmworkers. The Giemsa-stained blood smears were examined microscopically. The obtained DNA products, extracted from blood-spotted filter papers or from the thick blood smears, were analysed by nPCR to amplify the 18S ssrRNA Plasmodium-positive by microscopy, whereas 9.8 % were determined as Plasmodium-positive by nPCR. Of the positive Plasmodium specimens, 93.33 % were identified as P. vivax. Four out of the 15 specimens that were microscopically diagnosed as negative were Plasmodium-positive with nPCR. When compared to the microscopy, the sensitivity, specificity, and positive predictive values of the nPCR were determined as 100, 97.2 and 73.3 %, respectively. nPCR was determined to be more sensitive and specific than microscopy.Of the specimens, 7.2 % were determined as This study revealed that the accurate diagnosis of malaria by nPCR was compulsory in malaria-endemic Sanliurfa and nPCR should be applied routinely in laboratory studies. Plasmodium vivax and this ratio increases to 47 % outside the African continent [Malaria is the major cause of morbidity and mortality in adults and children worldwide. According to the World Health Organization (WHO), there were an estimated 198 million cases of malaria in 2013, of which approximately 82 % were in the African Region, followed by South-East Asia (12 %), and eastern Mediterranean regions (5 %). About 8 % of the globally estimated cases are due to Plasmodium species is present at low levels (<100 parasites/mL), or modified by anti-malarial drug treatment [Accurate diagnosis is an important tool in the fight against malaria and universal access to a parasitological test is part of the WHO objectives . Microscreatment , 7 and ireatment . Howeverreatment . Accordireatment , 9.The Southeastern Anatolia Region (GAP) is the most malaria-endemic region, where one of the two largest malaria epidemics of Turkey was occurred in 1994 with 84,345 cases . AccordiPlasmodium vivax is the only agent of indigenous malaria cases and only imported Plasmodium falciparum cases are seen in Turkey [Anopheles mosquitoes. As reported by the WHO, Anopheles mosquitoes breed in water and each species has its own breeding preference; for example, some prefer shallow collections of fresh water, such as puddles, rice fields, and hoof prints [In the GAP of Turkey, malaria transmission is seasonal, generally occurring between March and October, and shows a marked local distribution . Plasmodn Turkey –16. The f prints . The GAPThese environmental conditions and migration from neighbouring countries result in Sanliurfa being a malaria-endemic province. Generally, the diagnosis of malaria depends on microscopical examination of thick and thin Giemsa-stained blood smears in this study area. There were no data about the diagnosis of malaria using nested PCR (nPCR) in Sanliurfa. This was the first study to compare nPCR and microscopy in Sanliurfa, in Turkey.Plasmodium and a subspecies of Plasmodium using filter paper and compare the results of nested PCR (nPCR) with microscopy for accurate malaria diagnosis and present the epidemiological data in Sanliurfa, southeastern Turkey.In light of these data, the aim of this study was to detect This study was conducted between 2008 and 2011 in three towns  of Sanliurfa , where malaria was seen throughout the year with peaks during September to November. Average annual temperature is 19.83 °C and monthly minimal temperature −4.3 °C in January and maximal 44.2 °C in July. Average annual relative humidity is 45.27 % and rainfall is 518.91 mm [P. vivax, and in recent years, foreign originated P. falciparum cases were frequently seen in the region [After explaining the aim of the study, written informed consent was obtained from participants or their parents who work as seasonal farmworkers on agriculture of paddy, cotton, rice fields, etc., or the participants’ parents. All of the participants were asked to fill out a standardized questionnaire, which included sociodemographics and living conditions: age, gender, education, the story of malaria of the participant and his/her family in the past, history of travel abroad, presence of a chronic disease, and presence of a sewerage or stream close to their home. The main agent of the indigenous malaria transmission is due to e region .The investigators visited and actively screened Akcakale, Harran, and Siverek (by house-to-house screening) with malaria experts and technicians from the Malaria Eradication Centre of Sanliurfa. In this study, a total of 153 malaria-suspected farmworkers or farmworkers’ children with at least a few malaria symptoms, including fever, headache, chills, and vomiting were determined.®31ETCHR filter paper , air-dried, and stored at room temperature until DNA extraction.An aliquot of venous blood (100–200 µL) was taken by finger prick from 153 malaria-suspected farmworkers or their children, adsorbed onto WhatmanThin and thick Giemsa-stained blood smears were prepared during the collection of the blood specimens. All of the blood smears were examined by two experienced microscopists  blinded to each other’s results according to the WHO competency assessment protocol . The bloDNA was extracted from filter blots using the QIAmp DNA Blood Mini Kit  according to the manufacturer’s instructions.Before the extraction of DNA, the slides were cleaned to remove oil residues. Approximately 20 µL of Tris–EDTA (TE) buffer was put on the thick blood smear. Whatman filter paper was cut into strips and placed on the slide to absorb the buffer. The filter paper absorbed the blood from the thick blood smear, and was held by sterile forceps and put into a 1.5 mL centrifuge tube. Next, the DNA from the filter paper was extracted using the QIAmp DNA Blood Mini Kit  according to the manufacturer’s instructions.P. vivax, P. falciparum, Plasmodium ovale, and Plasmodium malariae, in which specific primers were used, as described by Snounou et al. [The nPCR amplification strategy was used for genotyping the 18S ssrRNA genes of u et al. . For theu et al.  , 2.5 mM MgCl2, and 0.4 units of Taq DNA polymerase .The cycling parameters for the PCR were as follows: step 1, initial denaturation at 95 °C for 5 min; step 2, annealing at X °C for 2 min ; step 3, extension at 72 °C for 2 min; step 4, denaturation at 94 °C for 1 min; step 5, repeat steps 2–4 for a total of 25 cycles (Nested 1) or 30 cycles (Nested 2); step 6, final annealing at X °C for 2 min ; step 7, final extension at 72 °C for 5 min; and step 8, reducing the temperature to 4 °C. The PCR products were stored at 4 °C until analysis.P. vivax, P. falciparum, P. malariae, and P. ovale, and stained with ethidium bromide for visual detection by ultraviolet transilluminator.The amplified products were electrophoresed on 2 % agarose gels performed in Tris–borate-EDTA for All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee of the Faculty of Medicine at Firat University (22.09.2011/13/13).Data entry and analyses were performed using Statistical Package for the Social Sciences 11.5. The descriptive data was given as a means with standard deviations, frequency counts, and percentages. Considered the gold standard in the diagnosis of malaria diagnostic methods based on microscopic diagnosis of malaria in the nPCR analysis of validity and reliability of the methodology for the determination of epidemiological research methods were used. nPCR analysis’ sensitivity, specificity, reliability, positive predictive value (PPV), negative predictive value (NPV) were analysed.The study sample comprised 153 suspected malaria patients, aged between 1 month and 77 years, from three provinces of Sanliurfa between 2008–2011  were determined as Plasmodium-positive using nPCR with genus specific primers   of the specimens were detected as 1 of the reatment .Fig. 1AgPlasmodium and 93.33 % of them were P. vivax by nested PCR; three samples from Siverek, eight from Harran in 2008, three from Harran in 2009 and one from Akcakale in 2011. Although these samples were limited in small number size (n = 15), they were included in 26.8 % of total samples (n = 56) in study provinces: 43 in 2008, eight in 2009, four in 2010, one in 2011 , and 53.3 % were male.A total of 15 samples were diagnosed as Plasmodium specimens, 14 of 15 (93.33 %) were identified as P. vivax (Fig. P. vivax, P. falciparum, P. malaria, P. ovale, respectively. One of the 15 Plasmodium-positive samples (Table Of the vax Fig.  using sp4 of 15 9.33 % werPlasmodium with nested-PCR (Table Four out of the 15 specimens that were microscopically diagnosed as negative were positive for CR Table .Table 4CWhen compared to the microscopy, the sensitivity, specificity, and positive predictive values of the nPCR were found as 100, 97.2 and 73.3 %, respectively.The results of this study indicated that the examination of thick and thin blood smears by microscopy were insufficient for the diagnosis of malaria in this region. This study supported the idea that sensitivity decreases with microscopical tests as parasitaemia falls below 100 parasites/mL and false negatives are observed , 26, 27.Plasmodium-positive samples, 93.33 % were detected as P. vivax and 6.77 % was not identified by targeting the 18S rRNA gene with the species-specific primers in the present study. According to the microscopical examination of the thick blood smear, it was identified as P. vivax. This result might be explained mostly in three ways: first, this patient was diagnosed by the technicians of the Sanliurfa National Malaria Eradication Centre, who only prepare thick and thin Giemsa-stained blood smears. They did not allow the adsorption of the patient’s blood onto the filter paper. They only sent the thick and thin blood smears to us for confirmation of the diagnosis of malaria. Hence, the investigators were obliged to extract DNA from the thick blood smears. This might be as a result of using DNA extraction from the thick blood smear. Because, most recently, Scopel et al. reported that the use of DNA extracted from thick blood smears resulted in poor detection of malaria parasites, particularly with parasite densities of less than 20/µL [Plasmodium. When the patient was asked whether he had travelled to southeast Asia or not, it was seen that the patient had never travelled. So the idea of fifth species of Plasmodium is highly remote moreover the morphology of this parasite is quite distinct from that of P. vivax, which was confirmed microscopic diagnosis. Third, it might have been a case of the variant P. ovale as the oligonucleotide primer pairs used did not include those that can amplify this variant (Fig. Of the detected Plasmodium by nPCR might prevent misdiagnosis, incorrect treatment, false positives, false negatives, the emergence and spread of drug resistance, and the transmission of Plasmodium parasites from a malaria-endemic region to other provinces of Turkey. The nPCR was not affected by the subjectivity of the observers. In light of these data, nPCR might be a good and useful complement for clinically suspected but microscopically-negative malaria cases and epidemiological studies of P. vivax infections. nPCR might be a huge chance for the detection of low density of malaria parasites, mixed infections and identification asymptomatic carriers and reservoirs of parasites. nPCR in malaria suspected people might be a promising alternative to thick smear for screening for malaria in endemic region. Since, asymptomatic malaria carriers might be the parasite reservoir and responsible for the transmission of malaria in endemic region as it had previously been reported [Plasmodium and Plasmodium species, detection of Plasmodium DNA by nPCR from filter paper is easy to use and slightly invasive complement in malaria endemic region. As a result of the findings, nPCR is compulsory in Sanliurfa and it should be applied routinely in laboratory studies.This study emphasized that nPCR is an excellent method for obtaining accurate epidemiological data in malaria endemic Sanliurfa Fig. . The diareported , 35. TheThe results of the survey presented indicate that some cases of malaria might be missed by conventional microscopy and warrant the use of sensitive molecular techniques for surveillance; this should be placed in the current context where a large influx of displaced persons from neighbouring countries and some malaria-endemic countries are located in the GAP region. This raises a major concern that a potential re-introduction of malaria might occur in areas where it can rapidly disseminate into the local population. For instance, in the 2012 outbreak registered in the province of Mardin, a single index case of imported malaria with 218 autochthonous cases due to the passing of international truck drivers through intensive control and surveillance studies .


The effects that artemether-lumefantrine (AL) has on gametocyte dynamics in the short-term have recently been described. However there is limited long-term longitudinal data on the effect of AL on gametocyte dynamics in asymptomatic children.An epidemiological study was conducted in Kombewa, Western Kenya, in which 270 asymptomatic children aged between 12 and 47 months were enrolled. The subjects were randomized to receive either a course of AL or placebo at enrolment. Active follow-up was conducted for one year.The gametocyte prevalence and density dynamics throughout the study period mirrored that of the asexual forms. The proportion of initially parasitaemic subjects becoming gametocytaemic was significantly lower in the AL arm for the first 12 weeks following randomization. The geometric mean gametocyte density was lower in the AL arm for 2 weeks following randomization. None of the variables of interest had a statistically significant effect on the duration of gametocytaemia. There is no effect seen in subjects who are not parasitaemic at the time of drug administration.The treatment of asymptomatic parasitaemic subjects with AL results in a significant reduction in the proportion of subjects who become gametocytaemic for at least 12 weeks. Plasmodium falciparum infections in endemic countries is high, with microscopically detected prevalence reported to be as high as 39%[Both symptomatic and asymptomatic malaria infected persons tend to harbour gametocytes, the sexual stage of the malaria parasite that mediates its transmission from the infected human host to a susceptible mosquito. Becauseh as 39%. A largeh as 39%,5. Studih as 39%-8. Interh as 39%.Gametocyte carriage in naturally infected individuals in a population is influenced by many factors such as the age group,11-13, tData on duration of gametocytes carriage in natural infections is limited because such longitudinal studies are challenging to conduct. In a loP. falciparum carriers directly benefits the carriers given that the clearance of the asexual parasite load prevents anaemia, which in turn is associated with reduced work capacity, poor pregnancy outcomes and reduced cognitive function[P. falciparum carriers has on gametocyte dynamics. To describe this, data collected from a longitudinal cohort study of the epidemiology of paediatric malaria in Kombewa Division, Western Kenya was analysed.Treatment of asymptomatic asexual function. Adoptiofunction-32. Artefunction. HoweverData was obtained from the longitudinal cohort study of the epidemiology of paediatric malaria in Kombewa Division, Western Kenya. The study was conducted in order to estimate the prevalence of asymptomatic and symptomatic malaria as well as to describe the temporal pattern of episodes of symptomatic malaria among young children living in Kombewa Division. An additional aim was to assess whether administration of a curative dose of an anti-malarial treatment at the beginning of the study leads to a large change in the pattern of development of episodes of malaria during the subsequent follow-up period. The information obtained from this study was for the planning and design of a paediatric malaria vaccine trial that was to be conducted in the same area.Kombewa Division is a 361 square kilometre rural area located near Lake Victoria in the western part of Kenya, with a population of approximately 70,000 and an under-five population of approximately 8,500. The climate in Kombewa is characterized by two rainy seasons – the long rains (April through June) and the short rains (August through October). The vegetation is largely savanna in nature. Kombewa has year round transmission of malaria, with the most intense transmission occurring during the long rains and the short rains. For the purpose of organizing recruitment and follow-up of the volunteers, the field site was divided into 22 subdivisions. Within each subdivision in which a participant resided there was a satellite field site that was staffed 24 hrs/day, 7 days/week. Each household participating in the study was thus located within a 1-mile radius of a satellite field site that was accessible during the rainy and dry seasons. The study protocol was reviewed and approved by the Ethical Review Committee of the Kenya Medical Research Institute (KEMRI) and the Human Use Review Committee of the Walter Reed Army Institute of Research (WRAIR) and by the Human Subjects Research and Review Board of the Surgeon General of the US Army at Fort Detrick, Maryland. Parents or guardians of participants were provided written, informed consent before screening and enrolment and had to pass an assessment of understanding.This was an epidemiological follow-up study, in which a total of 270 initially healthy, asymptomatic children aged between 12 months and 47 months living within the study area were enrolled in three age cohorts of 12–23 months, 24–35 months and 36–47 months. Inclusion criteria included being 12 to 48 months of age, verifiable by appropriate documentation. Subjects with clinical signs of malaria and other significant medical conditions such as homozygous sickle cell disease and malnutrition were excluded. Samples for a baseline malaria blood film (MBF) and a full blood count were collected just before randomization; the results of MBF were not used to determine eligibility, and were not made available to the investigators. The children were then randomized to receive either a course of artemether-lumefantrine (Coartem®) or placebo using a list prepared in advance using a blocked randomization scheme. All study staff with the exception of the study pharmacists and the study coordinator were blinded to the allocations. The treatment was prepared by the study pharmacist at the clinic and its administration directly supervised by study team members – the first, third and fifth doses were administered at the clinic, while the second, fourth and sixth doses were administered by field workers at home. Each of the children was then followed up for one year. The follow-up consisted of active surveillance and passive surveillance. Active surveillance consisted of weekly visits by field workers to the participants’ homes and scheduled monthly visits by the participants to the study clinic, during which samples for malaria blood films were collected. Haemoglobin determination was done during the monthly visit at the clinic. The results of the MBFs collected during the active surveillance were not availed to the investigators unless the subject was unwell during a scheduled contact. Passive surveillance consisted of unscheduled sick visits by the study participants to the study clinic whenever the participants had specific complaints – during these visits, laboratory assessments including malaria blood films were done whenever the clinical condition warranted. The results of the MBFs taken during sick visits were made available to the investigators. Subjects who were symptomatic and were diagnosed with malaria as per the laboratory assessment during the follow up period were treated with AL, regardless of the randomization group.Duplicate thick and thin blood smears were made and air dried. Thin blood smear was methanol fixed and both smears were stained with 5% Giemsa in phosphate buffer for 30 minutes. With high power objective the smears were examined, the number, species and stage of the parasites were recorded. The total numbers of parasites, both asexual and sexual forms per high power field (HPF) were counted against 200 white blood cells. For samples that had high density parasitaemia, the asexual forms were counted against 2,000 red blood cells. A total of 100 HPF were examined before a slide was considered negative, and a minimum of 25HPF were examined on a positive smear. Two independent microscopists examined the MBFs, and a mean of their findings calculated. Discrepant results were resolved by having a third reader, and the two closest results were taken. The microscopists were proficient and worked under the guidance of malaria diagnostic centre within the programme.6 respectively. The duration of gametocytaemia was calculated by assuming that a single gametocyte positive slide represented gametocytes circulating in the peripheral blood for at least 2.5 days. If participants presented with gametocyte positive MBFs on successive visits, the duration of gametocytaemia was estimated as the interval between the visits plus 2.5 days[P. falciparum and Plasmodium malariae, or P. falciparum and Plasmodium ovale) and whether or not the child was pre-treated with AL at randomization. The baseline characteristics data is presented of the three age groups; 12–23, 24–35 and 36–47 months in that order respectively. The association between the time to the first or only episode of gametocytaemia, as well as the risk for multiple events of gametocytaemia, and the variables of interest was analysed using Cox regression analysis. The association between continuous outcomes  and the outcomes of interest were evaluated using robust regression models.All data were entered and verified in Microsoft Access then imported into STATA software version 10 . Malaria parasite densities were converted from parasites per 200 white blood cells and parasites per 2,000 red blood cells to parasite/μL, assuming a WBC count of 8000 or a RBC count of 4×102.5 days,35. CovaA total of 539 children were screened, 270 met the eligibility criteria and were enrolled into the study where 135 were randomized to receive placebo and 135 randomized to receive AL. Of the 270 volunteers that were enrolled into the study, 69% (187) completed the study which the follow-up went on for a year. Of those that completed the study, 53% (99 of 187) were in the placebo arm and 47% (88 of 187) in the AL arm. Of the 83 volunteers that did not complete the one year follow-up, 81% (67) withdrew their consent, with 55% (46 of 83) participants citing their discomfort with the weekly blood draws as the reason for consent withdrawal. Migration out of the study area accounted for the remainder of those who did not complete the study. No deaths were recorded among the study cohort for the duration of the study. However, two serious adverse events (SAEs) were recorded where one child in the AL arm was hospitalized with a diagnosis of severe malaria 19 weeks after randomization - this particular participant had defaulted on clinic visits, while the other child in the placebo arm was hospitalized with severe pneumonia. Both children recovered. All children completed the six doses of the study treatment.The baseline characteristics of both treatment arms were generally well balanced Table . InsectiP = 0.002). This effect was observed for the first 12 weeks following randomization . Being in the older age category, AL administration at randomization and bed net use were protective against gametocytaemia, while being in the second age category, having a positive smear at enrolment and hyper-parasitaemia were significantly associated with gametocytaemia (Table P < 0.001 and P = 0.034). A positive malaria smear at enrolment and mixed infection were associated with faster appearance of the first or only episode of gametocytaemia (Table P = 0.016 and P = 0.001). A positive smear for malaria at enrolment and anaemia was statistically significantly associated with the development of multiple events of gametocytaemia (Table P < 0.001), while the second age category, hyper-parasitaemia and mixed infection seemed to reduce the risk for multiple events (Table P = 0.01 and P = 0.007).The gametocyte prevalence dynamics throughout the study period were similar to the asexual parasite prevalence dynamics, with the gametocyte prevalence peaks coinciding with the asexual parasite prevalence peaks Figure . For subP = 0.008) and negatively associated with haemoglobin levels (Table P < 0.001). The mean gametocytaemia duration was 7.4 days (range 2.5 – 41.5) in the lowest age category, 5.6 days (range 2.5 – 37.5) in the middle age category and 6.2 days (range 2.5 – 44.5) in the oldest age category. There was no statistically significant difference in the gametocytaemia duration between the age categories groups as determined by one-way ANOVA  = 2.11, P = 0.12).The gametocyte density dynamics were similar to the asexual parasite density dynamics, with the peaks coinciding with the asexual parasite density peaks Figure . The geoP = 0.8). None of the variables of interest had a statistically significant effect on the duration of gametocytaemia in the regression model.The duration was similar in those who had mixed infection versus those who did not . BA and JM analysed the data. BA, JM, EK wrote the manuscript. BO reviewed the manuscript. All authors read and approved the final manuscript.


Children with recent or acute malaria episodes are at increased risk of invasive bacterial infections (IBI). However, the exact nature of the malaria-IBI association is still unclear. Young children have an age-related spleen immunologic immaturity, mainly due to the still ongoing development of the marginal zone (MZ) B cell subset. By mounting a rapid antibody response against encapsulated bacteria, these cells are critical for the defence against highly pathogenic microorganisms that do not elicit classical T cell-dependent responses. There is increasing evidence that the anatomy of the spleen becomes disorganized during malaria infection, with complete dissolution of the MZ and apoptosis of MZ B cells. Correspondingly, a reduction in the frequency of the peripheral equivalent of the MZ B cells has been found in malaria endemic areas. A remarkable similarity exists in IBI susceptibility between African children with malaria and hyposplenic or splenectomized patients. However, studies specifically assessing the immune function of the spleen in controlling bacterial infections in young children with malaria are scarce.Plasmodium falciparum malaria infection constitutes a detrimental factor in the still immature spleen function of young children, resulting in a factually hyposplenic state during malaria episodes, putting children with malaria at a high risk to develop life-threatening bacterial infections. Studies to confirm or reject this hypothesis are greatly needed, as well as the development of affordable and feasible tools to assess the immune spleen function against encapsulated bacteria in children with malaria.Here, it is hypothesized that  Plasmodium species were the causative agent of malaria. Later, this method became accepted by the World Health Organization to be used in malaria surveys [For years, in malaria-endemic settings the percentage of children with splenomegaly has been correlated with intensity of malaria transmission in a community. The method to evaluate malaria endemicity by spleen measurement is named the Hackett score, and it was introduced by Dempster in India in 1848 before it was known that  surveys .The spleen is a complex lymphoid organ with several important functions that starts its development in foetal life and reaches full maturation during early childhood, around age two to three years –5. The cBH), innate lymphoid cells type 3 (ILC3), CD4+ T lymphocytes, MZ B cells and memory B cells, where TI-1 and TI-2 responses take place; (3) the perifollicular zone (PFz), separating the MZ from the red pulp (RP) and containing RBCs, pericapillary macrophages, NBH, and ILC3; (4) the RP-containing macrophages, DCs, NBH, plasmablasts, being the place where RBCs are efficiently filtered from the circulation and where reticulocytes mature [To accomplish its functions, the spleen encompasses the following anatomic subunits Figure : 1) thes mature , 13–16. s mature , 18. Mos thes mats mature , 19, 20.Plasmodium falciparum malaria causes alterations in the function of the cells of the innate immune system such as macrophages [P. falciparum infection impairs the immunological response of the spleen against encapsulated bacteria in young children, a postulate built upon the following observations.rophages , neutroprophages  and dendrophages . It alsorophages –26 and trophages . Regulatrophages  could imrophages . CurrentP. falciparum malaria [Streptococcus pneumoniae, Haemophilus influenzae type b, Staphylococcus aureus, Escherichia coli and other gram-negative bacteria [S. pneumoniae, characterized by a particularly high mortality [S. pneumoniae at rates 30–100 times higher than those expected in healthy children from the same age group [One of the risk factors to develop IBI in Africa is  malaria –33. Bactbacteria , 34–37. bacteria , 38–44. bacteria , 43. Rembacteria –55. For ortality . Of noteortality . The reaortality . Conversortality , 56, altortality –59. Inteortality . Childrege group . Splenic99mTc-labelled autologous RBC scintigraphy to measure functional splenic volumes (FSV). The immunological function was assessed by studying the response to polysaccharide vaccines. As expected, SS and SC individuals had a significantly diminished anti-polysaccharide response. Interestingly, HJB were indicative of splenic dysfunction; however, its absence did not necessarily indicate a normal functioning spleen tissue. Even more interesting, the FSV was strongly correlated with the amount of MZ B cells in peripheral circulation, being significantly reduced in SS and splenectomized patients, thus corroborating previous findings [Routinely, hyposplenism has been diagnosed by haematological rather than immunological methods . Neverthfindings . These rfindings , 61, 62.findings .Leishmania parasite phagocytosis by macrophages [Additionally, in the spleen tuftsin is synthetized, an opsonin that activates the alternative complement pathway , 17, androphages , and wherophages .Salmonella spp. [P. falciparum malaria, with ages between four and eight years, it was found that the neutrophil dysfunction was a consequence of induction of a haem-degrading enzyme (haem oxygenase-1) in neutrophil progenitors in the bone marrow [Patients with malaria infection and SCD present with neutrophil dysfunction, characterized by defective locomotion, phagocytic processes and bactericidal performance , 66, 76,lla spp. . In chile marrow . The dyse marrow , here ite marrow , but it BH to produce IgM and for class switching from IgM to IgG or IgA in a T cell-independent manner [As aforementioned, one of the anatomic subunits of the spleen tissue is the MZ. The MZ of the spleen is strategically positioned between the lymphoid compartment of the spleen, the WP, and the more innate, scavenging RP compartment Figure . Most oft manner . Other it manner . NKT celt manner ; howevert manner .Plasmodium chabaudi AS infection mouse model [P. falciparum malaria although not significant [P. chabaudi AS infection in mice causes apoptosis of transitional T2 and MZ B cells during and subsequent to the control of the first wave of parasitaemia [Plasmodium infection and TNF production, that mediates programmed cell death in immune cells [Depletion of cells in the MZ during acute malaria infection was reported in the se model . Later onificant . Recentlsitaemia . These fsitaemia –89. A prsitaemia , functiositaemia , 13, andne cells .+CD27+IgM+ B cells  in infants aged one to 24 months living in a malaria-endemic region when compared with a similar infant population from an unstable malaria transmission region [+CD27+IgM+B cells at the time of acute malaria, not seen in subjects with a lower incidence [Plasmodium infections . Here, it is hypothesized that the drop of the MZ B cells in peripheral circulation of patients with malaria is a result of the disruption in spleen architecture observed in the referred histology studies. Furthermore, the MZ B cell deficiency along with some other possible defective immune spleen functions appears to be responsible for a transient hyposplenism in children with malaria, increasing their risk to IBI. Importantly, children with malaria have been reported to show a lower antibody response against group C meningococcal polysaccharide vaccines when compared to children from the same setting but without parasitaemia, supporting this hypothesis [As explained, the development of peripheral B cells takes place during infancy. However, in malaria-endemic settings some alterations in B cell subsets independent of age have been reported . For insn region . Supportncidence . A recenpothesis .Taking into account that the spleen MZ has not yet reached maturity in young children, it can be speculated that the threshold of MZ dysfunction  that is needed to develop immune hyposplenism is lower in children when compared to the adult population, probably explaining children’s increased susceptibility to bacteraemia in the context of malaria infection when compared to the immune-competent adult population.It is important to clarify that the peripheral equivalent of the MZ B cells have different nomenclatures in the research literature: MZ-like B cells, IgM memory B cells , splenicSalmonella-specific antibodies facilitate specific T cell responses via augmentation of bacterial uptake and induction of apoptosis in macrophages [Salmonella colonization of splenic macrophages, markedly reducing bacteraemia by antibodies induced during the infection in mice [NTS bacteraemia most commonly occurs in African children below two years of age. A relative sparing of infants younger than four months old has been found coincidently within the period during which maternal antibodies are still present . Sera frrophages . Moreove in mice . Therefo51Cr-labelled RBC and presented with a lower mean haematocrit than patients without splenomegaly [A study in Sudanese children reported that SMA was associated with a larger spleen, longer fever duration and lower parasitaemia than cerebral malaria (CM) . In thesnomegaly . Filtratnomegaly . HoweverSalmonella typhimurium or S. pneumoniae bacteria with parasitic co-infection of the spleen. Apparently, co-infection by a non-lethal Plasmodium strain and S. typhimurium in mice does not alter the compartmental distribution of malaria iRBC and bacteria in the spleen [Leishmania donovani infection  were co-infected with S. pneumoniae. In this case, in the absence of MZ macrophages, bacteria were located in the RP instead of the MZ, and phagocytosed by RP macrophages, showing a relocalization of the bactericidal functions in the spleen [P. falciparum, this relocalization will not occur due to overload of RBC in the RP, among other factors. In human P. falciparum malaria, similarly to the L. donovani-S. pneumoniae co-infection, the MZ would be significantly reduced. Nevertheless, some important differences with this animal model would be: 1) the chronicity of infection in this L. donovani animal model, in contrast with acute P. falciparum malaria in humans, perhaps implying that the total phagocytic capacity of the spleen RP is not necessarily overwhelmed in chronic Leishmania infections - even though in massive splenomegaly cases of L. donovani in humans, the important structural changes in the spleen might impact its immunological function; 2) the high parasitaemia and splenic overload with iRBC and uRBC in the case of P. falciparum malaria, added to the increased erythrophagocytic capacity in young children, that will demand maximum phagocytic capacity from the macrophages in the RP and the MZ [S. typhimurium organisms preferentially phagocytose damaged RBC, presenting a defective bactericidal capacity. The reasons are not clear. It is postulated that inhibition of bacterial phagocytosis may be mediated by competition mechanisms between damaged RBC and bacteria for phagocytic sites on macrophages, or that macrophages are just overloaded temporarily [Of note, several studies have shown that children with SMA have a higher prevalence of IBI co-infections (largely NTS bacteraemia) compared with children with CM who infrequently have bacterial co-infections , 33, 103e spleen . Malariae spleen  , therefore not causing a major impact on MZ function. A possible hypothesis to explain this phenomenon could be that P. vivax malaria, by infecting reticulocytes, is protected by their CD47 membrane protein, a potent “do not eat me” signal to macrophages in the spleen [P. vivax infection. In addition, CD47 has been shown to undergo a conformational change after oxidation, a state that will lead to an “eat me” configuration, which will result in phagocytosis by RP macrophages [Plasmodium infection leads to reactive oxygen species (ROS) formation, which may lead to oxidation of CD47 and thus the “eat me” configuration of this protein. Reticulocytes, however, have a far better capacity to handle oxidative damage than mature RBC. Thus, infection of reticulocytes by P. vivax may not lead to CD47 oxidation and the switch from “do not eat me” to “eat me” signal would not take place, whereas during infection of mature RBC CD47 may be well oxidized. Together, the higher expression of CD47 and the better protection of reticulocytes against ROS generation might explain the accumulation of a big amount of infected reticulocytes in the spleen and the consequent splenomegaly in P. vivax malaria, due to the inability of RP macrophages to phagocytose them. In the case of this hypothetical incapacity of the RP to phagocytose P. vivax–infected reticulocytes, it is possible that different immunological pathways are triggered when compared to P. falciparum malaria, perhaps affecting in a different way or to a lesser extent the MZ immune cell compartment of the spleen.If splenomegaly and the excessive B cell stimulation in the spleen are the cause of immunological hyposplenism in young children with malaria, it should be expected that patients with other parasitic diseases that cause splenomegaly also develop an increased susceptibility to IBI. There are few reported cases in the literature of increase susceptibility to IBI by NTS in cases of leishmaniasis , 125 andchildren . Howeverablished –134. Howfections . What coe spleen . Interese spleen , 138. The spleen . Thereforophages . It is wAdditionally, it is important to clarify that the human spleen has remarkable differences compared to the murine spleen, for instance regarding its microcirculation  and anatomic characteristics  , 13, 94.P. falciparum malaria infection. However, malaria-dependent hyposplenism could possibly affect older children in sub-Saharan Africa as well. This hypothesis is based upon several observations in human spleen histology studies and immune cells in peripheral blood, indicating alterations in the structure and function of the spleen, particularly of the MZ. Animal models, with their acknowledged limitations, were analysed as well in detail and corroborated these findings, opening new questions that need to be urgently addressed in the human host. Testing the dysfunctional spleen hypothesis postulated here would have important implications for bedside care of African children with malaria.There is ample evidence in the scientific literature to support the hypothesis postulated here that African children younger than three years of age might present with immunological spleen dysfunction triggered by P. falciparum infection, it is necessary to evaluate the role that adjuvant intravenous g-globulin and/or tuftsin treatment could play in helping them overcome their splenic opsonization failure when encountering malaria-IBI co-infections. In this regard, it should be a main concern to accelerate the introduction of several vaccines missing in the expanded programme on immunization in most African countries. In addition, the risk of malaria-IBI co-infection in children from these countries should be taken into account by international organizations and funding sources supporting vaccine introduction in poor countries to orientate their immunization strategies, as well as to increase their investment in research and development of vaccines against NTS.For instance, the development of simple and affordable methods to assess the immune spleen function in children with malaria and other parasitic diseases that colonize the spleen should be a priority because current tools are largely oriented to evaluate the haematological but not the immunological function of this organ. Importantly, as young children lack protective antibodies against several encapsulated bacteria when they face a S. pneumoniae infection [Salmonella, optimizes its response against this micro-organism and mediates pathogen clearance [Assuming that bacteria colonize mucosal surfaces in children and, in the presence of malaria infection, translocate to the bloodstream, it would be important to evaluate the role of probiotics in improving the immune response to IBI in very young children at the first line of defence against these pathogens: the mucosal barrier. Some studies have shown that oral probiotic treatment importantly reduced late onset sepsis in newborns , remarkanfection . Of notenfection , 141, anlearance . On the P. falciparum malaria should be treated as hyposplenic patients is advised.The time gap between generation of knowledge and its application where is greatly needed is unfortunately long. The aim of this review is to reduce this time gap as much as possible by sharing information considered of value for the malaria research community. The proposed dysfunctional spleen hypothesis needs to be confirmed or rejected by studies in human subjects directly addressing the immune spleen function, since no studies of this kind have ever been performed in African children with malaria. Meanwhile, a general awareness to the health care providers that young children with


Galectin-9  is a β-galactoside-binding lectin that interacts with sugar moieties on glycoproteins and glycolipids of cells and pathogens. Gal-9 is known as an immune modulator that induces cell death via interaction with T cell immunoglobulin and mucin domain-3 (Tim3), a co-inhibitory receptor, and it inhibits production of several pro-inflammatory cytokines  and enhances production of IL-10. To understand the immune pathology of malaria, the Gal-9 in plasma was measured.Plasma samples and clinical parameters were obtained from 50 acute malaria cases (nine severe and 41 uncomplicated cases) from Thailand at three time points: day 0, day 7 and day 28. Gal-9 levels were determined by ELISA. A total of 38 species of cytokines and chemokines were measured using a BioPlex assay.rs = −0.73 and rs = −0.46, respectively). In both UM and SM cases, Gal-9 was significantly associated with pro- and anti-inflammatory cytokines and chemokines such as TNF, IL-6, IFN-α2, IFN-γ, IL-1Ra and IL-10. These correlations were observed at day 0 but disappeared at day 28.Gal-9 levels were higher at day 0 compared to day 7 and day 28 (P < 0.0001). Gal-9 levels were also higher in severe malaria (SM) cases compared to uncomplicated (UM) cases at day 0 and day 7 . Median Gal-9 levels were higher in patients with blood urea nitrogen to creatinine ratio (BUN/creatinine) ≥20 (mg/dL) than in patients with BUN/creatinine <20 (mg/dL) at day 0 . Gal-9 was inversely significantly correlated with chloride levels in both SM and UM cases (Gal-9 is released during acute malaria, and reflects its severity. This elevation of Gal-9 in acute malaria infection raises the possibility of its role in termination of the immune response by binding to Tim-3, a receptor of Gal-9. Plamosdium falciparum is responsible for the most severe cases of malaria and the greatest number of deaths. Detection and monitoring of severe cases are remaining issues for patient treatment [Malaria infection is still a big challenge for public health; around 214 million new cases of malaria worldwide occurred in 2015 (range 149–303 million) [Galectin-9  is member of the galectin family of β-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD) . Gal-9 w+ Th1 cells [It has been reported that PD1, a co-inhibitory receptor play a major role for inactivating a protective immunity in malaria infection in mice . Gal-9 bh1 cells , 18. Galh1 cells , which ih1 cells . Activath1 cells . These eh1 cells . Recentlh1 cells , 23; howIn this study, the plasma levels of Gal-9 in malaria patients were measured at three time points  to examine its kinetics and association with cytokines, chemokines and clinical parameters.P. falciparum. Categorization of malaria cases was performed using WHO criteria [Samples at three time points  were obtained from 50 malaria patients, of which 41 were acute uncomplicated falciparum malaria (UM) and nine were severe falciparum malaria (SM) cases. The median age of patients enrolled was 23 (range 15–50) years old. There were 29 males and 21 females. Patient body temperatures were >38.5 °C upon admission. Diagnosis was performed by thick and thin blood smear in patients presenting malaria symptoms and malaria cases were defined by positive asexual forms of criteria . Blood wAt enrollment, clinical information was taken and entered in standardized forms. Clinical and bedside physical examinations were performed. Complete blood counts (CBC) in addition to kidney and liver function tests were examined.Gal-9 concentration in plasma was measured using an ELISA  as previously described [Thirty-eight cytokine and chemokine species were measured using a commercially available kit  by Luminex methods as previously . The assP < 0.05.The distribution of data did not generally show a normal distribution by using the Kolmogorov–Smirnov test. Therefore, the data were expressed as the median and range. Friedman test, a non-parametric statistical test, was used to assess differences of Gal-9 and other biomarkers levels between different time points. The Mann–Whitney test, also a non-parametric test, was used to assess differences in biomarkers and clinical parameters between SM and UM cases. Correlation was assessed using Spearman’s rank correlation coefficient test. These statistical analyses were performed using GraphPad Prism 6 software . A significant difference was assumed when The median of haemoglobin were not significantly different between UM and SM cases at day 0 Table . However). At day 28, there was no significant difference in the Gal-9 levels between the two groups  compared to day 7 (398 pg/mL) and day 28 (243 pg/ml). The median levels of Gal-9 on day 7 was found to be higher compared to day 28 but the difference was not significant Fig. a. Medianups Fig. b.Fig. 1PAmong the 38 biomarkers measured, pro-inflammatory cytokines of IFN-α2, IL-1α, TNF and IL-6, Th1-related cytokine of IFN-γ, regulatory cytokines of IL-10 and IL-1Ra, growth cytokines of G-CSF, GM-CSF, EGF, FGF-2 and VEGF, and chemokines of IP-10, MIP-1α, MIP-β, MCP-1, IL-8, eotaxin, GRO, Fractalkine and MDC were detectable. All of them showed significant difference at day 0 compared to day 7 or day 28 except VEGF, MIP-1α, eotaxin and GRO (Table s = 0.82). TNF and IL-6 were positively correlated with Gal-9 in both UM  and SM cases . IFN-γ was positively associated with Gal-9 in both UM (rs = 0.33) and SM cases (rs = 0.75)  and SM cases .Numerous cytokines and chemokines were correlated with Gal-9 levels in malaria patients at day 0 Table . At day s = 0.35 and rs = 0.32, respectively) and SM cases . Gal-9 also correlated with MCP-1 in UM group (rs = 0.32) and Fractalkine in SM cases (rs = 0.72) , and were positively correlated with creatinine (rs = 0.44) and BUN (rs = 0.45) in the UM cases. The lack of correlation between these kidney parameters and Gal-9 in SM cases might be due to the small number of cases in that group (nine patients). Among the electrolytes tested, sodium levels were negatively associated with Gal-9 levels (rs = −0.48) also only in the UM cases. Chloride levels were negatively associated with Gal-9 in both UM (rs = −0.46) and SM cases (rs = −0.73)  Table .Table 4CThis study revealed the presence of Gal-9 in plasma in malaria patients and showed that plasma levels of Gal-9 accurately reflect the status of inflammation and recovery during malaria infection for the first time though the tissue expression of Gal-9 and Tim-3 was demonstrated in murine malaria model , 22. TheP. falciparum infection, and produced Gal-9 was assumed to inhibit their activation [It has been suggested that Gal-9 production might be stimulated by the response of mounting Th1 cells that occurs during tivation , 14. Thitivation . This as+ ThGal and Treg, which express high levels of IL-10 mRNA [The correlation of Gal-9 with IL-10 could be related to the effect of Gal-9 on differentiation of naïve T cells to Gal-9-10 mRNA . Though -10 mRNA . Another-10 mRNA . In mala-10 mRNA –32, and -10 mRNA , 33. How-10 mRNA . Gal-9 s-10 mRNA , 34. HMG-10 mRNA . In cont-10 mRNA , HMGB-1 -10 mRNA , 37. TheBeside immune modulatory activities, Gal-9 is released from infected or damaged cells, and could act as danger signal molecules . MoreoveLeishmania major Poly-β-galactosyl epitope by Gal-9 [The released Gal-9 can also bind to galactosides of pathogens, pathogen-associated molecular pattern (PAMP), as pathogen recognition receptors (PRRs), as shown recognition of the by Gal-9 . Future Taken together, this study shows an increase of plasma Gal-9 levels during malaria infection, which is capable of identifying severe cases, and is tracking the inflammation process. Therefore, it may be used as a novel biomarker of malaria infection.


Background. Laboratory diagnosis of malaria is the key for effective disease management. Diagnosis of malaria infection requires rapid, sensitive, and specific test methods with an affordable cost. This study was aimed to assess the diagnostic performance of Partec rapid malaria test with reference to light microscopy for the diagnosis of malaria in Northwest Ethiopia. Methods. A total of 180 febrile patients were tested for malaria using Giemsa stain microscopy and Partec rapid malaria test from June to July 2013 at Gendewuha health centers, Metema district. Data were analyzed using SPSS version 20 statistical software. Odds ratio with 95% CI was calculated. Result. The sensitivity and specificity of Partec rapid malaria test were 93.8% (95% CI = 87.1%–100%) and 87.9% (95% CI = 79.7%–96.1%), respectively, while the positive predictive value and negative predictive value were 6.4% (95% CI = 77.2%–95.5%) and 94.6% (95% CI = 88.7%–100%), respectively. There was also an excellent agreement between two tests with Kappa value of 0.811 (95% CI = 0.625–0.996). Conclusion. Partec rapid malaria test showed good sensitivity and specificity with an excellent agreement to the reference light microscopy. Therefore PT can be considered as alternative diagnostic tools in malaria endemic areas. Plasmodium falciparum and P. vivax are the dominant species of malaria in Ethiopia, with 60% and 40% relative frequencies, respectively. P. falciparum is the most dominant species in endemic areas and causes severe and complicated disease and death [Malaria is the most common fatal disease in the world. According to 2013 WHO malaria report, an estimated 3.4 billion people were at risk of malaria globally and populations living in sub-Saharan Africa have the highest risk of acquiring malaria. Approximately 80% of cases and 90% of deaths are estimated to occur in the African region [In Ethiopia half a million microscopically confirmed cases of malaria are reported to the Federal Ministry of Health (FMOH) in each year from basic health services. However, the actual number of malaria cases in the country is estimated to be more than 5 million each year . So, choClinical diagnosis is the most widely used diagnostic method in rural areas and where laboratory facility does not exist. It is inexpensive to perform and requires no special equipment or supplies . HoweverA laboratory diagnosis of malaria is one possibility in the management of a patient presenting with fever. To improve the quality care of the patients, many diagnostic procedures have been developed which aim to have accurate diagnosis, to reduce the time of preparation and training needed , 4. Even Plasmodium species DNA in human blood. It uses readily prepared and ready-to-use slides labeled with an unspecific DNA-binding fluorescent dye (4′-6-diamidino-2-phenylindole (DAPI); emission 365 nm) that detects plasmodial DNA. It is ultra compact and robust microscope design and has connector for optionally available CCD camera upgrade for visualization of the slides on any PC Windows with USB connection [The Partec rapid malaria test (PT) is newly innovative fluorescence microscopy method for the detection ofnnection , 11. The Plasmodium species. But the prevalence of P. falciparum is very high. The health center is serving around 18,000 people in Gendewuha town and surrounding areas.A study was conducted at Gendewuha health center found in Metema district, North Gondar Zone, Amhara Regional State, from June to July 2013. The district is found at the Ethio-Sudan border. It is far 925 km from Addis Ababa  and found at 180 km west of Gondar town. The mean annual temperature ranges from 22°C to 28°C and the daily temperature reaches as high as 43°C during the months of March to May. Mean annual rainfall ranges from 850 mm to 1100 mm and has unimodal pattern. The altitudes range from 550 to 1068 meters above sea level . The disOne hundred and eighty malaria suspected patients attending at Gendewuha health center during the study period were included and screened for malaria infection using light microscopy and Partec rapid malaria test. Patients who had received antimalarial drugs during the past two weeks, children under 1 year, and critically ill patients who were unable to respond to the interview were excluded from the study.Sociodemographic and clinical data of the study participants were collected using structured questionnaire. Three milliliters of venous blood was collected with EDTA tubes by laboratory technicians from each study subject for smear preparation of light microscopy and PT.After collecting blood samples, thick and thin blood smears were prepared and stained with 10% Giemsa working solution for 10 minutes. Blood films were observed under 100x objectives for the detection of malaria parasites and the result was reported as positive if asexual malaria parasites were seen or negative if malaria parasites were not seen after observing 100 fields of the thick smear. All blood films were reexamined by an experienced microscopist at University of Gondar Hospital laboratory who was blinded to initial light microscopy result.μL blood was placed on the dye labeled area of a slide and cover slipped, incubated at room temperature for a minute, and observed with 40x objective under LED UV light (365 nm). The presence of bright shiny intracellular tiny dots against a dark background observed under the UV light indicated the presence of malaria parasites in the erythrocytes as shown in Partec rapid malaria test was carried out according to the manufacturer's instructions. Briefly, on the collected EDTA blood, 10 The data was entered into SPSS version 20 software for analysis. Odds ratio, sensitivity, specificity, positive predictive value, negative predictive value, and Kappa value were calculated at 95% CI.Ethical clearance was obtained from University of Gondar, School of Biomedical and Laboratory Sciences Ethical Committee, and permission was obtained from Gendewuha health office and Gendewuha health center authorities to conduct the study. After informing about the objective of the study, written consent was taken from all study participants or parents/guardians. Participants who were positive to malaria parasite were given the appropriate treatment at the health centre.A total of 180 malaria suspected patients were diagnosed for malaria. About 99 (55%) of the study participants were males and 81 (45%) were females. The mean age of the participants was 19 ± 12.49 years and majority of the participants 60 (33.3%) were within the age range of 20–29 years, and 42.8% of the participants were illiterate. The rural residence has statistically significant association with malaria infection  . Plasmodium falciparum accounts for 76 (93.8%), P. vivax were 4 (4.9%), and mixed infection  was 1 (1.3%). There were 2 discordant results between the first (study site) reader and the final reading by experienced microscopist; thus the final report was based on the final reading. Turnaround time of Giemsa stain microscopy was more than an hour.The overall prevalence of malaria using light microscopy was 81 (45%). From thisThe parasite positivity of malaria by using Partec rapid malaria test was 88 (48.9%). Seventy-six slides were positive with Partec rapid malaria test and the gold standard Giemsa stain microscopy. Twelve slides were positive with Partec rapid malaria test and negative with light microscopy, while 5 slides were negative with Partec rapid malaria test and positive with light microscopy and 87 slides were negative for both .Using light microscopy as standard test for diagnosing malaria, the sensitivity and specificity of Partec rapid malaria test were 93.8% (95% CI = 87.1–100) and 87.9% (95% CI = 79.7–96.1), respectively. There was also an excellent agreement between light microscopy and Partec rapid malaria test with Kappa value of 0.811 (95% CI = 0.625–0.996) .In addition, operational characteristics were observed. Compared to light microscopy PT is more sensitive, less labor intensive, and faster to use and has a less turnaround time than the LM (an average of 5 minutes). PT also requires very little training and there is no need of reagent preparation. Furthermore, the CyScope florescence microscope is battery operated; the battery can be kept on for 6 hours after full charge making it best for field work. It is easier to use and to adjust and can be switched from UV light to bright field. However, the PT is not suitable for species identification and parasitic load determination and blood films cannot be stored for a long period of time.The current study revealed a high sensitivity and specificity of Partec rapid malaria test. The high sensitivity and specificity are in agreement with the reports in Sudan . However Plasmodium DNA [Partec rapid malaria test had detected 12 cases which were negative by light microscopy. Unlike Giemsa stain light microscopy, PT has sensitive fluorescent dye, 4,6-diamidino-2-phenylindole (DAPI), which can be detected in such low level of parasites. In addition, the presence of artifacts such as nonspecific aggregated DAPI, immature erythrocytes, or bacterial cells might have been misinterpreted asdium DNA . On the Determination of parasitemia with Partec rapid malaria test is difficult, because the red blood cell may overlap each other and make the parasites invisible under the UV light. This finding is in line with the finding of the study conducted in Ghana . Though,The overall prevalence of malaria in the study area was very high, as detected by both the LM (45%) and the PT (48.9%). This result is higher than the report from other regions in Ethiopia , 19. ThePartec rapid malaria test showed good sensitivity and specificity with an excellent agreement to the reference light microscopy. PT has very short turnaround times, requires little training, and is applicable under field conditions. Therefore PT can be considered as alternative diagnostic tools in malaria endemic areas. Further studies are needed to determine the cost-effectiveness and long term performance of the Partec rapid malaria test in diagnosing malaria.