Plasmodium knowlesi, a simian malaria parasite can be life-threatening to humans. Long-tailed macaques, which are widely distributed in Malaysia, are the natural hosts for simian malaria, including P. knowlesi. The aim of the present study was to determine the prevalence of simian malaria parasites in long-tailed macaques in the district of Hulu Selangor, Selangor, Malaysia.Malaria is a vector-borne parasitic disease which is prevalent in many developing countries. Recently, it has been found that Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.A total of 70 blood samples were collected from Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P.fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.Thirty-five (50 %) of the 70 samples were positive for This study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria. Plasmodium which has been affecting humans since ancient times [Plasmodium, namely Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax were known to infect humans. However in 2004, Singh et al. [Plasmodium knowlesi, a simian malaria parasite. This resulted in the suggestion that P. knowlesi could be considered the fifth species of Plasmodium known to infect humans [Malaria remains a public health problem in many tropical and sub-tropical regions. It is caused by the blood protozoan parasites of the genus nt times , 2. Accont times . Traditit humans .Plasmodium knowlesi mainly circulates among long-tailed (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) [P.knowlesi in human was reported later in 1965 in an American traveller who returned home from peninsular Malaysia [estrina) . In 1932estrina) . The firMalaysia .P. knowlesi, namely: Singapore [P. knowlesi infections from the Andaman and Nicobar Islands of India which are geographically nearby these Southeast Asian countries [Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium inui, Plasmodium coatneyi, and Plasmodium fieldi are the five common species of simian malaria parasites in M.fascicularis and M. nemestrina [Over the past decade, most countries in Southeast Asia and some in Asia have reported the presence of ingapore , Thailaningapore , Myanmaringapore , Philippingapore , Indonesingapore , 13, Vieingapore , Cambodiingapore , Brunei ingapore , China [ingapore . Recentlountries . To dateountries –22. Plasmestrina .P. falciparum and P. vivax whereas a significant increase in microscopy-diagnosed P. knowlesi/P. malariae cases was reported [P. knowlesi was the predominant (38 %) species followed by P. vivax (31 %) in 2012 [P. knowlesi malaria cases were reported from Pahang [P. knowlesi/P. malariae infection in humans was shown to be highest in Hulu Selangor district, one of the nine districts in Selangor [Malaysia is considered endemic for malaria, mainly in the forested, mountainous and inaccessible areas of Malaysian Borneo comprising the states of Sabah and Sarawak, and peninsular Malaysia. A Malaria Eradication Programme was started in Sabah and Sarawak in 1961 and in peninsular Malaysia in 1967. Since then, there has been a significant reduction in the transmission of human malaria from reported –26. Acco in 2012 . Most ofm Pahang , 29. Them Pahang . In the P. vivax  % in 201Selangor .P. knowlesi malaria in Hulu Selangor district. The study mainly focused on the epidemiology of P. knowlesi by determining the human infection and vector distribution. However, the limitation of the study was that there was no information regarding infection in the macaques from that same study area. In view of this gap of knowledge, it is imperative to understand the infection rate of simian malaria in macaque hosts. Hence, the main objective of this study was to determine the prevalence of malaria parasites in macaques, namely P. knowlesi, P. cynomolgi, P.coatneyi, P. inui, and P. fieldi in Hulu Selangor. The findings from this study have strengthened the evidence that Hulu Selangor is a potential risk area for knowlesi malaria transmission due to the presence of infected macaques. Besides, other malaria parasites such as P. cynomolgi and P. inui which have potential to infect humans were also present.In recent years, an experimental analysis was carried out by Vythilingam et al.  to eluciIn June 2014, a total of 70 macaque blood samples from Hulu Selangor  according to manufacturer’s protocol. Purified DNA was eluted from the column with 100 μL elution buffer and stored at −20 °C until further use.Plasmodium by using conventional PCR with the thermal condition and primers sequences as described [2 (Promega), 0.2 mM of each deoxynucleoside triphosphate (Promega), 0.3 µM of each primers (PlasF5′-AGT GTG TAT CAA TCG AGT TTC T-3′ and PlasR5′-CTT GTC ACT ACC TCT CTT CTT TAG A-3′) [Blood samples were screened for 18S ribosomal RNA genes for detection of escribed . The PCRAG A-3′) , 0.2 U oPlasmodium positive samples were subjected to nested PCR to detect the different species using the primer sequences as reported previously [2 (Promega), 200 mM each-dNTP (Promega), 0.25 µM of each primers, rPLU1: (5′-TCA AAG ATT AAG CCA TGC AAG TGA-3′ and rPLU5: (5′-CCT GTT GTT GCC TTA AAC TCC-3′) [All eviously , 33, 34.The PCR products were visualized over gel electrophoresis using 2 % agarose gel and the amplified products from the gel was purified and sequenced on an ABI 3730xl sequencer  using the PCR primers.The sequence data were analysed using the BLAST program  and the M. fascicularis) blood samples were screened using conventional PCR to detect malaria parasites, among which 35 (50 %) samples were Plasmodium positive. From the positive samples, five species of Plasmodium parasites , P. cynomolgi (two) P. coatneyi (two) and P. fieldi (one). None of the macaques had mono-infection with P. inui. A total of 11 macaques (31.4 %) had dual infection, amongst them the prevalence of infection with P. knowlesi and P. inui was highest (36.4 %) followed by P. knowlesi and P. cynomolgi (9.1 %), P. cynomolgi and P. inui (27.3 %), P. coatneyi and P. inui (18.2 %), P. cynomolgi and P. coatneyi (9.1 %)  with P. knowlesi, P. coatneyi and P. inui (33.3 %), P. knowlesi, P. cynomolgi and P. inui (33.3 %), P. cynomolgi, P. coatneyi and P. inui (25 %), P. knowlesi P. cynomolgi and P. coatneyi (8.3 %)  followed by P. knowlesi (60 %). The high infection rate indicates a high intensity of transmission occurring among long-tailed macaques in Hulu Selangor. This is similar to a study conducted in Kapit, Sarawak where the prevalence of P. inui infection in long-tailed macaques (82 %) was predominant compared to other species [P. inui was the most common parasite in both M. fascicularis and M. nemestrina [P. knowlesi was the predominant species (68.2 %) among long-tailed macaques [P. knowlesi, 13.7 % were positive [P. knowlesi infection detected in Selangor from 41 macaques captured in urban areas and two macaques from Kuala Lumpur [Plasmodium. In addition, Ho et al. reported that 64.5 % of 107 blood samples from long-tailed macaques were Plasmodium positive and among these 23.3 % were positive for P. knowlesi in Selangor [The screening of macaques in Hulu Selangor illustrated that ~50 % of the macaques were positive and five common species of simian  species . In Sabamestrina . Howevermacaques . In Kualpositive . Howevera Lumpur , which ma Lumpur . It mustP. knowlesi in the macaques; only P. inui and P. coatneyi were found [P. inui (38.9 %) was highest compared to P. coatneyi (16.7 %) followed by P. knowlesi at 5.6 % [P. inui is similar to the findings from the current study (65.7 %), although at a lower rate. Jones-Engel et al. reported one monkey (3.2 %) was infected by P. knowlesi among 31 in Indonesia [P. knowlsei. The results are summarized in Table In a study from Thailand, in 2008 they did not find any re found . In a suat 5.6 % . The higndonesia . It alsoP. knowlesi infections are on the increase in Selangor. The recent emergence of P. knowlesi infections in humans may be due to close proximity of competent vectors, together with monkeys and humans, leading to conditions favourable for inter-species transmission to occur. This has occurred with deforestation and human encroachment on the natural habitats of the monkeys increasing the chance of human infection by simian malaria [Anophelesintrolatus has been incriminated as a vector for P.knowlesi in Hulu Selangor [P. knowlesi is an admixture of two divergent parasite populations associated with the two different macaque hosts dwelling in the forest [P. knowlesi parasites in patients from Sarawak [P.knowlesi from long-tailed and pig-tailed macaques [Simian malaria parasites are of great interest in Hulu Selangor as  malaria . RecentlSelangor . The pree forest  and stud Sarawak . In Sabamacaques .P. cynomolgi parasite had been discovered after an accidental infection in humans by mosquito bites in the laboratory [P. cynomolgi in long-tailed macaques was 51.4 %. In addition, this species has attracted a lot of attention recently as one case of natural infection in humans has been reported from Hulu Terengganu, Malaysia [P. inui was infectious to humans in laboratory studies [The zoonotic potential of boratory . The preMalaysia . Besides studies  and thisThe findings of this study has demonstrated the prevalence of five simian malaria parasites in their natural host, further highlighting the need to deliberate new strategies to prevent the potential zoonosis of the other simian malarias. Efforts are ongoing to study the genetic diversity of these malaria parasites as recent studies have indicated their importance , 46. AnoPlasmodium species were present in long-tailed macaques and occurred commonly as multiple infections. The study has provided evidence that P. inui was the most prevalent (65.7 %) malaria parasite followed by P. knowlesi in long-tailed macaques in Selangor. The significance of zoonotic simian malaria parasites cannot be overlooked in the assessment of accumulative interfaces between humans and wild animals in the development of urbanization. Potentially more samples should be collected in order to obtain a better understanding on the prevalence of simian malarias in monkeys all over Malaysia. These findings would be important for the planning and control of malaria strategies in the future. The genetic diversity of these parasites should also be studied.This study showed the prevalence of simian malarias in the local monkey population of Hulu Selangor. It found that four simian


Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies.The ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria  to ensure they met the criteria to permit product release according to GMP requirements.Good Manufacturing Practices (GMP)-grade P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers.The P. falciparum blood-stage malaria cell banks represents a suitable approach for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials.The production of cultured The online version of this article (doi:10.1186/s12936-015-0663-x) contains supplementary material, which is available to authorized users. Significant advances have been made in the control of malaria, yet half of the world’s population remains at risk. In 2012, there were over 200 million cases of malaria and 627,000 malaria-attributable deaths, highlighting the need for continued development of malaria control strategies .Plasmodium falciparum-infected mosquitoes on study participants -hypoxanthine (Amersham Pharmacia) for up to 36 hours. Plates were then placed at −80°C overnight. Following thawing, cells were harvested onto glass fibre mats (Perkin Elmer) using a Filtermate cell harvester (Perkin Elmer) and 3[H]-hypoxanthine incorporation was determined using a Microbeta2 Microplate Counter (Perkin Elmer).Incorporation of escribed  with somP. falciparum NF54 and 7G8 cell bank parasites was determined by measuring the cloning efficiency via a limit dilution approach with positivity determined by production of PfHRPII. This was undertaken using the SD Malaria Antigen P.f ELISA kit (Standard Diagnostics), a sandwich enzyme-linked immunosorbent assay for the qualitative detection of P. falciparum histidine rich protein II (HRP-II).Quantitative viability of the P. falciparum NF54 and 7G8 cell banks were thawed, as outlined above. Cell pellets were re-suspended in RPMI-1640 (Gibco) supplemented with 10% heat inactivated human serum and 0.01 mg/ml gentamicin (Gibco). Based on the recorded parasitaemia of the cell bank when it was cryopreserved, the cell bank parasites were plated out in 96-well flat bottom plates at 2% haematocrit at theoretical concentrations of 40, 4, 2, 1, 0.5 parasites/well with 100 μl of parasite suspension added/well. Each concentration was plated out in replicates of 30 and non-parasitized erythrocytes were included as a negative control. Plates were placed in an atmosphere of 90% N2, 5% O2, 5% CO2, at 37°C for 7 days. On Day 4, the media was removed from each well and replaced with fresh media. On Day 7, plates were processed for detection of P. falciparum HRPII. The manufacturer’s instructions were followed, with minor modifications. Following mixing, 50 μl of the parasite suspension from each well was transferred to the corresponding well of an uncoated microplate. Seventy-five microlitres of the working enzyme conjugate  was then added into each well of the uncoated microplate. Plates were incubated at ambient temperature for 30 minutes. One hundred microlitres was then transferred from each well of the uncoated microplate to the corresponding well of the pre-coated microplate. Plates were incubated at 37°C for 90 minutes. Following this, plates were washed 6 times with the wash buffer and 100 μl of tetramethylbenzidine substrate was added to each well. Plates were incubated at ambient temperature for 10 minutes, after which 100 μl of a 1 N Hydrochloric acid stop solution was added to stop the reaction. Plates were read on an xMark microplate spectrophotometer (Biorad) at 450 nm, with a reference wavelength of 620 nM. Detection of P. falciparum HRPII was used to define a parasite positive well, with the cut-off for positivity calculated as the mean + 3 standard deviations of the wells containing normal erythrocytes alone. A limiting dilution analysis was undertaken to determine the frequency of viable parasites -hypoxanthine growth inhibition assay [3H]-hypoxanthine (0.2 μCi/well) added at trophozoite stage approximately 24 hours from the start of the experiment. For proguanil and pyrimethamine assays, the initial parasitaemia was 0.5% and parasite cultures were exposed to the compounds for 96 hours with [3H]-hypoxanthine added after the first 48 hours.The on assay . Brieflyvs parasitic hypoxanthine incorporation to determine inhibitory concentrations. The in vitro anti-malarial activity of the compound was defined as inhibitory concentrations (IC50) and (IC90) that cause 50% and 90% inhibition of parasite growth.Following incubation, plates were frozen, then subsequently thawed and harvested using Tomtech Harvester 96 Mach II and radioactive counts were obtained using Wallac TriLux 1450 Microbeta Liquid Scintillation Counter . All assays were performed in triplicate. For each compound, the data from at least two independent experiments were obtained and analysed using Graph Prism Software . Drug concentration values were transformed into logarithmic values. After subtracting the background values, the data from drug-treated wells were normalized against drug-free control wells. Non-linear regression analysis was carried out on the log values of the compound’s concentration P. falciparum 3D7 cell bank parasites was performed using similar methodology with minor modifications. The anti-malaria compounds used were: amodiaquine, artemisinin, chloroquine, dihydroartemisinin, mefloquine, pyronaridine, quinine and sulphadoxine.Testing of the P. falciparum cell banks immediately prior to cryopreservation were examined and read to define the parasite stages present and the parasitaemia of the cell banks. The P. falciparum NF54 cell bank contained 80% of parasites at ring stage with a 4.5% ring stage parasitaemia. The P. falciparum 7G8 cell bank contained 78% of parasites at ring stage with a 1.4% ring stage parasitaemia. Similarly, the P. falciparum 3D7 bank contained 80% of parasites at ring stage with a 3% ring stage parasitaemia.Thin blood smears that had been prepared from the P. falciparum NF54 cell bank and the P. falciparum 3D7 cell bank were not tested to confirm their identity as the parental cell banks, from which they were derived, have a well characterized provenance and were manufactured in a cleanroom environment. Thus, the possibility of cross-contamination with another malaria parasite was considered negligible.The Griffith University P. falciparum 7G8 cell bank to confirm its identity, as it was derived from research-grade material. Following a primary amplification of the block 2 region of MSP1, a nested reaction using the R033 allelic type-specific primers was performed. Analysis of the sequence data for the nested amplicon revealed 100% identity between the Griffith University P. falciparum 7G8 cell bank parasites and the P. falciparum 7G8 reference strain.Genotyping was undertaken on the Griffith University Following thawing of the cell bank vials, the viability of the cell bank parasites was assessed in 3 ways. For all three cell banks, blood smears were prepared from cultures on the day of thawing and days 1–3 following initiation of culture. Growth of the cell bank parasites, indicating viability, was confirmed by regular microscopy of the cultures. Passage of the parasites into subsequent asexual cycles was demonstrated by confirming the appearance of the ring stage parasites in significantly increased numbers.P. falciparum NF54 and 7G8 cell bank parasites, viability was also assessed by incorporation of [3H]-hypoxanthine into newly synthesized parasitic DNA. Radioisotope uptake was demonstrated for the parasites in these P. falciparum cell banks, in comparison with the non-parasitized erythrocyte control  of the 10 compounds evaluated against the P. falciparum NF54 and 7G8 cell bank parasites are shown in Tables P. falciparum D6 was tested as a reference strain, in parallel to P. falciparum NF54, due to its known drug sensitivity profile [P. falciparum D6 line  as well as to atovaquone, mefloquine and lumefantrine  were manufactured in cleanrooms suitable for the production of sterile products using screened blood components utilising robust standard operating procedures. The cell banks were subsequently tested according to specific criteria and as required by local guidelines of the Australian TGA, and the results evaluated to determine whether the cell banks could be released for subsequent use in clinical malaria studies.The aim of the present study was to establish methodology for the production of GMP-grade, cultured malaria cell banks, as a new approach for producing material that can be used in human malaria blood-stage challenge studies to evaluate anti-malaria drugs and malaria vaccine candidates. Three blood-stage malaria cell banks, derived from three different starting materials , together with the parasitaemia of the cells banks immediately prior to cryopreservation, can be used to calculate the volume of the cell bank suspension that is required to deliver a defined dose of viable parasites in clinical studies.All of the P. falciparum 7G8) was tested by PCR for blood borne viruses and was negative for all tested agents. This gives confidence in the biosafety of the product itself i.e. that when administered to human volunteers they will not result in an unintended infection with any of the tested adventitious agents.Sterility and endotoxin testing, performed using validated techniques, demonstrated that all cell banks were negative for bacterial contamination. Similarly, all three cell banks were tested to comply with applicable region-specific regulations and guidelines and were demonstrated to be free of contamination with mycoplasma and the donors of the erythrocytes and serum were shown to be negative for a broad panel of viral agents. One of the produced banks  and with artemether/lumefantrine (known as Coartem® or Riamet®) [P. falciparum 7G8 parasites exhibited resistance to a number of the drugs tested, restricting the treatment options. Only mefloquine (Lariam®) has been used to treat this parasite in clinical trials [P. falciparum NF54, but is highly susceptible to atovaquone, so atovaquone/proguanil may be effective also. The P. falciparum 3D7 parasites demonstrated susceptibility to all compounds tested, so all would be appropriate treatment.Establishing the Riamet®) ,13,28,29l trials . HoweverP. falciparum malaria cell bank is the ability to select the parasite strain. Currently, few P. falciparum strains are available with which to evaluate the strain-transcending nature of the immune response induced by vaccine candidates, prior to larger and more expensive field-based trials in malaria-exposed individuals. Strain or allele-specific immune responses induced by sub-unit vaccine candidates may have contributed to the disappointing efficacy observed thus far, when vaccine candidates have been evaluated in a field setting. The selection of numerous, defined parasite strains for the early evaluation of vaccine candidates will ensure that only the most promising are channelled into the more expensive field-based trials for evaluation of efficacy.One of the advantages of this culture-based approach to generating a An important aspect of this culture-based approach is the possibility to take research-grade material through to a final, GMP-grade product. Thus, the lack of access to GMP-grade starting material will not impose restrictions on the selection of parasite strains for a cultured cell bank. While the conversion of research-grade material to GMP-grade is an unexplored approach for clinical malaria research, there is precedence for this in clinical stem cell research . It invoin vitro characterization and release of these P. falciparum blood-stage malaria cell banks in these current studies, future work will entail the characterization of these cell banks in vivo. As a result of these processes, the necessary regulatory and ethical clearances have been obtained to examine the infectivity of these cell banks in malaria-naïve human volunteers, as has been previously undertaken for non-cultured P. falciparum and P. vivax cell banks that have been derived from malaria infected volunteers [Following the successful lunteers ,15,16.The current study describes the first alternate methodology for the production of blood-stage malaria cell banks for use in controlled human malaria infections in CHMI studies. The ability to undertake these studies is critical for advancing the development of a malaria vaccine and novel anti-malaria drugs. Unlike existing published methods, this alternate method is culture-based and is not restricted by access to volunteers who have been deliberately infected with malaria or who contracted malaria infection following travel to/residence in a malaria endemic area.


Motivated by the success in malaria control that was documented over the last decade Ethiopia is aiming at malaria elimination by 2020 in selected districts. It is currently unknown if asymptomatic, submicroscopic malaria parasite carriage may form a hurdle to achieve elimination. The elimination effort may further be complicated by possible glucose-6 phosphate dehydrogenase (G6PD) deficiency which would hinder the use of 8-aminoquinolines in the elimination efforts.Plasmodium falciparum and Plasmodium vivax with microscopy, rapid diagnostic test (RDT), and nested polymerase chain reaction (nPCR). Multiplicity of P. falciparum infections was determined based on genotyping the polymorphic merozoite surface protein-2 (MSP-2) gene. Individuals were also genotyped for mutations in the gene that produces G6PD.In February 2014 a community-based cross-sectional survey was conducted in Malo, southwest Ethiopia. Finger-prick blood samples (n = 555) were tested for presence of P. falciparum mono-infection in 5.2% (29/555), P. vivax mono-infection in 4.3% (24/555) and mixed infection in 0.2% (1/555) of individuals. All parasitemic individuals were afebrile (axillary temperature <37.5°C). None of the study participants carried mutations for the G6PD African A-(202GA) and Mediterranean (563CT) variants. All infections, except one, were single-clone infection by MSP-2 genotyping.All study participants were malaria infection negative by microscopy and RDT. Nested PCR revealed The detection of a substantial number of subpatent malaria infections in an apparently asymptomatic population without evidence for malaria transmission by conventional diagnostics raises questions about the path to malaria elimination. It is currently unknown how important these infections are for sustaining malaria transmission in the study sites. The absence of G6PD deficiency indicates that 8-aminoquinolines may be safely deployed to accelerate elimination initiatives. Over the last decade, several malaria-endemic countries have made major progress in their fight against malaria. Between 2000 and 2013 estimated malaria mortality rates fell by 47% worldwide and by 54% in sub-Saharan Africa [Encouraged by the remarkable success, several malaria-endemic countries have adopted elimination strategies . To achiIn its current national malaria strategic plan (2014–2020), Ethiopia declared to achieve elimination in low malaria transmission settings by the end of 2020 . Three qPlasmodium falciparum gametocytes and Plasmodium vivax liver-stages [G6PD) genotype was also assessed. Primaquine administration in G6PD deficient individuals is associated with a dose-dependent risk of 8-aminoquinolines-induced haemolysis that could be life-threatening in specific cases [The aim of this study was to assess the degree of asymptomatic and submicroscopic malaria parasite carriage in two low endemic settings of Malo-Koza district in southwest Ethiopia. To support future policy considerations that may involve treatment with primaquine for both r-stages , glucoseic cases .kiremt) and minor (belg) rainy seasons, respectively.The study was conducted in Malo (6°26′0″ N latitude and 36°38′0″ E longitude), a mountainous area in southwest Ethiopia that is located 650 km from the capital, Addis Ababa. Inhabitants of Malo occupy the middle Omo river basin. The study was conducted specifically in two villages: Salayish Mender 4 (SM4) and Tatta-qirchiqircho (TQ). Malaria transmission in both sites is seasonal with peaks in transmission in September to mid-November and April to May following the major  and earth floor without cement; the floor and wall are plastered with animal dung. TQ is a midland area situated in the escarpments of two perennial rivers (Mitsilito and Tullo rivers).Project approval was granted by the Institutional Research Ethics Review Board of the College of Natural Sciences, Addis Ababa University. Prior to sample collection informed written consent was obtained from adult participants and parents/legal guardians for children below the age of 18 years.® malaria Antigen pLDH/HRP2 P.f. and Pan Combo Card Test; Premier Medical Corporation Ltd, Dist. Valsad, India) and dried blood spots on Whatman 3MM filter papers . Thick and thin blood smears were prepared, Giemsa-stained and microscopically scanned for malaria parasites. A slide was declared malaria-negative when Plasmodium was not detected in 100 high power fields examined by oil immersion (100×); all slides were read by two independent microscopists with a third reader being consulted in case of discordant results. Body temperature was measured for all individuals. Those with an axillary temperature ≥37.5°C and a positive blood slide or RDT were defined as symptomatic malaria patients.This community-based survey was conducted in February 2014, in the dry season. Community members residing in the study sites for at least 2 years were invited to participate and members who gave their informed written consent were included in the study. No formal sample size calculation was performed and the objective was to sample the largest possible fraction of the population. Finger-prick blood samples were collected for malaria microscopy, RDT  and P. vivax Malaria Reference Laboratory positive control  were included on every PCR plate as positive controls, alongside a negative water sample control. Samples were visualized on a 2% agarose ethidium bromide gel by electrophoreses and results were subsequently visualized on UV-imager.DNA was extracted using Saponin-Chelex extraction as previously described  from twoP. vivax . The limP. falciparum-positive samples, the complexity of infection was determined based on the polymorphic merozoite surface protein-2 (MSP-2) [For all 18S  (MSP-2) . In brieG6PD: 202GA (rs1050828) and G6PD:563CT (rs5030868). For the 202GA allele the forward primer was 5′-CTGGCCAAGAAGATCTACCC-3′ and the reverse primer was 5′-GAGAAAACGCAGCAGAGCACAG-3′ [Extracted DNA samples were genotyped for SNPs in CACAG-3′ . For theCACAG-3′ . All priCACAG-3′  using thMSP-2 data were analysed using Peak Scanner . Analyses focused on describing prevalence of subpatent infection, multiplicity of infection and risk factors such as G6PD deficiency. Two-sample Wilcoxon rank-sum (Mann–Whitney) test was used to test the differences in continuous variables between the two study sites. Fisher’s Exact tests were used to test for differences in parasite prevalence between populations. The significance level was set at P < 0.05.Statistical analysis was conducted using STATA 12 . For SM4, there was a local census report published in 2013 indicating the presence of 432 households with an estimated total population of 1,633 inhabitants. Of these, 156 households and 298 individuals  participated in the study; the mean family size was 4.8 (range: 1–15). In TQ, 78 households were included with a total of 259 participants ; the mean family size was 3.7 (range: 1–12). In the SM4 area the average age of participants was 20.2 years (range: 0.6–95 years) and participants of the TQ area had an average age of 14.3 years (range: 0.4–76 years). The male to female ratio was 0.89 and 1.7 for TQ and SM4 sites, respectively (p = 0.10). Study participants in TQ were significantly younger than in SM4 (P < 0.001).P. falciparum and P. vivax prevalence by nPCR was 5.8% (15/257) and 7.4% (19/257), respectively  while P. falciparum parasite prevalence was not significantly different between sites (p = 0.57). Plasmodium falciparum parasite prevalence (p ≥ 0.13) and P. vivax parasite prevalence (p ≥ 0.61) were not significantly associated with age in categories.No cases of symptomatic malaria defined as axillary temperature ≥ 37.5°C and a positive blood slide or RDT were detected. In the TQ site was 5.8% 5/257 andP. falciparum were genotyped for the MSP-2 gene. The success rate of MSP-2 genotyping, that may be less sensitive than the 18S nPCR [MSP-2 block was four: two different alleles for each of the 3D7 and Fc27 family, 3D7_263 bp and 3D7_330 bp, and Fc27_365 bp and Fc27_424 bp, respectively. The vast majority of infections were single-clone infection by MSP-2 genotyping  with only one sample (5.9%) from TQ site having two MSP-2 alleles.All samples that were positive for 18S nPCR , was 58.G6PD A- (202GA) variant that predominates in sub-Saharan Africa [G6PD mutations [G6PD A- 2GA variaG6PD A- 2GA variaP. falciparum and P. vivax infections were detected in two Ethiopian villages while conventional diagnostics did not detect any malaria infections. Genotyping for P. falciparum MSP-2 gene suggested that most infections were single-clone in nature. No evidence was observed for G6PD deficiency (G6PDd) in the study populations.In the current study, a substantial number of submicroscopic P. falciparum and P. vivax. Similar results were reported for P. falciparum from Solomon Islands [MSP-2 genotyping that revealed a high proportion of single-clone infections, which is expected in low transmission settings [The presence of submicroscopic infections in low endemic settings is increasingly well documented . The cur Islands  where 13 Islands , 24 and  Islands  settings Islands . This st Islands . In the settings . It is psettings . HoweverIt is widely acknowledged that asymptomatic individuals that carry microscopically detectable infection often harbour gametocytes and therefore play an active role in ongoing transmission . HoweverP. vivax. Plasmodium vivax contributes towards 40% of reported malaria cases in Ethiopia next to P.falciparum (60%) [P. vivax cases globally  [P. vivax a difficult malaria species to eliminate [P. vivax will require a different strategy than P. falciparum.The other prevailing challenge in Ethiopia, unlike most of Africa, is the high prevalence of um (60%) . In 2011665,813) . The coeliminate . There aliminate . EliminaP. falciparum [P. vivax [G6PD A- (202GA) frequency estimates considerably lower than those generally found in sub-Saharan Africa [G6PD. These findings highlight the need for detailed studies on G6PD enzyme activity in combination with G6PD genotyping that may need to look beyond the mutations determined in the current study. The currently available evidence suggests that primaquine may be used in Ethiopia without considerable safety concerns [Deployment of drugs such as primaquine which is the only licensed drug that is active against the mature transmission stages of lciparum  and whicP. vivax  is cruciP. vivax . In the n Africa  ranging n Africa , 40 confn Africa . In agren Africa , the Afrn Africa  with a sn Africa , 45. Theconcerns . This isconcerns  with no P. falciparum and P. vivax infections in low endemic regions in Ethiopia. The importance of these infections for onward disease transmission is unknown. The apparent absence of G6PDd suggests that primaquine may be used in combination with schizonticidal treatment to clear P. vivax hypnozoites and P. falciparum transmission stages.The current report indicates a considerable proportion of submicroscopic


Plasmodium falciparum (Pf). Naturally acquired immunitydevelops over time after repeated infections and the development of antimalarialantibodies is thought to play a crucial role. Neonates and young infants are relativelyprotected from symptomatic malaria through mechanisms that are poorly understood. Theprevailing paradigm is that maternal antimalarial antibodies transferred to the fetus inthe last trimester of pregnancy protect the infant from early infections. Theseantimalarial antibodies wane by approximately 6 months of age leaving the infantvulnerable to malaria, however direct evidence supporting this epidemiologically basedparadigm is lacking. As infants are the target population for future malaria vaccines,understanding how they begin to develop immunity to malaria and the gaps in theirresponses is key. This review summarizes the antimalarial antibody responses detected ininfants and how they change over time. We focus primarily on Pf antibody responses andwill briefly mention Plasmodium vivax responses in infants.Malaria is one of the most serious infectious diseases with most of the severe diseasecaused by  Plasmodium falciparum (Pf) malaria is one of the most important paediatricinfectious diseases estimated to kill over 600 000 people annually, most of whom arechildren younger than 5 years of age , with detection of asexual parasites in cord blood or in neonatal peripheral bloodwithin the first week of life  than in children olderthan 12 months (3500 parasites µL−1)  is characterized by low parasitaemia, often <100parasites in vitro , method for detectingparasitaemia, location and transmission intensity. A few of these studies have demonstratedan association between maternal antimalarial antibodies and protection from infection were associated withdelayed onset of first infection among a cohort in western Kenya and with protection ofinfants against clinical disease in Liberia , MSP2, ring-infected erythrocyte surface antigen(Pf155/RESA) and crude schizont antigen) were positively associated with infection  domain cysteine-richinterdomain region 1α (CIDR1α) conferred protectionagainst malaria in infants during the first 6 months of life  canhave devastating effects on both mother and fetus, including anaemia, stillbirth, lowbirthweight, intrauterine growth retardation and pre-term delivery  make less functional MSP 119 invasion inhibitoryantibodies implicated in apical reorientation of merozoite, and erythrocyte-binding proteins  and reticulocyte-binding homologues (e.g. Rh4 and Rh5) thought tobe important in erythrocyte invasion which uses transgenic parasites, by 12 months of age, infants had detectableMSP119 IIA that gradually increased by 24–30 months after birth  which quantifies antibody-mediated activity againstparasites et al.In summary, infants acquire antimalarial antibodies with serologically measurableantibodies possibly acquired earlier than VSA or GIA measured antibodies which may reflectthe late and complex acquisition of functional antibodies. Serologically measured antibodiesmay be a biomarker of exposure and may not protect the young child until a certain magnitudethreshold is reached. These observations coupled with the immaturity of the infant immunesystem until 2 years of age (Jaspan in utero may have an important effect on infant immune responsesto malaria and other pathogens. In infants and young children, antibodies detected byserology may be a biomarker of exposure, with protection afforded only after a criticalthreshold is reached. As infants are a target population for future malaria vaccines,understanding the complexity of achieving protection in the context of an immature immunesystem must be considered. Further studies and development of novel assays are needed toanalyse the functional capabilities of these antibodies and to evaluate the maternal,fetal and neonatal factors leading to the development of protective immunity ininfants.Early infancy is a critical time in the development of immunity to malaria in childrenborn in malaria endemic areas. Antibodies are known to play an important role in hostdefence against malaria, but the acquisition of antimalarial humoral immunity in infantsis not yet well understood. Passively transferred maternal antibodies thought to protectinfants younger than 6 months from clinical disease may actually be a biomarker ofexposure and risk of infection rather than a correlate of protection. Infants withmaternal antimalarial antibodies still become infected with low-grade parasite densities.As maternal antibodies wane, older infants gradually acquire antimalarial antibodies, withincreasing breadth and magnitude of responses with age and exposure. Fetal exposure tomalaria


Malaria prevalence and transmission intensity in Tanzania is heterogeneous with spatial and temporal variations between geographical areas and ecological systems. The objective of this study was to determine the prevalence of malaria, anaemia and nutritional status in relation to livelihoods, ecosystem and health systems in Kilosa District in central Tanzania.This study was conducted in four villages, two characterised by rice irrigation ecosystem and the other two by dry savannah ecosystem and pastoral livelihoods. In each ecosystem, one of the villages had a healthcare facility. Schoolchildren were screened for malaria infection using malaria rapid diagnostic test (mRDT) and microscopy and they were assessed for their anaemia and nutritional statuses.Plasmodium falciparum infection was 10.6 % and 4.5 % by mRDT and microscopy, respectively. Children from pastoral villages had lower (2.9 %) prevalence of malaria than their counterparts (18.2 %) in the rice irrigation villages. A significantly high risk of malaria was observed among children in rice irrigation than in the pastoral ecosystem . Children living in areas with health care facilities had a low odd of malaria infection by 45 % . Overall, the prevalence of anaemia in the district was 43.4 % (n = 775); and 58.3 % of those with severe anaemia were among children from the pastoral villages. Anaemia was significantly higher among children not using mosquito nets (p = 0.049); and among those with malaria infection (p <0.001). The majority (96 %) of the children had Body Mass Index less than 18.5 kg/m2 which indicate high proportion of underweight.A total of 1,019 school children (age = 4–16 years) were screened for malaria infection. The overall prevalence of There are significant variations in the risk of acquiring malaria infection between different ecosystems and livelihoods. These findings suggest that malaria control programmes must take into account ecosystems and livelihoods of the targeted population through an integrated management of malaria and nutrition approach. Malaria is the single most important and widespread mosquito-borne disease in Tanzania. The disease is endemic in most parts of the country, and remains as a major cause of hospital attendance, admission and death . HoweverMalaria represents a complex, multi-dimensional health problem with a host of interacting variables. Such complex health problems are difficult to solve without understanding ecosystems, livelihoods and health delivery systems contexts. This is because, health is a social construct negotiated in the context of a better understanding of the constraints and opportunities provided by the three variables of which people are integral parts . EcosystMalaria is mostly a disease of rural communities in Tanzania, where agriculture is the backbone of the household economy. Studies in Sub-Saharan Africa have shown that the tropical climate and land use changes have either a positive or negative effect on mosquito reproduction and survival and exposure of humans to infectious mosquito bites , 19. CroA good understanding of the nature and dynamics of certain ecosystem and livelihoods variables and their relationship to malaria prevalence and transmission intensity is crucial in identifying and addressing interventions that may reduce malaria while increasing household productivity. This study was, therefore carried out to determine malaria prevalence, anaemia and nutritional status among schoolchildren in areas representing different ecosystems and livelihoods in Kilosa District in central Tanzania.2 and a population of 489,513 people living in 105,635 households with an average household size of 4.2 people [The study was carried out in Kilosa District  in central Tanzania. The district has a total surface area of about 14,400 km2 people . The disThis study was conducted in four villages, namely Tindiga, Malui, Twatwatwa and Mbwade. Tindiga and Malui are in the south-eastern part of the district and characterised by swampy flatland and wetlands lying on the Kilangali alluvial basin. Most of the communities in Tindiga and Malui are small-scale farmers of rice using the traditional ground flooding irrigation practice. Mbwade and Twatwatwa are located in the north-eastern part of the district and are characterised by dry savannah type of vegetation, with most of the areas covered with short grasses, trees and shrubs that provide a wide range of pasture for livestock grazing Fig. . Mbwade This study was carried out in May 2012 and involved schoolchildren. Each individual child was screened for the presence of malaria parasites using both microscopy and malaria Rapid Diagnostic Test (mRDT) . Thick and thin blood smears were stained with Giemsa and examined with a binocular microscope with an oil immersion lens to quantify the parasitaemia. Parasitaemia was measured by counting the number of asexual parasites against the number of leukocytes in the blood film, based on a putative count of 8,000 leukocytes per microlitre. The number of asexual parasites was counted against 200 leukocytes using a hand tally counter. A slide was considered negative if no malaria parasite was seen after scanning 200 fields. The blood films were examined by experienced microscopists without reference to the results of the malaria rapid diagnostic test.A sub-sample of the blood was used to determine haemoglobin concentrations by using a HemoCue haemoglobinometer . All children were examined for splenomegaly through the palpation of the spleen in a horizontal position by the research physician  and bodyTo determine the nutritional status, the body weight of each child was measured and recorded to the nearest 0.1 kg. Height was measured with a child standing upright using a stadiometer  and was recorded to the nearest 0.1 cm. The measurement was recorded while the child was standing without shoes on a horizontal flat plate.t-test and proportional test where applicable. Regression model was fitted to assess risk of malaria infection considering all possible relevant factors. Effect was considered at 5 % level of significance.All malariometric data were entered and verified in Microsoft Excel and analysed using STATA statistical analysis software package version 9 . Simple associations were tested using Chi-square test, The mRDT sensitivity was estimated as true positives/ , specificity as true negatives/ , positive predictive value as true positives/ , negative predictive value as true negatives/  and the positive likelihood ratio as sensitivity/ (100 – specificity). Inter-observer variability was assessed using Kappa statistic  while .2), normal weight (18.50-24.99 kg/m2) and overweight (≥25.00 kg/m2). Temperature readings were categorized into fever and non-fever status with a cut-off point of 37.5 °C.The World Health Organization age-adjusted cut-off for haemoglobin (Hb) was used to classify anaemia in children . For chiThis study received an ethical approval from the Medical Research Coordinating Committee of the National Institute for Medical Research. Before the survey began, the objectives, methods and benefits of the study were explained to the district authority, school teachers and parents in a meeting convened by the village authority. An informed oral consent was sought from parents/guardians of the children involved in the study. An oral assent was asked from each child enrolled in the study. Any child found to be infected with malaria was treated using artemether-lumefantrine tablets according to National Guidelines on Malaria Treatment.Plasmodium falciparum was the only (100 %) malaria parasite species observed. The overall prevalence of malaria infection was 10.6 % by mRDT and 4.5 % by microscopy . Within the rice irrigation ecosystem, malaria prevalence was higher in Malui (22.5 %) than in Tindiga (13.8 %) . There were no significant difference in malaria prevalence between villages within the pastoral villages . Only three (0.23 %) children were found with gametocytes . Six (0.6 %) of the children had an enlarged spleen; and all were from rice irrigation villages.A total of 1,019 school children (mean age = 9.4 ± 2.2 years) were examined for malaria infection. Of the children, females accounted for 54.6 %, while males accounted for 45.4 %  indicating moderate agreement.In all the four villages, higher malaria prevalence was detected by the use of mRDTs. The prevalence of malaria infection by microscopy was 2.4 lower than that found using mRDT. Among the 46 individuals who tested positive by microscopy, 17.4 % were found negative by mRDT. Amongst those tested positive by mRDT only 34 % were found infected with malaria parasites using microscopy. The mRDT specificity and sensitivity were 94.18 % and 82.61 %, respectively. Inter-observer variability between the results of the two tests gives a Kappa value of 0.45 . Comparing between the two villages in the rice irrigation ecosystem, the prevalence of malaria was 38.5 % higher in Malui  than in Tindiga .There were 14 children (1.4 %) with fever (temperature >37.5 °C). Ten (71.4 %) of them were from villages with health facilities. Among them, six 42.9 %) were from Tindiga; others were from Twatwatwa (28.6 %), Malui (21.4 %) and Mbwade (7.1 %). A total of 12 children with fever had no malaria infection. The proportion of those with fever but no malaria infection was highest in Tindiga (1.75 %) and lowest in Mbwade (0.42 %). The proportions of children with fever in the other villages were 1.28 % in Malui and 1.58 % in Twatwatwa ; and among those with malaria infection .Overall, the prevalence of anaemia in the district was 43.4 % (n = 775). Schoolchildren from the pastoral community of Twatwatwa village had the highest prevalence of anaemia  indicating severe thinness. The majority (96.6 %) of the children had BMI less than 18.5 kg/m2 which indicate high proportion of underweight. Only 3.4 % had normal weight and most of these (52.9 %) were from Malui village. Tindiga had the highest proportion (98.8 %) of children with underweight. The proportions of underweight in the other villages were 92.9 % in Malui, 96.6 % in Mbwade and 98.1 % in Twatwatwa. There was no significant difference in individuals with normal weight between those from rice and pastoral communities .The overall mean body mass index (BMI) was 14.9 kg/mAnopheles gambiae [The findings of this study indicate that children from rice irrigation villages had higher prevalence of malaria than their counterparts in the pastoral villages. Higher malaria prevalence among communities living in rice irrigation ecosystems have been reported elsewhere in Tanzania and in Sub-Saharan Africa , 23, 24. gambiae . The var gambiae . Similar gambiae , 26.Our results also indicate significant variations in the level of parasitaemia even between villages within the same agro-ecosystem. This variation was attributed to availability of health care facility. The fact that malaria prevalence was higher among children living in villages without health facilities confirms previous findings elsewhere in Tanzania , 17. It Although there was no significant difference in malaria prevalence between sex, age or possession of mosquito net, the likelihood of acquiring malaria infection was lower among those who were using mosquito nets. This indicates the protective effect of the nets. In this study, it was noted that a high proportion of children with fevers were from villages with health facilities. In addition, the observation that a proportion of children with fever had no malaria infection indicates that non-malaria fevers are prevalent in the area. Fevers of bacteria or viral origin are prevalent in Tanzania and are the most likely causes of febrile illnesses in most cases . In a reIn this study the mRDT specificity and sensitivity were high. Other studies in Tanzania have also reported similar higher sensitivity and specificity of histidine-rich protein-2 mRDTs , 31. On Anaemia prevalence in the district was 43.4 %, with the highest prevalence among children in the pastoral villages. Similar high prevalence of anaemia has been observed in the neighbouring district of Mvomero . In thisThe majority of the children in this study were underweight, with most of them from rice growing communities, where malaria prevalence was higher. Some reports already indicate that underweight among children in developing countries is due to lack of food and the presence of diseases, mostly malaria . In a stIn this study, strong effect of the ecosystem, livelihoods and health systems on malaria was observed. Rice farming communities were at higher risk of malaria as a result of their routine farm activities that involves irrigation that supports mosquito productivity . In a paThe findings of this study indicate that in Kilosa district, there are variations in terms of risk to malaria, anaemia and nutritional statuses between communities living in different ecosystems and with different livelihoods practices. The variability of the prevalence of malaria parasitaemia by ecosystems and livelihoods illustrates the existing of different micro-ecological zones and the contribution of human activities on transmission factors. It is therefore important, that malaria control measures are designed taking into consideration the specific local conditions and needs. The links between malaria, ecosystem, livelihoods and food insecurity needs to be addressed in tandem using an ecohealth approach.


Pklr deficiency in mice reduces the severity  of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that  P. vivax on erythrocytes and its absence in the Duffy negative blood group prevents parasite entry in erythrocytes and protects against P. vivax malaria  in 6-well tissue culture dishes. Recombinant PKLR construct DNA was mixed with Opti-MEM (Life Technologies) and Lipofectamine 2000  prior to being added to cells supplemented with fresh DMEM + 10% FBS. Transiently transfected cells were harvested 18 hrs later. In some experiments, transfected cells were plated in complete medium supplemented with 0.5mg/mL Geneticin , and G418-resistant colonies were isolated 14 days later. Transfected cells were washed in 1xPBS , then sonicated in PBS (6x15sec) on ice . The total protein concentration within the cell lysate was quantified using a Bradford assay , and equal amounts of protein lysate were separated on a 10% SDS-PAGE gel, followed by immunoblotting against HA.11 Clone 16B12 purified monoclonal antibody (mouse)  and β-actin antibody (mouse) (Covance Inc.) as a loading control. The secondary antibody, ECLTM Anti-mouse IgG, Horseradish Peroxidase linked whole Ab (from sheep) , was used in conjunction with SuperSignal WestPico Chemiluminescent substrate (ThermoFisher Scientific) for protein detection.Twenty-four hours prior to transfection, 5 x 1035S] methionine-cysteine . Cells were then washed twice with PBS and incubated in chase medium  for up to 24 hours. For immunoprecipitation, equal amounts of cell lysates were incubated with the mouse anti-HA antibody overnight at 4°C, followed by incubation with protein A/G-sepharose  for 4 hrs. Beads were washed three times in RIPA buffer and proteins were eluted with 50μl of 3X Laemmli sample buffer. The radiolabeled proteins were separated by electrophoresis using 7.5% SDS polyacrylamide gels. The gels were then fixed, impregnated with Amplify , dried and exposed to film. Densitometric analysis was performed using NIH ImageJ software .For protein stability assays, cells were incubated in methionine- and cysteine-free DMEM media containing 10% dialyzed FBS, and 55 μCi of -methionine labelled protein in pulse-chase studies  failed to detect a major effect of the R41Q mutation on basal enzymatic activity in such over-expressing cells (data not shown). We also examined the effect of the R41Q mutation on stability of the transiently transfected [ studies . In threPlasmodium parasite, and variants in certain erythrocyte proteins have been found enriched in malaria-endemic areas. Although homozygosity for such variants causes disease on their own , heterozygosity confers various degrees of protection against malaria [Pklr) in mice reduces the severity of infection with the blood stage parasite P. chabaudi AS [ex vivo with P. falciparum, homozygosity for PK-deficiency alleles causes a reduction in parasite replication, while parasitized erythrocytes from heterozygotes are phagocytized more avidly than those from normal individuals [PKLR gene, including the presence of rare, potential loss-of-function protein variants. Neutrality testing of PKLR haplotypes and variants suggested positive selection of the gene in the sub-Saharan African and Pakistani populations, possibly by the malarial parasite [Malaria kills one child every minute in Sub-Saharan Africa , 52. Ove malaria , 53, 54.baudi AS , 32. Likividuals , 33. Usiparasite . This hyPKLR gene, and haematological and malaria-associated phenotypes in three populations from Thailand and Senegal, living in areas of seasonal or year-long exposure to the malarial parasite. These populations were studied longitudinally for multiple phenotypes, including but not limited to frequency and intensity of infection by the malarial parasites, thereby providing added power to detect genetic effects of the host in response to the parasite. The Thai population has been used in the past to document the effect of G6PD deficiency  on differential susceptibility to infection with P. falciparum and P. vivax [Herein, we probed for the possible association between rare or common variants at the sequenced P. vivax .PKLR gene in subsets of Senegalese and Thai individuals identified a number of common variants (SNPs), nine of which were used to define PKLR haplotypes in these populations. Eight PKLR haplotypes were detected, two of which were common and representing approximately 86% of the Senegalese and 31% of the Thai populations. These common haplotypes are also present in various North, South, and Sub-Saharan African, Pakistani, Southeast Asian and European populations [PKLR further detected population substructure in the two Senegalese populations. The Dielmo community is composed primarily of the Serere and Mandinka ethnic groups [PKLR haplotypes were detected in these populations. Of particular interest is the Thai-specific haplotype 1  variant reduces infection with P. vivax, but not with P. falciparum in a population from South Asia, suggesting that Plasmodium vivax has been a driving force behind the strong selective advantage conferred by this mutation [Sequencing also identified three coding variants in the PKLR protein; these variants affected residues that were either highly conserved , or coded for a very significant change in amino acid property (V269F), both of which possibly result in altered protein function. The L272V and V269F variants were present at very low frequency in the two populations, and association with haematological and malaria traits could not be conducted with sufficient statistical power. Genotyping revealed that the R41Q mutation was present with a minor allele frequency of approximately 4.5% within the Thai cohort. We noted a statistically significant association between R41Q heterozygosity and a reduced frequency of  females . Althougfections . On the mutation .Pyruvate kinase is composed of an amino terminal (N) domain with a helix-turn-helix motif, followed by a bi-partite A domain  that contains residues implicated in binding the phosphoenolpyruvate (PEP) substrate, as well as a conserved B domain linker, followed by the C-terminal domain that binds the allosteric regulator fructose-1,6-bisphosphate (FBP) . StructuIn addition to basal activity, there are number of structural and functional aspects of PKLR that may be affected by R41Q. PKLR activity is regulated in part by phosphorylation of serine 43, which causes increased affinity for allosteric inhibitors ATP and alanine, and decreased affinity for PEP and the allosteric activator FBP , 59. TwoPKLR mutations do not affect enzymatic activity of the purified protein. For example, the R510Q mutant in a patient with nonspherocytic hemolytic anemia shows wild-type kinetic activity and regulatory properties when expressed in bacteria [Finally, it has been observed that some disease-associated bacteria : Howeverbacteria . AdditioPlasmodium infection in humans, with protective alleles possibly retained in populations living in areas of endemic malaria. The cumulative data supporting this proposal include: a) the demonstration that homozygosity for loss of function at Pklr cause complete protection against blood-stage malaria (P. chabaudi AS) in mice [PKLR cause a reduction in parasite replication and increase phagocytosis of parasitized erythrocytes in studies with primary erythrocytes infected ex vivo with P. falciparum [PKLR gene shows highest sequence diversity in regions where malaria is or has been endemic, when compared to regions where it has not been prevalent [Although our study requires additional validation in other cohorts where longitudinal data on haematological malaria phenotypes are available, results obtained so far strongly suggest that allelic variants at PKLR modulate susceptibility to  in mice , 32, witlciparum ; c) the revalent ; d) a unS1 Tableet. al. [Adapted from Berghout et. al. .(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TablePrimers were designed to have at least 10 nucleotides flanking the single point mutation (in bold).(DOCX)Click here for additional data file.S5 TableDesigned within the PKLR cDNA insert and pcDNA3 vector, these primers were utilized in confirming the sequence of the wild-type and mutant constructs.(DOCX)Click here for additional data file.


Plasmodium falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical host genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity and improve vaccine development initiatives.P. falciparum transmission in Cameroon. Thirty-four malaria candidate polymorphisms, including the sickle cell trait (HbS), were assayed on the Sequenom iPLEX platform while plasma TGF-β and IL-10 levels were measured by sandwich ELISA.The effect of single nucleotide polymorphisms (SNPs) and plasma transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) levels on malaria pathology was investigated in a case–control study of 1862 individuals from two major ethnic groups in three regions with intense perennial HbS against severe malaria and also reveals a protective effect of SNPs in the nitrogen oxide synthase 2 (NOS2) gene against malaria infection, anaemia and uncomplicated malaria. Furthermore, ADCY9 rs10775349 (additive G) and ABO rs8176746 AC individuals were associated with protection from hyperpyrexia and hyperparasitaemia, respectively. Meanwhile, individuals with the EMR1 rs373533 GT, EMR1 rs461645 CT and RTN3 rs542998 (additive C) genotypes were more susceptible to hyperpyrexia while both females and males with the rs1050828 and rs1050829 SNPs of G6PD, respectively, were more vulnerable to anaemia. Plasma TGF-β levels were strongly correlated with heterozygosity for the ADCY9 rs2230739 and HBB rs334 SNPs while individuals with the ABO rs8176746 AC genotype had lower IL-10 levels.The study confirms the known protective effect of Taken together, this study suggests that some rare polymorphisms in candidate genes may have important implications for the susceptibility of Cameroonians to severe malaria. Moreover using the uncomplicated malaria phenotype may permit the identification of novel pathways in the early development of the disease. Plasmodium falciparum infections progress to severe or fatal episodes ; severe malaria anaemia (SMA) ; hyperpyrexia (axillary temperature ≥ 40°C); hyperparasitaemia ; convulsions before/during admission; respiratory distress (RD)  and hypoglycaemia (blood glucose <2.2 mmol/L/40 mg/dl). Participants with co-existing severe or chronic medical conditions  unrelated to a severe malarial infection were excluded. UM was defined as a clinical illness characterized by an axillary temperature ≥37.5°C associated with a Plasmodium positive blood film, haemoglobin ≥ 8 g/dL and full consciousness, in the absence of clinical signs and symptoms of severe malaria and/or evidence of vital organ dysfunction.Controls (n = 891) consisted of apparently healthy children  and adults  also belonging primarily to the Bantu and Semi-Bantu ethnic groups  admitted in nine health facilities in the Centre, Littoral and South West regions of Cameroon . The 971Ethical and administrative clearance for the study was obtained from the University of Buea Institutional Review Board and the South West Regional Delegation of Public Health respectively. Authorization to conduct the surveys in designated primary schools was obtained from the Regional Delegation of Basic Education or the Catholic Education Secretariat of the South West Region. Individuals who fulfilled the specific inclusion criteria and volunteered to participate after adequate sensitisation on the project objectives, methods and possible benefits/risks were enrolled in to the study. A health facility or school was only investigated with the approval of its Director or Head Teacher and study participants were only enrolled if they or their caregivers/guardians gave written informed consent/assent.Plasmodium species was identified on the thin blood smear. A smear was only considered negative if no malaria parasites were seen in 50 high power fields. With each positive smear, the level of parasitaemia was estimated by counting the parasites against at least 200 leucocytes and using the corresponding leucocyte count to calculate the number of parasites/μl blood  and from SMA   while two of the assays did not pass the quality filters , leaving 30 SNPs that could be analysed for their association with malaria phenotype   and UM   while heterozygosity for the rs8176746 SNP in the ABO locus was associated with protection against hyperparasitaemia  , hyperparasitaemia , hyperpyrexia  and SMA   and females with rs1050828  and rs1050829  SNPs of G6PD were more susceptible to anaemia  and rs461645  SNPs respectively in the EMR1 gene were more likely to develop hyperpyrexia. Furthermore, individuals with the RTN3 rs542998 SNP  were also independently associated with susceptibility to hyperpyrexia (Table GBP7), CM (CD40LG), hyperparasitaemia (NOS2), hyperpyrexia , SMA (EMR1) and UM   and hyperparasitaemia  respectively. Conversely, the association between G6PD rs1050828 with anaemia in females  was only observed in the Bantu ethnic group.Additional file ADCY9 rs2230739 (p = 0.039) and HBB rs334 (p = 0.002) SNPs , with AC genotypes, however, having lower levels of the cytokine compared to their AA and CC counterparts. Nevertheless, no other SNPs were associated with levels of these cytokines.Plasma TGF-β levels were strongly correlated with heterozygosity for the falciparum malaria and its association with the β-globin SNP underlying the haemoglobin S sickle-cell trait [The clinical outcome of human malaria infection is highly variable and heterogeneous, depending on many factors including age, transmission intensity, parasite strain virulence, co-infections with other pathogens, socio-economic status as well as the genetic background of the human host . The humll trait . This mull trait  yet explll trait . Many unABO and RTN3 against severe malaria. It further provides the first evidence that polymorphisms in NOS2 and EMR1 may be associated with mild malaria, G6PD with anaemia while SNPs in ADCY9 and EMR1 may be linked with severe malaria in the Cameroonian population. Data reported here provide additional understanding on human candidate genes that may contribute to malaria susceptibility, help define the basis of individual and population variation in susceptibility to the disease and thus eventually facilitate efforts to develop vaccines and treatments to fight this infectious disease. However, it is possible that an apparent association with malaria can arise from linkage disequilibrium between the typed SNP and a primarily associated polymorphism. Constructing a detailed map of polymorphisms around these candidate genes and describing a profile of linkage disequilibrium among them will be helpful to identify the causal variant(s). In addition, an appropriately designed family-based study may provide useful linkage information, have more power to detect association and allow haplotypes to be constructed with more confidence.Genetic association studies utilising polymorphic markers in candidate genes have been successful in identifying a number of genes that are associated with susceptibility to malaria, or severe malaria. A large case–control study of severe malaria in children across different regions and major ethnic groups in Cameroon has the potential to confirm previously reported malaria/genetic associations or permit the identification of new ones following the assessment of association of SNPs of human genes with clinical phenotypes. This study focused on genes whose association with malaria remain controversial as well as those reportedly associated with susceptibility to other infectious diseases. This study suggests that the protective effect of the sickle cell trait may be linked to the raised level of TGF-ß and provides additional support for a role of NOS2 SNP is quite interesting since, no previous associations between rs2297518 and malaria have been reported. The SNP has, however, been linked with non-Hodgkin lymphoma [NOS2 to produce NO. The constitutively high NOS activity in children with the rs2297518 A allele will provide an advantage in resisting hepatic parasite stages without the need for additional stimuli. Nitric oxide is also thought to be effective in reducing blood-stage parasite density by antibody-dependent cellular inhibition [Although the CCTTT pentanucleotide microsatellite repeat in iNOS promoter is thought to play a key role in the pathogenesis of severe malaria , the prolymphoma  and infllymphoma . It is plymphoma . Parasithibition  triggereADCY9) is an interesting candidate gene since it is critical in neuronal signalling [ADCY9 gene variation with mood disorders. Although the gene has polymorphisms with large allele frequency differences (FST) between HapMap YRI and CEPH, the reported rs10775349 association with hyperpyrexia is very interesting. Nevertheless, further work is needed to strengthen these findings.Adenylate cyclase (AC) type 9 gene 1 polymorphisms and hyperpyrexia, severe malaria anaemia and uncomplicated malaria is quite interesting. cascades . Gene ex without  but no gABO variants on cytokines level.Polymorphisms in the ABO blood group were associated with hyperparasitaemia and hyperpyrexia  but not with the other more prevalent phenotypes. Previous studies have shown that cerebral malaria cases are less likely to be of blood group O ,50 but mGNAS) polymorphisms with severe malaria [Functional studies have demonstrated that a host erythrocyte G protein signal pathway may be a critical component in parasite invasion and that erythrocyte G-alpha-s protein and the malaria parasite interact at a cellular level . It howe malaria , althougReticulon is thought to be involved in malaria pathogenesis since its gene expression significantly increases in malaria and other infections  while chADCY9 rs2230739 with plasma levels of transforming growth factor-beta as well as ABO rs8176746 with interleukin-10 is quite interesting since resistance to severe malaria has been linked to the ability to produce these immuno-regulatory cytokines [ADCY9 rs2230739 may act to upregulate TGFβ transcription, with the heterozygotes providing some selective advantage since the raised TGF-β levels will down-regulate proinflammatory cytokines, such as TNF, and protect against severe malaria [Cytokine polymorphisms have previously been linked with their differential production and expression in malaria ,6, with ytokines ,23. How ABO and RTN3 against severe malaria. It also provides the first evidence that polymorphisms in NOS2 and EMR1 may be associated with mild malaria, G6PD with anaemia while SNPs in ADCY9 and EMR1 may be linked with severe malaria in the Cameroonian population. Polymorphisms in human genes have important implications for the outcome of paediatric malaria in Cameroon. Moreover using mild malaria clinical phenotypes may permit the identification of novel pathways in the early development of disease.This study suggests that the protective effect of the sickle cell trait may be linked to the raised level of Transforming Growth Factor beta and provides additional support for a role of The author(s) declare that they have no competing interests; that they had financial support from MalariaGEN, EVIMalaR and CANTAM for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work; no other relationships or activities that could appear to have influenced the submitted work.TOA, KAR, EAA, DPK conceived and designed the experiments. TOA, AJK, NC, RNM, ANN performed the experiments. TOA, TGC, MalariaGEN analysed the data. DPK, EAA, MalariaGEN, TGC contributed reagents, materials, analysis tools. TOA, AJK, NC, KAR, EAA wrote the paper. All authors read and approved the final version of the paper.Allelic and genotype associations between selected SNPs and malaria. Genotypes were tested for Additive, dominant, recessive and heterozygous advantage and then adjusted for age, sex, ethnicity and HbS, but only the most statistically significant result is presented.Click here for fileGenotype associations between selected SNP and syndromes of malaria in the two major ethnic groups. Genotypes were tested for Additive, dominant, recessive and heterozygous advantage and then adjusted for age, sex and HbS, but only the most statistically significant result is presented.Click here for file