Malaria continues to impose a tremendous burden in terms of global morbidity and mortality, yet even today, a large number of diagnoses are presumptive resulting in lack of or inappropriate treatment.Plasmodium spp. and differentiate Plasmodium falciparum infection from the other three human malaria species . To achieve this goal, red and blue colours were encoded to two markers on a single test line of strips, for simultaneous detection of PfHRP2 (red), a marker specific for P. falciparum infection, and pLDH (blue), a pan-specific marker for infections by all species of Plasmodium. The assay performance was first optimized and evaluated with recombinant malarial proteins spiked in washing buffer at various concentrations from 0 to 1000 ng mL−1. The colour profiles developed on the single test line were discriminated and quantified: colour types corresponded to malaria protein species; colour intensities represented protein concentration levels.In this work, a two-colour lateral flow immunoassay (LFA) system was developed to identify infections by −1 and 7.8 ng mL−1, respectively. To further validate the efficacy of the assay, 25 human whole blood frozen samples were tested and successfully validated against ELISA and microscopy results: 15 samples showed malaria negative; 5 samples showed P. falciparum positive; 5 samples showed P. falciparum negative, but contained other malaria species.The limit of detection  and the limit of colour discrimination (the limit to differentiate pLDH from PfHRP2) were defined for the two-colour assay from the spiked buffer test, and the two limits were 31.2 ng mLThe assay provides a simple method to quickly identify and differentiate infection by different malarial parasites at the point-of-need and overcome the physical limitations of traditional LFAs, improving the multiplexing potential for simultaneous detection of various biomarkers. Over 40% of world’s population live in malaria-endemic areas, and malaria is found in more than 100 countries in Africa, Latin America, the Caribbean, Southeast Asia, the Eastern Mediterranean, the Western Pacific, and parts of Europe . DespitePlasmodium falciparum histidine-rich protein2 (PfHRP2), and parasite aldolase (pAldo) [P. falciparum only, and pLDH pan-specific to all Plasmodium species. The concurrent detection of PfHRP2 and pLDH allows the discrimination of P. falciparum infection from the other three human malaria species .Early diagnosis and prompt, appropriate treatment is essential for improving patient outcomes, preventing overuse of malarial drugs, and minimizing development and spread of anti-malarial drug resistance . The cur (pAldo) , 8. The  (pAldo) . PfHRP2 P. falciparum infection, particularly when treatment is delayed [Severe malaria leads to significant mortality and is mainly attributed to  delayed . PatientP. vivax and P. ovale malaria [In addition to malaria species and severity, the treatment approach of malaria also depends on special risk groups, such as infants and pregnant women . For chi malaria . For mal malaria –15. Thus malaria .Here, a quantitative, multiplexing lateral flow immunoassay using two-colour latex particles to overcome current limitations of RDTs is reported. The red and blue latex particles were first functionalized with antibodies to PfHRP2 and (pan) pLDH, respectively. In the LFA, nitrocellulose membranes were pre-coated with one test line and one control line. The test line contains a mixture of antibodies to PfHRP2 and (pan) pLDH, and the control line contains antibodies to mouse IgG, as shown in Fig. The 400 nm blue and red latex particles  were functionalized with anti-(pan)pLDH antibodies , and anti-PfHRP2 antibodies , respectively, according to the manufacturer’s instruction.−1 anti-(pan)pLDH antibody  and 1 mg mL−1 anti-PfHRP2 antibody  in PBS, and 10 mg mL−1 anti-mouse IgG antibody  in PBS were dispensed to the nitrocellulose membrane , respectively, with a dispenser  and a syringe pump , followed by being dried overnight and stored at 37 °C until use. One cm × 12 cm glass fibre conjugate pad  was fully immersed in 800 μL conjugate buffer, with a mixture of 0.05% (v/v) blue and 0.05% (v/v) red latex particles in 2 mM borate buffer with 5% sucrose, followed by being dried overnight and stored at 37 °C. The conjugate pad, sample pad , and absorbent pad  were assembled onto the adhesive parts of the nitrocellulose membrane. Then the LFA was cut to 4 mm × 6 cm in dimension and was caged in the plastic cassette.To prepare the test and control lines, a mixture of 1 mg mL−1. For simultaneous detection, pLDH and PfHRP2 antigens were mixed at the ratio of 1 to 6, followed by twofold serial dilution with washing buffer.The LFA performance was demonstrated by detecting recombinant malaria antigens, including pLDH , and PfHRP2 . A series of twofold dilutions of each protein of pLDH, and PfHRP2 in washing buffer  were prepared at various concentrations from 3.9 to 1000 ng mLhttps://imagej.nih.gov/ij/plugins/color-profiler.html) was employed for obtaining colour profiles in the test strip images. The region of interest (ROI) was selected on the images where both test and control lines were included using rectangular selections on the ImageJ menu bar. The same ROI was applied to all acquired images. The red and blue colour intensity profiles along with the distance of the strip were plotted, and the peak areas at the test regions were calculated using Simpson’s 3/8 rule.Thirty microliters of the sample was dispensed on a sample pad of the LFA strip, followed by being washed by 70 μL washing buffer to allow the sample liquid to flow through the full length of the nitrocellulose membrane. After 15 min, images of the strips were acquired and saved using iPad Air2 which was equipped with a 3D printed accessory iPad case. To bypass the challenges of naked-eye detection, a plug-in command called ‘Color Profiler’ in ImageJ software  to get quantitative results to evaluate the performance of the LFA strips.In the LFA, when the sample liquid is dispensed on a sample pad and flows to the conjugate pad, the blue and red latex particles capture pLDH and PfHRP2 antigens, respectively. The antigens bound to the latex particles are subsequently transported through the strip, and are detected at the test line where a mixture of detection antibodies to (pan)pLDH and PfHRP2 are functionalized Fig. . The chaP. falciparum infection [The performance for the two-colour LFA was first demonstrated by detecting recombinant malarial antigens spiked in the washing buffer at various concentrations. Three different diagnostic scenarios were tested: pLDH only, PfHRP2 only, and concurrent pLDH-PfHRP2 infection. For the co-detection, pLDH and PfHRP2 was mixed at a molar ratio of 1 to 6, to mimic clinical samples of nfection . FifteenTo implement the quantitative and qualitative method in the assay, the intensity profiles of the LFA strips were analysed. The images of the strips were acquired using an 8-megapixel rear-facing camera of an iPad Air 2 under the same white LED lighting conditions. The distance between the test line and control line was about 200 pixels, and width of the line was about 50 pixels in the images. To obtain the RGB color profiles, the images were opened using ImageJ software and executed “Color Profiler” command. For simplicity, only the red and blue intensity profiles were analysed, since green intensity profiles did not significantly affect the red and blue colour discrimination, and provided an auxiliary value in colour images. The nitrocellulose membrane of the test strip was white, resulting in high background intensities. The colours with contrast at the test and control lines generated the peaks decayed from the background intensities Fig. .Fig. 3ReTwo intriguing features in the colour profiles were observed. First, when the blue test lines appeared on the strips, the red intensity peaks were more decayed than blue peaks in the colour profiles. This is because the blue colour retained relatively higher blue pixel values than red values. Figure The other feature in the colour profiles was the non-specific colour peaks in Fig. To further analyse the strips, avoid subjectivity, and confirm visual limit of detection, decay areas of red and blue peaks were calculated from Fig. non-target + 3SD) of the blank sample signal. The LoD at which all red and blue signals were distinguishable from the blank sample signals was estimated to be 31.2 ng mL−1 in all detection scenarios .Step 1: ImageJ is being used for calculating the red and blue decay areas for the test line, then the ratio of red to blue decay areas was calculated and defined as ratio_cal.Step 2: Since pLDH is present in all positive clinical samples, assume the sample contains pLDH only and correlate the red decay areas measured in Step 1 with pLDH concentrations, with the calibration curve in Fig. Step 3: Find the value of the ratio of red to blue decay areas (y-axis) on the top blue curve in Fig. P. falciparum, but malaria positive . In this scenario, the estimated pLDH concentrations from Step 2 are valid since the samples contain pLDH only; (2) If ratio_cal < ratio_ref, then the sample falls into the region between the top blue curve and the bottom red curve in Fig. P. falciparum positive. In this scenario, the estimated pLDH concentrations from Step 2 are not valid since the samples contain pLDH and PfHRP2, and the calibration curve in Fig. Step 4: will consist of two scenarios: (1) If ratio_cal ≥ ratio_ref, then the sample falls into the region above the top blue curve in Fig. For the 15 negative samples tested, the colour intensities are below the LoD for both pLDH and PfHRP2 and therefore are regarded as malaria negative. To distinguish infection types and estimate antigen concentrations for the 10 malaria positive samples, colour discrimination was conducted with the RGB values from ImageJ analysis. Since pLDH is pan-specific and binds to all malaria species, the presence of pLDH can be expected in all malaria positive samples. The pLDH concentration can be estimated by its corresponding red decay areas with the calibration curve in Fig. P. falciparum positive and P. vivax positive samples were presented in Tables The red and blue decay areas and colour ratios of the P. falciparum positive by microscopy examination. Using the four-step trial and error method illustrated above, the red to blue reference ratio (ratio_ref) was estimated at the corresponding red decay area by the pLDH calibration curves and compared with the red to blue ratio of clinical samples . Since ratio_cal < ratio_ref for all the 5 samples listed in Table P. falciparum positive. For example, sample No. 25 is confirmed as P. falciparum positive by microscopy. The red decay area and blue decay area at test line are 917.11 and 499.20, respectively. The red to blue ratio of sample No. 25  is therefore 1.84. The red to blue reference ratio at the corresponding red decay area (917.11) (ratio_ref) is calculated by the pLDH calibration curve in Fig. P. falciparum positive by LFA colour discrimination. This agrees with both microscopy and ELISA results.As shown in Table P. vivax positive by microscopy examination. Similarly, ratio_ref and ratio_cal were obtained. Since ratio_cal > ratio_ref for all the 5 samples listed in Table P. falciparum. In this case, the pLDH calibration curve in Fig. P. vivax positive by microscopy. The red decay area and blue decay area at test line are 631.10 and 299.22, respectively. The red to blue ratio of sample No. 471  is therefore 2.11. The red to blue reference ratio at the corresponding red decay area (631.10) (ratio_ref) is calculated by the pLDH calibration curve in Fig. P. falciparum negative by LFA colour discrimination. This agrees with both microscopy and ELISA results.As shown in Table −1, respectively, since 3.35 ng mL−1 is already beyond the LoD of LFA for PfHRP2 detection.For all the samples in Table The multiplexed LFA that employs two different colours of latex particles for detecting pLDH and PfHRP2 simultaneously at a single test line was demonstrated. The colour developed at the test regions varies from antigen types and concentrations. The assay was capable of distinguishing malaria positive from malaria negative samples. A colour discrimination protocol was also developed for discriminating malaria species if the assay was proved malaria positive.−1 for both pLDH and PfHRP2, and this performance was similar to that of conventional gold nanoparticle LFAs [The assay was first validated by testing recombinant malaria antigens in washing buffer. Based on the buffer test, the LoD for the assay to differentiate both pLDH and PfHRP2 from blank samples was defined, to determine whether a sample is malaria positive or negative. The LoD of the LFA was 31.2 ng mLcle LFAs .P. falciparum infection from the other three human malaria species , for malaria positive samples. The limit of colour discrimination of the assay in buffer test was 7.8 ng mL−1.The limit of colour discrimination to differentiate pLDH from PfHRP2 was also defined, to differentiate P. falciparum positive, 5 samples showed P. falciparum negative but contained other malaria species, and 15 samples showed malaria negative, which agreed with ELISA and microscopy results. The pLDH concentrations for P. vivax positive clinical samples were also quantified and compared with ELISA results. The discrepancies could be attributed to the differences in standard curves for buffer and whole blood samples.Twenty-five malaria clinical samples in whole blood were tested and the assay performance was validated by ELISA and microscopy results. For the clinical samples tested, 5 samples showed P. falciparum infection from the other three human malaria species was developed, with a single test line on the strips with pre-coded colours for different analytes. Co-infections of different malaria species, though uncommon, need to be further examined to distinguish from the single infection of P. falciparum. For the practical use in peripheral settings, the image analysis algorithm can be implemented in the image reader [A two-colour LFA to differentiate e reader , 22. It


Dear Editor: We read with interest the paper by Sonden et al. [Plasmodium vivax cases diagnosed in Europe during the years 2014 and 2015, mainly due to newly arrived Eritrean migrants. Using the GeoSentinel surveillance network data, we recently published similar trends with regards to P. vivax in Eritrean migrants presenting in Europe but also in Israel [P. vivax infections was 84.2% (123/146), followed by P. falciparum , which does not reflect the local epidemiology in Eritrea, where there is a large predominance of P. falciparum reported [P. ovale  and one case of P. malariae  when the proportion of these two species is 0.02% of all malaria cases in studies conducted in Eritrea [P. vivax/P. falciparum infections or unknown species. Among 100 patients with information available, 69 (69%) presented with symptoms after arrival and the median time between arrival in the host country and presentation was 39 days; 31/100 (31%) of migrants reported malaria symptoms while in transit. Among P. vivax infections, we also reported 4% (5/123) cases of severe P. vivax malaria.n et al.  confirmi 84.2% 12/146, folP. vivax likely occurred somewhere underway, possibly in Sudan or Ethiopia, or perhaps even in camps in Libya, a country that is currently considered malaria-free; this question is of medical and epidemiological importance. Competent malaria vectors are present in Libya and as camps are populated with migrants, some of whom may be infected by Plasmodium spp. including P. vivax, the possibility that malaria may be transmitted in such camps should be considered – and investigated by entomological surveys, despite the challenge that it may represent.This situation raises several important questions – where do the Eritrean migrants get infected with malaria on their migration route? Our study  proposedEarly diagnosis and correct management of malaria in Eritrean migrants is necessary to avoid severe disease and the potential reintroduction of malaria in receptive European areas. Primaquine treatment i.e. 15(−30) mg base per day for 14 days, is indicated to prevent relapses by hypnozoite elimination; it is considered standard of care to test for G6PD deficiency in all persons before use of primaquine. We conducted a survey among GeoSentinel clinics regarding availability of primaquine and G6PDH deficiency testing. Our results were that in several European countries, primaquine is only available through hospital pharmacies or through an international pharmacy with delays in procurement and results of testing may take days (1-14 days) . Point oBased on published literature , the inf


However, the evidence is inconsistent, provided from different regions, and insufficient to perform an aggregated analysis such as a meta-analysis. In addition, few candidate gene, genome-wide association or epistasis studies, which have been made possible with the use of big data in the post-genomic era, have investigated HbE. The biological pathways linking HbE and malaria infection have not yet been fully elucidated. Therefore, further research is necessary before it can be concluded that there was positive natural selection for HbE due to protection against malaria.It is known that there has been positive natural selection for hemoglobin S and C in humans despite negative health effects, due to its role in malaria resistance. However, it is not well understood, if there has been natural selection for hemoglobin E (HbE), which is a common variant in Southeast Asia. Therefore, we reviewed previous studies and discussed the potential role of natural selection in the prevalence of HbE. Our review shows that Lay summary: Our review shows that evidence largely supports an involvement of natural selection in the evolution of HbE and a protective role of HbE against malaria. However, the evidence is not consistent. Further research is necessary before it is concluded. The evolutionary connection between malaria and hemoglobin (Hb) variants was proposed in the 1940s by J.B.S. Haldane . It posiPlasmodium, which spread through infected anopheline mosquito [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi [P. falciparum is the most dominant species (66%) followed by P. vivax (34%) [P. falciparum is responsible for almost all malaria cases (99.7%), whereas P. vivax is rare (0.3%) [P. falciparum) include coma, metabolic acidosis, anemia, hypoglycemia, acute renal failure and acute pulmonary edema [In 2016, 216 million people were estimated to be infected with malaria, and 445 000 people were estimated to have died from malaria in the world . Southeamosquito . There aknowlesi . In Soutax (34%) . In Afriia cases 9.7%, whery edema . If a sery edema . It has ry edema .HBA1 and HBA2) and one β-globin gene (HBB) [Human hemoglobin is formed by two alpha-globin and two beta-globin chains which are encoded by two α-globin genes (ne (HBB) . Hemoglone (HBB) . Quantitne (HBB) . These ane (HBB) . Some stne (HBB) , while sne (HBB) .HBB gene variants, which exhibit balanced polymorphism, where a disadvantage of a homozygous status is offset by a heterozygous advantage against malaria [Humans have evolved several polymorphisms in human genomes and some, like glucose-6-phosphate dehydrogenase (G6PD) deficiency, are under selection by malaria to protect against malaria infection . Another malaria . Erythro malaria   analysis and epidemiologic studies including a genetic association study.We have identified four major potential mechanisms explaining how HbE can protect against malaria.in vitro studies that investigate the connection between HbE and malaria resistance. Chotivanich et al. performed a mixed erythrocyte invasion assay and calculated the susceptibility ratio (SR), which is denoted as (stained infected RBCs/stained RBCs)/(unstained infected RBCs/unstained RBCs). Median SR of HbAA = 0.88 (range = 0.53–1.4) > Median HbEE = 0.69 (range = 0.13–0.98) > Median HbAE = 0.34 (range = 0.05–0.64) [in vitro studies [P. falciparum [et al. which had samples of HbAA (n = 34), HbAE (n = 20) and HbEE (n = 22). There are two important considerations for interpreting in vitro studies such as a mixed erythrocyte invasion assay. First, if the malaria strains used for the in vitro studies are different from those prevalent in the Southeast Asian region for the past thousands of years, they can easily produce false negative results. Second, the results of in vitro studies do not directly represent what occurs in vivo.First, reduced probability of parasite merozoite invasion into the RBCs can provide resistance to malaria . In othe05–0.64) . This fi studies , 60. On lciparum . Howeverlciparum  such as P. falciparum was ameliorated in the presence of HbE [P. vivax asexual parasite densities in HbAE heterozygotes compared with HbAA P. vivax patients (P < 0.05) [Second, after the entry of malaria into an erythrocyte, remodeling the host cell structure and the growth of the parasite are important for the next steps of replication and egress . Therefoe of HbE , and othe of HbE . In addi < 0.05) . However < 0.05) .Plasmodium parasites showed decreased deformability [Third, an alteration in the morphology of erythrocytes in response to parasite infection plays an important role in the removal of infected cells . Erythromability , resultimability , 72, whimability . One notmability .P. falciparum-infected erythrocytes such as excrescences, knobs [Fourth, infected HbE cells are more susceptible to phagocytosis by monocytes . The surs, knobs , new ants, knobs , modifics, knobs , membrans, knobs  and binds, knobs  were sugs, knobs . One recs, knobs .Lastly, there are several other suggested protective mechanisms. For example, HbE is associated with high antimalarial antibody titers , 80. In In sum, we have identified and summarized various biological mechanisms of HbE. However, a list of the potential mechanisms is not sufficient to discuss the involvement of natural selection in the evolution of HbE. It has not been fully examined which mechanism can make a difference in human survival and reproduction or which mechanism makes HbE advantageous in an environment.LD is a non-random association of certain alleles that are coinherited with a higher probability than predicted by random chance within a population, and this can be due to selection or founder effects , 87. Theet al. [P. falciparum malaria. All subjects were recruited from the Suan Phueng District in the Ratchaburi Province , which is 200 km west of the Bangkok metropolitan area and near the border with Myanmar. Additionally, they performed a statistical test for detecting reduced haplotype diversity and ran forward in time computer simulations under a variety of selection models [et al.’ study with other populations.Ohashi et al.  performen models . The stun models . The stuAs stated in the prior section, different human populations could have developed the same or similar hemoglobin mutations despite different origins: there are five haplotypes of HbE with three Southeast Asian and two European origins , and at et al.’s study using the Thai population used a robust study design and provided clear evidence, a single study using a single population is not sufficient to draw a conclusion. A larger number of evolutionary genetics studies using various relevant populations from the malaria-affected regions must be conducted to confirm the findings of the Ohashi et al.’s study.In sum, the evolutionary genetics studies such as an LD analysis have potential to provide strong evidence supporting natural selection for HbE. Although the Ohashi et al. [P. falciparum infection. This analysis needs to be carefully interpreted as one case study showed a strong protective effect, while the other showed a null effect. There is a large heterogeneity within the small-scale meta-analysis [Taylor et al.  showed uet al. , 89 thatanalysis . Given tanalysis , 91, theP. falciparum infection  can be more relevant. Kar et al. [P. falciparum parasitemia in HbAE/EE (4.3%) compared with AA (12.2%)  (P < 0.001). Their results are also partially supported by a recent study, Shannon et al.  [P. falciparum. One recent study reported that groups with HbAE (P = 0.006) and HbEE (P = 0.004) had significantly lower odds of presenting with acute P. vivax infections using the HbAA group as the reference  [et al. pointed out that the time frame of HbE origination (between 1240 and 4440 years ago) [Studies examining an association of HbE status with the presence of r et al.  reportedgladesh)  which sh border) . Additio border) . Recentl border) , which i border) . In addiars ago) .0-thalassemia [Genetic association studies, including genome-wide association studies (GWAS), are another significant way to explain host genetics and parasite interactions and genetic correlations with common diseases , 99. Sevlassemia , 102.P. falciparum, whereas Southeast Asian populations also suffer from P. vivax. Future studies should investigate the influence of P. vivax and include more data from Southeast Asian populations.Epistatic interactions, where the presence of one gene influences the phenotypic expression of another gene , are expIn sum, epidemiologic studies have repeatedly shown a positive association of HbEE or HbAE with reduced malaria cases. However, the evidence is inconsistent and reported from different countries and regions. Available data is not sufficient to perform an aggregated analysis such as a standard meta-analysis and a meta-regression. Furthermore, there are variations in the study designs and the reported outcomes of the above-mentioned studies. Genetic association studies including a SNP for HbE have not been often implemented.Based on the literature review, we conclude that HbE seems to be a good candidate for fulfilling the malaria hypothesis , 49, 106et al.’ study [Therefore, readers should be careful regarding the interpretation. While the current research suggests that HbE is a good candidate for fulfilling the malaria hypothesis, richer data with more robust statistics are necessary before drawing a conclusion. Specifically, future research should look for more evidence of a selective sweep near the HbE mutation. Ohashi .’ study  only looSince the ongoing 1000 Genomes Project includes two potentially relevant populations: Yunnan, China (CDX) and Ho Chi Minh, Vietnam (KHV), this can be an important data source for future HbE research . Other dP. vivax, while HbE carriers are mainly populated in the regions where both P. vivax and P. falciparum are endemic.Furthermore, few genetic studies, including genetic association studies and epistasis studies, have focused on HbE or populations where HbE is common. For example, one multi-country study  had beenIt appears that there is a research gap in the malaria and hemoglobin literature. One of the potential reasons why research on HbS and HbC have been well-funded and supported, while HbE is not, is that the US government set a priority on researching sickle cell disease in response to pressure by African American advocacy groups starting in 1971, which led to a large allocation of funds for studying sickle cell diseases . HoweverP. falciparum [P. vivax [Although the present article and many of the cited references focus on the association of HbE, malaria and natural selection in Southeast Asia, new trends have occurred during the past century—migration and urbanization. These may change the evolutionary consequences of HbE. First, there has been a significant Asian migration and Asian births in the USA, many of whom were HbE carriers . In the lciparum , 119 andP. vivax  endemiciBoth migration and urbanization can alter the level of fitness of HbE in particular environments, which depends on the benefit of protection against malaria and negative health consequences of HbE. Quantifying the influence of migration and urbanization can be one of the directions of future research. Even if it is true that natural selection favors HbE in particularly malaria-rich regions and populations, this may not be the case in other regions and populations, especially in urbanized regions and migrated populations.


Plasmodium spp. in deer since its first identification, recent research has rediscovered Plasmodium on ungulates in Africa, Asia, North America and South America, including Central Brazil. Here, a captive herd was evaluated in southern Brazil using light microscopy and PCR. DNA samples were tested for fragment amplification of two Plasmodium spp. genes: mitochondrial cytochrome b and small subunit ribosomal RNA.Despite malaria epidemiology has been extensively studied in primates, few studies were conducted in ungulates. After half a century without descriptions of Plasmodium sp. infection. Further monitoring and/or multiple sequential sampling may improve the success rate of detecting parasites. Moreover, although this survey of Plasmodium represents the first molecular study on ungulate malaria parasites from Southern Brazil, further analysis of samples from different ungulate species is important for characterizing the epidemiology of Plasmodium of these mammals in this region.All analyses were negative. However, the tests were performed on samples that were collected at a single time point, and parasitemia may fluctuate over the parasite’s life cycle. Thus, the possibility of occult infection cannot be ruled out. Despite the negative results of all of the methods applied, it cannot be categorically stated that these animals are free from  Plasmodium species had been thought to be limited to the Old World. In particular, cervids had not been considered to be a vertebrate host due to the absence of parasites in blood smear investigations [Plasmodium parasite (P. odocoilei) in a blood smear from a splenectomized white-tailed deer (Odocoileus virginianus) in Texas, United States of America (USA) [Malaria parasites have been described in a wide range of hosts in the Americas, including humans , monkeysigations –8. This ca (USA) , 9.Plasmodium parasites, the cross-continental dispersion of these parasites and the role of ungulates as malaria reservoirs. Specifically, the recent identification of Plasmodium-positive deer in Central Brazil [Surprisingly, recent reports have “rediscovered” malaria parasites in cervids and other ungulates throughout the world –14. ThesMazama nana) is protected in the Bela Vista Biological Sanctuary (BVBS), Foz do Iguaçu, Brazil. Red brocket deer (M. americana) and marsh deer (Blastocerus dichotomous) are also present. BVBS  is a zoo and animal rehabilitation center located in a national protected area in southern Brazil and shares borders with Argentina and Paraguay. Cervids are kept in fenced areas that are covered and surrounded by native vegetation, making contact with free-living wild animals possible. Capybaras (Hydrochoerus hydrochaeris) are often seen moving freely through zoo areas. Our research group first reported Plasmodium sp. infection in capybaras that were captive animals at the BVBS, with findings of infection in 1/11 via microscopy and 3/11 via molecular testing [A large herd of captive Brazilian dwarf brocket deer  positive for Neospora caninum and 1/32 (3.1%) positive for Leptospira interrogans. Additionally, all animals tested negative for Brucella abortus, bovine viral diarrhea virus, foot-and-mouth disease virus, infectious bovine rhinotracheitis, Eastern equine encephalitis, Western equine encephalitis and Venezuelan equine encephalitis [Plasmodium species.In Brazil, the Brazilian dwarf brocket deer, red brocket deer and marsh deer have been screened for pathogens, with 27/31 (87%) infected by sma ovis , 5/32  that were previously surveyed for other pathogens [Blood samples from 32 cervids  under the Protocol Number 22.158-2  and was conducted in accordance with IBAMA’s ethical rules.Peripheral blood smears were stained with May-Grünwald-Giemsa stain and were examined using optical microscopy at ×1000  .cytb) gene [Plasmodium spp. was performed using two previously described protocols: one targeting the small subunit RNA gene (SSU rRNA) [cytb) of the parasite [Plasmodium amplification, and the species of Plasmodium is determined by the similarity to the genomic sequences deposited in GenBank. The primers DW2 and DW4, used in Plasmodium cytb gene amplification [Plasmodium screening from ungulates [DNA was extracted using a commercial kit , in accordance with the manufacturer’s instructions. DNA quality was assessed through amplification of the mitochondrial cytochrome b (tb) gene , using tSU rRNA) , 24 and parasite . Both PCfication , have alngulates –12, 14. Plasmodium-like parasites were observed through direct examination of deer blood smears, and no DNA amplification was detected by means of PCR for either the SSU rRNA or the cytb gene  [Plasmodium infection, despite the negative results obtained in the present study.No ene Fig. . Howeverbubalis) . TherefoPlasmodium sp. Conversely, previous studies have failed to detect Plasmodium in other ungulate species, including elk (Cervus canadensis), pronghorn (Antilocapra americana) and mule deer (Odocoileus hemionus) [Tragelaphus spekii), red river hog (Potamochoerus porcus) and water chevrotain (Hyemoschus aquaticus), also did not detect Plasmodium sp. [Ozotoceros bezoarticus) samples from Central Brazil [Mazama gouazoubira) and marsh deer (B. dichotomus) were negative for Plasmodium species [Nested PCR-based screening of white-tailed deer in 45 counties in the USA  found that 41/308 (13.3%) of the animals were infected by emionus) . Additiodium sp. . More rel Brazil . However species , indicatPlasmodium spp. divergence have shown that the clade including P. odocoilei is likely to have diverged from other clades between 2.3 and 6 million years ago [Plasmodium is an ancient parasite of deer. Hence, co-evolution of both species could have made deer a well-adapted host for the parasite. Other previous investigations on Plasmodium spp. in deer failed to detect the parasite [Plasmodium species have been discovered only after a host has been splenectomized [Recent molecular clock estimates of ears ago , thus suparasite , and somctomized .The global health and ecological impact of malaria in wild animals is still unknown. Therefore, active surveillance providing epidemiological information regarding health status, mortality rates and geographic distribution of malaria infection in domestic and wild ungulates, both free-living and captive animals, including deer, is important to develop strategies for the management and control of malaria infection, thus improving the health and wellbeing of these animals.Plasmodium sp. infection. Further monitoring and/or multiple sequential sampling may improve the success rate of detecting parasites. Moreover, although this survey of Plasmodium represents the first molecular study on ungulate malaria parasites from Southern Brazil, further analysis of samples from different ungulate species is important for characterizing the epidemiology of Plasmodium of these mammals in this region.The tests in this study were performed on samples that were collected at a single time point. Low parasitemia has been previously described for infected ungulates, and parasitemia may fluctuate over the parasite’s life cycle. Thus, the potential parasite life cycle at the time of blood collection may have had a direct impact on the negative findings. As the possibility of occult infection cannot be ruled out, despite the negative results of all of the methods applied, it cannot be categorically stated that these animals are free from


Plasmodium falciparum malaria. However, these methods lack sensitivity to detect low parasitaemia. Polymerase chain reaction (PCR), on the other hand, enhances the detection of sub-microscopic parasitaemia making it a much-needed tool for epidemiological surveys, mass screening, and the assessment of interventions for malaria elimination. Therefore, this study sought to determine the frequency of cases missed by traditional methods that are detected by PCR.Accurate diagnosis of malaria is important for effective disease management and control. In Cameroon, presumptive clinical diagnosis, thick-film microscopy (TFM), and rapid diagnostic tests (RDT) are commonly used to diagnose cases of P. falciparum by microscopy, RDT and PCR. The hospital records of participants were reviewed to obtain data on the clinical diagnosis made by the health care worker.Blood samples, collected from 551 febrile Cameroonian patients between February 2014 and February 2015, were tested for The prevalence of malaria by microscopy, RDT and PCR was 31%, 45%, and 54%, respectively. However, of the 92% of participants diagnosed as having clinical cases of malaria by the health care worker, 38% were malaria-negative by PCR. PCR detected 23% and 12% more malaria infections than microscopy and RDT, respectively. A total of 128 (23%) individuals had sub-microscopic infections in the study population. The sensitivity of microscopy, RDT, and clinical diagnosis was 57%, 78% and 100%; the specificity was 99%, 94%, and 17%; the positive predictive values were 99%, 94%, and 59%; the negative predictive values were 66%, 78%, and 100%, respectively. Thus, 41% of the participants clinically diagnosed as having malaria had fever caused by other pathogens.Malaria diagnostic methods, such as TFM and RDT missed 12–23% of malaria cases detected by PCR. Therefore, traditional diagnostic approaches  are not adequate when accurate epidemiological data are needed for monitoring malaria control and elimination interventions. Malaria remains a major public health threat, particularly in sub-Saharan Africa, where about 191 million new infections and 395,000 deaths were reported in 2015 [Plasmodium parasite and requires a competent microscopist. Also, the presence of sub-microscopic parasitaemia greatly reduces the accuracy of malaria diagnosis by TFM. Unlike TFM, RDT detect malaria antigens, not malaria parasites, which gives them an added advantage in the ability to diagnose malaria in patients with low-grade parasitaemia below the detection limit of TFM [P. falciparum, is limited when the parasite is cleared and antigens remain in circulation for about 28 days  [The traditional practice by health care workers (HCW) in malaria-endemic countries has been to diagnose malaria based on a history of fever  –6. The st of TFM , 8. HoweAs malaria-endemic countries move towards malaria elimination, there is a need for rapid and accurate diagnostic tools for malaria. Active monitoring of the performance of various diagnostic methods for malaria at the country level is necessary to guide policy on the diagnostic methods to use for malaria diagnosis and elimination. In this study, the performance of RDT, TFM and PCR in the diagnosis of malaria in Cameroon was compared in order to illustrate the number of cases missed by traditional methods  of diagnosis.This study was conducted in three Regions of Cameroon, Far North, Centre and North West with varied climatic conditions and altitudes Table . In the A cross-sectional study was conducted in selected health facilities in Maroua, Nkolbisson, and Bamenda. Inclusion criteria were age > 6 months and axillary temperature >37.5 °C at the time of recruitment or fever within 24 h preceding recruitment. A written informed consent was obtained from all study participants ≥ 18 years of age. Parents or legal guardians of children < 18 years gave a written informed consent on behalf of their children.Ethical approvals were obtained from the Committee on Human Subjects of the University of Hawaii (protocol number CHS 21724) and the National Research Ethics Committee of the Ministry of Public Health, Cameroon (protocol number 2014/04/442/CE/CNERSH/SP). Administrative approvals were obtained from the Ministry of Public Health, Cameroon and the Directors of the participating facilities.An easy-to-read questionnaire was used for the collection of demographic and clinical data. After obtaining informed consent, the research or clinic staff took axillary temperatures and recorded the reported signs and symptoms. Venous blood, 2–5 mL, was collected from each participant, dispensed into ethylenediaminetetraacetic acid (EDTA) tubes and stored in cold boxes until transported to the research laboratory where they were stored at 4–8 °C. TFM and RDT for malaria were conducted for all participants, and both results were presented to the consulting health care worker (HCW). At the end of the hospital visit, an exit survey was conducted to obtain information on (1) the clinical diagnosis made by the HCW, and (2) determine if anti-malarial drugs had been prescribed. This information was used to determine how frequent anti-malarial drugs were used to treat malaria-negative patients. When exit survey information was not available, the hospital record was consulted for the information.P. falciparum parasite was identified. Parasitaemia was determined by counting the number of parasites per 200 white blood cells and assuming that each subject had 8000 white blood cells/μL of blood. Two readings were conducted for each slide and discrepancies greater than 10% were resolved by a third reading by an independent trained microscopist. The slides for this study were read by well-trained and experienced scientists who have worked in malaria-research for a number of years.Thick blood films were prepared and stained using 10% Giemsa for 15 min. A slide was considered positive if at least one asexual blood-stage P. falciparum HRP-II and Plasmodium lactate dehydrogenase, which is a glycolytic enzyme common to P. falciparum, Plasmodium ovale, Plasmodium vivax and Plasmodium malariae asexual-stage parasites.Approximately 5 μL of blood was used to diagnose malaria using the Ag Pf/Pan malaria RDT kit , following the manufacturer’s instructions. This RDT is a qualitative immunochromatographic test that detects P. falciparum, as previously described [P. falciparum visualized on a UV transilluminator after electrophoresis on a 2% agarose gel stained with ethidium bromide.DNA was extracted from 200 μL of whole blood using the mini-prep spin-column technique  following the manufacturer’s instructions. Detection of malaria parasite DNA was based on nested PCR amplification of the 18 s rRNA gene in a reaction that used 2 μL of the extracted DNA, 10 μL of GoTaq polymerase and master mixes , 0.25 μM each of upstream and downstream primers, and 6 μL of nuclease free water in a total reaction volume of 20 μL. The first PCR encompassed genus-specific primers and the second nested PCR run encompassed the species-specific primers for escribed . The prekappa value) were calculated with confidence intervals by age groups. Multivariable logistic regression was conducted to identify correct diagnosis comparing other test methods (TFM and RDT) to PCR. P < 0.05 was considered statistically significant. All the other analyses were conducted using SAS version 9.4 . The figure was generated using R version 3.4.2.Data were entered into Microsoft Office Excel and analysed using StatPlus 5.9.80  and Prism 6.0  for descriptive statistics. Diagnostic test performance for clinical diagnosis, TFM and RDT for the diagnosis of malaria was analysed using MedCalc 16.8 . Descriptive statistics are represented as frequencies and medians. Sensitivity, specificity, positive and negative predictive values, accuracy and percentage of agreement  in this age group in Maroua, and 180 (57%) in Nkolbisson. In Bamenda however, 69 (61%) of the study participants were over 17 years of age  of the participants were diagnosed with clinical malaria (temperature range 36.5 to 39.7 °C), of which 41% (209/551) were negative for malaria by PCR  positive by PCR but negative by microscopy, ii) positive by PCR but negative by RDT, and iii) positive by RDT but negative by TFM. A total of 128 (23%) febrile patients were positive for P. falciparum by PCR, but negative by TFM  patients were positive for malaria by RDT but negative by PCR. This shows that, 23% of malaria cases were missed by TFM while RDT detected 15% more malaria infections compared to TFM but failed to detect 12% detected by PCR.Sub-microscopic  of 128 2% febrileAcross the different age-groups, RDT and PCR detected more malaria infections as compared to TFM Fig. . MoreoveAlso, across the different age-groups, the sensitivity and specificity of TFM and RDT were similar Table . This meUsing PCR as reference standard, 298 participants were positive for malaria while 253 participants were negative for malaria  are not adequate when accurate epidemiological data are needed to monitor malaria control and elimination interventions. PCR permitted the detection of 23% of malaria cases missed by TFM and RDT further, confirming it as a valuable tool for epidemiological surveys, mass screening, and the assessment of interventions for malaria elimination. Therefore, the development and standardization of a rapid and sensitive molecular-based test capable of detecting sub-microscopic malaria infection are warranted for the global elimination of malaria. Furthermore, continual training and proficiency testing should be instituted for laboratory technicians on malaria microscopy and post-market surveillance to assure the quality of malaria RDT since high-performance microscopy and quality assured RDTs will suffice for the clinical management of patients with suspected malaria.


Plasmodium falciparum may evade complement-mediated host defense by hijacking complement Factor H (FH), a negative regulator of the alternative complement pathway. Plasma levels of FH vary between individuals and may therefore influence malaria susceptibility and severity.P. falciparum malaria and in 173 healthy control children. We compared FH plasma levels between children with malaria and healthy controls, and between children with severe (n = 82) and uncomplicated malaria (n = 67). We determined associations between FH plasma levels and laboratory features of severity and used multivariate analyses to examine associations with FH when accounting for other determinants of severity.We measured convalescent FH plasma levels in 149 Gambian children who had recovered from uncomplicated or severe P < .0001). FH plasma levels correlated with severity biomarkers, including lactate, parasitemia, and parasite density, but did not correlate with levels of PfHRP2, which represent the total body parasite load. Associations with severity and lactate remained significant when adjusting for age and parasite load.FH plasma levels differed significantly between controls, uncomplicated malaria cases, and severe malaria cases (mean [95% confidence interval], 257 [250 to 264], 288 [268 to 309], and 328 [313 to 344] µg/mL, respectively; analysis of variance P. falciparum might be useful for malaria prevention or treatment.Natural variation in FH plasma levels is associated with malaria susceptibility and severity. A prospective study will be needed to strengthen evidence for causation, but our findings suggest that interfering with FH binding by  Plasmodium falciparum infections account for most of this burden, and most attributable deaths occur in young children in sub-Saharan Africa. Three clinical syndromes of severe malaria, occurring alone or in combination, predict the majority of deaths in children: cerebral malaria (characterized by coma), severe anemia, and respiratory distress  [Malaria accounts for a huge global burden of disease. tatemia) . Why somP. falciparum is vulnerable to complement-mediated defenses when parasites exit host cells to invade new erythrocytes or undergo sexual reproduction [The complement system is activated as part of the innate immune response to malaria . P. falcoduction . Rapid aoduction . To prevoduction .Neisseria meningitidis, for which high FH plasma levels increase susceptibility to invasive disease [P. falciparum can also recruit FH to its surface, protecting parasites from complement and raising the possibility that variation in plasma levels of FH may also influence outcomes in malaria [FH plays an essential role in regulating complement activity, and its genetic deficiency results in atypical hemolytic uremic syndrome . However disease , 5. P. f malaria .P. falciparum malaria as a proxy for the steady-state situation preceding infection. We compared levels with healthy community controls and between those who had suffered uncomplicated vs severe malaria.Although in vitro studies on the mechanisms of FH binding to the malaria parasite demonstrate a plausible mechanism of evading host defense, clinical studies are needed to determine the relevance to disease outcomes. As it has previously been shown that complement activation levels return to normal within a month after a malaria episode , we deteP. falciparum malaria with a parasite density >5000/µL [Subjects were Gambian children (<16 years old) recruited from the Greater Banjul region, where malaria transmission is seasonal and relatively low . Convale>5000/µL . Severe >5000/µL . SamplesEnzyme-linked immunosorbent assays (ELISAs) to measure Factor H and PfHRP2 were performed as previously described , 15.t test or 1-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Correlations were assessed using a nonparametric Spearman’s correlation test. Logistic and linear regression models were developed using the GLM function in R. The best model was assessed by the Akaike Information Criterion.Analysis was performed with GraphPad Prism v7.02 . Differences in mean values between groups were assessed by unpaired rs = –.034, P = .54), as was expected based on a previous study in healthy children [To determine whether FH levels may associate with malaria susceptibility and severity, we measured FH in convalescent samples from children with uncomplicated (n = 67) and severe (n = 82) malaria and in healthy community controls (n = 173). Because severe malaria is more common in younger children we first tested whether FH levels were associated with age and found that they were not significantly correlated   . This inSevere malaria can manifest in several overlapping syndromes; therefore, we examined whether the higher levels of FH in those with severe malaria were driven by any particular clinical phenotype. FH levels were similar in all major severe malaria phenotypes and were all significantly higher than in uncomplicated malaria, with the exception of the small group with SA  .To further investigate the association between FH and severity, we assessed relationships between convalescent FH and several host- and parasite-derived markers measured on admission. Lactate concentrations correlated significantly with FH levels, but platelets and hemoglobin did not . MarkersMeasures of circulating parasite load do not quantify additional cyto-adherent parasites sequestered in the tissue microvasculature. Total body parasite load can be assessed using the plasma concentration of the soluble parasite protein PfHRP2, which reflects both sequestered and circulating parasite numbers and is more predictive of clinical outcome of infection , 17. SurIn logistic regression models, age, PfHRP2, and FH all predicted severity, and a multivariate model incorporating all 3 indicated that that they have independent associations with outcome . Linear Despite the huge evolutionary pressure malaria has exerted, surprisingly few host factors have been convincingly shown to influence susceptibility or severity. Of the host factors with the strongest evidence, almost all involve the erythrocyte . Our resP. falciparum infection, but not all of these infections will result in symptomatic malaria. The likelihood of an infection causing symptomatic disease is dependent on parasite density [All children in malaria-endemic countries are potentially susceptible to  density . As we oBy comparing healthy children with those who had suffered a definite malaria episode, we expected that even relatively small differences in FH between the groups would indicate an association between FH and susceptibility to symptomatic malaria: the outcome of a new infection in the controls could be either asymptomatic or symptomatic, whereas our malaria cases are all symptomatic. Thus our results indicate that higher levels of FH are associated with susceptibility to symptomatic malaria. Among subjects with malaria, the associations of FH with clinical features of severe disease, lactate concentration and circulating parasite load, are consistent with the hypothesis that higher levels of FH also predispose to severe malaria. Indeed, multivariate models suggested that the association of FH with severity was independent of both age and parasite load.Although high FH levels may allow parasites to evade complement-mediated clearance, high levels may also limit endothelial activation and expression of adhesion molecules involved in sequestration of parasitized erythrocytes . If highAlthough total body parasite load is a strong predictor of severity, it is not the only determinant . The rapThe main limitation of our study is that we used convalescent plasma samples, which we believe are representative of the pre-infection status of our subjects. We cannot exclude that differences in FH levels could represent a residual effect of infection, although we think this is unlikely because of the lack of association with PfHRP2 concentrations. A large prospective study would be required to confirm that natural variation in FH levels definitely does predict susceptibility, severity, and mortality from malaria. Further studies will be needed to clarify the relative roles of complement activation and regulation in malaria, but interference with parasite FH binding might be considered as an adjunctive approach to limit parasite growth, enhance parasite clearance, and tackle drug resistance.


Plasmodium falciparum infection. However, other species of Plasmodium  can also cause malaria. Therefore, accurate identification of malaria species is crucial for patient management and epidemiological surveillance. This study aimed to determine the different Plasmodium species causing malaria in children under 5 years old in two provinces (Kinshasa and North Kivu) of the Democratic Republic of Congo (DRC).Worldwide, the highest malaria mortality is due to Plasmodium species identification.From October to December 2015, a health-facility based cross-sectional study was conducted in General Reference Hospitals in Kinshasa and North Kivu. Four hundred and seven blood samples were collected from febrile children aged ≤ 5 years. Nested polymerase chain reaction assays were performed for Plasmodium spp. and P. falciparum was the most prevalent species (99.2%). Among those infected children, 124 had a mono infection with P. falciparum and one with P. malariae. Mixed infections with P. falciparum/P. malariae and P. falciparum/P. vivax were observed in 6 (1.5%) and 8 (2.0%) children, respectively. The prevalence of infection was higher in females (64.8%) than in males (35.2%), p < 0.001. The age-specific distribution of infection showed that children of less than 2 years old were less infected (18.4%) compared to those aged above 2 years (81.6%), p < 0.001.Out of 407 children, 142 (34.9%) were infected with P. falciparum, the findings demonstrate the existence of non-falciparum malaria, especially P. malariae and P. vivax among children aged ≤ 5 years living both Kinshasa and North Kivu Provinces in DRC.Although this study showed clearly that the most prevalent species identified was  Plasmodium [Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and Plasmodium knowlesi.Malaria is the major cause of morbidity and mortality in many tropical and sub-tropical countries, with half of the world’s population at risk . Malariaasmodium , among wPlasmodium falciparum is responsible for major morbidity and for mortality due to severe clinically forms [Plasmodium vivax has been reported from time to time in populations in sub-Saharan Africa, especially when polymerase chain reaction (PCR) assays have been used [P. vivax. Plasmodium ovale and P. malariae are relatively common in many parts of East and West Africa causing a significant proportion of febrile malaria episodes [Plasmodium species is subject to change. Since anti-malarial treatments depend of the parasite causing the disease, the monitoring of the distribution of Plasmodium species is essential [P. falciparum represent the main species responsible of morbidity and mortality [Plasmodium species have been reported in DRC, but these observations were based on microscopy, not always appropriate for species distinction.ly forms . Howeverly forms . Despitely forms , 6. Plaseen used , 8, but episodes  and asymssential , 11. In ortality . Others Plasmodium species distribution using sensitive methods, such as molecular techniques, in samples coming from two DRC provinces (Kinshasa and North Kivu).The aim of the present study was to determine Kinshasa Province is an urban area in the western DRC, which hosts up 15% of the DRC’s 79 million people . KinshasNorth Kivu province is located in the eastern DRC. This province is mountainous, including a large portion of the Virunga National Park Fig. . Some paFrom October to December 2015, a cross sectional study was conducted in Kinshasa and North Kivu provinces in the DRC. Febrile children aged ≤ 5 years, seeking care for suspected malaria at the selected General Reference Hospitals, were systematically enrolled, once written consent was obtained and subject eligibility assessed.Blood samples were obtained from finger prick, collected on filter paper (Whatman No 1), dried, stored in individual plastic bags with desiccant and, then transferred to the National Institute for Biomedical Research in Kinshasa for molecular work.DNA extraction from the blood spots on filter paper was carried out according to the protocol of Musapa et al. . BrieflyPlasmodium genus. The PCR mixture had a total volume of 25 µl, containing 1X buffer, 200 μM dNTPs, 10 µM of each primer, 2.5 mM MgCl2, 1 unit of Taq DNA polymerase  and 2 μl of DNA solution. The thermoprofile consisted of an initial denaturation step at 94 °C for 5 min, followed by 25 cycles at 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 1 min, and a final extension step at 72 °C for 5 min. Amplicons were subjected to electrophoresis for approximately 40 min at 100 V on standard 2% agarose gels. The expected size was 1200 bp   Table . The PCRData were entered and analysed using Epi-info 7 and SPSS 21 (Chicago USA), respectively. Descriptive statistics were used to summarize data . Chi square was utilized for proportion comparison. Ninety five (95) % confidence interval and p value set at 0.05 were used for significance.The study was approved by Institutional Ethics Committee of the School of Public Health of Kinshasa/DRC. Written signed informed consent was obtained from the parents or legal tutors of each child before his/her enrollment in the study. Malaria positive cases were treated according to national malaria diagnosis and treatment guidelines .In total, 407 children aged ≤ 5 years old with the clinical suspicion of malaria were enrolled. Of the 407 selected children, 206 (50.6%) were female with sex ratio of 0.765 Table . Median Plasmodium sp. infection. Plasmodium falciparum was the most prevalent species and was found in 141 (99.2%) of the positive samples  than in males (35.2%) (p < 0.001). The age-specific distribution of infection in children showed that children of less than 2 years old were less infected (18.4%) compared to those aged above 2 years (81.6%), p < 0.001.Out of 407 samples analysed, 142 (34.9%) were positive for Plasmodium spp. infection including P. falciparum and P. vivax cases. Among the positive cases, the study revealed 6 (3.3%) mixed infection cases of P. falciparum and P. vivax (Table Kinshasa province reported 75 (41.7%) positive cases for vealed 6 .3% mixedPlasmodium spp. infection including P. falciparum, P. vivax and P. malariae cases with 2 (0.8%) mixed infection cases between P. falciparum + P. vivax and 6 (2.6%) mixed infection cases of P. falciparum + P. malariae  cases were positive for ae Table .Plasmodium falciparum is known to induce more severe infections [P. falciparum is more susceptible to anti-malarial drugs than other species [Malaria is still a major public health concern in most endemic areas in sub-Saharan Africa, especially in the DRC, where 14 million cases have been reported by the WHO in 2014 [fections , but oncPlasmodium could be achieved using the parasitological methods whereby different species of Plasmodium were discriminated by the morphology of the parasite and its position in the red blood cell. However, misinterpretations cannot be ruled out. Thanks to the development of molecular techniques, PCR with species-specific primers can be used with more accuracy [In the past, species characterization of accuracy . These taccuracy , 18 and,P. falciparum as previously reported in Tanzania and DRC [Plasmodium species circulating in children during the same period and in the same area, including the co-circulation of P. malariae and P. vivax. In general, the distribution of P. malariae coincides with that of P. falciparum in malaria endemic areas of Africa, explaining the high frequency of mixed infections of P. malariae with P. falciparum infections [P. falciparum and P. malariae have been found in 1.5% of samples.As expected results from study revealed a high prevalence of  and DRC , 20. HowP. vivax infecting children from DRC (Fig. Plasmodium species in DRC using molecular tools. Although the public health importance of P. vivax is overshadowed by P. falciparum, it has been shown that there is a strong association of P. vivax with certain severe malaria symptoms and it accounts for about 390 million clinical cases annually in Asia and South America [P. vivax is rare in Central Africa but it is known to be endemic in some populations of Sudan, Somalia, Ethiopia, Djibouti [Plasmodium species in this area.The current study revealed also the presence of DRC Fig. . This is America , 22. IndDjibouti . Thus itP. vivax malaria [P. vivax PCR-positive, Duffy-negative individuals have been made [P. vivax has broken through its dependence on the Duffy antigen for establishing human blood-stage infection and disease. In DRC limited studies have been conducted on erythrocyte polymorphisms associated with malaria susceptibility. So, further study will be necessary to investigate the Duffy antigen status among our P. vivax infection to elucidate its role in DRC Malaria cases.Most research on Duffy antigen in malaria cases over the past 20 years has showed that people with erythrocyte Duffy blood group-negative people were unable to develop  malaria , 25. Hoween made . This isP. malariae and P. vivax among children living in DRC should make the determination of the infecting Plasmodium species important in terms of treatment because certain Plasmodium infections can cause rapidly progressive severe illness or death while the other infection of Plasmodium are less likely to cause severe manifestations or have different drug resistance patterns in differing geographic regions [This presence of  regions , 10, 23.P. falciparum, the current study documented the presence of the non-falciparum malaria parasites in Kinshasa and North Kivu Provinces of the DRC. These findings suggest further surveys in other settings of DRC and a regular surveillance of these non-falciparum sp prevalence.Although the highest number of malaria cases involving


Malaysia has declared its aim to eliminate malaria with a goal of achieving zero local transmission by the year 2020. However, targeting the human reservoir of infection, including those with asymptomatic infection is required to achieve malaria elimination. Diagnosing asymptomatic malaria is not as straightforward due to the obvious lack of clinical manifestations and often subpatent level of parasites. Accurate diagnosis of malaria is important for providing realistic estimates of malaria burden and preventing misinformed interventions. Low levels of parasitaemia acts as silent reservoir of transmission thus remains infectious to susceptible mosquito vectors. Hence, the aim of this study is to investigate the prevalence of asymptomatic submicroscopic malaria (SMM) in the District of Belaga, Sarawak.In 2013, a total of 1744 dried blood spots (DBS) were obtained from residents of 8 longhouses who appeared healthy. Subsequently, 251 venous blood samples were collected from residents of 2 localities in 2014 based on the highest number of submicroscopic cases from prior findings. Thin and thick blood films were prepared from blood obtained from all participants in this study. Microscopic examination were carried out on all samples and a nested and nested multiplex PCR were performed on samples collected in 2013 and 2014 respectively.Plasmodium vivax by PCR. Additionally, of the 251 samples, the most prevalent mono-infection detected by PCR was Plasmodium falciparum 50 (20%), followed by P. vivax 39 (16%), P. knowlesi 9 (4%), and mixed infections 20 (8%).No malaria parasites were detected in all the Giemsa-stained blood films. However, of the 1744 samples, 29 (1.7%) were positive for Plasmodium by PCR, among samples previously undetectable by routine blood film microscopic examination, in local ethnic minority who are clinically healthy. SMM in Belaga district is attributed not only to P. vivax, but also to P. falciparum and P. knowlesi. In complementing efforts of programme managers, there is a need to increase surveillance for SMM nationwide to estimate the degree of SMM that warrant measures to block new transmission of malaria.This research findings conclude evidence of  Plasmodium vivax and Plasmodium falciparum are prevalent, in recent years, Plasmodium knowlesi has been recognized as the infective agent for an increasing number of cases, especially in Malaysia. In 2017, the country reported a total of 508 cases  of the human type of malaria, down substantially from 6141 cases in 2010 [P. knowlesi, while P. vivax and P. falciparum accounts for 7% and 4% of total cases, respectively. The remaining 0.7%, 0.4% and 0.1% accounts for P. ovale, P. malariae and mixed infections respectively [Malaysia declared its aim to eliminate malaria with a goal of achieving zero local transmission by the year 2020. However, malaria continues to be a public health burden especially in the interior parts of the country, which accounts for a considerable number of cases treated at health clinics and admissions to hospitals. Although both P. knowlesi). Kapit Division consists of three districts . Malaysia is home to many isolated indigenous tribal groups that do not generally have the same level of access to health care as the rest of the population. Many indigenous people use traditional remedies before seeking care in a health facility, which can delay treatment. Many of these groups live within the forest or forest-fringe areas, where the vector ecology and transmission patterns of malaria present a unique challenge for vector control management [Despite the decline in the number of malaria cases in the country, there have been reports of vivax and falciparum malaria cases in low endemic areas especially in the rural settings in Sarawak . Nonethenagement .Plasmodium transmission, there are individuals with submicroscopic infection present in the population. The relative proportion of submicroscopic and microscopic infections varies between settings, depending on factors such as age, transmission intensity and immunity. In low transmission settings, submicroscopic infections may represent a significant fraction of infections, but the major determinants of the contribution of submicroscopic infections to malaria transmission are not clear. Submicroscopic infections are prevalent both in “stable” low endemic areas and in those areas experiencing recent reductions in transmission. It has previously been reported that in low transmission settings, the proportion of asymptomatic individuals is less than that in areas of greater transmission severity. Nonetheless, even in low transmission areas the asymptomatic cases make up for 60% of the infected population.At all levels of Despite rarely causing clinical disease, submicroscopic malaria infections can contribute to malaria transmission. Experimental evidence has demonstrated that individuals with submicroscopic infections are capable of infecting mosquitoes; while these individuals may infect fewer mosquitoes than individuals with higher parasite counts, the high numbers of individuals with low-density infections may lead them to contribute substantially to malaria transmission . UnderstData on submicroscopic parasitaemia is needed to better understand disease progression and its impact on targets set in the National Malaria Elimination Programme. Current evidence is insufficient for understanding the contribution of low-density SMM infections to onward transmission to human populations. Intervention trials to directly assess the effect of identifying and treating low-density infections are warranted. Presently, limited studies on asymptomatic submicroscopic malaria are carried out in Malaysia. Both parasite and host factors will influence whether infections remain asymptomatic or become symptomatic and potentially life threatening. In order for Malaysia to achieve the national goal of malaria elimination by 2020, it is important to understand that asymptomatic submicroscopic infection plays an important role in malaria transmission and that interventions to target this parasite reservoir may be needed in both low- and high-transmission areas. Currently, there is a lack of information and investigation carried out on the outcome of submicroscopic asymptomatic malaria in the district of Belaga and in this region. Hence, the objective of this study is to investigate the prevalence of submicroscopic malaria among asymptomatic individuals from a low transmission area in the District of Belaga, Sarawak.P. vivax detected during the 2013 outbreak investigation activity. However, the residents from Long Malim and Long Wat were currently relocated to a new Tegulang Resettlement Site. The total population of Long Malim and Long Wat in 2014 was 253 and 321, with an incidence rate of 4.0 and 0 per 1000 population, respectively. In 2014, both longhouses reported zero malaria incidence by microscopic examination. A total of 112 and 139 blood samples in EDTA and blood films were collected from Long Malim and Long Wat, in October 2014 respectively.In January 2013, the Vector Borne Disease Control Programme and Sarawak Department of Health has requested the Parasitology Unit of Institute for Medical Research (IMR) to assist in the detection of suspected SMM during a malaria outbreak investigation carried out in the district of Belaga, in Kapit, Sarawak. Belaga is located in the upper reaches of the Rajang River, some 120 km northeast of Kapit but considerably further on the river and slightly less than 100 km from the South China Sea coast near Bintulu. Belaga is considered a good place to start exploring the Sarawak’s interior. There are many ethnic minorities co-habituating riversides such as the Kenyah and Kayan longhouses along the Balui and Belaga rivers, and along the Rejang Rivers are the Punan, Sekapan, Kejaman and Tanjung longhouses. The total population in Belaga in 2013 was 39,400, with the malaria incidence rate of 2.9 per 1000 population. Subsequently, in 2014 and 2015, the malaria incidence rate was reduced to 2.7 and 1.6 per 1000 population, respectively. Despite continuous efforts to eradicate and eliminate malaria in this district, the infection persists. With the aim to demonstrate the presence of submicroscopic malaria in Belaga, a total number of 1744 dried blood spot samples (DBS) were collected during the routine Active Case Detection (ACD) from 8 localities within the district of Belaga in Sarawak, namely Long Malim, Long Wat, Long Jaik, Long Tanyit, Lusong Laku/SK Lusong Laku, Rh Udau Tedong, Uma Pawa Tr Eric, and Simpang Uma Nyaving  were spotted on 3MM Whatman filter paper , air dried, individually kept in zip-lock plastic bags with desiccants and were sent to the Parasitology Unit Laboratory for DNA extraction from the first investigation. For the second investigation, approximately 1–5 ml of venous blood was drawn in a syringe and transferred to a vacuum tube containing anticoagulant (EDTA). EDTA blood samples were stored in a 4 °C refrigerator until being processed. The remaining blood in the syringe was spotted on 3MM Whatman filter papers, air dried and individually kept in zip-lock plastic bags with desiccants for future use. For each of the finger-prick and venous blood sample, a portion of the blood was utilized directly to prepare thick and thin blood smears for microscopic analysis in both investigations. All individuals and all age groups were invited to participate in the study. Signed informed consents were obtained from all participants before enrolment. Interviews were performed with parents and/or legal guardians for participants 7 years or younger. The participant’s history of previous malaria infection, occupation, age and gender, were recorded at the time of enrolment for the second set of sampling.Thin and thick blood films for the initial investigation were prepared and examined by personnel and microscopists from the Sarawak Health Department, MOH. On the other hand, thin and thick blood films from the second investigation were prepared in separate slides and labelled accordingly at the sites. Thin blood films were dried and fixed using absolute methanol while thick blood films were left to dry thoroughly. Both blood films were stained with 3% Giemsa for 45 min to 1 h. Blood films were examined by two trained microscopists who had over 10 years of experience in reading malaria slides and their proficiency were periodically evaluated by local/regional quality assurance programs of the Ministry of Health Malaysia. Identification of malaria species was done using the thin blood smear while parasitaemia was examined using thick blood smear.® Blood & Tissue Kit following manufacturer’s protocol with slight modification. Three 3-mm2 filter paper punches, equivalent to approximately 10–15 µl blood was excised using a puncher and transferred into a 1.5 μl tube individually. DNAs from 200 µl of venous blood samples collected in Vacutainer® EDTA tubes on the second study were extracted using the QIAamp® Blood Kit following manufacturer’s recommendation.DNA was extracted from DBS collected in the initial study using the QIAGEN DNeasyPlasmodium species and the primers are listed in Table Plasmodium species. This method is a semi-nested multiplex PCR using a single reaction for the second amplification with a mixture of five specific primers for each human and one zoonotic Plasmodium species, and a universal Plasmodium primer. The nucleotide sequence . The DBS were collected from 8 localities in the district of Belaga , followed by Rh. Udau Tedong, Lusong Laku/SK Lusong Laku, Long Wat, Long Jaik, Simpang Uma Nyaving and Long Tanyit with 0.3% (5/1744), 0.2% (4/1744). 0.2% (3/1744), 0.2% (3/1744), 0.1% (2/1744) and 0.05% (1/1744), respectively. From the total of 251 blood samples obtained in the second study, 118 (47%) samples were tested positive for Plasmodium sp. by PCR , followed by P. vivax, P. knowlesi and mixed infections with 18 (32.7%), 9 (16.4%) and 2 (3.6%), respectively. In Long Wat, the prevalence of P. falciparum was also the highest with 24 cases (38.1%) followed by P. vivax and mixed infections with 21 (33.3%) and 18 (28.6%), respectively. There were no mono-infections of P. knowlesi detected from Long Wat. There is no significant difference between the prevalence of malaria parasites in both localities (P = 0.55). Within Long Malim (P = 0.22) and Long Wat (P = 0.32), there was no significant difference between the prevalence of malaria species in both gender. Both genders are equally at risk of harbouring submicroscopic malaria within the population.No malaria parasites were seen by microscopic examination of Giemsa-stained thick and thin blood films collected from the 2013 and 2014 studies  efforts, and the Malaysian National Malaria Control Programme. In 2018, Malaysia had approximately 4630 malaria cases, where P. knowlesi accounted for 89% of the total number of cases reported and 11% for the human malaria cases (imported and introduced). The substantial decrease of P. falciparum and P. vivax cases gives rise to P. knowlesi, and this trend threatens malaria elimination. Although SMM have been reported from Southeast Asian countries  with the prevalence rate of between 1.7 and 36% [P. knowlesi, a zoonotic malaria parasite maintained by macaques, that has been described throughout Southeast Asia is now the most common cause of human malaria in Malaysian Borneo [P. falciparum, 170 cases of P. knowlesi, 4 cases of P. malariae and 7 cases of P. vivax. The number increased substantially in 2018 with 51 cases of P. falciparum, 267 P. knowlesi, and 43 cases of P. vivax. This data shows that despite efforts taken to control and prevent the disease, the number is still increasing. However, there is lack of evidence for or against a substantive role of submicroscopic malaria for transmission in this country. This makes the ethics of treating asymptomatic and submicroscopic parasitaemia unclear and dependent on the risk–benefit ratios of the treatment and/or prevention strategies being implemented. While Malaysia has a malaria elimination goal of zero indigenous transmission for all human malaria species by 2020, achieving malaria elimination requires targeting the human reservoir of infection, including those with asymptomatic infection which requires the combination of universal coverage of interventions with implementation of a robust surveillance system that collects, transmits, and analyses data about cases and programme activities in real time to inform rapid response strategies. The current study adds to the available evidence on the presence of submicroscopic parasitaemia in this country. Accurate diagnosis of asymptomatic SMM is important for providing realistic estimates of malaria burden and preventing misinformed interventions. However, it is not an easy task as low levels of parasitaemia may act as silent reservoir of transmission thus remains infectious to susceptible mosquito vectors. In this study, Since the transmission of malaria parasites from humans to mosquitoes requires the presence of gametocytes, any strategy that interferes with the development or persistence of gametocytes should help to interrupt transmission [Currently, the Malaysia is in the elimination phase of the human-only  and 36% , 16–22,  and 36% , 18. In n Borneo . In 2013smission –26. In msmission .While a systematic review indicated a relationship between microscopy and polymerase chain reaction (PCR) prevalence, the proportion of all infections in a population which are submicroscopic is not predictable in any given situation, particularly in areas of low transmission. Thus, when quantification of submicroscopic infections is needed, this has to be measured directly. Submicroscopic cases are often associated with asymptomatic malaria parasite carriers especially adults. Microscopic diagnosis using thick and thin blood smears plays an important role in malaria diagnosis because of its ability to diagnose and differentiate each species of malaria, and so it is used as the gold standard for any new detection tool or technique . NonethePlasmodium species. Meanwhile, the semi-nested multiplex method developed by Rubio et al. [The WHO recommends prompt parasitological confirmation of diagnosis by either good quality microscopy or good quality RDTs before treatment is being administered . Nonetheo et al. –15 has ao et al. .P. falciparum and P. vivax infections are common in both low and high transmission settings. Use of NAA methods in malaria programmes should be considered for epidemiological research and surveys to map sub-microscopic infections in low transmission areas. NAA methods might also be used for identifying foci for special interventions in elimination settings. According to Okell et al. [In general, challenges in developing multiplex PCR systems include difficulties in designing primers and optimal conditions for a highly sensitive and specific assay. The specificity of the primers for each species is essential in order to get good results in the identification of each species and avoid false negatives and misidentifications , 36–38. l et al. , there il et al.  and the l et al. , and it Plasmodium parasites leads to asymptomatic carriers that provide a fundamental reservoir of parasites, contributing to the persistence of malaria transmission [P. vivax infections do contribute to ongoing transmission but data are very limited [P. vivax infection because of the large proportion of submicroscopic infection in low transmission settings.Asymptomatic SMM has rarely been a major research focus. In malaria endemic areas, asymptomatic malaria parasite carriers especially adults are not uncommon and, as potential gametocyte carriers, represent an important reservoir for malaria transmission . Asymptosmission . Particusmission , 43, 44.smission –48. Ther limited , 50. It P. falciparum and P. vivax were not carried out. Since the transmission of malaria parasites from humans to mosquitoes requires the presence of gametocytes, any strategy that interferes with the development or persistence of gametocytes should help to interrupt transmission. In most malaria endemic areas, the majority of parasite carriers are asymptomatic. Asymptomatic individuals carrying gametocytes remain available as a reservoir for transmission by mosquitoes, contributing to the persistence of malaria transmission within local populations. It is vital to determine the gametocyte carriage potential of individuals followed without treatment and to identify surrogate markers of latent P. vivax infection because currently there is no diagnostic tool which is able to directly detect latent P. vivax. Studies of duration of infection, submicroscopic gametocyte carriage, and gametocyte production potential need to be carried out in submicroscopic infections in low transmission settings where elimination is being considered. This can be done, either by retrospective molecular analysis of prior cohort studies where only microscopy was used, or prospectively, in the context of cohort studies and national surveys, in which microscopy-negative but PCR-positive subjects are followed and not treated unless they become symptomatic or develop patent parasitaemia.Due to time constraint, the detection of gametocytaemia of For malaria elimination settings it is critical to detect all infections, including those with low and sub-microscopic parasite densities in asymptomatic carriers as they represent a parasite reservoir in the community capable of effectively transmitting infections to mosquitoes , 25 and P. knowlesi infections were also detected among the population. It remains unknown if and how much humans contribute to the infectious reservoir for P. knowlesi transmission. While the submicroscopic parasite densities of other malaria species have been shown to be capable of infecting mosquitoes, all experimental infections with P. knowlesi have been from clinical malaria cases. As demonstrated by the data from this study, the most common cause of asymptomatic SMM is P. falciparum and followed P. vivax, despite the fact that P. knowlesi is present. These results illustrate the possibility that by the time a person is symptomatic, the parasite load would have risen by many folds. This is an important aspect to be considered by public health personnel as well as clinicians on whether there is need to heighten surveillance or to make PCR detection routine. The occurrence of other possible zoonotic species also needs to be further elucidated. Unfortunately, this study did not cover that aspect and since this is one of the first community-based studies, more studies needs to be conducted to confirm this. Entomological and primatological studies are also needed to evaluate the presence of potential P. knowlesi vectors in these village environments and the proximity of infected reservoirs.Submicroscopic infections are thought to be important contributors to maintaining malaria transmission. However, there is currently no direct evidence that specifically targeting this low-density parasite reservoir will hasten progress towards elimination. In most malaria endemic settings, asymptomatic infections outnumber symptomatic infections. The objective of this study was to detect the presence of submicroscopic malaria in low transmission area. It was achieved with by using molecular methods. The semi-nested multiplex PCR has proven to be a very useful tool in detection of asymptomatic submicroscopic malaria in both high and low transmission area. However, the WHO does not currently provide guidance to countries regarding the programmatic suitability of molecular diagnostics or guidelines on how to utilize the information that would emerge from their use. Quality assured microscopy is still officially considered the gold standard by the WHO, despite large bodies of evidence that shows that PCR and other nucleic acid amplification-based assays are more sensitive than microscopy. There is a need to develop guidance on indications for use, assay selection and quality assurance/control for PCR and other molecular diagnostic techniques for the specific conditions in which use of these malaria diagnostic tools may be appropriate. In this study,