Plasmodium spp infections. Many infected individuals are asymptomatic, and persistent asymptomatic Plasmodium falciparum infections are common in endemic settings. We aimed to quantify the contribution of symptomatic and asymptomatic infections to P falciparum transmission in Tororo, Uganda.Symptomatic malaria cases reflect only a small proportion of all P falciparum parasite density, gametocyte density, and genetic composition were determined molecularly using quantitative PCR (qPCR), quantitative reverse transcriptase PCR (qRT-PCR), and amplicon deep sequencing, respectively. Membrane feeding assays were also done to assess infectivity to mosquitoes. The contribution of different populations to the infectious reservoir was estimated for symptomatic infections, asymptomatic but microscopically detected infections, and asymptomatic but qPCR-detected infections; and for age groups younger than 5 years, 5–15 years, and 16 years or older.We did a longitudinal, observational cohort study in Tororo district, Uganda. We recruited participants of all ages from randomly selected households within this district. Participants were eligible if the selected household had no more than nine permanent residents and at least two members younger than 10 years, and the household was their primary residence, and they agreed to come to the study clinic for any fever episode and avoid antimalarial medications outside the study. Participants were followed-up by continuous passive surveillance for the incidence of symptomatic infections; routine assessments  were done at baseline and at routine visits every 4 weeks for 2 years. P falciparum was detected in 28 (5·3%) of 531 participants by microscopy and an additional 64 (12·1%) by qPCR and declined thereafter. In 538 mosquito feeding experiments on 107 individuals, 446 (1·2%) of 37 404 mosquitoes became infected, with mosquito infection rates being strongly associated with gametocyte densities . Considering both transmissibility of infections and their relative frequency, the estimated human infectious reservoir consisted primarily of asymptomatic microscopy-detected infections (83·8%), followed by asymptomatic submicroscopic infections (15·6%), and symptomatic infections (0·6%). Children aged 5–15 years accounted for more than half of the infectious reservoir (58·7%); individuals younger than 5 years (25·8%) and those 16 years or older (15·6%) contributed less. Samples from four children contribued to 279 (62·6%) of 446 infected mosquitoes after multiple mosquito-feeding assays.Between Oct 4, 2017, and Oct 31, 2019, 531 individuals were enrolled from 80 randomly selected households and were followed-up for 2 years. At baseline, Individuals with asymptomatic infections were important drivers of malaria transmission. School-aged children contributed to more than half of all mosquito infections, with a small minority of asymptomatic children being highly infectious. Demographically targeted interventions, aimed at school-aged children, could further reduce transmission in areas under effective vector control.US National Institutes of Health, Bill & Melinda Gates Foundation, and the European Research Council. Plasmodium spp infections, and many infected individuals are asymptomatic. Asymptomatic infections are common in all malaria-endemic settings,Plasmodium falciparum are particularly prevalent in school-aged children, who have a higher complexity of infection and more persistent parasitaemia than in younger children or adults.Despite global efforts to control and eliminate malaria, progress has plateaued. In some high-transmission settings, malaria burden is increasing.Evidence before this studyPlasmodium falciparum infected hosts. The study in Ethiopia observed that asymptomatic infections were responsible for 99·2% of all P falciparum-infected mosquitoes, whereas the study in Cambodia observed that only symptomatic infections resulted in P falciparum mosquito infections. The likelihood that an infection is interrupted by antimalarial treatment and the timing of this treatment relative to the appearance of infectious gametocytes are important factors in determining the relative contribution of symptomatic and asymptomatic infections. These factors are likely to be setting-dependent and are best examined in longitudinal studies. Only one study, done in Burkina Faso in 2007–08, did mosquito feeding experiments repeatedly on the same individual, with up to three observations per individual over 7 months. This study only included asymptomatic infections and did not examine the duration of infections but assessed gametocyte presence and infectivity in relation to seasonality.We searched PubMed for publications, without language restrictions, until Oct 1, 2020, using the search terms “plasmodium” AND “falciparum” AND “(“membrane feeding” OR “skin feeding” OR “direct membrane feeding” OR “mosquito feeding” OR “infectious reservoir”)” AND “”. We found one review of studies that assessed the infectious reservoir of malaria. This review identified ten studies done in 1955–2010 in different African and Asian settings. Nine of ten studies were done in areas of intense or moderate transmission intensity; none were longitudinal in study design. We also identified three additional recent cross-sectional studies, done between 2013 and 2016, that assessed the infectious reservoir in Burkina Faso and Kenya together, and in separate studies in Ethiopia and Cambodia. All three studies used mosquito membrane feeding assays and molecular assays to determine parasite and gametocyte densities. The study done in Burkina Faso and Kenya included only asymptomatic participants, whereas the two studies done in Ethiopia and Cambodia quantified infectivity to mosquitoes from both symptomatic and asymptomatic Added value of this studyOur longitudinal study was done in an area of previously high transmission that is now under intense malaria control and as such is representative of other African settings where malaria burden has been successfully reduced. Our findings suggest that asymptomatic infections are responsible for the majority of mosquito infections with symptomatic infections having a negligible role in sustaining transmission in our setting. Excellent access to care in our cohort, demonstrated by a high proportion of gametocyte-free (short-duration) symptomatic infections might have contributed to these findings. In other settings, where symptomatic infections might be less likely to receive prompt treatment, these cases might develop into longer-lasting infections that produce transmissible gametocytes. Although microscopy-positive asymptomatic infections were responsible for most infected mosquitoes, parasite densities fluctuated considerably during infections such that these were frequently missed by research-level microscopy. We further identified school-aged children as an important and accessible reservoir that could be targeted by malaria control interventions.Implications of all the available evidenceRecent studies have shown that asymptomatic low-density infections are common in areas of low endemicity and areas where control tools have reduced malaria burden. Our findings provide evidence that these asymptomatic infections, including low-density infections, are an important source of onward transmission to mosquitoes. Oscillations in the density of infections result in imperfect detectability by conventional diagnostics. Our findings show that although infectivity to mosquitoes is highest when asymptomatic infections are of sufficient density to be detected by microscopy, many infections that contribute to transmission are initially submicroscopic. This finding suggests that passive detection of symptomatic infections or community mass screening and treatment based on conventional diagnostics might be insufficient to reduce the infectious reservoir and prevent onward transmission in settings of declining transmission.A considerable debate is ongoing about the characteristics of the human infectious reservoir. Understanding the relative contributions of symptomatic and asymptomatic infections to onward transmission of malaria parasites to mosquitoes is essential for guiding interventions to reduce and eliminate transmission.14P falciparum infections in an all-age cohort of residents in Tororo, Uganda. We also aimed to quantify gametocyte carriage and infectivity to mosquitoes, and compared them between age groups as well as participants with symptomatic and asymptomatic infections.To better understand the contributions of different populations to the human infectious reservoir for malaria, we aimed to assess the density, genetic composition, and kinetics of naturally acquired P falciparum entomological inoculation rate was 238 infectious bites per person per year, which was markedly reduced to 0·43 infectious bites per person per year 5 years later.We did a longitudinal, observational cohort study in Nagongera subcounty (Tororo district) in eastern Uganda. Tororo was previously a high-transmission area that is now under effective malaria control because of broad implementation of long-lasting insecticidal nets and indoor residual spraying of insecticides. Before 2013, malaria control in the Tororo district focused on malaria case management with artemether–lumefantrine, distribution of long-lasting insecticidal nets during antenatal visits, and promotion of intermittent preventive treatment during pregnancy. Universal distribution campaigns of long-lasting insecticidal nets were done in November, 2013, and May, 2017. Sustained indoor residual spraying of insecticides has been maintained since December, 2014, starting with rounds of bendiocarb every 6 months, followed by annual rounds of pirimiphos-methyl  since June, 2016.In this context, from 2017 to 2019, we included participants of all ages into this study. Households were eligible if they had no more than nine permanent residents, at least two members younger than 10 years, no plans to move from Nagongera subcounty, and willingness to participate in screening for clinical and entomological surveillance. Households that met these criteria were selected; participants from these households were enrolled if their household was their primary residence and if they agreed to come to the study clinic for any episode of fever and avoid antimalarial medications outside the study.All eligible participants provided written informed consent. Ethical approval for the study was received from the Uganda National Council of Science and Technology (HS119ES), Makerere University School of Medicine, the University of California, and the London School of Hygiene & Tropical Medicine.At baseline and at routine visits done every 4 weeks for 2 years, a standardised clinical evaluation was done and blood samples were collected. Participants were encouraged to attend a dedicated study clinic, which was open daily for all medical care. Study participants found to have a tympanic temperature of more than 38·0°C or a history of fever in the previous 24 h had a thick blood smear read urgently. If the thick blood smear was positive for malaria parasites by microscopy, samples were taken and individuals were treated with a 3-day course of artemether and lumefantrine . Asymptomatic infections were not treated unless symptoms occurred.P falciparum parasite density, targeting the multi-copy conserved var gene acidic terminal sequence, with a lower limit of detection of 0·05 parasite/μL using 200 μL whole blood samples.Blood samples drawn at the onset of each symptomatic malaria episode and at routine visits were assessed by quantitative PCR (qPCR) for Anopheles gambiae ss mosquitoes were offered heparinised venous blood  via two glass feeders filled with 0·5 mL of blood each.P falciparum DNA (18S) was detected in infected guts by nested PCR. To assess parasite diversity over the entire follow-up period, DNA from study participants and infected mosquito midguts was analysed by amplicon deep sequencing of the apical membrane antigen 1 (AMA-1) gene, as described previously.Mosquito membrane feeding assays were done on selected individuals who were diagnosed with symptomatic malaria before drug administration, or at the time of routine visits if an individual was qPCR positive at their previous visit. For each experiment, between 60–80 P falciparum sporozoites in these mosquitoes with ELISA, as described elsewhere.Entomological surveillance was done throughout follow-up, with mosquito sampling every 2 weeks with Centers for Disease Control and Prevention light-traps in all rooms where cohort participants slept, and detection of Poisson regression with generalised estimating equations to account for repeated measures was used to estimate incidence of symptomatic and asymptomatic infections and 95% CIs. Multiple generalised linear models were used to model measures of associations and present regression coefficients (β). Where appropriate, subject-specific random intercepts were added to account for correlations between observations from the same individuals. For response variables (gametocyte positivity and proportion of infected mosquitoes), we used generalised linear models assuming a binomial distribution with a logit for gametocyte positivity and a log link for the proportion of infected mosquitoes.We modelled gametocyte prevalence using symptomatic status as a covariate including subject-specific random effects; the proportion of infected mosquitoes was modelled using the covariate gametocyte density.The contribution of different populations to the infectious reservoir was estimated for symptomatic, asymptomatic but microscopically detected infections, and asymptomatic but qPCR-only detected infections;We did all the statistical analyses using R (version 3.1.12). Full details about the statistical analyses are shown in the The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Between Oct 4 and Oct 31, 2017, 466 residents from 80 randomly selected households in Tororo were enrolled into the study cohort. An additional 65 residents were enrolled when they joined participating households during the follow-up period that ended on Oct 31, 2019. In total, from Oct 4, 2017, to Oct 31, 2019, 531 residents were enrolled and followed-up. 177 (33·3%) of 531 participants were younger than 5 years, 193 (36·3%) were aged 5–15 years, and 161 (30·3%) were 16 years or older. 62 (11·7%) of 531 participants were excluded from the study follow-up for the following reasons: moving out of the study area (n=41), absenteeism for more than 120 days (n=15), death (n=3), inability to comply with study protocol (n=2), or withdrawal of consent (n=1). Among the 469 participants actively in the study, 14 702 (99·6%) of 14 754 scheduled routine visits were successfully done.Anopheles mosquitoes were collected per sleeping room per night, with peaks following seasonal rains  were positive for P falciparum sporozoites. During the study follow-up, in which 12 728 qPCR assays were done, a decline in the number of asymptomatic infections detected per month was observed. Overall, 38 episodes of symptomatic malaria were diagnosed over 955 person-years  and 110 new asymptomatic infections were detected (asymptomatic infection incidence of 0·12 episodes per person-year [95% CI 0·096–0·139]). Mean parasite prevalence over the study period in participants younger than 5 years was 1·2% by microscopy and 4·0% by qPCR, in those aged 5–15 years was 3·3% by microscopy and 14·0% by qPCR, and in participants 16 years or older was 0·8% by microscopy and 10·8% by qPCR  of 531 participants and by qPCR in an additional 64 (12·1%) participants. On average, two female al rains . Of the  by qPCR . Geometr by qPCR . Among pOverall, 538 mosquito feeding assays were done with blood samples from 107 individuals diagnosed with either symptomatic malaria (n=24 mosquito feeding assays) or asymptomatic infections (n=514). Of the feeds done on asymptomatic infections, 100 were done when individuals were parasite-negative, but with a positive qPCR result on the preceding visit . Parasitvs 11 [28·9%] of 38; odds ratio [OR] 3·2, 95% CI 1·1–9·3; p=0·033). 18 (46·2%) of 39 infectious feeds and 273 (61·2%) of 446 infected mosquitoes were from individuals with microscopically detectable gametocytes at the time of mosquito feeding. Among samples that were both positive for parasites and gametocytes (n=895), gametocyte density was positively associated with concurrent total parasite density in asymptomatic infections , but not in symptomatic infections . Although 14 (35·9%) of 39 infectious feeds were done on samples from individuals with parasites below the concentration detected by microscopy, all infectious individuals were microscopy-positive for asexual parasites or gametocytes at least once during follow-up . Four 0.Figure 4P falciparum DNA was detectable by nested PCR in 414 (92·8%) of 446 infected mosquito midguts; amplicon deep sequencing was successful for 322 (72·2%) of the 446 samples, providing genotyping data from at least one mosquito for 33 (84·6%) of 39 successful feeds. Identification of genotypes over time, in relation to transmission to mosquitoes, is presented for the four most infectious individuals in the cohort , can be found European Research Council (ERC-2014-StG 639776), and is further supported by a fellowship from the Netherlands Organisation for Scientific Research (Vidi fellowship NWO project number 016.158.306). MC is supported by the Fogarty International Center (P0529898 and TW009343) and the Centennial Travel Award from the American Society of Tropical Medicine and Hygiene. JIN is supported by the Fogarty International Center (TW010365). BG is a Chan Zuckerberg Biohub investigator. The open-access clinical epidemiology database resource (ClinEpiDB) platform is supported by the Bill & Melinda Gates Foundation (OPP1169785). All other authors declare no competing interests.TB received a fellowship from the


Malaria remains a major public health threat and tools sensitive to detect infections in low malaria transmission areas are needed to progress elimination efforts. Pregnant women are particularly vulnerable to malaria infections. Throughout pregnancy they access routine antenatal care, presenting a unique sentinel population to apply novel sero-surveillance tools to measure malaria transmission. The aim of this study was to quantify the dynamic antibody responses to multiple antigens during pregnancy so as to identify a single or multiple antibody response of exposure to malaria in pregnancy.This study involved a secondary analysis of antibody responses to six parasite antigens  measured by enzyme-linked immunosorbent assay (ELISA) over the gestation period until delivery (median of 7 measurements/woman) in 250 pregnant women who attended antenatal clinics located at the Thai-Myanmar border. A multivariate mixture linear mixed model was used to cluster the pregnant women into groups that have similar longitudinal antibody responses to all six antigens over the gestational period using a Bayesian approach. The variable-specific entropy was calculated to identify the antibody responses that have the highest influence on the classification of the women into clusters, and subsequent agreement with grouping of women based on exposure to malaria during pregnancy.Plasmodium infection detected by light microscopy during pregnancy , defined as cases. The antibody responses to all six antigens accurately identified the women who did not have a malaria infection detected during pregnancy . Antibody responses to P. falciparum merozoite surface protein 3 (PfMSP3) and P. vivax apical membrane antigen 1 (PvAMA1) were the least dynamic. Antibody responses to the antigens P. falciparum apical membrane antigen 1 (PfAMA1) and PfVAR2CSA were able to identify the majority of the cases more accurately .Of the 250 pregnant women, 135 had a PfAMA1 and PfVAR2CSA, may be useful for sero-surveillance of malaria infections in pregnant women, particularly in low malaria transmission settings. Further investigation of other antibody markers is warranted considering these antibodies combined only detected 63% of the malaria infections during pregnancy.These findings suggest that the combination of antibodies, The online version contains supplementary material available at 10.1186/s12936-022-04111-y. Malaria is a major infectious disease causing around 229 million clinical cases and 409,000 deaths globally in 2019 , 4. MalaPlasmodium falciparum antigen (PfVAR2CSA), which is expressed on the surface of infected erythrocytes (IEs), and mediates sequestration of P. falciparum in the placenta via binding to placental chondroitin sulphate A (CSA) receptors ) and both P. falciparum and P. vivax (77% [24/31]) infections as cases, but performed poorly at classifying P. vivax only infections as cases (27% [12/44])  into the expected cluster in the univariate analyses, i.e. 75 Table . The resPfAMA1 and PfVAR2CSA were best for identifying exposure to malaria during pregnancy, suggesting that these two antibodies may be good candidates for sero-surveillance of malaria infections in pregnant women.Pregnant women may serve as an accessible sentinel population to estimate the burden of malaria  and potePlasmodium spp. blood-stage antigens, in particular PfVAR2CSA, are an attractive candidate for sero-surveillance of malaria in pregnancy as they have been demonstrated as a biomarker of recent exposure to malaria during pregnancy , which may reflect the specific epidemiology of malaria in pregnancy in low endemic settings [PfMSP3 and PvAMA1 were the least dynamic, influencing the classification of pregnant women into the low immunity cluster suggesting that they are less suitable serological markers of recent exposure to malaria during pregnancy. Conversely, these properties meant that they were the most useful antigens studied in classifying women as controls (i.e. parasite negative) so may have potential for use as part of a broader panel of antigens. Other biomarkers of exposure to P. falciparum and P. vivax infection have been identified in non-pregnant individuals and would be valuable to investigate in future studies [PfVAR2CSA antibodies may have a very long half-life (30–50 years), whereas PfAMA1 antibodies decay more rapidly  [PfVAR2CSA, should be interpreted with caution as markers of malaria exposure.Antibodies specific for iewed in ) and to iewed in . However malaria , 37. No settings . Antibod studies , 40. Thi3 years) . InclusiSeveral methods have been developed to cluster individuals based on longitudinal data, but the majority can only cluster a single longitudinal trajectory from each individual –45 or asP. vivax, PvAMA1, limits what can be understood about the immune response of pregnant women exposed to vivax malaria. The antibody responses to PvAMA1 remained relatively low compared to other antibodies . Parasite density is believed to have a proportional relationship with antibody maintenance and boosting [PvAMA1 antibody responses at low levels. Administration of chloroquine, preventing vivax episodes, could be a significant confounding factor in disrupting antibody responses to PvAMA1 [PvAMA1 responses was observed though to be in the counterintuitive direction, however, there were a greater proportion of women in the chloroquine prophylaxis arm with a history of P. vivax infection which increased marginally the PvAMA1 response. This study also only assessed six antibody responses and one domain of VARCSA, therefore, there may be other antigens that are better candidates for sero-surveillance.This study has several limitations to consider. First, the availability of only one antibody marker for boosting –50. Thatboosting  could poo PvAMA1 . The assAnother limitation of this study was that the detection of exposure to malaria was based on microscopy, thereby, failing to detect low density sub-microscopic infections . HoweverPfAMA1 and anti-PfVAR2CSA antibodies could be used as a potential biomarker of exposure to malaria during pregnancy. However, only 63% of the malaria infections during pregnancy were detected, therefore further investigation of other antibody markers is warranted. This combined antibody diagnostic tool may facilitate the detection of microscopic infections as an alternative to standard diagnostic methods, particularly in low transmission settings. This proposed malaria sero-surveillance tool may also enhance malaria control and progress efforts to eliminate malaria.This study demonstrates that the combination of anti-Additional file 1: Supplementary methods. Table S1. Number and species of infections. Table S2. Penalized expected deviance (PED) values of the models with mixture distributions ranging from K = 1, 2, 3, 4 clusters. Table S3. Characteristics of the pregnant women by low and high immune antibody response profile clusters. Table S4. Assessment of the importance of combining PvAMA1 and PfMSP3, respectively with other P. falciparum antibodies. Table S5. The importance of including at least PfMSP3 or PvAMA1 with other Plasmodium falciparum antibodies investigated. Table S6. Classification of pregnant women based on the joint antibody responses of PfAMA1 and PfVAR2CSA. Table S7. Classification of pregnant women based on the joint antibody responses of PfAMA1 and PfEBA175, and PfEBA175 and PfVAR2CSA, respectively. Table S8. Classification of cases by the type of infection, based on antibody responses to PfAMA1 and PfVAR2CSA. Figure S1. Longitudinal antibody levels against six antigens for all women free from malaria (controls) by the serostatus to schizont extract. Figure S2. Longitudinal antibody levels against six antigens for all women by gravidity (primigravidae versus multigravidae). Figure S3. Posterior cumulative distribution functions of the observed data deviances for models with K = 1, 2, 3, 4 clusters.


Plasmodium and is, still today, despite all efforts and advances in controlling the disease, a major issue of public health. Its clinical course can present either as the classic episodes of fever, sweating, chills and headache or as nonspecific symptoms of acute febrile syndromes and may evolve to severe forms. Survivors of cerebral malaria, the most severe and lethal complication of the disease, might develop neurological, cognitive and behavioral sequelae. This overview discusses the neurocognitive deficits and behavioral alterations resulting from human naturally acquired infections and murine experimental models of malaria. We highlighted recent reports of cognitive and behavioral sequelae of non-severe malaria, the most prevalent clinical form of the disease worldwide. These sequelae have gained more attention in recent years and therapies for them are required and demand advances in the understanding of neuropathogenesis. Recent studies using experimental murine models point to immunomodulation as a potential approach to prevent or revert neurocognitive sequelae of malaria.Typical of tropical and subtropical regions, malaria is caused by protozoa of the genus  Plasmodium and is transmitted by the bite of the female Anopheles mosquito. There are eight species causing human malaria: P. falciparum, P. vivax, P. malariae, P. ovale curtisi and P. ovale wallikeri  in protuberances of the infected red-cell surface that interacts with host adhesion receptors on endothelium, contributing to the inflammatory process  were associated with impaired cognitive ability in children  were unable to reveal the presence of cognitive impairment , treating mice at D4, before any neurological manifestation of CM. This model proved to be useful for detecting cognitive sequelae and late (D92) behavioral alterations following (82 days after parasite clearance) a single episode of non-severe malaria  improved recognition memory in homeostasis, recovered late deficits in cognitive function and inhibited the manifestation of an anxiety-like phenotype caused by a unique episode of non-severe malaria , Fiocruz is an Associated Laboratory of the Instituto Nacional de Ciência e Tecnologia (INCT) in Neuroimmunomodulation supported by the CNPq, and of the Rio de Janeiro Neuroinflammation Network financed by Faperj.FR-G and CD-R are supported by CNPq, Brazil, through a Productivity Research Fellowship, and CD-R is a “The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Anaemia, a common nutritional deficiency, is a public health problem in the Mount Cameroon area. This study determined the prevalence and possible risk factors of microcytic and malarial anaemia in children less than ≤15 years residing in the Buea and Limbe municipalities in the Mount Cameroon area.  A total of 566 children were clinically examined in a cross-sectional study from December 2018 to August 2019 for anaemia and malaria parasites. Blood samples collected were used in evaluating full blood count with the aid of an automated haemoanalyser, and malaria parasite was confirmed by microscopy. Anaemia was defined based on WHO standards while microcytic anaemia and malarial anaemia were defined as microcytosis + anaemia and malaria + anaemia, respectively. Factors that showed significance in the bivariate analysis were entered into a multinomial logistic regression to determine risk factors for microcytic and malarial anaemia. P=0.007), forest ethnicity (P=0.019), parents being farmers (P=0.038) or jobless (P=0.009), and having moderate malaria parasitaemia (P=0.048) while those for malarial anaemia were child age of 6–10 years (P=0.008), parents' age of 26–35 years (P=0.049), parents being jobless (P=0.023), and consuming plantains 3-4 times (P=0.024) a week.  The overall prevalence for anaemia, microcytosis, microcytic anaemia, and malarial anaemia was, respectively, 68.7%, 48.9%, 36.9%, and 19.6% with microcytic anaemia representing 53.7% of all anaemic cases. Risk factors for microcytic anaemia included child age of 1–5 years ( Microcytic anaemia is getting to be a severe public health concern while malarial anaemia is a mild public health issue in children residing in urban areas of Mount Cameroon. Parents' occupation was directly linked to all anaemia forms; hence, any intervention to curb anaemia should consider aspects that will raise the socioeconomic status of the population. Anaemia is still a public health problem in developing countries  across aAnaemia is not a standalone condition but results from lack of one or more micronutrients, infections such as malaria and hookworm, congenital haemolytic diseases, poor maternal health, socioeconomic status, and even demographic factors –12. MalaMicrocytic anaemia which is characterised by smaller than normal circulating red blood cell for age  is a comWhile several studies in the Mount Cameroon area have determined the occurrence of anaemia in conjunction with malaria and other infections in various setting, the type of anaemia which is invaluable in making informed decision when planning control measures has been infrequently examined. Therefore, the objective of this study was to determine the prevalence and identify factors associated with microcytic and malarial anaemia in children ≤15 years living in urban areas of Buea and Limbe in the Mount Cameroon area.The study was conducted in the Buea and Limbe I and III municipalities, both located in the Mount Cameroon area of Fako Division. Buea is situated and characterised as described by previous studies . It is dThe study population included children 1–15 years living in the study area whose parents or caregivers gave consent to their participation in the study. Those with known HIV status, with active haemorrhage, who had blood transfusion or were operated upon two months prior to the study, and who are or had been on antimalarials two weeks prior to the study were excluded.This cross-sectional study was carried out from December 2018 to August 2019. Following administrative and ethical clearances, participants were enrolled into the study at their various communities and at presentation to the hospital following education. Informed consent/assent forms were given to parents/caregivers explaining the purpose, benefits, and risks of the study. Structured questionnaires to obtain information on sociodemographic data, type of accommodation, clinical history, and dietary habits were administered, and clinical evaluation and blood samples were collected thereafter for malaria parasite determination and full blood count (FBC) analyses.n = z2pq/d2 [n was the required sample size; z was 1.96, the standard deviation for a 95% confidence interval (CI); P value was 62% which was the anaemia prevalence in the region [q was (1 − p); and d was the acceptable error margin set at 0.05. The optimum sample size for both health districts was 362. To allow for data loss due to incomplete data entry, the sample size was increased by 10% to 400 requiring an average of 200 samples per health district.A convenient multistage sampling method was employed in the study. Firstly, for representativeness of each health district, a health area was randomly selected from each of the 3 council areas that make up the Limbe Health District and 2 were selected from the 4 distinct urban settings recognised in the Buea Health District. This was followed by a random selection of representative health facilities and neighbourhoods in the selected health areas. Following education by the community relay agents, potential participants in the selected communities were invited to a specified data collection hall in the neighbourhood on programmed dates coordinated by the neighbourhood head. Concurrently, at presentation to the outpatient department of both Buea Regional and Bota District Hospitals, participants were enrolled prospectively as they fulfilled the inclusion criteria for the study. The sample size for the study was determined using the formula  z2pq/d2 , where ne region ; q was , weight-for-age (WA), and weight-for-height (WH) z-scores were computed based on WHO growth curves  using WHcore <−2 . DietaryμL) of blood as low (<1000 parasites), moderate (1000–4999 parasites), high , and hyper  [Approximately 4 mL of venous blood was collected using sterile techniques into EDTA and dry tubes. Labelled blood samples were transported on ice to the Malaria Research Laboratory of the University of Buea for further analysis. Thin and thick blood films were prepared immediately after dispensing blood into EDTA tubes. Thin films were fixed with absolute methanol and together with the thick films stained for 15 minutes with 10% Giemsa stain and then examined for the detection and identification of the malaria parasite following standard procedures . Slides rasites) .α haemoanalyser according to manufacturer's instructions to obtain values for WBC, red blood cell (RBC) and platelet (Plt) counts, haemoglobin (Hb), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), red cell distribution width coefficient of variation (RDW-CV), red cell distribution width standard deviation (RDW-SD), mean platelet volume (MPV), and platelet distribution width (PDW). Anaemia was classified based on WHO age-based classification [Blood in the dry tube was centrifuged at 3000 rpm for 5 minutes, and the aliquots were stored at −20°C until use. C-reactive protein (CRP) concentration was determined using the Thermo Fisher ELISA machine as per the manufacturer's instructions. Inflammation was confirmed when acute phase protein (APP) CRP was >5 mg/L [fication  as Hb < fication .χ2), Pearson's ranked correlation (r) for haematological bivariate association, and analysis of variance (ANOVA) to compare group means. Parasite density was log-transformed before analysis. Factors with a P value <0.2 in the bivariate analysis were entered into a multinomial logistic regression to determine risk factors for microcytic and malarial anaemia. Odd ratios (OR) and 95% confidence intervals (CIs) were computed, and P < 0.05 value was suggestive of statistical significance.Data was keyed into Microsoft Excel 2010 and exported into IBM-Statistical Package for Social Sciences (SPSS) version 20 . Continuous variables were summarized as means and standard deviations while categorical variables were reported as percentages and frequencies. Proportion difference was evaluated using Pearson's Chi square test , following an administrative approval from the Regional Delegation of Public Health (R11/MINSANTE/SWR/RDPH/PS/430/940). Authorisations from community heads were also obtained. Only children whose parents consented to their participation in the study and who responded to the questionnaires were enrolled after the purpose, risks, and benefits were clearly explained to them. It was stressed that the study was completely voluntary and that a parent had every right to stop/withdraw their child from the study. Even after the parent's consent to participate in the study, any child who was too scared of a needle was not forced to continue. Data was treated with utmost confidentiality by assigning codes to the patients' samples.A total of 614 participants were approached to participate in the study, and 589 of them were enrolled. After data curation, 566 participants were retained of which 47.2% were males and 52.8% were females, living in low (61.5%) and highland (38.5%) areas. The mean (SD) age was 6.4 (4.5) years of whom 51.4% were ≤5 years. As shown in P=0.003), those from low altitude , and those who presented themselves at the hospital  when compared with their respective contemporaries. The prevalence of malaria parasite and malnutrition was comparable with sex . However, significantly higher occurrence of malnutrition was observed in children aged 1–5 years , those living at low altitude , those whose parents were fishermen , and those of other ethnicity  as shown in Significantly higher prevalence of malaria parasite was observed in children aged 6–10 years . The weekly plantain consumption was significantly linked to parents' education level (P=0.049) with more children whose parents had no formal education consuming plantain ≥3 times weekly (55.8%) when compared with their counterparts as shown in On dietary habits, more children ≤5 years reported not washing hands before meals (19.9%) and consuming fewer plantain meals a week than their older peers. This difference was statistically significant (P=0.011) was observed in those with inflammation (79.1%) as shown in Overall, the prevalence of anaemia (68.7%) was highest in children living in low altitude (77.3%), examined in the community (81.2%), and living in homes with 6–10 occupants (71.4%) and whose parents were 25–35 years old (80.2%), had no formal education (75.8%), or were fishermen (86.4%) as well as those of forest ethnicity (100.0%) when compared with their compeers. The differences were statistically significant. In relation to clinical factors, the only significantly higher prevalence of anaemia , those living in low altitude (49.1%), those examined within the community (42.8%), those whose parents were 26–35 years old (39.7%) and were fishermen (56.3%), those of forest ethnicity (60.0%), those malaria parasite positive (49.7%), and those who had moderate parasitaemia (62.8%) when compared with their respective contemporaries. While malarial anaemia occurred in 19.6% of the study population, children aged 6–10 years (24.1%), those living at low altitude (23.9%), those whose parents were 26–35 years old (21.6%), and those who were feverish (28.7%) and were malaria parasite positive (68.8%) had significantly higher prevalence than their respective peers .P=0.010, P=0.047, and P=0.009) in children who were stunted (83.1%), were underweight (89.5%), and consumed plantains 3-4 times a week (85.3%), respectively, when compared with their equivalents. On the other hand, microcytic anaemia prevalence was significantly higher (P=0.048) in normal (43.7%) than wasted children, while only those who consumed plantains 3-4 times a week had a significantly higher (P=0.025) prevalence of malarial anaemia (20.6%) as shown in The prevalence of anaemia was significantly higher (P=0.017), those whose parents were farmers, worked privately, or were fishermen , and those who consumed meat 3-4 times weekly (P=0.026) and plantains (P=0.034) were significantly at risk of developing anaemia. They were, respectively, 4, 2.2, 2.7, 5.5, 2.5 and 2.5 times more likely to be anaemic than their counterparts. Significantly, children aged 1–5 years (P=0.007), those of forest ethnicity (P=0.007), those whose parents are farmers (P=0.038) or jobless (P=0.009), and those who had moderate parasitaemia (P=0.048) were 2.4, 15.7, 2.5, 3.4, and 2.6 times, respectively, more likely to have microcytic anaemia. With respect to malarial anaemia, the identified risk factors were the child's age (P=0.008), parent's age (P=0.049) and occupation (P=0.023), and child's weekly consumption of plantain (P=0.024). Children aged 6–10 years, whose parents were 26–35 years old or jobless, and who consumed plantains 3-4 times a week were 3.3, 2.6, 3.8, and 2.9 times, respectively, more likely to have malarial anaemia than their counterparts.As revealed in This study investigates the prevalence and risk factors for microcytic and malarial anaemia in children 1–15 years in urban settings of the Mount Cameroon area. The overall malaria prevalence of 27.7% is like that reported by Teh et al.  with theMore than half of the study participants reported washing hands before meal and consuming plantains more than twice weekly. Hand washing increased with age because younger children must be reminded frequently to wash their hands but will form the habit as they grow older. This finding is corroborated by studies elsewhere . In addiThe overall anaemia prevalence of 68.7% reveals that anaemia is a serious public health problem in children in this region as elsewhere in the country. This is higher than both the national anaemia prevalence of 60%  and thatThat anaemia was more prevalent at lower  than higher altitude  in this study shows the association between malaria, anaemia, and altitude which had long been established in previous studies , 33, 42.Plasmodium infection that was not determined and the prevalence of malaria parasite observed in these apparently healthy children in the community. It is established that malaria causes haemolysis of both parasitized and healthy red cells leading to anaemia [Contrary to findings by Sakwe et al.  who repo anaemia . Also, i anaemia  also cau anaemia .Parents' age, education level, occupation, and ethnicity as well as family size are sociodemographic factors that are intrinsically linked and significantly associated with childhood anaemia as reported in previous studies , 48, 49.Although not significant, observations from this study reveal that children who were feverish, were malaria parasite positive, and had moderate parasitaemia were more anaemic than their respective counterparts supporting findings from elsewhere , 56. FevMalnutrition affects all strata of the society but mostly children are more vulnerable because of high nutritional demands required for proper growth . AnaemiaRegarding microcytosis and microcytic anaemia, an overall 48.9% of the study population had microcytosis while microcytic anaemia prevalence was 36.7%. This proportion ranks it as a moderate public health concern according to WHO classification on anaemia burden . This prMales had higher microcytic anaemia prevalence (70%) than females although this observation may not be a surprise since the males equally had higher prevalence of malaria and anaemia than females. Age-wise, we would have expected children 6–10 years old to have the highest prevalence of microcytic anaemia as with both malaria and anaemia but that was not the case. Instead, the ≤5 years old had significantly higher prevalence of microcytic anaemia than their counterparts. Microcytosis and anaemia each have varied aetiologies, the result of which in combination may lead to microcytic anaemia. The observed microcytic anaemia in this case may have been due to anaemia resulting from nutritional deficiency and not malaria, as children in this age have high iron needs for growth. Furthermore, microcytosis may have been due to the body's protective mechanism to wade off infection with malaria parasite , or resuA significant association between occupation, ethnicity, and microcytic anaemia is like that observed with anaemia. Parents who were jobless, fishermen, and farmers had children with significantly higher prevalence of microcytic anaemia than their counterparts of other occupation. It is understandable that being jobless predisposes one to lower income and therefore inadequate financial resources to provide nutritious meals to meet the daily nutrient requirement of the household. However, children whose parents/guardians were fishermen and farmers were expected not to have microcytic anaemia because they have access to iron-rich food sources. Nevertheless, observations from this study, congruent to previous studies in this area , 34, shoPlasmodium falciparum infection has been associated with anaemia in other studies [The overall low prevalence of malarial anaemia in this population (19.6%) reveals that malaria may not be a major contributor to the overall anaemia prevalence. This proportion is lower than the 27.7% previously observed in the same area . Even th studies , 66 whic studies . NonetheWhile the findings of this study may be applicable in several areas in the region and elsewhere with similar environmental conditions and microclimates as well as cultural practices, there is a limitation in its applicability in areas nonendemic for malaria and having variable eating habits. However, the findings provide valuable information that could be used in the development of appropriate, context specific control measures against anaemia, microcytic anaemia, and malarial anaemia by public health authorities.With anaemia prevalence still well above the cut-off value of 40%, it is important to reevaluate childhood determinants that foster its presence and also revisit the implementation strategies that have so far been put in place. An anaemia-monitoring system, especially those aimed at reducing nutritional anaemia, will effectively help in curbing anaemia. While microcytic anaemia is closely moving to the red zone as a public health problem in urban areas of Mount Cameroon, malarial anaemia is of mild public health concern. The high microcytic anaemia prevalence in children aged ≤5 years, those living at low altitude and within the community, shows that some insidious factors other than malaria are at work. While age, parent's/guardian's occupation and ethnicity, and moderate parasitaemia were risk factors for microcytic anaemia, malarial anaemia had as risk factors child's age, parent's age and occupation, and a ≤4 times weekly consumption of plantains. Therefore, when planning interventions to reduce anaemia prevalence in the community, strategies that will elevate the socioeconomic status of the parents should be integrated to aid better and healthy food choices to reduce anaemia due to nutritional deficiencies.


One major issue that has set back the gains of the numerous malaria control interventions that national malaria control programs have implemented is asymptomatic malaria. Certain host genetic factors are known to influence symptomatic malaria; however, not much is known about how host genetics influences the acquisition of asymptomatic malaria. N = 109, and uninfected, N = 40) volunteers aged between 2 and 69 years from a high (Obom) and a low (Asutsuare) malaria transmission setting in Southern Ghana. Restriction fragment length polymorphism (RFLP) was used to determine polymorphisms at the MBL2 54, TNF-α 308, NOS2 954, and G6PD 202/376 gene loci.  Genomic DNA was extracted from whole blood collected from 60 symptomatic and 149 nonfebrile  and Obom (p=0.05). In Asutsuare, a low malaria transmission area, the allele G has significantly higher odds (3.15) of supporting asymptomatic malaria as against symptomatic malaria. There were significantly higher odds of TNF-α genotype GA being associated with symptomatic malaria as against asymptomatic malaria in both sites, Obom (p=0.027) and Asutsuare (p=0.027). The allele B of the G6PD gene was more prevalent in symptomatic rather than asymptomatic parasite-infected individuals in both Obom (p=0.001) and Asutsuare (p=0.003).  Polymorphisms at the MBL2 54 and TNF-α 308 GA genotype are more likely to exhibit symptoms of malaria when infected with the malaria parasite as opposed to harboring an asymptomatic infection. Also, the B allele of the G6PD gene is likely to prevent a P. falciparum-infected person from exhibiting symptoms and thereby promote asymptomatic parasite carriage. Individuals in Southern Ghana carrying the TNF- Plasmodium falciparum endemicity. Ghana is one of the countries hardest hit by malaria, accounting for about 2% of global malaria cases. Despite successes in malaria control in Ghana over the past decade, there were 161 cases per 1000 of the population at risk as of 2019 [Plasmodium falciparum  can result in different manifestations of malaria: asymptomatic and symptomatic. Symptomatic cases could be uncomplicated or complicated, and complicated cases could be severe malaria or cerebral malaria [Millions of individuals, particularly children and expectant mothers, suffer from malaria in areas of Africa known for  malaria , where t malaria . The use malaria , which oPlasmodium parasites in high transmission areas results in premunition  and the subsequent ability to maintain asymptomatic parasite carriage within a given population [P. falciparum infections that are detected during community surveys are asymptomatic [P. falciparum parasites in order to reduce the transmission and incidence of malaria.Persistent exposure to pulation . A very ptomatic . These aptomatic . It is tα) gene, and inducible nitric oxide synthase 2 (NOS2), on providing protection from or enhancing susceptibility to the disease have been identified [In symptomatic malaria, the effects of polymorphisms in some genes, including the beta globulin gene (HBB), glucose 6 phosphate dehydrogenase gene (G6PD), mannose-binding lectin gene (MBL2), tumor necrotic factor-alpha  and kills the potential pathogen by the membrane attack complex and complement-mediated phagocytosis [Mannose-binding lectin gene (MBL2) is a soluble pathogen-recognizing molecule that is involved in the activation of the complement system of the innate defence mechanisms. Point mutations in the promoter region and exon 1 of the MBL2 result in mannose-binding lectin (MBL) deficiency. Single nucleotide polymorphisms (SNPs) in MBL2, including G54 A, are known to result in reduced levels of functional MBL2 protein in circulation , which cocytosis  or assisocytosis , 25. Howocytosis , 21.α) is an inflammatory cytokine that results in apoptosis and necrosis and is also a mediator of tumor regression, an effector of cachexia amongst other functions [α gene is known to result in increased production of TNF-α [A polymorphism has less space for recurrent malaria [falciparum malaria [α has been related to antiparasitic activity, and persistently high levels of this cytokine lead to a rapid reduction in parasitaemia [Tumor necrotic factor-alpha  gene produces nitric oxide (NO), and polymorphisms in the promoter region, including a point mutation at position 954, have been suggested to exhibit enhanced NOS2 activity . There aThe study was conducted in two communities in the Greater Accra Region of Southern Ghana, Asutsuare and Obom . Obom isN = 84) and Asutsuare (N = 66). Another 60 study participants with microscopy-confirmed P. falciparum parasites and malaria-related symptoms, including a fever of ≥37.5 C at the time of sample collection, were recruited between March and September 2016 from the Obom Health Center (N = 40) and the Osodoku Community Health Center (N = 20) in Asutsuare.The samples for this study included 150 archived samples collected from afebrile  individuals as part of a cross-sectional community survey conducted in January/February 2016 (during the off-peak season) in both Obom (µl) of blood was used to spot the Urit 12  hemoglobin meter according to the manufacturer's instructions.Venous blood (1 ml) was collected from each participant into EDTA tubes. A drop  followed by a wash buffer (500 µl). Afterwards, 90 µl of elution buffer was added to the column to elute the DNA. The eluted genomic DNA (gDNA) was subsequently quantified using a nanodrop 2000C and either used immediately or stored at −20°C.Extraction of DNA was carried out on whole blood using the Quick-gDNA Mini Prep extraction kit  using the recommended manufacturer's protocol. Briefly, 400 Plasmodium falciparum species identification was done using nested PCR [µM with 30–50 ng of gDNA. The secondary nested amplification utilized P. falciparum species-specific primers . MRA102 g, gDNA extracted from the 3D7 P. falciparum strain was used as a positive control and distilled water (dH2O) as the negative control. The secondary PCR products were electrophoresed on 1.5% agarose gel containing 0.5 µg/ml ethidium bromide and subsequently visualized under UV, using a gel imager .sted PCR  as previsted PCR , 44 withα, G6PD, and NOS2 genes were amplified in a 25 µl PCR reaction mixture containing 15–30 ng of genomic DNA, 200 nM of each primer  , and 0.5 U of OneTaq Polymerase . The amplification was performed using the Mastercycler nexus  and annealing temperatures set at 61.6°C, 58°C, 60°C, 65°C, and 60°C, respectively. A list of all primers and restriction enzymes used is in Additional File Restriction fragment length polymorphism (RFLP) was used to determine all the gene polymorphisms, using previously published procedures , 44 withAll PCR amplicons were digested for 1 hr using 5 U of the appropriate restriction enzyme at the recommended temperature. The digested products were electrophoresed as described above, with the exception that the agarose gel was set at 2% . A total of 150 participants comprising 69% (58/84) and 77% (51/66) of the afebrile individuals from Obom and Asutsuare, respectively, tested positive for malaria parasites by microscopy and/or PCR and were classified as asymptomatic (AS). The remaining 31% (26/84) and 23% (15/66) of the afebrile individuals from Obom and Asutsuare, respectively, were free of malaria parasites and classified as uninfected (UN). The mean (SEM) age of the participants across the three malaria groups in both sites ranged between 20.9 (1.9) and 25.8 (2.4) years, with no statistical difference observed . The male to female ratio was also similar between the two sites (p=0.889). The prevalence of female participants ranged from 47% to 65% and was not significantly different amongst the three groups (p=0.311) (p < 0.05), and this was due to marked difference between the uninfected and the asymptomatic groups. The distribution of the participants amongst the three groups in both sites did not vary significantly . Hemoglobin data were available only for the asymptomatic and the uninfected group; the levels ranged between 12 g/dl and 13 g/dl and were uniformly distributed between the groups . All the participants with symptomatic malaria had a fever of 37.5°C or higher  ; howeverr higher .p=0.534) was observed in the distribution of allelic variants across the three groups (p=0.006). In Asutsuare, the MBL2 54 GA genotype had significantly higher odds of supporting asymptomatic infections compared to no infection (p=0.021) . In other words, allele G has significantly higher odds (3.15) of supporting asymptomatic malaria compared to symptomatic malaria in individuals from Asutsuare. In Obom, however, there was no significant difference in the odds of any of the MBL2 54 mutant genotypes or alleles supporting asymptomatic or symptomatic malaria  groups, and no significant difference (e groups . The wilp=0.021) . The odd malaria .α 308 variants amongst the three malaria groups (p=0.05) . There were significantly higher odds of TNF-α 308 genotype GA supporting symptomatic malaria compared to asymptomatic malaria in both sites, Obom (p=0.027) and Asutsuare (p=0.027) (p=0.034)  . In Asutp=0.027) . Additiop=0.034) .p=0.342). Also, in both Asutsuare and Obom, there were no significant differences in the odds of the three NOS2 954 genotypes supporting asymptomatic or symptomatic malaria compared to no infection ; however, a significant difference was observed in Asutsuare (p=0.042), most likely due to the very low prevalence of G6PD deficiency amongst the people in Asutsuare. In both Asutsuare and Obom, there was no significant difference in the odds of the G6PD “d” group supporting asymptomatic or symptomatic infections relative to no infection  participants as distributed in nfection . HoweverP. falciparum is a major obstacle to malaria control [P. falciparum parasite carriage [P. falciparum in asymptomatic infections. As such, polymorphisms in G6PD, MBL2, TNF-α, and NOS2 genes in a group of P. falciparum-infected and P. falciparum-uninfected adults and children living in a low and a high malaria transmission setting in Ghana were characterized.Asymptomatic carriage of  control , and the control , with vecarriage . This stSignificant variations in the distribution of MBL2 54 and G6PD variants were observed amongst the volunteers sampled from only the low transmission setting, Asutsuare. The variations observed in the distribution of G6PD variants are most likely due to the very low prevalence of G6PD deficiency amongst the people in Asutsuare, which could suggest that the prevalence of G6PD deficient (G6PDd) variants decreased with decreasing transmission intensity. Studies have shown a higher frequency of G6PDd variants in high endemic areas explaining the fact that G6PDd variants increased with increasing transmission intensity, whereas the prevalence of G6PD deficiency in the high transmission setting, Obom, was higher and did not vary significantly amongst the different groups of people sampled in this study.G support asymptomatic parasite carriage in Asutsuare, which is a low malaria transmission area, where the MBL 54 GA genotype was associated with a 4-fold increase in the likelihood of a person harboring malaria parasites in an asymptomatic infection compared to the individual not having an infection. The mutant G allele also increased the likelihood of individuals having an asymptomatic infection by 3-fold relative to exhibiting symptoms of malaria when infected with the malaria parasite. The association of the mutant allele with asymptomatic infection is not surprising because the mutant exhibits much lower functional activity compared to the wild type, and considering the role played by MBL2 in complement activation and innate defence mechanisms [P. falciparum transmission. Some studies have identified the TNF-α 308AA to be associated with severe malaria [α 308 mutations with malaria parasite carriage [α 308 GA and asymptomatic malaria amongst nonfebrile children [α 308 GA was not able to differentiate between asymptomatic and symptomatic malaria [α 308 GA mutation. TNF-α 308 GA resulted in a significant increase in the odds of a person infected with malaria parasites exhibiting symptoms (being symptomatic) in both low and high malaria transmission settings. This suggests that the TNF-α 308 GA mutation makes a person susceptible to symptomatic malaria episodes irrespective of the level of malaria transmission in the community. TNF-α 308 GA mutation increases the production of TNF [α 308 GA would immediately seek treatment for malaria as they would exhibit symptoms and not add to the transmission reservoir of asymptomatic carriers. This will subsequently result in a reduction of the infectious reservoirs associated with asymptomatic parasite carriage. Additionally, in the high malaria transmission area (Obom), the mutant allele A of TNF-α 308 was found to significantly reduce the risk of asymptomatic infection compared to no infection in afebrile individuals. This also supports a reduction in the prevalence of transmission reservoirs.The data show that MBL2 54 GA genotype and allele chanisms , it is echanisms  and in Ichanisms  show ass malaria , whilst carriage . In a stchildren , and anon of TNF , 22, andn of TNF –53, hencThe G6PD deficiency status of the volunteers in this study was not associated with malaria outcomes, symptomatic, asymptomatic, or uninfected. One likely reason for this outcome is that the G6PD genotype does not always correlate with the phenotype (enzyme activity), such that some people with a genetic deficiency possess normal enzyme activity , 55. ThiP. falciparum carriage, episodes of symptomatic malaria, and the development of immunity in the study volunteers is required to validate the observations made in this study.Polymorphisms at the NOS2 954 loci were similarly distributed in the three categories of volunteers and were not associated with disease outcomes. No reports exist on the association of NOS2 954 genotype with asymptomatic or symptomatic malaria; however, some findings point to its association with disease severity, though they are inconsistent , 58. ThePlasmodium falciparum-infected individuals living in both high and low malaria transmission settings who carry the TNF-α 308 GA genotype are more likely to exhibit symptoms of malaria, whilst those with the B allele of the G6PD gene are likely to remain asymptomatic. Significant differences in the distribution of MBL 54 variants were observed amongst volunteers from the low transmission area, where the MBL 54 GA genotype was found to be associated with a 4-fold increase in asymptomatic malaria parasite carriage amongst afebrile volunteers.Put together, our data suggest that host genetics may play a role in asymptomatic malaria parasite carriage and, in effect, the sustenance of malaria transmission. Thus, migration and the general movement of individuals into and out of especially low transmission settings can significantly influence the dynamics and patterns of malaria transmission.P. falciparum carriage by asymptomatic volunteers was not done beyond five days. This means that we could not rule out the possibility of some of the asymptomatic volunteers developing symptoms thereafter. The uneven distribution of females in the groups could have caused a bias in the distribution of G6PD genotypes identified in this study.Due to logistical constraints, the monitoring of


Currently 12 human polyomaviruses (HPyVs) have been identified, 6 of which have been associated with human diseases, including cancer. The discovery of the Merkel cell polyomavirus and its role in the etiopathogenesis in the majority of Merkel cell carcinomas has drawn significant attention, also to other novel HPyVs. In 2010, HPyV6 and HPyV7 were identified in healthy skin swabs. Ever since it has been speculated that they might contribute to the etiopathogenesis of skin and non-cutaneous human cancers.Here we comprehensively reviewed and summarized the current evidence potentially indicating an involvement of HPyV6 and HPyV7 in the etiopathogenesis of neoplastic human diseases. The seroprevalence of both HPyV6 and 7 is high in a normal population and increases with age. In skin cancer tissues, HPyV6- DNA was far more often prevalent than HPyV7 in contrast to cancers of other anatomic sites, in which HPyV7 DNA was more frequently detected.It is remarkable to find that the detection rate of HPyV6-DNA in tissues of skin malignancies is higher than HPyV7-DNA and may indicate a role of HPyV6 in the etiopathogenesis of the respected skin cancers. However, the sheer presence of viral DNA is not enough to prove a role in the etiopathogenesis of these cancers. Polyomaviruses (PyV’s) comprise a family of non-enveloped viruses containing a small genome of approximately 5.0 kbp in size of circular double-stranded DNA and are capable of infecting mammals and birds –3. PyV’sIn 2008, Merkel cell polyomavirus (MCPyV) has been linked to the etiopathogenesis of the majority (i.e. 80%) of the highly aggressive neuroendocrine Merkel cell carcinoma (MCC) –17. AlreIn 2010, HPyV6 and HPyV7 were identified from healthy skin swabs using the rolling circle amplification technique . The HPySeveral studies have assessed HPyV6 and HPyV7 serum antibody levels across a broad range of age groups of various populations , 37–41. Altogether, both HPyV6 and 7 were found to be ubiquitous and to infect all age groups, and HPyV6 seroprevalence is slightly higher than HPyV7. Seroprevalence significantly increases with age, and a substantial proportion of individuals 50 years of age and older has tested positive for HPyV6 and HPyV7. Approximately 52–93% of humans are seropositive for HPyV6, whereas 33–84% are seropositive for HPyV7 according to the seroprevalence studies Table In general, HPyV6 and 7 infections seem to be clinically rather asymptomatic, irrespective of the age at which the infection occurs. Increasing seroprevalence of these viruses is clearly age-related and reaches its peak in individuals beyond 40 years of age, possibly indicating a relationship of HPyV6 and 7 infections with immunosenescence. Regarding the methodology, the previous studies reported the seroprevalence of HPyV6 and HPyV7 in the sera obtained from blood donors or routine lab tests in various studies. Most of the studies used ELISA or Luminex platform antibody-binding assay methods to detect the antigen containing the structural viral capsid protein of the targeted viruses fused with VLP or glutathione S-transferase (GST) proteins Table Only a very limited number of studies have reported seroprevalence data of HPyV6 and 7 in cancer patients. Madeleine et al. recently reported that the seroprevalence of HPyV6 in immunosuppressed skin cancer patients 59.1%) was lower compared to healthy controls (66.2%), whereas the seroprevalence of HPyV7 in the same patient cohort was slightly elevated (52%) compared to the control group (41%) . It is i% was lowIn the past, studies on the seroprevalence of other human tumor viruses, e.g. Epstein-Barr virus or human papillomaviruses (HPV), have helped to indirectly indicate whether or not these viruses are involved in the etiopathogenesis of human cancers –48. MoreA limited number of studies have screened for the prevalence of HPyV6- and HPyV7-DNA in non-neoplastic diseases, with very low prevalence across a variety of specimens Table Studies conducted on patients with cutaneous diseases have reported the presence of both HPyV6- and 7-DNA in tissue specimens. Three dyskeratotic dermatosis cases tissue revealed 2 positives for HPyV6 and 1 positive for HPyV7, and in previously 12 HPV-induced warts tested showed that 3 positives for HPyV6 and 8 for HPyV7 , 53. In The prevalence of HPyV6- and 7-DNA in non-neoplastic non-cutaneous diseases appears to be much lower compared to non-neoplastic skin diseases. Our group reported the presence of HPyV7-DNA in  of non-malignant thymic hyperplasia . Other sAccording to the World Health Organization, the incidence of skin tumors has increased over the past decades, approximately 8500 new cases of skin cancers are reported daily in the United States , 67. Up One study reported the presence of HPyV6- and HPyV7-DNA 2/47; 4.3%) in malignant melanoma (MM) specimens using rt-PCR . In non-% in maliAdditionally, primary cutaneous lymphomas have been tested for the presence of HPyV6- and 7-DNA: cutaneous T-cell lymphoma (CTCL) patients, and all three studies showed that CTCLs were positive (5.20 to 20%) for HPyV6- and (0.90 to 8.6%) for HPyV7-DNA, while no cutaneous B-cell lymphoma was positive for either virus , 70, 71.It is remarkable that HPyV6-DNA is far more often detected than HPyV7-DNA in human neoplastic skin diseases Fig. , which mThe prevalence of HPyV6 and 7 have also been studied in a variety of non-cutaneous malignancies summarized in Table In body fluids specimens, Chan et al. investigated the presence of HPyVs in the bile fluid of patients with a variety of hepatobiliary malignancies . HPyV6 bOverall, among patients with different non-cutaneous cancers, HPyV7-DNA was more frequently detected than HPyV6-DNA in tissues. Yet, it remains unclear if and how HPyV7 might contribute to the tumorigenesis of these thymic epithelial tumors or cholangiocarcinomas, or that it rather is present in the context of viral latency.The frequent prevalence of HPyV6- and 7-DNA in non-neoplastic tissues along with their high seropositivity indicates that these viruses remain in long-term latency in humans. Therefore, it is important to unmask the host cells facilitating this long-term latency in healthy individuals to understand the host cell-type specific encoded proteins needed for viral entrance, replication, and transcription.So far, the underlying mechanisms of tissue tropism or the possible latency of HPyV6 and 7 are still unknown. Other HPyVs have been reported to remain in latency within the epithelium of the proximal tubule of the kidney (BKPyV) while JCPyV is found in the brain . In otheThe potential contribution of oncogenic viruses is either direct and/or indirect in the carcinogenesis of virus-associated tumors , 81. MCPHuman tumor viruses also can induce neoplasms by indirect mechanisms via the interaction between viral antigens, environmental factors, and other modifications to the host genome . InflammBesides the direct and indirect HPyVs-related carcinogenesis, the “hit and run” hypothesis has been proposed to explain a possible role of viral agents to mediate cellular transformation . The “hiIn addition, HPyVs may contribute to human tumorigenesis together with other oncogenic viruses assuming, that two or maybe more different viruses potentially infect the same cells and interact with each other in the etiopathogenesis of human cancers –100. PriThe HPyV6 and 7 contributions to human tumorigenesis are not yet elucidated. Therefore, several important questions regarding the pathogenesis of both viruses need to be addressed. For example, how to confirm the interaction of HPyV6 and HPyV7 in the inactivation of p53 and retinoblastoma protein (pRb), or in the transformation function. Rozenblatt-Rosen et al. namely proposed this mechanism in which HPyV6 and 7 both LTAg and sTAg proteins to bind p53 and inactivate it leading to tumorigenesis . MCPyV iThe presence of HPyV6- and 7-DNA and their association with human malignancies have commonly been tested by PCR and qPCR. However, the conclusions that can be drawn from these studies restricting their level of evidence to the proof of viral nucleic acids in homogenized tissue extracts are unfortunately very limited. The meaning of the presence of viral nucleic acids must remain inconclusive because these findings cannot be interpreted on the single-cell level in the histomorphological context of the specific disease. In addition, only limited reliable data are available on the viral protein expression, because the detection of antibodies by IHC in FFPE tissues are lacking. A possible screening approach might start with the use of the PAb416 antibody followed by specific HPyV6 and 7 PCR. In addition, studies definitely are needed in order to investigate next to the presence of HPyV6-DNA and 7-DNA, if these viruses are integrated into the tumor genome and possibly carry tumor-specific mutations within the viral helicase of LTAg in analogy to MCPyV , 17. ThiIn this review, we aimed to comprehensively analyze the literature for a possible role of the two closely related HPyV6 and 7 in human diseases and cancers. The seropositivity of HPyV6 was found to be higher than HPyV7 and both increased with age. The etiopathogenetical contribution of these viruses to human cancers remains unclear. Studies revealed that low HPyV6 and HPyV7 prevalence in non-neoplastic tumors resembled those in malignancies. Interestingly, HPyV6 prevalence was higher in skin malignancies than that of HPyV7. In contrast, HPyV7 was more frequently detected in non-cutaneous malignancies compared to HPyV6.Based on sero- and tissue-prevalence HPyV6 and 7 remain important putative candidates for contributing to the etiopathogenesis of human disease, including skin cancers. There is an urgent need to further define their presence within cutaneous lesions e.g., keratoacanthoma, squamous cell carcinoma, and basal cell carcinoma, and to apply additional molecular techniques to unravel the molecular basis of virus-host interactions and assess a possible role of viral integration or mutation of the LTAg in skin cancer development. The same applies for HPyV7-associated tumors e.g., cholangiocellular carcinomas or thymomas. Further studies are needed to understand the contribution of HPyV6 and HPyV7 to human cancers.


Since 2000, a good deal of progress has been made in malaria control. However, there is still an unacceptably high burden of the disease and numerous challenges limiting advancement towards its elimination and ultimate eradication. Among the challenges is the antimalarial drug resistance, which has been documented for almost all antimalarial drugs in current use. As a result, the malaria research community is working on the modification of existing treatments as well as the discovery and development of new drugs to counter the resistance challenges. To this effect, many products are in the pipeline and expected to be marketed soon. In addition to drug and vaccine development, mass drug administration (MDA) is under scientific scrutiny as an important strategy for effective utilization of the developed products. This review discusses the challenges related to malaria elimination, ongoing approaches to tackle the impact of drug-resistant malaria, and upcoming antimalarial drugs. Malaria control, elimination, and eradication are the long-term agendas of malaria endemic countries (MECs). Currently, almost all endemic countries are working to eliminate it with the ultimate goal of making the world free from this killer disease. Achievement of this goal depends on effective and integrated implementation of both preventive strategies and case management . HoweverIn order to deal with multi-drug-resistant parasites, Artemisinin-based Combination Therapies (ACT) has been recommended. However, an insufficient supply of affordable and quality-assured ACTs has opened the room for parallel circulation of falsified and substandard products—antimalarial drugs appearing among the top lists , 8. The The emergence of artemisinin resistance, its spread to Southeast Asia (SEA) countries and some resistance reports from African countries are the indicators of the current threat to malaria control and elimination effort , 15. ThiSeveral reasons have been forwarded as challenges, especially, for antimalarial drug discovery . Given tAs has well been recognized from the ACTs, optimization of treatment regimen through either dosage modification or combined use of drugs with different mechanisms of actions, favors antimalarial activity , 23. HowOne of the potential options is increasing the total dose given to patients, as has historically been done with several antimalarial drugs like chloroquine (CQ) . AnotherBased on the experience from the combination drug treatment of other infectious diseases, in addition to working on the dose and treatment duration of the usual ACT combinations, triple ACTs is also under way . In thisatovaquone on pyrimidine metabolism have led to the development of new compounds targeting dihydroorotate dehydrogenase (DHODH), DSM265, and dihydrofolate reductase (DHFR), P218, - overcoming resistance issues of pyrimethamine /high-[K+] in the cytosol. Inhibition of this target causes an increase in the parasite's cytosolic Na + level and the resulting osmotic water uptake leads to parasite death. Additional benefit of this target is its accessibility on parasite plasma membrane so that the drugs do not need to pass through the membrane.Following the discovery of the first spiroindolones compound, KAE609 , targeti(4) Phosphatidylinositol-4 Kinase (PfPI4K). Another new antimalarial drug target operating in all stages of the Plasmodium life cycle is phosphatidylinositol-4-kinase (PI4K). PfPI4K is a ubiquitous eukaryotic enzyme that phosphorylates lipids, at position 4 of the inositol ring, and subsequently synthesizes phosphatidylinositol 4-phosphate (PI4P) as depicted in Plasmodium species at each stage of infection. These compounds displayed potent preventive, therapeutic, and transmission-blocking activities in rodent malaria models as well as blood-stage field isolates. The activity of Imidazopyrazines is reported to likely stem from altered phosphatidylinositol 4-phosphate (PI4P) pools and disrupted Rab11A-mediated membrane trafficking because of the blockade of a late step in parasite development [Imidazopyrazines were the first class of compounds identified as potent inhibitors of PI4K that inhibit the intracellular development of multiple elopment . Subsequelopment .(5) DOXP Reductoisomerase (PfDOXPR). A mevalonate independent isoprenoid synthesis pathway, the 1-deoxy-d-xylulose 5-phosphate (DOXP) pathway or methylerythritol phosphate (MEP), which is probably located in the apicoplast of plasmodium falciparum, is essential for the production of isoprene derivatives and shown to be a potential target for malaria chemotherapy [C-methyl-d-erythritol 4-phosphate, which is catalysed by DOXP synthase and DOXP reductoisomerase.otherapy . In thisThe inhibition of one of the key enzymes of this pathway, DOXP reductoisomerase, with fosmidomycin topped with its absence in mammals makes this pathway one of the potential new drug targets with low toxicity , 70. Cur1 (PfCTYbc1)(6) Cytochrome bc. In erythrocytic Plasmodium falciparum, mitochondrial electron transport supports pyrimidine biosynthesis. During the biosynthesis, ubiquinone functions as the electron acceptor for dihydroorotate dehydrogenase (DHODH). Regeneration of ubiquinone should, therefore, be in place for the proper functioning of this essential enzyme in Plasmodium species. This metabolic function in turn requires active mitochondrial electron transport chain (mETC). Cytochrome bc1 complex provides oxidized ubiquinone to DHODH through catalysing the transfer of electrons from ubiquinol to cytochrome c as shown in atovaquone [ovaquone .b, Y268S, and atovaquone resistance. Such mutation is reported to cause late treatment failure thereby reducing hydrophobic interactions between Cyt bc1 protein complex and atovaquone. Following this there has been increased effort to identify alternative cytochrome bc1 inhibitors with limited or no cross-resistance with atovaquine and likely to be much less expensive. One of the potential drugs that has been reported to be selective and specific inhibitor of P. falciparum mitochondrial bc1 complex is decoquinate [ELQ-300 is also another drug being developed as a long-acting injectable chemo-protective agent. This parasiticidal drug is reported to inhibit cytochrome bc1 complex of Plasmodium parasites and bypass resistant mutations to atovaquone [Molecular analysis of samples collected from recrudescent malaria cases –75 and moquinate . As compoquinate . ELQ-300ovaquone , 80.The future goal of transmission blockade, elimination and eradication of malaria requires integrated approach involving improved diagnostics, vector control, vaccination, as well as chemotherapy and chemoprophylaxis . In addiOn top of treatment with effective antimalarials and mosquito control measures, to realize malaria eradication agenda, it is unwise to exclude the role of malaria vaccine. As a result, malaria vaccines have long been a research priority and currently, several vaccine trials, targeting one or more stages of the parasite life cycle, are underway. Based on the stages to be targeted, the vaccines can be categorized as preerythrocytic vaccines, blood-stage vaccines and transmission-blocking vaccines . There iP. falciparum malaria and conducted in African children. During the trial, it was administered with or without booster and prevented a substantial number of cases of clinical malaria over a three- to four-year period in young infants and children. Administration of a booster dose was reported to enhance the efficacy of the vaccine. It displayed 50% efficacy for clinical malaria in children aged 5–17 months old; while only 30% in target population, infants. Febrile seizures and other safety signals  were identified during the Phase 3 trial [The first vaccine to successfully complete a Phase III clinical trial and get a positive regulatory assessment is the RTS, S/AS01 vaccine , 82. It  3 trial .P. falciparum malaria vaccine candidates, targeting all stages of the parasite lifecycle, are in early-stage development with the most advanced in Phase 2 trials [This vaccine has demonstrated the possibility of malaria vaccine development and augments the strategic goals of updated malaria vaccine technology roadmap to be met by 2030. The goal is to have vaccines, by 2030, that can provide at least 75% protective efficacy against clinical malaria, reduce transmission of the parasite, and can be deployed in mass campaigns , 85. To 2 trials , 86.falciparum malaria to bring rapid reduction of malaria related morbidity and mortality through reduced transmission. In order to apply MDA, the following criteria are required to be in place: being close to interrupt transmission, vector control, effective surveillance, and access to case management are at high coverage, and importation of infection is minimal; or for malaria epidemics [Recently, there has been renewed interest in an old and often controversial approach called mass drug administration (MDA). It is also called targeted malaria elimination, targeted malaria treatment, or targeted parasite elimination . Notablypidemics . Predictpidemics .P. falciparum cases were observed for at least a year after application of three rounds of MDA (a three-day course of DHA-PPQ plus PQ) and the regimen was reported to be safe with high coverage in Cambodia [According to WHO, MDA consists of the administration of a full therapeutic course of antimalarial medicine to every member of a defined population or person living in a defined geographical area (except for those for whom the medicine is contraindicated) at approximately the same time and often at repeated intervals . Based oCambodia .However, rising PPQ resistance, PQ safety, and undefined first-trimester safety of products considered for MDA, which are being deployed to areas where pregnancies are not usually reported in the first trimester, may threaten the effectiveness of this approach , 26. FurPlasmodium as well as emergence and spread of resistance against the parasite underlines the urgent need for continuous pipeline of new medicines with novel mechanisms of action [P. vivax. Despite the long time it takes to make them available to the market, several compounds that can be partnered with other drugs in combination therapies are being rigorously tested and are progressing through the pipeline currently [Infectious nature of f action . On top f action . Furtherurrently , 33, 91.The evidence reporting Phase II trial result of KAE609, a new medicine from novel class of antimalarials called spiroindolones, has opened new milestone in antimalarial drug research . CurrentP. vivax malaria [All new medicines in phase I and II are blood schizonticidals; all but DSM265 have transmission blocking activity and the majority have chemo- protective potential . Those i malaria .Beyond discovering new and effective drugs, there is another challenge of identification of best possible partner drug to combine with the new one. Extreme potency and tolerance required from such a SERCaP combination products makes the problem tougher . CurrentDespite the threat related to ART-R, there is still considerable interest for endoperoxide compounds. This is mainly dictated by the clinical success of ART and the longer duration of the action of these compounds, which could potentially counteract the K13-mediated ART-R . As a reP. falciparum and P. vivax parasites in patients [Plasmodium falciparum cyclic amine resistance locus (Pfcarl), is active against both asexual and sexual blood stages as well as the preerythrocytic stage of the parasite [KAF156 (Ganaplacide) belongs to imidazolopiperazines class of antimalarial molecules and brings about rapid clearance of both patients , 96. KAFparasite . There iparasite .Plasmodium falciparum [P. vivax or P. falciparum malaria through inhibition of PfATP4. It has the shortest parasite clearance time (<1 h) than any other antimalarials discovered so far [DSM265, a triazolopyrimidine-based inhibitor of DHODH and the first inhibitor of this enzyme to reach phase II clinical trial, is also reported to be safe and efficacious, in a single dose, against both hepatic and blood schizonts of lciparum , 49. A dd so far . Cipargad so far . Modified so far .radical cure though elimination of P. vivax hypnozoite and also shares the risk of haemolytic side effects of PQ in case of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Unlike 14 day regimen with PQ, it will offer a single-dose cure for the liver-stage of P. vivax infections when administered with a standard 3-day CQ or potentially an ACT treatment regimen [P. falciparum and P. vivax across all stages of the parasite lifecycle but discontinued due to its teratogenic effect [The only investigational medicine that currently completed phase III studies is TQ, which has been studied by GlaxoSmithKline in partnership with MMV. TQ is the same 8-aminoquinoline scaffold of PQ that brings  regimen . MMV048, regimen , 105, wac effect , 91.atovaquone, on the other hand, failed to become a first-line treatment probably due to high cost and immediate emergence of resistance [Variety of factors compromised the utilization of previously developed antimalarials and also the current attempts to maintain the efficacy of ARTs. One of such challenges is the strict need for fixed dose combination, where modification of ART dose will lead to increase in partner drug dose resulting in increased toxicity and adherence issues. Extending the course of treatment would also raise adherence issue as well as increased cost. Furthermore, drugs to be partnered with ART are also coming up with their own problems like toxicity, low efficacy and cross-resistance. Risk of hepatotoxicity with PN and low efficacy of PN–artesunate combination has cast doubt about the usefulness of the regimen as universal first-line treatment , 107. Desistance .These flaws of earlier antimalarial drugs are, currently, raising the question about the promise of new candidates in the pipeline. Beside the history, there are also current lines of evidence to be sceptical about these products. For instance, there is a risk of earlier loss of KAE 609 and KAF 156 because of their low genetic barrier , 98 and In the face of resistance against first-line antimalarial drugs and other related bottlenecks, sustained effort in developing new malaria tools is critical to meet the goals of malaria elimination and eradication. The current portfolio on development of the tools seems healthy and on good progress. However, time will tell how many of these products will effectively reach to the world's poor and contribute for malaria eradication. In general, unless complemented by such new products, the current interventions might be hardly enough to eradicate malaria.