Plasmodium vivax malaria on the executive and cognitive functions of elderlies, in the Brazilian Amazon. A cohort study was conducted to evaluate executive and cognitive functions one week (T0), two months (T2) and eight months (T8) after the malaria episode. Mini-Mental State Examination (MMSE), Beck Depression Inventory II (BDI-II), Clock Drawing Test (CDT), Wechsler adult intelligence scale (WAIS-III), and Wisconsin Card Sorting Test (WCST) were used to assess executive and cognitive functions. One hundred-forty elderlies were enrolled . P. vivax malaria was associated with impairment of the executive and cognitive functions in elderlies for up to 8 months after acute P. vivax malaria. Prior history of malaria, recurrences and higher parasitemia were independently associated with various surrogates of executive and cognitive impairment. With the increase in life expectancy, elderlies living in malaria endemic areas will deserve more attention from health authorities, to guarantee improvement of their quality of life in the tropics.The exact path leading to cognitive impairment that goes beyond malaria is unclear, but it appears to be the result of interactive factors. Time of exposure to disease and recurrences are potentially major determinant variables. Cognitive impairment is described mainly in children, rarely in adults. The disease in high endemic areas usually does not affect elderlies, because of acquired immunity over time. However, this population is relatively more frequently sick in lower endemic areas, such as in the Amazon. This study assessed the effect of  Plasmodium vivax infection1. Although predominant globally, for a long time it was believed that P. vivax malaria was always associated with benign disease2. However, in recent decades, severe cases and deaths from P. vivax malaria have been reported3. The main complications of severe P. vivax malaria include pulmonary edema, severe acute respiratory syndrome, acute renal failure, severe anemia, hemorrhage, acidosis, and disseminated intravascular coagulation5. Cerebral malaria (CM), a potentially fatal condition associated with P. falciparum infection, has also been reported in P. vivax mono-infection in children, but is rare6.Approximately a third of the global population is at risk for P. falciparum (CMF), which could be a potential cause of short- and long-term neurological and cognitive deficit in Subsaharan African children7. Few studies documented the effects of CMF on adults’ cognitive performance and none of these studies tested all the five facets of cognition  making it difficult to identify patterns of cognitive impairment.A few studies address the association between malaria and cognition, particularly after the acute phase of the disease, during convalescence. Those few studies usually focus on cognition following the development of CM triggered by 8. Patterns of cognitive impairment may differ between children and adults, with age of exposure to disease being an important variable, as well as repetitive infections9.The exact path leading to cognitive impairment that goes beyond CM is unclear, but it appears to be the result of many potentially interactions. There are many risk factors, such as anemia, multiple infections, hippocampal dysfunction, damage to sub-cortical white matte, neurotoxins released from infected red blood cells, which may damage cortical areas of the brain, and cytokine stormP. vivax14 or P. falciparum malaria16, in non-severe malaria, the impact on adults is not well addressed, especially in vivax malaria. Four studies investigated the impact of infection due to non-severe vivax malaria on cognition. A study conducted in Sri Lanka and determined the short-term impact of malaria on the cognitive performance of 571 schoolchildren (ages 1–8 years)10. Oliveira-Ferreira et al.11, in a cross-sectional study carried out in the city of Careiro, Amazonas, with 198 students (aged 6–14 years) identified impairment in school performance. Brasil et al.12, in a study on the Marajó Island, Pará, with 17 schoolchildren (aged 2–10 years) demonstrated that children with a history of vivax malaria presented significant impairments in the cognitive, affective, instrumental domains (problem solving in activities of daily living) and in verbal comprehension . Tapajós et al.13, in a study with 219 schoolchildren (aged 2–7 years) in the community of Brasileirinho, Manaus, Amazonas, revealed that vivax malaria is a risk factor for low cognitive development.Thus, although few studies evaluate the cognitive impairment of children after P. vivax malaria on executive and cognitive functions in such age group, in the Brazilian Amazon, where about 89,3% of malaria cases are caused by P. vivax in 201914. Thus, due to the lack of knowledge on the subject and local epidemiological issues, it was decided to study exclusively P. vivax infection.To our knowledge, there is no report on the assessment of cognition in elderlies after malarial infection, especially vivax malaria. This study assessed the effect of Fundação de Medicina Tropical Dr Heitor Vieira Dourado (FMT-HVD) (CAAE: 71396317.7.0000.0005). All participants were informed about the objectives and risks of participation and signed informed consent terms. This study was conducted in accordance with the principles of the Declaration of Helsinki and the guidelines of Good Clinical Practice of the International Harmonization Conference.This study was approved by the Human Research Ethics Committee of This was a cohort study, with selection of malarial exposed and non-exposed without malaria, in which executive and cognitive functions were evaluated during an 8-month follow-up. Elderlies’ evaluations were performed during the first week after malaria infection (T0), two months (T2) and eight months (T8) later.15. The non-exposed group consisted of elderlies in the same age group, without malaria in the last six months of the interview, living in the same locality as the cases. Participants with any history of neurological or psychiatric impairment were not included in the study.The study was carried out from February 2018 to November 2020 at FMT-HVD, a reference institution for infectious diseases in Manaus, Amazonas State. After malaria diagnosis by thick blood smear, patients aged ≥ 60 years, both genders, were invited to participate. In case of interest in participating in the study, they received a domiciliary visit during the first week after the diagnosis. Vivax malaria patients were treated according to the Brazilian malaria treatment protocol1. All participants (exposed and unexposed) were instructed to seek the care unit if they manifested symptoms. In addition, all participants were followed in online official Malaria Epidemiological Surveillance Information System . At the time of assessment of executive and cognitive functions, in T2 and T8, peripheral venous blood was collected for assessment of asymptomatic infection by molecular techniques For DNA extraction, the miniblod Qiagen Kit was used, according to the manufacturer's instructions. Qmal Taqman PCR was used to detect Plasmodium spp. in DNA samples, as previously described16. Malaria recurrences over the study period have been confirmed in Sivep-Malária. Patients diagnosed with malaria were treated according to the Brazilian Ministry of Health's treatment guidelines.Thick blood smears were performed for the malaria diagnosis Clique ou toque aqui para inserir o texto. in all symptomatic patientsHepatitis C, HIV and syphilis were also investigated by serological rapid tests (Abbot) performed according to the manufacturer's recommended instructions, as potential concomitant causes of cognitive impairment at T0. At T2 and T8, blood samples were also collected to assess blood glucose and hemoglobin measurements, as uncontrolled diabetes and anemia are also potential confounders.17. Instruments used and their respective domains evaluated are summarized in Supplementary Table Participants answered to the socio-demographic cognition assessment form  at T0. At T2 and T8, MMSE, CDT, Beck depression inventory II (BDI-II instruments), subtests  of the Wechsler adult intelligence scale (WAIS-III), and Wisconsin Card Sorting Test (WCST) were applied. Global cognitive and executive functions were assessed by tests validated for the Brazilian population. The Addenbrooke’s cognitive examination III (ACE-III), a standard cognitive assessment, was undergoing validation during the study period18. BDI-II is a widely used self-report inventory measuring the severity of depression through 21-items19. CDT measures cognitive assessment and is used in the investigation of the impairment of some cognitive skills such as visual-constructive functions, visuo-spatial functions, symbolic and graphomotor representation, auditory language, semantic memory, and executive functions20.MMSE is an instrument that assesses mental state, more specifically the symptoms of dementia, through questions that assess orientation in time and place, registration and evocation of words, attention and ability to simple calculations, language, and visual constructive capacity21. As a measure of executive functions (EFs), the Working Memory Index (WMI) was used in these studies and was obtained through the weighted score of the subtests, digits in direct order and digits on the back, arithmetic and sequential numbers and letters. The correlation of their results with brain location has been the subject of many studies and has contributed significantly to the understanding of the action of the prefrontal cortex, specifically as dorsolateral and ventromedial subregions22.The WAIS-III is an advanced cognitive assessment instrument used to assess intellectual ability in adults and is composed of several subtests that measure different aspects of intelligence23. It evaluates strategic planning, organized search, the use of feedback from the environment to change cognitive strategies, the direction of behavior to achieve goals and the modulation of impulsive responses. The WCST assessment, therefore, can be used to assess and identify cognitive impairments and neurological conditions related to the frontal region of the brain that has deficits23. In this study, the WCST scores: "number of trials administered", "total number of errors"," trials to complete the first category", and "learning to learn", were not considered. The first three are redundant and the last one has the power to bias studies24.WCST is an internationally recognized instrument for assessing EFs and is frequently adopted in neuropsychological assessmentsP. falciparum in adults25. For the general population, the prevalence was assumed to be 6.1%26. Thus, considering a test of differences in proportions between two groups of the same size, 90% of power and 5% alpha error, 244 participants were required (122 per group). Adding 15% of losses, the final sample size was ~ 280 participants. Sample calculation was performed using software and R, version 4.0.5 27. Interim analysis was scheduled after the inclusion and follow-up of 140 patients.The sample size calculation assumed a 20% prevalence of cognitive impairment due to infection caused by Normal distribution was assessed for variables with the Shapiro–Wilk test. Differences between baseline characteristics were evaluated by ANOVA, for continuous variables with normal distribution, Wilcoxon rank-sum test for continuous variables not normally distributed, and Pearson's Chi Squared test was applied for categorical variables. Multivariate analysis included age, sex, and education variables, as well as depression (expressed by BDI-II indicators).3. Previous univariate analysis was performed for multivariable variable selection. Variables with a p-value lower than 0.2 were included. All analyses were conducted in Stata 13.0 .The main and subgroup analyzes were performed with Poisson regression. The main analyzes compared executive and cognitive fucntions between subjects with malaria and without malaria. Additional subgroup analysis was performed for malaria patients considering three variables: (1) malaria episodes before enrollment; (2) recurrences between T0 and T8; and parasitemia higher than the median number of parasites/mmOut of 470 subjects, 140 were considered eligible (70 exposed and 70 non exposed) . However, in the multivariate analysis, no differences were found , even though the marginal significance indicated a trend in worst performance in the exposed group. Otherwise, the malaria group had worse performance in cognitive  and executive functions , both at T2 and T8, independently of age, sex, education and depression  and T8 (WCST categories: p < 0.05) . In this study, we used tools with different sensitivities, which resulted in different results regarding the determination of cognitive and executive functions. However, it is important to emphasize that screening tests MMSE and CDT have high specificity and are used to signal mild cognitive impairment and a maximum number of suspected psychological processes and are suitable to safely exclude false positives. The other tests used WCST and (WAIS/WMI) are high sensitivity instruments and used to confirm any psychological condition and confirm true positives, and are used to signal mild cognitive impairment and a maximum number of suspected psychological processes8 and cytomegalovirus (CMV)37, HIV41, and bacteria, such as Chlamydia pneumoniae8 and Helicobacter pylori8 and Lyme neuroborreliosis42 have been associated with cognitive impairment. In older adults with pneumonia and urinary tract infection, for example, the risk of cognitive loss is about 1.4 times higher and previous episodes are positively associated with cognitive loss43. Inflammatory reactions have been highly related to cognitive decline and risk of dementia44. Inflammatory cytokines produced by the nervous system act on neural substrates to produce behavioral symptoms that can be related to cognitive changes45. Thus, like the diseases, vivax malaria is capable of significantly altering cognitive and executing functions for up to 8 months, which, in turn, may affect the quality of life of the elderly.Infections by herpes simplex virus type 1 (HSV-1)46. In the elderlies, we observed similar effects up to 8 months after the malaria episode. Parasitemia at T0, number of previous and recurrent malaria had a negative impact on cognition and executive functions.In children, parasitemia, the number of previous and recurrent malaria has a negative impact on cognition and executive functions, with a cumulative negative effect on school performance being observedPlasmodium berghei ANKA (PBA) strain, are a valuable tool for understanding the cognitive and neurological outcomes associated with this condition48. Studies with an animal model are focused on seeking to understand mainly the cognitive and neurological mechanisms, based on the implementation of CM. Neuroinflammation after PBA infection of mice influences neurotrophin expression, which impairs hippocampal neurogenesis and increases hippocampal cell death. This is associated with impaired memory, but specifically short-term memory after the CM course48. Inflammation of the central nervous system, because of CM, determines the release of inflammatory cytokines, which affect neurogenesis and reverberate in cognitive defects49. The study organized by Azevedo-Quintanilha et al. revealed that αDβ2 integrin deletion alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in the immunoinflammatory responses that mediate brain involvement50. In non-severe malaria, the cognitive impairment mechanisms are not known, but they may be associated with cytokine storms during acute infection51, the production of neurotoxins by infected red blood cells52, impaired coagulation that can impact the correct oxygen supplementation to the tissues of the central nervous system53, anemia54, multiple infections55.The cognitive mechanisms underlying malaria remain poorly understood and experimental models of severe malaria, particularly the murine model using the Plasmodium vivax can be associated with neurological and vascular impairment56. The predilection for reticulocytes, marked proinflammatory responses, and the reversible microvascular dysfunction typically associated with P. vivax infection may explain the pathogeny of CM in malaria vivax59. Vascular congestion, hypoperfusion, and localized hypoxia in CM occurs mainly in the occipital and parietal lobes, this in turn has important reflexes in visuospatial deficit63. In the present work, visuospatial deficits were evaluated with the MMSE and CDT tools and the exposed group's performance was worse than non-exposed group. Executive functions and visuospatial skills are two interrelated cognitive processes and these deficits, which are associated with a decline in the performance of daily activities65.67. Since only the patients exposed in this study had previous malaria, they are in an endemic area, where CQ has been the treatment of first choice for many years and low CQ resistance is defined, we consider that CQ may potentially play an important role in altering neurocognitive functions. CQ is reported as an inhibitor of autophagy in a variety of diseases, including Alzheimer's disease and cerebral ischemia. After inducing brain trauma, treatment with CQ significantly suppressed neuronal autophagy and reduced levels of expression of inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), in the hippocampus of the mouse (81) and this can reduce cognitive impairment. In contrast, patients treated with CQ (cases) showed worsening cognition and executing functions68. However, the half-life of the drug is highly unlikely to explain the long-term cognitive impairment seen in our patients at T8.It must be considered that chloroquine (CQ) used for the treatment of vivax malaria may have as an adverse event psychiatric and nervous system disorders, which may reflect on changes in cognitive and executive functions during the treatment of vivax malariaIn this study, we did not assess other potential co-infections associated with malaria or underlying diseases that could potentially interfere with cognition. A systematic approach to addressing these confounders would be important, however, the clinical and pre-clinical follow-up of these patients to define potential confounders was hampered in this study population, especially due to home care and to the fact that they present at the hospital unit exclusively for the malaria diagnosis. The use of secondary information from SIVEP is also an important limiting factor. Although the groups exposed and not exposed to malaria were selected from the same transmission area, other variables were not properly paired. The adjustment of variables not properly paired were included in multivariate analyzes and had no impact on cognition alone. Attention and language were evaluated by instruments of low sensitivity.P. vivax infection, and that factors such as number of previous malaria cases, recurrence, and parasitemia are important predictors of executive impairment in the second and eighth months after infection. In the context of a rapidly ageing population and the high rates of transmission of vivax malaria in subtropical and tropical areas, where vivax malaria is endemic, malaria can contribute with a substantial negative impact on cognition and executing functions that reflect on the life quality. This, in turn, adds to the deleterious events of the normal ageing process . The revelations obtained in this study should serve as a notice to the policy makers and professionals involved in the healthcare and improvement of the elderlies’ quality of life, considering that this population is vulnerable to cognitive damage.Our results suggest that malaria promotes cognitive, executive, and functional decline in the elderly population for up to 8 months after Supplementary Tables.


P. falciparum immunity evasion, and pathogenesis or virulence. There is limited updated information on P. falciparum msp polymorphisms and their impact on artemether-lumefantrine treatment outcomes in Tanzania. Therefore, this study is aimed at examining msp genetic diversity and multiplicity of infection (MOI) among P. falciparum malaria patients. The influence of MOI on peripheral parasite clearance and adequate clinical and parasitological response (ACPR) was also assessed.  The severity of malaria infection depends on the host, parasite and environmental factors. Merozoite surface protein (msp) diversity determines transmission dynamics,  Parasite DNA was extracted from dried blood spots according to the manufacture's protocol. Primary and nested PCR were performed. The PCR products for both the block 2 region of msp1 and the block 3 regions of msp2 genes and their specific allelic families were visualized on a 2.5% agarose gel. p = 0.669). A significant correlation between age and multiplicity of infection (MOI) for msp1 or MOI for msp2 was not established in this study . Similarly, there was no positive correlation between parasite density at day 1 and MOI for both msp1  and msp2 . The association between MOI and ACPR was not observed for either msp1 or mps2 .  The majority of the isolates, 58/102 (58.8%) for msp1 and 69/115 (60.1%) for msp2, harboured more than one parasite genotypes. For the msp1 gene, K1 was the predominant allele observed (75.64%), whereas RO33 occurred at the lowest frequency (43.6%). For the msp2 gene, the 3D7 allele was observed at a higher frequency (81.7%) than the FC27 allele (76.9%). The MOIs were 2.44 for msp1 and 2.27 for msp2 ( This study reports high polyclonal infections, MOI and allelic frequencies for both msp1 and msp2. There was a lack of correlation between MOI and ACPR. However, a borderline significant correlation was observed between day 2 parasitaemia and MOI. Plasmodium falciparum malaria, with eleven countries accounting for 70% of all cases and 94% of the recorded deaths [Plasmodium falciparum surface protein (Pfs47), and apical membrane antigen (AMA1) may influence treatment outcomes.Sub-Saharan Africa has the highest burden of d deaths . Malariad deaths . ParasitP. falciparum immunity, evasion and virulence. The merozoite surface proteins are regarded as a potential target for malaria vaccine development [Merozoite surface protein diversity determines transmission dynamics, elopment , 4 and aelopment , 6.P. falciparum strains. Among the blood stage surface antigens, merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) are the most commonly used markers for the identification of genetically distinct P. falciparum parasite populations [P. falciparum surface protein encoding a 185–215 kDa protein which is cleaved into several polypeptides during merozoite maturation and red cell invasion [N-terminal of the gene) is the most polymorphic part of the msp1 gene [Diversity in merozoite surface protein (msp) genes is also employed in the characterization of ulations . The mspulations  is a majinvasion . The mspsp1 gene  and is gP. falciparum malaria transmission dynamics and virulence [The msp2 gene is located on chromosome 2 and is encoding the merozoite surface protein 2 which is a glycoprotein with an approximately 30 kDa , 11. It irulence . GenotypP. falciparum strains coinfecting a single host [Multiplicity of infection (MOI), also referred to as complexity of infection (COI), is defined as the average number of distinct parasite genotypes concurrently infecting a patient  or the ngle host . MOI is gle host . Parasitgle host .P. falciparum diversity and population structure after many years of intensive use of insecticide treated nets (ITNs) and insecticide residual spraying (IRS) despite a decline in malaria transmission due to the above interventions [Individuals in areas of high transmission experience multiple mosquito bites associated with multiple clones per bite, unlike in low transmission areas where most of the mosquito bites are associated with monoclonal strains , 18. Theventions , 21. In P. falciparum treatment outcomes has been reported in very few areas [P. falciparum msp polymorphisms and their impact on transmission and treatment outcomes in Tanzania. Therefore, this study aimed at examining msp genetic diversity and MOI in a meso-endemic region and their association with peripheral parasite clearance and adequate clinical and parasitological response (ACPR) among P. falciparum malaria patients treated with artemether-lumefantrine.The frequencies of the msp1 and msp2 alleles have been extensively reported globally. However, the influence of msp polymorphisms and MOI on ew areas  with conew areas  in orderPlasmodium falciparum ) malaria as part of the efficacy study involving artemether-lumefantrine and dihydro-artemisinin piperaquine published earlier [This study was carried out at Karume Health Centre in Igombe, a semiurban and malaria meso-endemic area in Ilemela District, Mwanza region. The samples were collected during the rainy season of the year. Data for the study were prospectively collected from patients with uncomplicated  earlier . Only pa earlier . DetailsPatients were requested to return to the clinic for follow-up on days 1, 2, 3, 7, 14, 21, 28 and 35. Blood from finger pricks was collected on filter paper (FTA®Whatman paper) during the visits. The filter papers were then dried at room temperature and kept in sealed plastic bags. Thick and thin blood smears were stained by Giemsa (on each day of the visit) according to the standard protocol . ParasitParasite DNA was extracted from the dried blood spots (DBS) using the Life Sciences genomic DNA kit for dried spots according to the manufacture's protocol. Primers for genotyping of the block 2 for msp1, block 3 for msp2, specific allelic families for msp1  and specific allelic families for msp2 (FC27 and 3D7) were in accordance to Somé et al. . PrimaryThe WHO 2015 protocol  was usedp value less than 0.05 was considered statistically significant.Statistical analyses were performed using STATA version 13.1 . MOI was defined as the number of distinct parasite genotypes coexisting within a single infection  and was Alleles of msp1 and msp2 were classified according to the PCR amplified fragments. K1 was the predominant allele observed for msp1 (75.6%) and yielded 9 fragments (160–300 bp) as shown in High MOI (above 2) was observed for both msp1 and msp2, but the difference between the two groups was not statistically significant . The majrho = 0.1415, p=0.2164 and rho = 0.1415, p=0.2164, respectively) . However, there was a strong negative correlation between parasite density at day 2 and ACPR .A Spearman's correlation was run to assess the relationship between age and parasite density (at days 1 and 2). There was a negative correlation between age and parasite density at days 1 and 2, but this was not statistically significant (ctively) . Parasitrho = 0.074, p = 0.521 and rho = −0.1289, p = 0.261, respectively) as shown in rho = 0.133, p = 0.244)  (Significant correlation between age and MOI for msp1 or MOI for msp2 was not established in this study (= 0.244) . There w= 0.054) .rho = 0.043, p = 0.712)  .For msp2, there was no strong positive correlation between parasite density at day 1 and MOI, for which there was a statistical significance (= 0.712) . There w= 0.957) . The assP. falciparum is essential for the parasite to adapt to environmental changes, escaping host immunity through antigenic variation and develop resistance [P. falciparum is essential for monitoring and evaluating malaria control strategies and interventions. The present study focused on the allelic diversity of P. falciparum and its influence on treatment outcomes utilizing isolates from patients with uncomplicated malaria treated with artemether-lumefantrine.The genetic diversity of sistance , 30. Thesistance , 31. UndThe predominant alleles were K1 for the msp1 gene and 3D7 for msp2 the gene. These findings are similar to those in other parts of the world , 32, 33.Plasmodium falciparum infected malaria patients had high frequencies of polyclonal infection for both msp1 and msp2. The observed high polyclonal infection is not surprising in a malaria endemic and even meso-endemic area where it is common that patients will be infected by more than one distinct parasite genotype [P. falciparum mixed clone infections in humans also lead to cross-fertilization and recombination between parasite genomes in the mosquito vector [genotype . Multiclgenotype , 41. Thegenotype , 40 and genotype . The P. o vector . These cThe mean MOI for both msp1 and msp2 was high (between 2 and 2.5) at the study site, similar to findings from Sudan and Uganda , 44. TheThe correlation between MOI with age has been reported with conflicting results; some studies have reported an inverse association between age and MOI, showing lower MOI as age increases , 7, 47. P. falciparum allelic families or MOI and clinical outcomes, particularly ACPR similar to studies done in Sudan, Uganda and Gabon [The current study reports a lack of association between nd Gabon , 50, 51.nd Gabon . This coOur findings suggest a lack of correlation between MOI and parasite density (at days 1 and 2). The findings are in match with other previous studies in Africa , 53 but P. falciparum malaria patients.Findings from the present study serve as baseline data for future malaria epidemiological studies on malaria transmission at the study site and an evaluation of the influence of parasite genotypes on treatment outcomes among P. falciparum infection in the country. Inverse correlations between day 2 parasitaemia and ACPR and between haemoglobin values and MOI were reported in our study. No correlation was observed between parasite density or age and MOI. MOI did not influence ACPR among malaria patients. The observed high number of multiclonal isolates suggests a complex population structure of the parasite and may accelerate the spread of resistance over time.Most malaria patients harbour polyclonal infections harbouring multiple genotypes and the high MOI displays the high genetic diversity of


Furthermore, stunted children had significantly lower VO2max scores . Regarding the antimalarial drug resistance markers, the most prevalent Pfmdr1 86-184-1034-1042-1246 haplotypes were the NFSND in 47% (n = 88) and the NYSND in 52% (n = 98). The wild type Pfcrt haplotypes  were found in 99.1% (n = 219) of the samples. Malaria, stunting and parents’ or guardians’ illiteracy were the key attributable factors for anaemia in schoolchildren. Given malaria infection in schoolchildren is mostly asymptomatic; an addition of interventional programmes such as intermittent preventive treatment of malaria in schoolchildren (IPTsc) would probably act as a potential solution while calling for an improvement in the current tools such as bednet use, school food programme, and community-based (customised) health education with an emphasis on nutrition and malaria control.In Africa, children aged 5 to 15 years (school age) comprises more than 50% (>339 million) of the under 19 years population, and are highly burdened by malaria and anaemia that impair cognitive development. For the prospects of improving health in African citizens, understanding malaria and its relation to anaemia in school-aged children, it is crucial to inform targeted interventions for malaria control and accelerate elimination efforts as part of improved school health policy. We conducted a study to determine the risk factors for asymptomatic malaria and their association to anaemia. We explored the prevalence of antimalarial drug resistance as well as the association of asymptomatic malaria infection and anaemia on cognitive and psychomotor functions in school-aged children living in high endemic areas. This study was a comprehensive baseline survey, within the scope of a randomised, controlled trial on the effectiveness and safety of antimalarial drugs in preventing malaria and its related morbidity in schoolchildren. We enrolled 1,587 schoolchildren from 7 primary schools located in Muheza, north-eastern Tanzania. Finger-pricked blood samples were collected for estimation of malaria parasitaemia using a microscope, haemoglobin concentration using a haemoglobinometer, and markers of drug resistance processed from dried blood spots (DBS). Psychomotor and Cognitive functions were assessed using a ‘20 metre Shuttle run’ and a test of everyday attention for children (TEA-Ch), respectively. The prevalence of asymptomatic malaria parasitaemia, anaemia and stunting was 26.4%, 49.8%, and 21.0%, respectively with marked variation across schools. In multivariate models, asymptomatic malaria parasitaemia attributed to 61% of anaemia with a respective population attribution fraction of 16%. Stunting, not sleeping under a bednet and illiterate parent or guardian were other factors attributing to 7%, 9%, and 5% of anaemia in the study population, respectively. Factors such as age group (10–15 years), not sleeping under a bednet, low socioeconomic status, parents’ or guardians’ with a low level of education, children overcrowding in a household, and fewer rooms in a household were significantly attributed to higher malaria infection. There was no significant association between malaria infection or anaemia and performance on tests of cognitive function (sustained attention) or psychomotor function (VO2 max). However, a history of malaria in the past one month was significantly associated with decreased cognitive scores (aOR = -4.1, 95% CI -7.7–0.6,  Africa is the second most populous continent in the world, with over 1.3 billion people (by the year 2020), of whom 52% are below 19 years. Children aged 5 to 15 years (schoolchildren) comprise of more than 50% (over 339 Million) of the under 19 years population –6. TheseP. falciparum triggers the development of acquired immunity that controls blood-stage parasitaemia, thereby reducing clinical symptoms and life-threatening complications in older children and adults . However, participants at Pangamlima located at a mean altitude of 213.3 meters were not the least in altitude location but had the highest malaria prevalence over all schools (40%). STH was less prevalent in the study area, only 1% of the population had hookworm infestation. Overall, Kwakibuyu had high anaemia, STH and schistosomiasis infestations, 75%, 2.9%, and 30.3%, respectively. Songa Kibaoni primary school had the least anaemia prevalence at 24% and had a higher mean cognitive score (27.3) of all schools. Bwitini primary school had the least mean cognitive score of 11.8. The median age of all children in each school was similar at 9 years old. The VO2max was essentially stable across schools. Other variations are as seen on the There was variation across schools for different characteristics . Heinkelp = 0.01) of malaria infection compared to girls, but became non-significant on multivariate analysis. Children aged 10–15 years old had 1.4 adjusted odds  of having malaria parasite compared to those aged 5–9 years, this group attributed to 28% of malaria infection per age group, and attributed to 12% of malaria infection in the population. Not sleeping under a bednet attributed to 34% of having malaria infection , low socioeconomic status, low level of parents or guardian education (primary or none), not sleeping under a bednet, fewer rooms and increased number of children (overcrowding) in a household were significantly associated with high odds of malaria infection . Childrenfection . Having The malaria model was able to predict correctly about various factors in association with malaria infection by 72% depicted as area under the receiver operating characteristic (ROC) curve, developed following the logistic regression .Pfmdr1 86-184-1034-1042-1246 haplotypes were the NFSND in 47% (n = 88) and the NYSND in 52% (n = 98). The wild type Pfcrt haplotypes  were found in 99.1% (n = 219) of the samples, and CVIET in 0.9% (n = 2). No changes in Pfexo was found in codon 415, all samples being wild type E (n = 198)  were carrying the quintuple mutant haplotype, and 33.5% (n = 59) the sextuple mutant haplotype as shown in Pfdhfr triple haplotype  (51I-59R-108N-164I). The Pfdhps double (431I-436S-437G-540E-581A-613A) and triple mutant (431I-436S-437G-540E-581G-613A) were found at prevalence’s of 61.0% (n = 92) and 35.1% (n = 53), respectively  . For 176ectively . The Pfkound see .Schistosoma infestation or low socioeconomic status and location altitude did not have significant association to anaemia in multivariate analysis  of having anaemia compared to those with no malaria infection, and attributed to 61% of anaemia equivalent to 16% attribution of anaemia in the population. This was higher than all other factors in association with anaemia, and was followed by not using bednet and stunting that were attributed to 43% and 40%, respectively. The two factors attributed to 9% and 7% of anaemia in the population, respectively. Children whose parents/ guardian were less educated had high adjusted odds of having anaemia  compared with those with education, these attributed to 43% of anaemia in this group and 5% in the study population. analysis . ChildreThe anaemia model was able to predict correctly about various factors in association with anaemia by 73% depicted as area under the receiver operating characteristic (ROC) curve, developed following the logistic regression .p = 0.02), but could not have an effect on adjustment with other factors. All other factors did not show significant correlation to VO2max. Boys had high cognitive scores compared to girls . Malaria and anaemia did not have significant correlation with cognitive scores. History of confirmed malaria the previous one month was associated with reduction in cognitive scores at adjusted coefficient of -4.1 . Schools were included in the model to control any school specific confounders, their respective correlation to psychomotor or cognitive function model are as shown on Boys had significantly higher VO2max compared to girls . Stunted children had lower VO2max compared to normal children . Malaria and anaemia reduced VO2max but the correlation was not statistically significant. Schistosomiasis significantly correlated with reduction of VO2max at unadjusted coefficient of -2.63  and NYD (47%), and close to wild type fixation for PfCRT-CVMNK (99%) and PfExo- E (100%). Factors such as; age group (10–15 years), not sleeping under a bednet, low socioeconomic status, parents’ or guardian with low level of education, more number of children in a household, and less number of rooms in a household significantly attributed to higher malaria infection in the study area. Even though, anaemia has multiple causalities, in this study, asymptomatic malaria infection attributed significantly to 61% of anaemia, followed by stunting that attributed to 34% of anaemia among the study participants with malaria and stunting, respectively.Despite geographical variations across communities, schoolchildren shared some parameters for risk of infection where, children from households located at low altitude had more malaria infection than those in high altitude areas, this has also been reported in other studies ,52,54. HPreventive programs such as bednets and increased literacy level in the communities have attributed significantly to reduced malaria infection rates in the communities ,61. In op = 0.03) adjusted odds of malaria infection compared to children aged 5–9 years old. In addition, this age group (10–15 years) had an attributable fraction to malaria infection of 28%, and had a population attributable fraction of 12%. A Tanzanian national wide school malaria survey conducted in 2014 and 2019 showed a similar trend as the present study [P. falciparum infection among children aged 2–10 years (PfR2-10) cannot be precise when dealing with schoolchildren. When planning malaria control programmes implementation, the decision based on PfR2-10., should be improved by a PfR10-15 and or a PfR5-15.The shifting trend of asymptomatic malaria infection with increased risk from low age to young adolescents is obvious; where children aged 10 to 15 years had 1.4 . There was also resistance to Pfmdr1-NFD haplotype at 47%, normally associated with resistance to Lumefantrine (a partner drug in Artemether Lumefantrine-ALU) the first line antimalarial treatment drug in Tanzania [Pfdhfr and Pfdhps sulphadoxine pyrimethamine (SP) drug resistance, was expected as it was earlier documented [We also explored the prevalence of antimalarial drug resistance in schoolchildren. This gave us more information on malaria infection and management in the study community thereof. We saw high sensitivity to Amodiaquine (AQ) and Chloroquine (CQ) , STH and stations ,77. A ststations . Also a stations . Generalstations . STH andstations ,77, howeTanzania ,21,80, bTanzania , especiaMalaria related anaemia has been associated with impaired cognitive and psychomotor function as documented elsewhere ,53,82,83On the other hand, there was a significant association of low cognitive scores attributed to history of malaria infection in the past one month. This could be confirming the narrative that cognitive function derangement is an outcome of cumulative malaria infection as noted in Kenya  and sincOur study findings cannot escape the limitations of a cross sectional design especially on issues related to causality ,85. Yet et al., 2021 have summarised quite a number of interventions addressing malaria in schoolchildren, and proposed increasing access to testing and treatment in schools or providing chemoprevention to school-age children as possible approaches [Studies ,43,47,88proaches . We giveproaches  comparedproaches ,90–94, aThis study has highlighted the extent of attribution of each risk identified to malaria and anaemia. We have shown that malaria, illiteracy and stunting play a key role on anaemia, which would in turn, contribute to cognitive impairment. Current malaria control tools are suboptimal, especially for malaria control in school-aged children. Given malaria infection in schoolchildren is mostly asymptomatic; an addition of intervention programmes such as IPTsc would probably give potential solution while calling for improvement on the current tools such as bednet use and school food programme, community based (customised) health education with emphasis to nutrition and malaria control.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The Democratic Republic of the Congo (DRC) is the second most malaria-affected country in the world with 21,608,681 cases reported in 2019. The Kongo Central (KC) Province has a malaria annual incidence of 163 cases/per 1000 inhabitants which are close to the national average of 153.4/1000. However, the malaria prevalence varies both between and within health zones in this province. The main objective of this study was to describe the epidemiology and transmission of malaria among children aged 0 to 10 years in the 4 highest endemic health areas in Kisantu Health Zone (HZ) of KC in DRC.A community-based cross-sectional study was conducted from October to November 2017 using multi-stage sampling. A total of 30 villages in 4 health areas in Kisantu HZ were randomly selected. The prevalence of malaria was measured using a thick blood smear (TBS) and known predictors and associated outcomes were assessed. Data are described and association determinants of malaria infection were analysed.kdr-west resistance alleles showed the presence of both L1014S and L1014F with 14.6% heterozygous L1014S/L1014F, 84.4% homozygous 1014F, and 1% homozygous 1014S. The risk factors associated with malaria infection were ground or wooden floors aOR: 15.8 (95% CI: 8.6–29.2), a moderate or severe underweight: 1.5 (1.1–2.3) and to be overweight: 1.9 (95% CI: 1.3–2.7).A total of 1790 children between 0 and 10 years were included in 30 villages in 4 health areas of Kisantu HZ. The overall prevalence in the study area according to the TBS was 14.8% . The mean sporozoite rate in the study area was 4.3% (95% CI: 2.6–6.6). The determination of Malaria prevalence differed between villages and health areas within the same health zone. The control strategy activities must be oriented by the variety in the prevalence and transmission of malaria in different areas. The policy against malaria regarding long-lasting insecticidal nets should be based on the evidence of metabolic resistance.The online version contains supplementary material available at 10.1186/s12936-022-04415-z. The Democratic Republic of the Congo (DRC) is, after Nigeria, the most affected malaria-endemic country with more than 29 million estimated cases annually . The malAnopheles gambiae sensu lato (s.l.) is generally susceptible to bendiocarb (carbamate) and pirimiphos-methyl (organophosphate), but resistant to DDT (organochlorine), permethrin, deltamethrin, and alpha-cypermethrin (pyrethroids) [Vector control interventions are threatened by the emergence of insecticide resistance. In DRC, the primary vector, throids) –7. The ithroids) –10.In DRC, the physical durability and effectiveness of nets impregnated with deltamethrin (DawaPlus 2.0) and α-Cypermethrin (Duranet) is only two years and not three years as recommended by the World Health Organization (WHO) . Despite2 and a population of 153,188 inhabitants. In this area, the malaria incidence rate, in 2017, was 163/1000 Cases per person per year . The insecticide susceptibility testing conducted in the closest national entomological monitoring site, Kimpese (in the same province) in 2016 showed that An. gambiae s.l. was resistant to permethrin with 17% mosquito mortality in WHO susceptibility tests (95% CI: 11–26) and 76% to deltamethrin mortality (95% CI: 67–83)[This study was carried out in the four most malaria endemic health areas in Kisantu health zone (HZ) in Kongo Central (KC), one of 26 provinces of DRC. Kisantu is located in Lukaya district covering a surface area of 2400 kmI: 67–83).A mixed entomological and epidemiological study was carried out in 4 health areas of Kisantu health zone. The epidemiological study consisted of a stratified multistage cross-sectional study conducted in households with children aged 0 to 10 years. Informed and signed consent was obtained from the head of household. In case of illiteracy, fingerprint (thumb) consent in the presence of an independent witness was used.Anopheles and their susceptibility to some insecticide.The stratification took into account the model used in the strategic plan for the fight against malaria. This model divides the country in 3 zones according to the parasitic infection: hypo-endemic (<5%), holo-endemic (6–30%) and hyper-endemic zone (30%). The entomological study consisted of identifying species of The number of children (n) included in the study was calculated using the following formula described by Kish (1965) for proportion .\documen II, DRC); \documerand. In the third stage, a census was conducted to collect a list of all children in these 30 villages between age 0 and 10 years included. A systematic approach was then used to identify children aged from 0 to 10 years per village preceded by the calculation of sampling interval by dividing the total number of eligible children in the villages by the sample size calculated as illustrated in Fig. A multi-stage sampling was used. In the first stage, 4 of the 15 Health areas within Kisantu Health Zone with the highest malaria prevalence were selected based on the 2017 parasitological surveys (Database of DLM 2017). In the second stage, 30 villages were selected randomly from the list of all the 72 villages in the four health areas using the Excel function A questionnaire was administered to each household of the selected children. Information on demographic characteristics , socio-economic status (of the parents/guardian) and related variables were collected. To assess the socio-economic level of the households, observations were made on household characteristics .Plasmodium infection prevalence was calculated using the formula: total children with positive (Plasmodium infection) TBS / total children tested. The infection prevalence by village was used to group villages according to the DRC NMCP’s 2016–2020 strategic plans as: (i) hypo endemic ≤ 5%, (ii) meso -endemic 6–30% and (iii) hyper-endemic > 30% [A thick blood smear (TBS) and a thin blood smear were taken from selected children in the households following WHO procedures . Thick aHaemoglobin levels were measured on the spot in the field using a portable automated HemoCue®Hb 301 haemoglobinometer system according to the manufacturer’s instructions. Anaemia was classified as mild, moderate, severe and life-threatening when haemoglobin concentrations were 9.0–9.9 g/dl, 7.0–8.9 g/dl, 5.0–6.9 g/dl and ˂5.0 g/dl, respectively .The weight of children was measured using digital scales  to the nearest 0.1 kg. The height was measured using a mobile measuring Seca mat  for children 0 to 99 cm and a recumbent length board for children of height > 99 cm to the nearest centimeter. The outcome variable was undernutrition indicated by wasting, stunting and underweight status in children >6 months of age. Stunting (height/age), wasting (weight/height) and underweight (weight/age) were defined as per WHO child growth standards. Children with Z scores less than two standard deviations (≤ 2 SD) below the median of the WHO child growth standards were classified as malnourished , minus 3 SD indicates severe stunting, wasting, and underweight. Z scoreAnopheles were sampled from each of the same 30 villages in June 2018. Six households were randomly selected, of which human landing catches (HLC) were used in three of the houses for one night per house to estimate the biting rate. In the 3 other selected households, indoor resting mosquitoes were collected from the walls, roof and furniture using standard battery-powered Prokopack aspirators between 6:00 to 8:00 am. A total of 180 households were included in this survey .Adult The HLC collectors were placed indoor and outdoor with four collectors per household divided in two teams working six-hour shifts. The collectors were selected amongst the local population. After written informed consent, they were enrolled and trained on entomological activities. The collectors were referred to a health center and were treated free-of-charge if any illness occurred during the HLC activities and up to three weeks after collections.Kdr 1014 locus. Based on the biting rates and the Plasmodium falciparum prevalence, the P. falciparum entomological inoculation rate (PfEIR) was calculated the mean number of sporozoite-infected anopheles per house per night) and weighted to account for the proportion of collected anopheles processed for sporozoites. It is usually interpreted as the number of P. falciparum infective bites received by an individual during a night or season (aPfEIR) [The collected mosquitoes were morphologically identified  and subs(aPfEIR) .kdr 1014 locus, East and West together using the new developed technique from Huynh [The sex and physiological status  of the mosquitoes collected by Prokopack were determined. Subsequently they were dried on silica gel for the CSP by ELISA and the alleles of om Huynh  at CDC AAnopheles larvae and pupae were collected in all villages included in the study and brought to the laboratory where they were reared to adults. Adult Anopheles species aged 3–5 days were tested using WHO susceptibility tube tests against the diagnostic concentrations of deltamethrin 0.05%; permethrin 0.75%; alpha-cypermethrin 0.05%; DDT 4%; bendiocarb 0.1% and PBO 4% following WHO guidelines [idelines . After 2idelines .Anopheles gambiae s.l. and Anopheles funestus s.l. heads and thoraces were separated from the abdomen and P. falciparum sporozoite antigen was detected using ELISA according to standard protocols [kdr-w). SINE PCR was used to identify the species of 200 mosquitoes morphologically identified as An. gambiae s.l. [Kdr resistance alleles 1014F were also determined by PCR on the same 200 mosquitoes using the protocol described by Huynh et al. [rotocols . ELISA tiae s.l.  Kdr resih et al. . The PCRStata version 13.0 was used for statistical analysis. Descriptive statistics were used to summarize the characteristics of the study population. Continuous variables were reported using means with standard deviations when variables were normally distributed. If not, medians with ranges were reported. Categorical variables were summarized in frequencies or percentage. For the epidemiological study, the prevalence was calculated using all children who had a positive thick drop, over the total study population.2 test was used to search for the association between strata of malaria transmission and some characteristics of the households . Statistically significant associations were thereafter assessed in a logistic regression analysis to determine factors independently associated with malaria transmission. To take in account the cluster, the vce (cluster clustvar) option was used. The odds ratio (OR) with a corresponding 95% confidence interval was reported to quantify the strength of association. Significance was set at p-value of 0.05. Mapping of vector distribution and prevalence was also performed. Student’s t test was used to compare means of dead anopheles according to the insecticide.χAnopheles species tested) × 100The sporozoite rate (SR) =  = total number of Nightly entomological inoculation rate (EIR) = Nightly HBR × SR.The following formulas were used to calculate entomological indicators:A total of 1790 children were included in this study in 30 villages in the Kisantu health zone. Nearly half of the children were female (51.4%) or aged under five years (51.7%)  of houses visited was built with mud walls. The most frequent roofing material was steel sheeting (44.2%).The averages of the weight, height, and temperature of the children were 15.5 ± 5.8 kg; 100.5 ± 20.5 cm, and 36.7 ± 0.5 °C, respectively (Table Plasmodium infection according to the TBS was 14.8% (95CI:13.3–16.6) . Plasmodium malariae represented 3.8% (CI 1.9–6.7 and 0.77% (CI 0.1–2.5) were mixed infections P. falciparum and P. malariae.The overall prevalence of 8% 95CI:1.3–16.6  and having moderate or severe malnutrition  were associated to the emergence of Plasmodium infection  and malnutrition were associated with Anopheles funestus s.l. was the most abundant 58.2% (CI 95%: 55.1–61.3) species collected followed by the Anopheles gambiae s.l. 24.1% (CI 95%: 21.5–26.9)  for Anopheles nili, 1.8% (CI 95%: 1.1–2.9) for Anopheles paludis and 3.0% (CI 95%: 2.0–4.2) for Anopheles coustani. A total of 1049 Culicinae were also captured. Both An. gambiae s.l. and An. funestus s.l. indoor biting behaviors were like those of outdoor biting ,  per night was estimated at 0.04 infectious bites per night for The mosquito populations tested were resistant with insect mortality rates to permethrin of 25% (95% CI: 17–34), deltamethrin of 62% (95% CI:53–72%), alpha-cypermethrin of 48% (95% CI: 38–58), DDT of 11% (95% CI:5–17%) and fully susceptible to bendiocarb (100%) . In those samples, 14.6% are heterozygous (kdr-east (L1014S) /kdr-west (L1014F)), 84.4% are homozygotes kdr-west (L1014F), and ~ 1% are kdr-east (L1014S). The allele frequency was 0.99 for kdr-west and 0.16 for kdr west–east.  sentinel site 2017 through the President Malaria Initiative (PMI) entomological monitoring activities [An. gambiae s.l. and 4.5 per person per night for An. funestus s.l.The sporozoite rate found in this study is similar to that reported in other studies . From thtus s.l. . The preKinshasa  and elseKinshasa . Anopheltivities . The EIRAn. gambiae s.l. populations. This resistance to pyrethroids could have a negative impact on the efficacy of LLINs impregnated with pyrethroid [An. gambiae s.l. observed after pre-exposure to PBO showed the likely implication of metabolic resistance mediated by oxidases [An gambiae s.s. populations [The Phenotypic resistance to pyrethroids was observed in rethroid , 5. Susculations .An. gambiae s.s. could have a negative impact in LLINs efficacy [. The authors found that malnourished children were almost twice as likely to develop malaria compared to healthy children [Malaria control strategy in DRC is based on detection, treatment and prevention. The prevention is based mainly on intermittent preventive treatment (IPT) and LLINs. The observed resistance of efficacy . The reschildren , 33. Thechildren  and a deAn. funestus s.l and An. gambiae s.l. These vectors were resistant to pyrethroids, but more susceptible when PBO was used in pre-exposure phase. The policy against malaria regarding LLINs should be based on the evidence of metabolic resistance and the biting period including the peak which is around 23h00.In these selected Health areas, malaria infection prevalence was high in some villages compared to the other villages in the Kisantu health zone. House type was highlighted as a risk factor of having malaria infection. Malnutrition was associated with malaria endemicity. Malaria transmission vector is mostly Additional file 1. Figure S4. Mapping of distribution of Anopheles species in Kisantu Health Zone, Kongo-Central Province. Table S6. Human biting rate was determined from human landing collections in the different villages in Kisantu only An. gambiae sl and funestus sl. Table S7. Study sites and their epidemiological stratum in Kisantu Health Zone, Kongo-Central Province.


Plasmodium falciparum parasite populations in high malaria transmission regions. There has been a decline in malaria transmission intensity over 12 years of surveillance in the community in Kilifi, Kenya. This study sought to investigate whether there was a corresponding reduction in P. falciparum genetic diversity, using msp2 as a genetic marker.High levels of genetic diversity are common characteristics of msp2 and capillary electrophoresis fragment analysis.Blood samples were obtained from children (< 15 years) enrolled into a cohort with active weekly surveillance between 2007 and 2018 in Kilifi, Kenya. Asymptomatic infections were defined during the annual cross-sectional blood survey and the first-febrile malaria episode was detected during the weekly follow-up. Parasite DNA was extracted and successfully genotyped using allele-specific nested polymerase chain reactions for msp2 genetic diversity remained high. A high heterozygosity index (He) (> 0.95) was observed in both asymptomatic infections and febrile malaria over time. About 281 (68.5%) asymptomatic infections were polyclonal (> 2 variants per infection) compared to 46 (56%) polyclonal first-febrile infections. There was significant difference in complexity of infection (COI) between asymptomatic 2.3 [95% confidence interval (CI) 2.2–2.5] and febrile infections 2.0 (95% CI 1.7–2.3) (P = 0.016). Majority of asymptomatic infections (44.2%) carried mixed alleles , while FC27 alleles were more frequent (53.3%) among the first-febrile infections.Based on cross-sectional surveys conducted in 2007–2018, there was a significant reduction in malaria prevalence , however Plasmodium falciparum infections in Kilifi are still highly diverse and polyclonal, despite the reduction in malaria transmission in the community.The online version contains supplementary material available at 10.1186/s12936-022-04213-7. Plasmodium falciparum infections and highly diverse parasite populations ) were observed in the asymptomatic infections than the FC27 alleles (101 [24%]) that were predominant in the first-febrile infections  polyclonal (≥ 2) infections, with a mean COI of 2.3 (1–10) . The FCepisodes , 37, 38.episodes , 39. TheP. falciparum infections can act as precursors to symptomatic malaria [msp2 has previously been used to assess whether the development of symptoms is due to persistence of an existing clone or due to infection with a new clone [The high COI and large proportion of polyclonal asymptomatic infections is a result of the frequent and repeated exposure to genetically distinct malaria parasites in endemic areas, as described in previous studies . This le malaria . Genotypew clone . In thisew clone –47. Althmsp2 genetic diversity maintained across the study period was expected as Kilifi is a region of moderate to high malaria transmission. The 291 bp, 327 and 411 bp FC27 and 555 bp IC/3D7 fragment sizes were common in both asymptomatic and first-febrile infections. Strikingly, some of these genotypes have been reported in other countries, such as Mali [The high  as Mali , as the Malaria surveillance should also focus on asymptomatic infections, in addition to symptomatic infections, given the extensive genetic diversity and the impact they have on sustaining malaria transmission. Similar studies should be conducted to monitor the trends in parasite genetic diversity to associate this with changes in malaria transmission.Additional file 1: Table S1. Temporal changes in FC27 allele frequencies across asymptomatic and febrile infections in Kilifi, Kenya from 2007 to 2018. n refers to the number of successfully genotyped individuals per year. m denotes the total number of genotypes per year.Additional file 2: Table S2. Temporal changes in IC/3D7 allele frequencies across asymptomatic and febrile infections in Kilifi, Kenya from 2007 to 2018. n refers to the number of successfully genotyped individuals per year. m denotes the total number of genotypes per year.


Severe malaria is a medical emergency. It is a major cause of preventable childhood death in tropical countries. Severe malaria justifies considerable global investment in malaria control and elimination yet, increasingly, international agencies, funders and policy makers are unfamiliar with it, and so it is overlooked. In sub-Saharan Africa, severe malaria is overdiagnosed in clinical practice. Approximately one third of children diagnosed with severe malaria have another condition, usually sepsis, as the cause of their severe illness. But these children have a high mortality, contributing substantially to the number of deaths attributed to ‘severe malaria’. Simple well-established tests, such as examination of the thin blood smear and the full blood count, improve the specificity of diagnosis and provide prognostic information in severe malaria. They should be performed more widely. Early administration of artesunate and broad-spectrum antibiotics to all children with suspected severe malaria would reduce global malaria mortality. Severe malaria is important. It is a major cause of preventable childhood death in tropical countries. This large number of avoidable deaths justifies the substantial global investments in malaria control and elimination. But severe malaria is increasingly overlooked by the international agencies, donors and policy makers who determine the direction and support for global malaria initiatives.Plasmodium infection, as did the notorious “blackwater fever”. Until the 1980s, the majority of research on severe malaria was conducted in adults. It derived largely from war-time experiences in the military, or observations from colonial medical services. Specific anti-malarial treatment comprised the parenteral administration of quinine and, from the 1950s, chloroquine. When they became available, renal replacement therapies for adult patients with acute renal failure could also save lives [Severe malaria, or ague, was recognized long before discovery of the malaria parasite by Laveran in 1880. The Cinchona bark arrived in Europe nearly four hundred years ago providing, for the first time, a potential cure for the pervasive and dangerous illness that then affected most of the inhabited world. But, as today, the specificity of the clinical diagnosis of febrile illnesses was poor. Torti recognized that only some fevers could be cured by the bark [ve lives .Soon after Laveran’s discovery of the causative parasite, the pathological processes underlying severe malaria were elucidated by the great Italian malariologists Marchiafava and Bignami. They considered, correctly, that the sequestration of parasitized erythrocytes in the microvasculature, causing microcirculatory dysfunction, was the key pathological event in “malignant tertian”  malaria , 4. Begihyperparasitaemia was defined as > 5% parasitaemia (today this is 10%)after a convulsion, coma had to persist for 6 h (now 30 min),severe anaemia was defined as a haematocrit < 20% (now < 15%),jaundice  alone was a criterion ,‘fluid, electrolyte or acid–base disturbances requiring intravenous therapy’ was a criterion .Hyperpyrexia (> 39 °C), vomiting of oral treatment and haemoglobinuria were also included – none of which today are considered defining criteria.Before 1985, there was no standard definition of severe malaria. Cerebral malaria was defined as unrousable coma . After publication of the Glasgow Coma Scale (GCS) in 1976 [These definitions and descriptions have been generally referred to and referenced as “WHO definitions” although each successive version of the severe malaria review contains a disclaimer that the contents are the opinions of experts, and not those of the WHO itself.In 1988 a second informal WHO meeting was held to update the recommendations and to incorporate recent observations in African children with cerebral malaria  was endoThe third WHO meeting was held in Geneva in December 1995 to incorporate further experience from clinical research in African children –12. ThisThe hyperparasitaemia threshold was changed to 4% in low transmission settings, and to 20% in high transmission settings. The newly added “prostrate” criterion was very broad. It included many children with acute malaria who had no other signs of severity. This substantially expanded definition of severe malaria therefore encompassed a larger proportion of all children with acute malaria . The new inclusive definition ensured a high proportion of at-risk children would be managed appropriately (i.e. it had high diagnostic sensitivity), but it had low specificity in identifying potentially fatal infections. In clinical research use of the broader inclusion criteria obviously resulted in overall “better outcomes” as more children with a good prognosis were included within the broader definition of severe malaria. Recognizing the disparities with the earlier criteria some investigators continued with the stricter (i.e. more specific) earlier severe malaria criteria  in theirThe most recent WHO meeting on severe malaria was convened in 2013, again in Geneva –21. The Plasmodium vivax and Plasmodium knowlesi infections were reviewed specifically, and slightly modified definitions for severe malaria with these infections were proposed [Prostration was not included in the ‘research” definition, convulsions were “dropped”, the acidosis criterion was refined, the jaundice criterion was reintroduced with a parasite density > 100,000/µL, and the hyperparasitaemia criterion was changed (again!). In addition, severe proposed .P. falciparum and P. vivax) in elderly travellers, and of malariatherapy  in neurosyphilis, testifies to the lethal potential of acute malaria illness, whichever the infecting parasite species, in frail or debilitated persons [12. If severe malaria was defined as clinical and laboratory measures which are associated with > 5% mortality, then the current thresholds would conform, except for the anaemia criterion (see below) which would require a threshold of 3 g/dL rather than 5 g/dL.Strictly speaking, severe malaria is malaria with an increased risk of death at the time of assessment compared to everyone else in that community with malaria illness. How much higher this risk should be  has not been agreed upon. Mortality varies substantially as it depends on the infection, the host, the circumstances and the treatment. For the same admission severity, outcomes in well-equipped intensive care units (ICUs) with well-trained staff are better than in peripheral health centres. However, tertiary ICUs often receive the very sickest patients, often after long delays in referral – with consequently high mortalities. A frail and debilitated patient may die from a malaria infection that would be regarded as mild in a younger and fitter person. The high mortality of imported malaria . Acknow persons . This coMalaria is traditionally diagnosed by microscopy examination of a peripheral blood smear. Unfortunately, this diagnostic skill is being lost in many places as microscopy is replaced by the more ‘convenient’, but less informative, rapid diagnostic tests. In malaria microscopy, the parasites are speciated and their numbers counted. The result is reported either as the number of parasitized erythrocytes in a stained thin smear or, in a thick film, as the number of parasites seen in a fixed volume or while counting a certain number of white blood cells . The old semi-quantitative ‘cross’ system, in which density is graded from + to +  +  +  + , is no longer recommended. The thin film should be used for high parasite densities (> 0.2% parasitaemia).Plasmodium falciparum infected erythrocytes begin to stick (“cytoadhere”) to vascular endothelium. By 20 h the majority have cytoadhered. This “sequestration” is the fundamental pathological process in falciparum malaria [P. falciparum infections, although the tissue distribution of sequestration varies between patients. As a result, the parasite densities measured in blood films (reflecting circulating parasites only) variably underestimate the total malaria parasite biomass [P. falciparum entomological inoculation rate was approximately 0.5/year, the mortality of children with > 4% P. falciparum parasitaemia  was 3% [P. falciparum peripheral blood parasite densities over 200,000/uL may be tolerated with relatively few symptoms. Thus, the prognostic value of parasitaemia depends on the epidemiological setting and, overall, it is poor.In falciparum malaria the parasite count can be misleading. This is because after approximately 12 to 16 h (depending on core temperature) of intraerythrocytic parasite growth   malaria . It occu biomass –34. Neve biomass  ) was 3% , 36. In ) was 3% . As the ) was 3% –34. OtheThe three main clinical presentations of severe malaria in children are coma, metabolic acidosis  and anaemia , 22–26. The most characteristic syndrome of severe falciparum malaria is unrousable coma or cerebral malaria , 42. ThiMetabolic acidosis is a grave sign in both adults and children with severe malaria, , 47–49 , unless The definitions of anaemia in malaria vary widely . The mosImproved malaria control reduces the frequency of malaria infections and thus the prevalence of severe anaemia , 65. As Pulmonary oedema (ARDS) carries a very high mortality in falciparum malaria- even with positive pressure ventilation. It often occurs after the other severe manifestations have become evident. Pulmonary oedema results from increased pulmonary capillary permeability. Pulmonary oedema may also occur in vivax malaria, where the prognosis is much better . Liver dP. fragile, P. coatneyi), but unlike the other human malaria parasites, P. falciparum causes the infected erythrocyte to cytoadhere to vascular endothelium after the first third of the asexual blood cycle [P. knowlesi is potentially lethal in humans – but these parasites do not cause cerebral malaria [Similar to some primate malaria parasites , the difficulty in distinguishing malaria from sepsis in children means that both parenteral anti-malarials (i.e. artesunate 3 mg/kg stat for children < 20 kg and 2.4 mg/kg for larger patients) and broad-spectrum antibiotics should be given together as soon as the diagnosis is suspected . The mosIn low transmission settings where misdiagnosis is much less likely, it is reasonable in adults to treat only for severe malaria unless there is evidence for concomitant bacterial sepsis. However, antibiotics should be given to all adult patients with a very high parasitaemia (> 20%) , and shoMisdiagnosis of severe malaria is common. Its impact is underestimated. Misdiagnosis can result in incorrect treatment  and it dfHRP2) [The relationship between malaria and bacterial infections is complex , 102–109fHRP2) , 111 meafHRP2) , 109 (FifHRP2) .Fig. 6MiThe substantial overdiagnosis of severe malaria cannot be ignored in epidemiology, burden of disease, pathophysiology, genetic association and treatment studies. In the large evaluation of African children who had been admitted to leading research centres with a diagnosis of severe malaria (described above), mortality was higher in the likely misdiagnosed group, presumably because most had sepsis , 109. ThPlasmodium vivax. In endemic areas low density chronic P. vivax parasitaemia is common, and so it is not unusual for severely ill patients to have incidental low-density infections, particularly if PCR is used for parasite detection.Thus, it seems that some of the children with bacteraemia who are diagnosed as having severe malaria may genuinely have a high parasite biomass and extensive sequestration predisposing to bacterial sepsis—but the remainder have a primary bacterial infection and incidental or concomitant malaria. The interaction is complicated further as severe malarial anaemia predisposes to bacterial sepsis, and patients with uncomplicated malaria may have concomitant sepsis. At a population level, as malaria is controlled, the prevalence of sepsis declines  pointing to the important contribution of malaria to bacterial sepsis, both concomitantly and sequentially . It is vChildren who are admitted with severe malaria anaemia have a high mortality in the months following admission –41. Thisimmediately in all cases. As delay in receiving artesunate is a major contributor to death, it is important that referral to a facility capable of managing the sick patient should be as rapid as possible. Pre-referral rectal artesunate should be given to all children with suspected severe malaria [Overall, the consensus definitions of severe malaria described generally as “WHO criteria” have worked well to identify patients at risk and to inform research studies. From a practical case management perspective, specificity in the diagnosis is not as important as recognition that severe malaria could be the cause of the severe illness, and thus starting life-saving treatment with artesunate as soon as possible . A new s malaria , 98. The malaria  on recta malaria . Pre-refFor patients needing respiratory support, artificial ventilation has improved in recent years as the dangers of high inflation pressures have become evident . UnfortuPfHRP2  are not readily available [PfHRP2 RDT is not too difficult [PfHRP2 concentration of ≥ 1000 ng/ml had an estimated sensitivity of 74% and specificity of 93% in identifying true severe falciparum malaria [From a research or epidemiology perspective, the low specificity of the current definition of severe malaria in African children is a challenge Fig. . It has RP2 Fig.  or plasmvailable , 126—altifficult . Importaifficult . The proifficult , 87. Mos malaria   but they can cause the other potentially lethal manifestations of severe malaria. Morphologically the younger P. knowlesi parasites resemble P. falciparum, whereas the older forms are often mistaken for Plasmodium malariae. Indeed any P. malariae parasitaemia over 1% should be regarded as P. knowlesi until proved otherwise. Uncomplicated P. vivax infections in a non-immune subject are often worse than uncomplicated P. falciparum malaria infections, causing high fever, weakness, malaise and sometimes rigors and prostration. Some of these vivax malaria illnesses warrant hospital admission. In the past 20 years there has been a marked increase in the number of reports of “severe” vivax malaria, mainly from India [P. vivax malaria die, particularly if they are old or debilitated [P. vivax may sometimes cause acute pulmonary oedema-although the prognosis is better than in severe falciparum malaria [P. vivax endemic areas) Plasmodium vivax can cause severe illness, but the proportion of symptomatic cases which develop life-threatening illness is substantially less than for P. falciparum infections. However recurrent infections with P. vivax in areas of high transmission, such as the island of New Guinea, are associated with severe anaemia and substantial mortality both in the acute phase and over the longer term [P. vivax infections would help clarify the prognostic associations and risk factors. But overall, the mortality of acute P. vivax infections is substantially lower than that of P. falciparum infections.n humans , 129. Itom India –132. In ilitated , 134. P. malaria , 135. Buger term –138. FurThe apparent lack of progress in reducing the global death toll from malaria despite substantial investment suggests that we should reexamine the evidence, and review the current strategies to prevent and treat severe malaria . The mor


Haemoglobinopathies and G6PD deficiency are inherited disorders found mostly in malaria-endemic areas among different tribal groups of India. However, epidemiological data specific to Particularly Vulnerable Tribal Groups (PVTGs), important for planning and implementing malaria programmes, is limited. Therefore, the present community-based study aimed to assess the prevalence of haemoglobinopathies and G6PD deficiency among the 13 PVTGs found in the state of Odisha, reporting the maximum malaria cases in the country.Plasmodium species identification.This cross-sectional study was conducted from July 2018 to February 2019 in 12 districts, home to all 13 PVTGs, in an estimated sample size of 1461, selected two-stage sampling method. Detection of haemoglobinopathies was done by the variant analyser. Screening of G6PD deficiency was carried out using DPIP method followed by quantification using spectrophotometry. The PCR–RFLP technology was used to determine variant of G6PD deficiency and haplotype analysis of sickle cell, while ARMS-PCR and GAP-PCR was used for detecting the mutation pattern in β-thalassaemia and α-thalassaemia respectively. The diagnosis of malaria was done by Pf-PAN RDT as point of care, followed by nPCR for confirmation and S revealed the presence of Arab-Indian haplotype in all HbS cases and IVS 1–5 G → C mutation in all β-thalassaemia cases. In case of α-thal, αα/α-3.7 gene deletion was most frequent (38%), followed by αα/α-4.2 (18%) and α-3.7/α-3.7 (4%). The frequency of G6PD Orissa (131C → G) mutation was found to be 97.9% and G6PD Mediterranean (563C → T) 2.1%. Around 57.4% of G6PD deficient individuals and 16% of the AS were found to be malaria positive.The prevalence of sickle cell heterozygotes (AS) was 3.4%, sickle cell homozygous (SS) 0.1%, β-thalassaemia heterozygotes 0.3%, HbS/β-thalassaemia compound heterozygote 0.07%, HbS-α-thalassaemia 2.1%, G6PD deficiency 3.2% and malaria 8.1%. Molecular characterization of βPlasmodium vivax. Screening and counselling for HbS is required for the PVTGs of Odisha.The present study reveals wide spread prevalence of sickle cell anaemia, α-thalassaemia, G6PD deficiency and malaria in the studied population. Moderate to high prevalence of G6PD deficiency and malaria warrants G6PD testing before treating with primaquine (PQ) for radical cure of  Plasmodium falciparum malaria /[(d2/Z21-α/2*(N-1) + p*(1-p)] using OpenEpi software version 3.01. A two-stage sampling method was adopted to select the samples. Villages, the Primary Sampling Units (PSUs) were selected from a list of all the PVTGs villages from 12 districts with probability proportional to population size (PPS) sampling at the first stage followed by random selection of a sample of households within selected two PSUs from each PVTG at the second stage. The study was approved by the ethics committee of ICMR-Regional Medical Research Centre (RMRC), Bhubaneswar  by an automatic blood cell counter  on the day of arrival.At least 1 ml of venous blood was collected in BD VacutainerDiagnosis of haemoglobinopathies was done by using cation-exchange high-performance liquid chromatography  with the aid of the β-thalassaemia short programme.For molecular analysis, the genomic DNA was extracted from 200 μl of whole blood using QIAGEN mini DNA extraction kit following the manufacturer’s instructions. DNA samples of sickle cell diseases and trait cases were screened for β-globin gene haplotypes (the HindII (5′ε), HindIII (Gγ), HindIII (Aγ), HindII (5′ψβ), HindII (3′ψβ), AvaII(β), and HinfI (β-3′) by restriction fragments length polymorphism and common α-globin gene deletions  by using published primers and methods , 24.Five common mutations in the ß-globin gene including IVS 1–5 G → C, IVS 1 -1 G → T, Codon 41/42 (-TCTT), Codon 8/9, and the 619 bp in β-thalassaemia positive cases were carried out by ARMS-PCR technique using normal and mutation-specific primers .. [G6PD deficiency was detected by the Dichlorophenol-indophenol (DPIP) dye decolourization method and quantified by spectrophotometry following the method described by Dacie and Lewis . The sam. . At leasP. falciparum and other Plasmodium infections using 20 μl of blood sample at the study site. Genomic DNA from all RDT positive samples was extracted from blood using QIAamp® DNA Blood Mini Kit  following the manufacturers’ manual. The DNA extracted was further subjected to species-specific nested PCRs as described by Snounou et al. [P. falciparum: 205 bp, P. vivax; 120 bp, Plasmodium malariae; 144 bp, Plasmodium ovale; 800 bp) were visualized and recorded digitally using the Gel-Doc system .The Pf-PAN Rapid diagnostic kit  was used for the detection of u et al. . The PCRData obtained were analysed and statistically tested using the software SPSS-20. Descriptive statistics were used to analyse the data obtained. The association test was done using one way-ANOVA . The Hardy Weinberg Equilibrium (HWE) was tested using the Guo and Thompson method . Chi-squA total number of 1461 individuals  with a mean age of 32.59 ± 20.92 years were included in the study. Among them 25% (n = 365) were children from 1 to 14 years, 48.9% (n = 715) were between 15 and 49 years of age and 26.0% (n = 381) were 50 years and above of age. In the case of sickle cell and thalassaemia, 57.1% were female and 42.9% were male and a majority of them were ≤ 50 years. Whereas in the case of G6PD 59.5% were male and 40.4% were female and 44.7% of the cases were between 15 and 49 years of age. G6PD and malaria were found to be more among males than in females  were diagnosed as Sickle cell heterozygotes (AS), 0.1% n = 2) Sickle cell homozygous (SS), 0.3% (n = 5) β-thalassaemia heterozygotes, 0.07% (n = 1) HbS/β-thalassaemia compound heterozygote and 2.1% (n = 30) α-thalassaemia. Of the 50 HbAS individuals, 30 (60%) were found to be associated (co-inheritance) with the α-thal gene. Amongst the α-thalassaemia individuals, 4% (n = 2) had double (α-3.7/α-3.7), 38% (n = 19) had αα/α-3.7 and 18% (n = 9) had αα/α-4.2 single gene deletion. A sequence haplotype analysis revealed the presence of Arab-Indian haplotype in all the HbS cases. Of the 13 tribes studied, β Sickle cS and Saora for αthal , 39.3% were in the reproductive age group (15–49 years) and 42.8% were above or equal to 50 years of age  with an average erythrocytic enzyme activity of 1.26 ± 0.65 IU/g Hb in males and 3.8 ± 1.8 IU/g Hb in females, while the average enzyme activity for all the individuals was 7.4 ± 0.8 IU/g Hb. Out of 13 PVTGs, G6PD deficiency was found to be prevalent among nine tribes , the lowest in Didayi (1.1%) and the highest in Hill Kharia (10.4%). Gender-wise G6PD activity distribution of the entire population is shown in Fig. The average haemoglobin (Hb) level of the individuals having HbAS with α deletion was 12.2 g/dl (range: 10.8–13.1) whereas HbAS individuals without α gene deletion have 11.3 g/dl (range: 9.00–10.6), β-thal have 11.02 g/dl (range: 8.6–12.6) and the HbS/β-thal compound heterozygote individuals have 10 g/dl. The average haemoglobin in HbAS male and female was 10.8 ± 2.3 and 11.7 ± 2.1, respectively. The individuals with G6PD deficiency had significantly (P = 0.023) low levels of Hb  than the normal individuals . Further age-wise stratification of G6PD female individuals, the Hb level of female in reproductive age group (8.8 ± 0.5 g/dl) was found to be significantly lower compared to the females of other age group  as shown in Table P. falciparum and 24 (20.3%) were due to P. vivax, while 20 (16.9%) were mixed infections . The prevalence of malaria was high in Kutia Kondho, Dongoria Kondho, and Paudi Bhuyan. Out of 50 sickle cell individuals (48 HbAS and 2 HbSS), 8 (16%) were found to be infected with malaria  individuals were diagnosed to be positive for malaria. Amongst the malaria positives, 62 were due to Mayurbhanj, Keonjhar, Deogarh, Anugul, Sundargarh, Kandhamal, Rayagada, Malkangiri, Kalahandi, Gajapati, Ganjam and Nuapada) [Juang, Bondo, Didayi, Langia Saora, Saora, Kutia Kondho, Dongoria Kondho, Paudi Bhuyan, Birhor, Chuktia Bhunjia, Kharia, Lodha, Mankidia) found in Odisha. The study shows an overall allele frequency of the HbS gene as 0.019, β-thal as 0.0009, α-thal as 0.0113, and G6PD deficiency as 0.022 among the studied PVTGs, while the malaria test positive rate (TPR) was 8.1. The most important finding in the present study was α-thal gene among certain PVTGs not reported earlier.The PVTGs are the marginalized section of the scheduled tribes of India, who are relatively isolated, educationally and socio-economically backward, living in habitats far away from amenities and having stagnant or declining populations. Among the 75 enlisted PVTGs by the Government of India, the highest number are found in Odisha (13) with a population of around 0.25 million, who are residing in 12 districts (Nuapada) . This reS gene carrier (3.4%) was found in eight “primitive tribes” , βthal (0.3%) and βS-βthal compound heterozygote gene (0.07%) in one “primitive tribes” (Paudi Bhuyan) and αthal and βS gene co-inheritance in around 2.1% of the individuals in five  “primitive tribes”. The Hardy–Weinberg-Equilibrium (HWE) analysis calculated using the gene frequency of unrelated individuals shows genetic equilibrium in all PVTGs except in the Paudi Bhuyan tribe for βS and in Saora for αthal indicating the influence of genetic drift or genetic migration among Paudi Bhuyan and Saora tribes, whereas others are still isolated and maintain genetic homogeneity. DNA analysis has revealed that the majority (~ 56%) of αthal mutations in the study population is due to either αα/α-3.7 or αα/α-4.2 gene deletion similar to the observations made in other Indian tribes [S gene are linked to Arab-Asian haplotype and β−thal gene with IVS 1–5 mutations suggesting unicentric origin of the mutation in PVTGs of Odisha as predicted for other tribes in India [Since the first report of sickle cell disease by Lehman and Cutbush  among thn tribes , 41. Whein India .: 65.3, Malkangiri: 44.8 Kalahandi: 20.3, Gajapati: 18.3, Ganjam: 4.2 and Nuapada: 8.7) suggesting high transmission [thal and βS gene among some of the PVTGs is very significant, because αthal can reduce the amount of sickled RBC, increase the HbF/HbA2 level and reduce haemolysis eventually diminishing the severity of the disease [thal and βS heterozygotes, in this group of the population could be clinically advantageous and might be giving protection against P. falciparum. An intensive study is therefore, required in this setting to generalize the data to African populations in this regard [In spite of a higher carrier frequency of sickle cell mutation, detection of a low percentage of the sickle-cell homozygote (0.1%) cases in the studied PVTG population could be due to increased mortality during infancy and early childhood because of poor nutritional/haemoglobin status and malaria infection . As per  disease . Hence cs regard .P. falciparum and P. vivax. However, the risk of haemolysis in G6PD deficient individuals hampers the widespread use of PQ safely in malaria-infected patients. There has been a sharp decline in malaria cases in Odisha [P. falciparum: 12.7%, P. vivax: 32.6%, P. falciparum + P. vivax: 12.76%) indicating high transmission. Since G6PD deficiency is very common in malaria-endemic areas, WHO have recommended G6PD testing before giving PQ [P. falciparum and P vivax malaria.G6PD deficiency plays a central role in malaria elimination programme that use PQ as part of the treatment of choice for both n Odisha  since 20iving PQ . G6PD teiving PQ . The WHOIn conclusion, a high frequency of alleles such as G6PD deficiency, HbS, and α-thalassaemia associated with malaria are prevalent among the PVTGs of Odisha. Moderate to high frequency of G6PD deficiency indicates that the diagnosis and management of G6PD deficiency are necessary and an obligatory new-born screening programme, prenatal screening, and counselling for these genetic disorders, especially HbS, are required for the PVTGs. The most significant challenge in delivering the health services in PVTG areas is the poor physical access due to non-availability of health care service providers. In such a case, Mobile Health Units (MHU) at least once a week might be considered as an alternative option to address such health issues in PVTG-specific areas of Odisha.


Plasmodium falciparum in pregnant women attending antenatal care (ANC) in selected private hospitals in Onitsha metropolis South East Nigeria.Recent reports suggest that pregnant women living in holoendemic regions of sub-Sahara Africa die in great numbers annually due to malaria disease resulting from their higher susceptibility, reduced immunity and demographic associated factors. This work investigated the prevalence of Venous blood samples were collected from 270 pregnant women during ANC visits between October 2016 and December 2017. A questionnaire was used to collect demographic data, gestational age, knowledge of malaria and preventive measures while clinical presentations and symptoms were extracted from the physician’s clerking form. Laboratory diagnosis was done using microscopy. The effect of the demographic variables and other associated factors on prevalence and parasite densities was studied using Chi-square and ANOVA tests.P. falciparum prevalence was 42.6%. Prevalence varied with the maternal age, gestational age, preventive measures adopted by the pregnant women and clinical presentations. 27.8 % of the infected women were highly parasitized (>5000 parasites/μl); 67% had a moderate parasite density  and 5.2% showed a low parasite density (1-999 parasites/μl). We observed that 35.2%, 30%, 18.9% and 5.2% of the study cohorts preferred and used treated bed nets, insecticides, windows and door screening and non-treated bed nets respectively as malaria preventive measures. 5.9% did not use any protection.The overall The findings of this study revealed high prevalence of malaria among pregnant women living in Onitsha metropolis with high mean parasite densities despite strong adherence to use of sulphadoxine-pyrimethamine (SP) for intermittent preventive treatment in pregnancy (IPTp) and other malaria preventive measures. Malaria disease is considered a major public health concern among pregnant women especially in tropical and subtropical areas of the world and in sub-Sahara Africa, where malaria vectors are commonly found. Malaria in pregnancy results in adverse pregnancy outcomes, such as spontaneous abortion, neonatal death, and low birth weight. Chronic anemia, due to malaria may also affect a child’s growth and intellectual development. Pregnant women are particularly vulnerable to malaria infection as a result of reduced immunity .In malaria-endemic regions of Africa, pregnancies are threatened by malaria. Recently, prevalence of exposure to malaria during pregnancy was highest in Central Africa 40%), closely followed by West Africa 39%), while East and Southern Africa had a reported exposure prevalence of 24%  x Assumed WBC count.Samples were labeled negative if no parasites were detected after observing 200 fields of a thick blood film.All data were presented and calculated using SPSS statistical package (version 22). The effect of the demographic variables and other associated factors on prevalence and parasite densities was studied using Chi-square and ANOVA tests, respectively, with a significance level of p≤0.05 and confidence level of 95%.nd trimester and had enjoyed secondary education (47.8%).The demographic characteristics of the 270 women as captured by the questionnaire are shown in Nearly half of the participants (48.9%) had used SP for prophylaxis against malaria infection prior to participation in the study; nearly a third (29.4%) had not received any prophylaxis. Some participants used quinine (8%), amodiaquine (0.4%) or artemether-lumefantrine (AL) (13%), which are not usually recommended for use during pregnancy. The use of insecticide-treated bed nets and insecticides  was higher  than any other vector control measure adopted by the women.Clinical presentations as recorded on the ANC cards by doctors and nurses were extracted and are shown in P. falciparum prevalence with respect to age, gestational age, prophylaxis using SP, vector control measures and clinical symptoms is shown in Microscopy confirmed 115 of the blood samples as positive, giving an overall prevalence of 42.6% . ParasitPrevalence decreased with age. The highest prevalence (60.9%) was observed in the youngest age cohort (15-20 yrs). In contrast, 27-32 yrs group had the lowest prevalence  . Mean past trimester (1-3 months) than among those in 2nd (4-6 months) and 3rd trimesters (42.70% and 35.90% respectively)  of 4283±4827 and 3861±2594 respectively (p>0.05) (The frequency of parasite occurrence was high (48.20%) among pregnant women in their 1ctively)  at p>0.0(p>0.05) .P. falciparum infection decreased with increasing level of education among pregnant women  than those who had used AL (50%) and SP (48.7) at p<0.05  than among those who used windows and door nets (43.10%), treated bed nets (42.10%) and insecticides (IRS) (39.50%). The observed difference was not statistically significant at p≤0.05.It was further observed that the prevalence rate of The distribution of parasite density with respect to the type of malaria prevention method used by the pregnant women participating in the study is shown in P. falciparum among pregnant women with respect to the clinical symptoms was higher among those with headache and fever (78.50%) than in asymptomatic individuals who recorded only 16.10% of the P. falciparum infections. Furthermore, the mean parasite density was higher in those who indicated headache and fever (4931±4301parasites/μl) compared to those who indicated headache only (3764±1949 parasites/μl) or fever only (3644±3284 parasites/μl). Among the asymptomatic individuals, the mean parasite density was considerably low (1847±1319 parasites/μl). The observed effect was statistically significant at p≤0.05  are proven interventions adopted by Roll Back Malaria (RBM) partners in Nigeria to stem the high incidence of malaria [Also,  malaria .However, both prevalence and mean parasite density was higher in symptomatic pregnant women than among the asymptomatic individuals. This suggests that the symptoms and clinical presentation of malaria cases during pregnancy is signifcantly dependent on parasite load and can be useful for early detection. It further approves the use of clinical presentation as first line diagnosis for malaria in pregnant women and immediate treatment of patients with fever, headache, or headache and fever with antimalarial drugs, where laboratory screening and confirmation is not readily available. Treatment based on clinical presentation in pregnant women may help prevent complications associated with malaria in pregnancy as well as prevent potentially fatal disease among those living in rural settings.P. falciparum parasites which gives room for parasite immune evasion including drug resistance especially in areas of high transmission [P. falciparum isolates [Finally, a high mean parasite density was recorded contrary to the thought that pregnant women usually have reduced number of parasites in the blood circulation due to proliferation of parasites in the placenta ,29. Highsmission -32. Studisolates -36 suggeThe prevalence of malaria among pregnant women living in the Onitsha is considerably high. There is a strong adherence to the use of SP for IPTp among pregnant women attending ANC in selected hospitals. Also, there is a strong adherence to the use of treated bednets, and window and door screening as malaria prevention measures. Despite strong adherence to SP for IPTp and other malaria preventive measures, the findings of this study revealed a high mean parasite density among the  study cohort. Therefore, efforts should be made towards studying and understanding the molecular basis for the reported high prevalence and parasite density among pregnant women. Malaria elimination programmes should focus on strategies that will help pregnant women in poor and highly dirty environments to understand the risk of exposure to malaria infection as well as provide access to malaria prevention measures including ITNs and LLINs so as to reduce the malaria burden in this population.