Plasmodium species. Comprehensive control measures and surveillance programmes must be intensified. This study aims to determine the prevalence of microscopic and submicroscopic malaria in Langkat district, North Sumatera Province, Indonesia.Indonesia is progressing towards malaria elimination. To achieve this goal, intervention measures must be addressed to cover all A cross-sectional survey was conducted in six villages in Langkat district, North Sumatera Province in June 2019. Data were recorded using a standardized questionnaire. Finger pricked blood samples were obtained for malaria examination using rapid diagnostic test, thick and thin blood smears, and polymerase chain reaction.Plasmodium malariae infection, no positive RDT examination, and three (0.9%) were positive for P. malariae (n = 1) and Plasmodium knowlesi (n = 2). The distribution of bed net ownership was owned by 40% of the study participants. The participants had a house within a radius of 100–500 m from the forest (86.3%) and had the housing material of cement floor (56.1%), a tin roof (82.2%), wooden wall (35.7%), bamboo wall (28.1%), and brick wall (21.6%).A total of 342 individuals were included in the study. Of them, one (0.3%) had a microscopic Malaria incidence has substantially decreased in Langkat, North Sumatera, Indonesia. However, submicroscopic infection remains in the population and may contribute to further transmission. Surveillance should include the detection of microscopic undetected parasites, to enable the achievement of malaria elimination. Indonesia has shown significant progress towards a malaria-free zone within the last decade with a reduction in 50% of cases and 66% of deaths due to malaria . AggressMicroscopic examination is currently the gold standard for malaria diagnosis, however, misdiagnosis occurs in cases of low parasitaemia as well as incorrect species identification . Rapid dPlasmodium falciparum infection prevalence to an increase of non-P. falciparum infection prevalence. This is a concern as the current malaria elimination strategies are focusing on P. falciparum and Plasmodium vivax infections [In near to elimination settings, there are several challenges to approach its elimination goal. There is a shift in the trend of malaria infection with a decrease of fections , and thefections . In low fections , 11.The prevalence of submicroscopic malaria infection in Langkat was 33.5% with falciparum malaria infection being the most common type of malaria infection in that area . This stThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Medicine, Universitas Sumatera Utara (No. 179/TGL/KEPK FK-USU-RSUPHAM/2019). Survey subjects were informed by the local health district personnel of the purpose and procedures of the study, and a written informed consent was obtained from each adult participant at study registration. In the case of individuals < 18 years, written informed consents were sought from their parents or legal guardians.2 with an altitude of 4–105 m above sea level. The population of Langkat was estimated 1,041,775 inhabitants in 2019 [A cross-sectional study was conducted in six villages  in Langkat district, North Sumatera Province, Indonesia in June 2019 /e2, where z is the confidence interval which is set at 95% ; (p) is the expected prevalence of malaria infections of 34% from a previous study [The sample size for study participants was calculated using the following Cochran’s formula: N = zus study  and (e) Blood sample was collected from finger pricked and spotted on glass slides for thick and thin blood film, onto malaria RDT , onto the Whatman 3MM filter paper  for molecular screening, and onto a microcuvette for Hb examination using the HemoCue® Hb 301 . The malaria RDT kit and Hb examination were performed according to the manufacturer’s instructions and read on site. Similarly, thin and thick blood films were prepared on site, stored in slide boxes and transported to the Parasitology Laboratory, at the Faculty of Medicine, Universitas Sumatera Utara. Thin blood films were fixed with methanol and all films were stained with 3% Giemsa solution  for 30 min and examined under oil immersion (10× 100× magnification) by experienced microscopists. Blood films were defined as negative if no parasites were found after examining 1000 × high power microscopy fields. For any positive samples, malaria species were identified and asexual parasite forms were counted against 200 leukocytes. Gametocytes counts were separately recorded. Parasite density was estimated from parasite counts, assuming that there were 8000 leukocytes per μl of blood. All slides were independently re-examined by two experienced microscopists blinded to the first microscopy reading results. Discrepancies between the two readings were resolved by a third experienced microscopist.Plasmodium spp. identification was conducted following previously published methods targeting the 18S rRNA, and confirmation of P. knowlesi infection was done using PCR targeting the sicavar genes [For the detection by molecular screening, blood spotted filter papers were air-dried thoroughly at ambient temperature before sealed in individual zipped plastic bags and stored at the − 20 °C. The molecular analysis was performed at the laboratory in the Faculty of Medicine, Universitas Sumatera Utara, Medan. DNA was extracted using Chelex method , and PCRar genes , 15, 16.The data collected were double entered into Microsoft Excel (version 2016) spreadsheets and cross-checked for errors. Then, the data were processed and analysed using SPSS software . The data were presented as a frequency distribution table and analysed with Fisher's exact test (P ≤ 0.05) to identify the risk factors of submicroscopic malaria as well the correlation with related variables evaluated.A total of 342 participants were enrolled in this study Table . The majP. malariae by microscopy, with asexual and sexual parasite densities of 1160 and 2320 parasites µ/L of blood, respectively . None of the common human malaria parasites were detected, while P. knowlesi and P. malariae infections contributing to 1.2% positivity. All of the infections occurred in women aged > 18 years with asymptomatic clinical manifestation and one respondent had an anaemia (Hb level was 8.7 g/dl).The prevalence of malaria by microscopy, RDT and PCR is shown in Table P. falciparum and P. vivax, indicating the control interventions for these species are effective.This study was done as a follow-up study conducted at the same study sites in 2015. A significant decline was observed in the prevalence of microscopic malaria in the area from 16.5% to 0.3%. The dramatic reduction was also seen in the submicroscopic infection . This suThe malaria control programme named “Gebrak Malaria” was started in 2009 with several control measures, such as active and passive case finding, mass blood surveys and malariometric surveys, effective drug administration, vector control and surveillance. These interventions were conducted to reduced malaria burden and accelerate the target of Indonesia being malaria-free by 2030 [P. malariae and P. ovale by 2 and 6 times, respectively [Plasmodium malariae infection causes prolonged infection, but also reactivation after decades, and remain in the body as asymptomatic infection [P. malariae recurrences after 3-days treatment with ACT, suggesting that the universal treatment for malaria might not be appropriate enough to support human malaria elimination particularly for P. malariae [Both microscopic and submicroscopic examination in this study found non-falciparum malaria. Previously, a significant reduction of falciparum malaria infection in the population has been associated with an increased in the prevalence of ectively . Plasmodnfection . Furthernfection . Studiesmalariae –25.P. knowlesi has more complex factors contributing in its life cycle, involving humans, the mosquito vectors and the macaque hosts. Environmental factors such as deforestation and land-use change contributed to more competent vectors, increased breeding sites which in favour to an increase in knowlesi transmission [P. knowlesi specific control measures.The continuing trends of decreased incidence in human-only malaria in Sabah, and an increased in knowlesi malaria explains that the current control measures are mainly addressing human-malaria only. smission , 26, 27.P. knowlesi infection was detected among females, knowlesi malaria is mostly found in men [Plasmodium knowlesi infection is also more common in adults and usually among older individuals compared to falciparum and vivax malaria [There were several limitations in this study. First, all malaria infections detected occurred in females. The participants were screened for only one day in each survey area, and screening was done between 9 am and 4 pm. The study sites were located in the middle of the forest, and most of the male residents work in the forest and return to the village at dawn. This may have increased female participation (68%) and caused a sampling bias in this study. Although a low proportion of d in men , who havd in men . Plasmod malaria . The pre malaria .A very low prevalence of malaria was found in this study. Surprisingly, the only parasite detected by microscopy resulted in a negative PCR. One of the possibilities was DNA degradation due to improper collection and storage of the filter papers , 8. FiltPlasmodium species except P. ovale were detected [Despite the limitations of this study, the possibility of successful malaria control resulting in significant decline of malaria burden in these areas was could not completely ruled out. The incidence of microscopic and submicroscopic malaria in the same study areas in 2015 were 33.5% and 13.6%, respectively. Four detected . In contdetected . This stdetected , 13, thePlasmodium malariae and P. knowlesi were the only malaria species detected suggesting the control efforts for human malaria may be effective in reducing the malaria burden in the area. Additional strategies are still needed to accelerate malaria elimination by intensification of vector control, targeting the submicroscopic malaria, and the interruption of the non-human malaria parasite transmission, thus Indonesia can achieve its target to be malaria free.Indonesia aims to become malaria free area by 2030. Follow-up survey in Langkat district, North Sumatera province showed a substantial decrease of malaria prevalence to 1.2%. Submicroscopic infection contributed to 75% of these malaria cases.


To estimate the fraction of anaemia attributable to malaria and sickle cell disease (SCD) among children aged 6–59 months in Nigeria.Cross-sectional analysis of data from Nigeria’s 2018 Demographic and Health Survey (DHS).Nigeria.11 536 children aged 6–59 months from randomly selected households were eligible for participation, of whom 11 142 had complete and valid biomarker data required for this analysis. Maternal education data were available from 10 305 of these children.Haemoglobin concentration.We found that 70.6% (95% CI: 62.7% to 78.5%) of severe anaemia was attributable to malaria compared with 12.4% (95% CI: 11.1% to 13.7%) of mild-to-severe and 29.6% (95% CI: 29.6% to 31.8%) of moderate-to-severe anaemia and that SCD contributed 0.6% (95% CI: 0.4% to 0.9%), 1.3% (95% CI: 1.0% to 1.7%) and 10.6% (95% CI: 6.7% to 14.9%) mild-to-severe, moderate-to-severe and severe anaemia, respectively. Sickle trait was protective against anaemia and was associated with higher haemoglobin concentration compared with children with normal haemoglobin (HbAA) among malaria-positive but not malaria-negative children.This approach used offers a new tool to estimate the contribution of malaria to anaemia in many settings using widely available DHS data. The fraction of anaemia among young children in Nigeria attributable to malaria and SCD is higher at more severe levels of anaemia. Prevention of malaria and SCD and timely treatment of affected individuals would reduce cases of severe anaemia. This study uses individual-level data from a large, nationally representative survey that includes haemoglobin concentration, malaria testing and sickle cell testing.The relatively simple approach used here could be applied to Demographic and Health Survey and similar surveys.The data do not include measures of iron or inflammation, which would be needed to identify iron deficiency, a major cause of anaemia.We treated malaria rapid diagnostic test results, which are imperfect, as a proxy for malaria infection.http://ghdx.healthdata.org/gbd-results-tool), there were 8.3 million (95% CI: 5.5 to 12.1 million) years lived with disability worldwide among children under 5 years old due to anaemia in 2019, the majority of which  were due to moderate or severe anaemia. The highest rates of anaemia (adjusted haemoglobin concentration <110 g/L) and severe anaemia (<70 g/L) among preschool-aged children are reported from sub-Saharan Africa (SSA).1 2Anaemia is a major cause of morbidity and mortality among children. According to Global Burden of Disease (GBD) models , children may have several concurrent causes for their anaemia, each requiring a different treatment strategy, for example iron supplementation, chemotherapy, transfusion, antimalarials or deworming.6http://ghdx.healthdata.org/gbd-results-tool). Because most studies of anaemia are based on relatively small populations, national and regional estimates often need to extrapolate data from these geographically scattered studies to cover the rest of the population. Here, we have explored how Nigeria’s 2018 Demographic and Health Survey (DHS), a large, and uniquely detailed data source, could shed light on the contribution of Plasmodium falciparum (P.f.) malaria and sickle cell disease (SCD) to anaemia. For the first time in DHS history, this Nigerian survey not only tested a population-representative sample of children for malaria infection, but also tested them for haemoglobin subunit beta (HBB) type, including the alleles associated with sickle cell trait and disease.vs rural residence, maternal education) risk factors, we can provide a better estimate of the contribution of malaria and SCD to anaemia and explore the inter-relationships between SCD and trait, malaria and anaemia.Nigeria is Africa’s most populous country and experiences a high childhood anaemia burden. Previously, 60% of Nigeria’s moderate and severe anaemia among children under 5 years old has been attributed to iron deficiency and only 12% and 2% to malaria and sickle cell disorders, respectively (P.f. (HRP-II) malaria rapid diagnostic test (RDT), yielding 11 173 (96.9% of eligible children) RDT results. The same finger or heel prick was also used to evaluate HBB type using SickleSCAN point-of-care test from BioMedomics, yielding 11 186 results (97.0% of eligible children). This test determined the presence of the HbS and HbC alleles, allowing us to determine which children had normal HBB (HbAA), SCD (HbSS or HbSC), sickle cell trait (HbAS) or haemoglobin C trait (HbAC). The nine children who had a recorded HBB type of ‘other’ were excluded. In total, 11 142 children (96.6% of eligible children) with all three biomarker measurements were included in our analyses. Maternal education was not available for all of these children, so analyses that included maternal education had 10 305 (92.5%) of the children with all three biomarkers. The survey was approved by the National Health Research Ethics Committee of Nigeria and the ICF Institutional Review Board.We obtained data from Nigeria’s 2018 DHS.The DHS includes sample weights based on the survey design and on household response rates.We studied the relationships between anaemia and other factors using three thresholds for adjusted haemoglobin concentration: <110 g/L (anaemia), <100 g/L (moderate-to-severe anaemia) and<70 g/L (severe anaemia).82 approximation for the other characteristics .All the demographic and socioeconomic data came from the DHS. To compare demographic characteristics between children with and without moderate-to-severe anaemia, we applied the Wilcoxon rank-sum test to determine if the distribution of ages was significantly different and a proportions test with χhttps://r-survey.r-forge.r-project.org/survey/html/svyglm.html). The primary sampling unit is the cluster  and the strata are the DHS-defined strata . When there were no observations of one of the outcomes  in a category , we do not report the odds of the outcome. This effect did not have an impact on the model estimates for the other categories. To compute the effect of sickle cell status among RDT-positive children, we fit models using RDT-positive children with HbAA as the reference group rather than RDT-negative children.We fitted multivariate quasibinomial logistic and multivariate linear generalised linear models to predict anaemia status and haemoglobin concentration, respectively. We used the svyglm package to fit these models using survey weights , 67.9% were anaemic (adjusted haemoglobin concentration <110 g/L), 41.1% had moderate or severe anaemia (<100 g/L) and 3.0% had severe anaemia (<70 g/L) . Childre10.1136/bmjopen-2022-063369.supp1Supplementary dataChildren with moderate-to-severe anaemia were significantly younger and had lower proportions of females, malaria RDT+, HbSS, rural residence, lower three wealth quintiles and lower maternal education compared with children with mild-to-no anaemia .Malaria RDT positivity, HBB type, age and wealth quintile were significantly associated with all degrees of anaemia . Among cWe found the fraction of anaemia attributable to malaria to increase with the severity of anaemia, from 12.4% (95% CI: 11.1% to 13.7%) of overall anaemia (adjusted haemoglobin concentration <110 g/L) to 29.6% (95% CI: 29.6% to 31.8%) of moderate-to-severe anaemia (<100 g/L) and to 70.6% (95% CI: 62.7% to 78.5%) of severe anaemia (<70 g/L) . The fraAlthough RDT positivity is a major risk factor for anaemia at all levels of severity , malariaChildren with HbSS or HbSC were at high risk of anaemia , but somIf the information about HBB type is not used by the regression models used for prediction , then thEach year of age was associated with an increase in haemoglobin of 2.74 g/L . Among chttp://ghdx.healthdata.org/gbd-results-tool). We found that higher levels of household wealth and maternal education were associated with reduced risk of anaemia and higher average haemoglobin concentration in children, which is consistent with earlier analyses of the same datasetWe found that malaria RDT positivity and SCD were associated with a high risk of anaemia among children participating in Nigeria’s 2018 DHS. Our results support the hypothesis that malaria and SCD play a larger role in more severe compared with mild anaemia. Our estimate of the contribution of malaria to moderate-to-severe anaemia among young children in Nigeria of 30% is substantially higher than the GBD’s estimate of 12% , our analysis attributes 10.6% of severe anaemia (haemoglobin concentration <70 g/L) to SCD. We note that 11.6% of children with severe anaemia had HbSS or HbSC. Ideally all children with SCD should be identified through a newborn screening programme. However, such programmes are scarce in Africa but as an interim measure our findings indicate that, because SCD is so prevalent in our population-representative sample of children with severe anaemia, screening all young children with severe anaemia with a point-of-care test could identify undiagnosed SCD. Active management could then be started, which would improve their intellectual and physical development and could reduce their need for transfusions.16et al found a clinical malaria incidence rate ratio of 0.66 between children with HbAS versus HbAA, with protection peaking around 3–6 years of age20Although sickle trait was associated with an increased risk of mild anaemia, it was associated with protection against anaemia among malaria-positive children, with a greater degree of protection against severe anaemia. This is consistent with observations that sickle trait protects against severe malaria more than mild malaria illnessP. falciparum malaria were available in the DHS, not on P. vivax, though falciparum comprises most infections is in Nigeria. Even with additional data, the causes of anaemia are complex and can be interrelated, making it difficult to assign aetiology. Calis et al 2008 used structural equation modelling to account for these relationships.Our study has several limitations. Although we found that increasing age was associated with a reduced risk of anaemia, this finding could be partially attributed to the fixed thresholds used to define anaemia for all children regardless of age. Haemoglobin concentrations increased with age, which has been observed in healthy, disease-free children.Our analyses highlight the importance of malaria and SCD in the more severe stages of anaemia among young children in Nigeria. The majority of children in the DHS survey had mild-to-severe anaemia, most of which was not due to malaria or SCD. At the more severe end of the anaemia spectrum, malaria appears to be linked to 70.6% of severe cases and SCD to 10.6%. There are many ways to prevent or mitigate anaemia early, which could avert the development of severe anaemia and the need for blood transfusions. Malaria control has been observed to reduce anaemia prevalence.


Malaria burden among under-five children living in endemic areas of Yemen is largely unknown due to the lack of community-based studies. Therefore, this study determined the prevalence and risk factors associated with falciparum malaria among under-five children in rural communities of Al-Mahweet governorate, Yemen.Plasmodium falciparum and non-falciparum species using rapid diagnostic tests (RDTs), and duplicate Giemsa-stained thick and thin blood films were examined for malaria parasites. The density of asexual P. falciparum stages was also estimated. Data were then analysed, and the agreement between the results of thick-film microscopy and RDTs for diagnosing falciparum malaria was assessed using the kappa index. Statistical significance was set at a P-value of < 0.05.This community-based, cross-sectional study recruited 400 under-five children from two rural districts of Al-Mahweet governorate in December 2019. Demographic characteristics  and risk factors associated with malaria were collected through interviews with children’s caregivers using a structured questionnaire. Finger-prick blood was screened for Plasmodium falciparum was prevalent among 9.8% (95% CI 7.0–13.1) of under-five children in the rural communities of Al-Mahweet, with a median asexual parasite density of 763 ± 2606 parasites/μl of blood (range: 132–4280) and low-to-moderate parasitaemia levels. Approximately one-third of microscopy-confirmed cases were gametocyte carriers. Multivariable logistic regression analysis confirmed that age of three years or older , not sleeping under a mosquito net the previous night of the survey , sleeping outdoors at night , and absence of indoor residual spraying (IRS) during the last year  were the independent predictors of falciparum malaria among under-five children in the rural communities of Al-Mahweet. The observed percentage agreement between thick-film microscopy and RDTs was 98.5%, with a very good agreement (k-index = 0.9) between the two methods for falciparum malaria diagnosis that was statistically significant.P. falciparum based on microscopy and RDTs. Age of three years or older, not sleeping under mosquito nets, sleeping outdoors at night and absence of IRS can independently predict falciparum malaria among them. The very good agreement between thick-film microscopy and RDTs for diagnosing falciparum malaria in children supports the usefulness of using RDTs in such resource-limited rural communities.Approximately one in ten under-five children in rural communities of Al-Mahweet is infected with  Plasmodium falciparum [Malaria is a major public health problem in the world. According to the latest report by the World Health Organization (WHO), 241 million cases were estimated in 85 malaria-endemic countries, increasing from 227 million in 2019 [Under-five children are at high risk of contracting malaria and developing severe disease . Apart fP. falciparum in the mid-2000s, and falciparum malaria was recently reported among 8% of schoolchildren from Bajil district of Hodeidah governorate [In Yemen, community-based malaria prevalence in under-five children is largely unknown, with few published studies reporting on childhood malaria. In this regard, 18.6% of children in rural districts of Taiz governorate were confirmed to be infected with ernorate . Anotherernorate . With thernorate , childreA community-based, cross-sectional study was conducted among under-five children in two rural districts of Al-Mahweet governorate known to be highly endemic for malaria in December 2019. Children of both genders were included in the study if they were residents in the study districts over the year preceding the study and did not receive antimalarial drug(s) one month before the study, and if their parents/guardians gave written informed consent.2 and is inhabited by approximately 597,000 people [, P. falciparum was detected among 15.6% (3235/20764) of febrile patients examined by RDTs and/or microscopy at health facilities in the two districts; of whom, 908/5001 (18.2%) were under-five children according to the data derived from the Electronic Disease Early Warning System (eDEWS) of Yemen . Therefore, these two districts were purposively selected to conduct this study.Al-Mahweet is a small mountainous governorate at the coordinates of 15° 28′ N and 43° 32′ E, approximately 111 km to the northwest of Sana'a. It is about 2,382 km0 people . It is e0 people , Hodeida0 people . Of its www.OpenEpi.com) based on an expected prevalence of 13% [A minimum sample size of 348 children was calculated using OpenEpi software, Version 3.01  and risk factors associated with malaria were collected through face-to-face interviews with children’s caregivers using a structured questionnaire. Axillary temperature was measured using a thermometer. Children with a temperature of ≥ 37.5 °C were considered febrile. Blood drops were collected by finger prick for rapid malaria testing and preparing duplicate thick and thin blood films.P. falciparum and non-falciparum species using CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo RDTs , according to the manufacturer’s instructions. Such RDT kits are currently adopted by the NMCP for malaria diagnosis in the country. Thick and thin blood films were prepared, Giemsa-stained and examined microscopically using standard procedures [P. falciparum asexual stages against 200 leukocytes on thick blood films, assuming an average leukocyte count of 8000/µl of blood [Blood drops were screened for ocedures , 16. Thiof blood , 16. Thik) index, where the agreement was interpreted as poor (k < 0.20), fair (k = 0.20–0.40), moderate (k > 0.40–0.60), good (k > 0.60–0.80) or very good (k > 0.80) [P-value of < 0.05.Data were analysed using IBM SPSS Statistics, Version 23.0 . Categorical variables were expressed as frequencies and proportions, while continuous variables were expressed as the median and interquartile range (IQR) for non-normally distributed data. The prevalence of falciparum malaria was reported with its corresponding 95% confidence interval (CI), as determined by microscopy and/or RDTs. Univariate analysis with Pearson's chi-square or Fisher’s exact test was used to test the association of demographic and clinical characteristics as well as risk factors with falciparum malaria among under-five children, reporting the odds ratios (ORs) and corresponding 95% CIs of the associations. Multivariable logistic regression analysis was performed for significant factors in univariate analysis to identify the independent predictors of falciparum malaria among under-five children, together with their adjusted ORs (AORs) and corresponding 95% CIs. Agreement between the results of thick-film microscopy and RDTs for diagnosing falciparum malaria was assessed using the kappa ( > 0.80) . StatistThe majority of children included in this study were males (54.2%), aged three years or older (62.5%), with a median (IQR) age of 3 (2) years, and living in households of six members or more (63.5%). Most children’s fathers had primary education (37.0%) or were uneducated (37.0%) and were labourers (68.8%), while the majority of children’s mothers were uneducated (83.2%) and unemployed (99.2%) Table .Table 1CFalciparum malaria was prevalent among 9.8% (95% CI 7.0–13.1) of under-five children in rural communities of Al-Mahweet, where microscopy confirmed infection among 9.3% (95% CI 6.6–12.5) and RDTs were positive among 8.8% (95% CI 6.2–12.0) of children , with low and moderate parasitaemia levels being prevalent among 51.4% and 48.6% of infected children, respectively. Of children with falciparum malaria, 32.2% were gametocyte carriers (Table P < 0.001) than in younger children . In contrast, no significant association was observed between falciparum malaria and gender, household size, parental educational status, or parental employment status. Likewise, no significant association was observed between falciparum malaria and fever, sweating, chills, vomiting, or jaundice , sleeping outdoors at night , absence of indoor residual spraying (IRS) during the last year  were significantly associated with falciparum malaria among under-five children in Al-Mahweet governorate , not sleeping under a mosquito net the previous night of the survey , sleeping outdoors at night , and absence of IRS during the last year  were the independent predictors of falciparum malaria among under-five children in the rural communities of Al-Mahweet  between thick-film microscopy and RDTs for falciparum malaria diagnosis that was statistically significant  pandemic, the situation might be even worse as a result of the negative impact of the pandemic on the prevention and control of infectious diseases, including malaria. The prevalence of falciparum malaria among children in the present study is slightly higher than that (8%) recently reported among schoolchildren in Bajil district of Hodeidah [P. falciparum among children in two rural districts of Taiz in the mid-2000s [P. vivax among under-five children in the present study is consistent with the very low prevalence of less than 0.25% recently reported among schoolchildren in Bajil [The vision of the NMCP’s strategic plan was to make Yemen malaria-free by the end of 2018 [P. falciparum infection among children in the rural communities of Al-Mahweet for the true burden of infection to be uncovered. Compared to the present study, a higher prevalence was reported among under-five children in Tanzania (15.9% by RDTs) [The prevalence of falciparum malaria among under-five children in the present study is comparable to the microscopy-based prevalence of 11.7% reported among under-five children in southern highland Rwanda in community- and facility-based surveys . Nonetheby RDTs) , Uganda by RDTs) , sub-Sahby RDTs) , Nigeriaby RDTs)  and Malaby RDTs) . Howeverby RDTs) , 26.P. falciparum in the rural communities of Al-Mahweet poses a major challenge to malaria control and elimination efforts. This gametocyte reservoir may considerably contribute to the transmission of infection in the community [Gametocyte carriage by approximately one-third of under-five children with microscopically confirmed ommunity , 6, 27, ommunity . Becauseommunity . Howeverommunity , 31.P. falciparum in younger children could be partly explained by the greater care they are given, reducing their exposure to mosquito bites. Furthermore, age-related differences in infection of children with P. falciparum could be attributed, among other factors, to variations in acquired immunity, transmission intensity and seasonality [P. falciparum among under-five children in the present study could be explained by similar exposure patterns to mosquito bites among male and female children in rural communities. On the other hand, the non-significant association between the educational and employment status of fathers and mothers with falciparum malaria among under-five children is inconsistent with those reported for children infected with malaria in African countries [The independent prediction of falciparum malaria in children aged three years or older in the present study is consistent with those recently reported for under-five children in Rwanda, Uganda and Malawi , 21, 24 sonality , 33. Thesonality . Gender ountries , 34, 35.The absence of a significant association between fever or other clinical features and falciparum malaria among under-five children in the present study is inconsistent with the finding among schoolchildren in Hajr valley in Hadhramout , where fP. falciparum compared to those who did. Sleeping under mosquito nets was found to be significantly associated with reduced malaria prevalence among under-five children in African countries [Vector control through mass distribution of long-lasting insecticidal nets (LLINs) is one of the most important interventions to protect local communities at risk of malaria. In Yemen, LLINs were first introduced and distributed to pregnant women and under-five children in 2006 , and sinountries , 23, 24.ountries  and undeountries .P. falciparum compared to those living in sprayed households. The lack of IRS during the year preceding the survey independently predicted falciparum malaria among under-five children in Al-Mahweet rural communities, highlighting the need to assess the household coverage with IRS in targeted areas and take action accordingly. In agreement with the present study, lacking IRS was found to be significantly associated with a higher risk of malaria among Ugandan under-five children [In Yemen, IRS was scaled up as a vector control intervention in 2007 to reinforce efforts to control and eliminate malaria in endemic areas of the country . In the children . Combinichildren , and succhildren . Understchildren .P. falciparum among under-five children in the present study, with children sleeping outdoors being 4.4-fold more likely to have been infected compared to those sleeping indoors. Given that sleeping outside houses is a common practice in rural communities of malaria-endemic areas of the country because of the hot climate, understanding the exophilic/exophagic behaviours of mosquitoes would help prevent outdoor malaria transmission. Sleeping patterns in the community can affect vector-human contact and could significantly increase malaria transmission. Therefore, parents should be educated not to leave their children to sleep outdoors without using LLINs, particularly with the finding that none of the under-five children in the present study was reported to be protected with mosquito repellents. The absence of a significant association between residence close to water or garbage collections and infection of under-five children with P. falciparum in the present study could be attributed to the almost similar geographical and ecological aspects of rural areas. In contrast, residence near water collections was an independent predictor of falciparum malaria among schoolchildren in Bajil [Sleeping outdoors at night was an independent predictor of infection with in Bajil . In anotin Bajil .Given that the strongest risk factors of malaria in under-five children were related to malaria control measures rather than natural topographical or other reasons, there is a need to assess the factors negatively influencing the performance of the NMCP in implementing such interventions. With the great global emphasis on expanding the use of current malaria control interventions together with international funding for them, the strong association between malaria prevalence and poor malaria control measures should raise concerns about the status of malaria control in such rural districts. Accordingly, gaps in coverage, access and utilization of LLINs besides household coverage with IRS should be identified and addressed, considering the potential impact of armed conflicts and the humanitarian crisis on malaria control efforts.RDTs have become essential tools for malaria control and elimination in rural areas and primary healthcare settings, where microscopy is unavailable or impractical, by enabling immediate diagnosis and prompt treatment. The significant and very good agreement between thick-film microscopy and RDTs for diagnosing falciparum malaria among under-five children in rural communities of Al-Mahweet supports using RDTs in remote and rural areas of the governorate where electricity is unavailable. However, the difference between the results of microscopy and RDTs in six cases justifies the need to assess the sensitivity and specificity of RDTs, preferably compared to PCR. Apart from being time-consuming, the sensitivity of microscopy may overlook infections with low parasite densities and its specificity is affected by the individual capacities of microscopists , 45. AccThe present study is limited by adopting microscopy and RDTs for malaria diagnosis. As a result, overlooking sub-microscopic infections and false positivity of RDTs could not be ruled out. However, it provides preliminary data on the burden of malaria among under-five children in rural communities of Al-Mahweet. Another limitation could be introduced by the possible variations in cluster sizes because of having no information about the population size per cluster to adopt the probability proportional to size sampling approach. As a conservative approach, we used a sample design effect of 2.0 when calculating the sample size to account for the uneven distribution of the outcome in clusters. The findings of the present study can guide public health authorities in tailoring interventions to reduce malaria burden. Malaria prevalence among under-five children in the present study underscores the need for molecular surveillance of malaria among such an at-risk population to uncover the burden of sub-microscopic infections and develop appropriate elimination strategies accordingly.P. falciparum based on microscopy and RDTs. Age of three years or older, not sleeping under mosquito nets, sleeping outdoors at night and absence of IRS are the independent predictors of falciparum malaria among under-five children. The very good agreement between thick-film microscopy and RDTs for diagnosing falciparum malaria among children supports the usefulness of using RDTs in such resource-limited rural communities.Approximately one in ten under-five children in rural communities of Al-Mahweet is infected with


Plasmodium falciparum malaria is recognized as a major global public health problem. The malaria vaccine was important because the case fatality rate of falciparum malaria was high. Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the potential vaccine candidates, but the genetic polymorphism of PfCSP raises concerns regarding the efficacy of the vaccine. This study aimed to investigate the genetic polymorphism of PfCSP and provide data for the improvement of PfCSP-based vaccine .P. falciparum and returning from Africa to Henan Province during 2016–2018. The Pfcsp genes were analysed to estimate the genetic diversity of this parasite.Blood samples were collected from 287 Chinese migrant workers who were infected with Pfcsp in Henan Province, including insertion amino acids  and A → G . The number of repeats of tetrapeptide motifs (NANP/NVDP/NPNP/NVDA) in the central repeat region ranged mainly from 39 to 42 . A total of 14 nonsynonymous amino acid changes were found at the C-terminus. The average nucleotide difference (K) of imported Pfcsp in Henan Province was 5.719, and the haplotype diversity (Hd) was 0.964 ± 0.004. The estimated value of dN-dS was 0.047, indicating that the region may be affected by positive natural selection. The minimum number of recombination events (Rm) of imported Pfcsp in Henan Province was close to that in Africa. The analysis of genetic differentiation showed that there may be moderate differentiation between East Africa and North Africa (Fst = 0.06484), and the levels of differentiation in the other regions were very small (Fst < 0.05).The results showed that there were two mutations at the N-terminus of imported Pfcsp was relatively conserved, and the central repeat region and the Th2R and Th3R regions of the C-terminus were highly polymorphic. The gene polymorphism pattern among Chinese migrant workers returning from Africa to Henan Province was consistent with that in Africa. The geographical pattern of population differentiation and the evidence of natural selection and gene recombination suggested that the effect of polymorphism on the efficacy of PfCSP-based vaccines should be considered.The N-terminus of  Plasmodium spp. infections, is one of the most significant life-threatening infectious diseases to humans worldwide. According to the World Health Organization (WHO) Malaria Report 2021, the total number of malaria deaths worldwide reached 627,000, equivalent to one death from malaria every minute in 2020. The incidence of malaria increased 69,000 in 2020 compared with that in 2019. However, affected by COVID-19, the diagnosis of malaria has declined, and the number of malaria deaths in sub-Saharan Africa has increased by 13%. [Plasmodium falciparum is the most common parasite causing human malaria. It has the strongest pathogenicity and is also the main cause of severe malaria. Falciparum malaria patients with low immunity or untimely treatment easily develop severe malaria and even die [Malaria, caused by P. falciparum has become responsible for these infections.Malaria has historically been a major health problem in Henan Province . In 2010P. falciparum circumsporozoite protein (CSP) expressed on the surface of sporozoites, are leading malaria vaccine candidates undergoing phase III clinical trials in malaria-endemic areas [Pfcsp gene polymorphisms is an international research hotspot, but there have been few domestic research reports. This study aimed to determine the molecular characterization of falciparum malaria to produce a genetic characterization of Pfcsp, to understand the molecular evolution of the Pfcsp gene and to provide data for the improvement of PfCSP-based vaccines .Developing a malaria vaccine that provides durable protection against clinical disease and completely prevents infection will be critical for controlling and eliminating malaria. Anti-sporozoite vaccines, such as RTS,S, which target ic areas , 12. RTSic areas –15. PfCSic areas –19. PfCSic areas . PfCSP hic areas , 22. ThePlasmodium falciparum-infected blood samples from patients, including finger-prick blood samples and venous blood samples, were collected. Blood samples (2 mL each) were collected from symptomatic patients before treatment. All of the patients came from Africa in 2016–2018. The samples were confirmed by PCR and microscopy examination. Samples were collected in Ethylene diamine tetraacetic acid (EDTA) tubes and transported to the Henan Province Center for Disease Control and Prevention (Henan CDC) the next day. The samples were stored at − 80 °C until laboratory analysis. A structured questionnaire was used to collect sociodemographic and clinical data from the subjects.Plasmodium falciparum genomic DNA was extracted from the blood samples using a QIAamp DNA Mini kit  following the manufacturer’s instructions. TE buffer  was used to dissolve the DNA and it was stored at − 20 °C until use. A 1.5% agarose gel stained with ethidium bromide was used to check the quality of the DNA and it was visualized with UV illumination.Pfcsp gene. The primers were as follows: PfCSP – F (5’-CGTGTAAAAATAAGTAGAAACCACG -3’), PfCSP – R (5’-TGTACAACTCAAACTAAGATGTGTTC -3’) [2O. PCR was performed with the following conditions: 94 ℃ for 1 min, followed by 35 cycles of 94 ℃ for 30 s, 50 ℃ for 30 s, and 72 ℃ for 2 min and a final extension of 72 ℃ for 5 min. Sequencing was conducted by Shanghai DNA Bio Technologies Co., Ltd. . All PCR products were analysed using 1.5% agarose gel electrophoresis and were then they were purified and sequenced by using an ABI 3730 × L automated sequencer. To ensure the accuracy of the sequencing, at least two clones for each isolate were sequenced.A PCR amplification method was used to amplify the TTC -3’) . AmplifiSequence alignment and analysis were carried out using Bio-Edit software. The amino acid sequences were compared with the 3D7 strain (XM_001351086) as a reference sequence. The sequences of the amplicons were aligned with published data from the 3D7 strain from the NCBI database by BLAST analysis.Pfcsp neutrality, the rates of synonymous (dS) and nonsynonymous (dN) mutations were estimated and compared by MEGA 7.0.26 software [Nucleotide polymorphism, natural selection, and gene recombination were analysed using DnaSP 6.12.03 software . For onesoftware .Tajima's D test (α = 0.05) and Fu and Li's D and F test (α = 0.05) were used to evaluate the neutral theory of natural selection. Tajima's D and Fu and Li's D and F statistics were positive , indicating that it was a positive selection; A negative statistic  indicating that it was a negative selection .R represents the occurrence of gene recombination. Ra is the recombination probability between adjacent nucleotides of each generation; Rb refers to the recombination estimation of the whole gene, that is, the effective population size; and Rm is the minimum number of reorganization events.For population differentiation analysis, Arlequin 3.5.2.2 software and the R program were used to calculate the Fst index . Fst wasST represents the inbreeding coefficient of subgroup (S) relative to the total population (T); HT represents the expected frequency of heterozygotes in the total population under Harwin equilibrium; HS represents the expected frequency of heterozygotes in the subgroup under Harwin equilibrium.FP. falciparum returning from 27 countries of Africa to Henan Province during 2016–2018. The male: female ratio was 56.5:1 (282/5). The age ranged from 19 to 71 years old, of which the proportion of patients who were 18 to 55 years old was 98.61% (283/287). The imported patients all came from African countries, including countries in East Africa, West Africa, South Africa, North Africa, and Central Africa, which accounted for 7.66%, 40.07%, 28.23%, 1.05%, and 22.99% respectively. The positive rate of Pfcsp gene PCR amplification was 91.29% (262/287), and the size of the amplification product was 1100–1300 bp. After gene sequencing, 262 amplified positive products successfully obtained the full-length Pfcsp sequence. Ultimately, 201 full-length monoclonal Pfcsp sequences were analysed in this study, including 83 in West Africa, 57 in South Africa, 48 in Central Africa, 10 in East Africa, and 3 in North Africa, while 61 polyclonal Pfcsp sequences were excluded  , H1  was completely consistent with it. Two mutations occurred in H2, H3, and H4: one inserted a 19 amino acid fragment (NNGDNGREGKDEDKRDGNN) fragment after site 80 ; the second was an A → G change at the 98351086, HPfcsp had a different number of tetrapeptide repeat motifs—NANP/NVDP/NPNP/NVDA. The number of repeats of tetrapeptide motifs ranged from 38 to 43, of which 40  and 41  had higher frequencies. These different numbers of repeats led to the polymorphism of Pfcsp in the central repeat region.As shown in Fig. 314KHIKEYLNKIQNSL327), including K314Q, K317E/T, E318Q/K, N321K/Q, K322T/R/I/ E, Q324K/R, N325Y, L327/I. N352D/G, P354S, D356N, E357Q, D359N, while A361E/I were located in Th3R (352NKPKDELDYAND 363). Th2R and Th3R were identified as T-cell epitope regions  were identified in the C-terminal nonrepetitive region of imported PfCSP in Henan Province, of which H7  was completely consistent with the 3D7 reference sequence. The Th2R and Th3R regions were highly polymorphic, and 14 nonsynonymous amino acid changes were found. A total of 8 of 14 were located in Th2R  of the C-terminal nonrepeat region was analysed in the imported Pfcsp and African native Pfcsp were evaluated. The average nucleotide difference (K) of the imported Pfcsp in Henan Province was 5.719, and the haplotype diversity (Hd) was 0.964 ± 0.004. The estimated value of dN-dS was 0.047, indicating that the region may be affected by positive natural selection. Although the neutral test results were not statistically significant, the statistics of Tajima's D value , Fu and Li's D  and F values  were positive, indicating that the region may be experiencing positive selection. In addition, the Rm value of imported Pfcsp in Henan Province was close to that in Africa , the genetic differentiation among other regions was very small (Fst < 0.05), which cannot be considered  insertion. Two variations were found in the Pfcsp N-terminal region in this study. The study by Huang et al. demonstrated that A98G and 19 amino acid length insertions were universally popular while several novel mutations were found with low frequency [Pfcsp gene [Pfcsp was relatively conserved.The N-terminal region of PfCSP plays an important role in the process of sporozoite invasion into hepatocytes by mediating or promoting the interaction between sporozoites and host cells , 30. Therequency . Notablyrequency . Larger csp gene . The N-tPfcsp had between 40 and 41 repeats. In addition, two novel tetrapeptide motifs NPNP and NVDA were found. Other tetrapeptide motif forms have been reported in the literature, including NVVP, NAKP, NAHP, NAIP, NVNP, NANL, NVAD, NADP, KANP, and SANP. It was unclear how these tetrapeptide motifs changed and how different positions affected the antibody response to CSP. The central repeat region is important in the PfCSP-based vaccine . However, no studies indicated that the various number of tetrapeptide repeats can or may affect the effectiveness of the RTS, S malaria vaccine [The central repeat region of PfCSP played a key role in sporozoite formation and development . The res vaccine . TherefoAbundant polymorphisms were found in the C-terminal analysis of PfCSP, especially in the thrombospondin type-I repeats  region (including Th2R and Th3R), which confirmed T-cell immunogenic epitopes. The overall values of Hd (0.964 ± 0.004) in the C-terminal region of PfCSP were higher than those in previously reported studies , 21. TheThe population differentiation analysis revealed that the genetic relationship between PfCSP in East Africa, West Africa, South Africa, and Central Africa was very close, and there was almost no differentiation, while North Africa and East Africa showed slight differentiation, which may be due to the small sample size of North Africa and the non-representativeness. Thus, the imported PfCSP did not consider geographical differences.The limitation of this study was that there was no further study on the effects of the amino acid mutations on the structure or function of CSP to predict the effect of amino acid mutations on the efficacy of PfCSP-based vaccines.Pfcsp gene among the different regions may affect the efficacy of the RTS, S malaria vaccine. In this study, the analysis of the genetic diversity of imported PfCSP in Henan Province indicated that N-terminus non-repeat region was relatively conserved, but the central repeat region and the Th2R and Th3R regions of the C-terminus were highly polymorphic. According to natural selection and gene recombination, the maintenance and production of genetic polymorphisms were speculated. The gene polymorphism pattern was consistent with that in Africa. These findings filled in missing data of imported PfCSP data in Henan Province and provided valuable information for the improvement of the PfCSP-based vaccines .PfCSP is a main component of RTS,S, the most advanced malaria vaccine currently, but the genetic diversity in the


Iron deficiency (ID) is common in malaria-endemic settings. Intermittent preventative treatment of malaria in pregnancy (IPTp) and iron supplementation are core components of antenatal care in endemic regions to prevent adverse pregnancy outcomes. ID has been associated with reduced risk of malaria infection, and correspondingly, iron supplementation with increased risk of malaria infection, in some studies.Plasmodium falciparum, Plasmodium vivax) was determined by light microscopy, polymerase chain reaction, and placental histology. Multiple logistic and linear regression analyses explored the relationship of ID or ferritin levels with indicators of malaria infection. Models were fitted with interaction terms to assess for modification of iron-malaria relationships by gravidity or treatment arm.A secondary analysis was conducted amongst 1888 pregnant women enrolled in a malaria prevention trial in Papua New Guinea. Maternal ID was defined as inflammation-corrected plasma ferritin levels < 15 μg/L at antenatal enrolment. Malaria burden (Two-thirds (n = 1226) and 13.7% (n = 258) of women had ID and peripheral parasitaemia, respectively, at antenatal enrolment , and 18.7%  had evidence of malaria infection on placental histology. Overall, ID was associated with reduced odds of peripheral parasitaemia at enrolment ; peripheral parasitaemia at delivery ; and past placental infection . Corresponding increases in the odds of infection were observed with two-fold increases in ferritin levels. There was effect modification of iron-malaria relationships by gravidity. At delivery, ID was associated with reduced odds of peripheral parasitaemia amongst primigravid , but not multigravid women . A two-fold increase in ferritin associated with increased odds of placental blood infection  and active placental infection on histology amongst primigravid women only .Plasmodium species require further investigation.Low maternal ferritin at first antenatal visit was associated with a lower risk of malaria infection during pregnancy, most notably in primigravid women. The mechanisms by which maternal iron stores influence susceptibility to infection with Trial registrationThe online version contains supplementary material available at 10.1186/s12936-022-04177-8. Plasmodium falciparum and Plasmodium vivax. In 2018, approximately 11 million women were exposed to Plasmodium spp. infection  in moderate to high transmission areas in sub-Saharan Africa alone  ferritin for ease of interpretation) were used as exposure measures. Ferritin levels were corrected for inflammation using the ‘internal regression correction’ method devised by Namaste et al. [ID was defined as a plasma ferritin level < 15 μg/L at antenatal enrolment . ID and e et al. , and adje et al. . AdditioP. falciparum or P. vivax) by light microscopy and/or qPCR at enrolment or delivery; placental blood infection by light microscopy and/or qPCR; and active or past infection on placental histology [Outcome measures included peripheral malaria infection (istology .2) at enrolment with indicators of malaria infection at enrolment and delivery were assessed using multiple logistic or linear regression, as appropriate.Associations of ID or ferritin  had mixed P. falciparum/P. vivax infections, 20.2% (n = 52) had P. vivax infections, with the remainder having Plasmodium ovale or Plasmodium malariae infections (n = 4). Half of these infections were submicroscopic (n = 134)  were parasitaemic at antenatal enrolment. Amongst participants with parasitaemia, 73.6% (n = 190) had P. falciparum mono-infections, 2.5% (n = 3) had mixed P. falciparum/P. vivax infections, and 38.3% (n = 46) had P. vivax mono-infections. About half of these infections (n = 61) were submicroscopic. Placental blood parasitaemia was detected in 5.0% (n = 63) of women with complete data (n = 1253) for these analyses: 87.3% (55/63) of infections were caused by P. falciparum  had malaria parasites detected in peripheral blood: amongst them 59.2% (n = 71) had 0 had malP. falciparum infection  but not P. vivax infection at enrolment  . Women with ID had reduced odds of peripheral P. falciparum infection  and peripheral P. vivax infection   adjusting ferritin levels for CRP and AGP, but not concurrent malaria infection; and (b) excluding women with malaria parasitaemia at enrolment. The findings were very similar to those presented above . ID, defined as an inflammation and infection-corrected ferritin level < 15 μg/L at first antenatal visit, was associated with reduced odds of peripheral infection at enrolment; reduced odds of peripheral and placental blood infection at birth; and reduced risk of past infection on placental histology. At antenatal enrolment, when the highest burden of peripheral infection was detected, ID was associated with a substantially reduced odds of P. falciparum, but not P. vivax infection, and a more pronounced decrease in microscopic than in submicroscopic infections. By contrast, ID was associated with reduced odds of peripheral P. vivax infection and submicroscopic infection at delivery. Associations between indicators of maternal iron stores and malaria infection at delivery were modified by gravidity. Amongst primigravid women only, there were increased odds of malaria infection  associated with a two-fold increase in ferritin, and reduced odds of peripheral infection associated with ID. Collectively, these data provide further evidence in support of previously reported associations of indicators of maternal iron stores and the occurrence of peripheral and placental malaria infection during pregnancy [The present study was conducted in a regnancy , 37, 38.P. falciparum parasites in vitro, whilst their growth was enhanced in response to iron supplementation and consequential elevated erythropoiesis [P. vivax infection at delivery but not at enrolment. ID decreases production of reticulocytes, which P. vivax preferentially infects, so a protective effect might be envisaged. In addition to erythropoiesis-mediated impacts of ID on parasite growth, other mechanisms may be of relevance to iron-malaria interactions. Though ID may decrease susceptibility to disease it is worth acknowledging that it may also have a negative effect of the development of immunity. ID at the time of vaccination in young children is associated with decreased antibody to diphtheria, pertussis, and pneumococcal vaccines [P. falciparum-specific antibodies in iron-deficient African children [How does an iron-replete state (or supplementation) increase the risk of malaria infection? Suppressed erythropoiesis in women with ID anaemia was associated with reduced growth rates of opoiesis , 40. Depvaccines , and servaccines . Experimvaccines . This ischildren . ID may children .P. falciparum infection in pregnant women [P. falciparum infection by light microscopy compared to their iron-replete counterparts. Amongst multigravidae, malaria prevalence did not differ significantly between iron-deficient and iron-replete women [The size and the directionality of effects observed in our study were consistent with previous studies that assessed associations between ferritin and nt women . ID may te women . These dte women  may modite women , 46. In and concurrent peripheral parasitaemia would have resulted in misclassification of 25% of women as iron replete in the present cohort, which prompted a primary analysis using ferritin levels corrected for both inflammation and malaria infection. However, associations between maternal ID or ferritin levels with malaria infection were highly comparable in analyses using ferritin levels corrected for CRP and AGP only  and malaria in pregnant women and children [A 2014 meta-analysis exclusively reported iron-malaria interactions when CRP-corrected ferritin levels, but not other markers, were used to determine maternal iron stores . It may children , 49 suppchildren , 46. Howchildren .P. falciparum and P. vivax infection; adjustments of ferritin levels for concurrent inflammation and peripheral parasitaemia; and adjustments for potential confounders including maternal residence, a marker of infection exposure risk. Weaknesses include that the provision and uptake of iron supplementation was unmonitored and may have differed between iron deficient and iron replete women. Furthermore, residual confounding of ferritin-malaria interactions by the presence of subpatent placental infections at the time of ferritin measurement could not be ruled out.The strengths of the present research include comprehensive assessments of infection status in a large cohort of pregnant women at risk of both Plasmodium infection against reference methods to diagnose ID, such as bone marrow iron stores, are desirable in the future.There are substantial associations between markers of antenatal iron status and malaria infection during pregnancy. Whilst residual confounding due to misclassification of ID and unmeasured predictors of malaria infection cannot be ruled out, maternal iron status may influence malaria risk by altering parasite growth potential and maternal immune responses to malarial infection, including placental malaria. Prospective studies that assess the temporal relationships between pre-pregnancy and early pregnancy iron stores and iron supplementation during pregnancy with malaria incidence and the development of anti-malarial immunity could consolidate our understanding of iron-malaria relationships in pregnancy, alongside evaluations of the impact of placental infection on markers of maternal iron status. Broader validation of the approach to adjust ferritin levels for both inflammation and Additional file 1: Table S1. Association of maternal ferritin levels at antenatal enrolment with malaria infection at antenatal enrolment and at delivery, Madang, Papua New Guinea, 2009–2013. Ferritin levels were adjusted for concurrent inflammation (C-reactive protein and α-1-acid glycoprotein). Table S2. Association of ferritin levels at antenatal enrolment with malaria infection at delivery in women without peripheral Plasmodium parasitaemia at antenatal enrolment, Madang, Papua New Guinea, 2009–2013. Ferritin levels were adjusted for concurrent inflammation (C-reactive protein and α-1-acid glycoprotein). Table S3. Associations between maternal iron status (ferritin) at first antenatal visit and malaria infection at delivery, stratified by gravidity, Madang Province, Papua New Guinea, 2009–2013. Table S4. Associations between maternal iron status (ferritin) at first antenatal visit and malaria infection at delivery, stratified by malaria prevention regimen, Madang Province, Papua New Guinea, 2009–2013. Table S5. Associations between maternal iron status (ferritin) at first antenatal visit and malaria infection at delivery, stratified by haemoglobin status at antenatal enrolment, Madang Province, Papua New Guinea, 2009–2013.


Plasmodium spp. that cause human malaria appear similar under light microscopy, which raises the possibility that misdiagnosis could routinely occur in clinical settings. Assessing the extent of misdiagnosis is of particular importance for monitoring P. knowlesi, which cocirculates with the other Plasmodium spp. We performed a systematic review and meta-analysis of studies comparing the performance of microscopy and polymerase chain reaction (PCR) for diagnosing malaria in settings with co-circulation of the five Plasmodium spp. We assessed the extent to which co-circulation of Plasmodium parasites affects diagnostic outcomes. We fit a Bayesian hierarchical latent class model to estimate variation in microscopy sensitivity and specificity measured against PCR as the gold standard. Mean sensitivity of microscopy was low, yet highly variable across Plasmodium spp., ranging from 65.7%  for P. falciparum to 0.525%  for P. ovale. Observed PCR prevalence was positively correlated with estimated microscopic sensitivity and negatively correlated with estimated microscopic specificity, though the strength of the associations varied by species. Our analysis suggests that cocirculation of Plasmodium spp. undermines the accuracy of microscopy. Sensitivity was considerably lower for P. knowlesi, P. malariae, and P. ovale. The negative association between specificity and prevalence imply that less frequently encountered species may be misdiagnosed as more frequently encountered species. Together, these results suggest that the burden of P. knowlesi, P. malariae, and P. ovale may be underappreciated in a clinical setting.The five major  Plasmodium falciparum and Plasmodium vivax are the predominant parasites responsible for human malaria worldwide, contributing nearly 241 million clinical cases in 2020.P. falciparum and P. vivax remain the primary focus of public health efforts aimed at achieving malaria control, elimination, and eradication.Plasmodium species—P. knowlesi, P. malariae, and P. ovale—also cause clinical episodes of malaria, although their overall burden remains uncertain due in part to routine misdiagnosis in a clinical setting.Plasmodium spp. causing the infection. However, morphological similarities across the five Plasmodium spp., such as the resemblance of early blood-stage P. knowlesi parasites to P. falciparum parasites and late blood-stage P. knowlesi parasites to P. malariae parasites,,Plasmodium infections.Diagnosis of clinical malaria is routinely performed using light microscopy (LM). In addition to challenges presented by low or submicroscopic parasite densities,Plasmodium spp., routine misdiagnosis by light microscopy has consequences for clinical decision-making and malaria control. The choice of therapeutic is specific to the Plasmodium spp. causing the infection,Plasmodium spp. infections as well as ineffective or no treatment of liver-stage parasites for P. vivax and P. ovale infections. Additionally, misdiagnosis may present challenges for monitoring Plasmodium spp. transmission, particularly for the emerging zoonosis P. knowlesi. Currently, the extent of human-to-human transmission of P. knowlesi is unknown,,In settings with co-circulation of the Plasmodium spp. parasites.,–Consequently, measuring the extent of misdiagnosis by light microscopy is of importance to public health, and past studies have directly compared the diagnostic performance of light microscopy to polymerase chain reaction (PCR) in settings with co-circulation of Plasmodium spp. We identified studies that compared the diagnostic performance of light microscopy to PCR and fit a hierarchical Bayesian latent class model to estimate study- and species-level sensitivities and specificities of light microscopy. Finally, to assess whether the perceived prevalences of the five species might affect diagnostic outcomes of light microscopy, we explored the relationship between the estimates of sensitivity and specificity that we obtained and the observed PCR prevalences in each study.To synthesize across these studies, we performed a systematic review and meta-analysis of the diagnostic performance of light microscopy in settings with co-circulation of all five Plasmodium spp. infections. We identified studies using the following search strategy:  AND knowlesi AND ((microscopy OR microscopic OR blood film OR thick film OR thin film) AND (PCR or “polymerase chain reaction”)).In this systematic review and meta-analysis, JHH searched MEDLINE and Web of Science for studies published in English before April 30, 2021, that compared the diagnostic performance of LM and PCR for Plasmodium spp. that naturally infect humans . Furthermore, studies were only included if they used a PCR protocol that was capable of amplifying DNA from all five Plasmodium spp. Before 2004, the public health burden of P. knowlesi was underappreciated,Studies met the criteria for inclusion if they compared the performance of LM and PCR on clinical samples collected in areas with endemic transmission of all five The data were extracted from each study that met the criteria for inclusion. Because the data in each study were anonymized and reported in aggregate, ethical approvals were not required according to a determination from the University of Notre Dame Institutional Review Board that this study did not constitute human subjects research.Plasmodium spp. To model the diagnostic performance of LM and PCR, we constructed a Bayesian hierarchical latent class model of clinical diagnosis.Plasmodium spp. Plasmodium spp. Plasmodium spp. k among participants in study In Eq. , seLM, Thailand (N = 2), Indonesia (N = 1), Myanmar (N = 1), and China (N = 1). Among the 7,999 total patients enrolled across the 12 studies, the pooled percentage positive by PCR was 19% for P. falciparum, 25% for P. vivax, 52% for P. knowlesi, 1.1% for P. malariae, and 0.48% for P. ovale  in which we estimated LM diagnostic performance.Of the 176 unique studies identified using MEDLINE and Web of Science, 10 studies met the inclusion criteria, and their data comparing LM and PCR diagnostic performance were extracted . The stuP. ovale . The stuSupplemental Figure 1), we applied our hierarchical latent class model to the data from the 10 studies identified in the systematic review. Our fitted hierarchical latent class model captured the variation in the observed data  and revealed differences in LM diagnostic performance across Plasmodium spp.  for P. vivax, and 0.525% (95% CI: 0.0210–3.11%) for P. ovale. It was not possible to estimate group-level mean sensitivities for the pooled P. knowlesi and P. malariae LM diagnosis due to identifiability concerns, yet the mean of study-level posterior median estimates were 44.9% for P. knowlesi parasites and 45.3% for P. malariae parasites. Species-level mean specificity of LM was consistently high across Plasmodium spp. and was estimated at 98.2% (95% CI: 96.6–99.1%) for P. falciparum, 98.6% (95% CI: 97.3–99.3%) for P. vivax, and 100% (95% CI: 100%–100%) for P. ovale. For the pooled P. knowlesi and P. malariae LM diagnosis, the mean of study-level posterior median estimates of specificity were 95.6% for P. knowlesi parasites and 95.9% for P. malariae parasites. These results were robust to the assumption of PCR as the gold standard .After validating on 200 simulated datasets , suggesting that the range of sensitivities that fell within one standard deviation of the mean was 60.9% to 85.1%. By comparison, the estimated logit-transformed standard deviation of LM specificity for P. falciparum was 0.967 (0.753–1.23), corresponding to a range of specificities within one standard deviation of the mean of 95.4% to 99.3%. Across Plasmodium spp., study-level estimates of LM sensitivity and specificity for P. falciparum, P. vivax, P. knowlesi, and P. malariae exhibited greater variation than for P. ovale.The variance in the hierarchical distributions of LM diagnostic performance in nsidered . In geneP. vivax  and P. falciparum  and weaker, positive correlations for P. knowlesi  and P. ovale , although the latter two were not statistically significant associations. By contrast, for P. malariae, there was a negative correlation between PCR prevalence and LM sensitivity, although this estimate was also not statistically significant. Across Plasmodium spp., increased PCR prevalence of P. knowlesi was associated with reduced sensitivity of LM for P. falciparum  and P. vivax , highlighting a possible source of misdiagnosis. Additional negative associations were observed between PCR prevalence and LM sensitivity across P. falciparum, P. knowlesi, and P. malariae—Plasmodium spp. that are commonly misdiagnosed, although these quantities were not statistically significant.Because the estimates of LM diagnostic performance varied across the unique studies, we then assessed its relationship with observed PCR prevalence . We obsePlasmodium spp., PCR prevalence was negatively associated with LM specificity , and additional weaker, yet statistically nonsignificant relationships were observed for P. falciparum, P. vivax, and P. ovale. The opposite relationship was observed for P. malariae, although this quantity was not statistically significant. Across Plasmodium spp., increased prevalence of P. malariae was associated with reduced specificities of LM for P. falciparum and P. vivax, and increased prevalence of P. knowlesi was associated with reduced specificity of LM for P. malariae, though these effects were not statistically significant.Generally, for a given cificity . For P. Plasmodium spp. that cause human malaria. Our analysis revealed variable sensitivity of light microscopy to diagnose Plasmodium spp. infection. Sensitivity of light microscopy was estimated to be considerably lower for P. knowlesi, P. malariae, and P. ovale than for P. falciparum and P. vivax. This suggests that the burden of P. knowlesi, P. malariae, and P. ovale in a clinical setting may be appreciably underestimated, consistent with a recent study of P. ovale in health clinics in Kenya.In this systematic review and meta-analysis, we estimated study- and species-level variation in the malaria diagnostic performance of light microscopy in epidemiological settings with cocirculation of the five major Plasmodium spp. and the sensitivity of light microscopy for that Plasmodium spp. This supports a phenomenon whereby the more a microscopist encounters samples of a Plasmodium spp. infection in a clinical setting, the more likely they are to accurately identify the Plasmodium spp. causing that infection. By contrast, increased PCR prevalence of a Plasmodium spp. was associated with high false-positive probabilities for that Plasmodium spp. This tendency to misdiagnose malaria infections as what is most commonly encountered in that clinical setting could lead to ineffective monitoring of less commonly encountered Plasmodium spp., such as P. knowlesi, P. malariae, and P. ovale, and especially could present challenges in settings where a Plasmodium spp. is newly emerging.Post hoc analyses revealed relationships between the diagnostic performance of light microscopy and the epidemiological setting. In general, we noted a positive association between the PCR prevalence of a P. knowlesi and reduced sensitivity of LM for P. vivax. This suggests that in areas with P. knowlesi, infections of P. vivax may not be correctly diagnosed and therefore may not receive radical cure therapy to clear hypnozoites and prevent relapse.P. malariae was associated with reduced specificity of LM for P. vivax, indicating that many P. malariae infections may be misdiagnosed as P. vivax. This form of misdiagnosis could unnecessarily subject individuals to radical cure therapy, potentially increasing the risk of acute hemolysis among individuals deficient of the glucose-6-phosphate dehydrogenase enzyme.The high probabilities of misdiagnosis estimated in this analysis could have important consequences for malaria treatment and control. Specifically, we observed an association between increased prevalence of P. knowlesi transmission and burden. We estimated variable sensitivity of LM to diagnose P. knowlesi and P. malariae infections, given the morphological similarities of P. falciparum, P. knowlesi, and P. malariae parasites,P. knowlesi among microscopists in the health systems. Although in practice P. knowlesi and P. malariae infections diagnosed by light microscopy are confirmed by PCR in many settings,P. knowlesi infections misdiagnosed as P. falciparum, for instance, would not be subject to confirmatory PCR. This could lead to an underestimate of P. knowlesi burden, affecting epidemiological assessments of P. knowlesi infection risk.Routine diagnosis of clinical malaria by LM may prevent effective monitoring of Plasmodium spp. However, studies that included subsets of the five Plasmodium spp. could still be informative of species-level diagnostic performance. Our latent class model assumed conditional independence of diagnosis across Plasmodium spp. Future extensions of this work could account for the correlation structure of LM diagnosis across the Plasmodium spp., due to shared morphologies of the parasites. Additionally, due to the data available for this analysis, it was not possible to estimate sensitivity and specificity as a function of Plasmodium parasitemia. Future work could estimate the relationship between diagnostic performance and parasitemia, should such data be made available. Finally, to address nonidentifiability issues, we assumed PCR to be the gold standard. However, varying limits of detection and the complexity of multiplex assays due to amplification issues, parasitemia, and laboratory settings among other factors may weaken this assumption. Although sensitivity analyses revealed that our species-level estimates were robust to this assumption, future work could jointly estimate the diagnostic performance of both methods, providing a more complete characterization of misdiagnosis of Plasmodium spp. in settings with co-circulation of multiple Plasmodium species.Our analysis is subject to several limitations. Our systematic review may not have identified all studies with relevant data, including those studies not indexed by MEDLINE and Web of Science. We limited our review to studies that tested for all five Plasmodium spp. may be limited in settings of cocirculation. Such misdiagnosis could have implications for malaria treatment and control, so more sensitive diagnostics, such as PCR, may be warranted in settings where Plasmodium spp. parasites routinely co-circulate.This analysis estimates that the diagnostic performance of light microscopy for Supplemental materials


Macaca fascicularis, are a known reservoir of Plasmodium knowlesi, the agent of simian malaria which is the predominant zoonotic species affecting humans in Malaysia and other Southeast Asian countries. Recently, a naturally acquired human infection of another simian malaria parasite, P. cynomolgi has been reported. Thus, it is crucial to study the distribution of simian Plasmodium infections with particular attention to the macaques. Four hundred and nineteen (419) long-tailed macaques (Macaca fascicularis) were trapped in selected areas where human cases of P. knowlesi and P. cynomolgi have been reported. Nested polymerase chain reaction (PCR) was conducted to identify the Plasmodium spp., and circumsporozoite protein (CSP) genes of P. knowlesi samples were sequenced. Plasmodium cynomolgi infection was shown to be the most prevalent among the macaque population (68.4%). Although 50.6% of analyzed samples contained single infections either with P. knowlesi, P. cynomolgi, P. inui, P. coatneyi, or P. fieldi, mixed infections with double, triple, quadruple, and all 5 species were also detected. Infection with P. cynomolgi and P. knowlesi were the highest among Malaysian macaques in areas where humans and macaques are in close contact. The risk of zoonotic infection in these areas needs to be addressed since the number of zoonotic malaria cases is on the rise. With the elimination of human malaria, the risk of humans being infected with simian malaria is very high and steps should be taken to mitigate this issue.Macaques,  Plasmodium that cause malaria in humans, simian malaria such as Plasmodium inui , while the license for macaque trapping and blood collection was obtained from the Wildlife Department [PERHILITAN: PHL&TN(IP):80-54/2 Jld 21 (8)].Macaca fascicularis were trapped in selected areas in the states of Kedah, Pahang, Kelantan, Terengganu, Selangor, Kuala Lumpur, Putrajaya, and Sabah  were also collected. Thick and thin blood films were also prepared for malaria parasite examination by microscopy (BFMP). Each macaque was marked and then handed over to the Wildlife Department for release into the deep forest or areas of less conflict with humans. The blood samples in EDTA tubes were kept at 4 °C during transportation to the Parasitology Laboratory, IMR. Thin blood smears were fixed with methanol. Both thick and thin blood smears were then stained with 3–10% KaryoMAX® Giemsa Stain  for 30–60 min, examined and results were recorded. Blood samples in EDTA tubes were subjected to DNA extraction.Trapped macaques were sedated with 0.3–0.5 mL ketamine  via intramuscular injection before blood collection (1–5 mL). The blood samples were transferred into ethylenediaminetetraacetic acid (EDTA) tubes . Blood spots on filter paper (WhatmanTwo hundred microlitres of each EDTA blood sample was subjected to genomic DNA (gDNA) extraction using a DNeasy Blood and Tissue Kit , following the manufacturer’s protocol. The concentration and purity of extracted DNA were determined using a NanoDrop™ 2000/200c Spectrophotometer .Plasmodium small subunit ribosomal RNA (SSU rRNA) using the primer pair rPLU1 and rPLU5 in a 50 μL reaction volume. In the second stage, PCR was carried out for species-specific identification using primer pairs Kn1f and Kn3r for P. knowlesi, CY2F and CY4R for P. cynomolgi, PinF2 and INAR3 for P. inui, PfldF1 and PfldR2 for P. fieldi, and PctF1 and PctR1 for P. coatneyi, and the PCR product from the first stage as the DNA template. The master mix contained 1x reaction buffer , 20 nM of each primer, 200 ng DNA template, and nuclease-free water in a 50 μL reaction volume.Nested PCR was conducted to identify the genus and species of the parasites present in the blood samples pre-determined as malaria-positive by microscopy (BFMP). In all, 10% of malaria-negative samples by BFMP were selected randomly for validation by nested PCR. PCR runs were performed according to the protocol from Singh  to Lee  with sliP. knowlesi circumsporozoite protein (CSP) genes were amplified by PCR using the primers PKCSPF2 and PKCSPR3, cloned and sequenced by the protocols described by Singh  followed by P. knowlesi [68 (38.6%)], P. coatneyi [67 (38.1%)], P. inui [34 (19.3%)] and P. fieldi [33 (18.8%)] (All 202 samples that tested positive for the (18.8%)] . Single P. knowlesi, including 10 samples from Pahang and one each from Kelantan, Terengganu, and Sabah. The reason that only P. knowlesi cases were sequenced is mainly because at this point in time, infections with P. knowlesi in humans were increasing and were more of a public health concern. Meanwhile, infection with other zoonotic simian malaria was very low or none. Phylogenetic analysis was limited to a single infection of P. knowlesi. The phylogenetic analysis was constructed to compared the genetic profile of CSP genes in human and macaques’ samples.The CSP genes were sequenced for samples with a single infection of P. knowlesi obtained from GenBank references in The 13 CSP genes were amplified, cloned, and sequenced successfully. The PCR products ranged in size from 1050 to 1200 bp. The Neighbour Joining (NJ) method, Bayesian method, and Maximum-likelihood method inferred that malaria parasites isolated from these samples clustered with the reference P. cynomolgi (67.6%) as the predominant species. However, the prevalence of P. knowlesi and P. inui, both of which have been shown to infect humans, is rather significant . A previous study by Akter and co-workers identified P. inui as the most predominant species in 70 samples collected from Hulu Selangor, a district in the state of Selangor [P. knowlesi cases in Peninsular Malaysia from 2019 to 2020 and infection could have been transmitted and spread to the neighboring districts and states . Similar results were also obtained from M. fascicularis samples in Sarawak [M. fascicularis plays a significant role as a large reservoir of simian malaria parasites including those capable of zoonoses. This is in agreement with earlier twentieth-century parasitologists who suggested that macaques are the predominant simian host for malaria parasites [This study showed that simian malaria parasites are abundant in Malaysian macaques in certain states, with  Sarawak  and Saba Sarawak . In our P. knowlesi infections, which includes reports on the detection of P. knowlesi in humans especially in East Malaysia [P. knowlesi is widely distributed in Malaysia, where the infection can potentially be life-threatening [P. knowlesi in East Malaysia. Moreover, past studies focused more on the infection in humans with very limited critical assessments on the aspects involving macaques as the initial host. For example, Lee and co-investigators tackled the distribution of P. knowlesi infection in humans and Plasmodium spp. in macaques, but sample collection was limited to the state of Sarawak [Previous studies have documented an increasing number of malaria cases in Southeast Asia, with Malaysia having the highest number of Malaysia , 42, 46 Malaysia . Cox-Sineatening , 14. How Sarawak  which doPlasmodium spp. in wild-caught macaques from three states of Peninsular Malaysia namely Pahang, Johor, and Perak [Macaca nemestrina, whereby, consistent with our findings, P. cynomolgi was the most prevalent species [Plasmodium species in the survey in comparison to ours [Very recently, a survey was conducted to identify the prevalence of nd Perak . The sur species . The sam to ours . Nonethe to ours . The datP. knowlesi in Thailand, the Malaysian states neighboring Thailand, namely Kedah and Kelantan, were included as the sampling sites. The analysis of these samples could provide insights into the origin of infection according to geographical distribution. Interestingly, a similar study carried out among macaque populations in Southern Thailand showed the presence of only P. inui and P. coatneyi [P. knowlesi considering the life-threatening human disease it may cause. The phylogenetic analysis of P. knowlesi CSP genes indicated a high similarity to those of macaques, mosquitoes, and human samples isolated from the east coast region of Peninsular Malaysia and Thailand. Nevertheless, these results create a positive preliminary profile of the distribution of P. knowlesi in Malaysia and provide avenues for analysis of other simian malaria species in macaques with a high probability of future zoonotic transmission.Due to an increasing number of human cases of coatneyi , 35. In P. cynomolgi has been considered less important compared to P. knowlesi, the increasing evidence of its contribution to human health, as seen here, warrants further validation. For example, P. cynomolgi has long been known to be capable of infecting humans experimentally but in 2014, Ta and colleagues reported the first naturally acquired P. cynomolgi infection in humans [P. vivax.Although n humans . Howevern humans , 31, 37.P. brasilianum had been identified in one of the malaria-endemic regions in the Venezuelan Amazon, whereby 12 patients were found to be infected with P. brasilianum identical to those detected in macaques from the species Alouatta seniculus, as confirmed by sequencing of their CSP genes. Furthermore, an outbreak of P. minimum infection was reported in the Atlantic Forest in Rio de Janeiro following the conclusion that the cases of P. vivax for the past 50 years in the same areas were caused by P. minimum [Plasmodium to adapt rapidly to drug pressure and challenges by the immune system [Meanwhile, the first naturally-acquired infection with  minimum . Malariae system .P. knowlesi cases in humans was high, including Pahang, Kelantan, and Sabah [Plasmodium spp. Correspondingly, in states with low cases of human P. knowlesi, such as Kedah and Terengganu [Plasmodium spp. in the respective trapped macaques was very low. While human cases of P. knowlesi infection were also detected in Selangor [Plasmodium spp. This finding is also supported by a study by Braima and colleagues in 2013 [Anopheles maculates. However, none of the mosquitoes collected were shown to be positive wither with oocyst or sporozoites. The establishment of the relationship between the vectors  and the host still needs further investigation. Although the first naturally transmitted P. cynomolgi human infection was reported in Terengganu [P. cynomolgi but were infected with either single or mixed infection by P. knowlesi, P. inui, P. coatneyi, and P. fieldi. This may be due to the sampling areas. According to the report, the patient had been living and working in Hulu Terengganu, a district considered malaria-free [Anopheles cracens (the mosquito vector for P. inui and P. cynomolgi) were found to co-exist in the same area [P. knowlesi in Peninsular Malaysia.An interesting finding in our study is that, in states where the number of nd Sabah , most ofrengganu , the detSelangor , none ofrengganu , macaqueria-free . Howeverame area . Out of P. knowlesi infections were often misdiagnosed as P. malaria or P. falciparum [. Plasmodium brasilianum can also be identified as P. malariae, while P. cynomolgi and P. simium have a high resemblance to P. vivax [P. knowlesi cases and recent reports of infection by P. cynomolgi, P. minimum, and P. brasilianum, indicate that this may be due to conflicts between humans and macaques following rapid deforestation and land clearing for agriculture, infrastructure, real estate, and industry, as well as eco-tourism. Consequently, human populations have encroached into the vicinity of macaques’ natural habitat. On the other hand, improvements in molecular diagnostic methods and increased surveillance have increased detection of the infection, especially in endemic regions. Taken together, these preliminary results revealed a significant trend that could be helpful in learning and predicting the disease burden in the near future.The importance of identifying the distribution of simian malaria in macaques and the prevalence of zoonotic species lies in the risk of transmission to human populations, especially when there is clear evidence of vectors co-habiting in the same geographical areas. For instance, lciparum . PlasmodP. vivax . The incP. knowlesi and now with P. cynomolgi. Technical reports by Vector-Borne Disease Centre (VBDC), Ministry of Health Malaysia in 2020 showed an increasing number of cases of P. knowlesi from 2008 to 2020 with mean cases of more than 3000. The highest number of cases was reported in 2018 with 4131 and slightly reduced in 2020 (2609 cases), which may be due to Movement Control Orders (MCOs) during the Covid-19 pandemic. The latest report on Malaria cases for the year 2000–2021 showed that total cases had increased from 2838 in 2020 to 3688 in 2021. In addition, more recent studies reveal that human infection with simian malaria was unquestionable and worrying, especially with P. knowlesi and P. cynomolgi [Macaca fascicularis is found to be the perfect host for this zoonotic Plasmodium, including for P. knowlesi and P. cynomolgi. This study has therefore strengthened the evidence in the past that implicated macaques as the potential source of increasing human infections. In addition, the information gathered has enhanced the evidence that previously implicated macaques are a possible source of rising human infections. If P. knowlesi is considered human malaria, failure to address these issues promptly may affect malaria elimination efforts Our findings could be exploited in any situation where predictions of outcomes are important. Furthermore, new research methodologies such as the use of a mathematical model, data mapping, and study on the sub-microscopic, could also be applied with caution to predict the upcoming disease burdens of simian malaria. This can be achieved by integrating all information on surveillance, the epidemiological link between disease-host-vector, genetic surveillance, and flexible modelling approach. Thus, it has become evident through our findings that certain precautions must be taken to be prepared for the upcoming disease burdens of simian malaria that, only in a matter of time, may have the potential to cause an outbreak. However, it remains to be further clarified whether our findings could be applied by policymakers to give stakeholders greater roles in planning necessary measures for malaria disease control and elimination. An integrated control strategy and multi-disciplinary agencies including experts from research institutes, universities, the wildlife department, the forestry department, and state government are required to discuss the strategy to manage this zoonotic disease, and analyze the situation at the human-animal-ecosystem interface. Without cooperation with all departments, these issues will be prolonged and new zoonotic malaria might be introduced to humans. Jessica Scott in her review in 2020 has suggested integrated control for zoonotic malaria where she highlighted some of the main issues that we need to take into consideration if we want to control or reduce zoonotic malaria [P. knowlesi are high and wear protective clothing, especially during peak mosquito biting hours.From this study we were able to provide insights on the prevalence of simian malaria in the long-tailed macaques collected, which may correlate to the increase of malaria zoonotic infection is in Malaysia, especially infection with ynomolgi , 49. Thi malaria . The imm