Plasmodium falciparum malaria and acquire protective antibodies over successive pregnancies. Most studies that investigated malaria antibody responses in pregnant women are from high transmission areas in sub-Saharan Africa, while reports from Latin America are scarce and inconsistent. The present study sought to explore the development of antibodies against P. falciparum and Plasmodium vivax antigens in pregnant women living in a low transmission area in the Brazilian Amazon.Pregnant women have increased susceptibility to P. falciparum, 96 had infections with P. falciparum and P. vivax, and 201 had no Plasmodium infection) were used to measure antibody levels. Levels of IgG and opsonizing antibody to pregnancy-specific variant surface antigens (VSAs) on infected erythrocytes (IEs), 10 recombinant VAR2CSA Duffy binding like (DBL domains), 10 non-pregnancy-specific P. falciparum merozoite antigens, and 10 P. vivax antigens were measured by flow cytometry, ELISA, and multiplex assays. Antibody levels and seropositivity among the groups were compared.In a prospective cohort study, plasma samples from 408 pregnant women  had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other , but not with antibody to VSA or history of infection. Infection with either malaria species was associated with higher seropositivity rate for antibodies against P. vivax proteins, adjusted odds ratios (95% CI) ranged from 5.6 , p < 0.0001 for PVDBPII-Sal1 to 15.7 , p < 0.0001 for PvTRAg_2.Antibodies to VSAs on P. vivax antigens, but only the latter varied with infection. Apparent antibody prevalence is highly dependent on the assay platform used.Pregnant Brazilian women had low levels of antibodies to pregnancy-specific VSAs that increased with exposure. They frequently recognized both VAR2CSA DBL domains and The online version contains supplementary material available at 10.1186/s12936-022-04402-4. Plasmodium in sub-Saharan Africa, which resulted in about 872,000 low birthweight deliveries [Plasmodium falciparum infected erythrocytes (IEs), leading to poor outcomes for both mother and infant, including maternal anaemia, miscarriage, stillbirth, low birthweight, and preterm birth [P. falciparum erythrocyte membrane protein 1 (PfEMP1) family member, VAR2CSA [Plasmodium vivax does not sequester in the placenta [Malaria during pregnancy remains a major public health problem in tropical and sub-tropical regions. In 2018, an estimated 11 million pregnant women were infected by  VAR2CSA , that bi VAR2CSA , a glyco VAR2CSA . Althougplacenta , it has placenta . The reaplacenta ).P. falciparum infection [P. vivax generally dominates, and the region is characterized by lower malaria transmission with some foci of higher endemicity and a prevalence of malaria in pregnancy generally below 10% [P. falciparum infection [In high transmission areas, primigravidae are at highest risk of nfection . By contelow 10% ; [12, 13nfection .With repeated exposure, pregnant women acquire antibodies against VAR2CSA that block adhesion of IEs to CSA , 18, whiP. vivax antigens. One group reported a high prevalence (> 60%) of antibodies to VAR2CSA recombinant protein in both pregnant women and in never-pregnant women, men, and children [P. vivax Duffy-binding protein (PvDBP) [Studies of antibodies in pregnant women from Latin America have given inconsistent findings, and few have included antibody to children , 24. The (PvDBP) , 26. A s (PvDBP) . The pre (PvDBP) . The lac (PvDBP)  could reP. falciparum antigens and P. vivax proteins in a cohort of pregnant Brazilian women. Correlations between different antibody responses were sought. The effects of gravidity and number of episodes of Plasmodium infection on the seroprevalence of these antibodies were analysed, and the effect of time since last Plasmodium infection on antibody levels was investigated.To address some of these knowledge gaps and contradictory findings, the current study examined antibody to VAR2CSA expressed on whole IEs, including antibody that opsonizes IEs for phagocytosis, and measured IgG to recombinant VAR2CSA proteins, non-pregnancy malaria-associated P. vivax [Plasmodium sp. were recruited during their first antenatal care visit and followed until delivery. Malaria was diagnosed from thin and thick blood smears by 2 experienced microscopists and confirmed by PET-PCR [A prospective cohort study was conducted in the Amazonian region of the Alto do Juruá valley, Acre, Brazil. In a previous study in this location, 8.9% of pregnant women had microscopically documented malaria infection, over 60% of which were due to P. vivax . In tota PET-PCR . IsolateP. falciparum, 96 had infections with P. falciparum and P. vivax concurrently or at different time points during the current pregnancy, and 201 had no Plasmodium infection during the pregnancy. Samples from women with P. vivax mono-infection were not available.A total of 192 women who presented with other infections  were excluded . Of the P. falciparum lines, CS2 (which constitutively expresses VAR2CSA and binds to CSA) [P. falciparum line E8B-ICAM (which adheres to intercellular adhesion molecule (ICAM)–1 but not CSA) [Two pregnancy malaria-associated  to CSA)  and 3D7  to CSA) ) and a nnot CSA)  were usenot CSA) . Cell cunot CSA)  and subjnot CSA) . Adhesio5 cells/ml in 75 cm2 cell culture flasks  stored upright in a humidified 37°C incubator with 5% CO2, as previously described [Pro-monocytic THP-1 cells  were maiescribed .P. falciparum infected Malawian multigravid women, after subtraction of the negative control .IgG antibody levels to pregnancy-specific VSAs on CS2 and 3D7 IEs were measured by flow cytometry as previously described . In brie7/ml and opsonized with heat inactivated test plasma (1:10). Opsonized IEs were then incubated with THP-1 cells in a 5% CO2 humidified incubator at 37°C. Phagocytosis was stopped and non-phagocytosed IEs were lysed before cells were resuspended in 2% PFA. Test samples were then analysed by flow cytometry  and opsonizing antibody was determined as proportion of THP-1 cells positive for DHE and expressed as a relative percentage of positive control . Samples were defined as seropositive if the relative percentage was more than two standard deviations (SD) above the mean relative percentage of the unexposed controls .The opsonic antibodies against CS2, 3D7 and E8B-ICAM were measured as previously described in . In briePlasma IgG levels to VAR2CSA DBL domains  Tween-20) in 96-well flat-bottom plates for 30 min at room temperature on a plate shaker. Following the incubation, microspheres were washed thrice with PBS containing 1% BSA and 0.05% (v/v) Tween-20. Then, anti-human IgG detector antibody conjugated with PE (Jackson ImmunoResearch (1 mg/ml), 1:100 dilution) was added to each well and incubated for 15 minutes at room temperature on a plate shaker.IgG levels to After washing and resuspending in PBS containing 1% BSA and 0.05% (v/v) Tween-20, plates were read on a Bio-Plex MAGPIX multiplex reader (Bio-Rad) and the MFI values were reported. Samples were defined as seropositive if the MFI was more than two SD above the mean MFI of the negative controls .P. falciparum MSP1-19 were measured by enzyme-linked immunosorbent assay (ELISA), as described elsewhere [Escherichia coli as previously described [P. falciparum CS2-IEs [PfMSP1-19).Antibody responses to schizont extract and lsewhere , 41. PfMescribed  and schi CS2-IEs . ResultsPlasmodium infection and the number of episodes of P. falciparum infection on antibody seropositivity. Multivariate linear regression assessed the association between the time since the infection was diagnosed and antibody levels. P-values of less than 0.05 were considered statistically significant.Demographic, clinical variables and antibody measurements were summarized with number and percentages for categorical variables and median and interquartile range (IQR) for continuous variables. Pearson chi-squared test was used to compare frequencies of seropositive individuals between groups, and Kruskal-Wallis rank sum tests followed by Dunn's multiple comparison test were used to compare the antibody levels amongst multiple groups. Spearman's rank test assessed the correlations between antibody to multiple antigens measured by different assays. Multiple logistic regression analysis was performed to determine the effect of Plasmodium spp. infection at some point in the current pregnancy. Of these, 111 had at least one P. falciparum infection, and 96 women had both P. falciparum and P. vivax infections. Maternal age, gravidity, and gestational age at delivery were similar across the three groups , 11.5 g/dL , and 11.4 g/dl , respectively, p = 0.008). Birthweight tended to be lower in women with Plasmodium infection during pregnancy than in those without infection (p = 0.05)  had a P. falciparum IEs were measured, and antibody seroprevalence was compared between women with P. falciparum infection, both P. falciparum and P. vivax infections, and without infection. When stratified by infection status 4–16% of women were seropositive for antibody to VSA on pregnancy-specific CSA binding CS2-IEs and 15–22% of women had opsonizing antibodies against pregnancy-specific VSA on CS2-IEs. By contrast, 43–63% of women had opsonizing antibodies to the non-pregnancy-specific VSAs of E8B-ICAM  for DBL5-isolate to 21–27% (depending on exposure group) for DBL6-IT4  P. vivax proteins also correlated significantly with each other  and showed weak to moderate correlation with antibodies to schizont extract  and P. falciparum MSP1-19 . Moreover, weak but significant correlation was observed between antibodies to P. vivax proteins and opsonic antibodies; CS2 phago , 3D7 phago , and E8B phago . Notably, the level of IgG to P. vivax proteins did not correlate well with the level of IgG against VAR2CSA DBLs .The correlation between the antibodies measured by different assays was investigated Fig. . AntibodP. falciparum and P. vivax antigens . However, no gravidity dependent acquisition was observed for all other tested P. falciparum only and both P. falciparum and P. vivax infections) and uninfected.To investigate whether malaria infection was a significant determinant of seropositivity, multivariate logistic regression was performed after adjusting for gravidity, maternal age, and body mass index (BMI) at enrollment. The participants were stratified into two groups: infected (women with Plasmodium-infected women were more likely to have antibodies against pregnancy-specific VSAs on CS2 IEs (AOR (95% CI) = 4.1 , p = 0.001) than uninfected women, and they were twice as likely to be seropositive for opsonic antibodies against E8B-ICAM IEs compared to their uninfected counterparts , p < 0.0001). However, the proportion of women with opsonic antibodies to VSA on placental binding IEs did not vary between infection groups , p < 0.0001) and 9.7 , p < 0.0001, respectively)  ranges from 5.6 , p < 0.0001 for PVDBPII-Sal1 to 15.7 , p < 0.0001 for PvTRAg_2; Table To investigate the association between from 5.6 .2, 9.7, P. falciparum infection during the current pregnancy. Individuals with both P. falciparum and P. vivax infections or without Plasmodium infection were excluded from this analysis to investigate the influence of only P. falciparum. Out of 111 women infected with P. falciparum, 81 had a single infection, and 30 had multiple infections during the current pregnancy. In adjusted analyses, women who had multiple P. falciparum episodes during the current pregnancy were more likely to be seropositive for antibodies against VSAs on CS2 IEs (AOR (95% CI) = 4.0 , p = 0.028) than women who had a single infection. The seropositivity against all other tested P. falciparum antigens did not differ significantly by the number of P. falciparum episodes  ranged from (median (IQR), 0.43  for P. vivax infection to 7  for P. falciparum infection. Generally, the levels of antibody against P. falciparum antigens were not significantly associated with the time since the P. falciparum infection was diagnosed. The exception was for antibodies to PfMSP1-19, which declined as time since the infection increased (coefficient (95% CI) = 1.5 , p = 0.001), suggesting that anti-PfMSP1-19 antibodies could be a marker of recent malaria exposure . Similarly, there were no significant associations between the time since P. vivax infection and the antibody levels against P. falciparum antigens, except for antibody to DBL3-7G8 which increased with increasing time.Multivariate linear regression was performed to investigate whether antibody levels decline from the time the infection was diagnosed until delivery. Time since last infection date was available for 97 of 111 women infected with P. vivax proteins tended to be negatively associated with the time since last P. vivax infection, but these trends did not reach statistical significance (p > 0.05). Similarly, no significant association was found between the antibody levels to P. vivax proteins and time since P. falciparum infection  as markers of P. vivax infections in the prior 9 months [The relationship between the time since infection and the antibody level at delivery was also investigated. There were no significant associations between the time since either nt women , althoug9 months , althougP. falciparum and P. vivax infection, the longitudinal study design and the examination of antibody response using multiple experimental approaches. Weaknesses include the lack of sequential samples, which could have allowed more definitive conclusions regarding antibody decline following infections, and the relatively small number of women with multiple episodes of infection during pregnancy. Timing of infection during pregnancy relative to sampling varied quite widely. Infections were not genotyped, so it was not possible to confirm whether they were recrudescent or new infections. Inclusion of women with P. vivax alone would have added another dimension to the study.Strengths of the study included the large number of women with documented P. falciparum antigens, recombinant DBL proteins, and P. vivax antigens. The lack of correlation between antibody to VAR2CSA DBL domains and antibody to surface antigen on IEs, or between antibody to VAR2CSA DBL domains and gravidity or malaria infection as markers of exposure, suggests they have limited utility in measuring immunity in this setting. Further studies are needed to define antibody responses that are robust markers of recent P. falciparum infection and/or may be correlates of protection from adverse pregnancy outcomes in low and unstable malaria transmission settings such as the Brazilian Amazon.In summary, pregnant women in this region of Brazil had limited acquisition of antibodies to pregnancy-specific antigens on surface of whole IEs, despite having relatively high levels of antibodies to non-pregnancy associated Additional file 1: Table S1List of P. falciparum VAR2CSA domains and P. vivax proteins tested in Luminex assays.Additional file 2: Table S2The association between gravidity and antibody prevalence at delivery in women with and without history of malaria infection during current pregnancy.Additional file 3: Table S3The association between the frequency of P. falciparum infections during current pregnancy and malaria antibody prevalence at delivery.Additional file 4: Table S4The association between time since malaria infection was diagnosed and malaria antibody levels at delivery.Below is the link to the electronic supplementary material.


In Ethiopia, malaria is a major concern to the health, and socio-economic development of the country because of its occurrence at the peak agricultural activities. Factors such as environmental, human host, parasite, and vector determine malaria transmission. Therefore, the present study was conducted to determine the prevalence and associated factors of malaria among febrile patients who visited selected health centres.Institutional-based cross-sectional study was conducted between October 2020 to July 2021 in eight selected health centres located in Lake Tana and its surrounding areas. A simple random sampling technique was used to select febrile patients. Thick and thin blood films were prepared and processed according to the WHO guidelines. Socio-demographic and malaria risk factors were collected from study participants who could read and write using a self-administered questionnaire, whereas face-to-face interview was used to collect information from those participants who could not write and read. The strength of association between risk factors and malaria was assessed using univariate and multivariate logistic regression models.Plasmodium falciparum (72.5%) followed by Plasmodium vivax (23.7%) and mixed-species (3.8%). The highest prevalence was recorded in Kidist Hana (51.5%) followed by Robit (34.8%), Gorgora (30.3%), and Wusha Tiris (25%) health centres. In terms of months, the highest prevalence (37.5%) was detected in October whereas the lowest (14%) was in March. Logistic regression analysis revealed that gender (p = 0.023), educational level (p = 0.025), study month (p = 0.036), presence of eave in the house (p = 0.002) and wall openings (p = 0.041), not using bed nets (p = 0.001), sleeping in the same house with cattle (p = 0.031) and the distance between mosquito-breeding site and living house (p = 0.020) were explanatory risk factors significantly associated with malaria among studied participants.Of the total (531) febrile patients, 75.3% were malaria negative and 24.7%  were malaria confirmed cases. Most of the infections were caused by In this study, we confirmed that the occurrence of malaria prevalence was high and continued against the Ethiopian malaria elimination plan of 2021–2025. Therefore, to meet the goals of this plan, the current prevention and control efforts should be stepped up even better in the coming years. Plasmodium [Anopheles mosquito [Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae are known to infect human beings in Ethiopia [Plasmodium species, P. falciparum is more severe than others in terms of morbidity and mortality, followed by P. vivax [Mosquito-borne diseases are the world’s major causes of illness and death, particularly in tropical and subtropical countries . Among tasmodium , 3, thatmosquito , 4. AmonEthiopia . From thP. vivax  with proP. vivax .Globally, nearly half of the population lives in areas at risk of malaria transmission . ChildreMost malaria cases and death occur in sub-Saharan Africa (SSA) , 14, 15.The distribution and transmission pattern of malaria in Ethiopia differ from place to place depending on climate, rainfall patterns, and altitude , 23. TheAnopheles arabiensis was reported [In Ethiopia, malaria control strategies are very complex and influenced by various factors. Among these strategies, indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are the most important malaria vector control strategies . Additioreported , 31, 32.The transmission and incidence rate of malaria infection is determined by different factors such as environmental, human host, parasite, and vector , 27. In Malaria has continued to be one of the major public health problems in Amhara Regional State. It accounts for 31% of Ethiopia’s malaria burden . In 2012The weekly reports of each district health office indicated that many people who lived in and around Lake Tana suffered from malaria. This is due to the presence of suitable altitude, temperature and habitats for breeding and development of malaria vectors in Lake Tana and its surrounding localities. The present study was aimed to assess the prevalence and associated factors of malaria among febrile patients visiting eight selected health centres in Lake Tana and surrounding areas.This prospective malaria prevalence study was conducted in different locations of Lake Tana and its surrounding areas. Lake Tana is the source of the Blue Nile and it is the largest lake in Ethiopia, which contributes up to 60% of the Nile’s water and 50% of the country’s freshwater. The lake is located in Amhara Regional State at latitude of 11°36' N and a longitude of 37°23' E. The major portion of the total annual rainfall is observed between June and September. The minimum mean temperatures of the study area is 10.9 °C and the maximum is 29.2 °C . Lake TaIslands, peninsulas and mainland areas are found in Lake Tana scattered all over the five districts, i.e., Semein Achefer, Bahir Dar Zuria, Fogera, Libokemkem and Chuahit and one city (Bahir Dar city administration), and these were selected purposively based on accessibility, suitability and proximity of areas to the local inhabitants. The data were collected from one island (Dek), two peninsulas , and five surrounding mainland areas  Fig. .Fig. 1MaAn institutional-based cross-sectional study was employed to determine the current malaria prevalence in eight purposively selected health centres. The clinical data were collected for ten consecutive months from October 2020 to July 2021.The source population of the study was all patients coming to selected health centres for treatment during the study periods. All symptomatic or febrile patients visiting the governmental health institutions for treatment during the study period were considered as a target population.All symptomatic or febrile patients (body temperature above 37.5 °C) visiting the selected health centres during the study period and willing to participate were included in the study. Individuals taking anti-malarial drugs or those who took anti-malarial drugs for three weeks before the study period were excluded.α/2)2 *p*(1-p)/d2, where: n = the total sample size, P = prevalence of malaria in Bahir Dar town health centres (38.3%), Zα/2 is the critical value of normal distribution at 95% CI (1.96), d = the desired precision of the estimate/margin of error (5%). After adding 5% to compensate for the non-responses and 1.5-design effect, the total sample size was 571. This sample size was allocated in each health centre based on the number of populations residing in the catchment areas.The sample size was calculated using a single population proportion formula. The prevalence of malaria among the general population (38.3%) in Bahir Dar town health centres was used to determine the sample size . The samThe Zones, districts, Kebeles  and health centres were selected purposively based on accessibility, suitability and proximity of areas to the local inhabitants. However, study participants (symptomatic or febrile patients) coming to the health centres for blood film examination were selected using a simple random sampling method to reduce sampling bias during sampling. This is happen due to each member of the population has an exactly equal probability of being chosen in a basic random sampling process. The sample was collected monthly until the final sample size was reached. Dependent variable consist of malaria prevalence whereas the independent variable consists of socio-economic characteristics and other malaria-associated risk factors.The following data collection methods were used to accomplish this study.The socio-demographic and other malaria risk factors were collected using pre-tested structured questionnaires. For those study participants who had difficulty in reading and filling out the questionnaires, face-to-face interviews, based on questionnaire, were made by trained laboratory technicians. A self-administered questionnaire was used for adult study participants aged above 18 years who were capable of reading whereas a face-to-face interview was made with parents/guardians of children.Plasmodium parasites and species identification, respectively, according to WHO guidelines [Plasmodium infection and species identification. If there was controversy on the results observed by the two, a third person resolved the disagreement. Microscopy or Rapid Diagnostic Test  was used for diagnosis of study participants.Finger-prick blood samples were collected using a strictly disposable sterile blood lancet. Thick and thin blood films were prepared for the purpose of checking the presence of idelines . All bloBefore data collection, an ethics clearance letter was received from the Ethics Clearance Committee of the College of Science, Bahir Dar University with Ref. No. PRCSVD/08/2020. A permission letter or supportive letter was obtained from Amhara Public Health Institute. The objective, potential risks and benefits of the study were explained in detail for health centre heads, adult febrile patients and their parents/guardians. Questionnaire-based data and blood sample collection were made after obtaining written consent from adult febrile patients and parents/guardians of children under 18 years of age.Data was entered and analysed using SPSS 20. Descriptive statistics were used to describe the prevalence, frequency and proportion of study participants in connection to different risk factors. The association of malaria prevalence and risk factors were assessed using univariate and multivariate logistic regression statistical models. In univariate logistic regression analysis, those variables with less than 0.25 p-values were entered into a multivariate logistic regression model . The strThe data for this malaria prevalence was gathered from different health centres located in Lake Tana and its surrounding areas. In the study period, a total of 531 febrile patients were included and most of them were diagnosed using a microscope . Males, teenagers and middle-aged people, widowed ones, rural residents, private occupations, farmers, and students were more prone to infection. Infection levels looked similar among education levels of patients and religions. Infection levels were less on patients who earned less income. RDT tested patients were found more prone to be positive than microscope examination , P. vivax for 31 (23.7%), and mixed-species  for 5 (3.8%) . The highest prevalence was recorded in Kidist Hana (51.5%) followed by Robit (34.8%), Gorgora (30.3%), and Wusha Tiris (25%) health centres  malaria suspected patients, 400 (75.3%) were malaria negative and 131 (24.7%) were malaria confirmed cases. The overall prevalence was 24.7%. Out of this, 17.9% were accounted for by 31 malarires Fig. .Fig. 5ToPlasmodium parasites than females . The probability of malaria infection was 100 times higher in patients who had eave openings in their houses than in those who had not eave openings in their houses . Similarly, all family members who did not use bed nets at night were at higher risk of malaria compared with those family members who used bed nets . Additionally, participants who live within < 1 km distance of mosquito breeding site had a 223-fold higher probability of Plasmodium infection than those who lived > 2 km . Furthermore, the result of this analysis showed that elementary school education level (p = 0.025), living in the study areas in the month of October (p = 0.036), staying with cattle all night in the house (p = 0.031), and presence of wall openings in the houses were significantly associated with malaria.A multivariate logistic model was used to analsze risk factors that had p < 0.25 in the univariate logistic regression analysis result Table . Only eiAnopheles mosquito, altitudinal differences, and microclimate variations. Moreover, the reasons might be a lifestyle change, geographical area, communities awareness difference, development of dams or irrigation, economic status, and the type of malaria diagnosis methods used [In Ethiopia, malaria is a major concern and it can cause much damage to the health, and socio-economic development of the country due to the occurrence of malaria during harvesting seasons . The resods used .The relatively lower malaria prevalence in the current study might be connected to malaria eradication and control programs, such as the use of long-lasting insecticide-treated nets (LLIN), insecticide residual spray (IRS), the introduction of rapid diagnostic tests, and the use of artemisinin-based combination therapies, might have led to a decrease in a load of malaria . FurtherMalaria prevalence was more predominant in the Kidist Hana health centre (51.5%) compared to other health centres (p = 0.002). However, the least malaria prevalence was recorded in Kunzila (p = 0.033). This dissimilarity is due to community awareness differences about the use of malaria intervention activities, geographical location, altitude difference, and temperature . In addiPlasmodium parasite detected was P. falciparum (72.5%), followed P. vivax (23.7%) and mixed-species (3.8%). This result is in line with other previous studies carried out in different parts of Ethiopia [P. falciparum proportion is much lower than those of P. vivax in Ethiopia [P. ovale [In this study, the predominant Ethiopia , 60–62. Ethiopia , 64. ThiEthiopia . AdditioEthiopia . In contP. ovale .In multivariate analysis, gender of study participants, elementary school education level, living in the study areas in the month of October, the presence of eave and wall openings in the house, family members who use bed nets, staying with cattle all night in the house and mosquito breeding site distance from the house were significantly associated with malaria prevalence. On the other hand, age, income, sleep under bed nets, outdoor sleeping, outdoor activity, residence area, and sleeping with cattle assumed to be associated factors in the univariate analysis did not consider as explanatory factors in multivariate analysis.The current study showed males (29%) were more susceptible to malaria than females (17.7%) (p = 0.004). The finding of this study is similar to studies carried out in different parts of Ethiopia , 67–69. Plasmodium infection than other participants who joined college and above educational level (p = 0.025). Educational level was significantly associated with malaria prevalence in studies carried out in different parts of Ethiopia [Elementary school study participants were more likely to be infected with Ethiopia , 71, 72.Ethiopia , 73.In this study, the highest malaria prevalence was reported in October 37.5%) whereas the lowest was in March (14%) (p = 0.036). This is due to October is one of the major malaria transmission seasons that occurs following the rain from June to August , 27. Thi.5% whereIn Ethiopia, environmental management is the key component of vector control among the national malaria prevention and control strategies . In thisAmong Ethiopian malaria control strategies, long-lasting insecticidal nets (LLINs) are the most important malaria vector control strategies , 74, 75.The result of this study showed sleeping in the same house with cattle was the main determinant factor for malaria (p = 0.031). This finding is in agreement with other similar study conducted in Malawi . The avaThe current study showed malaria prevalence was much greater than the pooled prevalence of malaria among adults and pregnant women in Ethiopia as well as it is continued to go against the Ethiopian malaria elimination plan of 2021–2025. Gender of study participants, elementary school education level, living in the study areas in the month of October, the presence of eave, and wall openings in the houses, family members who did not use a bed net, staying with cattle all night in the house and mosquito breeding site distance from the houses were confirmed to be the risk factors for malaria prevalence. Hence, to avoid these problems, the current prevention and control methods such as health education, environmental management practice, larviciding, use of LLIN and application of IRS for controlling the vector, prompt diagnosis and treatment, and use of ACT should be effectively implemented in the study area.


Age-adjusted antibody responses were analyzed using a reversible catalytic model to calculate seroconversion rate (SCR). Malaria parasite was not detected in any of the 345 participants. The overall malaria seroprevalence was 72.2% for PfAMA-1, 18.8% for PfMSP-119, 32.5% for PvAMA-1, and 4.4% for PvMSP-119. The proportion of seroprevalence for P. falciparum and P. vivax antigens was significantly highest in Ban Koh  and Don Yang , respectively. For all parasite antigens except PvMSP-119, the proportion of seropositive individuals significantly increased with age (P < 0.001). Based on the SCRs, there was a higher level of P. falciparum transmission than P. vivax. Regarding KAP, almost all respondents showed adequate knowledge and awareness about malaria. Nevertheless, significant effort is needed to improve positive attitudes and practices concerning malaria prevention measures. Multivariate regression analyses showed that living in Ban Koh was associated with both P. falciparum  and P. vivax  seropositivities. We also found significant associations between age and seropositivity against P. falciparum and P. vivax antigens. The data suggest that seroepidemiological surveillance using AMA-1 and MSP-119 antigens may provide further evidence to reconstruct malaria exposure history. The absence of weak evidence of recent malaria transmission in Mukdahan Province is promising in the context of the disease elimination program.Malaria remains a major public health challenge in Thailand. Continuous assessment and understanding of the behavior and perceptions related to malaria exposure in the high-risk group are necessary to achieve the elimination goal. This study aimed to investigate the parasite prevalence, seroprevalence rate, knowledge, attitudes, and practices (KAP), and malaria risk factors in rural communities living close to a forested area in the northeastern part of Thailand. A community-based cross-sectional survey was conducted in three forest-goer communities  located in Khamcha-i district, Mukdahan Province, Thailand, from July to August 2019. Demographic, socioeconomic information and KAP data were collected using a structured questionnaire. Parasite prevalence was determined by microscopy. Seroprevalence was determined  Anopheles mosquitoes. Globally, malaria is the top mosquito-borne disease that causes infection and death. In the World Health Organization (WHO) Southeast Asia region, approximately 200 million people in nine countries are at high risk of acquiring malaria  antibody responses in an asymptomatic population of forest-goer communities living in rural areas in Mukdahan Province, one of Thailand’s upper northeastern provinces. Three endpoints were assessed to characterize malaria burden in the study population: (i) malaria seroprevalence of individuals measured by detection of antibodies to P. falciparum and P. vivax, (ii) knowledge, attitudes, and practices (KAP) regarding malaria prevention and control, and (iii) potential risk factors associated with malaria seropositivity.The primary objective of this cross-sectional study was to determine malaria . It had a population size of 342,200 in 2019 . To the east, it borders the Mekong River, across which lies the Savannakhet Province of Lao People’s Democratic Republic (Lao PDR). West of the province is the Phu Phan mountain range, covered with thick dry dipterocarp and evergreen forests, including several national parks and other protected areas. This mountain range is also home to two non-human primate populations, namely, the rhesus macaque (Macaca mulatta) and the pig-tailed macaque (Macaca nemesterina) , Ban Khok , and Dong Yang  located at the Khamcha-i district of Mukdahan Province, Northeastern Thailand Figure 1sterina) . The typPlasmodium infections, of which 11.1% and 3.6% were due to P. vivax and P. falciparum, respectively . Furthermore, the incidence of malaria is generally higher in Mukdahan Province due to its location along the international border of Savannakhet Province in Lao PDR and relatively porous, with a high potential for malaria importation. Among the 18 provinces of Lao PDR, Savannakhet has the third highest malaria incidence . A recenectively . In addiectively .A cross-sectional survey was carried out between July and August 2019. All Thai nationals of both genders and residing in the study villages were eligible. A convenience sampling strategy was used, whereby residents were asked to come to the selected survey points such as temples or clinics. Participants were briefed on the study procedure, and their consent was documented. Individuals who could not communicate or were unwilling to provide informed consent were excluded. Furthermore, axillary body temperature was also measured using a digital thermometer , and those with temperatures exceeding 37.5°C were considered febrile.A standardized questionnaire was developed and administered to each participant to gather data on demographic, socioeconomic status and KAPs on malaria. The questionnaire included a mixture of closed-ended and open-ended questions. A pre-tested and validated pilot survey was conducted in communities in other settings to evaluate the questionnaire’s clarity, adequacy, and comprehensibility. In addition, preliminary interviews were conducted to ensure standard interviewer methods and relevant interview questions were used. The interviews were conducted anonymously by trained interviewers with on-site supervision to minimize the courtesy bias and improve the accuracy of participants’ self-reporting. Before conducting the interviews, the lead researchers (SC and ZMI) conducted a one-day training for the surveyors to explain each question and how to conduct the interviews. The interview was conducted face to face using the Thai language, and all questions were open-ended. A knowledge score was calculated according to the correct answers.A blood sample was obtained by finger prick for thick and thin blood smears, and three spots of blood (approximately 100 µl per spot) were collected on Whatman ET31 Chr filter paper . All blood smears were stained with Giemsa solution and examined for the presence of malaria parasites under a 100X high-power field by experienced microscopists as described previously . Blood sFour 3-mm disks were punched from each dried blood spot, and serum was eluted in reconstitution buffer in 0.5-ml deep-well plates  as described previously . The rec19), i.e., P. falciparum (PfAMA-1 and PfMSP-119) and P. vivax (PvAMA-1 and PvMSP-119), as described previously  with 0.05% Tween 20 (PBS/T) and blocked using 1% (w/v) skimmed milk solution  in PBS/T for 3 hours. After washing, 50 µl of reconstituted blood spot solution was added in duplicate at final dilutions of 1:1,000 for both MSP-119 antigens and 1:2,000 for AMA-1 and incubated overnight at 4°C. A positive control consisting of a pool of hyper-immune sera (i.e. pooled sera from infected humans) was included in each plate. The plates were washed, and 50 µl of horse-radish peroxidase (HRP)–conjugated rabbit anti-human IgG antibody  were added to all wells at a dilution of 1:15,000 in PBS/T and incubated for 3 hours. After further washes, 100 µl of the substrate solution 3,3′,5,5′-tetramethylbenzidine (TMB)  was added. Reactions were stopped after 15 min with 100 µl per well of 0.3M H2SO4. The optical density (OD) at 450 nm was read using a Multiskan Go ELISA reader .All sera were tested for IgG antibodies by an indirect quantitative enzyme-linked immunosorbent assay (ELISA) to recombinant apical membrane antigen-1 (AMA-1) and 19-kDa fragment of recombinant merozoite surface antigen-1  and cross-checked for errors. Data were processed and analyzed using Stata/SE version 13.1 for Windows . Data were presented as frequencies and percentages for categorical variables, whereas data for continuous variables were presented as the median and interquartile range (IQR). Duplicate ELISA OD values were averaged and normalized against values from blank wells to adjust for background reactivity . SeroposP = 0.212). The median age of the respondents was 57 years (IQR: 47–64). More than 90% of the respondents were over 30 years. At enrollment, the mean axillary temperature of the population differed across the three villages (P < 0.001), but no febrile illness (axillary temperature > 37.5°C) was recorded. Majority of the respondents were farmers (94.2%) and most had a monthly household income of Thai Baht (THB) ≤8,000 (~$260). There was no evidence of recent malaria transmission in the study areas, as evidenced by negative microscopy results in the three studied villages.A total of 345 respondents in Mukdahan Province participated in this study Table 1.via mosquito bites (P < 0.001). However, 96% of the respondents recognized that fever, headache, and chill were symptoms of malaria, with significant differences between study sites (P < 0.001). A small proportion of respondents (3.8%) reported not taking any medication after contracting malaria. Respondents from Ban Khok showed the highest proportions of knowledge about drug resistance due to incomplete adherence to the antimalarial drug , using bed net to prevent malaria , and the importance of malaria examination after returning from the forest (100%). However, they were lowest on the knowledge of relapsing fever due to malaria .Generally, almost all respondents (97.7%) were knowledgeable about malaria transmission to bites Table 2.P = 0.015), reported living within 500 m of the forest. Although the villagers live near a forested area, respondents from Don Yang  and Ban Khok (71.4%) reported the highest proportions of spending the night and entering the forest for the past 6 months, respectively. Most respondents (75.7%) did not own bed nets as part of the practice to prevent the spread of malaria; however, only respondents in Ban Khok responded well to this practice (63.2%). Nevertheless, there was remarkable compliance for nightly bed net usage among those who owned them (100%). Similarly, all respondents reported 100% indoor residual spraying (IRS) coverage by the health authority at least once during 2019. Moreover, approximately 60% of the respondents reported seeking immediate treatment at the nearby clinic when malaria symptoms developed, with the significantly highest number of respondents from Ban Koh (P < 0.001). In contrast, respondents from Ban Khok claimed the highest proportion of waiting out the symptoms until well . Only a small proportion still considered buying medication directly from the local shop (3.2%).Concerning attitudes, most respondents (73.1%), with significant predominance from Ban Khok . 19, 32.5% for PvAMA-1, 4.4% for PvMSP-119, 79% for any P. falciparum antigens, and 40.4% for any P. vivax antigens. Between the study areas, the proportion of seropositivity for any P. falciparum and P. vivax antigens was significantly highest in Ban Koh  and Don Yang , respectively. Within the study areas, the proportion of PfAMA-1 was significantly higher than other parasite antigens , ranging from 45.3% to 90.2%. For all parasite antigens, except for PvMSP-119, the proportion of seropositive individuals significantly increased with age . Similarly, most parasite antigens showed a similar trend in the age-specific proportion of seropositivity within the study areas, i.e., highest in the 46–60 age group and lowest in the ≤16 age group.Antibody responses to PfAMA-1, PfMSP-1P. falciparum transmission than P. vivax using all tested antigens, ranging from 0.007 year−1 (0.002–0.026) to 0.060 year–1 (0.031–0.119) for P. falciparum and 0.001 year−1 (0.000–0.001) to 0.008 year-1 (0.007–0.009) for P. vivax. The SCRs were statistically significant between species-specific antigens, evidenced by the non-overlapping confidence intervals.The annual probability of malaria seroconversion curves in Mukdahan Province is shown in P. falciparum– and P. vivax–specific antigens are shown in P. falciparum– and P. vivax–specific antigens. For P. falciparum antigens, a significant association was observed in Ban Koh  and Ban Khok . In contrast, for P. vivax antigens, the increased seropositivity was significant in Ban Koh  and Don Yang . Interestingly, the likelihood of seropositivity increased significantly with age for only P. falciparum antigens , whereas for P. vivax antigens, the increase in seropositivity was not significant with age . No association was found between gender, occupation, income level, and seropositivity to any P. falciparum– and P. vivax–specific antigens.Univariate and multivariate logistic regression analyses to identify factors associated with seropositivity to any Plasmodium species : insights into KAP and risk factors of malaria exposure among forest-goer communities in rural areas of Mukdahan Province, Thailand. The results showed an apparent heterogeneity of antibody responses for AMA-1 and MSP-119 antigens of both species, with AMA-1 demonstrating a higher prevalence of 4- to 8-folds compared to MSP-119. The relationship between age and seroprevalence rates was similar in all studied areas, increasing with age, peaking at 46–60 years, then decreasing with age. In addition, seroprevalence and SCRs were higher in the older age group, indicating that the age-specific antibody response reflects cumulative exposure or population behavioral differences  acknowledged that mosquito bite transmits the malaria parasite to humans, and this is consistent with the reported 100% bed net use by those who owned them. A comprehensive systematic review by Nofal et al. reported that forest-goer respondents in the Greater Mekong Subregion (GMS) countries, including Thailand, often described that mosquito bites were the cause of malaria . Neverthc dishes . In addic dishes .Local variation in attitude toward malaria transmission was also observed among communities in the province. Almost a quarter of the respondents spent the night in the forest within the last 6 months, but it varied between settings . According to a Cambodian report, forest-goers spent a maximum of a week per month in the forest, and the malaria risk varied among different communities . FurtherThe application and full coverage of long-lasting insecticidal nets (LLINs)/insecticide-treated bed nets (ITNs) are effective methods for malaria prevention and control . AccordiNearly 42% of respondents did not go to the clinic or health facilities when they experienced malaria-like symptoms. It may be due to the remote location of the villages, causing difficulty for villagers in obtaining health assistance. Also, the problem of getting public transport is a factor contributing to the low percentage of those who seek medical treatment in health facilities. A study in Myanmar also reported that the long distance between villagers’ houses and local health facilities was a factor in the community’s reluctance to seek malaria treatment . FurtherPlasmodium species . The risk of seropositivity for P. falciparum and P. vivax was discovered in Ban Khok and Ban Koh, respectively, which corresponded to higher adjusted odd ratios. Moreover, it was interesting to note that the increasing age of the population is a risk factor for P. falciparum exposure but not for P. vivax. It may be due to differences in risk behavior, such as occupational activity involving forestation or agricultural activities, which increase their exposure to malaria vectors between the older and younger age groups. In addition, P. vivax seroprevalence was extremely low; therefore, the lack of any correlation was most likely a result of statistical restrictions due to the small number of seropositive samples. Another possibility is that P. vivax infections cause lower antibody responses or shorter-lived responses not detectable by the current assay  insufficient sample size for statistical analysis. Although small sample sizes may be adequate to detect a significant decline in SCR, they inevitably contribute to poor estimation precision of current SCR and restrict the possibility of identifying substantial changes in malaria transmission over time for the reverse catalytic model. (ii) While the convenient sampling was efficient and cost-effective, it has inherent selection bias. The survey was conducted mainly during the weekdays; thus, the younger adults were disproportionally represented. This group of individuals is typically away from home during weekdays, and under-representing them may likely underestimate the actual data. (iii) The assessment was based on a questionnaire, thus not representing the participants’ actual behavior. (iv) The cross-sectional study design provides information about a specific point in time, and the response of participants may not be the same in different seasons. Thus, we are unable to determine rates of change or stochastic variation. (v) The validity of the information provided by respondents may be questionable in the absence of independent checks or attempts to minimize errors of recall; however, this limitation is difficult to overcome due to inadequate funding.This study provides critical insight into the seroepidemiological surveillance of malaria in low transmission settings in Mukdahan Province, Northeastern Thailand. The present study also demonstrated that history of exposure can be constructed in the situation of missing baseline data and predictions can be made in the absence of active transmission. Furthermore, although most populations living in the study area have an acceptable level of knowledge about malaria, positive attitudes and practices concerning managing malaria require marked improvement. Finally, given the relatively low ownership of bed net, there is a need for future studies to engage the community directly through education and awareness programs on the importance of mosquito bed net. Continued malaria surveillance, vector control, education, and information campaigns are important not only among the forest-goer community but also among other vulnerable communities in the country.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ministry of Health of Mukdahan Province, Thailand (ref. no.101.0032.2562) and the Medical Ethics Committee, National University of Malaysia (ref. no. JEP-2019-148). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.ZMI, SC, INDL, and PCSV conceived and designed the study. MAFAR, SC, PC, LP, PK, NDD, and ZMI performed the fieldwork. AK and KKAT expressed proteins. MAFAR, SC and NDD performed data cleaning and analyzed the data. MAFAR, SC, INDL, PCSV, AK, KKAT, and ZMI interpreted the data. MAFAR, SC, and NDD wrote the first draft of the manuscript. ZMI, INDL, PCSV, AK, and KKAT provided critical revision of the manuscript for important intellectual content. All authors have contributed to the final version of the manuscript.This work (P.I: ZMI) was supported by the ASEAN Science Technology and Innovation Fund  from the ASEAN Secretariat and Geran Pembiayaan Sepadan (FF-2019-124/1) from the Faculty of Medicine, Universiti Kebangsaan Malaysia.We would like to extend our gratitude to the communities and community leaders for their support and participation in the survey. We wish to sincerely thank all members of the field team. We are grateful to the Khamcha-i District Public Health Office director and the director of Sub-District Health Promoting Hospital at Ban Koh for their support throughout the study. We are grateful to Thiti Srihanam, Sudarat Chaisangrat and Sujitrapa Surasan for their assistance in the survey. We sincerely thank Professor Rahmah Noordin for her critical reading of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malaria is one of the most common causes of hospital admission and death in children under the age of five. The World Health Organization (WHO) has issued guidelines for the identification and treatment of severe malaria. Evidence has shown that adherence to standardized malaria treatment protocols improves outcomes. As a baseline assessment in preparation for a malaria treatment quality improvement project, this study aimed to determine adherence to the WHO severe malaria treatment guidelines in children at a Ugandan Regional Referral Hospital.A retrospective review was performed on a convenience sample of children discharged between June 2021 and March 2022 from the Mbale Regional Referral Hospital Paediatrics Ward with a diagnosis of severe malaria. Data were collected using a standardized case report form. Demographics, presenting symptoms, laboratory results, treatments, length of stay, and mortality were extracted. Comparison of treatments received to items recommended in the WHO guidelines was undertaken to determine adherence.147 patients were included. The median age was 5 years (IQR 2–7 years), and 55% were male. The most common features of severe malaria were haemoglobinuria (49%), haemoglobin < 5 mg/dL (34%), and altered mentation (24%). Median hospital length of stay was 3 days (IQR 2–4 days), and the mortality rate was 27% (n = 40). Overall adherence to all aspects of the WHO severe malaria guidelines was achieved in 3% (n = 4) of patients. The most common areas of deficiency were not testing to confirm malaria diagnosis (34%) and inadequate administration of artesunate (82%). Fewer than the three recommended doses of artesunate occurred in 22% of patients. Additionally, a delay in the administration of the second dose occurred in 67% (n = 78) and in the third dose in 77% (n = 71) of patients. While the recommended time between doses is 12 h, the median interval between dose one and dose two was 15 h (12–20) and the median interval from dose two to dose three was 17 h (14–25).Current adherence to severe malaria treatment guidelines in children at this Ugandan regional referral hospital is poor, but this study has identified target areas for improvement. According to the 2021 World Health Organization (WHO) World Malaria Report, there were 241 million cases of malaria in 2020 in 85 malaria-endemic countries. These cases led to 627,000 deaths, 77% of which were in children under 5 years of age. Uganda accounted for 5% of all malaria cases, making it the 3In order to address these issues, the WHO has issued guidelines for the identification and treatment of malaria , 4. ThesThis is a retrospective descriptive study of children with severe malaria based on a convenience sample of children discharged between June 2021 and March 2022 from the Mbale Regional Referral Hospital Paediatrics Ward. This study was approved by the institutional review board at Nationwide Children’s Hospital, USA and the Mbale Regional Referral Hospital Research Ethics Committee, Mbale, Uganda.The setting of this study is a government-funded regional referral hospital. As a public hospital, patients do not need to pay for care received at this hospital, nor do they pay for any medications, supplies, or laboratory tests that are available at the hospital. Similar to other regional and district hospitals in sub-Saharan Africa, supplies available at the hospital are limited and subject to frequent stock-outs. Long-term employees report that quarterly deliveries of supplies typically last 1–2 months. The paediatrics ward at this hospital serves 100–200 children each day on average. The ward is staffed by clinical officers, intern physicians, resident and attending paediatric physicians, qualified nurses, as well as medical students and nursing students. Physicians are present on the acute section of the ward from approximately 7:00am to 11:00 pm and otherwise available to be called in if necessary. From 11:00 pm to 7:00am there is usually one qualified nurse staffing the acute section of the ward. The providers rotate frequently, usually on a weekly basis. The acute section of the ward has two cardiorespiratory monitors and oxygen tanks available. The ward is equipped with a glucometer and malaria rapid diagnostic tests (RDTs), though glucometer strips and RDT materials frequently suffer from stock-outs. The hospital lab is able to perform complete blood counts, microscopy for malaria parasites, complete metabolic panels, urine and stool microscopy, and urinalyses. This laboratory is open during daytime hours on weekdays, and limited weekend hours. The most commonly prescribed medications and fluids used for patients described in this study are provided by the hospital when in stock. Similar to other hospitals in sub-Saharan Africa, a patient caregiver must be present to assist in care for the patient. The ward staff provide all intravenous medications and appropriate monitoring, but any oral medications, nutritional needs, and hygiene needs are the responsibility of the caregivers accompanying the patient.The sample was selected on review of paper charts of children who had been discharged from the paediatrics ward. All paper charts of paediatric patients are stored within the ward after patient discharge for several months before entering long-term storage. Discharged patient’s charts available on the ward at the time of initiation of the study were assessed, and those with documentation of a diagnosis of severe malaria were further reviewed and data abstracted. Initial screening identified 172 children that had a clinician reported diagnosis of “severe malaria,” but on further review of diagnostic criteria, only 147 of these children had documented evidence of severe malaria following WHO criteria  and for each guideline individually as it applied to that patient. Adherence to diagnostic testing guidelines was considered appropriate if the patient had a documented positive test for malaria before receiving their second dose of artesunate in order to account for emergency situations where the first dose was given at the time of presentation before a test result was available. A patient could have either a positive malaria rapid diagnostic test (RDT) or positive microscopy to be considered adherent. Adherence to artesunate administration guidelines was considered appropriate if the patient received the 3 minimum doses recommended and the doses were given at the recommended interval of 12 h. Based on local guidelines that recommend medications be provided within 1 h of ordered time, administration outside of this 1-h time window (greater than 13 h) was considered “delayed”. Adherence to artesunate administration guidelines in patients who died before the 3 minimum recommended doses were received was assessed only on those doses that should have been received prior to death. Adherence to haemoglobin monitoring guidelines was considered appropriate if the patient had a documented haemoglobin result, as this is recommended for all patients with severe malaria. Adherence to blood transfusion administration guidelines was considered appropriate if patients who had a haemoglobin < 5 mg/dL received a transfusion of packed red blood cells 10 ml/kg or whole blood 20 ml/kg, or if children who did not have a haemoglobin result were deemed clinically to require blood transfusion received the appropriate dose of products. Adherence to hypoglycaemia management guidelines was considered appropriate if patients either had a documented blood glucose result or received empiric treatment with 5 ml/kg of 10% dextrose fluids when presenting with altered mental status or seizure activity when a blood glucose result was not available.Adherence to oxygen administration guidelines was considered appropriate if patients with respiratory distress, severe anaemia, altered mental status, seizure activity, or documented poor perfusion were placed on oxygen therapy. Adherence to seizure management guidelines was considered appropriate if patients with documented seizure activity received appropriate doses of diazepam or phenobarbital. Adherence to guidelines on nasogastric tube use in unconscious children was considered appropriate if patients noted to have altered mental status had a nasogastric tube placed. Adherence to guidelines concerning empiric antibiotic use was considered appropriate if patients received an empiric course of ceftriaxone. Complete adherence to all guidelines was defined as having all of the above relevant criteria met for each patient. Continuous data are reported as median (IQR). Categorical data are reported as percentages.Among 147 patients who were included, 55% were male and the median age was 5 years (IQR 2–7 years). Table The most common features of severe malaria were haemoglobinuria (49%), haemoglobin < 5 mg/dL (34%), and altered mentation 24%)  of patients reviewed  had testing ordered that was either not completed or completed but not documented. Fewer than the three recommended doses of artesunate occurred in 22% of eligible patients (n = 32). Additionally, a delay in the administration of the second dose occurred in 67% (n = 78) of patients who received 2 doses, and in the third dose in 77% (n = 71) of patients who received 3 doses. While the recommended time between doses is 12 h, the median interval between dose one and dose two was 15 h (IQR 12–20 h) and the median interval from dose two to dose three was 17 h (IQR 14–25 h).Other areas of weakness included irregular monitoring of haemoglobin, inadequate administration of transfusions for severe anaemia, inconsistent hypoglycaemia management, lack of oxygen therapy for patients at high risk of hypoxemia , and sporadic use of nasogastric tubes for unconscious patients Fig. . In partThis study sought to determine the current level of adherence to severe malaria treatment guidelines for children at a Ugandan regional hospital and identify opportunities for intervention. Current adherence to severe malaria treatment guidelines was poor at 3%. The most significant areas of deficiency were in malaria testing and artesunate administration. There are resource limitations that contribute to these issues—there are regular stock outs of malaria rapid diagnostic tests and artesunate, the hospital lab is only operational during limited hours, and nurse: patient ratios can be as extreme as 1:100 overnight making proper administration of medications difficult.Increased supplies of rapid diagnostic tests would allow for more consistent testing prior to antimalarial treatment. Indeed, it has been shown in several studies that the ready availability of testing is a major factor in adherence to the “test and treat” strategy recommended by the WHO , 10–14. Despite the resource barriers, there are likely potential opportunities for improvement within the existing system. As demonstrated by the research team at Koidu Government Hospital in Sierra Leone, implementing and adhering to a standardized protocol for treatment of severe malaria in a rural hospital with limited resources was effective at decreasing malaria-specific mortality . ThroughBased on this successful model and the particular issues identified in this study, findings were presented to key hospital stakeholders and care providers to develop initial quality improvement interventions. Several recommendations to consider attempting to improve compliance were produced. One is the use of an admission checklist for patients suspected of having severe malaria. This checklist would include confirmation of diagnostic testing and document its results. The checklist also highlights the recommended timing for administration of the three artesunate doses. While rapid diagnostic tests for malaria are not always available, the hospital lab is able to perform microscopy to confirm the diagnosis. To improve the proportion of patients that receive this testing, another change in the admission workflow was proposed—include acquisition of patient blood samples for thick and thin smears by the provider at the time of admission. This will allow for confirmation of malaria diagnosis once the laboratory is open even when the patient is admitted overnight. Lastly, it was proposed to use patient caregivers to assist in timing medications by informing them during ward rounds how many doses are remaining for the child and the times those doses should be administered. This may allow for better caregiver advocacy for drug administration and improved compliance.Resource limitations also contribute to the other areas of weakness that were identified in this study. Limited laboratory hours and lack of point of care testing decrease the consistency of haemoglobin monitoring despite a high prevalence of severe anaemia and high demand for blood transfusions. Short supply of blood available for transfusions contributes significantly to deaths from severe anaemia. At a public health level, these investigators and hospital stakeholders hope that there can be increased efforts to increase community blood donation and make this vital resource more available. Inadequate supply of glucometer test strips often limits testing for hypoglycaemia. While this will be a difficult barrier to overcome, a different focus for improvement could be on the empiric administration of 10% dextrose for patients with altered consciousness, seizure activity, and poor oral intake. Treating providers acknowledge that oxygen is often provided without being documented, and the same is true for placement of nasogastric tubes in patients with altered consciousness, so documentation of utilization of these items should be improved to better assess algorithm adherence.This study has several limitations. It is a retrospective review of a convenience sample, and therefore cannot be certain to be representative of all cases at this facility. There can be no assessment of true malaria-related mortality rates as this sample did not include all patients admitted with malaria during this time period. The data obtained were also subject to the quality of documentation in the paper charts, which was incomplete in some cases. It is possible that patients received additional interventions or had test results that were not documented in the reviewed records and, therefore, actual adherence may be higher than what is presented here. The clinical significance of adherence to each component of the guidelines in this study is not known, but given that adherence to standardized treatment protocols has been shown to improve outcomes, this data is still relevant.Despite the limitations, the data provided here have identified areas of focus for the ongoing malaria treatment quality improvement initiatives in the Paediatrics Wards at Mbale Regional Referral Hospital. The increased awareness that this data has provided for the treatment team allows for increased vigilance in the areas of weakness. Future work will focus on improvements in adherence following implementation of the proposed interventions noted above.Current adherence to severe malaria treatment guidelines in children at this Ugandan regional referral hospital is poor, but this study has identified target areas for ongoing malaria treatment quality improvement initiatives. Implementation of an admission checklist for patients suspected of having severe malaria and adjustments to workflow may improve rates of testing for malaria and adherence to anti-malarial administration guidelines.


Plasmodium falciparum malaria has rarely been reported in a returning traveler with no prior history of malaria infection. Here, we report an imported case of afebrile P. falciparum malaria infection from Tanzania in a returning traveler to the Republic of Korea, following an earlier COVID-19 infection without previous history of malaria infection. Our case suggests the hypothesis that severe symptoms of P. falciparum malaria infection might be prevented by cross- immunity from previous COVID-19 infection. Malaria is well-known as one of the most common causes of fever among travelers returning from endemic areas such as tropical African countries. However, afebrile  Plasmodium falciparum malaria in a returning traveler with no prior history of malaria infection has rarely been reported. Here, we report an imported case of afebrile P. falciparum malaria infection from Tanzania in a returning traveler to the Republic of Korea following an earlier coronavirus disease 2019 (COVID-19) infection, without previous history of malaria infection.Malaria is well-known as one of the most common causes of fever among travelers returning from malaria-endemic areas such as tropical African countries . In addi9/L), platelets (294 × 109/L), aspartate transaminase (33 IU/L), alanine transaminase (44 IU/L), total bilirubin (0.77 mg/dL), urine analysis (urine red blood cell < 1 per high-power field and urine white blood cell < 1 per high-power field), prothrombin time (11.3 s), and activated partial thromboplastin time (35.9 s), were within the normal ranges, except for mildly anemic hemoglobin (12.8 g/dL). The inflammatory marker of C-reactive protein was also unremarkable (0.2 mg/dL). However, atypical material suspicious for malaria gametocytes was found from the routine complete blood-count test result review process by a laboratory technician. For further investigation, a peripheral smear for blood cell morphology test was performed, which showed crescentic gametocytes, indicating P. falciparum malaria infection with parasitemia (952/µL)  . TherefoP. falciparum malaria has not been reported in Korea since 1945 [P. falciparum malaria-naïve. Reported cases of P. falciparum malaria infection in Korea were classified as imported malaria infections [P. falciparum malaria as he had not previously been diagnosed with malaria infection. Despite the likelihood of developing symptomatic P. falciparum malaria infection, as would be expected in the P. falciparum malaria-naïve individuals, such as our case patient, he was afebrile and did not present the typical features of P. falciparum malaria infection. The pathogenesis of asymptomatic P. falciparum malaria infection in our patient is not clearly understood; however, there are several factors to consider. First, our patient had COVID-19 infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in January 2021, approximately 4 months prior to the diagnosis of P. falciparum malaria infection. There have been reports of the common immunodominant epitopes between SARS-CoV-2 and P. falciparum [P. falciparum infection of the host through invasion of the erythrocyte [P. falciparum malaria infection, leading to protection against the dissemination and progression of P. falciparum malaria infection. Second, SARS-CoV-2 is known to have various glycoproteins (GPs): membrane GPs, spike GPs, and GPs that have properties of hemagglutination [P. falciparum malaria infection.As endemic lciparum ,7. Thesethrocyte . Furtherthrocyte . As SARSthrocyte , SARS-Cotination ,11,12. Ftination . In additination . Interestination . TherefoP. falciparum malaria infection in the individual from non-endemic areas is rare. However, our case suggests that severe symptoms of P. falciparum malaria infection might be prevented by cross-immunity from previous COVID-19 infection. With this case report, we would like to draw attention to the diagnostic challenge presented to clinicians by the atypical presentation of illness, possibly influenced by the current ongoing COVID-19 pandemic. Further studies, involving a larger number of patients, are needed to better define the pathogenesis of afebrile P. falciparum malaria infection following COVID-19 infection.In summary, afebrile


Plasmodium falciparum and Plasmodium vivax malaria infection are from 2007. To inform global malaria prevention and control efforts, we aimed to estimate the global distribution of pregnancies at risk of malaria infection from 2007 to 2020.The most recent global estimates of the number of pregnancies at risk of P falciparum and P vivax transmission, combined with country-specific demographic data on women of reproductive age, fertility rates, induced abortions, and stillbirths, to derive the annual number of pregnancies overall, by parasite species, and by endemicity strata from 2007 to 2020. The definition of endemicity strata was based on the parasite point prevalence in individuals aged 2–10 years for P falciparum and 1–99 years for P vivax. We also did a sensitivity analysis in which we considered most of sub-Saharan Africa endemic for P vivax.We used estimates from the Malaria Atlas Project on the total population living in areas of P falciparum transmission declined by 11·4% globally, despite an overall 7·0% increase in pregnancies, representing a decrease of 100·0% in the WHO Europe (EURO) region, 52·6% in WPRO, 51·5% in AMRO, 23·9% in EMRO, and 17·2% in SEARO, and a 25·4% increase in AFRO. Pregnancies in P vivax transmission areas fell by 42·8%, representing a decrease of 100·0% in EURO, 89·8% in WPRO, 48·4% in AMRO, 32·4% in EMRO, and 10·0% in SEARO, and a 25·8% increase in AFRO. Our sensitivity analysis suggests that the number of pregnancies at risk of P vivax infection could be seven-fold higher for AFRO if the whole of sub-Saharan Africa was considered endemic for P vivax.In 2020, 121·9 million pregnancies occurred in malaria transmission areas, resulting in an estimated 70·9 million (58·1%) livebirths. The total number of pregnancies at risk of malaria was 52·9 million in the WHO South-East Asia (SEARO) region, 5·1 million in the Western Pacific (WPRO) region, 46·1 million in the Africa (AFRO) region, 11·1 million in the Eastern Mediterranean (EMRO) region, and 6·7 million in the Americas (AMRO) region. Between 2007 and 2020, pregnancies in areas of P falciparum or P vivax malaria than in 2007. This increase in the number at risk in AFRO comes despite the decline in malaria rates due to the rapidly rising population and the corresponding number of pregnancies in endemic areas. These estimates should guide priority setting for resource allocation to control malaria in pregnancy.Between 2007 and 2020, substantial declines in the number of pregnancies at risk of malaria were seen globally. However, in AFRO, 25·4% more pregnancies were at risk of Bill & Melinda Gates Foundation and Telethon Trust. Plasmodium falciparum and Plasmodium vivax are the major contributors to adverse maternal and pregnancy outcomes.Malaria infection carries serious risks for pregnant women, their fetuses, and their newborn babies. Pregnant women are at higher risks of malaria-related complications and death than aged-matched women who are not pregnant.P falciparum and P vivax are from 2007.The most recent global estimates of the number of pregnancies at risk of malaria for Evidence before this studyMapping pregnancies at risk of malaria is crucial for informing control strategies and targeting interventions for malaria in pregnancy. The most recent update on the number and global distribution of pregnancies at risk of malaria is for 2007, which showed that the number of pregnancies at risk in the Asia-Pacific region was much higher than in other regions. However, new estimates are needed to reflect population growth, changes in fertility rate, and major reductions in malaria transmission since 2007. We searched PubMed and Google Scholar for articles related to quantifying the number of pregnancies at risk of malaria published between Jan 1, 2010, and Sept 29, 2022, using the search terms “ AND  AND (risk) AND humans[MeSH])”, with no language restrictions. No relevant publications were identified. The WHO World Malaria Report shows the number of pregnancies resulting in livebirths at risk of malaria in Africa only. This manuscript expands these estimates beyond Africa to all pregnancies at risk of malaria globally, including those resulting in spontaneous pregnancy loss and induced abortions.Added value of this studyPlasmodium falciparum and Plasmodium vivax prevalence relative to previous 2007 estimates by the Malaria Atlas Project. It is also the first report of time trends on the changing pattern of pregnancies at risk of malaria between 2007 and 2020 by geographical regions, endemicity levels, and parasite species. The study estimates a 28·8% decrease in pregnancies at risk of P falciparum or P vivax malaria despite a 7·0% increase in the number of pregnancies globally, reflecting the shrinking map of malaria transmission.This study provides the most up-to-date estimates of the global distribution of pregnancies at risk of malaria. The study is based on improved estimates of populations at risk of malaria with substantially expanded data sources for Implications of all the available evidenceP falciparum or P vivax malaria infection in sub-Saharan Africa reflects rapid population growth, even as these areas are progressing towards controlling and eliminating malaria. These data provide valuable information for researchers, policy makers, and programme managers to allocate targeted resources to control malaria in pregnancy, monitor the effect of malaria control efforts, and inform priority areas for research.The findings highlight the gains made in the Asia-Pacific regions since 2007. The increasing number of pregnancies at risk of either Malaria Atlas Project (MAP), a WHO collaborating centre for geospatial malaria monitoring. Annual country-level estimates of the population at risk of P falciparum or P vivax transmission were derived from MAP for 2007 to 2020 by WHO malaria endemicity strata. We based endemicity class on the parasite prevalence rate in the population of individuals aged 2–10 years for P falciparum and 1–99 years for P vivax.We used model-derived estimates from the P falciparum and P vivax transmission using surveillance data on the number of malaria cases, nationally representative cross-sectional surveys, and satellite imagery data of environmental predictors of the prevalence of Anopheles mosquitoes.P falciparum were generated by MAP using a geostatistical modelling framework. The parasite rate model used household surveys as the response variable and environmental and malaria intervention datasets as predictor variables.P falciparum and P vivax. For the high-burden African countries, the maps of P vivax prevalence were created using the high-resolution P falciparum parasite rate maps and the proportions of P vivax cases using reported country-specific and year-specific ratios of P vivax to P falciparum.MAP provides estimates of the total population living within the global limits of Plasmodium and pixels set to no cases based on lower-level  administrative subunits that reported no cases by routine surveillance in the same year. A third exception relates to Africa, as MAP only considers P vivax to be endemic in the horn of Africa despite evidence indicating that there might be P vivax transmission elsewhere in sub-Saharan Africa.P vivax.MAP's current approach to estimating the total population at risk for malaria is based on a combination of environmental datasets, all available routine survey data, and WHO endemicity status. This approach results in transmission limits that change over time as regions eliminate malaria or oscillate between reporting no and some malaria cases across annual surveys. To avoid contradicting routine surveillance data, MAP's parasite rate maps consider populations at risk in administrative units that reported malaria cases that year. Two exceptions to the previous statement are pixels that are set to zero malaria cases due to environmental suitability for The total population at risk estimates were split into endemicity strata data on the basis of the prevalence maps. Hypoendemic transmission was defined as areas with a parasite prevalence of 10% or less, mesoendemic as higher than 10–50%, hyperendemic as higher than 50–75%, and holoendemic as higher than 75%.P falciparum, P vivax, and risk of either or both species) because the spatial extent of malaria endemicity was fixed according to routine surveillance data.Uncertainty intervals (UIs) were calculated by MAP for estimates of population at risk stratified by species and endemicity level only. The 95% UIs for the malaria endemicity classes were created by processing a set of 100 realisations  drawn from the posterior distribution of the geostatistical model) for each year. This process generated 100 estimates for the population at risk within each class for each country each year. From these 100 estimates, we derived the mean and upper UIs as 98th ranked estimates and lower UIs as 3rd ranked estimates. UIs could not be derived for overall risk estimates  using different data sources for livebirths, stillbirths, and induced abortions.WHO Global Health Observatory (GHO) and used mid-year resident numbers. The GHO derived these data from the World Population Division of the UN. When multiple data sources were available from the UN, we used civil registration systems and population censuses as preferential sources. If these data were not available, we used household surveys.For annual number of livebirths for each malaria-endemic country, we used country-specific total fertility rate (TFR) data (which is defined as the sum of age-specific fertility rates) and demographic data of the number of women of reproductive age  and SPSS (version 26 or later).The estimates of pregnancies at risk of malaria were determined by multiplying the total number of pregnancies each year by the proportion of the population living in areas with malaria transmission. The estimates of pregnancies at risk of malaria was also done by pregnancy outcomes, endemicity strata, and parasite species. 95% UIs for the estimates of pregnancies occurring in areas of The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.P falciparum or P vivax malaria transmission and were therefore at risk of malaria, representing 77·7% of all pregnancies in malaria-endemic countries and 49·2% worldwide. An estimated 70·9 million (58·1%) of these 121·9 million pregnancies at risk of malaria were estimated to have resulted in livebirths. We estimated the remainder to have resulted in pregnancy loss, with 1·4 million (1·1%) ending in stillbirths, 33·5 million (27·5%) in induced abortions, and 16·1 million (13·2%) in miscarriages. There were 67·7 million pregnancies at risk of both P falciparum and P vivax malaria transmission  were in 85 countries where malaria was endemic . Of thessmission .Table 1E·7 million (5·5%) in the Americas  to estimate populations at risk suggests that estimates for the population at risk of P vivax would be seven-times higher (36·6 million) for AFRO than in the main analysis.Of the 121·9 million pregnancies at risk of malaria, 52·9 million (43·4%) occurred in countries in the WHO regional offices for South-East Asia (SEARO), 5·1 million (4·2%) in the Western Pacific (WPRO) region, 46·1 million (37·8%) in the Africa (AFRO) region, 11·1 million (9·1%) in the Eastern Mediterranean (EMRO) region, and 6as AMRO;  region.  in 2020 . The senP falciparum in 2020 occurred in hypoendemic transmission areas, 25·5 (23·1%) in mesoendemic transmission areas, 3·2 (2·9%) in hyperendemic transmission areas, and 0·5 (0·5%) in holoendemic transmission areas (P vivax transmission occurred exclusively in areas of hypoendemic transmission.81·0 million (73·6%) of 110.8 million pregnancies at risk of on areas . P vivaxP falciparum transmission areas fell by 11·4%, from 125·1 million in 2007 to 110·8 million in 2020, and those in P vivax transmission areas fell by 42·8%, from 137·5 million in 2007 to 78·7 million in 2020. The reduction in pregnancies in P falciparum transmission areas was seen in all WHO regions except AFRO, where the number increased by 25·4% . In the AMRO region, pregnancies at risk fell by 51·5% (from 9·3 million to 4·5 million) in AMRO, 23·9% (from 13·7 million to 10·5 million) in EMRO, and 17·2% (from 55·5 million to 46·0 million) in SEARO.Comparison over time showed that, globally, the number of pregnancies at risk fell by 28·8%, from 171·2 million in 2007 to 121·9 million in 2020 . PregnanP vivax transmission fell in all regions except AFRO, which saw a 25·8% increase (from 4·2 million in 2007 to 5·3 million in 2020). There has been no P vivax transmission in the EURO region since 2014. The second largest reduction, 89·8%, occurred in WPRO (46·4 million to 4·8 million), followed by 48·4% in AMRO (10·5 million to 5·4 million), 32·4% in EMRO (15·4 million to 10·4 million) and 10·0% in SEARO (58·6 million to 52·8 million). The largest reduction in pregnancies at risk of P vivax globally, 26·3%, occurred between 2014 (119·0 million) and 2015 (87·7 million).Similarly, pregnancies at risk in areas of P falciparum transmission reduced in holoendemic settings  and hyperendemic , whereas the number at risk increased in mesoendemic  and hypoendemic areas . The number of pregnancies at risk in areas of mesoendemic transmission also declined by 54·2% (from 0·014 million to 0·007 million). In the EMRO region, pregnancies at risk of P falciparum infection increased from 0 to 0·002 million for hyperendemic areas, doubled (from 0·2 million to 0·4 million) in mesoendemic areas, and decreased by 25·9% (from 13·5 million to 10·0 million) in hypoendemic areas. In the EURO region, P falciparum transmission declined to 0 in 2010. In the SEARO region, the number of pregnancies at risk in hypoendemic settings reduced by 16·7%, from 55·2 million in 2007 to 46·0 million in 2020. Those in mesoendemic settings reduced by 84·6%, from 0·34 million to 0·01 million. In the WPRO region, the number of pregnancies in hypoendemic transmission areas reduced by 52·5%, from 7·9 million to 3·7 million and in mesoendemic areas by 63·7%, from 0·05 million to 0·02 million  occurred in areas of P vivax transmission (from 137·5 million to 78·7 million), compared with only 11·4% for P falciparum (from 125·1 million to 110·8 million).We estimate that 121·9 million pregnancies occurred in malaria transmission areas in 2020, resulting in 70·9 million livebirths. Although sub-Saharan Africa has the most malaria-associated adverse pregnancy outcomes, it represents only 37·8% of pregnancies at risk of P falciparum or P vivax in the AFRO region increased by 25·4% between 2007 and 2020. However, considering the 28·2% increase in the number of pregnancies in AFRO in the same period, the data reflect positive movement towards shrinking the malaria transmission map in this region. For P falciparum, stratification of time trends by malaria endemicity showed that pregnancies at risk fell in holoendemic (by 80·4%) and hyperendemic (by 48·7%) settings and increased in mesoendemic (by 53·7%) and hypoendemic (by 46·7%) areas. These data correspond to about 5·1 million fewer pregnancies in people living in the highest transmission areas (holoendemic and hyperendemic) in these 14 years between 2007 and 2020. This change reflects a region-wide decline in P falciparum transmission, as seen by the substantial declines in the case burden of malaria in the general population.P vivax transmission occurred only in the lowest endemicity strata over the study period. Our primary estimates for the number of pregnancies at risk of P vivax transmission in Africa were restricted to the horn of Africa. This restriction might represent a conservative estimate of the actual number of pregnancies at risk as this model's geospatial boundaries for P vivax transmission were derived from documented P vivax surveillance data, which are mostly based on microscopy and rapid diagnostic tests. However, many P vivax infections are not detectable by these standard diagnostic tools, which do not detect vascular or extravascular subpatent , or latent infections seated in inaccessible tissues.P vivax in AFRO could be close to seven-times higher than reported in the main analysis, because, with this alternative method, all of sub-Saharan Africa is considered endemic for P vivax, not just the horn of Africa. Better diagnostic tools and data are needed to estimate the true global distribution of P vivax endemicity.Pregnancies at risk of P falciparum and P vivax that resulted in livebirths for the WHO AFRO region (31·2 million in 2020) are consistent with the latest estimates by WHO for 2020 (33·8 million).24Our estimates for pregnancies at risk of P falciparum or P vivax (171·2 million) in 2007 is 36·7% higher than our previous estimate for 2007 (125·2 million).Our current estimate of the pregnancies at risk of The strengths of this study include the use of the most up-to-date estimates of the global spatial limits of malaria transmission by species and endemicity strata from MAP. Furthermore, the multiple data sources for livebirth, miscarriage, stillbirths, and induced abortion estimates were cross-referenced with alternative data sources.P vivax transmission in sub-Saharan Africa could be underestimated. In the future, estimates based on advanced diagnostics might be able to account for subpatent infection, improving determination of geospatial distribution and pregnancies at risk of P vivax infections.P falciparum and P vivax malaria or to incorporate uncertainty for some pregnancy covariates, such as TFR and miscarriage, which have their own uncertainties.The study has several limitations that could affect the accuracy of our estimates. First, the data on the population living within the global spatial limits of malaria transmission is affected by the same survey and routine surveillance data limitations reported previously.Our study provides the most up-to-date contemporary estimates and trend in estimates of pregnancies at risk of malaria by parasite species and endemicity at regional and global levels. In this study, we also provide an informed platform to estimate the disease and economic burden of malaria in pregnancy and its spatial distribution and to monitor the effect of malaria control efforts. Our findings might guide priority setting for resource allocation for the control of malaria in pregnancy to areas of greatest need.https://malariaatlas.org/data-directory/; number of women of reproductive age (country level) and total fertility rates (country level) at https://www.who.int/data/gho; stillbirth rates (country level) at https://data.unicef.org/dv_index/; and induced abortion rates (country level) at https://osf.io/5k7fp/.All aggregated country-level data collected during this analysis will be made available for a period of 5 years after publication. Access to data will be provided following the investigators’ approval of a proposal after considering the overlap between the proposal and any ongoing efforts. Proposals should be directed to the corresponding authors. People requesting data will need to sign a data access agreement and the database will be transferred electronically. The study protocol will be made available on request. For raw data: pixel-level and administrative-level summaries of populations at risk of malaria are available for visualisation and download at We declare no competing interests.