Malaria is among the leading causes of mortality and morbidity among under five children in developing countries. Ethiopia has set targets for controlling and eliminating malaria through at-risk group interventions. However, the disease remains a serious public health concern in endemic areas like in Wollo, Northeast Ethiopia. Therefore, this study aimed to determine malaria prevalence, risk factors and parasite density among under five children in Ziquala district.A facility—based cross-sectional study was conducted in Ziquala hospital, and Tsitsika, Mishra and Hamusit health centers in Ziquala district, Northeast Ethiopia, from January 2022 to April 2022. The study enrolled a total of 633 under five children using a systematic sampling technique. A capillary blood sample was collected from each child to prepared thin and thick blood smears. Smears were then stained with 10% Giemsa and examined under light microscope. A pretested structured questionnaire was used to collect on socio-demographic data, parental/caregiver knowledge, and malaria determining factors. Bivariable and multivariable logistic regression analysis was done to identify factors associated with malaria.Plasmodium falciparum, P. vivax, and mixed infection (both species) accounted for 57.1%, 38.5%, and 4.5% of the cases, respectively. Regarding to parasite load, moderate parasitemia was the most common, followed by low and high parasitemia with the proportion of 53.8%, 31.4% and 14.7% parasite density, respectively. Malaria infection was linked to irregular utilization of insecticide-treated bed nets , staying outside at night , and parents not receiving malaria health education in the past six months .The overall prevalence of malaria among children visiting Ziquala district health institutions was 24.6% (156/633). Malaria was prevalent among children under the age of five enrolled in the study. The local government should focus on regular insecticide treated net utilization, reducing the risk of mosquito bites while sleeping outdoors at night and increasing public understanding of malaria prevention and control through health education would also help to minimize the burden of malaria. Plasmodium species [Malaria is one of the most serious public health concerns and a life threatening parasitic disease caused by the  species . Malaria species –4. Child species .Between 2000 and 2019, global malaria prevention and control activities were ramped up, and the World Health Organization African Region achieving significant success in reducing its malaria burden, particularly in Sub-Saharan Africa . HoweverPlasmodium falciparum causes 60% of malaria cases, while the rest is caused by P. vivax [Despite strong investments to decrease malaria incidence rates over the past years, infection rates remain significant in sub-Saharan Africa. Children under the age of five in sub-Saharan Africa, where the great majority of malaria deaths occur each year, are particularly vulnerable to the disease . Every dP. vivax .To prevent and reduce malaria infections, several intervention activities such as the distribution of insecticide-treated nets (ITNs), indoor residual spraying (IRS), artemisinin-based combination therapy (ACT), and health information dissemination have been carried out in Ethiopia. These efforts notwithstanding, malaria remains a serious public health concern in endemic areas, particular among children under the age of five . EthiopiMalaria is one of the top causes of morbidity and mortality in both adults and children under the age of five in Ziquala district in northeast Ethiopia, according to a district health office report. However, a study on the epidemiology of malaria in children under the age of five had not been conducted.Knowing the prevalence, parasite density, and determinants of malaria in children under the age of five in health facilities is critical for scaling up and designing effective intervention programs. Therefore, this study fills in an important knowledge gap by identifying potential risk factors for malaria infection among under five-year children in Ziquala District.P. falciparum. Subsistence farming and livestock breading are the main occupations in the area. Water conducive to mosquito breeding is available throughout the year because of rivers such as the Tekezie and Tela rivers. In the district, there is one primary hospital, five health centers, and fifteen health posts. All health facilities provide diagnostic and treatment services to the community.A multi-site facility-based cross-sectional study was conducted in Ziquala hospital, and Tsitsika, Mishra and Hamusit health centers in Ziquala district, Northeast Ethiopia, from January 2022 to April 2022. Ziquala district is one of the eight districts of Wag Hemra Zone, which is located at a distance of 780 km from capital city of the country, Addis Ababa. The district has an altitude of 1462 meters above sea level. Its annual average rainfall and temperature are 255 mm and 22°C, respectively . The areThe sample size was calculated using a single population proportion formula, the 95% confidence limits (Zα/2 = 1:96), and 5% margin of error (d) with a maximum proportion of 50%.2 P (1- P)/ d2, (1.96)2 *0.221 (1–0.221)/ (0.05)2 = 384.Sample size = Z(α/2)Accounting for a 10% non-response rate and implementing design effect of two, the final sample size was 633. Study health facilities were chosen by stratifying the district’s health facilities as malaria-endemic and non-endemic based on their altitude. Then, among the resulting six health facilities in the district, four were selected for data collection by simple random sampling technique. The number of study participants to be enrolled at each health facility was determined based on the number of febrile under five children at each health facility during the same period (January 2021 to April 2021) of the previous year. Hence in this period, a total of 1240 febrile under five children were screened for malaria from Ziquala Hospital (482), and Tsitsika (376), Mishra (251) and Hamusit (131) health centers. Accordingly 246, 192, 128 and 67 febrile patients were included from Ziquala Hospital and Tsitsika, Mishra and Hamusit health centers, respectively. Finally, a systematic random sampling was applied to select study participants at each health facility.The source population consisted of all children under the age of five who visited the designated health facilities during the study period. The study only included children whose parents or guardians signed a written consent form. Children who had undergone antimalarial chemotherapy 42 days before to data collection were excluded because the parasite is thought to be cleared from the blood within 42 days of treatment, and re-infection can happen after that.To collect data on socio-demographic and related risk factors for malaria, a standardized questionnaire was developed in English. It was then translated into Agewgna, the indigenous language. Data of socio-demographic and environmental were collected from their parents/caregivers using structured and pretested questionnaire by trained data collectors.Plasmodium infections, while thin smears were employed for parasite species identification using an oil immersion objective.At Ziquala hospital, and Tsitsika, Mishra and Hamusit health centers laboratories; capillary blood was drawn aseptically from the children’s fingers using a blood lancet. Thin and thick blood films were prepared and air dried. After that, the thin blood film was fixed with absolute methanol and allowed to air dry. Both thin and thick blood films were stained with 10% Giemsa stain for 10 minutes. The slides were then air dried after being washed with distilled water. Finally, laboratory technologists at the health facilities examined the stained slides. Thick smears were used to detect To measure parasitemia, the asexual forms of the parasites, such as trophozoites and schizonts, were manually counted until 200 WBCs from thick smear using a tally counter. The number of parasites per microliter of blood was calculated using the formula below, with the patient’s total leukocyte count assumed to be 8000/μl of blood. Parasite load is equal to the parasites counted per 200WBC X 8000WBC/μl blood. The parasitemia was then classified as low (<1000 parasites per microliter of blood), intermediate (1000–4999 parasites per microliter of blood) and high (>5000 parasites per microliter of blood) .Plasmodium parasite by two laboratory technologists at each site. A colour atlas was used during microscopic examination. To assure quality of the microscopic examinations, all positive and 10% of the negative slides were re-examined by a third reader to remove discrepant result.Data collectors were trained for two days by the investigators. The Giemsa stain was tested on known positive and negative malaria slides. During the microscopic examination, a slide was regarded as negative after 200 fields had been examined without finding of Data were entered and analysed using SPSS version 22 after data collection. The prevalence rate and determinant factors were estimated using descriptive statistics. Bivariable and multivariable logistic regression models were used to find correlations with malaria infection. The multivariable logistic regression analysis included variables with a p-value of less than or equal to 0.25 in the bivariable regression. A p-value <0.05 was considered to determine statistical significance. Finally, the strength of the association between factors was determined using adjusted odds ratios (AOR) with a 95% confidence interval (CI).A total of 633 febrile children under the age of five years old took part in the study, with a 100% response rate. Of the total participants more than half (53.9%) of individuals were males and 46.1% of them were females. Regarding to their age group , the higPlasmodium parasite prevalence was higher in younger children, with a statistically significant difference (P = 0.001). The malaria parasite was shown to be more prevalent in children living in rural areas as compared to children in urban areas. Home village location was not associated with malaria infection (p = 0.455). In this study, the prevalence of P. falciparum and P. vivax was 57.1% and 38.5%, respectively, while that of mixed infection was 4.5% . Of these, 60.3% were males and 39.7% were females. Malaria prevalence in sex was not statistically significant difference (P = 0.065). Likewise, the higher number of malaria cases was detected a mong the age group of 12–23 and less than 12 months with the prevalence of 45.5% and 24.4%, respectively. was 4.5% .Regarding to parasite density, moderate parasitemia was the most common, followed by low and high parasitemia, which accounted for 53.8%, 31.4%, and 14.7% individuals respectively. Males were found to have a higher prevalence of moderate malaria parasitemia than females, accounting for 57.1% of the total, however there was no statistical difference in parasite density (p = 0.683). Likewise, moderate parasitemia had the highest parasite density in the 12–23 age group, and there was statistical significance in parasitemia across age groups (p = 0.001). Moreover, there was a statistically differences (p = 0.001) between rural and urban residents, with 70.2% of rural residents having moderate parasitemia .The highest numbers of children who participated were from Ziquala hospital (38.9%), while the lowest were from Hamusit health center (10.6%). There was also a considerable variation in the prevalence rate between the health facilities, ranging from 19.4% at Hamusit health center to 28.0% at Ziquala hospital .In this study, 72.0% of the parents/caregivers had no ability to read or write, whereas 28.0% had attained various levels of formal education. Of the total parents/caregivers, 91.9% believed that malaria is a curable disease and 72.0% had knowledge of malaria transmissibility between individuals. The majority of the respondents (78.0%) were aware that mosquitoes are a vector for malaria transmission, whereas the remaining (22.0%) had misconceptions about the mode of transmission. Despite the high prevalence of malaria, 70.3% of participants had knowledge about the breeding site of mosquitoes. Meanwhile, 94.0% of the respondents knew that malaria is a preventable disease. When asked about malaria prevention measures, the majority of the respondents 73.0% mentioned that insecticide treated nets (ITNs) as a malaria preventive method while only 2.5% mentioned that malaria can be prevented through indoor residual spraying (IRS). On the other hand, children under five and pregnant women were accurately identified as the most susceptible segments of the population by 32.5% and 19.6% of the respondents, respectively. Furthermore, a majority (84.0%) of the children sought treatment at least within 24 hours after the onset of fever. Early diagnosis and treatment, within 24 hours after the onset of symptoms, were recognized as critical components of malaria control .In this study, 94.0% of the respondents possessed an ITNs, and 96.1% had received IRS service in the previous 12 months. In terms of ITNs utilization, 58.0% of under-five children slept in an ITNs on a regular basis during the previous two weeks. In bi-variable logistic regression, the following variables were chosen and entered into the forward stepwise multivariable logistic regression model: stagnant water near the house, under five children sleeping under ITNs regularly for the last two weeks, number of under five children per household, staying outside at night, and health education on malaria in the previous six months.In the final adjusted model, children under the age of five who did not sleep under an ITNs regularly for the last two weeks were more likely to be infected with malaria  than those who did. Children who stayed outside at night had a 2.1-fold increased chance of contracting malaria than children who did not . In addition, children whose mother/caregiver had never received malaria health education in the previous 6 months also had a greater risk of infection  than those whose mother/caregiver had received malaria health education .The present study showed that malaria is a public health concern among children under five years old in the study area. The overall prevalence of malaria was 24.6% 95% CI = 21.1–28.1) among children visiting health institutions in Ziquala district. This result was comparable to a study conducted in Arsi Negele, Ethiopia (22.8%) , Arba Mi.1–28.1 aHowever, the prevalence of malaria in this study was higher than the prevalence determined by studies conducted in Wogera district, Ethiopia 8.7%) , Sherkol% , SherkPlasmodium falciparum and P. vivax were the two species identified in the blood of the children, accounting for 57.1% and 38.5% of infections, respectively. This result is in line with the national Plasmodium species distribution [P. falciparum over P. vivax can be explained by several factors. These include P. falciparum’s high proliferation in the host cell, the parasite’s capacity to infect all ages of red blood cells, the parasite’s resistance to first line treatment [P. falciparum is a widespread species in the lowlands.ribution  and otheribution , Kombolcribution  and Dembribution , Ethiopireatment  and the Plasmodium infection decreases with increasing age of a child. This is might be due to these children live in stable malaria transmission endemic areas and can develop age-related protective immunity as the result of continuous exposure to infective mosquito bites [In this study similar to a study done in Wogera district, Ethiopia , the numto bites . The majto bites . Howeverto bites , while ato bites . The immto bites .Plasmodium falciparum was the predominant species found in all health facilities except Hamusit health center. This variability in the prevalence of malaria among different locations is in line with studies undertaken in Wolkite, Ethiopia [Regarding to the health institutions, Ziquala hospital has the highest rate of malaria infection, followed by Tsitsika and Mishra health centers. Ethiopia , lowlandEthiopia  and CameEthiopia . This coAbout 78.0% of the parents/caregivers had knowledge that mosquitoes are a vector for the transmission of malaria. This result was relatively lower than other studies done in Damot Gale, Wolayta Zone (85.0%) and Gondar (94.5%) Ethiopia , 41. HowWhen asked about malaria prevention measures, 73.0% of responders identified ITNs, whereas only 2.5% chose IRS. The knowledge of using ITNs (73.0%) and IRS (2.5%) for malaria prevention was lower than a study done in Damot Gale, Ethiopia . AccordiPlasmodium parasite It was similar with prior studies conducted in South Gondar and Arbaminch Zuria District, Ethiopia [This study revealed that those children who did not reguraly use ITNs were 5.04 times more likely than those who did to be infected with the Ethiopia , 33. InsEthiopia . TherefoChildren who stayed outdoors at night were more likely to be exposed from malaria infection compared to those that did not. Staying outside during the night showed a statistically significant association with malaria. This finding was in line with the previous studies done in Armachiho, in Dembia district and Sherkole refugee camp in Benishangul-Gumuz, Ethiopia , 46, 47.The prevalence of malaria in under-five children attending at selected health facilities of Ziquala district was high. The highest prevalence of malaria was found in those aged between 12 and 23 months old, and a higher proportion of moderate parasitemia was also observed in this study. Irregular use of bed net, staying outside at night and parents’ not receiving malaria health education were the main correlates of malaria. The local government and other concerned bodies should give focus on regular ITNs utilization, infection associated with staying outdoors at night, environmental management and changing attitudes towards malaria prevention and control through health education to minimize the burden of malaria.S1 Data(SAV)Click here for additional data file.


Plasmodium falciparum  malaria. A longitudinal cohort study performed in the Southern part of Benin, identified a group of infants who were able to control asymptomatic malaria infections (CAIG).Immunoglobulin G (IgG) antibodies are thought to play important roles in the protection against IgG antibodies against distinct merozoite antigens were quantified in plasma from Beninese infants. Functionality of these antibodies was assessed by the merozoite-phagocytosis assay using THP-1 cells and primary neutrophils as effector cells. Gm allotypes were determined by a serological method of haemagglutination inhibition.Purified IgG from infants in CAIG promoted higher levels of merozoite-phagocytosis than did IgG from children who were unable to control asymptomatic infections . High level of merozoite-phagocytosis activity was significantly associated with high levels of IgG against AMA1  and GLURP-R2 . Moreover, infants of the G3m5,6,10,11,13,14,24 phenotype showed higher merozoite-phagocytosis activity  than those presenting other G3m phenotypes.The results of the present study confirm the importance of antibodies to merozoite surface antigens in the control of asymptomatic malaria infection in Beninese infants. The study also demonstrated that G3m phenotypes impact the functional activity of IgG. This last point could have a considerable impact in the research of candidate vaccines against malaria parasites or other pathogens.The online version contains supplementary material available at 10.1186/s12936-022-04361-w. Plasmodium genus. Of these, Plasmodium falciparum causes the most severe forms of malaria and is the primary cause of malaria related mortality and morbidity in children under 5 years of age in sub-Saharan Africa [P.falciparum malaria after repeated infections [Malaria is caused by parasites of the n Africa . Immune fections , 3. Howefections , 5.P. falciparum parasitaemia below the detection threshold of thick blood smear (CAIG) as determined by monthly thick blood smears (TBS) [To study the development of immunity against clinical malaria during infancy, samples and data were collected from children residing in Tori Bossito in Benin from birth to 18 months of age. The analyses of clinical and parasitological data obtained during the follow-up allowed us to identify a group of infants who were able to clear asymptomatic infections or at least to reduce the rs (TBS) . These crs (TBS) .P. falciparum malaria infections while other correlated these IgG to malaria risk or exposition [P. falciparum-infected red blood cells (iRBC) in vivo, free circulating merozoites and occasionally trophozoites [It is generally accepted that antibodies of the cytophilic subclasses (IgG1 and IgG3) may be particularly important in protection against clinical malaria –12. Accoposition , 13–15. position . In FcgRposition , 17, 18.position . Neutrophozoites –23.http://www.imgt.org/). Gm allotypes have been shown as implicated in malaria susceptibility [Gm allotypes are allelic variations in the CH1, CH2 and CH3 IgG heavy chain, that induce alterations in the amino acid sequences which may affect their binding to FcgRs . Gm allotibility , 26, 27,Here, samples and data were used from a well-established birth-cohort from Benin, to investigate the functional activity of serum IgG antibodies. Infants were typed with respect to their Gm allotypes and specific antibodies  against a panel of merozoite-antigens were quantified by ELISA. The functional activity of IgG was investigated in the merozoite-phagocytosis assay. IgG levels and specific G3m phenotypes were strong predictors of the merozoite-phagocytosis activity by monocytes and neutrophils, respectively.The study was conducted in the district of Tori Bossito in southwest Benin between July 2007 and January 2010, where 567 children were followed-up from birth to 18 months of age. This follow-up consisted in an epidemiological, parasitological and immunological follow-up detailed elsewhere –28. HealP. falciparum vaccine antigen candidates AMA1, MSP1, MSP3, MSP2-3D7, MSP2-FC27, GLURP-R0 and GLURP-R2 were quantified at 6, 9, 12, 15 and 18 months by ELISA [TBS were also performed monthly to detect asymptomatic malaria infections. In addition, mothers were invited to bring their infants to the health centre, at any time, in case of fever (suspected by the mother) or clinical signs, whether or not related to malaria, and the same protocol was applied. Venous blood samples were collected quarterly for hematological and immunological measurements. Venous blood was centrifuged for plasma isolation and genomic DNA extraction. IgG1 and IgG3 responses against P. falciparum parasites or at least maintain P. falciparum at very low parasitaemia, below the detection threshold of thick blood smear [P. falciparum but not able to control asymptomatic malaria infection (IG group) and a group of 23 children for which P. falciparum has not been detected by TBS examination during the survey (NPfDG group). These two groups were composed of all other infants for whom sufficient plasma volume was available.The control of asymptomatic malaria infection was defined as follow: the clearance of blood od smear . CAIG gr™ columns containing protein G coupled to Sepharose were used to purify IgG from 60 μl of plasma diluted 1/10 in phosphate buffered saline (PBS), as per manufacturer's instructions (Sigma-Aldrich). IgG concentration was measured using a NanoDrop®ND-1000 spectrophotometer, and samples were stored at −80 °C. Purified IgG samples were diluted in PBS to obtain a final concentration of 50 ng/mL before performing OP assays. Pooled IgG from malaria-exposed hyperimmune Liberian adults or malaria-non immune Danish blood donors served as internal controls [Ab SpinTrapcontrols .P. falciparum line NF54 was cultured and merozoite isolation was performed as described previously [l-glutamine, 0.02 g/l hypoxanthine, and 25 μg/ml gentamicin) at 37 °C in a humidified 5% CO2, 2% O2 and 93% N2 atmosphere. The parasitaemia was monitored by examination of Giemsa-stained thin blood smears, and parasite cultures were synchronized with 5% sorbitol treatment for 10 min. Synchronized trophozoite-stage parasites were harvested using a magnetic separation column (Vario MACS) and subsequently treated with 10 μM of epoxysuccinyl-l-leucylamido (4-guanidino) butane (E64) for 6–8 h to allow schizonts to mature without rupture. Segmented schizonts were centrifuged, resuspended in 4–6 ml RPMI 1640 medium, and then filtered through a 1.2 μm/32 mm syringe filter to obtain merozoites. The filtrate was passed over an LS MACS Column twice to remove the haemozoin. Free merozoites were stained with 10 μg/ml of ethidium bromide (EtBr) for 30 min before used in OP assay.eviously . Briefly4Cl, 10 mM KHCO3 and 0.1 mM EDTA) and incubated for 10 min.Primary neutrophils were purified from freshly drawn blood from a healthy Danish donor as described previously . Brieflyl-glutamine and 25 μg/ml gentamicin) and distributed in 96-well, U-bottom plates (1 × 105 cells/150 µl/well). The human monocytic cell line THP-1 was maintained as described previously [5 cells/150 µl/well).The cells were centrifuged and further purification of neutrophils was carried out with the EasySep Human Neutrophil Isolation Kit (StemCell Technologies) according to the manufacturer’s protocol. For OP assay, neutrophils were resuspended in cell medium , 4 nM eviously . Likewis2 humidified incubator. To stop phagocytosis, plates were centrifuged for 5 min at 400 g at 4 °C, followed by 2 washes with cold fluorescence-activated cell sorting (FACS) buffer (PBS + 0.5% BSA + 2 mM EDTA). Then, cells were fixed with 200 μl of FACS fixative  and kept at 4 °C until analysed in a CytoFLEX S flow cytometer (Beckman Coulter). Viable neutrophils or THP-1 cells were gated by forward scatter and side scatter properties, and EtBr-positive events were enumerated using primary neutrophils or THP-1 cells alone [The OP assay was performed as described in detail . Brieflyls alone . The perThe determination of Gm allotypes was performed before our study and included in our statistical analyses. Detailed description on the determination of IgG Gm allotypes of the Tori Bossito cohort is available elsewhere . BrieflyConsidering G3m allotypes, no child presented G3m16 or G3m21, both located on the CH2 domain of the IgG heavy chain, and absent in populations from sub-Saharan Africa. Similarly, due to the uncertain location of the Gm28 allotype on either IgG1 or IgG3 sub-classes among sub-Saharan Africans , this alIgG quantification was performed before our study and included in the statistical analyses.The Enzyme Linked Immunosorbent Assay (ELISA) was used to assess antibody levels against seven merozoite antigens. Details about the recombinant proteins and the procedure are described elsewhere , 29. BriPfDG groups using Chi-square test for categorical variables and Mann–Whitney U-test and Kruskal–Wallis test for quantitative variables. Kruskal–Wallis test was used to determine if the distribution of OP values was different between groups while the Chi-square test compared the proportion of infants with low/high OP  between groups.The statistical analysis was carried out using the Stata software version 13. Comparisons of demographic and clinical characteristics were investigated between the CAIG, IG and NPfDG), antibody levels against P. falciparum merozoite antigens and G3m phenotypes carriages were studied by multivariate regressions. Multivariate regressions studied the correlation between OP values, groups, antibody levels and G3m phenotypes using control variables. In this study, only control variables with P < 0.20 among demographic and clinical characteristics were specifically used. The correlation between OP values and groups  was studied using a multivariate ordered logit regression model; NPfDG, IG, and CAIG was coded 0, 1 and 2, respectively, according to OP values observation. If the coefficient and P value was significant and positive, the OP values increased with groups in the order 0, 1 and 2 otherwise the OP values did not increase with groups. The association between OP values, IgG levels and G3m phenotypes were studied using a generalized linear multivariate regression model where only control variables with P < 0.20 among demographic and clinical characteristics were specifically used. In these models, validity assumptions  have been checked and also the deviance and Pearson were shown as regards the model fitting.Finally, the correlation between OP values, groups  with the possibility to withdraw at any time.PfDG) and 80 children tested parasite-positive at one or more of the monthly visits. Of the 80 parasite-positive children, 27 were able to clear asymptomatic malarial infections or to reduce P. falciparum parasitaemia under detection threshold of thick blood smear (CAIG) while 53 had P. falciparum infection with clinical symptoms (IG) .Infants from CAIG had more asymptomatic infections P < 0.001) when compared to infants from IG while there was no difference regarding symptomatic infections between the two groups  (Table The distribution of bed net use and gender was similar between the three groups (P = 0.429). In contrast, the age of the mothers P = 0.001) and the number of antenatal visits (P = 0.005) was highest in N and the Merozoite-phagocytosis assays are typically performed with primary cells or with the THP1 cell line –35. To cPfDG groups , respectively  against AMA1, MSP2-3D7, MSP2-FC27, MSP1, MSP3, GLURP-R0, and GLURP-R2 were quantified by ELISA  and MSP1 IgG1  while levels of AMA1 IgG3 showed a modest association . Second, we investigated the association between merozoite-phagocytosis by neutrophils and specific IgG levels  . However, a significant low level of IgG3 against MSP2-3D7 was found in infants carrying G3m5,10,11,13,14  (Table PfDG groups. OP was previously correlated to malaria protection and judged as a reliable vaccine evaluation criterion through analysis of antibody responses [The results of the present study showed a higher ability of antibodies from infants included in the CAIG group to stimulate phagocytosis of monocyte cell line THP-1 or primary neutrophils compared to infants belonging to IG and Nesponses , 19, 36,PfDG groups (P ≤ 0.001, Additional file P. falciparum infections in Papua New Guinea [Infants from the CAIG presented higher IgG concentrations against AMA1, MSP1 and GLURP-R2 compared to infants belonging to IG and Nw Guinea . IgG1 tow Guinea  and suggin vitro OP activity observed in CAIG group could reflect the antibody involvement in the control of parasite growth. However, it is difficult to affirm that OP activity observed in this group is responsible for parasitemia control observed during the follow-up. Unfortunately, certain constraints such as the presence of maternal antibodies until 12 month of age and the quantity of available plasma at 15 month of age did not allow us to perform a prospective study.The pattern of malaria infections in CAIG group during the survey was very different from the one of IG group. In addition to have the capacity to control their parasitaemia, individuals included in CAIG group presented more asymptomatic infections than IG group Fig. . InfantsP. falciparum malaria after repeated infections. The correlation of high concentrations of IgG subclasses against AMA1 and MSP1 to high levels of OP may reflect the repeated exposure of these children to malaria which can lead to a better antibodies’ memory responses and phagocytosis capacity. Moreover, in this cohort, it has been shown that the levels of IgG against P. falciparum merozoite antigens reflected malaria exposure [PfDG group had lower levels of phagocytosis compared to CAIG and IG groups. Note that a slightly higher bed net possession was observed in NPfDG group, however other factors might be involved in lower exposition to P. falciparum infections. However, it is possible that a proportion of infants in NPfDG group could also be protected from malaria infections by other mechanisms.Immune epidemiological studies have shown that children living in malaria endemic areas may develop naturally acquired immunity against exposure . Thus, t. [. [The finding that the levels of total IgG to GLURP-R2 were associated with a high OP and not significant when considering specific IgG1 or IgG3 may suggest a potential implication of IgG2 and/or IgG4. Indeed, the total IgG level to GLURP was associated with a reduced risk of malaria by Nebie et al.  or Kana . [.  while an. [Indeed, IgG3 and IgG4 against GLURP were associated with a reduced risk of clinical malaria by Nebie et al.  while on. –42. In a. . IgG2 anP. falciparum merozoites. The effector functions of IgG3 are numerous among which activation of complement pathways, neutralization, antigen recognition and cell activation via FcgR receptors [As regards the influence of G3m phenotypes on OP, our results showed a high level of OP associated with the carriages of G3m5,6,10,11,13,14,15 and G3m5,6,10,11,13,14,24. No work previously investigated associations between Gm phenotypes and opsonizing antibodies to eceptors , 44. Amoeceptors , 45. The. demonstrated a protective effect of the Gm5,6,13,14; 1,17 phenotype carriage in Benin [P. vivax PvMSP1-19 and PvAMA-1 antigens in Brazilian Amazon endemic area [Facer  was the in Benin  while thmic area . Moreovemic area . G3m5,6,mic area , 48 or qThis work showed that G3m5,6,10,11,13,14,24 and G3m5,6,10,11,13,14,15 phenotypes were associated with high OP using primary neutrophils while only G3m5,6,10,11,13,14,24 phenotype was associated with high OP in THP-1 cell lines with a decreased significance. Other factors such as FcgR polymorphisms may also impact OP activities. The genotypes of primary neutrophils and THP-1 cells used in this study were respectively FcgRIIA 131 RR/FcgRIIIB NA2NA2 and FcgRIIA 131 RH/FcgRIIIB NA1NA2. It is worth noting that FcgRIIA 131 RR/FcgRIIIB NA2NA2 was previously associated with malaria protection and FcgRIIA 131 RH/FcgRIIIB NA1NA2 with a trend of protection .However, this aspect deserves further studies, performed on diverse populations in high number in order to explore this potential role of Gm phenotypes in the functionality of IgG, which could have a considerable impact in the research of vaccine candidates against malaria.This study shows that the group of infants able to control infection had probably a better functionality of IgG through a high OP compared to a control group. It showed also that the inherent polymorphism of the IgG heavy chain represented by the Gm phenotypes play an important role in IgG functionality, opening up avenues for the efficacy of antibodies in the search for anti-malarial vaccines.Additional file 1: Table S1. IgG levels in the study group.


Plasmodium vivax have proven diagnostic performance to identify people exposed in the previous 9 months.The landscape of malaria transmission in the Peruvian Amazon is temporally and spatially heterogeneous, presenting different micro-geographies with particular epidemiologies. Most cases are asymptomatic and escape routine malaria surveillance based on light microscopy (LM). Following the implementation of control programs in this region, new approaches to stratify transmission and direct efforts at an individual and community level are needed. Antibody responses to serological exposure markers (SEM) to P. vivax in peri-urban (Iquitos) and riverine (Mazán) communities of Loreto, communities that have seen significant recent reductions in malaria transmission. Socio-demographic, geo-reference, LM and qPCR diagnosis data were collected from two cross-sectional surveys. Spatial and multilevel analyses were implemented to describe the distribution of seropositive cases and the risk factors associated with exposure to P. vivax.We measured antibody responses against 8 SEM to identify recently exposed people and determine the transmission dynamics of P. vivax in Mazán (56.5%) than Iquitos (38.2%). Age and being male were factors associated with high odds of being seropositive in both sites. Higher antibody levels were found in individuals >15 years old. The persistence of long-lived antibodies in these individuals could overestimate the detection of recent exposure. Antibody levels in younger populations (<15 years old) could be a better indicator of recent exposure to P. vivax.Low local transmission was detected by qPCR in both Iquitos (5.3%) and Mazán (2.7%); however, seroprevalence indicated a higher level of (past) exposure to The large number of current and past infections detected by SEMs allows for detailed local epidemiological analyses, in contrast to data from qPCR prevalence surveys which did not produce statistically significant associations. Serological surveillance will be increasingly important in the Peruvian Amazon as malaria transmission is reduced by continued control and elimination efforts. Plasmodium vivax is its relapsing nature that can contribute up to 80% of the P. vivax cases. The detection of this source of transmission remains crucial for focused interventions in settings attempting to achieve elimination. Antibody responses to serological exposure markers to P. vivax have been validated to detect recent exposure and likely hypnozoite carriers in low transmission settings. To evaluate the benefits of adding serology to the detection of infection in areas where malaria transmission is rapidly reduced, we measured the prevalence of P. vivax by qPCR and serology in two sites of the Peruvian Amazon. This study shows that antibody responses to P. vivax antigens can detect more recently exposed people than molecular testing, providing more statistical power for surveillance and critical additional epidemiological knowledge for regions attempting to eliminate malaria transmission.A challenge for eliminating  Peru is considered a low-endemic transmission setting for malaria, with 24,000 cases reported in 2019  and the there was a noteworthy increase in the number of malaria cases [Over the last 20 years, the case incidence of malaria in Peru has fluctuated, influenced largely by the prevailing intervention programs. After the cessation of the Global Fund Malaria Project “PAMAFRO” in 2010, ia cases , with a ia cases . In threia cases . In its Plasmodium vivax causes the majority of malaria cases in Peru. The detection of cases is based on light microscopy (LM) diagnosis; this may substantially underestimate prevalence as greater than 50% of infections that are detectable by qPCR may be missed by microscopy [croscopy . Being bcroscopy  and hypnP. vivax have been developed and validated for their use in low transmission settings [New uses of malaria serological tools have been proposed to inform the impact of such control measures . Ideallysettings . These ssettings .P. vivax through antibody responses to serological markers in the Peruvian Amazon. Whereas previous studies in the Peruvian Amazon only focused on the analyses of antibodies to individual protein such as PvAMA1 [19 [P. vivax: PvMSP119, PvTRAg_2, PvTRAg_28, PvMSP8, PvMSP3.10, PvRAMA, PvRBP2b and PvEBPII. This provided an excellent opportunity to evaluate the benefits of adding serology to infection detection in monitoring and understanding changes in malaria epidemiology in the context of rapidly intensifying elimination efforts.Given that the Malaria Zero program has been implemented in the Peruvian Amazon region, new tools are needed to capture transmission patterns, detecting hot-spots and individuals likely contributing to transmission. In 2018, cross-sectional surveys were executed in two contrasting geographical areas of Loreto: Iquitos (peri-urban communities) and Mazán  to determine the exposure level to s PvAMA1 , PvMSP11AMA1 [19 , PvMSP8 AMA1 [19 , or PvMSAMA1 [19 , the advSamples were collected from consenting individuals through the CAM study (SIDISI 101645/2017) and the ICEMR study (SIDISI 101518/2018); both studies were approved by the Institutional Ethics Committee of Universidad Peruana Cayetano Heredia (UPCH). UPCH also approved the use of the serum samples collected in those studies and the use of Peruvian negative controls at the Institut Pasteur (SIDISI 100873/2017).Two cross-sectional studies were carried out in two districts of Maynas province, Department of Loreto in 2018: Iquitos and Mazán.Iquitos is the capital of Loreto and is the most populated metropolitan area in the Amazon Region. It is accessed only by air or by river and the majority of the population are mestizo (very mixed-race identity). Three peri-urban communities of the district of Iquitos were surveyed for this study: Rumococha (RC), Santo Tomás (ST), and Quistococha (QC). RC is located 8 km from Iquitos city; ST is 11km from Iquitos city and surrounded by the Nanay River. QC is located 12km from Iquitos city and close to the Nauta-Iquitos Road. The most common occupations are commerce, mototaxi and being a housewife .Mazán is a district of Maynas province located 55–60 km northeast of Iquitos. It is comprised of many small communities, and the community of Mazán is surrounded by the Mazán and the Napo Rivers . The only access is by speedboat (1 hour from Iquitos). Gamitanacocha (GC), Libertad (LI), Primero de Enero (PE) and Puerto Alegre (PA) are located on the Mazán River. Salvador (SA), Lago Yuracyacu (LY) and Urco Miraño (UM) are located on the Napo River. The main economic activities are farming, timber extraction and fishing .Malaria transmission in Maynas is perennial, with a peak between February and July . While iP. vivax according to the MINSA reports of 2017, and due to ease of sample transport to the reference laboratory installed in Iquitos city. The enrolled individuals represented a total of 358 households. From the enrolled participants, 895 individuals consented to provide a blood sample for LM and qPCR diagnosis, and 880 individuals accepted donating a plasma sample for serology purpose (In April 2018 (mid transmission season), 933 individuals were enrolled to participate in the Circles of Research on Arboviruses and Malaria (CAM) study executed in the 3 communities of Iquitos mentioned above. In this study, purposive sampling was implemented to the household members presented at the moment of survey. These communities were selected due to their high incidence of  purpose .In the district of Mazán, a representative population-based cross-sectional survey was conducted in July 2018 (end of transmission season), where 1127 individuals were enrolled to participate in the baseline survey by the Amazonia International Center of Excellence in Malaria Research (ICEMR) team . A totalA population survey and geo-referencing of households were implemented in communities from both areas, and data were collected through surveys using tablets and Open Data Kit (ODK) ,21. AxilWhole blood samples (5 mL) were collected by a venous puncture in citrate tubes. Serum was separated by centrifugation and kept at -80°C until processing. Red blood cell pellets were stored at -20°C prior to molecular diagnosis.Thick and thin blood smears were stained for 10 min with a 10% Giemsa solution. A slide was declared negative if no malaria parasite was found after examining 100 microscopy fields . Slides DNA was isolated from parasitized red blood cell pellets using the E.Z.N.A. Blood DNA Mini Kit , according to the manufacturer’s instructions. Subsequent amplification was performed by a real-time quantitative PCR (qPCR) method using PerfeCTa SYBR Green FastMix 1250  as previously described .P. vivax were measured using a Luminex platform, as described elsewhere [P. vivax has been previously validated [19), PVX_096995 , PVX_112670, PVX_097625 , PVX_097720 , PVX_087885 , PVX_094255  and KMZ83376.1d . The additionally proteins screened in this study were: PVX_090240 , PVX_110810 , PVX_000930  and PVX_082700 (merozoite surface protein 7 (MSP7), putative, PvMSP7). The complete IgG dataset generated is available on IgG antibody responses to 12 proteins of lsewhere . The panalidated  and consTo account for inter-plate variation, a standard curve was prepared using a plasma pool from hyper-immune adults from Papua New Guinea. Relative Antibody Units (RAU) or dilutions were obtained extrapolating the Median Fluorescence Intensity (MFI) in a standard curve by a 5-parameter logistic model written in R.χ2 test or Fisher’s exact test when required. Continuous covariates were compared using Mann-Whitney U or T- test when required.Demographic data was collected from the databases of the CAM study and the baseline survey of the cohort executed by the Amazonian ICEMR. Categorical covariates were compared using the P. vivax by a seropositivity classifier based on the combined antibody responses to SEM. The seropositivity classifier was a Random Forests based classification algorithm previously validated in low P. vivax transmission contexts: Thailand (n = 826), Brazil (n = 925), the Solomon Islands (n = 754), and negative controls (n = 274) [https://github.com/MWhite-InstitutPasteur/Pvivax_sero_dx.Participants were classified as seropositive  or seronegative  to n = 274) . The diaP. vivax SEM was estimated as the proportion of individuals classified as seropositive stratified by age. The seropositive rate of individuals per household was estimated to visualise the spatial distribution of seroprevalence to P. vivax. For the spatial data processing and visualisation, QGIS 2.16  was used.The proportion seropositive for P. vivax exposure (binary) and P. vivax parasitaemia (binary) by qPCR were assessed by mixed-effects logistic regression models with random intercepts per households and communities. The following potential risk factors were assessed as fixed effects: age, sex, fever, travels in the last month, outdoor occupation, education level, livestock inside dwelling, indoor spray, and housing type. Factors in final models were selected by manual backward elimination guided by the minimisation of the Akaike information criterion. Three levels were included in these models: individuals within households, households within communities and communities. Intra-class correlation (ICC) was estimated as a measure of correlation and homogeneity at each level [https://www.r-project.org/).The association of epidemiological factors with ch level . The ICCch level . Statistp < 0.001). Outdoor occupations such as logger, farmer and fisher were more common in Mazán (40.9%) than in Iquitos (8.9%) (p < 0.001). Farmer and housewife were the most common economical occupation in adults older than 18 years old in Mazán (38.1%) and in Iquitos (30.8%), respectively. Only 2.5% of participants from Iquitos and 1.2% from Mazán had fever (axillar temperature > 37.5°C) at the moment of survey (p = 0.045).Of the 933 surveyed inhabitants in the CAM study, 880 individuals from 358 households (HH) were included in the analysis Figs  and S1. P. vivax in Mazán [p = 0.004). Having livestock inside dwelling was seen in 24.6% of the HH from Iquitos and 26.6% from Mazán (p = 0.565).Crowded households  were slightly more frequently reported in Mazán than in Iquitos with no significant differences. Open houses have been associated as a high risk factor for in Mazán ,19 and iin Mazán . 16% of P. vivax prevalence by LM in Iquitos was not significantly different from Mazán . Although P. vivax prevalence by qPCR was higher in Iquitos than in Mazán ; Mazán  showed higher seroprevalence than Iquitos  (Tablep mean < 0.001). Participants from Iquitos had 1.2 times higher antibody levels to PvMSP119 than participants from Mazán (p = 0.013).P. vivax by community, we found high heterogeneity within study sites (p = 0.005) and Quistococha  (p < 0.001). Conversely, seroprevalence was higher in Santo Tomás  than Rumococha  (p < 0.001) and Quistococha  (p < 0.001).When exploring the prevalence of dy sites . Both LMdy sites . For betIn Mazán, Libertad had the highest qPCR prevalence among all the communities , followed by Gamitanacocha , Puerto Alegre , Urco Miraño , and Lago Yuracyacu . Salvador and Primero de Enero did not have any positive qPCR sample in the survey (0%). On the other hand, Gamitanacocha was the community with higher seroprevalence , followed by Libertad with 75% (138/184) of seroprevalence, Primero de Enero , Urco Miraño , Puerto Alegre , Salvador  and Lago Yuracyacu .p mean < 0.001) (p = 0.006) (Antibody levels and seropositivity rate increased with age in both study sites  Tables. < 0.001) . There w< 0.001) . This di= 0.006) . Antibod= 0.006) .Within each study area, villages had different seropositive rate-age patterns . The lowIn order to detect spatial patterns of exposure within communities, a seropositivity rate per household was estimated for both areas. The household-level spatial distribution of individuals is represented in In the peri-urban areas of Iquitos, Santo Tomás had the highest proportion of households with seropositive rate ≥ 0.75 (40%), in contrast to Rumococha (20%) and Quistococha (20%) . HoweverA more heterogeneous transmission landscape was found in the communities of Mazán, probably related to the geographical location of households. Mazán district is crossed by two rivers: Mazán River and Napo River. The highest proportions of seropositive individuals at household level were detected in communities along the basin of Mazán River: Gamitanacocha (88%), Libertad (63%) and Primero de Enero (60%). Although also located in the basin of Mazán River, only 31% of the households in Puerto Alegre had ≥ 0.75 seropositive rate. While the proportion of households with high seropositive rate in the communities of the basin of Napo River were the following: Urco Miraño (45%), Salvador (20%) and Lago Yuracyacu (4%) . The expP. vivax parasitaemia output was measured by PCR positivity. The effect of households and community levels on individual outcomes were measured by the intra-class correlation (ICC). The ICC quantifies the size of the general contextual effect, so the more relevant the context is for understanding individual differences in the outcome, the higher the ICC [Due to the multilevel data structure of this study, we implemented multilevel multi-community logistic regression models at three levels: individual, households, and communities. The exposure output was measured by seropositivity to SEM, whereas the  the ICC . The estP. vivax increased with increasing age , being male  and working outside . Having domestic animals was associated with a low risk of being seropositive . There was no association with fever symptom and seropositivity, as well with the travel history in the last month and the type of house . There was an association between being male and being seropositive to P. vivax . Having a high education level (either secondary school or higher) was a protective factor . History of travel in the past month was not associated with seropositivity. The ICC values indicated that the household variable explains considerably more variance of seropositivity to P. vivax. This possibly indicates a heterogeneous individual exposure that could be related to job- related habits rather than to community factors  identified more exposures (both current and previous) than molecular testing in two low transmission regions of the Peruvian Amazon. The measures of P. vivax seroprevalence resulted in greater statistical power for epidemiological analyses than qPCR parasite prevalence in such settings with low transmission rates. P. vivax SEM will be a key tool for serological surveillance as malaria transmission is reduced by continued control and elimination efforts in the Peruvian Amazon.Here we show that IgG antibody responses to 8 P. vivax in the last 9 months. The seroprevalence of P. vivax was higher in the riverine communities of Mazán than in the peri-urban communities of Iquitos. Seroprevalence increased with age in both sites, with a significant increase in participants older than 15 years old, although the distribution by age was more heterogeneous among communities from Mazán. The distribution of seropositive cases did not show a spatial structure in communities of Iquitos but did it in Mazán.We used a Random Forest algorithm that incorporated the combined antibody responses to the 8 SEM to classify surveyed individuals into seropositive or seronegative, i.e., recent exposure to P. vivax seropositivity in both areas, while having higher education level and livestock inside dwelling were low-risk factors in Iquitos and Mazán, respectively. Age is a factor that can be linked to i) lifetime cumulative exposure  or ii) labor exposure . Our previous work showed that antibody levels increased with the recency of infection, the number of blood-stage infections and age [P. vivax prevalence in the community of Urco Miraño [P. vivax seropositivity in Mazán only, which is in line with previous reports of the malaria epidemiology in the area [Age and being male were factors associated with high  and age . However and age . The preo Miraño . Previouo Miraño . This opo Miraño . From outhe area .The high seroprevalence in Iquitos and Mazán is consistent with recent intense exposure in both sites ,19,28. OP. vivax could have a significant impact on the control measures implemented by health authorities. For instance, in population screening studies in Mazán, only 23% of the infections were detected by LM-based active case detection, compared to qPCR [P. vivax in Iquitos and Mazán, respectively. However, our study showed that in areas that after long periods of moderate-to-high transmission rapidly goes to low transmission, antibodies remain at high levels for several years after transmission has been reduced. As a consequence, they are not well suited to measure short-term changes in transmission in areas where populations have established high levels of immunity. This could impact the use of antibodies to measure the impact of interventions [The choice of tools to detect exposure to  to qPCR . Also, o to qPCR . In low  to qPCR . In our ventions ,31. We tSome limitations of this study are the lack of information about recent malaria episodes and time of residence in the study survey to better explain the communities’ transmission patterns and the relationship with seropositvity. The differences in the timing of execution of each study could have influenced the LM and qPCR prevalence in both sites, i.e., in Iquitos, samples were collected during transmission months. In contrast, in Mazán, samples were collected after the transmission peak. The low proportion of participants sampled per household in Iquitos could have overestimated the seropositive rate per household and thus the differences between households detected by our multilevel logistic model. This sampling bias may have also affected the distribution of seropositive cases or maps of seropositivity.Overall, this study confirms that malaria transmission in the Peruvian Amazon is heterogeneous and that geographical, socio-demographic and human behaviour are important factors for the maintenance of disease burden in this region. Our findings have implications for designing appropriate interventions against malaria in the region; these novel serological markers could be deployed as a tool to target individual and community interventions, but care needs to be taken with their use in areas where transmission has been reduced rapidly from high to low transmission.S1 FigCAM: Circles of Research on Arboviruses and Malaria study, ICEMR: Amazonia International Center of Excellence in Malaria Research, HH: Households, Ind: Individuals.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigP. vivax detected by antibody responses to serological exposure markers.A) Proportion of individuals with ongoing infections detected by Light microscopy (LM). B) Proportion of individuals with ongoing infections detected by qPCR. C) Proportion of individuals exposed to (TIF)Click here for additional data file.S4 FigA) Age curve of antibody titers in Iquitos and Mazán. B) Distribution of antibody titers stratified by seropositivity given by the Random Forests based classification algorithm. Note the controls are grouped together regardless of age.(TIF)Click here for additional data file.S5 FigSeropositive rate per household was estimated as the proportion of seropositive individuals classified per number of household members participating in the study.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Malaria in pregnancy is associated with significant morbidity and mortality, and requires early diagnosis and intervention. Plasmodium falciparum is responsible for 98% of malaria cases in Zimbabwe and causes the most severe disease. Abnormal haematological parameters are a frequent finding in patients with malaria; however, they are rarely the sole presenting feature. We present the case of a 32-year-old woman in her fifth pregnancy, with a history of one previous caesarean section, who presented for caesarean section and was incidentally noted to have severe thrombocytopenia. Subsequent investigations at a tertiary institution revealed a pancytopenia with thrombocytopenia as the most prominent feature in an asymptomatic patient. The unavoidable caesarean section done under platelet cover was eventful, with severe intractable haemorrhage necessitating an emergency hysterectomy. However, the patient made a full recovery with antimalarial treatment and blood product transfusions. This case is presented to illustrate the need to consider malaria as a differential diagnosis in pregnant patients from malaria-transmitting areas who have thrombocytopenia. Previous studies have shown that thrombocytopenia can be a predictor of malaria in patients who present with fever, and a marker of disease severity, but has no utility in prognostication and follow-up. Plasmodium falciparum causes the most severe malaria, and accounts for over 98% of the cases [This case is reported according to the CARE guidelines . Estimathe cases . Pregnanhe cases ,7, owinghe cases ,3,8. Malhe cases . The cashe cases -12. The P. falciparum malaria in advanced gestation, which was only suspected and diagnosed because of severe thrombocytopenia noted when patient was being prepared for caesarean section (C/S). We present the case to illustrate the possibility of missing such a potentially fatal diagnosis, and the need for continued vigilance by attending clinicians.Malaria is a haematological disease, with anaemia and thrombocytopenia as frequent features ,14. HowePatient information: a 32-year-old woman in her fifth pregnancy with three children alive and one first trimester miscarriage was referred to our hospital, a tertiary institution, from a provincial hospital. She had a previous C/S, and had presented late at an estimated menstrual gestational age of 42 completed weeks and four days. On work-up for C/S at the referring hospital, she was noted to have a severe thrombocytopenia of 34x103/µL. The reasons for referral were the absence of blood products and appropriately qualified anaesthesiologists at that hospital. On systemic enquiry at presentation, she reported generalised body weakness in the preceding three-to-four days that she attributed to advanced pregnancy and did not bother to seek medical attention. She had no fever, chills and rigors, joint pains nor headaches, and the rest of her systemic enquiry was non-contributory. The pregnancy was booked, and she had six antenatal care (ANC) visits. She was HIV negative, and had no other chronic illnesses. No ultrasound scan (USS) was done due to financial constraints. She was on hematinic supplements from the booking visit; however, she did not receive IPT for malaria, and did not take any other medicines. In the first trimester, she was treated for slide-positive symptomatic malaria with a complete course of co-artemether (artemether/lumefantrine). She had a history of one C/S performed for fetal distress in her last pregnancy in 2015, after two vaginal deliveries and a first trimester miscarriage. She stayed with her family in Burma Valley, which is malaria-endemic.Clinical findings: she looked rather unwell, pale and dehydrated, with no jaundice or lymphadenopathy. The blood pressure was 110/56 mmHg with a heart rate of 119 beats per minute, respiratory rate of 14 breaths per minute and a temperature of 36.5 °C. Chest, cardiovascular, central nervous system and skin examinations were unremarkable, and she did not have epigastric tenderness on abdominal examination. She had a Pfannenstiel scar. The symphysis-fundal (SFH) was 38 cm, with a singleton foetus in longitudinal lie and cephalic presentation. The foetal heart rate was 132 beats per minute.Diagnostic assessment: the problems noted were the severe thrombocytopenia, one previous C/S and post-term pregnancy. Laboratory investigations revealed a pancytopenia, and numerous ring forms of P. falciparum. However, no level of parasitemia was provided. The white cell count was 2.0x103/µL, haemoglobin level was 9.8 g/dl and platelet count was 29x103/µL, with a mean corpuscular volume of 81.1 fL. The urea, electrolytes and liver enzymes were all normal with an alanine transaminase (ALT) of 13 iU/l and an aspartate transaminase (AST) of 36 iU/l. Both total and direct bilirubin were within normal limits. Bedside clotting time was prolonged at 13 minutes. Despite low normal BPs, urinalysis was done and was negative for proteins.Therapeutic intervention: diagnoses of P. falciparum malaria, one previous C/S and post-term pregnancy were made. Treatment for malaria with intravenous (IV) artesunate 2.4 mg/kg body weight once daily, ceftriaxone 1 g IV once daily, and paracetamol 1 g IV three times a day was commenced. An emergency C/S was done under platelet cover and tranexamic acid 1 g infusion, and a live baby weighing 3195 g with Apgar scores of 8/10-9/10 was delivered. However, post C/S the patient was noted to be actively bleeding per vagina, and a re-laparotomy was done, proceeding to a supra-cervical hysterectomy after she had continued bleeding despite uterotonics, traexamic acid infusion and uterine massage. The total estimated blood loss was 2500 ml, and the patient was transfused three units of packed cells, 6 units of platelets and 6 units of fresh frozen plasma.Follow-up and outcomes: post-operatively, the patient remained anaemic, with a haemoglobin level of 7 g/dl, and she was transfused two more units of packed cells. She had a good urine output of 1800 mls of clear urine in the first 24 hours, and started mobilising on the first day. The repeat malaria thin slide was negative, and she was discharged on co-artemether (artemether/lumefantrine), paracetamol and oral antibiotics on the fifth day. For further postnatal follow-up, she was referred back to the referring hospital to minimise the costs and need for travelling.Patient´s perspective: the patient was very grateful that both her and the baby were well on discharge, and was also grateful that she had been diagnosed of malaria before she had developed fatal complications.Informed consent: the patient provided signed written informed consent for case publication. However, the Medical Research Council of Zimbabwe and the Joint Research Ethics Committee do not require approvals for case reports.et al. noted 909 recurrent episodes of P. falciparum malaria out of 700 pregnant women treated with several regimens in Thailand, with 481 novel infections, and 428 recrudescent infections, confirmed by polymerase chain reaction (PCR) genotyping [We have presented a maternal near-miss case due to malaria-induced haematological dysfunction in a patient who had no overt malaria symptoms. A history of residence in an endemic area, previous treatment of malaria in current pregnancy and a severe isolated thrombocytopenia prompted investigations for malaria. Recrudescence and recurrence of malaria after treatment have been reported, regardless of treatment regimen. In a retrospective analysis, Laochan notyping . Periodsnotyping . Comparanotyping . Our patP. falciparum malaria. In some reports, the frequency is 24-94% in patients with acute malaria [et al. reported a case of a primigravida who had pancytopenia and hepatosplenomegaly without fever at 26 weeks of gestation [An unavoidable C/S was done in a patient with a previous C/S and a post-term pregnancy under adequate platelet cover, and with the administration of the antifibrinolytic tranexamic acid. It is recognised that patients with thrombocytopenia at surgery may bleed despite platelet cover, and a pro-active approach to minimise bleeding was undertaken, hence the prophylactic administration of tranexamic acid. Thrombocytopenia is well-recognised complication of  malaria . It rareestation . Due to estation .3/µL, and the condition is usually clinically inconsequential.Thrombocytopenia is the second most common haematological derangement after anaemia in pregnancy and the reported frequency is 6.6-11.2% . SeveralP. falciparum and P. vivax malaria at a hospital in South Ethiopia, thrombocytopenia and anaemia were noted in 84% and 67% of malaria patients respectively [et al. noted similar findings in another cross-sectional study in Buea, Cameroon [Evidently, malaria is a haematological disease, with frequent severe anaemia, coagulopathies, leucocyte numerical or functional changes and spleen involvement . In a crectively . An inveectively . NdamukoCameroon . They noet al. in a retrospective analysis in Ghana, observed lymphopenia (56.78%), anaemia (55.51%), thrombocytopenia (47.46%), eosinopenia (45.76%), neutropenia (29.24%), monocytosis (21.19%), and leucocytosis (17.37%) in infected patients [P. malariae (12.7%), and this may have altered the clinical picture. Nevertheless, the study adds to the pool of evidence that malaria is a haematological disease. Even studies beyond Africa corroborate these findings. Lopez-Perez et al. conducted an observational study in Colombia, Latin America in a low-malaria transmitting area and observed mild-to-moderate anaemia in 68% of their study participants, and thrombocytopenia in 41% of their study participants [P. vivax infections however, are more prevalent in this region, compared to Africa where P. falciparum accounts for the majority of the cases, hence the results may not be directly comparable [Pancytopenia, like in the case we presented, has also been observed frequently. Sakzabre patients . Howeverpenia 56.8%, anaempenia 56.8%, anaemP. falciparum and 32% P. vivax) [et al. noted that thrombocytopenia is a marker of disease severity in P. falciparum malaria, though its value in prognostication, triage and management is limited [et al. in their study noted the median time for recovery from malaria associated thrombocytopaenia to be 7 (range 2-14) days and did not differ by treatment regimen [Studies have been carried out to evaluate the predictive value of thrombocytopenia for malaria. In a longitudinal study of 228 patients who presented to hospital with fever and thrombocytopenia, 53% tested positive for malaria (68% . vivax) . In mala limited . There i regimen . HoweverP. falciparum malaria. However, attending clinicians must also rule out other causes of thrombocytopenia in pregnancy, corroborating clinical history, findings and diagnostic evaluations, as there are other causes of thrombocytopenia in pregnancy, which are also quite as fatal. Prospective studies evaluating the utility of platelet counts in patients must continue being carried to inform evidence-based medical practice.Pregnant women from malaria-transmitting areas who have thrombocytopenia must be routinely investigated for malaria. Thrombocytopenia is a predictor of malaria, and can be a marker of disease severity in patients with


Persons with submicroscopic malaria infection are a major reservoir of gametocytes that sustain malaria transmission in sub-Saharan Africa. Despite recent decreases in the national malaria burden in Kenya due to vector control interventions, malaria transmission continues to be high in western regions of the country bordering Lake Victoria. The objective of this study was to advance knowledge of the topographical, demographic and behavioral risk factors associated with submicroscopic malaria infection in the Lake Victoria basin in Kisumu County.Plasmodium infection. Persons who were PCR positive but blood smear negative were considered to harbor submicroscopic infections. Topographical, demographic and behavioral risk factors were correlated with community prevalence of submicroscopic infections.Cross-sectional community surveys for malaria infection were undertaken in three eco-epidemiologically distinct zones in Nyakach sub-County, Kisumu. Adjacent regions were topologically characterized as lakeshore, hillside and highland plateau. Surveys were conducted during the 2019 and 2020 wet and dry seasons. Finger prick blood smears and dry blood spots (DBS) on filter paper were collected from 1,777 healthy volunteers for microscopic inspection and real time-PCR (RT-PCR) diagnosis of P. falciparum, whereas RT-PCR also detected P. malariae and P. ovale mono- and co-infections. Submicroscopic infection prevalence was associated with topographical variation . The highest prevalence was observed in the lakeshore zone  followed by the hillside  and highland plateau zones . Infection prevalence varied significantly according to season . The highest prevalence was observed in residents of the lakeshore zone in the 2019 dry season  and 2020 and 2019 rainy seasons . In both the rainy and dry seasons the likelihood of submicroscopic infection was higher in the lakeshore  and hillside  than in the highland plateau zones. Residence in the lakeshore zone (p<0.0001), male sex (p = 0.025), school age (p = 0.002), and living in mud houses (p = 0.044) increased the risk of submicroscopic malaria infection. Bed net use (p = 0.112) and occupation (p = 0.116) were not associated with submicroscopic infection prevalence.Out of a total of 1,777 blood samples collected, 14.2%  were diagnosed as submicroscopic infections. Blood smear microscopy and RT-PCR, respectively, detected 3.7%  and 18%  infections. Blood smears results were exclusively positive for Topographic features of the local landscape and seasonality are major correlates of submicroscopic malaria infection in the Lake Victoria area of western Kenya. Diagnostic tests more sensitive than blood smear microscopy will allow for monitoring and targeting geographic sites where additional vector interventions are needed to reduce malaria transmission. Plasmodium infections are submicroscopic, with densities too low to be detected using standard diagnostic methods such as blood smear microscopy. Concerns have been raised that asymptomatic submicroscopic infections sustain ongoing Plasmodium transmission, particularly if microscopy is used exclusively for Plasmodium parasite detection [Kenya is currently ramping up malaria control interventions in order to reduce disease burden and eventually eliminate the disease. The primary vector control methods are long-lasting insecticide-treated nets (LLIN) and indoor residual spraying (IRS) . Despiteetection , 8.Plasmodium infection risk, information regarding variables that may contribute to submicroscopic infection throughout the year in western Kenya near the Lake Victoria basin is meager.Topography is a major determinant of malaria transmission and endemicity because it affects local hydrology and the stability and productivity of vector breeding habitats. It is also linked to the spatial distribution of malaria exposure in the human population . Local pSeasonality has also been shown to influence malaria burden, with rainfall having a positive correlation with infection prevalence in both wet and dry seasons. Declines in transmission observed in prolonged dry seasons are most likely due to the reduced numbers of vector breeding sites . The higThe study was carried out in the Nyakach Sub-County of Kisumu County in western Kenya near the shore of Lake Victoria at latitude -.333333° and longitude 34.991°. The area includes three eco-epidemiological zones: lakeshore, hillside and highland plateau. The lakeshore zone is characterized by a flat plain at an elevation of 1100-1200m above sea level. It is prone to flooding during the rainy season. The highland plateau has elevations ranging from 1450 to >1600m. The hillside zone is located at 1300-1450m, between the lakeshore and Nyabondo highland plateau. Larval habitats are unstable and permanent habitats scarce in this zone. Nyakach sub-County has an area of approximately 327 square kilometers and a population of 168,140 people living in 35,553 households at a population density of 460 people per square km .Cross-sectional community based surveys were performed in June and November in 2019 and 2020. Malaria transmission generally peaks after the long rainy season in June. The dry season in November is associated with minimal transmission in western Kenya , 19. A dA total of 1,777 finger-prick blood smears and filter paper DBS samples were collected for parasite examination via microscopy and RT-PCR, respectively. Blood samples were transported to the International Centre of Excellence for Malaria Research (ICEMR) laboratories at Tom Mboya University College located in Homa Bay, Kenya for further analysis.Plasmodium parasites by microscopic inspection. The thick and thin blood films were stained with 10% Giemsa. Two expert microscopists examined the slides in immersed oil to identify the parasite species. If at least one asexual blood-stage malaria parasite was found on a slide, it was considered positive. Discrepancies in slide readings were confirmed by a third experienced microscopist for quality control.Finger prick blood smears were examined for Plasmodium species were used to target 18S ribosomal RNA [® qPCR ToughMix™, Low ROX™ Master mix (2X), 0.4 μL each of the forward and reverse species-specific primers (10 μM), 0.5 μL of the species-specific probe, 0.1 μL of double-distilled water and 2 μL of parasite DNA were used in the PCR reaction. The following RT-PCR cycling conditions were used: 50°C for 2 minutes,  for 45 cycles . Nested PCR to detect P. vivax DNA was not performed since recent studies of finger prick blood samples from ~1,000 local residents failed to detect a single P. vivax infection. Submicroscopic malaria infection was defined as an infection detected by RT-PCR but not by microscopy.The Chelex resin (Chelex-100) saponin method was used with minor modifications . Primersomal RNA . Six μL The study received ethical approval from Maseno University’s Ethics Review Committee (reference number: MSU/DRPI/MUERC/00778/19) and the University of California, Irvine’s Institutional Review Board (HS# 2017–3512). Administrative approval to conduct the study was obtained from the Kisumu County Director of Health and the Nyakach Deputy County Commissioner. The survey was open to all community residents who were willing to participate in the study. Residents who declined to participate in the study or changed their willingness to participate at any time were excluded from the study analysis. All study participants provided written informed consent. Minors provided assent with informed consent from parents or guardians.Plasmodium species and seasonality were performed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. The adjusted agreement between microscopy and RT-PCR results were measured using Cohen’s kappa statistic, sensitivity, specificity, positive predicted value, negative predicted value, and diagnostic accuracy. Univariate binary logistic regression and multivariate mixed effect binary logistic regression analyses were used to identify the risk factors associated with submicroscopic infection prevalence. In the univariate analysis, all risk factors were tested, and only those with p≤0.50 were chosen and included in the multivariate analysis. In the multivariate analysis, variables with p<0.05 were considered significant risk factors. Topography, gender, age, bed net usage, wall type, occupational, education level, household population size, bednet type, symptoms, and seasonality were among the variables investigated. Chi-square test and odds ratio (OR) with p values <0.05 were considered significant.IBM SPSS Software Version 21.0 was used to analyze the data. The Chi-square test was used to assess the significance association in malaria infection prevalence across seasons and topography. Multiple comparisons between Cross-sectional community based surveys were conducted in the June 2019 and June 2020 wet seasons  and the November 2019 and November 2020 dry seasons .p = 0.667) (p = 0.039), equal to and older than 15 years (p<0.0001), and occupied a household that included three or less residents (p<0.0001). Reported use of long lasting insecticide treated bed nets was 95.4% at the time of study enrollment. There were minor changes in bed net use according to season and year, but self-reported bed net use was greater than 90% in all four cross-sectional surveys  . Study p surveys .Plasmodium parasitemia detected by microscopic inspection of blood smears and RT-PCR, respectively, varied from 2.3% to 4.8% (p = 0.2355) and 12.8% to 24.6% (p<0.0001) over the two rainy and dry seasons. Blood smear microscopy exclusively identified P. falciparum infections, whereas RT-PCR identified P. malariae and P. ovale mono-infections and co-infections with P. falciparum. Plasmodium malariae and P. ovale were detected in less than one percent of blood samples  with high infection in the dry season of November 2019 , followed by the rainy season of June 2020 , then the rainy season of June 2019 , and the dry season of November 2020  , with microscopy and RT-PCR detecting 3.7%  and 18%  infections, respectively . The ove samples . Submicrn = 475) .P. falciparum infection prevalence showed significant topographical variation , with highest value in the lakeshore zone (20.6%) followed by the hillside zone (13.6%) and highland plateau zone (9.1%) (Submicroscopic e (9.1%) . Furthere (9.1%) . The hige (9.1%) .Residency in the lakeshore zone, age ≤4 years and 5-<15 years, male sex, household wall type consisting of mud and cement, subjective fever and elevated body temperature recorded at the time finger prick blood sample was obtained, and the 2019 dry season were associated with an increased risk of submicroscopic malaria infection by univariate analysis . All risp = 0.002). Furthermore, school-aged children (5-<15 years old) in the hillside  and highland plateau zones  had a lower risk of submicroscopic infection than those in the lakeshore zone (p = 0.042), but the hillside (p = 0.102) and highland plateau (p = 0.812) zones had no effect on gender likelihood of submicroscopic infection (p = 0.018) and highland plateau  zones compared to the lakeshore zone. Males in the hillside  and highland plateau  zones were less likely to be infected than males in the lakeshore zones (p = 0.022), there was no significant difference in the likelihood of infection between bednet users and non-users in the lakeshores (p = 0.523) and hill side zones (p = 0.958) (p = 0.002) and highland plateau zones , than in the lake zone. Residency in mud wall houses on the hillside and highland plateau had a lower risk of submicroscopic infection than those who lived in lakeshore zones, with risk factors of 0.48  and 0.38 , respectively (p = 0.0.613), hillside (p = 0.125) and the highland plateau (p = 0.710) (p = 0.002).Children under the age of 5 years old in the lakeshore zone had a lower risk of submicroscopic infection than residents over the age of 15 years old  . The risectively . The lik= 0.710) . The raip = 0.009)  .p<0.0001). Topography, gender, wall type, occupation, symptoms, and seasonality, on the other hand, were not associated with submicroscopic infection in the malaria infection  of the malaria infections. Among the RT-PCR positives, only age group was significantly associated with submicroscopic infection  with the overall prevalence by microscopy and RT-PCR at 3.7%  and 18%  despite the bed net coverage of 95%. The infection showed topographical and seasonal variations with lower altitudes areas and the wet seasons having the highest carriage of infection. In addition, there was a strong association with carriage of submicroscopic infection if one lived in the lowland lake zone, or was a male, or was of school going age and lived in a mud walled house. Bed net ownership and resident’s economic activity did not influence the carriage of submicroscopic infection.p = 0.007). As rivers and streams run along the valley bottoms and the majority of breeding habitats are confined to the valley bottoms as hillside gradients provide efficient drainage, the hillside zone has unstable larval habits. As a result, heterogeneous distribution of larval breeding habitats is likely to affect adult vector distribution and heterogeneity exposure of the human population to malaria [The prevalence of submicroscopic infection varied along the altitudinal transect, according to the current study. When compared to the highland plateau zone, the lakeshore zone, which is adjacent to Lake Victoria, had 3-fold higher likelihood of submicroscopic infection. The findings are consistent with other studies that show a higher prevalence of malaria in lowland areas than in highland areas –13. The  malaria , 14. In  malaria . The hil malaria , 14, 28. malaria . Studies malaria , 30, howMalaria is expected to be present throughout the year in endemic areas of western Kenya, with seasonal peaks during the rainy season , 31–34. P. falciparum infection and disease transmission, necessitating the scaling up of intervention strategies [p = 0.115). The findings contradict previous research that found long-lasting insecticide-treated nets to be especially important in reducing malaria incidence [p = 0.022), those who did not use bed nets in the lake and hillside zones had equal chances of infection. Non-compliance with LLINs is linked to an increased risk of malaria [p = 0.115), which could be due to behavioral changes in malaria vectors, with outdoor transmission occurring earlier in the evenings and mornings when people are not protected by LLIN [Existing control and treatment tools have not suppressed mosquito-borne rategies . Despitencidence –42. Furt by LLIN –45, and  by LLIN , though Plasmodium exposure as a result of inadequate bed net use [P. falciparum parasite, and this is related to age [Plasmodium infection, as children on the highland plateau may have a slower ability to suppress parasite density than children in lowland areas with well-established transmission [In the current study, school-aged children were 1.5 times more likely than ≥15 year olds to have sub-patent infection. The current study supported previous research that found that school-going children had an increased risk of submicroscopic infection, acting as reservoirs of infectious gametocytes and thus maintaining infection in the community , 47. Thi net use . Individd to age . Adults smission , 47. Whip = 0.025). According to the current study, doing business during the dry season increased the risk of developing submicroscopic infection by 2.68 times. This could be because of increased exposure to infection risk as a result of favorable weather, as opposed to business disruption caused by rain, which caused the business to close early. The main economic activity in Lakeshore zone is fishing and reed cutting for mats, which causes residents to stay close to water for longer periods of time, exposing themselves to mosquito breeding sites, resulting in higher biting rates and high infection levels. The main economic activity in the highland plateau is farming and brick-making. The brick-making sites have pits that, when filled with water, provide potential breeding grounds for mosquitoes. However, further investigation revealed that the type of occupation had no effect on the likelihood of developing submicroscopic infection across the topography.Gender bias in infections could be attributed to socioeconomic differences that kept them awake late at night and early in the morning, adult males engage in nocturnal outdoor gatherings without the protection of the IRS and treated nets, exposing themselves to mosquito bites , 49. ThiThe current study found that socio-demographic factors such as housing type was linked to submicroscopic infection. The characteristics of the house have an impact on submicroscopic infection. The current study discovered that houses constructed with mud, or mud and cement increased the risk of submicroscopic infection, which is consistent with the findings of other studies , 25, 26.P. falciparum infections, whereas RT-PCR identified P. malariae and P. ovale mono-infections and co-infections with P. falciparum. The low sensitivity of sensitivity of microscopy has been reported in western Kenya [The current study blood smear microscopy exclusively identified rn Kenya . The rourn Kenya  and in srn Kenya –59. The A substantial proportion of submicroscopic malaria carriers were found in the population living along lakeshores and in mud houses, as well as among males and school-aged children. However, bed net use and population occupation had no effect on submicroscopic infection. To maintain progress toward parasite reservoir elimination and case malaria management, strategies for identifying malaria transmission determinants within sub-counties should be developed to guide the formulation of targeted interventions in different eco-epidemiological settings.S1 TableDependent Variable: sub-miscroscopic. Tukey HSD. Based on observed means. The error term is Mean Square (Error) = .120.*. The mean difference is significant at the 0.05 level.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.