Malaria is a major public health problem in sub-Saharan Africa, and children are especially vulnerable. In 2019, an estimated 409,000 people died of malaria, most  were young children and 94% of the cases and deaths were in Africa. Prior studies in Ethiopia focused on the adult population and high transmission areas. Hence, this study aimed to determine the prevalence and associated factors of malaria in children under five years in low transmission areas.A facility-based cross-sectional study was conducted among 585 under-five children who attended public health facilities in the Wogera district from September to October, 2017. Health facilities were selected by stratified cluster sampling, and systematic random sampling was held to select study participants from the selected facilities. Multivariable logistic regression was used to identify correlates of malaria.Plasmodium species were P. falciparum 33 (65%) and P. vivax 18 (35%). Regularly sleeping under long-lasting insecticide treated nets (LLIN) was associated with decreased odds of malaria , and an increased odds of malaria was observed among children who live in households with stagnant water in the compound  and children who stay outdoors during the night .Of 585 children who provided blood samples, 51 (8.7%) had malaria. The predominant The prevalence of malaria in the study population was high. Environmental and behavioral factors related to LLIN use remain potential determinants of malaria. Continued public health interventions targeting proper utilization of bed nets, drainage of stagnant water, and improved public awareness about reducing the risk of insect bites have the potential to minimize the prevalence of malaria and improve the health of children. Plasmodium species are responsible for malaria, only a few of them cause most infections.In sub-Saharan Africa, infectious diseases remain the primary public health threat . MalariaPlasmodium falciparum accounted for 99.7% of estimated malaria cases in the World Health Organization (WHO) African Region, 50% in the WHO South-East Asia Region, 71% in the Eastern Mediterranean, and 65% in the Western Pacific. P. vivax is the predominant parasite in the WHO Region of the Americas, representing 75% of malaria cases [P. falciparum and P. vivax are the major malaria parasites [In 2018, ia cases . In Ethiarasites ,5.Children under five years are one of the most vulnerable groups affected by malaria. Severe anemia, hypoglycemia and cerebral malaria are features of severe malaria more commonly seen in children than in adults . ChildreAccording to the WHO 2016 report, the global prevalence of malaria among under-five children was 16% . In the The Ethiopian government developed a National Malaria Control Strategy (NMSP) for the years 2017–2020 that was envisioned to be aligned with the country’s four-year health sector transformation plan (HSTP) 2015/16–2019/20. The proposed goals for the 2017–2020 NMSP include: maintaining near-zero malaria deaths  by 2020, reducing malaria cases by 40% by 2020, and eliminating malaria from Ethiopia by 2030 [Even though malaria is one of the leading causes of under-five morbidity and mortality in Ethiopia, prior studies focused only on the adult population and were done in malaria-endemic transmission areas. Nevertheless, it is a potential threat in non-endemic regions . There hkebeles , of which 15 kebeles (35.7%) are malaria-endemic. In the Wogera district, there was 1 hospital, 10 health centers, 42 health posts, and 4 private health institutions. It shares borders with Dabat and Tach-Armacho in the North, Misrak-Belesa and Janamora in the West, Merab Belesa in the South and Lay-Armacho in the East [A health facility-based cross-sectional study was conducted from September to October, 2017 in the Wogera district. Wogera is one of the districts in the North Gondar zone. It has an average altitude of greater than 2050 meters above sea level, with an estimated total population of 274,384, of which 37,152 (13.5%) are children under five years old. The district has 42 rural and one city the East . AccordiAll children whose age was five years or below visiting the selected health facilities during the study period were the source population.The calculated sample size was 266 using a single population proportion formula as well as a power approach using a double proportion formula based on previous studies . Adding First, we stratified the health facilities as malaria-endemic and non-endemic based on their altitude. Then, we randomly selected five health centers  and one hospital  from the non-endemic clusters by using a lottery method. The calculated 585 sample size was proportionally allocated to the selected health facilities. Finally, a systematic random sampling technique was used to reach under-five clients who attended the selected health facilities.A structured questionnaire was used for data collection. The tool contained socio-demographic, environmental, and malaria prevention related questions. The questionnaire was initially developed in English and translated into Amharic for data collection. A face-to-face interview of the parents/guardians of the under-five children was conducted to collect the data.Plasmodium species. A positive result was defined as the presence of one or more asexual stages  of plasmodium [After the interview was completed, following the Federal Democratic Republic of Ethiopia Ministry of Health National Malaria Guidelines, blood was taken from a finger prick to prepare thick and thin blood film smears . Using aasmodium .Six laboratory technicians  and two supervisors from the district health office were trained for two days by the investigators. Each filled questionnaire was checked thoroughly for completeness and consistency, and the necessary feedback was given to data collectors. Recruitment was preceded by obtaining informed written consent from parents or caregivers of the children. To assure the quality of the microscopic examinations, all positive and randomly selected five percent of the negative slides were checked blindly by another experienced medical laboratory technologist.Bed net utilization: was self-reported ownership and regular use of bed nets. A 15-day recall period was used to measure whether each child regularly slept under long lasting insecticide treated nets (LLIN) or not.Malaria: was defined as a positive thin or thick blood film for the Plasmodium parasite.After data collection, data were entered using Epi info version 7 and then exported to SPSS version 20 for analysis. The correlates of malaria were identified using bivariate and multivariate logistic regression models. Variables which had a P-value of <0.2 in the bivariable regression were included in the multivariable logistic regression analysis. A P-value <0.05 was considered to determine statistical significance. Finally, adjusted odds ratios (AOR) with a 95% confidence interval (CI) were used to determine the strength of association of variables.Ethical approval was obtained from the ethical review committee of the Institute of Public Health, College of Medicine and Health Science, University of Gondar, Ethiopia. Permission was gained from the Amhara Regional Health Bureau, North Gondar health department, and Wogera health office. The caregivers were given detailed explanations about the study’s objectives, procedures, and potential risks and benefits, and written consent was obtained following that. The interview of each study participant took place in a separate room after the children gave blood samples. Appropriate treatment was given to children who tested positive.In this study, 585 children from five health centers and one district hospital participated: Gedebgie health center (HC) 178 (30.4%), Ambagiorgis HC 114 (19.5%), Tirgosgia HC 111 (19%), Selarie HC 98 (16.8%), Ambagiorgis hospital 37 (6.3%) and Chichiki HC 47 (8%). Three hundred twenty-three (55.2%) were males and 218 (37.3%) were below 12 months. About 370 (63%) of the respondents live in rural areas, and 305 (54%) of the caregivers can not read and write .Only 131 (22.4%) of the respondents had LLIN. Of the respondents who possessed LLIN, 90% of respondents reported that their children had regularly slept under LLIN in the last 15 days .In this study, the prevalence of malaria by microscopy among under-five children was 8.7% (51). There was a considerable variation in the prevalence rate between the health facilities, ranging from 0% at Wogera hospital to 21% at Selarie health center .Both bivariable and multivariable binary logistic regression analyses were done to identify the determinants of malaria infection. In bivariate analysis, factors with a P-value of <0.2 were: place of residence, stagnant water around the home, staying outside during the night, possession of an LLIN and regularly sleeping under an LLIN for the last 2 weeks. However, place of residence, sex of the child, age of the child, age of the mother/guardian, educational status of the mother/guardian, presence of radio/television, child having a regular sleeping area, construction material of the house and incidence of IRS within six months had a P-value of >0.2 in the bivariate analysis and were not included in the final model.In the final adjusted model, children who stayed outside at night had 5.5 times higher odds of malaria infection than children who did not stay outside at night . Children who regularly slept under a LLIN had 92% lower odds of infection than those who did not sleep regularly . Children who lived in households with close to stagnant water had—4 times higher odds of malaria infection than children who did not live in those homes with nearby stagnant water  .In this study, we estimated the prevalence of malaria among under-five children in the low-risk area and its determinant factors, and the results showed that the malaria prevalence in under-five children was 8.7%, which is in line with the study conducted in Dilla, Southern Ethiopia, where the prevalence of malaria in under-five children was identified to be 7.1%  and a stThis finding is much higher when compared to the national malaria indicator survey in 2015 that identified a prevalence of 0.6% among under-five children . This coPlasmodium species, with the predominant occurrence of P. falciparum infections at 65% over P. vivax at 35%. This estimate is approximately similar to the study conducted by the Carter Center in Amhara, Oromia, and Southern Ethiopia, where P. falciparum accounted for 56.5% and P. vivax for 43.5% [P. falciparum accounted for 15.6% of the participants, which was threefold higher than P. vivax in the seven-year trend [P. falciparum = 41.2%, P. vivax = 57.1 and Mixed = 1.8%) [P. falciparum = 25.5%, P. vivax = 71.7% and Mixed = 2.8%) [P. falciparum = 26.8%, P. vivax = 62.5%, and Mixed = 10.7%) [In Ethiopia, there is spatial and temporal variability in the occurrence of malaria. The current findings also demonstrated similar spatial variations in the proportion of or 43.5% , and a 7ar trend . However = 1.8%) ; Hadiya  = 2.8%)  and DillSleeping under LLIN for the last two weeks was found to be protective against malaria. This evidence is supported by other similar studies conducted in East Shewa , Amhara,In this study, malaria was highly prevalent among children living in households with stagnant water in the compound compared to their counterparts. This is consistent with a facility-based cross-sectional study conducted in a low transmission area of the Hadiya zone, south Ethiopia . This isAs a limitation of this study, since it is a cross-sectional study, it only captures the point prevalence and can not account for seasonal trends in transmission. All surveys are self-report with no confirmation of bed net ownership or use. RDTs with PCR confirmed were not conducted, nor are there details on the life stages of detected parasites observed–gametocytemia, parasitemia.The prevalence of malaria in under-five children attending health care facilities in Wogera district was high. Regularly sleeping under a bed net, staying outside during the night, and stagnant water around the household were the main correlates of malaria. Focusing on LLIN distribution, environmental management, and changing attitudes towards malaria prevention and control through health education would help minimize the burden of malaria.


Control efforts in Zanzibar reduced the burden of malaria substantially from 2000 to 2015, but re-emergence of falciparum malaria has been observed lately. This study evaluated the prevalence of malaria and performance of routine diagnostic tests among hospitalized fever patients in a 1.5 years period in 2015 and 2016.From March 2015 to October 2016, paediatric and adult patients hospitalized with acute undifferentiated fever at Mnazi Mmoja Hospital, Zanzibar were included. The malaria prevalence, and performance of rapid diagnostic test (RDT) and microscopy, were assessed using polymerase chain reaction (PCR) as gold standard.Plasmodium falciparum infection, except for one case of Plasmodium ovale. Ten malaria patients had no history of visiting mainland Tanzania. The RDT had a sensitivity of 64% (36/56) and a specificity of 98% (561/575), and microscopy had a sensitivity of 50% (18/36) and  a specificity of 99% (251/254), compared to PCR. The malaria parasitaemia was lower in patients with false negative results on RDT  and microscopy  compared to those with true positive RDT  and microscopy .The malaria prevalence was 9% (63/731). Children under 5 years old had lower malaria prevalence  than older children  and persons aged 16 to 30 years , but not different from persons over 30 years old . All cases had The study emphasizes that malaria was a frequent cause of febrile illness in hospitalized patients in Zanzibar in the years 2015-2016, particularly among school age children and young adults. We found evidence of autochthonous malaria transmission in Zanzibar. Compared to PCR, both RDT and microscopy had low sensitivity, and false negative results were associated with low parasitaemia. While low parasitaemia identified only by PCR in a semi-immune individual could be coincidental and without clinical relevance, clinicians should be aware of the risk of false negative results on routine tests. Successful control efforts reduced the burden of malaria in Zanzibar substantially from 2000 to 2015 . In the Commercially available malaria rapid diagnostic tests (RDTs) differ widely in sensitivity and specificity , and accThe main objective of this study was to evaluate the prevalence of malaria identified by PCR, and the performance of the routine tests RDT and microscopy, in febrile patients admitted to Mnazi Mmoja Hospital (MMH), Zanzibar.From 17th March 2015 to 4th October 2016, we consecutively enrolled patients with acute undifferentiated febrile illness admitted to the Department of Internal Medicine and the Department of Paediatrics at MMH. With 544 beds, this hospital in Zanzibar city is the referral hospital for the 1.3 million population of the Zanzibar Archipelago . InclusiBlood for on-site RDT and microscopy, and blood in EDTA tubes was obtained, the latter stored at − 20 °C and shipped on dry ice to Norway for malaria-PCR to be done later. Malaria microscopy was performed if requested by attending clinician, while PCR and RDT was performed on all patients for the sake of the study.PCR was defined as gold standard for assessment of prevalence and for evaluation of performance of routine diagnostic tests.For microscopy, a 10% Giemsa solution was used to stain both thick and thin blood films, in accordance with hospital procedures. The RDT First Response Malaria Ag pLDH/HRP2 Combo Card Test  was used until 20.08.2016. At this time, for the remaining 6 weeks, it was replaced by CareStartTM Malaria HRP2/pLDH (Pf/PAN) Combo  due to stock-out. 96% (685/714) of the patients were tested with the first RDT.Plasmodium DNA was assessed applying a genus-specific PCR, targeting cytochrome b (cytb) on the mitochondrial genome, and quantitative analysis (q-PCR) was performed using a customized plasmid, as previously described [cytb target exists in about 20–160 copies depending on the different development stages. One mitochondrion harbours about 20 copies of the Plasmodium genome. It is reported that early ring stage parasites have one mitochondrion, while mature gametocytes have up to eight fold higher quantity of the mitochondrial genome (about 80–160 copies) [Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Previously published primers [P. falciparum, 100 nM for P. vivax, 300 nM for P. ovale, and 200 nM for P. malariae. The species-specific amplifications were performed using the following cycling parameters: Step 1, 50 °C for 2 min; step 2, 95 °C for 10 min; step 3, denaturation at 95 °C for 15 s and step 4, annealing at 60 °C for 1 min; steps 3–4 repeated 40 times. All reaction mixtures, both genus-/ and species-specific, contained 2 µL template (DNA), and 12.5 µL SYBR Select Master Mix , at a total volume of 25 µL. To identify species in samples not detected by species-specific PCR, relevant genus-specific positive PCR-products were sequenced in one direction applying primer PgMt19 F3, sequences run by BLAST, and specific polymorphisms confirmed, as previously described [DNA was extracted from 500 µL whole blood using MagNA Pure 96 DNA and Viral NA Large Volume Kit . Presence of escribed . Parasit copies) , 13. Due primers , 15 wereescribed .Dichotomous variables were assessed by Chi-square test, and by binomial logistic regression for factors with multiple levels (i.e. age groups). Continuous variables such as age and level of parasitaemia, were assessed by pairwise Wilcoxon rank-sum test for two groups and by Kruskal Wallis test for multiple groups. Analyses were performed in R version 4.1.2, Rstudio version 2021.09.1  . Among 1044 patients fulfilling the inclusion criteria, we excluded 207 neonates < 15 days, 17 patients from whom we could not obtain blood for testing and 89 patients lacking a result for PCR, resulting in a study population of 731 . All positive cases had P. falciparum infection, except for one, who had P. ovale identified by sequencing . Malaria RDT was performed in 631 patients, of whom 8% (50/631) had positive test results. Compared to PCR, the RDT had a sensitivity of 64% (36/56), a specificity of 98% (561/575), a positive predictive value (PPV) of 72% (36/50) and a negative predictive value (NPV) of 97% (561/581)  Fig. . The med Fig. . T3 copies/µL, IQR 8 × 102 – 7 × 104) than the true positive ones . Similarly, patients with false negative RDT had significantly lower parasitaemia  than the true positive ones  in males and 7% (22/310) in females, however, this difference was not statistically significant . The prevalence of malaria among children under 5 years old  was significantly lower compared to children aged 5 to 15 years  and young adults aged 16 to 30 years , but not different from that in people over 30 years old . Monthly variations in malaria prevalence are shown in Fig. Travel history was recorded for 33% (243/731). Malaria patients were more likely to have travelled to mainland Tanzania within the past six months , than those testing negative . Ten of the 19 malaria patients with known travel history had not visited the mainland, four of these had negative RDT. However, all ten cases of presumed autochthonous malaria were positive on PCR with a median parasitaemia of 5.0 × 10A malaria prevalence of 9% was identified by PCR in this study on patients hospitalized for acute febrile illness in Zanzibar in 2015–2016. This is higher than in a survey in 2009 to 2010 at three hospitals in Pemba, the second largest island of the Zanzibar archipelago, where only 1% of febrile patients were positive for malaria with RDT and/or microscopy .There are no previous PCR-based malaria prevalence studies in hospitalized patients in Zanzibar. Previous community-based studies in Zanzibar reported a prevalence below 3% up to 2015, including PCR-based studies –20. In 2In line with that publication , the preAnopheles gambiae, and increasing resistance of vectors to pyrethroid [Malaria was strongly associated with travel to mainland Tanzania within the past six months. This is also shown in recent molecular studies substantiating malaria import from the mainland , 22. Howrethroid , re-intrThe finding of rising malaria prevalence shortly after rainy periods is in line with the study from Zanzibar  and numeThe present study indicates that malaria had resurged as an important cause of febrile illness in Zanzibar by 2015. While fifteen years of comprehensive malaria control greatly reduced malaria incidence in the archipelago, it may also have rendered school age children and young adults with less immunity and increased the susceptibility to malaria. The finding of higher malaria prevalence in these age groups underlines the risk of severe malaria in a non-immune population.P. falciparum parasites per µL was scored 85% and 90% for the tests used in the present study  Combo Test) [The RDT showed a slightly poorer performance compared to PCR than reported previously from Zanzibar . The lowbo Test) . In compbo Test)  had a sebo Test) . Howeverbo Test)  could, abo Test) . In the bo Test) .The superior sensitivity of PCR compared to microscopy is well known , and mayWith its high sensitivity, PCR may be useful in malaria surveillance, including reactive case detection in elimination programs as shown in recent publications from Zanzibar , 32. In The study emphasizes the importance of malaria as a cause of febrile illness in patients admitted to hospital in Zanzibar, and confirms autochthonous malaria-transmission in Zanzibar in the years 2015 and 2016. The higher malaria prevalence in school age children and young adults could partly be related to waning immunity during the last decades of strict malaria control, in addition to factors such as behaviour and mosquito exposure. The study shows that currently used routine diagnostics may miss up to one-third of malaria positive patients in Zanzibar. Low sensitivities of routine diagnostic tests were related to poor test performance in patients with low parasitaemia.


Plasmodium falciparum malaria cases from travelers to the UK and the Republic of Ireland (RoI).Our objective was to systematically investigate false-negative histidine-rich protein 2 rapid diagnostic tests (HRP2-RDT) in imported in vitro approaches.Five imported malaria cases in travellers returning to the UK and RoI from East Africa were reported to the PHE Malaria Reference Laboratory as negative according to histidine-rich protein (HRP2)-RDT. The cases were systematically investigated using microscopic, RDT, molecular, genomic, and in P. falciparum. Further analysis revealed that the genes encoding HRP2 and HRP3 were deleted in three of the five cases. Whole-genome sequencing in one of these isolates confirmed deletions in P. falciparum chromosomes 8 and 13. Our study produced evidence that the fourth case, which had high parasitemia at clinical presentation, was a rare example of antigen saturation (‘prozone-like effect’), leading to a false negative in the HRP2-RDT, while the fifth case was due to low parasitemia.In each case, HRP2-RDT was negative, whereas microscopy confirmed the presence of P. falciparum are concerning. Our findings emphasise the necessity of supporting the interpretation of RDT results with microscopy, in conjunction with clinical observations, and sets out a systematic approach to identifying parasites carrying pfhrp2 and pfhrp3 deletions.False-negative HRP2-RDT results with  Plasmodium falciparum histidine-rich proteins  are the most widely used RDTs in endemic countries. In non-endemic countries, these are typically used for initial diagnostic screening and/or to confirm the findings of blood film microscopy, which is considered the gold-standard test. The availability of reference laboratory confirmatory diagnosis by microscopy or PCR, in addition to RDT, is strongly recommended  are point-of-care immunochromatographic lateral flow assays which detect malaria antigens in blood from patients with suspected malaria. RDTs play a key role in the diagnosis and appropriate timely treatment of malaria throughout malaria-endemic regions . Formatsommended . This isantigens . This phantigens .P. falciparum parasites that fail to express the histidine-rich antigens are known to carry deletions of the pfhrp2 locus on chromosome 8 (gene ID: PF3D7_0831800) and/or the pfhrp3 locus on chromosome 13 (gene ID: PF3D7_1372200). Parasites with one or both loci deleted have been identified in the Americas, Africa, and some parts of Asia  between 2019 and 2021. We also report the successful culture adaptation of one pfhrp2/3-deleted parasite isolate of Sudanese origin.We have previously identified nd Sudan . In the Blood samples and fixed blood films from suspected or confirmed malaria patients are submitted by laboratories to the PHE Malaria Reference Laboratory (MRL) at the London School of Hygiene and Tropical Medicine (LSHTM) for confirmatory diagnosis. Films are examined by microscopy, as previously described , and molpfhrp2 and pfhrp3 are suspected when P. falciparum parasites are identified by microscopy or when P. falciparum DNA is confirmed using molecular analysis, and the sending laboratory has made an incorrect diagnosis based on RDT findings alone, such as reporting a non-falciparum malaria infection as the result.Deletions and the presence of genetic polymorphisms located in Plasmodium species ; Binax-NOW Malaria  and OptiMAL-IT .Where pfhrp2 and pfhrp3 as well as parasite reference and human house-keeping genes. Details of the genes and components of the qPCR have previously been described (DNA was purified by a robotic DNA extraction system (Qiagen) following the manufacturer’s instruction, as previously described . Moleculescribed .pfhrp2/3 deletion and determine the extent of chromosome deletions, whole-genome sequencing (WGS) was performed using a previously described selected whole genome amplification (sWGA) method (To confirm the ) method . The met) method . All seqP. falciparum isolates to in vitro culture are detailed in the supplementary material.The methods for whole-genome sequence data analysis and adaptation of Ethical approval for the samples from MRL patients was obtained from the NHS England Research Ethics Committee (18/LO/0738). Ethical approval for the laboratory work involving MRL samples was obtained from LSHTM Ethical Review Committee (#14710).In March 2019, a 72-year-old Sudanese female presented to the emergency department at a hospital in England with a 1-day history of symptoms suggestive of malaria. The patient had recently returned from visiting family in Sudan.P. vivax.Blood films examined by the sending laboratory suggested malaria parasites which, following testing by Carestart RDT, were found to be positive for LDH antigen (pan-genus) only. The sample was referred to the MRL at LSHTM for confirmation of In September 2019, a 17-year-old British male of Somali heritage presented to the emergency department at a hospital in London with a 10-day history of symptoms suggestive of malaria, having returned from a 2-year stay in Uganda the day before clinical presentation. The patient had started feeling unwell in Uganda, presented to hospital, and been treated with antibiotics. He reported not receiving antimalarial treatment because his malaria diagnostic test was negative.P. vivax was made after testing by Binax RDT, which was found to be positive for aldolase antigen (pan-genus) only. However, following microscopy, this diagnosis was revised to severe P. falciparum with > 20% parasitemia, and the patient was admitted to the intensive care unit, where a course of appropriate treatment was started. The hospital laboratory performed a Carestart RDT, which, despite the presence of microscopy-confirmed P. falciparum hyperparasitemia, generated only a weak positive signal for PfHRP2, but was positive for pan-LDH antigen. Blood films and an aliquot of EDTA blood were referred to the MRL for confirmation of species and investigation of inconsistency in RDT results. Biochemical data indicated that the patient had multiple markers of severe malaria (data not shown).A provisional diagnosis of P. falciparum were reported despite a negative result using Binax RDT. The sample was sent to the Hospital for Tropical Diseases (HTD) in London for confirmation, because it operates a 24-h out-of-hours malaria diagnosis service. The sample was initially reported as P. malariae following on-call microscopy together with a Carestart RDT result negative for the P. falciparum HRP2/3 antigens, but subsequent examination of the blood-film by HTD microscopists on the next working day reverted the previous diagnosis to P. falciparum infection and suspected false-negative RDT. The sample was sent to the MRL for molecular confirmation.In January 2020, a 28-year-old male returned to the UK from a 1-week trip to Ethiopia to visit friends and family. He presented to the emergency department at a hospital in London 3 days after arrival, reporting a 5-day history of symptoms consistent with malaria. Scanty trophozoites of In March 2020, a 55-year-old male returned to the RoI following a 3-week trip to Sudan, where he had been visiting friends and family. He reported presenting for medical care in Sudan, with symptoms suggestive of malaria, 6 days prior to his return to RoI. Tests for malaria were reported to be negative. The patient then presented to the emergency department at a hospital in the RoI 3 weeks after arrival, a delay likely to have been caused by the rapidly emerging SARS-CoV-2 pandemic. Malaria parasites were seen on blood films and a Carestart RDT was positive for pLDH (pan-genus) antigen only, indicating a non-falciparum infection.P. falciparum was diagnosed by microscopy and confirmed by molecular tests. RDTs were performed, confirming the sending laboratory’s findings that the sample was negative for the HRP2/3 antigens. The sample was received within 72 h of being taken from the patient, and an aliquot was successfully established in long-term culture, designated as the parasite line HL2004.Samples from the patient were received by the MRL for confirmation; In January 2021, a 51-year-old female returned to the UK from 6-week stay in Zambia where she has been visiting family. The patient presented to a hospital in England in February and was diagnosed microscopically with malaria, species unknown, with scant parasites seen. A Carestart RDT was negative. Slides and an EDTA blood sample were sent to the MRL for confirmation and species diagnosis.P. falciparum was diagnosed in the MRL by microscopy: early and late trophozoites with Maurer’s clefts were seen. The sample was tested with Carestart, OptiMAL, and Binax RDTs and all three were negative for Plasmodium antigens, while Alethia™ Malaria LAMP (Meridian Bioscience) was positive. The qPCR indicated that the infection below 200 p/μL. An aliquot was successfully established in long-term culture, designated as the parasite line HL2005.P. falciparum, inconsistencies in RDT results reported by the sending laboratory were also found by the MRL. These included weak or negative results obtained for HRP2/3 antigens detection by Carestart or Binax test kits, despite positive results for the optimal PfLDH antigen. We therefore suspected possible deletions of the respective genes in all five cases, and proceeded to assess the genomic status at both the pfhrp2 and pfhrp3 loci using multiplex qPCR.Diagnostic test results and investigations for the five blood samples are summarized in pfhrp2, pfhrp3, and pfldh) and a human gene (HumTuBB) in a single reaction. All samples with suspected pfhrp2/3 deletions showed good amplification of the pfldh target gene  showed amplification signals with Ct values of 22.0 and 28.6, respectively, for each of the genes, confirming the presence of both pfhrp2 and pfhrp3  produced a good amplification signal for both samples (data not shown). This confirmed that both culture-adapted isolates reflected the results seen in the original patient isolate .Given that qPCR analysis of the parasite DNA from S160 confirmed the presence of intact genes coding for both HRP antigens, sample dilution was performed to test the hypothesis that antigen saturation (a ‘prozone-like effect’) may have accounted for the inconsistent RDT results for HRP antigen detection in this sample. The undiluted blood sample gave inconsistent results with HRP RDT, with a weak positive signal by Carestart™ Malaria Rapydtest® and a negative result by Binax-NOW. The original EDTA sample was diluted 1:10 in uninfected human blood. This dilution tested positive with both RDT formats, confirming the presence of both pfhrp2/3 loci . DN is funded by the Public Health England Malaria Reference Laboratory. The Malaria Reference Laboratory is funded by 10.13039/501100002141Public Health England (research contract awarded to CJS). The funders did not have any role in study design, data collection, data analysis, interpretation, or writing of the report.This work was supported by a 10.13039/100004423World Health Organization. Other authors declare no conflicts of interest.KBB declares financial support from the


Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum–specific pLDH (PfLDH), P. vivax–specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium Knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07–5.16) and 4.56 (3.43–6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56–2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37–2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation. Plasmodium falciparum in sub-Saharan Africa and to a lesser extent P. vivax in Asia and South America.Plasmodium species—such as P. falciparum, P. vivax, P. malariae, and P. ovale spp. —that cause natural human malaria via parasite speciation and treatment of infection.–P. malariae and P. ovale spp. are prevalent in Africa and other malaria-endemic regions but receive little attention because of clinically mild symptoms.,Global malaria programs have been focused largely on P. knowlesi and P. cynomolgi. P. knowlesi, commonly found in natural hosts including long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) in Southeast Asian countries, has been documented in humans as a result of increased human–macaque contacts as a result of habitat disruption and destruction by extensive population growth and deforestation in the areas where natural hosts and the anopheline mosquito vectors carrying P. knowlesi exist.,P. cynomolgi, often detected with other simian parasites such as P. inui, P. coatneyi, or P. fieldi in the macaque population in Southeast Asia, is a close relative of P. vivax.,P. cynomolgi has been reported in Southeast Asia.–In addition, malaria can be caused in humans by simian parasites such as P. knowlesi infection.–P. knowlesi has a relatively short asexual life cycle of approximately 24 hours, compared with approximately 48 hours for P. falciparum, P. vivax, and P. ovale, and 72 hours for P. malariae.P. knowlesi infection can rapidly replicate to reach a high parasite density and progress to severe malaria similar to that from P. falciparum if there is no prompt medical treatment.,P. knowlesi  may have been prevalent at low levels for many decades, a relative surge has arisen as a result of a dramatic reduction in prevalence of P. vivax and P. falciparum over the past decade.,P. knowlesi by microscopy is unreliable largely because of its similar morphological features with P. falciparum at the early trophozoite stage and to P. malariae at the other stages, notably trophozoite band forms.,Severe malarial symptoms and even death have been described with human P. knowlesi, there are no rapid diagnostic tests (RDTs) specifically designed for P. knowlesi identification. Currently available malaria RDTs use several key biomarkers to differentiate between species: P. falciparum-specific histidine-rich protein 2 (HRP2) or/and lactate dehydrogenase (PfLDH) for P. falciparum, and P. vivax-specific lactate dehydrogenase (PvLDH) for P. vivax. Some malaria RDTs detect pan-malarial biomarkers such as the pan-Plasmodium lactate dehydrogenase (pLDH) or Plasmodium aldolase (pAldo) that can, in principle, detect but not differentiate all Plasmodium species.® , Entebe Malaria Cassette, BinaxNOWTM Malaria , and Paramax-3TM ) between parasites of two major Plasmodium species and P. knowlesi parasites, and poor sensitivity in cases of low parasitemia with P. knowlesi infection, revealing the need for caution in data interpretation if confirmatory testing is not conducted.,P. knowlesi, several groups reported the stronger performance of molecular techniques, such as nested polymerase chain reaction (PCR) assay, loop-mediated isothermal amplification (LAMP), and real-time PCR assays.–P. ovale- and P. malariae-specific RDTs, also leading to the underreporting of these more widely spread forms of human malaria.,Plasmodium species.,P. cynomolgi (PcLDH) and P. vivax (PvLDH) indicated the close similarity of pLDH isoforms, whereas the absence of reactivity of the P. falciparum-specific antibodies with other simian parasites except P. knowlesi suggested the possible use of these antibodies to distinguish P. knowlesi from the other simian parasites.In addition to the limitations of microscopy to accurately identify P. vivax diagnosis with RDTs.hrp2/hrp3 gene deletions in P. falciparum infections leading to false negative RDT results, pLDH is increasingly a key biomarker for RDT-based diagnosis of malaria.,Plasmodium species to the different pLDH assays used in RDTs—namely P. falciparum pLDH, P. vivax pLDH, and PanLDH. In previous studies, we described the development and validation of a laboratory-based multiplex platform, the Human Malaria Array , that can quantify HRP2 and pLDH from P. falciparum and P. vivax.,Plasmodium species, may increase our understanding of the performance of current RDTs in these species.The pLDH is the primary target analyte for P. malariae and P. ovale and by using culture-adapted strains of both P. cynomolgi and P. knowlesi.,P. falciparum and P. vivax parasites, and the time-course expression of P. knowlesi-specific pLDH (PkLDH) protein in culture by a multiplex reference assay.Here, analysis of cross-reactivity of this reference assay was investigated with clinical samples of P. falciparum, 62 P. vivax, 17 P. malariae, and 8 P. ovale) from febrile patients with malaria mono-infection were obtained from the Foundation for Innovative New Diagnostics (FIND) Specimen Bank. Nested PCR with microscopy was used to identify the species of human malaria parasites in the samples. Parasite density in clinical samples was confirmed by either microscopy or quantitative PCR (qPCR) methods.A total of 116 clinical blood samples  gradient solution 4 hours apart, retaining the upper brown portion in the first centrifugation and the lower RBC portion for the second centrifugation, to achieve the highest synchronization rate. After synchronization, synchrony was confirmed, and parasite density in the culture was determined by staining thin-film smear using a Hemacolor® staining kit  and counting parasites in a minimum of 2,000 RBCs using a microscope. Following the second synchronization, the culture was incubated and harvested at different time points. For kinetics of PkLDH, infected RBC (iRBC) cultures that were incubated in the culture tubes in three replicates were harvested at 0, 1, 2, 6, 19, 21, 25, 27, 28, 29, and 30 hours. After centrifugation, the cell culture supernatant was collected, and the remaining iRBC pellets were washed with phosphate-buffered saline (PBS) and resuspended in PBS at the original culture volume. The total number of iRBCs was counted using an Accuri™ C6‐UV flow cytometer The Plasmodium cynomolgi parasites were cultured in complete RPMI media supplemented with GlutaMAXTM  and 200 µM hypoxanthine  as described previously.2, 5% CO2, and 5% O2. Three lots of in vitro culture as both iRBC pellet and culture supernatant were prepared on the second day after synchronization and frozen prior to shipment to PATH  for the antigen quantification.Plasmodium species-specific pLDH proteins and human LDH protein for use as reference proteins. Based on the DNA sequence information available from GenBank, the full-length ldh genes specific for P. falciparum, P. vivax, P. ovale curtisi, and P. malariae and the human full-length ldh gene were synthesized and cloned into the prokaryotic expression vector pOPIN-NHis3C to express in RosettaTM (DE3)pLysS competent cells . Bacterial culture was performed in 1 L TB media (Novagen) containing antibiotics at 30°C for around 24 hours. Culture was harvested by centrifugation at 5,000 × g for 30 minutes at 4°C and resuspended in lysis buffer. After lysing using sonication, the lysate was centrifuged at 20,000 × g for 30 minutes at 4°C, and the supernatant representing the soluble fraction was isolated. The soluble fraction was subjected to an immobilized metal affinity chromatography using a HisTrapTM Fast Flow column  followed by size-exclusion chromatography (SEC)® 26/600 Superdex® 200 column . The SEC elution fractions were analyzed by resolving on 12% SDS-PAGE. The SEC elution fractions of interest were pooled and concentrated using an Amicon® centrifuge filter . The sample was filtered through a 0.22 µm filter and stored at −80°C. The amount and purity of His-tag-containing recombinant pLDH and human LDH proteins were estimated by absorbance at 280 nm using NanoDropTM  spectrophotometer and SDS-PAGE, respectively. All recombinant pLDH and human LDH proteins showed more than 95% purity  and were kept at −80°C for long-term storage until future use.PATH collaborated with the University of Queensland Protein Expression Facility  to develop PfLDH, PvLDH, and PanLDH in iRBC pellets, culture supernatant, clinical samples, and samples spiked with recombinant pLDH proteins were tested using the Q-PlexTM Human Malaria Array  to target five different analytes—HRP2, PfLDH, PvLDH, PanLDH, and C-reactive protein (CRP)—according to the manufacturer’s instructions.P. vivax and P. falciparum parasites were individually spotted for the capture of PvLDH and PfLDH, respectively, with a third spot representing a capture antibody to Plasmodium LDH as a PanLDH determinant. A broadly reactive PanLDH antibody was used as the detector antibody for all three assays . For comparison of reactivity pattern, iRBC pellets of synchronized P. cynomolgi and P. knowlesi cultures and P. vivax clinical samples were diluted at 200 and 40 p/µL of parasite density and tested on the 5-Plex. For the samples at high concentration, which falls outside the standard range, the samples were subsequently diluted 1:20 and 1:400 with assay diluent, and the dilution factors were applied for the final concentration of antigens.The presence and quantification of /PfLDH and PanLDH/PvLDH are presented as mean ± SD. Student’s t-test was used to determine significant differences between ratio values of PanLDH and species-specific pLDH of recombinant and native proteins, estimated by reference assay. In addition, a two-way analysis of variance (ANOVA) with Sidak multiple comparison test was used to establish statistical significance between groups (P < 0.05).All data were analyzed using GraphPad Prism . Pearson’s correlation coefficient was calculated for the PanLDH level based on the 5-Plex and for parasite density based on microscopy or qPCR. The results relative to ratios of PanLDHP. falciparum, P. vivax, P. malariae, and P. ovale curtisi, and clinical whole blood samples infected with each of four Plasmodium species were tested on the 5-Plex to examine the presence and level of reactivity to the assays for PanLDH, PfLDH, and PvLDH. Analysis showed that good specificity on PfLDH and PvLDH spots was found with recombinant PfLDH and PvLDH proteins, respectively . Interestingly, recombinant P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) proteins showed reactivity with both PanLDH and PfLDH spots of the 5-Plex. However, a notable difference was observed with the relative signal from the PanLDH spot, in which both PoLDH and PmLDH recombinant proteins showed relatively less signal, resulting in lower measured concentration as compared with recombinant PfLDH or PvLDH proteins for a given concentration . Furthermore, the relation between level of PanLDH and parasite density was assessed with clinical samples infected by each Plasmodium species , P. vivax  and P. malariae  but relatively poor correlation with parasite density for P. ovale .For the reactivity to native pLDH from four  species . The corPlasmodium species . The PanLDH concentration measurement in the 5-Plex is calibrated to recombinant PfLDH and so the ratio of close to 1 for PanLDH/PfLDH is to be expected.The ratio of PanLDH to species-specific pLDH was similar for both native pLDH from clinical samples and the same recombinant proteins across the four  species . SpecifiP. knowlesi and P. cynomolgi on each assay for PanLDH, PfLDH, and PvLDH, a series of dilutions (200 and 40 p/µL) of whole blood sample spiked with synchronized P. cynomolgi and P. knowlesi iRBC pellets were tested with the 5-Plex. The results were comparable to that of a clinical P. vivax blood sample. The results showed the similar reactivity between PkLDH and PvLDH proteins for binding to the PanLDH and PvLDH spots, and with comparable PanLDH/PvLDH ratio values between these two Plasmodium species at 2.7 for PkLDH and 2.8 for PvLDH at 200 p/µL of parasite density , similar to the previously published finding of 27 hours.PkLDH was found in iRBC pellets rather than in the supernatant, both pellets and supernatant showed progressively increased concentration of PkLDH (∼8 times more after 19 hours compared with 1 hour for both pellets and supernatant) during ring and trophozoite stages . The maximal amount of PkLDH is reached in the pellet during the schizont stage (21 to 27 hours), but a continuous accumulation of PkLDH released from the schizont rupture in the supernatant was observed over this period of time. Interestingly, during the first life cycle, parasite density seems to be decreased during the schizont stage, and the number of parasites in the second life cycle is increased after the released cells infect the fresh cells.For the time-course study,  results  demonstrPfLDH, and PvLDH and a qualitative measure for all malaria species (PanLDH) as well as measure the human biomarker CRP.PfLDH and PvLDH,hrp2/hrp3 deletions both in whole blood pelletsP. falciparum and P. vivax, using the described biomarkers.,Plasmodium species such as P. malariae and P. ovale, and two simian Plasmodium species, P. knowlesi and P. cynomolgi, which have been recognized to cause human infection, has not been fully explored. As the 5-Plex includes a PanLDH spot detecting all Plasmodium species, in this study, we first evaluated the potential cross-reactivity of pLDH proteins from these parasites against pan-malaria and species-specific spots using both recombinant and native proteins.The human malaria array (5-Plex) was developed to provide quantitative measurement of the malaria antigens HRP2, P. falciparum, P. malariae, P. ovale curtisi, and P. vivax offer great diagnostic value for the appropriate evaluation of the assay performance when there are limited clinical blood samples. Our study demonstrated that these recombinant pLDH proteins behave similarly to native pLDH proteins from corresponding Plasmodium species as demonstrated by PanLDH/species-specific pLDH ratio while being distinct from the equivalent host LDH protein .The recombinant pLDH proteins of  protein . HoweverPlasmodium species, but obtaining native proteins from live parasites is a major challenge, especially with species of P. vivax, P. ovale, and P. malariae, for which continuous culture systems have not been established unlike P. falciparum strains.–P. falciparum parasites and their respective native proteins.The significant variation of PanLDH values in the estimated concentration of recombinant pLDH proteins suggest two possibilities: 1) some minor variance in the amino acid sequence of the conserved region or that of the surrounding area that enhances or reduces the antigen–antibody reaction, and 2) inefficient protein folding during protein expression/purification might have occurred when using a prokaryotic expression system. The second possibility may be addressed by evaluating the native pLDH proteins purified from different Supplemental Figure 3). The results suggest that this could be an artifact derived from the protein purification processes.The pLDH amount measured as PanLDH correlates to parasite density equally across species, as shown by the normalized binding reactivity of native pLDH proteins with respect to the given parasite number . It will therefore be of interest to investigate the extent of the level of cross-reactivity, which may have a significant influence on interpreting diagnostic test results. The PanLDH/species-specific pLDH ratio values for P. malariae, P. ovale, and P. vivax were significantly different from that for P. falciparum  and found a conserved region (the region forming the loop structure) as the potential site recognized by pan-malaria antibodies and several species-specific regions as described previously .–PcLDH protein as compared with three other parasite species including P. falciparum, P. vivax, and P. knowlesi that would explain our observation. It would be interesting to explore binding characteristics of commercially available pan-malaria antibodies to the laboratory-adopted P. cynomolgi strain or recombinant PcLDH reagents and investigate the extent of sequence conservation within the conserved region of PcLDH protein from the cultured P. cynomolgi in a future study.The reactivity patterns of pLDH from LDH spot . UnfortuPkLDH exhibited significant cross-reactivity to both the PanLDH and PvLDH spots, but no or little cross-reactivity with the PfLDH spot on the 5-Plex. A previous study looking at cross-reactivity between pLDH proteins from different Plasmodium species and different antibodies to pLDH highlighted that PkLDH can react to antibodies specific to both PfLDH and PvLDH.PkLDH and the PvLDH antibody pair was observed. Additionally, the PanLDH/PvLDH ratio for P. knowlesi was similar to that seen when measuring PvLDH from the P. vivax clinical samples. Although the PanLDH and PvLDH spots on the 5-Plex can detect the presence of the PkLDH antigen, they were unable to distinguish P. knowlesi from P. vivax infection. To differentiate P. knowlesi from other Plasmodium species, development of an expanded array that hosts a P. knowlesi-specific antigen assay is required.PkLDH in a culture system. The results exhibited accumulation of PkLDH over time in culture, except with a slight decrease in PkLDH concentration seen in samples from the late schizont stage at around 25–28 hours. The pellet fraction contains the largest total amount of PkLDH until 29 hours after synchronization. At an inflection point where more PkLDH is contained in the supernatant, the erythrocytic cycle is completed through the rupturing of the schizonts to create new ring-stage parasites. The overall decrease in PkLDH seen starting at hour 25 may be explained by the lability of PkLDH and consequently an overall drop in PkLDH concentration until the parasites mature again. It should also be noted that the reactivity pattern of PkLDH proteins as a function of PanLDH/PvLDH ratio was maintained at 2.11 ± 0.24 and 2.35 ± 0.24 in both cell pellet and supernatant compartments, respectively, throughout all development stages .This is the first study to demonstrate the dynamic kinetic expression of Plasmodium species investigated and observed in this study across the 5-Plex PfLDH, PvLDH, and PanLDH assays echoes results from previous studies investigating both antibody reactivity to pLDH and RDT reactivity to pLDH.,,,P. ovale, P. malariae, and P. knowlesi, and also to the less-common species known to infect humans, including P. cynomolgi and P. simium. This will be particularly relevant to countries approaching elimination and/or that have a significant relative burden of P. knowlesi.,hrp2/hrp3 deletions in P. falciparum infections and pLDH as the most commonly used target analyte for non-P. falciparum malaria RDTs, understanding the dynamics and cross-reactivity of pLDH between the different human Plasmodium species is essential. Additionally, given the known cross-reactivities between different Plasmodium species, new diagnostic markers for better differentiating Plasmodium species should be explored.The differential binding of pLDH from the different Supplemental materials


We hypothesized that asymptomatic malaria parasitemia increases susceptibility for bacterial infections through an effect on the innate immune system. We measured circulating cytokine levels and ex-vivo stimulated with S. aureus, E. coli LPS and Salmonella Typhimurium; cytokine concentrations  were measured on supernatants using ELISA.Data were collected from asymptomatic participants <5 years old with and without positive malaria microscopy, as well as from hospitalized patients <5 years old with clinical malaria, bacteremia, or malaria/bacteremia co-infections in a malaria endemic region of Burkina Faso. Circulating cytokines  were measured using multiplex assays. Whole blood from asymptomatic participants with and without positive malaria microscopy were Ex-vivo whole blood cytokine production to LPS and S. aureus was significantly lower in asymptomatic malaria compared to controls. Whole blood IFN-γ and IL-10 production in response to Salmonella was also lower in asymptomatic malaria.Included were children with clinical malaria (n=118), bacteremia (n=22), malaria and bacteremia co-infection (n=9), asymptomatic malaria (n=125), and asymptomatic controls (n=237). Children with either clinical or asymptomatic malaria had higher plasma cytokine concentrations than controls. Cytokine concentrations correlated positively with malaria parasite density with the strongest correlation for IL-10 in both asymptomatic (r=0.63) and clinical malaria (r=0.53). Patients with bacteremia had lower circulating IL-10, TNF-α and IFN-γ and higher IL-6 concentrations, compared to clinical malaria. ex-vivo bacterial stimulation are downregulated. Further studies are needed to explore if the suggested impaired innate immune response to bacterial pathogens also translates into impaired control of pathogens such as Salmonella spp.In children with asymptomatic malaria, cytokine responses upon  Plasmodium falciparum infections are a risk factor for bacterial bloodstream infections (bBSI)   bloodstream infection is estimated at 20% (s (bBSI) , 2, especa (SSA) –5, both d at 20% . PatientSalmonella infection , a rural research facility situated in the central-west region of Burkina Faso, at approximately 85km from the capital city Ouagadougou . With anly found , 16. SeaThe objective of this study was to assess the effect of (asymptomatic) malaria on cytokine responses to bacterial pathogens among children below the age of five, since children of this age group are most likely to suffer from severe malaria and iNTS infection. We hypothesized that patients with clinical malaria and asymptomatic malaria have a lower cytokine response to bacterial pathogens compared to patients who are malaria microscopy slide negative. To assess our hypothesis, we aggregated data from two studies simultaneously being carried out at CRUN on respectively a healthy population  and a population presenting with acute febrile illness (AFI) . For the current study, all participants from either study who were less than 5 years old, were selected.ex-vivo whole blood stimulation. Supernatants were stored at -80°C and shipped to the Netherlands for cytokine analyses.The first study was an explorative cross-sectional study among healthy volunteers of 1 year and older living in the Health and Demographic Surveillance System (HDSS) area of Nanoro. The HDSS area of Nanoro covers a population of approximately 65.000 inhabitants distributed over 24 villages. Study methods are described in detail elsewhere . Briefly, healthy participants of 1 year and older were randomly selected from the 24 villages of the HDSS catchment area. Informed consent was obtained from their parents or legal guardians. After inclusion, an extensive electronic questionnaire concerning health and demographic data was obtained. Finally, EDTA anti-coagulated and heparinized blood was collected and transported back to CRUN in a cool box for hemocytometry, malaria diagnostics and The second study was a phase-three diagnostic accuracy study to assess the diagnostic performance of two novel technologies to differentiate malaria, bacterial infections, and viral infections. Study methods are described in detail elsewhere . In summUpon arrival at the clinical research laboratory of CRUN, the EDTA blood samples were analyzed using an XN-1000 automated hematology analyzer . Complete blood counts (CBC) and leukocyte differential were recorded.Plasmodium parasites according to the World Health Organization (WHO) procedures  the results of a third blinded microscopist was decisive. Malaria diagnostics and hematology analysis were done within 6 hours after sample collection.Thick and thin blood films were made using 10μL EDTA-anticoagulated blood. Slides were stained with 3% Giemsa solution and examined for presence of ocedures . Resultsex-vivo whole blood stimulations. The whole blood stimulations were done at the research laboratory of CRUN within 12 hours after blood collection. Three stimuli (10ng/ml) Escherichia coli LPS , 1x10ˆ6/ml heat killed Staphylococcus aureus  and 1x10ˆ6/ml heat-killed Salmonella Typhimurium  and a negative control medium  supplemented with gentamicin 50μg/ml, glutamax 2mM, pyruvate 1mM) were used for each participant.A heparinized whole blood sample was collected from participants in the cross-sectional study for 6/ml and incubated at 37°C. After 48 hours, plates were centrifuged at 400g for 5 minutes. Supernatant aliquots of 300 µl were collected and stored in Micronics tubes  at -80°C. After completion of the trial, all supernatants were transported to Radboudumc Nijmegen on dry ice for batch analysis of cytokine levels. Participants with an IL-1β and TNF-α response in the RPMI control sample were considered contaminated and were therefore excluded from analyses.Blood was diluted at 1/5 in RPMI and distributed over four wells per participant in a final concentration of 500 µl. Each well was stimulated with one of four stimuli at an end concentration of 1x10Staphylococcus aureus were measured on day one, Salmonella Typhimurium and Escherichia coli LPS were measured on day two. Samples were allowed to thaw at 4°C one day prior to analysis. Plate set-up was randomized in terms of participant age, presence of asymptomatic malaria and month of storage to decrease batch-effect. Reported cytokine concentrations are based off standard curves which were ran on each plate.Cytokines were assessed using commercial ELISA kits for human TNF-α, IFN-γ, IL-1β, IL-10  and human IL-6  according to manufacturer’s instructions. All assays were performed in a timespan of two consecutive days: RPMI and Circulating cytokines of patients participating to both studies were batch-tested, using stored EDTA plasma samples. Plate set-up was randomized as previously described. The pro-inflammatory cytokines IL-6, TNF-α, IFN-γ and anti-inflammatory cytokine IL-10 were quantified using the MAGPIX technology  with the Human High Sensitivity Cytokine Premixed Magnetic Luminex Performance Assay kits from Bio-Techne (R&D systems). Manufacturer’s recommendations were followed during the assay.Salmonella co-infection” in case of combined malaria and Salmonella. Anemia was defined by age and sex according to WHO classification. Malnutrition was recorded as patients with a low weight-for-height (wasting) or height-for-age (stunting) according to WHO guidelines.Asymptomatic malaria was defined as one or more parasites in thick smear malaria microscopy without fever or other acute symptoms. Clinical malaria was defined as febrile patients with one or more malaria parasites in thick smear microscopy and without other possible explanations for the fever. Bacteremia was defined as a blood culture grown with a pathogenic bacterium. Combined malaria and bacteremia was recorded in case of simultaneous blood culture grown with a pathogenic bacterium and one or more malaria parasites in malaria thick smear microscopy. Patients with a combined malaria and bacteremia are hereafter referred to as “co-infection” or “ex-vivo stimulated cytokine production between participants with and without asymptomatic malaria were corrected for using a multivariable regression, multicollinearity was assessed using a correlation matrix. Data were log-transformed for analysis in case of non-normal distribution. For normally distributed cytokine measurements a linear regression model was used. A Tobit regression was used for analyses with results outside of the upper or lower quantification limits. The association between cytokine production and parasite density was measured using a Pearson’s correlation or Spearman’s correlation as appropriate. Graphs and figures were created using Graphpad . A p-value of.05 was considered statistically significant. Results were corrected for multiple testing using a Bonferroni correction.As appropriate, means or medians were compared using a two-sample t-test, Mann-Whitney U test or Kruskal-Wallis. Differences in Both study protocols were approved by the national health ethics committee of Burkina Faso (ref 2016-01-006 and 2015-01-006 respectively). The diagnostic accuracy study was furthermore approved by the internal review board of IRSS (ref A03-2016/CEIRES) the ethical committee of the Antwerp University Hospital (ref 15/47/492) and the institutional review board of the Institute of Tropical Medicine Antwerp (ref 1029/15).A total of 510 participants were included in this study: 361 from the cross-sectional study among healthy participants, and 149 from the clinical diagnostic accuracy study Figure 1The cross-sectional study enrolled 1000 participants between June and October 2016, of whom 449 (44.6%) had asymptomatic malaria. Out of 399 eligible participants, a total of 361 (90.5%) participants were included in analysis; reasons for exclusion were missing malaria microscopy data (n=33), missing demographic data (n=2) and missing supernatant samples (n=3). Among included participants, 125 (34.6%) had asymptomatic malaria.Salmonella spp n=18, streptococci n=5, Escherichia coli n=3, Haemophilus influenzae n=2, Neisseria meningitidis n=1 and one double infection) and 9 patients had a co-infection . Residual blood was available from 149/171 (87.1%) patients; n=118 (69.0%) with clinical malaria, n=22 (73.3%) with bacteremia and n=9 (69.2%) with a co-infection.The diagnostic accuracy study enrolled 930 patients between March 2015 and July 2016, of whom 461 (49.6%) were between three months and five years old. In total 171 of them were eligible for inclusion in the current study: 171 patients had clinical malaria, 30 patients had bacteremia , Reticulocyte count  and lower platelet count  compared to slide negative participants, but we found no significant difference in age, height, weight, middle upper arm circumference (MUAC), axillary temperature, white blood cell count or neutrophil count between both groups.Baseline characteristics are presented in Among participants in the diagnostic accuracy study, differences between patients with clinical malaria, bacteremia and co-infection were more pronounced, though this was also more difficult to interpret due the variability in numbers of cases. Patients with bacteremia were in median less than 1.5 years old, whereas patients with malaria and those with co-infection were approximately 2 years old. The height-for-age among patients with co-infection was considerably lower compared to patients with malaria and patients with bacteremia, suggesting a correlation with chronic malnutrition. Patients with co-infection typically presented at the hospital earlier (1.9 day after onset of fever) compared to patients with clinical malaria (mean 2.4 days) and bacteremia (mean 2.8 days) and had a slightly higher temperature at presentation. As expected, patients from the diagnostic accuracy study had higher neutrophil counts compared to participants to the cross-sectional study. The median numbers of white blood cells were comparable between both groups.versus 0.00088, and for TNF-α 0.0014 versus 0.010.We first assessed circulating cytokine  concentrations among healthy participants with- and without asymptomatic malaria and patients with clinical malaria. All cytokines were highest among patients with clinical malaria Table 2 These results indicate that apart from IFN-γ, all other tested cytokine concentrations are upregulated in asymptomatic malaria, as in clinical malaria. Cytokine concentrations were significantly higher in clinical malaria, whereas the IL-10 and TNF-α over parasite density ratio were higher in asymptomatic malaria.We next assessed circulating cytokine concentrations in acute febrile patients below the age of five diagnosed as malaria, bacteremia, or malarial bacteremia co-infection. Results are presented in i.e. Gram positive , Gram negative , Salmonella spp.) showed a lower IL-6 concentration among patients with Salmonella bacteremia compared to the patients suffering from infections with other pathogens. Patients with Salmonella bacteremia also had the lowest circulating concentrations of IL-10, but the highest concentration of IFN-γ. There was no significant correlation between parasite density and cytokine levels among patients with co-infection, or Salmonella co-infection. These results suggest that the anti-inflammatory response (IL-10) is upregulated during clinical malaria infections while pro-inflammatory IL-6 (and TNF-α) are particularly downregulated during Salmonella bacteremia.Further analysis of the different pathogens involved in bacteremia (Staphylococcus aureus (S. aureus), heat-killed Salmonella Typhimurium and Escherichia coli LPS (LPS). Samples from 180 (49.7%) participants were excluded due to contamination of the sample during the stimulation assay. The excluded participants are further detailed in versus n=59, respectively p=.38).To explore whether circulating immune cells of asymptomatic patients with or without malaria parasitemia differed in their capacity to produce cytokines, whole blood was stimulated with various antigens, including heat killed S. aureus, Salmonella Typhimurium and LPS, after correction for age, sex, weight, MUAC, leukocyte count, monocyte count, platelet count, hemoglobin levels and ELISA-plate layout to correct for batch effect. Height caused multicollinearity and was therefore not corrected for.Staphylococcus aureus were observed among participants with asymptomatic malaria compared to controls. Participants with asymptomatic malaria had also lower IFN-γ and IL-10 responses after Salmonella Typhimurium stimulation, while monocyte cytokines  were not statistically different between slide negative participants and participants with asymptomatic malaria. Likewise, we found that cytokine responses to Salmonella Typhimurium were generally considerably higher compared to the other two stimuli.Significantly lower cytokine responses  to LPS and Salmonella Typhimurium did not significantly correlate with parasite densities, which corresponds to the ex-vivo cytokine stimulation results.In the whole-blood stimulations, parasite density was strongly negatively correlated to production of most cytokines Figure 5S. aureus, while Salmonella Typhimurium downregulates the mostly lymphocyte-derived IFN-γ but had limited effect on monocyte-derived cytokines.These results suggest that asymptomatic malaria downregulates the cytokine production capacity of circulating immune cells after stimulation with LPS and heat killed ex-vivo bacterial stimulation in children with asymptomatic malaria are altered compared to children without asymptomatic malaria. Further studies should be done to explore whether the observed downregulation of pro-inflammatory cytokines impairs the control of bacterial infections, in particular intracellular infections such as invasive Salmonella, confirming the non-benign nature of chronic malaria parasitemia  may have influenced results, seeing as part of the clinical malaria samples were collected prior to the introduction of seasonal prophylaxis whereas the asymptomatic cases were collected after introduction.The amount of blood sampled was too low to perform all analyses in duplicate. Since anemia is common among children in the study site, to sample more blood would have been unethical. However, the fact that we did not use duplicate measurements may have introduced a bias.Salmonella serotype used in the stimulation experiments was obtained from DR Congo , both in asymptomatic  and clinComparing circulating cytokine concentrations among asymptomatic participants, we also found significantly higher pro- and anti-inflammatory cytokine concentrations in malaria microscopy positive participants compared to microscopy negative controls . The larPlasmodium induces immune hyporesponsivity to bacterial antigenic stimulation. Metenou et al  studying filarial infected patients with and without asymptomatic malaria also found evidence using flow cytometry of a lower cytokine production capacity of immune cells in case of concurrent malaria parasitemia. Other previous research focused on clinical malaria, demonstrating that circulating blood samples of clinical malaria patients produced a lower cytokine response . The diagnostic accuracy study was furthermore approved by the Internal Review Board of IRSS (ref A03-2016/CEIRES), the Ethical Committee of the Antwerp University Hospital (ref 15/47/492), and the Institutional Review Board of the Institute of Tropical Medicine Antwerp (ref 1029/15). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.BK, AP, MB, QM, and AV were responsible for study design and execution of the cross-sectional study. AP, BK, SD, HT, JJ, QM, and AV were involved in study design of the diagnostic accuracy study. BK, SD, and OT were responsible for study management in Burkina Faso. RA, MN, and LJ were involved in preparation of laboratory techniques and quality control. Analyses and writing were done by AP, BK, MB, QM, and AV. All authors contributed to the article and approved the submitted version.AV and QM have a non-restricted research grant from SYSMEX, which funded the current study. Both the diagnostic accuracy study and the cross-sectional study were funded by a non-restricted research grant from SYSMEX company.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.


Glucose 6-phosphate dehydrogenase deficiency (G6PDd) plays a central role in readiness assessment for malaria elimination in Myanmar by 2030 that includes primaquine (PQ) use. The risk of hemolysis in G6PDd individuals hampers the widespread use of primaquine safely in malaria-infected patients. In the pre-elimination era, it is important to screen initially for asymptomatic malaria in combination with G6PD deficiency by applying more sensitive diagnostic tools. Therefore, this study examined the proportion of G6PDd and the distribution of G6PD genotypes among malaria-infected national groups in Myanmar before initiation of malaria elimination strategies.A cross-sectional study in one township each with high malaria burden from two states in the western part of Myanmar, was conducted during 2016-2018, and 320 participants  were recruited. We used RDT and ultrasensitive polymerase chain reaction (us PCR) method to confirm malaria infection, and a G6PD RDT(CareStart) to detect G6PDd and PCR/restriction fragment length polymorphism (RFLP) method to confirm the variant of G6PDd for genotyping. G6PD enzyme activity was measured by G6PD Biosensor (CareStart).Malaria positivity rates detected by RDT were lower than those detected by us PCR in the combined samples [13% (42/320) vs. 21% (67/320)] as well as in the Rakhine samples [17% (28/164) vs. 25% (41/164)] and in Chin samples [9% (14/156) vs. 17% (26/156)]. G6PD deficiency rates were approximately 10% in both the combined samples and specific national groups. For G6PD enzyme activity in the combined samples, G6PDd  was 10% (31/320) and severe G6PDd (< 10% of AMM) was 3% (9/320). Among malaria-infected patients with positive by both RDT and usPCR, G6PDd was less than 20% in each national group. G6PD genotyping showed that the G6PD Mahidol (G487A) was the major variant.P. vivax by primaquine for radical cure. Establishing a field monitoring system to achieve timely malaria elimination is mandatory to observe the safety of patients after PQ treatment.The varying degree of G6PDd detected among malaria-infected national groups by advanced diagnostic tools, strongly support the recommend G6PD testing by the National Malaria Control Program and the subsequent safe treatment of  Plasmodium vivax is increasing relative to Plasmodium falciparum  as well as in the specific samples of Rakhine national group [17% (28/164) vs. 25% (41/164)] and Chin national group [9% (14/156) vs.17% (26/156)]. Regarding species prevalence, p = <0.05).The G6PD deficiency rate as determined by CareStart RDT was 10% (32/320) in the combined sample, and similar rates were noted for both Rakhine and Chin national groups [9.8% (16/164) vs. 10.3% (16/156)] respectively. In the combined sample, males were two times more likely than females to identify as G6PD deficient [13.3% (22/165) vs. 6.5% (10/155)] and more than two times more likely to be malaria-infected than females  as 6.6 (IQR 5.3-7.9) for the combined samples and as 6.3 (IQR 4.6-7.5) in the male population. The adjusted male median (AMM) value of G6PD enzyme activity was calculated after exclusion of G6PD enzyme activity values of 7 male patients having < 10% of male median value. AMM was 6.4 U/gHb (IQR 5.0-7.6) Table . ApplyinAccording to Table PCR/RFLP method was used to genotype all 22 male samples with G6PD RDT positive and all female samples. Nineteen out of 22 male samples harbored Mahidol variant (G6PD G487A), and 3 samples were wild type.Among 155 female samples consisted 10 G6PD RDT positive and 145 G6PD RDT negative samples. Overall, 85.8% of female participants harbored wild type genotype (133 out of 155) and mutant G6PD was found in 22 samples (14.2%). Mutant female samples included 10 G6PD RDT positive and 12 G6PD RDT negative. G6PD RDT positive 10 samples showed homozygous mutation of Mahidol variant and 9 out of 10 samples had G6PD enzyme activity < 30% of AMM and one sample had 39% of AMM. In the remaining 12 samples, one sample was heterozygous Chinese-4 variant and 11 were heterozygous Mahidol variant. In relation to enzyme activity, 4 out of 12 samples showed intermediate deficiency (30-80% of AMM) and 8 samples had normal G6PD enzyme activity (> 80% of AMM)  which was consistent with the Thailand study [. Biosensor) was negligible (9.7% vs. 10%). The performance of CareStart RDT is satisfactory to identify individuals with < 30% of G6PD enzyme activities with high negative predictive value , suggesting its applicability to guide PQ treatment [Among those with G6PD deficiency in the combined samples in this study, 68.8% were male   which waAmong the female samples, mutation was detected in 22 samples (14.2%) which were comprised of 21 G6PD Mahidol and one Chinese-4 variant. All G6PD RDT positive female samples (n = 10) harbored homozygous Mahidol type. In relational to enzyme activity measured by Bisensor, 9 out of 10 samples had G6PD enzyme activity < 30% of AMM and one sample showed enzyme activity 39% of AMM. G6PD enzyme activity is presented as IU per gram of existing hemoglobin. Very low level of Hb may result in higher enzyme activity than actual. This may be the reason for the discrepancy in G6PDd rates between Biosensor and other diagnostic methods. Future studies with a larger sample size could explore the distribution of other G6PD variants to explore novel mutations among population. As WHO recommends for G6PD testing for the safe use of primaquine , G6PD deThis is the first study in Myanmar exploring the prevalence of G6PD deficiency with exact measurements using advanced molecular tools among two national groups in the western part of the country with high malaria burden to inform NMCP for its applicability in moving toward malaria elimination. The FST Gold standard testing for G6PD deficiency was unable to be applied in this study as the facility was limited in the study areas.P. vivax for radical cure. Establishing the field monitoring system to achieve timely malaria elimination is mandatory to observe the safety of patients after PQ treatment.The varying degree of G6PD deficiency among malaria-infected national groups supports the strongly recommend G6PD testing by the National Malaria Control Program and the subsequent safe treatment of


Malaria is one of the leading causes of morbidity and mortality especially in pregnant women and under-five-year-old children. However, data on the prevalence among delivering mothers, potential fetal transmission, and associated birth outcomes is lacking in Ethiopia. Plasmodium infection from peripheral, placental, and cord blood samples among delivering mothers in Kuch health center, Northwest Ethiopia.  To assess the prevalence of  An institution-based cross-sectional study was conducted among 218 delivering mothers from February to May 2021 in Kuch health center. Data on sociodemographic characteristics and clinical and obstetric history of mothers were collected using a structured questionnaire. Giemsa stained blood films from maternal capillary and placental and umbilical cord blood were examined for plasmodium infection. Data were analyzed using Statistical Package for the Social Sciences version 23 software package. Plasmodium falciparum and Plasmodium vivax accounted 3.7% (8/218) and 2.8% (6/218), respectively, in maternal peripheral blood but only Plasmodium falciparum was detected in placental and umbilical cord blood samples. Maternal malaria had significant association with primigravida  and low birth weight . Placental malaria was also significantly associated with low birth weight .  The prevalence of maternal, placental, and umbilical cord malaria was 6.4% (14/218), 2.3% (5/218), and 0.5% (1/218), respectively.  The prevalence of malaria among delivering mothers was considerable. Maternal peripheral malaria had a significant association with gravidity and birth weight. Placental and umbilical cord malaria also had a significant association with birth weight. Pregnant mothers should be examined for malaria and receive appropriate treatment to prevent adverse birth outcomes. Plasmodium. Five species of Plasmodium, Plasmodium (P.) falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, are known to cause malaria. Plasmodium is transmitted by female Anopheles mosquitoes, which bite mainly between dusk and dawn. Malaria is a global public health problem that there were an estimated 229 million cases and 409,000 deaths in 87 malaria-endemic countries in 2019. In Africa, there were an estimated 215 million cases and 384,000 deaths in the same year [Malaria is a febrile illness caused by protozoan parasites of the genus P. falciparum [Pregnant women and under-five-year-old children living in malaria-endemic areas are at risk of acquiring malaria depending on the degree of endemicity , 6. In alciparum . On the lciparum .P. falciparum adheres to placental villi and reduces the placental blood flow to cause birth defects like LBW [One of the main features of malaria in pregnancy is that women frequently presented with parasitized placenta during delivery . This relike LBW , 11, 12.like LBW .Placental and congenital malaria are common in the first pregnancy and decline in consecutive pregnancies due to immunity development , 10. Nul″ N and 37° 01′ 24″ E. There are six kebeles in the catchment of Kuch health center. The area has an altitude of 2038 m above sea level. The rainfall significantly varies with 80% of the annual rainfall occurs in June-August. The highest temperature occurs in January up to 32°C and the lowest in July and August reaching up to 19°C according to the woreda profile. According to data obtained from Bure Zuria district health office, the catchment population of Kuch health center in 2020/2021 is about 33,925 with 16,928 males and 16,997 females. From the total population, 10,558 live in urban and 23,367 live in rural areas. In the 2020/21 season, about 1143 pregnant women were estimated to give birth according to Bure Zuria Woreda health office. Malaria has minor and major transmission seasons in the study area, the major transmission season being from September to December while the minor transmission season is from April to June. However, despite the magnitude varies, malaria is transmitted throughout the year in the district.A facility-based cross-sectional study was conducted in Kuch Health Center from February to May 2021. Kuch is a subtown of Bure Zuria district in West Gojjam Zone, North Western part of Ethiopia. The area is located at geographical coordinates of 10° 29′ 00n) with the following assumptions: malaria prevalence (p) of 15.2% among delivering mothers according to a previous study from Wolkitie, Ethiopia [d) of 0.05, and 95% confidence level (Zα/2 = 1.96). A single population proportion formula was used to calculate sample size (After adding 10% (20) for nonrespondents, the final sample size was 218. Participants who fulfill the eligibility criteria were enrolled by convenient sampling technique. All delivering mothers in Kuch Health Center who were volunteer to participate were included in the study. Delivering mothers who took antimalarial or antibiotic treatment within a month before data collection, who were not permanently living in the catchment area of Kuch health center, who delivered twins, and those who were referred to hospital were excluded from the study.Data on sociodemographic characteristics and clinical data were collected using a structured questionnaire in the labor ward at the postpartum period. The questionnaire was adapted from previous similar studies in Ethiopia . QuestioMaternal capillary blood was collected, first by wiping the ring or middle finger with 70% alcohol swab and then piercing with sterile lancet. The first drop of blood was wiped away, and the second drop was used for thin and thick blood film preparation following standard protocol . ImmediaData collectors were trained about the data collection tools and the study procedure. Standard operating procedures were strictly followed during the laboratory test procedures. Each smear was examined by two laboratory technicians who are blind to each other's results. A malaria microscopy expert from the Amhara Public Health Institute reexamined slides with discrepant results among the two technicians. In such cases, results of the expert were reported as final results.Data was coded, entered, cleaned, and analyzed using statistical package for the social science (SPSS) version 23 software packages. Descriptive statistics was performed to describe study participants in terms of sociodemographic characteristics and clinical data. Prevalence of malaria in maternal peripheral, placental, and umbilical cord blood was also computed. Chi-square test was used to assess the association between malaria and sociodemographic and clinical factors.A total of 218 study participants whose age ranged from 19 to 44 years old with a mean age of 29.4 and standard deviation (SD) of ±5.7 were included in the study. One hundred ten (50.5%) study participants were in the age group of 19-28 years old. Among 218 participants, 148 (67.9%) and 135 (61.9%) were farmers and participants who did not attend formal education, respectively .Plasmodium species were detected from peripheral blood of 14  participants. Similarly, placental blood smears from 5  participants were positive for Plasmodium infection. Plasmodium species was detected in umbilical cord blood of a single delivering mother . Maternal peripheral blood film examination revealed that P. falciparum and P. vivax monoinfections were detected in 8 (3.7%) and 6 (2.8%) participants, respectively. Regarding to parasite stages, only trophozoites were detected in blood film of 9 (4.1%) participants while in both trophozoites and gametocytes, and only gametocytes were detected in the blood film of 4 (1.8%) and 1 (0.5%) participants, respectively. Plasmodium falciparum was the only species detected in placental and cord blood smears. Out of five placental malaria detected, four had maternal malaria parasitemia. Only trophozoites were detected in 4 (4.1%) placental blood smears while both trophozoites and gametocytes were detected in 1 (0.5%) placental blood smear. The range of parasitemia in maternal peripheral and placental blood films was 480-4480 parasites/μl and 200-1120 parasites/μl, respectively. Cord blood malaria-positive blood films revealed a parasite load of 80parasites/μl of blood. The mean maternal peripheral and placental parasite counts were 1290 parasites/μl with SD of ±1196 parasites/μl and 660 parasites/μl with SD of ±487 parasites/μl, respectively . Delivering mothers who had no full ANC follow-up were 36.2%, but there was no significant association between peripheral malaria and ANC follow-up . Among the delivering mothers who gave LBW (<2.5 Kg) neonate, maternal peripheral malaria was detected in 27% of the mothers while 5.3% of malaria was detected among mothers who gave normal birth weight babies. There was a significant association between birth weight and peripheral blood malaria . There was no significant association between previous malaria attack history and peripheral malaria   .χ2 = 32.255, p ≤ 0.001) and , respectively   but it i studies , 12. ThiThe prevalence of umbilical cord malaria was 0.5%. It was from a participant who had placental malaria but not peripheral malaria. This result is in agreement with a previous study conducted in eastern Uganda 0.95%) , but it 5% , but The prevalence of LBW in the present study (5%) was comparable to previous finding in Wolkitie Ethiopia which was 7.4%  and Nigeχ2 = 12.611, p = 0.002). It was similar to a result reported by Oringanje, in Cross River State in Nigeria in 2010 where primigravida mothers were more vulnerable to peripheral malaria [P. falciparum infection than women of higher gravidities which, in turn, higher risk of adverse birth outcomes. Immunity developed in multigravid women in their third or later pregnancy, but not in primigravidae, suggesting that variant-specific immune responses develop following exposure during pregnancy [In this study, gravidity had a significant association with maternal peripheral malaria . There were significant associations between maternal history of malaria and low birth weight in previous studies [χ2 = 32.255, p ≤ 0.001). This is in line with a study result from Wolkitie, Southern Ethiopia, where placental malaria had a significant association with LBW [P. falciparum-infected erythrocytes. Monocytes in the placenta induce altered cytokine profiles and complement activation that causes placental tissue injury and leads to placental insufficiency. Thus, it results in fetal growth restriction and LBW [χ2 = 18.905, p ≤ 0.001). The result is in consistent with the study result in rural Malawi [Birth weight had a significant association with maternal peripheral malaria ( studies –29. The with LBW . Placent and LBW , 31. Umbl Malawi . HoweverPlasmodium falciparum erythrocyte membrane protein 1 is the main adhesion receptor that adheres to the trophoblastic villous endothelium mainly through circumsporozoite antigen [In the present study, out of 5 participants who had placental malaria, one had both trophozoite and gametocyte stages. Even though trophozoites commonly sequester in the placenta , 34, pre antigen .The prevalence of malaria among delivering mothers was considerable. Maternal peripheral malaria had a significant association with gravidity and low birth weight. Placental malaria and umbilical cord malaria also had a significant association with low birth weight. Hence, pregnant mothers living in endemic areas should be routinely screened for malaria and receive appropriate treatment to prevent adverse birth outcomes. As an alternative, intermittent preventive chemotherapy should be implemented in malaria endemic areas. We recommended further large-scale studies covering diverse geographical locations and using more sensitive molecular diagnostic methods.