Plasmodium falciparum impact cellular aging by inducing high levels of inflammation and redox-imbalance; and that cellular senescence reversed after treatment and parasite clearance. This study provides insights into the complex relationship of telomere length, cellular senescence, telomerase expression and aging-related processes during a single malaria infection.Cellular aging is difficult to study in individuals with natural infection, given the diversity of symptom duration and clinical presentation, and the high interference of aging-related processes with host and environmental factors. To address this challenge, we took advantage of the controlled human malaria infection (CHMI) model. This approach allowed us to characterize the relationship among cellular aging markers prior, during and post malaria pathophysiology in humans, controlling for infection dose, individual heterogeneity, previous exposure and co-infections. We demonstrate that already low levels of  The clinical consequences of acute and chronic malaria are well recognized, however, there might be a hidden long term cost for the host. Studies in birds have shown that mild chronic malaria infections accelerate telomere shortening, leading to decreased fitness and reduced life span5. A follow up study in travelers with imported malaria showed that an acute Plasmodium falciparum infection also affects cellular aging markers in humans1. While acute malaria infection has a pronounced effect on cellular aging, it remains elusive whether an extremely low parasitic P. falciparum infection is proficient to impact cellular aging kinetics.Cellular aging, the decline in the function, growth and division of cells is believed to be involved in the onset of various age-related diseases. Previous research suggests that infections are possible key-drivers of accelerated aging6. There are several hallmarks of cellular aging including telomere length (TL), cellular senescence and telomerase activity7. Telomeres, the non-coding repetitive sequences, play an essential role in chromosomal integrity, decrease with age and are linked to cellular senescence9. The effect of telomere shortening can be counteracted by the reverse transcriptase, telomerase; an enzyme expressed primarily in stem and germ cells10. Existing studies have linked accelerated telomere shortening to several human pathologies13. Besides malaria, shorter telomeres were shown in patients with HIV, Epstein–Barr virus (EBV), chronic hepatitis-C virus (HCV) and cytomegalovirus (CMV)17; with significant correlation of HCV and CMV to changes in the expression of the cellular senescent marker CDKN2A/p16INK4a19.Cellular aging is defined as progressive loss of physiological integrity and thus, the driving intrinsic component of increased age-specific mortalityP. falciparum parasitaemia triggers an array of host responses in both innate and adaptive immune system, including the expansion of helper and effector CD4+ T cells, governing the cell-mediated immune response through the production of pro-inflammatory cytokines 21, and generating free radicals to clear parasites. Inflammation and oxidative stress are further involved in age-related conditions22, by inducing age-associated functional losses23. However, the exact mechanisms of oxidative stress induced aging and its correlation to cellular senescence (CDKN2A/p16INK4a) is still not clear.Asexual 24. Moreover, the majority of studies in human are cross-sectional or cohort studies in individuals with acute or chronic infections with various clinical presentations and infection duration25. Hence, studying cellular aging kinetics in disease optimally requires a sample prior to infection and a well-controlled study setting.The study of aging markers in natural infections is challenging, as our knowledge is limited about whether accelerated aging is a direct consequence of infection, or whether aging markers increase susceptibility to disease. One previous study in human volunteers showed that individuals with longer telomeres were less prone to developing symptoms when infected with a respiratory virus, however aging markers were not followed over time after infection26. Here we used a CHMI, where healthy volunteers were infected with Plasmodium falciparum parasites using a standardized protocol with a known inoculation dose, well-defined pre and post infection follow up time points, close monitoring and prompt treatment upon the onset of symptoms and/or detection of parasite levels above protocol standards to examine the effect of low level parasitaemia on cellular aging. We characterize cellular aging markers together with a wide panel of inflammatory cytokines and anti-oxidant gene expression, blood biochemical analysis before, during and after infection, and further evaluate aging-associated loss of function, using biochemical liver function assays.Over the past decade, controlled human malaria infection (CHMI) has become a highly valuable model to study malaria pathophysiology and immunology, providing an efficient way to test the efficacy of new drugs and vaccines26. The Central Committee for Research involving human subjects (CCMO) located in the Netherlands, and the Western Institutional Review Board (WIRB) located in the USA approved the study protocol for the trial. Informed consent was obtained from all study participants. The study procedure corresponds to principles expressed in the Declaration of Helsinki and Good Clinical Practice standards and is registered in the database Clinical Trials.gov (NCT02836002), and all applied methods were carried out in accordance with relevant guidelines and regulations. The malaria challenge study recruited 16 healthy, malaria-naïve Dutch individuals from June until November 2016, and was conducted pursuant to the standard CHMI described in Sauerwein et al.27. Majority of the study participants were female (75%), age ranged from 20 to 29 years (median = 22), and bodyweight ranged from 57 to 90 kg (median = 73). General and baseline characteristics of the CHMI participants are described elsewhere26.The CHMI study was conducted at the Radboud University Medical Center, Nijmegen, the NetherlandsP. falciparum (Pf) infected mosquitoes for 10 min each, and monitored twice daily upon day six post infection (PI). Upon parasite density reaching ≥ 5000 parasites per milliliter blood , the initial sub-curative treatment of 500 mg/25 mg sulfadoxine–pyrimethamine  or 480 mg of piperaquine phosphate  was administered and participants were monitored twice daily for four days. Consequent to parasite density reaching ≥ 1500 Pf/mL (0.00004% iRBC), the second treatment was administered, and monitoring was pursued daily for three days, followed by three times a week until final treatment with curative regiment (ET) of atovaquone/proguanil on day 42. Study participants were followed until day 64 post infection and blood samples were collected on every respective visit to monitor the development of parasite density and blood safety parameters  was used in our analysis, and was interconverted to percentage of infected red blood cells (% iRBC).Parasitological assessment was performed by microscopy of thick blood smears and quantitative real-time PCR (qPCR)An overview of the number of study participants and time-points, and the measured markers per individual are found in supplementary materials Table .Total nucleic acid was extracted from in total 481 whole blood samples from the respective 29–31 time-points in 16 study participants, using an automated extraction system .1. This approach was chosen in respect of the large number of samples (N = 481), considering the time consumption of other quantitative measurements (TRF and STELA). DNA was purified from total nucleic acid, using Ambion RNase I , and diluted to a concentration of 2 ng μl−1 to measure TL on a QuantStudio 5 qPCR instrument. A telomere-specific amplicon set of primers  was used, together with a single-copy gene amplicon set of primers  to control for total amount of DNA in each reaction30. The total qPCR reaction contained 25 μl, including 5 μl DNA (2 ng/μl), 10 μl Supermix , 0.2 μl of each tel and HBG primer (10 μmol/L) and ddH2O. The primer sequences for tel are found in O’Callaghan and Fenech36 and sequences of single-copy gene primers are described elsewhere31. Each plate included samples, a Gold standard (an arbitrary sample included on each plate to control for inter-plate variability), a serial dilution of sample (to produce standard curve), and a non-template control. All samples were run in duplicates for each primer set, and each primer set was run on a separate plate. Average quantitative values of duplicates were normalized across the plates by dividing them by the plate value of the Gold control to avoid inter-plate variability and to obtain plate adjusted amounts of telomere and single-copy gene sequence. Relative TL was calculated by dividing the normalized telomere sequence (T) by normalized single copy gene sequence (S). This approach corresponds to ΔΔCT values, as – log2 of T/S is equal to ΔΔCT32. Initially, our samples were run to analyze T/S ratio, while a genomic DNA sample  was included as an additional reference sample on later analysis of 92 samples to compare T/S values with kb values. Our T/S ratio was then extrapolated for each sample based on the formula (T/S ratio 1 = 538 bp), to calculate TL in kb for all samples, referring to our human genomic DNA reference sample. Inclusion of gDNA sample on every qPCR plate (N = 24) resulted in non-significant variation in quantitative values . Moreover, our method showed high intra- and inter-plate repeatability, ICC > 0.98, which was in line with our previous studies33.Telomere length was assessed by quantitative real-time PCR (qPCR) by following the method described in Asghar et al.RNA was isolated from whole blood as total nucleic acid at three time-points C-1 (pre-infection), DT+1 (day after treatment 1) and C+64 (day 64 post-infection) for all study participants, with one participant lacking the sample for C+64. Total nucleic acid was treated with DNase to remove DNA contamination, using the TURBO DNA-free Kit . After RNA assessment, 100–150 ng RNA was converted to cDNA applying the SuperScript VILO cDNA Synthesis Kit , according to the manufacturer´s instructions.CT method by calculating the CT values of the target gene against the CT values of our reference gene (GAPDH)1. Our target gene and endogenous control (GAPDH) were amplified in same wells, run in duplicates and respective CRT values were averaged before performing the ΔCT calculation (ΔCT = CTTarget – CTGAPDH). The corresponding ratio refers to “expression of gene of interest”. Fold change was calculated as follows, DT+1/C-1 and C+64/C-1.Real-time PCR was performed on the corresponding cDNA synthesized from samples C-1, DT+1 and C+64. Relative gene expression of CDKN2A, telomerase and anti-oxidants was determined using the comparative Δ2O. TaqMan GAPDH Assay  was added to each run as an endogenous control.CDKN2A expression was measured using TaqMan Gene Expression Assay  on a QuantStudio 5 qPCR instrument. The total qPCR reaction of 20 µl contained 5 µl cDNA, 10 µl TaqMan Multiplex Master Mix , 1 µl GAPDH TaqMan Assay , 1 µl CDKN2A GAPDH TaqMan Assay,  and ddH2O.Telomerase expression was measured using the human hTERT TaqMan Assay .Relative gene expression was measured for five genes, including superoxide dismutases , catalase (CAT # Hs00156308_m1), glutathione S-transferase Kappa (GSTK1 # Hs01114170_m1) and nitric oxide synthase (NOS3 # Hs01574665_m1). Each 20 μl qPCR reaction contained, 5 μl cDNA, 10 μl TaqMan Multiplex Master Mix , 1 μl GAPDH Assay, 1 μl of either SOD1/SOD2/CAT/GASTK1or NOS3 Assay and ddH34. This kit was used to analyze 13 cytokines, including IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 simultaneously, as described in Reuling et al.35.Cytokine levels in citrate-plasma at three time-points C-1, DT+1 and C+64 were quantified using the LEGENDplex Multi-Analyte Flow Assay Kit , a bead-based immunoassay referring to the basic principle of sandwich immunoassays, according to manufacturer´s instruction35. Liver function test abnormalities (#LFT) were assessed at three time-points , and were graded based on grade 1: 1.1.–2.5X ULN, grade 2: 2.6–5.0 × ULN, grade 3: > 5.0X ULN, using an adaptation of the World Health Organization (WHO) Adverse Event Grading System 2003, as described in Reuling et al.26.Standard biochemical blood tests were performed to assess liver enzymes and renal function during the challenge, and included aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γGT), alkaline phosphatase (ALP), lactate dehydrogenase (LD), and total bilirubin. All clinical laboratory data refer to standard tests performed on peripheral whole blood and reference laboratory values are found in Table 36. Splines were randomized and fitted to three knots using the Stata statistic software, version 15 . To investigate whether the level of parasite density was influenced by any host factor, we ran a separate cubic spline mixed model fitted by restricted maximum likelihood, including parasite density as outcome variable. Parasite density was log-transformed as values were not normally distributed. A paired t-test was used to investigate the change of TL, gene expression and cytokine levels on C-1, DT+1 and C+64. All variable values were log-transformed before paired t-test and for all further analysis. To investigate the potential correlation between variables, we used the mathematical and topological features of Spearman’s correlation (rs) and visualized it as arc diagram using R-studio, version 1.1.442, http://www.R-priject.org37.A cubic spline mixed model with restricted maximum likelihood was used to investigate telomere dynamics in association with host factors , including individual ID as a random factor. These adjustments were made for each variable separately in a univariate model, and jointly for all variables in a multivariate model. Time was modeled using restricted randomized cubic splines, connected by knots and constricted to follow a linear distribution before the first and after the last knotNext, we performed a three-step analysis. First, we used Hierarchical Clustering to obtain a dendrogram that assembles variables based on similarities of variables according to time-points . We included the variables that showed significant change between C-1 and DT+1. Second, we used the Principal Component Analysis (PCA) approach to reduce the number of variables to avoid multiple co-linearities among the variables, using the two-clusters based on hierarchical cluster analysis. PCA is an unsupervised learning algorithm providing dimensions (linear correlations) along with the data that are separable, and reduces the noise associated with data whilst increasing its robustness. From the PCA analysis, we have selected the first three PCA axes , in total explaining 78.6% of the variance. Third, we did Factor analysis, based on our PCA results with three Factors, using Varimax method . We used the individual Factor values on each of the three axes as dependent variable in a cubical spline mixed model to investigate how these values varied as a function time, age and sex. Time was fitted as cubical spline function, age and sex as fixed factors and individual ID as random factor.P. falciparum parasites by exposure to five infected Anopheles mosquitoes, and followed until day 64 post inoculation (PI). Blood samples were collected before infection and twice a day during day 6–15, daily during day 16–24, then every second day until day 45, and on day 64, respectively. P. falciparum parasites were detected by real time qPCR in peripheral blood from day 7–9 and reached a maximum level of 22,092 Pf/mL (median parasites/mL blood) between days 10–13 post challenge. After administration of the first treatment (DT+1) between days 9–13, very low levels of parasites were detected, while after the second treatment between days 20–23 all participants were qPCR negative 1, peak parasite density in study participants of the CHMI ranged from 0.000062 to 0.0049% iRBC (median 22.09 Pf/µL), indicating the low parasitic CHMI protocol. No serious adverse events were reported during the study period.Sixteen healthy, malaria-naïve individuals were infected once with 2 = 26.13, N = 139, p < 0.001, Fig. P. falciparum infection accelerated telomere shortening, with the shortest telomeres observed between day 10–13 post challenge . Telomere length and parasite density were negatively correlated  was measured in peripheral blood n = 481) from 29 to 31 samples per participant 26. As described in Reuling et al., this study noted one grade three abnormality . All study participants showed mild to severe ALT/AST elevations; 5/16 (31%) mild (grade 1); 3/16 (19%) moderate (grade 2), and 8/16 (50%) participants showed severe (grade 3) (up to 25 × ULN) ALT/AST elevations26. Besides the positive correlation of #LFT with inflammatory parameters, as reported in Reuling et al., #LFT were further positively correlated with parasite density and CDKN2A levels, while negatively correlated with TL  and day post treatment 1 (DT+1), with a significant decrease in TL  and post infection (C+64), and highlighting the distinctive difference to the marker profile during infection (DT+1). This difference is characterized by an increase in cytokine levels and CDKN2A expression, higher #LFT, lower anti-oxidant gene expression and reduced TL at DT+1 Fig. A, compar2 = 690.09, df = 103.89, p < 0.001), PC 2 explains 15.4% variance  and PC 3 explains 9.1% variance  and aging-related processes (inflammation and oxidative stress) during a single malaria infection. Existing studies in malaria are often restricted in individual heterogeneity, history of malaria exposure and the effect of environmental factors, such as co-infections; key factors that are well-known to influence TL and other cellular aging processes24. The overproduction of pro-oxygen compounds can trigger activation of leukocytes, enhancing the expression of cytokines, chemokines and other inflammatory mediators38. An imbalance between oxidants and anti-oxidants, and a lack of anti-inflammatory defense systems has been shown to cause cellular damage and aging39. In terms of cellular aging, increase in pro-inflammatory cytokines has been shown to induce a reinforced senescence status, impacting cell plasticity and stemness features40. In this study, secretion of several inflammatory cytokines was elevated, while cytokine levels of IL-1β and IL-6, key players in clinical malaria41, together with TNFα were not detected. The absence of IL-1β and IL-6 and TNFα during CHMI is not unusual as reported in other controlled malaria studies43. This might be due to strict monitoring and early drug treatment of parasitaemia, compared to high parasite levels and severe clinical pathology in natural malaria infection. Another explanation could be the use of citrate plasma for cytokine measurement, which is known to compromise the IL-6 detection44.Both, cells of the innate and adaptive immune response produce free radicals, reactive oxygen species (ROS) and inflammatory factors for the defense and destruction of pathogens. Inflammation and oxidative stress are interlinked processes, which play an important role in invoking an immune response, but also contribute to age-related loss of homeostasis and health21, inflammation induced by the malaria parasite and the host’s production of ROS can give rise to pronounced DNA damage45, with telomeres being preferential targets for oxidative damage-leading to a senescence- associated inflammatory response, including elevated IL-6, IL-8 and INF-γ levels46. Critically short telomeres or damage in these non-coding sequences are known mechanisms inducing cellular senescence47. Accumulation of senescent cells in turn, leads to the acquisition of the senescence-associated secretory phenotype (SASP), which has a deleterious effect on the tissue microenvironment by further increasing pro-inflammatory cells, most prominently inducing IL-649. The pro-inflammatory phenotype consequently leads to an imbalance of the cellular redox state, generating even higher amounts of oxidative stress. It is well established that deprivation of anti-oxidant mechanisms along with oxidative damage play a major role in several pathways leading to cellular senescence50, mainly reasoned by declining levels of IL-10 that cause inflamm-aging  inflammatory processes initiated by the host’s response to infection53, (b) reactive species produced directly by the parasite52, (c) Plasmodium infected erythrocytes producing twice as much hydroxyl radicals compared to uninfected erythrocytes54 and (d) anti-malaria treatment57.Results from this study show that anti-oxidant gene expression was down regulated for SOD1, SOD2, NOS3, CAT and GSTK during infection, which is in line with results published by Reuling et al.58, suggesting that these might be abundant cell type changes. However, we did not find that change in cell composition  was associated with telomere length in our previous study of naturally acquired malaria in travelers1.In this study, all markers  were assessed by extracting RNA from whole blood. In previous studies, using an exploratory approach with mass cytometry, followed by targeted flow cytometry in individuals with naturally acquired malaria, we have shown an expansion of T helper cells and memory B cells35 reported an association between parasitic density, inflammation parameters and liver injury (including data from this study) during an uncomplicated malaria infection. Here, we further explored the relationship between liver injury and cellular senescent markers (telomere length and CDKN2A). The association of liver function test abnormality score with cytokines, anti-oxidant enzymes and CDKN2A expression in this study suggests that increased cytokine production and low anti-oxidant gene expression in whole blood might also reflect DNA damage (including telomere shortening and elevated CDKN2A expression) in human hepatic cells60. These findings are concurrent with an experimental malaria study in birds showing that malaria infection causes systemic stress that shortens TL in multiple body tissues, including blood, liver, lung, kidney, spleen, brain and heart6.Recently, Reuling et al.1. An adjacent hypothesis of telomere restoration and increased telomerase activity is the activation and differentiation of T lymphocytes61, as part of humoral immune response. Majority of activated cells die during immune activation or drug treatment, however, memory T and B cells with longer telomere length remain in circulation for longer time, which could partially explain telomere restoration in whole blood , which might be enough to restore TL (Fig. In conclusion, using a novel approach of controlled human malaria infection with daily samples, we characterized the impact of low-density malaria infection on cellular aging markers, and their complete restoration after successful treatment. Our work presents an integrative model of hallmarks of aging during infection, combining data of aging marker dynamics with inflammation and oxidative stress, generated by extremely low levels of malaria parasitaemia. Our findings stress that even short term exposure to human pathogens generates a high host response, given the fact that even low parasite exposure impacts cellular aging. These results further urge the need to explore the impact of malaria in endemic areas, considering that continuous exposure to the parasite, repeated clinical episodes, and chronic asymptomatic infections could potentially lead to permanent long-term consequences on cellular aging and immune function, which may cause irreversible health consequences in human. We therefore stress future studies to evaluate infection-induced long term consequences in human living under chronic disease exposure, to not only emphasize the need of malaria eradication, but also imply intervention methods based on the parasite-altered cellular mechanisms in the host.Supplementary Information.


In Ethiopia, anti-malaria treatment is initiated after parasitological confirmation using blood film microscopy at health centers and hospitals, or serological rapid diagnostic tests at health posts. At health posts, the diagnosis is performed by health extension workers using rapid diagnostic tests after little training. However, there is paucity of data about the health extension workers’ performance on rapid diagnostic tests. Hence, periodic monitoring of the performances of health extension workers on malaria rapid diagnostic tests and predicted factors plays a pivotal role for the control of malaria.A cross sectional study was conducted in May 2020, among 75 health extension workers working at health posts in Bahir Dar Zuria district, Northwest Ethiopia. Their performance on malaria rapid diagnostic tests was assessed by distributing known positive and negative samples as confirmed by investigators using both rapid diagnostic test and blood film microscopy. Test results from health extension workers were then compared with that of investigators. Procedural errors committed while performing the tests were assessed using observational checklist. Data were analyzed using SPSS software version 20.Plasmodium species were 96.8% and 98.7%, respectively with 97.3% result agreement between the health extension workers and investigators . The most common procedural errors committed by health extension workers was ‘not checking expiry date of the test kits’ followed by ‘not adhering to the appropriate time of reading results’ that 70.7% and 64% of the participants committed these errors, respectively. Total number of procedural errors committed by those who have got in-service training was decreased by 47.3% as compared to those without in-service training.The overall sensitivity and specificity of health extension workers in detecting Health extension workers had high performance on malaria rapid diagnostic tests. However, in-service training and periodic supervision should be given in order to maximize performance on these tests. Plasmodium [Plasmodium falciparum , Plasmodium vivax (P. vivax), Plasmodium malarae , Plasmodium ovale , and Plasmodium knowlesi (P. knowlesi) naturally infect humans [P. falciparum and P. vivax are the most prevalent species globally [Malaria is a vector borne disease caused by protozoan parasites of the genus asmodium . Five spt humans . Of thesglobally .According to World Health Organization (WHO) 2019 report, malaria is one of the most common diseases with major global public health problem. About 228 million malaria cases occurred in 2018 causing 405, 000 deaths worldwide. Most malaria cases and deaths occur in African Region, which accounted for 93% (213 million) and 94%  of all malaria cases and deaths, respectively in 2018 [P. falciparum and P. vivax are the two dominant species accounted for 69% and 30% of infections, respectively [In Ethiopia, about 68% of the population is at risk of the disease and there are 1–2 million annual confirmed malaria cases. There were 356 reported deaths in 2017 [ectively . MalariaPlasmodium species antigens. Hence, they are based on the detection of either histidine-rich protein 2 (HRP2), a protein synthesized by P. falciparum only and lactate dehydrogenase (LDH) or aldolase, enzymes produced by all human Plasmodium species [The WHO recommends that anti-malarial treatment should be initiated after parasitological confirmation of suspected cases . As a re species , 9.Rapid diagnostic tests have a significant impact on the reduction of malaria burden in the world . Since tKebele (village). Health extension workers are recruited from the communities in which they will work according to specific criteria: they are female , at least 18 years old, have completed at least grade 10 in education, and speak the local language. Females are selected because most of the HEP packages are related to issues affecting mothers and children; since communication is thought to be easier between mothers and female HEWs [The HEP is implemented at health post or community level which is the lowest level of the Ethiopian health tier system. The HEWs are community health workers who are trained for one year in order to provide basic health services such as family planning, vaccination, health education, diagnosis and treatment of certain diseases like diarrhea and malaria. In this program, two female HEWs are assigned to a health post in each ale HEWs .The main objective of the HEP in Ethiopia is to improve access to essential health services provided at the village and household levels . DespiteDespite malaria RDTs are easy to perform with little training, strictly following the manufacturer’s instructions is important to have reliable results. Hence, periodic monitoring of HEWs while performing the RDT procedures is mandatory to take timely corrections, in case errors are committed. However, there is paucity of data about the HEWs’ performance on malaria RDTs in Ethiopia. Therefore, the present study aimed to assess the performances of HEWs on malaria RDTs and predicting factors in Bahir Dar Zuria district, where malaria is a public health problem.kebeles and 9 health centers and 36 health posts where 81 HEWs have been employed.A cross-sectional study was conducted at all health posts in Bahir Dar Zuria district, Northwest Ethiopia in May 2020. Bahir Dar Zuria district is one of the 14 districts in West Gojjam Zone, which is located at a distance of 560 km from capital city of the country (Addis Ababa) and it is situated surrounding Bahir Dar city, capital city of Amhara National Regional State. The altitude of the district ranges from 1700–2300 meters above sea level . The disAll HEWs who were actively on duty during data collection period and gave consent to participate in the study were included.Data on predicting factors for committing procedural errors while performing RDTs were collected using structured questionnaire. The questionnaire was designed to collect information about HEWs’ age (in years), prior experiences in performing malaria RDTs (in years), prior in-service refreshment training on malaria RDTs (yes/no), supply of RDTs to the health post so that the service has not been interrupted (yes/no), frequency of supervisions per quarter year by the malaria officer or other responsible body assigned to support HEWs in the diagnosis and treatment of malaria (number of visits per every 3 months), trust in RDT result by HEWs or their feelings in the accuracy of RDT results (trust/not trust).P. falciparum (pf), P. vivax (PV) and P. falciparum/P. vivax (pf/PV) mixed infections and from apparently healthy  individuals. Each of the collected blood samples was transferred into Ethylenediamine Tetra Acetic Acid containing test tubes. Seven blood samples  confirmed by both microscopy and RDT were distributed to 34 health posts and HEWs tested all samples using their own RDTs. Then data on the performance of each procedural step were collected using observational checklist prepared for this purpose. The prepared checklists divided the RDT procedure into 8 steps in three domains: procedural (checking RDT expired date), safety (discarding the pipette in the sharps box), as well as accuracy . Among the seven samples to be run by each HEW, one RDT procedure (4th sample) was strictly observed by principal investigator, noting whether the HEWs performed each step correctly or not.The HEWs’ performances on RDTs were evaluated using known positive and negative samples as confirmed by investigators using both RDT and blood film microscopy. Two ml of venous blood was collected by the principal investigator from laboratory confirmed malaria cases for Data were checked for completeness and entered and analyzed in statistical package for social science (SPSS) software version 20. Descriptive statistics like frequency and percentage were manipulated to explain the study participants and their test performance. Multivariable Poisson regression was fit to test for associations between error count data and pre-specified covariates . Selected pre-specified covariates were included in the analysis based on the hypothesized relationship to the outcome. P-value < 0.05 in the multivariate analysis was considered as statistically significant at 95% confidence level. Kappa value was also calculated to show the strength of test agreement between ‘HEWs and investigators’ and interpreted as follow: values ≤ 0 as indicating no agreement and 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreement .Performance of HEWs’ on malaria detection and species identification using RDTs were tested by computing the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) against investigators’ results using Med-Calc software version 19.3. The software calculates the value for each parameter with the sense of the following equations.sensitivity = a/n1×100specificity = b/n2×100positive predictive value = a/n3×100negative predictive value = b/n4×100where n1 = number of true positives identified by investigator;n2 = number of true negatives confirmed by investigator;n3 = number of samples identified as positives by HEWs;n4 = number of samples identified as negatives by the HEWs;a = number of cases identified as positives by investigator and by the HEWs;b = number of samples identified as negatives by investigator and by the HEWs.The research was carried out after ethical approval was obtained from the institutional review committee of College of Medicine and Health Sciences, Bahir Dar University with reference number CMHSc009/2020. Additionally, supportive letters were obtained from Amhara public health institute, West Gojjam Zone health department, and Bahir Dar Zuria district health office and permission was obtained from each health center authorities where clinical samples used to evaluate HEWs were collected. Information obtained at any course of the study was kept confidential. Informed verbal consent was obtained from participating HEWs and no personal and health facility identifier was included as participants were given a unique code. According to the research ethics guideline of Bahir Dar University, informed verbal consent is accepted to conduct researches where the data collection doesn’t include invasive procedures (like aspirate or biopsy samples). Hence, the verbal consent for the present study was approved by the institutional review committee. Health extension workers who committed procedural errors while performing RDTs were given feedback on the spot about the procedural errors they committed and how those errors could be avoided.Among the total of 81 HEWs employed in Bahir Dar zuria district, 75 participated in the study. The remaining 6 HEWs were not actively on work during data collection period due to different reasons. In this study, age of the participants ranged from 25–34 with mean age of 29.23 (± 2.077 SD) years. Forty-five (60.0%) participants have taken in-service training on malaria RDTs. The participants had work experience ranged from 5–14 years with mean experience of 8.96 (±2.5 SD) years .From a total of 75 HEWs participated in the study, all the 8 procedural steps was carried out correctly by more than 70% of the participants, except for steps 1,5 and 7. The median number of total and accuracy steps completed correctly was 6 and 4, respectively. The number of errors observed ranged from 0 to 5 errors per HEW. The steps most commonly performed incorrectly were step 1 and 7 .P. falciparum. From the 150 negative samples, 148(98.7%) were correctly reported as negative  samples were tested by HEWs. Of these, 511(97.3%) were correctly reported. Among 375 positive samples, 363 (96.8%) were correctly reported as positive and the rest 9(2.4%) were falsely reported as negative. Out of 75 samples with mixed infection, 72(96.0%) were correctly reported as mixed and the remaining 3 (4.0%) were falsely reported as negative .Plasmodium species were 96.8% and 98.7%, respectively while the overall positive and negative predictive values were 99.5% and 94.3%, respectively. The test agreement between the HEWs and investigators was 97.3% with kappa value of 0.949  for getting in-service training was 0.527(95% CI 0.365–0.763), which corresponds to the mean number of total errors for those who took in- service training being 47.3% (95% CI 23.7–63.5) lower when compared to those without training .Malaria rapid diagnostic tests have been important alternatives to diagnose malaria at health posts in Ethiopia as the tests can be performed by HEWs with little training. However, monitoring of the RDTs performance of HEWs in conducting the tests is indispensable to maximize the diagnostic quality of RDTs. In the present study, the performance of HEWs on RDTs were evaluated and reported for the first time in Ethiopia as far as our knowledge is concerned.Plasmodium species. The sensitivity of the present study was higher than the study reported in Western Kenya (92%). Such higher sensitivity in the present study might be due to variations in duration of experience in performing RDTs. However, the specificity of the present finding was comparable with previous result of 97.0% in Western Kenya [Health extension workers performed and interpreted RDT test results almost perfectly with sensitivity and specificity of 96.8% and 98.7%, respectively on detection and identification of rn Kenya .P. falciparum and one P.vivax), nine positive samples as false negative, and three samples with mixed infection as P. falciparum mono infection. These false positive and false negative results might be due to shipment and storage conditions of the RDT kits such as direct exposure to sunlight. Study conducted in Burkina Faso documented that transportation and storage condition of RDTs affect the test performance [As compared to the investigators’ result, there were two negative samples reported as false positive  as compared to those without training. This finding is supported by previous studies showing that training on RDTs is associated with improved performance , 18. MorPerformance of HEWs in patient preparation and sample collection were not evaluated. The optimal condition of RDT such as storage conditions was not observed. Moreover, the presence of observers may have impacted HEWs’ performance that formal observation can lead to a state of anxiety that decrease or increases in performance during supervision and evaluation , 29.Health extension workers in the present study area had good performance on malaria RDTs. However, the errors seen during observations are significant when viewed from the number of patients screened by each HEW every day. The most common procedural errors committed by HEWs were ‘not checking expiry date of the test kits’ and ‘not adhering to the appropriate time of reading results’. Prior training on malaria RDTs significantly affects the performance of HEWs in correctly completing each step of the test procedure. Regular supervision and in-service training should be given for HEWs on RDT procedures by cluster health centers in order to reduce errors observed in the present study. Further research should be conducted to investigate performance of HEWs’ in patient preparation and sample collection.S1 File(ZIP)Click here for additional data file.


Plasmodium falciparum and Plasmodium vivax infection.Malaria-associated anaemia, arising from symptomatic, asymptomatic and submicroscopic infections, is a significant cause of morbidity worldwide. Induced blood stage malaria volunteer infection studies (IBSM-VIS) provide a unique opportunity to evaluate the haematological response to early P. falciparum (Pf3D7), 15 with an artemisinin-resistant P. falciparum strain (PfK13) and 46 with P. vivax. Factors associated with the fractional fall in haemoglobin (Hb-FF) were evaluated, and the malaria-attributable erythrocyte loss after accounting for phlebotomy-related losses was estimated. The relative contribution of parasitized erythrocytes to the malaria-attributable erythrocyte loss was also estimated.This study was an analysis of the haemoglobin, red cell counts, and parasitaemia data from 315 participants enrolled in IBSM-VIS between 2012 and 2019, including 269 participants inoculated with the 3D7 strain of P. vivax, respectively. The median Hb-FF was 10.3% (IQR 7.8–13.3), 14.8% (IQR 11.8–15.9) and 11.7% (IQR 8.9–14.5) in those inoculated with Pf3D7, PfK13 and P. vivax, respectively, with the haemoglobin nadir occurring a median 12 (IQR 5–21), 15 (IQR 7–22), and 8 (IQR 7–15) days following inoculation. In participants inoculated with P. falciparum, recrudescence was associated with a greater Hb-FF, while in those with P. vivax, the Hb-FF was associated with a higher pre-treatment parasitaemia and later day of anti-malarial treatment. After accounting for phlebotomy-related blood losses, the estimated Hb-FF was 4.1% (IQR 3.1–5.3), 7.2% (IQR 5.8–7.8), and 4.9% (IQR 3.7–6.1) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. Parasitized erythrocytes were estimated to account for 0.015% (IQR 0.006–0.06), 0.128% (IQR 0.068–0.616) and 0.022% (IQR 0.008–0.082) of the malaria-attributable erythrocyte loss in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively.The median peak parasitaemia prior to treatment was 10,277 parasites/ml , 71,427 parasites/ml , and 34,840 parasites/ml  in participants inoculated with Pf3D7, PfK13, and P. falciparum and P. vivax infection resulted in a small but significant fall in haemoglobin despite parasitaemia only just at the level of microscopic detection. Loss of parasitized erythrocytes accounted for < 0.2% of the total malaria-attributable haemoglobin loss.Early experimental The online version contains supplementary material available at 10.1186/s12936-021-04003-7. Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae, and in all three species increases the risk of death  in PfK13 and 84/254 [33%] in Pf 3D7), and 7/46 (15%) in the P. vivax group.The overall median PPP. falciparum, the median haemoglobin fell from a baseline of 149 (IQR 139–156) g/L to a nadir of 132 (IQR 123–140) g/L, representing a median fall of 17 g/L and a fractional fall of 10.6% (IQR 7.9–13.8%)  and those inoculated with PfK13 . In participants with PfK13, the haemoglobin fell by a median 18 (IQR 12–23) g/L compared to 17 (IQR 11–20) g/L in the Pf3D7 group, representing a median fractional fall of 14.8% (IQR 11.8—15.9%), compared to 10.3% (IQR 7.8–13.3%) in the 3D7 group (p = 0.001)  g/L to 129 (IQR 121–140) g/L, with a total median fall of 18 (IQR 14–21) g/L and a median fractional fall of 11.7% (IQR 8.9–14.5), which compared to 10.6% (IQR 7.9–13.8) in the P. falciparum group (p = 0.07). The median fall in haemoglobin prior to treatment contributed to 45% (IQR 27–56) of the total fall. The haemoglobin nadir occurred a median of 8 (IQR 7–15) days following treatment and returned to baseline levels a median of 20 (IQR 18–25) days following treatment.In participants inoculated with P. falciparum, 99 (37%) participants who recrudesced had a fractional fall in haemoglobin of 12.0% , compared to 10.2%  in those who did not (p = 0.006). In the P. vivax group, the median haemoglobin fractional fall was similar in those who recrudesced  compared to those who did not .In participants inoculated with P. falciparum participants, there was no significant difference observed in the haemoglobin nadir or fractional fall in haemoglobin between age groups   as well as a lower haemoglobin nadir .P. vivax, where day of treatment was more variable, the median haemoglobin nadir was lower in participants treated on Day 10–14 , compared to those treated on Day 8 . The median fractional fall in haemoglobin was also greater in those treated at Day 10–14 (12.4% [IQR 9.7–14.6]), compared to those treated on Day 8  , artefenomel (n = 8) or artemether/lumefantrine (n = 14), with no significant differences in the haemoglobin parameters observed between treatment groups  and fractional fall of haemoglobin in the overall Pre  and PMR  and the fractional fall in haemoglobin in the PfK13 group, although all (15/15) PfK13 participants recrudesced , there were no correlations between any of the parasitaemia variables and haemoglobin nadir or fractional fall in haemoglobin, even after adjusting for drugs , the PPPre correlated with the fractional fall in haemoglobin , with this correlation remaining significant when adjusted for drugs (p = 0.041). The TPBPre correlated with the haemoglobin nadir  and fractional fall in haemoglobin , with both correlations remaining significant after adjustment for drug   × 1012 erythrocytes/L, with this loss being greater in those inoculated with PfK13 (median 0.7 [IQR 0.6–0.8] × 1012/L) compared to Pf3D7  (Table P. vivax (median 0.5 [IQR 0.4–0.6] × 1012 erythrocytes/L) was similar to those inoculated with Pf3D7.In participants inoculated with 5) Table . The ery12/L in participants inoculated with P. falciparum, or ~ 42% of the total erythrocyte loss. Again, this loss was greater in the PfK13 group  compared to Pf3D7 group . In the P. vivax group, the median malaria-attributable erythrocyte loss was 0.21 (IQR 0.4–0.33) × 1012/L, similar to the erythrocyte loss in the Pf3D7 group  × 10–0.36 × 1P. falciparum, parasitized cells accounted for an estimated 0.015% (IQR 0.006–0.060%) of the malaria-attributable erythrocyte loss. This proportion was significantly higher in the PfK13 group (median 0.128 [IQR 0.068–0.616] %) compared to the Pf3D7 group . In those inoculated with P. vivax, parasitized cells accounted for an estimated 0.022 (IQR 0.008–0.082) % of the malaria-attributable erythrocyte loss. The loss of parasitized cells as a percentage of the total malaria-attributable erythrocyte loss was associated with the TPBPre in participants inoculated with each species  and with each P. falciparum inocula .In participants inoculated with P. falciparum, the reticulocyte count increased from a median of 55 (IQR 44–68) × 109/L at baseline to 68 (IQR 54–90) × 109/L at EOS, representing a median 22% increase (p = 0.0001)  but not in the in the P. vivax group . In the P. falciparum group, there was a correlation between age and the reticulocyte response , although this was not observed in the P. vivax group.In those inoculated with 1) Table . A similP. falciparum and P. vivax infection is described. The analysis demonstrates that in both P. falciparum and P. vivax, experimental infection results in an ~ 11% fractional fall in haemoglobin, approximately half of which occurs prior to treatment. The haemoglobin nadir occurred ~ 12 days after treatment in participants inoculated with P. falciparum and 8 days in participants inoculated with P. vivax, returning to normal by 28 days in P. falciparum and 20 days in P. vivax.In this study, the haematological response to early experimental P. vivax, after accounting for phlebotomy related losses. It should be noted that in experimental malaria the duration of parasitaemia prior to treatment is only a few days, in contrast to endemic regions where low-level asymptomatic parasitaemia may persist in partially immune individuals for prolonged periods, thus the haemoglobin loss may be expected to be substantially greater. This is supported by recent studies from endemic regions, which have demonstrated a significant burden of anaemia associated with submicroscopic parasitaemia [Volunteer infection studies are associated with frequent blood sampling, and it was estimated that phlebotomy-related losses accounted for ~ 60% of the total fractional fall in haemoglobin. Nonetheless, the malaria-attributable loss from early experimental malaria, with parasitaemias only just reaching the limit of detection by microscopy, still accounted for a fractional fall in haemoglobin of ~ 4% in Pf3D7, ~ 7% in PfK13 and ~ 5% in sitaemia , 11.P. falciparum, the fractional fall in haemoglobin was greater in those inoculated with an artemisinin-resistant PfK13 strain, compared to those inoculated with the artemisinin-sensitive Pf3D7 strain. Contributing factors that may have accounted for this greater fractional fall include a later day of treatment and higher parasitaemias in participants inoculated with PfK13 compared to Pf3D7, as well as the slower parasite reduction ratio, and the recrudescence that occurred in all of the PfK13 participants following treatment with artesunate. The day of haemoglobin nadir also occurred later in the PfK13 participants, and no participant recovered their haemoglobin prior to the end of study, again likely reflecting delayed parasite clearance and recrudescent parasitaemia. In the PfK13 participants the parasite reduction ratio was significantly and inversely correlated with the fall in haemoglobin, with slower parasite clearance associated with greater haemoglobin fall. Although this study was not designed to directly compare haemoglobin losses in participants inoculated with PfK13 vs Pf3D7, the results reported here are consistent with data from clinical studies reporting a greater incidence of anaemia in patients with drug-resistant P. falciparum [In participants inoculated with lciparum , 55 and lciparum –59. ThusConsistent with the greater fall in haemoglobin in participants inoculated with PfK13, all of whom recrudesced, within the Pf3D7 group those who recrudesced also experienced a significantly greater fall in haemoglobin. These data are consistent with previous studies which have also demonstrated higher rates of anaemia associated with parasite recrudescence .It is also possible that the greater fractional fall in haemoglobin observed in participants inoculated with PfK13 may have been due in part to the fact that these participants were treated with artesunate. Post-artesunate anaemia is well described, and results from delayed clearance of once-infected erythrocytes . HoweverP. vivax, we found that delaying day of treatment was associated with a greater fall in haemoglobin. This is also consistent with clinical studies reporting correlations between the time since symptom onset and degree of anaemia [In participants inoculated with  anaemia –64, and P. vivax, a correlation between parasitaemia  and the fractional fall in haemoglobin was observed. This is as expected, and consistent with clinical data demonstrating a correlation between parasitaemia and anaemia [In participants inoculated with  anaemia , 64. UneP. falciparum malaria in over 4000 children and adults in Thailand during 1990–1995, and estimated that parasitized cells accounted for 7.9% of the total malaria-attributable loss. A similar estimate was obtained by Jakeman et al. [P. falciparum parasitized cells accounted for ~ 10.5% of erythrocytes lost. In neurosyphilis patients inoculated with P. vivax, Collins et al. [P. vivax. This suggests that in submicroscopic infections, the relative contribution of the loss of unparasitized cells to malarial anaemia is likely much greater than that seen in higher parasitaemia infections.In this study we attempted to quantify the loss of parasitized cells as a proportion of the overall malaria-attributable erythrocyte loss. Similar analyses have been conducted previously, including in clinical malaria in endemic regions , and in n et al. , who uses et al.  estimateThe mechanisms mediating the loss of unparasitized cells at such low parasitaemias remain incompletely understood. In malaria volunteer infection studies, despite the low parasite counts, participants still experience a significant inflammatory response, with elevated levels of IFN-γ and IL-6 –67 potenP. vivax infection studies, the calculation of total parasite burden prior to antimalarial treatment did not account for parasite sequestration. Recent studies have suggested that in chronic P. vivax infection, a very high proportion of parasitized erythrocytes are sequestered in the spleen [P. vivax infection [This study had a number of limitations. First, there was substantial heterogeneity between and within the individual malaria volunteer infection studies, including the drug used, the day of treatment, and the study duration, making it difficult to account for factors associated with haemoglobin loss. Second, in the e spleen , and it nfection . It is pnfection . FinallyIn summary, this study demonstrates that a small but statistically significant fall in haemoglobin occurs in experimental malaria infection, despite parasitaemias that are only just at the level of microscopic detection. This study adds to studies from endemic regions reporting a significant burden of anaemia from asymptomatic and submicroscopic infections, highlighting the importance of treating these groups to reduce the overall burden of anaemia. Finally, this detailed description of the expected haemoglobin loss in malaria volunteer infection studies can be used as a baseline against which to compare the haemoglobin losses that may occur when new antimalarial drugs are evaluated in this model.Additional file 1. Additional tables.


P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes.There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses .The gene encoding KAHRP was deleted from 5 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B ; one subject developed mild symptoms on day 28 ; and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo.None of the subjects who were administered with 1800 or 1.8 × 10P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses.This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A ACTRN12617000824369; date: 06 June 2017).Australian New Zealand Clinical Trials Registry (number: The online version contains supplementary material available at 10.1186/s12916-021-02150-x. There is currently a strong commitment to eradicate malaria in the 21st century. This ambitious goal will be facilitated by the development of an effective vaccine. The past 20 years have seen over 100 clinical trials of malaria vaccine candidates, with the vast majority testing sub-unit vaccines . Given tLive, whole cell vaccines offer several potential advantages over subunit vaccines against a range of pathogens including malaria parasites. These include exposure to a greater range of antigens that are delivered to the correct anatomical compartments for immunological priming, as well as providing the correct pattern recognition signals to the immune system, which are specific for the pathogen class . VariousP. falciparum erythrocyte membrane protein 1 (PfEMP1) is a polymorphic adhesion ligand displayed on knob-like structures on the surface of P. falciparum-infected erythrocytes, with specific types binding a variety of host receptors present on the vascular endothelium [var genes per genome [var gene allele, an approach that may be technically challenging. Further, if such a result were achieved it would result in no expression of PfEMP1, a protein which has been proposed to be the target of strain-specific immunity [othelium . This arothelium . Furtherothelium . PfEMP1 othelium  and has othelium . PfEMP1 r genome . Thus, cimmunity .P. falciparum clone selected under in vitro culture was investigated in Aotus monkeys by Langreth and Peterson [The in vivo virulence and immunogenicity of a knobless Peterson . After mP. falciparum-infected erythrocytes lack knob-structures and cannot correctly display PfEMP1 [Aotus monkeys, we hypothesized that the loss of KAHRP would result in a significant loss of fitness in vivo due to enhanced parasite clearance accompanied by priming of the immune system from lack of parasite sequestration and increased passage to the spleen. The production of the blood-stage GAP vaccine under Good Manufacturing Practice (GMP) by targeted deletion of the gene encoding KAHRP has been described previously [P. falciparum kahrp– blood-stage GAP vaccine in healthy volunteers.Our approach to develop a blood-stage GAP vaccine was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. In the absence of KAHRP, y PfEMP1 . Buildineviously . Here weP. falciparum. However, it was decided not to proceed with the second part due to the lack of complete attenuation of the GAP vaccine, and alloimmunization events, observed in the first part (see the “Results” section). The study design and methodology for the second part of the study are not described here.This phase 1 study was planned to be composed of two parts. The first part, performed as planned, was an open-label, dose-escalation study to determine the safety, infectivity, and immunogenicity of single doses of the GAP vaccine administered intravenously. The second part was planned to provide preliminary information on the efficacy of the GAP vaccine by determining the protective immune response of GAP vaccination to subsequent challenge with wild-type blood-stage Healthy malaria naïve males aged 18 to 55 years were eligible for inclusion in the study . A sentinel dosing strategy was used for the first three cohorts whereby the first subject in each cohort was inoculated with the GAP vaccine at least 24 h before the second subject. Subjects in cohort 4 were inoculated with the GAP vaccine concurrently since no significant safety signals had been observed in the preceding cohorts.P. falciparum 18S rRNA [Parasitemia was monitored throughout the study by collecting blood samples and performing quantitative PCR (qPCR) targeting the gene encoding 18S rRNA . SubjectP. falciparum 3D7 wild-type parasites routinely used in previous controlled human malaria infection (CHMI) studies using the induced blood-stage malaria (IBSM) model [5 viable parasites for cohort 2 (100-fold increase from cohort 1) and 3 × 106 viable parasites for cohort 3 , but with the antimalarial treatment criteria amended with a longer follow-up to allow for better characterization of parasite infectivity. A planned protocol deviation was performed for cohort 3 due to the unexpected results observed; subject 6 was administered antimalarial treatment on day 35 when parasitemia reached 5000 parasites/mL instead of the protocol stipulated day 28. This was considered reasonable since the subject was asymptomatic and consented to the additional period of follow-up. The QIMR Berghofer Medical Research Institute Human Research Ethics Committee was notified of this decision.The GAP vaccine dose administered to cohort 1 was 1800 viable parasites which is the dose of M) model –23. A foMI study ). Based Safety assessments were performed throughout the study and included adverse event (AE) recording, physical examinations, vital signs monitoring, electrocardiographs (ECGs), clinical laboratory evaluation , and malaria clinical score recording . The sevP. falciparum merozoite surface protein 2 (MSP2) and to whole parasite lysate  at any time-point during the study , although parasitemia was only detected very briefly and at a very low level (around the limit of detection) in one of these subjects . The other three subjects developed significant parasitemia, the progression of which was highly variable between subjects. Antimalarial treatment with artemether/lumefantrine was initiated on day 17, day 28, and day 35 for these subjects at a parasitemia of 14,022 parasites/mL, 19,956 parasites/mL, and 5151 parasites/mL, respectively  or cohort 2 (administered 1.8 × 106 viable parasites) that were considered to be possibly related to the GAP vaccine. Twelve of these AEs occurred in one subject (subject 7) and were mild symptoms/signs frequently associated with malaria . However, this subject was confirmed by PCR to be positive for influenza B at the time of antimalarial treatment (which coincided with the onset of the majority of the signs/symptoms listed above); thus the symptoms and signs observed may not have been malaria-related. Another subject (subject 5) experienced mild fatigue (24 h duration) which was considered possibly related to the GAP vaccine.A total of 44 adverse events (AEs) were reported in this study, the majority of which were of mild severity (Table Alloimmunization to minor erythrocyte antigens was observed in two subjects (subject 7 and subject 8) at the end of the study visit on day 90, which was considered related to the GAP vaccine. Subject 7 developed low titer anti-C and anti-P1 antibodies, while subject 8 developed low titer anti-c antibodies. Retrospective testing was performed on blood samples collected on day 21 (subject 7) and day 31 (subject 8). Subject 7 was positive for anti-P1 on day 21 (there was an insufficient sample to test for anti-C at this time point); subject 8 was negative for anti-c on day 31. The blood group and phenotype of these subjects, as well as the donor red blood cells used to manufacture the GAP master cell bank, are presented in Additional File 6 parasites  also developed a malaria-specific antibody response. The antibody response to a key immunogenic blood-stage antigen (MSP2) was measured. The timing of the anti-MSP2 IgG response , to elevated expression of most of the var repertoire in two subjects (subject 5 and subject 6) and a less broad but still fairly diverse pattern of expression in subject 7 .This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. We demonstrate that culation , 46, 47.P. falciparum kahrp– parasites are capable of generating a significant infection in humans was somewhat unexpected based on the well-characterized role of KAHRP in mediating the adherence of infected erythrocytes to the microvascular endothelium, which is an important process in promoting parasite survival by evading splenic clearance [Aotus monkeys challenged with multiple doses of knobless P. falciparum parasites (up to 1× 107 parasites) [P. falciparum 3D7 parasites [The fact that learance . The in rasites) . The delarasites , there avar genes during in vivo growth. Parasites administered to subjects on day 0 expressed a single dominant group C var gene. This is consistent with previous evidence that, in the absence of phenotype selection, parasites often switch to expression of a group C var gene which appear to have a slower rate of silencing than other var gene types [var gene, suggesting selection in the naïve, human host, possibly for an adhesion phenotype. The increased diversity of var expression could also reflect the absence of selection for cytoadhesive variants, because the infected erythrocytes could not cytoadhere. If this were the case, then the altered environment within the host stimulated the de-repression of much of the var repertoire. Interestingly a similar observation of broad var de-repression has been made in CHMI studies using sporozoites [var genes, consistent with the broad, epigenetic reprogramming that occurs through sexual reproduction in higher order eukaryotes. The data we present suggests that the human host environment itself may stimulate var gene de-repression during the parasite’s asexual lifecycle.There was evidence that parasites switched their expression of ne types . Followirozoites . In thatP. falciparum clones were found to adhere to CD36 at a level comparable to the parent 3D7 strain under static conditions, but dramatic differences were observed under flow conditions [P. falciparum kahrp– parasites exhibiting some adhesion to CD36 under static conditions  and almost no binding under flow conditions. These results are supported by recent research using engineered human capillaries which demonstrated that erythrocytes infected with knob-minus parasites can accumulate in the post-capillary space where shear rates are lower [P. falciparum kahrp– parasites were able to bind to the microvascular endothelium in areas where shear rates were low and avoid splenic clearance. This may have been facilitated by the switch in var gene expression observed during in vivo growth to present a range of PfEMP1s on the cell surface. An alternative hypothesis is that parasites may have accumulated in the spleen and were cleared by the immune system slowly enough to maintain an infection.Despite the well-characterized role of knobs in the cytoadherence and sequestration of parasite-infected erythrocytes, there is some evidence that erythrocytes infected with knob-minus parasites may be capable of binding weakly to the microvascular endothelium. Knobless nditions . Similarre lower . In cont6 viable parasites). Immunogenicity appeared to be dependent on parasite replication, evidenced by the fact that the subject administered the highest dose who did not develop parasitemia failed to generate a detectable antibody response. This finding may be consistent with evidence that killed whole parasite malaria vaccines require an adjuvant to induce robust immunity [Immunogenicity results indicated that the GAP vaccine was capable of eliciting a malaria-specific antibody and cell-mediated immune response when administered at the highest dose  suggests that the dose of parasitized erythrocytes may be a factor. The total dose of erythrocytes (including non-parasitized) administered was similar to that routinely administered (approximately 1 × 108 erythrocytes). We speculate that altered antigen presentation by parasitized erythrocytes with increased passage to the spleen may contribute to a higher probability of alloimmunization with the GAP vaccine. Of note, one of the 8 subjects administered a chemically attenuated blood-stage P. falciparum parasite vaccine in a pilot study also developed anti-c alloantibodies [Although no serious safety concerns prevented completion of this stage of clinical development of the candidate vaccine, two considerations resulted in the vaccine not progressing to testing for protective efficacy in a challenge study with unattenuated parasites. Firstly, the fact that an immune response only developed in subjects who developed patent parasitemia some weeks after inoculation, and this parasitemia required termination by timed antimalarial chemotherapy, would impose significant practical limitations on the clinical development of this vaccine in the absence of other modifications to render the parasite avirulent. Secondly, two subjects administered the highest dose of GAP vaccine developed erythrocyte alloimmunization (anti-C and anti-P1 antibodies in one subject and anti-c antibodies in the other subject). This was unexpected based on our previous experience with CHMI studies using the IBSM model where alloimmunization attributed to the malaria challenge agent has not been reported previously (approximately 400 subjects in 32 studies). The fact that alloimmunization was observed in subjects who were administered a dose of parasites approximately 1000-fold higher than is typically administered in CHMI studies using the IBSM model involving wild-type P. falciparum kahrp– strain as a blood-stage GAP malaria vaccine, they do contribute valuable new information on the pathogenicity of P. falciparum. Additionally, this study acts as a proof-of-concept for the use of genetically modified blood-stage malaria parasites in humans. We have previously developed IBSM models for P. falciparum [P. falciparum [P. vivax [P. malariae [Although the results of this study overall do not support the further clinical development of the lciparum , artemislciparum , P. vivaP. vivax , and P. malariae . The useAdditional File 1. Supplementary methods and results for subject eligibility and safety assessments. Text S1: Subject eligibility criteria. Table S1: Incidence of adverse events by subject. Table S2: Red blood cell alloantibody test results for Cohort 4 and phenotype of donor red blood cells used for GAP master cell bank manufacture.Additional File 2. Supplementary methods and results for immunogenicity assessments of GAP vaccine. Text S1. Methodology for determining antibody mediated immune response to GAP vaccine. Text S2. Methodology for determining cell mediated immune response to GAP vaccine. Table S1. Antibodies used for flow cytometry. Figure S1. The gating strategy used to identify CD4+ T cells and subsets, as well as the expression of T cell activation markers. Figure S2. The anti-MSP2 IgM response over the course of the study in each subject. Figure S3. The IgG and IgM response to whole parasite lysate over the course of the study in each subject. Figure S4. The frequency of CD4+ T cells, Treg cells, Th1 cells and Tfh cells in peripheral blood mononuclear cells over the course of the study in each subject.Additional File 3. Supplementary methods and results for in vitro assessments of GAP vaccine. Text S1. Methodology for parasite genotyping. Text S2. Methodology for cytoadherence assays. Text S3. Methodology for scanning electron microscopy. Text S4. Methodology for var gene expression analysis. Table S1. Duration of in vitro culture of P. falciparum prior to harvest for RNA extraction for var gene transcription analysis. Text S5. Results of parasite genotyping. Text S6. Results of scanning electron microscopy analysis of parasite-infected erythrocytes. Text S7. Results of cytoadherence assays. Text S8. Results of var gene expression analysis. Table S2. Most abundantly expressed var genes.


Sickle cell disease (SCD) is the most common genetic disease in sub-Saharan Africa. The signs and symptoms of SCD usually begin in early childhood. Characteristic features of this disorder include anaemia, repeated infections, and periodic episodes of pain. Malaria is one of the infections that can occur in patients with SCD in endemic countries. Many guidelines recommend antimalarial chemoprophylaxis in these patients, although the debate on which drug should be used is still ongoing. Hydroxyurea (HU), which is considered a safe and effective treatment for both children and adults with SCD, seems to affect the incidence and severity of malaria, although these impacts have yet to be fully demonstrated. We report a case of an eight-and-a-half-year-old Sudanese boy with SCD treated with HU admitted for suspected severe malaria who showed a recrudescence after first-line treatment. Although he had undergone splenectomy and thus belonged to a category of patients at high risk for infectious complications, he was not receiving any malaria chemoprophylaxis. This case emphasises the importance of the routine administration of malaria prophylaxis to children with SCD living in endemic areas, even when they are treated with HU, and especially if they are at high risk for infectious complications because they have undergone splenectomy. There is an urgent need for further research to evaluate the most appropriate regimen and its optimal duration. S mutation called sickle cell anaemia (HbSS) [S mutation and one of several other abnormal HBBs [Sickle cell disease (SCD) is a chronic multi-system disease caused by an inherited haemoglobin disorder . People a (HbSS) . Other fmal HBBs .SCD is the most common genetic disease in sub-Saharan Africa, with 300,000 neonates born with SCD annually ,5. NeverWe report a case of an eight-and-a-half-year-old Sudanese boy with SCD treated with HU admitted to the Emergency NGO Paediatric Clinic in Port Sudan for severe malaria who relapsed after first-line treatment. Although he had undergone splenectomy and thus belonged to a high-risk category, he was not receiving any malaria chemoprophylaxis.3/µL, a C-reactive protein (CRP) level of 12 mg/dL, a platelet count of 213 × 103/µL, normal kidney function and mildly altered liver function , 59 UI/L; aspartate aminotransferase (AST), 33 UI/L). His bilirubin level was within normal limits. His urine was clear. A rapid diagnostic test for malaria (histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (pLDH)-based) was positive, and the blood smear confirmed the presence of Plasmodium falciparum with hyperparasitaemia (++++). A diagnosis of suspected severe malaria was made in this patient affected by SCD treated with HU with a history of splenectomy.An eight-and-a-half-year-old male patient from Sudan presented to our outpatient department in Port Sudan with a high-grade fever (39 °C), vomiting and episodes of shivering resembling convulsions. This patient was already being followed in our clinic for SCD (he showed an homozygous state for HbS), for which he started treatment with oral HU at a dose of 15 mg/kg/day due to a history of acute chest syndrome and several painful crises with a significant time absent from school. It is of note that one year prior, this patient had undergone splenectomy in a private hospital in Khartoum, Sudan. Despite living in a malaria-endemic area, the patient did not start malaria prophylaxis. On arrival at our hospital, the child appeared in good condition; he was febrile (39 °C), with vital signs within the acceptable limits according to age and a Glasgow Coma Scale (GCS) of 15/15. Blood tests revealed a Hb level of 7.0 g/dL , a white blood cell (WBC) count of 11.4 × 10The child was admitted to our paediatric ward and started on IV artesunate  and IV ceftriaxone (70 mg/kg/day). HU was stopped according to the protocol. The child had a fever for the first 24 h, with normal vital signs and normal GCS scores. Because of his improved general condition and apyrexia, he was started on a 3-day course of oral artemisinin-based combination therapy on the third day of hospitalisation, and subsequently, he was discharged with instructions to complete antibiotic therapy with oral amoxicillin/clavulanic acid for 7 days at home. At discharge (on day 3), his Hb was reported as stable with a value that was normal for the patient (7.0 g/dL); however, due to the risk of the worsening of his anaemia, we asked the family to attend our clinic for a follow-up and a Hb assessment in a week.P. falciparum ++, gametocytes of P. falciparum ++). The complete blood count showed a decreased Hb level (6.1 g/dL), increased WBC count (38.1 × 103/µL), a platelet count of 401 × 103/µL, an increased lactate dehydrogenase level , a CRP level within normal values and normal kidney and liver function. Given the suspicion of a partially treated case of severe malaria, the patient was started on IV ceftriaxone 100 mg/kg/day and quinine 10 mg/kg IV three times per day. The patient completed 7 days of IV therapy. His low-grade fever disappeared completely 48 h after admission. The child underwent a blood transfusion (on day 5) because of the risk of worsening anaemia. At discharge, his Hb level was 9.1 g/dL, his WBC had normalised, and his CRP level had decreased to 6 mg/dL from the peak of 96 mg/dL (on day 8). The malaria test was repeated before discharge (on day 12), and the blood smear showed one plus for gametocytes only.Surprisingly, two days after discharge (on day 5), the child was brought back to our clinic because of a relapse of fever (39.5 °C), episodes of vomiting and shivering. Clinically, the patient looked unwell, although he had vital signs within the normal limits for his age. The blood smear was positive for malaria (ring stage of Here, we report a representative case of recrudescent suspected severe malaria in a Sudanese patient affected by SCD who underwent a splenectomy at the age of 7 years. The patient was receiving regular treatment with HU and was therefore not receiving malaria prophylaxis. He developed suspected severe malaria with a recrudescence after a first-line treatment with artesunate IV, that required 7 days of treatment with IV quinine.P. falciparum in SCD patients, and some studies have reported a higher risk of severe malaria and a higher incidence of parasitemia after splenectomy [Subjects homozygous for HbS are at greater risk of suffering from severe malaria and have a higher mortality rate than non-SCD subjects . The risenectomy ,14. Therenectomy . NeverthP. falciparum, facilitating parasite adhesion to the endothelium [Chronic therapy with HU has been shown to have clinical benefits with regard to reducing the incidences of vaso-occlusive crisis and acute chest syndrome and the need for blood transfusion in patients with SCD ,7,8,15. othelium . On the othelium ,18. Howeothelium . Furtherothelium ,21. In aothelium ,23. The othelium . All parothelium . The secP. falciparum [Currently, there is no consensus on the most appropriate malaria chemoprophylaxis regimen for children with SCD. The current clinical practice in most African countries includes the use of insecticide-treated nets and the early diagnosis and treatment of malaria ,5. Chlorlciparum . A doubllciparum . Additiolciparum . Eke et lciparum . Chemoprlciparum . A recenlciparum . Considelciparum .This case emphasises the importance of administering regular malaria chemoprophylaxis to children with SCD living in endemic areas, even when they are treated with HU, and especially if they are at high risk for infectious complications because of splenectomy. There is, however, an urgent need for further research to evaluate the most appropriate regimen and its optimal duration.


Plasmodium falciparum is genetic polymorphism within parasite population. Understanding the genetic polymorphism like block 2 region of merozoite surface protein-1 (msp-1) gene of P. falciparum enlighten mechanisms underlining disease pathology, identification of the parasite clone profile from the isolates, transmission intensity and potential deficiencies of the ongoing malaria control and elimination efforts in the locality. Detailed understanding of local genetic polymorphism is an input to pave the way for better management, control and elimination of malaria. The aim of this study was to detect the most frequent allelic variant of the msp-1 gene of P. falciparum clinical isolates from selected health facilities in Adama town and its surroundings, Oromia, Ethiopia.One of the major challenges in developing an effective vaccine against asexual stages of msp-1 gene using specific primers. Nested PCR amplification was conducted targeting K1, MAD20, and R033 alleles followed by gel electrophoresis for fragment analysis. Based on the detection of a PCR fragment, infections were classified as monoclonal or multiple infections.One hundred thirty-nine clinical isolates were successfully amplified for msp-1 gene were identified in the study, with 67(48%) MAD20, 18 (13%) K-1 and 18 (13%) RO33 allelic family. Whereas, the multiple infections were 21(15%), 8 (5.8%), 4(2.9%), 3(2.2%) for MAD20 + K-1, MAD20 + RO33, K-1 + RO33, and MAD20 + K-1, RO33, respectively. The overall Multiplicity of infection (MOI) was 1.3 and the expected heterozygosity (He) was 0.39 indicating slightly low falciparum malaria transmission.19 different size polymorphism of msp-1 allele size polymorphism, MOI and He observed in the study revealed the presence of slightly low genetic diversity of P. falciparum clinical isolates. However, highly frequent MAD20 allelic variant was detected from clinical isolates in the study area. Moreover, the driving force that led to high predominance of MAD20 allelic variant revealed in such malaria declining region demands further research.The status of The online version contains supplementary material available at 10.1186/s12936-021-03914-9. Plasmodium falciparum and Plasmodium vivax accounts for 70% and 30%, respectively [P. falciparum is prominent. High genetic diversity is not only an indicator of its evolutionary success [Despite an enormous effort to control and eventually eliminate malaria, studies reveal that it is still a major public health problem, especially in sub- Saharan Africa (SSA) where more than 90% of the disease burden prevails , 2. Abou success  but also success  that pos success .P. falciparum enables us to investigate the genetic diversity of infection with consideration of various factors, such as disease phenotype, age and host immunity [P. falciparum is usually determined through genotyping of the polymorphic regions block 2 of msp-1 [P. falciparum blood-stage malaria vaccine targets [msp1 gene located on chromosome 9 and contains 17 blocks of sequences flanked by conserved regions [msp1 during invasion has not been fully evaluated, and its macromolecular characterization is incomplete [Molecular characterization of immunity . Geneticof msp-1 , 8, 9. M targets –12. MSP1 regions , 14. Thecomplete .msp-1 markers are useful to investigate genetic diversity, multiplicity of infection (MOI) and parasite carriage. Polymorphism in msp1 and msp2 have been frequently reported from different parts of the world. Of the 17 blocks of msp1, block 2 is the most polymorphic region characterized into three allelic families . Based on the variation in length and sequence diversity, this region is a commonly targeted part in determining genetic diversity and MOI in clinical isolates of P. falciparum.P. falciparum has been extensively studied in different parts of the world, limited data are available from Ethiopia. The aim of this study was, to assess genetic diversity of block 2 region of msp-1 gene of P. falciparum clinical isolates from three districts in central Ethiopia.Even though genetic diversity of Health facility based cross-sectional study conducted at Adama, Modjo, Wonji, Awash Malkasa and Olanciti towns from September 2019 to August 2020. These sample collection sites include Adama city administration, Adama district which includes Wonji and Awash Malkasa, Modjo town capital of Lume district, Olanciti the capital of Boset district. The location of these study sites is as shown in , and various micro ecological factors that favour mosquito breeding [Adama and its surrounding area is a well-known malaria endemic region in central Ethiopia. The major factors that account for such malaria endemicity are; its physical location in Rift Valley area of Ethiopia, latitudinal location which varies from1436 to 1850 m above sea level, rainfall patterns, average annual temperature that varies from 16–32 °C that is favourable for the breeding of breeding .P. falciparum infected patients were included in the study. The age of study participants were ranged from 1 to 66. The inclusion criteria for the study were uncomplicated malaria patient with the history of fever onset since 24 h of the clinical examination. Blood samples were collected through finger prick for dry blood spot (DBS) preparation was held from September 2019 to August 2020 by trained medical laboratory technologists from the catchment area of those selected health facilities in the study area. After consent of the patients or guardians, the spotted blood on Whatman TM 3MM filter paper was allowed to air dry in dust free area. The DBS were placed in a zip-lock bag with silica gel temporarily stored at 0–4 °C to prevent DNA degradation. For longer time storage of DBS was kept in deep freeze (− 20 °C) at Adama regional research laboratory.One hundred thirty-nine microscopically-confirmed P. falciparum and its parasite density. In case of discordance, a third laboratory technologist read the slides. In addition, parasite density was estimated by counting and recording the asexual stage of the parasite per 200 white blood cells (WBC) in thick film. Moreover, when the sexual form (gametocytes) have been seen, the slides were excluded from the count. The parasite density of the asexual stage was estimated by counting the number of WBC by field examined assuming that 8000 WBC were present in 1 µl of blood. Thus, parasite density per microlitre (μl) of blood was calculated by using the following formula [Microscopic examination was conducted for both thick and thin blood smear following the national malaria microscopy protocol. All slides examined by two independent laboratory technologists to determine species identification for  formula , 18, 19:For comparison with ranked order variables, parasitaemia were categorized in to five levels: L1 (< 50 parasite/µl blood), L2 (50–499 parasite /µl blood), L3 (500- 4999 parasite /µl blood), L4 , and L5  .msp-1 were conducted at malaria research laboratory, Akililu Lemma Pathobiology Institute, Addis Ababa University (AAU). Genomic DNA of P. falciparum extracted from approximately 200 µl of frozen blood samples spotted on Whatman 3 filter paper for nested PCR amplification. 0.5% Tween® 20  was used to lyse RBC; tracked by treatment with 6% chelex ® 100  and heat treatment in water bath at 96 °C following the optimized standard operating procedure (SOP) to free the parasites DNA [Extraction of the parasite genomic DNA and genotyping the polymorphic region of 18 srRNA gene was held by using specific primer pairs for molecular detection of P. falciparum from the isolates [P. falciparum msp-1 gene (block 2) was used as a genetic marker for the genotyping of parasite populations. The primers and PCR conditions used during this study were slightly modified from the previously described work [P. falciparum msp-1. Multiplicity of infection (MOI) was defined as the average number of detected P. falciparum genotypes per infected patient. Allele for each family were considered the same when the fragment size is less than 20 bp [Nested PCR amplification targeting the unique sequence of isolates . In the bed work , 22 . To examine endemicity or potential importation of the To test the correlation of two variables, Pearson correlation test was used. Pearson Chi square test was also conducted for statistical comparison of categorical variables. P < 0.05 was used to test the level of statistical significance to accept or reject the hypothesis.The expected heterogeneity Ethical approval of the study was obtained from Institutional Ethical Review Board of ASTU, certificate reference number RECSoANS/BIO/01/2019 and approval of Oromia Regional State Health Bureau. In addition, written informed consent obtained from parents or guardian prior to recruitment.P. falciparum patients were successfully analysed for msp-1 allelic diversity. A total of 68.3% of the study participant were males. The age of the study participants ranged from 1 to 66. Mean ± SD (27.0 ± 13.6*) years. Asexual parasite density ranged from 64 – 104,320 parasites/µl with a geometric mean of 5,654 parasites/µl. Of all study participants 83 (60%) were from urban inhabitants, and only 15 (11%) were having recent travel history to other malarious area. Of all the study subjects, by occupation 74% P. falciparum malaria cases were detected from students, daily labourers and farmers  carry disproportionate burden of the infection  and age of the patient with parasite density  , sex (X2 = 0.56), residence (X2 = 0.2), travel history (X2 = 0.9), educational level (X2 = 0.8) and occupation (X2 = 0.5)  and 2% triclonal (MAD20 + K1 + RO33) infections. From all P. falciparum msp-1 gene amplified by nested PCR for block 2 region, 19 different alleles were identified of which 8 alleles were MAD20 (160–280 bp), 6 alleles were K-1(100–270 bp), and 5 alleles of RO33 type (100–200 bp). The overall MOI was 1.3, with the expected heterozygosity of 0.39  in the study area. Moreover, 33 (92%) patients with polyclonal infection were having no travel history to other malaria endemic places. Thus, there was no statistically significant variation in the distribution of allelic variants in relation to patient’s travel history in the study area (X2 = 0.9)  were detected during the major malaria season (September to December) and the rest were from the isolates of minor malaria season in the region. No statistically significant variation in the seasonal distribution of polyclonal infection  were from the urban locality, and the rest were from rural area  and (X2 = 0.57), respectively.In this study, of all ea Table . Allelicmsp-1 allelic variants and MOI from the study sites has shown that 65 (47%), 18 (13%), 17 (12%), 18 (13%), 21(15%) isolates were from Adama, Modjo, Wonji, Malkasa, and Olanciti sites, respectively. The spatial variation of the distribution of msp-1 allelic variant across sample collection sites was significantly related (P = 0.000) , 18 1%, 17  and relatively stable afterwards Fig. . In addi2 = 0.5). This finding is in congruent with the report from Burkina Faso [In the present study, multiple infections slightly increased with age group Fig. B, althouina Faso  and Tanzina Faso , where tina Faso –30. OtheP. falciparum with seasonal variation of malaria incidence and travel history of patients , 2.0 MOI and He (0.43), respectively. This shows that malaria transmission in the study under report exhibits slightly low genetic diversity, compared with northwestern and southwestern Ethiopia. This could be due the locational advantage of central Ethiopia to better health services, differences in local epidemiology, demographic and environmental conditions that might have resulted in observed reduced genetic diversity pattern in Adama and its surroundings. In the present study, from 139 samples 19 different length polymorphism of msp-1 allelic variant was revealed; 8 MAD20 (160–330 bp), 6 K-1 (100–270) bp, and 5 RO33 (100–220 bp). This shows the level of size polymorphism of msp-1 alleles in the study area. However, the number of alleles identified may have been under estimated due to a number of limitations like sensitivity of PCR technique used, inability to differentiate minor fragments, the possible existence of similar size fragments and the same size fragment having different amino acid motifs [In this study, 26% of the isolates having multiple genotype infections. The overall MOI of 1.3 and the expected heterozygosity of 0.39  were monoclonal infections, whereas the remaining 36 (26%) was poly-allelic type, with 15% for (MAD20 + K-1), 5.7% for (MAD20 + RO33), 2.8% for (K-1 + RO33), and 2.1% were MAD20 + K-1 + RO33 type. The proportion of monoclonal infection was 48% MAD20, 13% K-1 and 13% RO33 , and seasonality of transmission (P = 0.8). This could be due to the characteristic feature of low transmission settings in such malaria endemic regions [Size polymorphism of Ethiopia  and HumeEthiopia . This waEthiopia , but morEthiopia , and BobEthiopia . The majining 36 6% was poamplicon 4% were mntensity , 40, countensity . In addi regions , 43. On  regions  and the msp-1) of P. falciparum population in the study area. However, further characterization of this gene needs to be designed by increasing the sample size, use of the most powerful techniques, such as microsatellite DNA sequencing and capillary electrophoresis that would provide strong molecular evidence for malaria parasite genetic profile.This study is the first attempt to analyse the most polymorphic gene (P. falciparum clinical isolates found in the study area. Moreover, high frequency of MAD20 allelic variant form was detected. The driving force for such selective advantage for this allele under declining malaria prevalence in our study area demand further investigation. Thus, this information will serve as a baseline molecular evidence for further research on areas having similar malaria epidemiology for targeted interventions to make the control and elimination efforts more effective (Additional files The study revealed that slightly low genetic diversity of Additional file 1. Primer design.Additional file 2. msp-1 allelic fragment size using 50 bp ladder identified by gel electrophoresis.