Plasmodium vivax is widely predominating even though Plasmodium falciparum is still present due to imported cases mainly from Africa. In FG, rapid diagnostic test  is based on the detection of pan-pLDH, PfHRP2, and PfHRP3 antigens, while in South America, the share of deletion of PfHRP2 gene is significantly increasing. Accordingly, the study questions the reliability of RDTs in the Amazonian context.French Guiana (FG) is a French overseas territory where malaria is endemic. The current incidence rate is 0.74‰ inhabitants, and The study is retrospective. It is conducted over 4 years and analysed 12,880 rapid diagnostic tests (RDTs) compared to concomitant Blood Film Tests (BFTs) sampled for malaria diagnosis.P. falciparum (88%). Overall, the concordance rate between RDT and BFT  was 99.5%. The PPV of the RDT in the follow-up of patients diagnosed with P. falciparum was the lowest during the first 28 days. The PPV of the RDT in the follow-up of patients diagnosed with P. vivax was the lowest during the first 21 days. The global sensitivity of SD Malaria Ag P.f/Pan® test was, on average, 96% (88.2–100) for P. falciparum and 93% (90.6–94.2) for P. vivax. The global specificity was 99.8% (99.5–100) for all included species.The global assessment of the accuracy of SD Malaria Ag P.f/Pan® in the diagnostic of malaria shows both Positive and Negative Predictive Values (PPV and NPV) higher than 95%, except for PPV in the diagnosis of malaria to SD Malaria Ag P.f/Pan® is a reliable rapid test used for the first-line diagnosis in remote healthcare centres. The test results should be interpreted in the light of patient’s recent medical history and the date of arrival to FG. Plasmodium vivax [WHO classified malaria as a public health concern. In 2017, malaria was the most prevalent parasitic disease, with 1.4 billion people at risk worldwide  with 75%um vivax . Indeed,Plasmodium falciparum was accountable for the majority of cases. However, within the last 20 years, the distribution of Plasmodium species changed with a vast rate of diagnosed P. vivax [Previously in FG, P. vivax , 8. DespP. falciparum infections is a crucial point for surveillance studies to steer malaria control strategies in countries where malaria is near to almost elimination [P. falciparum, and causing false negative RDT results [P. falciparum can present PfHRP3 deletion (4.5% of cases) but no PfHRP2 deletion [Among laboratory methods used for malaria parasite detection and exposure, only rapid diagnostic tests (RDT) and blood films tests (BFT) are currently available for daily practice. Molecular detection of low-density mination . RDT is  results . In Sout results . In FG, deletion .This study aims to assess the relevance of SD Malaria Ag P.f/Pan® in the diagnosis of malaria in French Guiana.Our study is a retrospective analysis conducted over 4 years (January 2016 to December 2019) in the microbiology laboratory of the Cayenne General Hospital. It includes all RDT and BFT sampled for malaria diagnosis. Cayenne General Hospital is a 742-beds health facility that provides first-line medical care for an urban population of 150,000 inhabitants. It manages 18 delocalized prevention and healthcare centers providing care for almost 50,000 inhabitants. Thereby, it is also a referral centre for a larger population from all over French Guiana and other border countries .Data were collected from the computerized database of the microbiology laboratory of the Cayenne General Hospital. They include the date of the tests, and the results of the RDT and the concomitant BFT. In a first step, files with a RDT and a concomitant BFT were included and files with a BFT without a concomitant RDT were excluded. Also files where concomitant RDT/BFT were sampled during the six months following the malaria diagnosis were separately analysed.RDT was based on the SD Malaria Ag P.f/Pan® (Standard Diagnostics Inc.) which detects the presence of pan-pLDH and PfHRP2 antigens . BFT is Thick and thin blood films are prepared within one hour of blood collection. Thick blood films are stained with Giemsa diluted at 10%, while thin blood films are stained using a rapid method . Two hundred fields of the thin blood film are examined before classifying the thin smear-negative, and 1000 counted white blood cells (WBCs) from the thick smear are observed before classifying the sample as negative . The parResults are reported as mean and standard deviation, or numbers and percentages. We calculated the sensitivity (Ss), specificity (Sp), positive and negative predictive values (PPV and NPV), Youden test, and the Q coefficient of Yule to assess the diagnostic accuracy of RDT in the diagnosis of confirmed malaria by BFT. All statistical analyses were carried out using Excel  and IBM SPSS Statistics for Windows, version 24 .The study is retrospective that did not require individual consent according to the French law regarding research conforming to MR-003 (JORF no. 0160 du 13 juillet 2018. texte no. 109). The database has been registered at the Commission National de l'Informatique et des Libertés (registration no 2219819), in compliance with French law on electronic data sources.During the study period, 12,984 samples of blood films for the diagnostic of malaria were recorded. A concomitant BFT and RDT were sampled in 12,880 cases. Amongst them, 10,873 files (84.4%) fulfilled our inclusion criteria  and P. vivax in 648 cases (83.9% of positive tests). BFT was performed in 10,873 cases and was positive in 791 cases (7.3%) with identification of P. falciparum in 105 cases (13.3% of positive tests), P. vivax in 673 cases (85.1% of positive tests), both of them in 10 cases (1.3% of positive tests), and Plasmodium ovale in 3 cases (0.4% of positive tests).The average number of RDTs and BFTs per year was 2718 ± 394 tests. The average number of RDTs and BFTs per month was 1073 ± 105 tests. RDT was performed in 10,873 cases and was positive in 773 cases (7.1%) with identification of P. falciparum (88%). Figure Plasmodium species identification.The global assessment of the accuracy of RDT in the diagnosis of malaria shows an overall PPV and NPV higher than 95%, except for PPV in the diagnosis of malaria to P. falciparum and P. vivax is reported in Table P. falciparum shows a drop-down in the PPV in 2017, but an increase after that  and 100% in 2018–2019. The concordance rate between RDT and BFT in the diagnosis of P. vivax was 99.3%. It was at 99.1% in 2017 (the lowest) and 99.5% in 2016 and 2019.Overall, the concordance rate between RDT and BFT  was 99.5%. It was the highest in 2016 (99.8%) and the lowest in 2017 (99.1%) Fig. . The conP. falciparum was the lowest during the 28 first days. The PPV of the RDT in the follow-up of patients diagnosed with P. vivax was the lowest during the 21 first days  in 2007 cases 15.6%). The PPV value of RDT in this context was the lowest during the 42 days of follow-up  [. In the current study, the average share of confirmed P. falciparum malaria cases decreased significantly with 13.3% of positive tests (minus 17.7%), while confirmed P. vivax cases increased to 85.1% of positive tests (plus 16.6%).In FG, the implementation of malaria control strategies allowed to reduce the number of recorded cases by 82% during the last years , 19. In ectively . In 2019P. falciparum and 93% (90.6–94.2) for P. vivax. The sensitivity can be affected by a low rate of parasitaemia [P. falciparum and P. vivax with 88% (80.4–1) and 98% (94.7–99.1), respectively.The current study analysed all performed RDTs and compared them to BFTs regardless of whether they were first-line diagnostic tests or monitoring tests during treatment. The global sensitivity of SD Malaria Ag P.f/Pan® test was 96% (88.2–1) for sitaemia . The gloPlasmodium in the blood. In this study, we have not collected clinical information. Consequently, immunological factor or infectious agents explaining the calculated PPV were not investigated [Most of RDTs carried during the follow-up period were performed during the two first years of the study, which can explain the annual variation of the PPV. Furthermore, there is a significant variation of the global PPV week after week during the first 28 days of follow-up. It is also explained by the number of false positive cases related to the persistence of the protein-encoding for stigated , 22. ThuP. falciparum and P. vivax. It has been reported that a low parasitaemia could affect the accuracy of the test with false-negative results [The NPV of SD Malaria Ag P.f/Pan® in the malaria diagnosis was, 99.7% (99.3–99.5) without a significant difference between  results , but thi. Contrarily, pLDH is quickly eliminated from the blood within 1 week of treatment [. Thus, the initial parasite density influence false-positive results and PfHRP2 persistence [In the current study, 2007 concomitant RDT/BFT were carried during the follow-up period of patients under treatment. The results have shown that the RDT remains positive up to 28 days, even though parasites were no longer detectable with BFT. Previous findings suggest that PfHRP2 RDTs remain positive after treatment for longer than the combined or pLDH RDTs . This issistence .Furthermore, RDTs have a higher probability of remaining positive in patients receiving artemisinin-based combination therapy (ACT) than in those receiving non-ACT drugs . In pracgarimpeiros) inside the rainforest and native populations on border areas between Suriname and Brazil are at risk of malaria resurgence [P. falciparum and P. vivax, 47.9% and 37.2%, respectively with 10.6% co-infections. Thus, there is a risk of periodic reintroduction of the disease in FG [During the last 2 years, the majority of malaria cases in FG were autochthonous  while the remaining cases were from Brazil (range 9 to 14%), Suriname (range 2 to 5%), or Africa (range 2 to 7%) [surgence , 29, 30.surgence . Most casurgence . In FG, se in FG . It is eP. falciparum HRP3 deletion was recorded in 100% of cases [It is worth noting that, in the Brazilian Amazon basin, the lack of PfHRP2 protein varies from region to region. It is the highest in Acre state (31.6%) and absent in Para state . Howeverof cases . In 2013of cases . Moreoveof cases .Plasmodium and impact the efficiency of the test.SD Malaria Ag P.f/Pan® is a reliable rapid test used for the first-line diagnostic of malaria in remote healthcare centres in FG. However, it must no longer be used during the follow-up period of patients diagnosed with malaria. The test reading should be interpreted with caution while considering the recent medical history of patients and their arrival date in FG. BFT must always confirm RDT results. In FG, uncontrolled migratory flows might increase the risk of importing new variants of


Plasmodium falciparum is an important pathogen that causes severe malaria in humans. The malaria parasite carries unique epigenetic signatures, which are required for optimal gene expression in the parasite during its development in red blood cells (RBCs)  and that its presence on various transcripts in the parasite is highly dynamic  protein that specifically interacts with m6A-containing mRNA of P. falciparum  –3. A rec dynamic . Subsequlciparum . The PfYlciparum . Importalciparum . The firlciparum . The apiarasites –6. We st


Plasmodium vivax and its risk factors could be used to prevent and control malaria infection. Therefore, the aim of this study was to characterize the distribution and risk factors of P. vivax, and to compare them with Plasmodium falciparum occurrence in south-central Ethiopia.Better understanding of the distribution of P. vivax episode.A cohort of 34,548 individuals were followed for 121 weeks between 2014 and 2016 as part of larger cluster randomized controlled trial to evaluate the effect of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) on malaria prevention in Ethiopia. Weekly home visit (active search) and patient self- report to health post (passive search) between the weekly home visits were used to register malaria cases. A blood sample was collected by finger prick and malaria was diagnosed using rapid diagnostic test (RDT). Generalized estimating equation (GEE) Poisson model that accounts for repeated measure of malaria episodes was applied to assess the risk factors of P. vivax was 7.4 episodes per 1000 person-years of observation. The study showed households closer to the lake Zeway and Bulbula river  were more at risk of P. vivax infection (incidence rate ratio (IRR): 1.33; 95% CI = 1.23–1.45). Furthermore, the age group under 5 years , the age group 5–14 years , households with less educated household head  and house roof made of thatch/leaf  were at higher risk for P. vivax. Similar explanatory variables such as distance from the breeding sites, age group (under 5 years but not 5–14 years old), educational status and type of housing were also found to be the predictors of P. falciparum incidence.The overall incidence rate of P. vivax. The result of this study can be used for tailored interventions for malaria control and prevention by prioritizing those living close to potential mosquito breeding site, enhancing bed net use of children less than 15 years of age, and improving housing.Households living closer to a mosquito breeding site, age group under 15 years, less educated household heads and thatch/leaf roof housing were the risk factor for The online version contains supplementary material available at 10.1186/s12936-021-03790-3. Anopheles mosquito bite [Plasmodium vivax accounts for about 3% (5 million) of all the estimated cases. More than 85% of the global P. vivax cases occurred in six countries: India, Afghanistan, Pakistan, Ethiopia, Papua New Guinea and Indonesia [Malaria is an infectious disease mainly transmitted by female ito bite . Globallito bite . The samndonesia , 3.Plasmodium  [Plasmodium falciparum is a cause of severe malaria, and most prevalent  in Africa [P. vivax has the widest geographic distribution of the four malaria parasites, and can also cause severe malaria in children [P. vivax in most of sub-Saharan Africa is low (1%), it accounts for about 40% of malaria cases in Ethiopia and outside Africa, mainly Latin America and Asia [Malaria is caused by five species of nowlesi) . Plasmodn Africa . Whereaschildren , 6. AlthP. vivax (40%) and P. falciparum (60%) are the two dominant causes of malaria infection [In Ethiopia, nfection . The trenfection , 10. Hownfection . For exanfection .P. vivax infection. However, the observed relationship particularly between distance of living house from mosquito breeding site and P. vivax transmission varied from one area to another, and need further investigation. For example, some reported increased risk of P. vivax in individuals living closest to breeding site [P. vivax with increase in distance from breeding site [P. vivax [.Studies have shown that altitude , 13, ageing site , other ring site , and othP. vivax .P. vivax declines slower than that of P. falciparum infection, and remains the main cause of malaria [Plasmodium vivax is more difficult to eliminate than P. falciparum. This could be because P. vivax remains dormant in human liver for weeks, months or years, the source of relapse infection; the parasite develops in the vector at lower temperatures than P. falciparum, and it has wider geographic distribution and extending to highland areas [P. falciparum, the gametocytes of P. vivax are released into the bloodstream as soon as a patient becomes ill. Therefore, a patient infected with P. vivax transmits the parasite to mosquitoes before the clinical symptoms appear [P. falciparum and P. vivax using data from cluster randomized controlled malaria prevention trial (the same data source as the current study) [P. vivax and risk factors could be used to prevent and control P. vivax malaria infection. Therefore, the objective of this study was to characterize the distribution and risk factors for P. vivax compared to P. falciparum in the Great Rift Valley of Ethiopia, from a large cluster randomized controlled malaria prevention trial carried out in south-central Ethiopia.In areas where the two infections co-exist, the incidence of  malaria . Plasmodnd areas . Moreoves appear . In a prt study) . The undkebeles (the lowest government administrative unit), and a kebele is further divided into villages (“Gare”). Lake Zeway, one of the Ethiopian Rift Valley lakes borders the district. The residents use Lake Zeway and Bulbula River (coming out of Lake Zeway) for irrigation to cultivate crops. Furthermore, there is a large flower farm that use water from the lake and the river. Such water based development were reported to be potential malaria vector breeding sites [kebele. The health extension workers provide malaria diagnosis and treatment, prevention and control services including basic health services  is located about 160 km south of the capital Addis Ababa at latitude of 7°56′N and longitude of 38°42′E with an altitude of 1600 m above sea level. According to the 2007 National Census [ng sites . Malariakebeles were randomly selected. Distance of the households from the lake and river border  was calculated using a proximity analysis tool in ESRI ArcMap 10.3.1. A village or cluster has on average population size of 175 residents. The villages or Gare  were randomly assigned to the four malaria intervention arms .This cohort study is a part of large cluster randomized controlled trial to evaluate the effect of LLINs and IRS on malaria prevention in Ethiopia, short named “MalTrials project”. More information about the details of the design, participants and some results have been published elsewhere , 22–25. As presented elsewhere , the samCensus and weekly data were collected by diploma graduates non health professionals. Blood sample collection for malaria diagnosis and treatment was carried out by diplomaed nurses. The data collectors and nurses were trained before starting the trial and also refresher training was given periodically during the follow-up study. The structured questionnaires were obtained from malaria study conducted at Chano Mille in south Ethiopia ; were mokebles were enumerated. Identification number was given to the houses using metal plates, and unique number was given for all household members. Baseline data were collected to list household members, socio-demographic, economic, history of malaria illness and malaria prevention. Furthermore, the household geographic coordinate was taken using a Global Positioning System (GPS) device . The census was repeated after one year to enumerate those people newly joined the cohort  and left the cohort (death and lost to follow-up).All households within 5 km of Lake Zeway from 13 Weekly home visits (active case search) were carried out to identify person with clinical sign of malaria and to assess LLINs utilization practice by the household members. Any person identified with the history of fever within the previous 48 h during the home visit was referred to health post with referral slip. Moreover, family members were advised to visit health post if encounter fever (passive case search) between the weekly visit.® Malaria Pf/Pv combo test .Blood sample from patients presented with history of fever within the previous 48 h was taken using finger prick by trained clinical nurses employed by the MalTrials project. Rapid diagnostic test (RDT) was performed in the health post using CareStartP. vivax or P. falciparum was calculated by dividing number of each species episodes per 1000 person years of observation. ArcMap 10.3.1 software was used to calculate the distance in kilometres between household and nearest main potential mosquito breeding site (the shore of Lake Zeway and Bulbula river) [P. vivax or P. falciparum episode was count variable that follows a Poisson distribution, and to account for the within subject correlation due to repeated measurements of malaria status generalized estimating equation (GEE) was applied. The GEE procedure is the extension of general linear model [2) was the scale parameter, and robust estimator was the specified covariance matrix. The term used to build the reported model was the main effect, and the parameter estimation method was a hybrid with a maximum Fisher scoring iteration of 1. Finally, Kernel was specified for the log quasi-likelihood function. The potential predictors of P. vivax or P. falciparum were age group, gender, educational status of the head of households, wealth status, intervention arms, distance from mosquito breeding place, and roof of the housings. The association between P. vivax or P. falciparum and the predictor variables was assessed using bivariable and multivariable Poisson log-linear model, and incidence rate ratio (IRR) with 95% confidence interval was reported.Data were entered and cleaned using SPSS version 24 , and analysed using STATA version 15 . Descriptive statistics was carried out to summarize the data. A principal component analysis method  was useda river) . The outar model . The repIn this study 34,548 individuals from 6071 households were followed. Half (50.2%) of the participants were male, and in the age group of 15 years and above (50%). Fifty one percent of the participants were illiterate, ~ 33.2% were poor, 69% of them had more than 5 household size, around 26% of the households received both LLINs and IRS, and median (interquartile range) of household distance from breeding site was 1.66 (0.70–2.95) km , two episodes , three episodes  and four episodes . As shown in Fig. P. falciparum. The incidence rate of P. falciparum and P. vivax was higher during the major malaria transmission seasons (September–December).Overall, 531 episodes of P. vivax was 7.4 episodes per 1000 person-years of observation. Plasmodium vivax incidence was high among children under 5 years old (9.6 per 1000 person-years), less educated (read and write) household heads (11.4 per 1000 person-years), house roof made of thatch or leaf (7.9 per 1000 person-years) and living within 1 km from mosquito breeding site (10.8 per 1000 person-years). Whereas, the overall incidence rate of P. falciparum was 12.3 episodes per 1000 person-years of observation. The incidence of P. falciparum was high among children under 5 years old (15.9 per 1000 person-years), less educated (read and write) household heads (10.88 per 1000 person-years), living within 1 km from mosquito breeding site (17.00 per 1000 person-years) and house roof made of thatch or leaf (14.22 per 1000 person-years) . Furthermore, age group under 5 years , age group 5–14 years  and less educated (read and write) household head  were the predictors of P. vivax. The study also showed that peoples living in a house roof made of thatch/leaf were more at risk of experiencing P. vivax  than corrugated iron roof housing  was lower than a pilot study that was conducted prior to the main trial in the same study area from August 2013 to December 2013, in which the average incidence rate was 4.6 episodes per 10,000 person-weeks of observation (approximately 24 episodes per 1000 person-years) [The observed incidence rate of n-years) , and stun-years) . This con-years) .P. vivax and P. falciparum. The presence of water bodies, where female mosquitoes lays their eggs, is critical for the life of the mosquito [Anopheles mosquito carrying eggs need human or animal blood and thus visit nearby houses to take their blood meal. Previous studies reported higher mosquito density in households closest to mosquito vector breeding areas [P. vivax infection than the group furthest away [P. falciparum [P. vivax among children living close to the dam [Plasmodium infection among residents living close to rice growing areas [P. vivax infection among residents living far from dam [In this study household proximity to potential mosquito breeding site was the risk factor for both mosquito . The femng areas , 32. Theest away . Furtherlciparum . Another the dam . Similarng areas . In contfrom dam . This coP. vivax compared to those in the age group of 15 years and above. In study from the same setting and time period by Solomon et al. demonstrated that those in the age group 15 year and above use LLINs more frequently compared to those less than 5 years old [On the other hand, children under the age of 5 years and 5–14 years old were more at risk of ears old . This LLP. vivax incidence decreases more slowly than P. falciparum [P. vivax increases and becomes the dominant causes of malaria. In line with earlier finding [P. vivax but not P. falciparum among children 5–14 years of age.When malaria incidence declines, and both species co-exist, lciparum . Also, i finding , this stP. vivax infection. This could be due to the presence of eves, poorly fitted doors, and holes in walls of roofs made of thatch/leaf. Studies have shown that modern houses, roof made of corrugated iron and others are associated with decreased risk of malaria infection by reducing mosquito entry to house [In this study, materials from which the roof of the house made, thatch/leaf was observed as risk factor for to house .P. vivax infection than those who had received a formal education. This could be explained by the fact that household heads with formal education better understand and use malaria prevention tools, that could reduce risk of infection than less educated household heads. Study from same setting in the same time period has shown that household heads with a formal education were more likely to use LLINs compared to less educated ones [Furthermore, less educated household heads (read and write) were more at risk for ted ones .P. vivax episode was a new infection or a relapse. In the event of relapse, the P. vivax infection detected may be months or years old. Therefore, lack of knowledge on the actual place of residence at the time of initial infection could lead to overestimation of malaria episode in evaluating the association between distances from mosquito breeding site and P. vivax episodes.This cohort study was based on a random selection of clusters or villages from rural community for cluster randomized controlled trial. The study involved large sample with adequate power for long follow up (121 weeks) period in a drought prone area. Even though the sample is representative of a population in a similar setting in Ethiopia’s rift valley, the generalizability of this study may be harmed by the extreme drought that hit the study region. Therefore, this circumstance should be taken into account while interpreting the finding of this study. The other limitation is that it was not established whether a P. vivax. The result of this study can be used for tailored interventions for malaria control and prevention by prioritizing those living close to potential mosquito breeding site, enhancing bed net use of children less than 15 years of age, and improving housing.In this study, household living closer to a mosquito breeding site, age group under 15 years, less educated household heads and thatch/leaf roof housing were the risk factor for Additional file 1: Table S3 Risk factors of P. vivax and P. falciparum episodes Generalized log-linear model, Adami Tullu District, south-central Ethiopia.


Plasmodium falciparum. However, false negative results of RDT caused by genetic variation of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes.Rapid diagnostic tests (RDT) are commonly used for the diagnosis of malaria caused by pfhrp2/3 were purified and sequenced.A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger-prick blood samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of The PfHRP2 amino acid repeat type isolates were less conserved compared to the PfHRP3 repeat type. Eleven and eight previously characterized PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 proteins. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3% in PfHRP3 sequence isolates at parasitaemia levels > 250 parasites/µl. Using the Baker model, 100% of the isolates in group A (If product of types 2 × type 7 repeats ≥ 100) and 73.7% of the isolates in group B (If product of types 2 × type 7 repeats 50–99) were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/μl.P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in The online version contains supplementary material available at 10.1186/s12936-021-03928-3. Plasmodium falciparum has remained a life-threatening infectious disease despite significant advances made towards malaria control and elimination in most malaria endemic countries in the world [Plasmodium falciparum is the most common species that causes complicated malaria in humans. The genetic variation in the genome of P. falciparum has the potential to make pathogenesis virulent and challenge the accuracy of diagnosis [Malaria caused by he world . Plasmodiagnosis . One of iagnosis .P. falciparum, has been shown to share high homology with PfHRP2 [P. falciparum infections by PfHRP2-based RDTs [Rapid diagnostic tests (RDTs) are widely and routinely used in remote and resource limited settings. Currently available malaria RDTs utilize parasite-specific antigens produced by malaria parasites for diagnosis . The mosh PfHRP2  and playsed RDTs .pfhrp2 and pfhrp3 genes are located in the subtelomeric region of chromosome 8 and 13, respectively. Both pfhrp2 and pfhrp3 genes consist of a single intron and two exons [pfhrp2 and pfhrp3 encodes amino acids, which exhibit high levels of homology, suggesting that these genes were originated by duplication and divergence from a common ancestral sequence [The wo exons , 9. Exonsequence , 10. Thesequence . Baker esequence , 12.Pfhrp2 and pfhrp3 genes [pfhrp2/3 gene cause a lack of target antigen in the diagnosis of P. falciparum malaria [However, frequent recombinations that occur at the subtelomeric regions contribute to vast genetic variation in the p3 genes , 13, 14.p3 genes , 12, 15. malaria –18. Gene malaria , 20. Ult malaria .pfhrp2/3 gene in Assosa zone, northwest Ethiopia. Information on the abundance of PfHRP2 and PfHRP3 peptides repeats in P. falciparum isolates from geographically distinct regions is vital to monitor the diagnostic performance of PfHRP2 RDTs. Thus, the aim of this study was to determine the extent of genetic variation of pfhrp2 and pfhrp3 in clinical isolates, where P. falciparum is highly prevalent and PfHRP2 RDT is used for front-line diagnosis.Before this study, there were no data on the genetic variation of the This study was carried out during the low and high transmission seasons in four selected health facilities: Assosa, Bambasi, Kurmuk and Sherkole Health Centres in Assosa Zone, Benishangul-Gumuz regional state, northwest Ethiopia, April to December 2018. The study area is a high malaria transmission setting. The Assosa Zone is located on the border of Sudan, where transboundary transmission of malaria likely occurs. The map of the study area is indicated in Additional file pfhrp2/3 in clinical isolates. The study populations were residents of the Assosa Zone which includes all patients with clinical suspicion of malaria aged ≥ 5 years in the selected health facilities during the study period. The inclusion criteria were all study participants with clinical suspicion of malaria who gave their written consent and/or assent. High-quality sequence data were included for molecular analysis of pfhrp2/3 genetic variation. Study subjects who worked or lived outside the Assosa Zone catchment area and or who were unwilling to participate were excluded from the study. Data with poor quality sequences were also excluded.A cross-sectional health facilities based study was conducted to assess genetic variation of 2p (1 − p)/d2 [The sample size was calculated based on the single population proportion formula n = z − p)/d2 ; Where,  − p)/d2 . As a reThe study area, Assosa Health Centre, Bambasi Health Centre, Kurmuk Health Centre and Sherkole Health Centre, was selected using a simple random sampling technique among eight districts in the Assosa zone. Then, allocation of the study participants to each selected health centre was performed based on proportion of confirmed malaria case in each selected district/Woreda.In a single finger-prick, capillary blood samples were collected from malaria suspected individuals for microscopy, malaria RDTs as well as dried blood spots (DBSs) for molecular assay. Overall work flow of this study indicated in Fig. The CareStart™ malaria RDTs (Pf/PV HRP2/PLDH) were used following manufacturer’s instructions. Thick and thin blood smears were stained with a 10% buffer-diluted working solution of Giemsa for microscopic detection and the measurement of parasite density according to World Health Organization recommendations .P. falciparum identification was confirmed by SYBR Green quantitative PCR (qPCR) assay after amplification of DNA coding for 18S ribosomal RNA using species-specific primers [P. falciparum positive samples, all PCR reaction of pfhrp2 exon2 and pfhrp3 exon2 were performed at 25 µl total with 2 × Promega Hot Start Master Mix , 0.4 µl each of forward and reverse primers, and 3 µl of extracted template DNA using PCR conditions previously described [pfhrp2 exon2 and pfhrp3 exon2 were separated by electrophoresis on a 2% agarose gel, visualized in a UV transilluminator, and the size of the expected amplicons compared to 1 kb DNA ladder. Primer sequences, PCR conditions, and expected amplicon size of pfhrp2 exon2 and pfhrp3 exon2 are provided in Additional file Genomic DNA was extracted from DBSs using the chelex-saponin method as described previously . P. falc primers . After cescribed , 21, 27.pfhrp2 exon 2 and 88 pfhrp3 exon 2 high quality sequence data were included for molecular analysis of pfhrp2/3 genetic variation. Pfhrp2/3 sequence of this study deposited in the NCBI Gen Bank database (with Accession number: MZ050658-MZ050792). All amplicons were cleaned using Exosap and sequenced using Sanger technology with ABI BigDyeTM Terminator v 3.1 chemistry  and ran on a 3130 Genetic Analyzer . Sequences were then cleaned and analysed using CodonCode Aligner Program V.6.0.2 . Nucleotide sequences were inputted into the ExPASy Translate Tool  and translated into corresponding amino acids sequence using the correct open reading frame. The amino acid repeat sequences in pfhrp2 and pfhrp3 were given a numeric code in the system as described previously [A total of 48 eviously .P. falciparum isolates were analysed using Statistical Package for Social Sciences (SPSS) version 20. The association between PfHRP2 RDT results (sequenced samples could be RDT positive or negative) and certain group of amino acid repeat was tested by Chi square and Fisher’s exact test. P-values < 0.05 were interpreted as statistically significant. Bakers’ model [The proportions of each amino acid repeat of PfHRP2 and PfHRP3 in the s’ model , 11 usedpfhrp2 exon 2 sequences varied between 453 and 873 base pairs (bp) in DNA and 150 to 290 residues in amino acids (aa). A total of 11 known PfHRP2 amino acid repeat types were identified  of the distinct PfHRP2 amino acid sequence occurred only once, while five PfHRP2 amino acid sequence patterns (I–V) were found in more than one isolate , as well as types 12 and 13 . By contrast, types 9 and 11 were not identified in any PfHRP2 amino acid sequences in this study  and Bambasi   between types 7 and 20. Most of the isolates  had a similar unique PfHRP3 repeat motif with structural organization of repeat types 15, 16, 20, 17, and 18, and this motif was found in all PfHRP3 patterns except type VII  analysis reveals a range of 88.50–98.82% similarity with DD) and type 10  repeats were reported previously  was previously reported  were detected as positive by PfHRP2 RDT. Among these PfHRP2 RDT positive samples, 82.9% (29/35) was found with a parasitaemia level > 250 parasite /μL. However, all PfHRP2 RDT negative samples (13/48) were submicroscopic  was detected by PfHRP2 RDT and 84.3% (59/70) of these positive samples had parasitaemia levels > 250 parasites/µl , repeat 2 (P = 0.456), repeat 6 (P = 0.95), repeat 7 (P = 0.486), and repeat 10 (P = 0.273) observed in the samples were not statistically associated with one another. Likewise, the performance of PfHRP2 RDT and variation of PfHRP3 amino acid repeat 1 (P = 0.928), repeat 4 (P > 0.05), repeat 7 (P > 0.05), repeat 15 (P > 0.05), repeat 18 (P = 0.436), and repeat 20 (P > 0.05) were not statistically associated. Interestingly, significant association was detected between performance of PfHRP2 and the number of PfHRP3 repeat 16 (P = 0.031) and repeat 17 (P < 0.001)  that had the novel variants were detected negative by PfHRP2 RDT  followed by group A . Among these isolates, 100% (8/8) in group A and 73.7% (14/19) in group B were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/μl. About 86% (6/7) of P. falciparum diagnosis. The structural organization of PfHRP2 amino acid sequences were less conserved than PfHRP3 among the Ethiopia isolates. The sequence length and the number of amino acid repeat types of PfHRP2 and PfHRP3 in this study were comparable to those previously reported in Yemen [The present study provides valuable information on genetic variations in the PfHRP2 and PfHRP3 amino acid repeat types, which could affect the performance of PfHRP2 RDT for in Yemen  and Madain Yemen , but lowin Yemen  and Ghanin Yemen  and highin Yemen .P. falciparum isolates, similar to previous reports in Kenya [All PfHRP2 repeat types started with repeat type 1 in the Ethiopian in Kenya , Yemen [in Kenya , Senegalin Kenya , and outin Kenya . The majin Kenya , 29, 32 in Kenya . On the in Kenya . Two typin Kenya , 29.P. falciparum isolates in Ethiopia, similar to those reported in Africa [Eleven different PfHRP2 repeats and eight different PfHRP3 repeats were found at Sherkole, Bambasi, Kurmuk and Assosa with slight difference in the frequency of the repeat type within and between n Africa , 30 and n Africa .The PfHRn Africa –30, Asian Africa , 31, andn Africa , 35. BLApfhrp2 /3 throughout the world [On the contrary, PfHRP2 repeat types obtained within this study showed certain difference from African and global reports in a number of ways. First, PfHRP2 type 12 repeat was found in a few isolates (10.5%) this did not align with previous studies from Kenya , Ghana  and Centhe world .Concerning PfHRP3, the majorities of sequences started with type 1 and ended with a type 4 repeats in the Ethiopia isolates, which agrees with previous studies , 31. MorDD, type 10-AHHAATHHATD) and one unique repeat type (type-AHHAAAHHAND) were found with low frequency in Ethiopia and also in a previous study from Ghana [G) was detected with high frequency in Ethiopia as well as in Kenya [Interestingly, of all the novels repeat types identified in this study, 15 in PfHRP2 and 12 in PfHRP3 have not been reported elsewhere. Among all novel PfHRP2 repeat types, two unique repeat types  and shared repeats in PfHRP2 and PfHRP3  were not statistically associated with the performance of PfHRP2 RDT, consistent with previous study . Instead malaria . Moreove malaria . As resuThis study had two limitations. First, the samples represented a limited geographical area from Assoa, Ethiopia. Second, this study did not assess cross reactivity of the possible epitopes based on the amino acid repeat sequence of PfHRP2 and PfHRP3 using specific monoclonal antibodies.P. falciparum isolates within and between the study sites in Ethiopia. There is a need to closely monitor the performance of PfHRP2 RDT and examine the distribution of novel repeat type and shared repeat in PfHRP2/3, and unique repeat found in PfHRP3 broadly in Ethiopia.The present study indicated, for the first time, the presence of extensive existence of genetic variability of PfHRP2 and PfHRP3 amino acid repeats including novel repeats in Additional file 1. The map showing the study area in Assosa zone. The map generated using ArcGIS version 10.0 software.Additional file 2. Primer sequences, PCR conditions and expected amplicon sizes of Pfhrp2exon2 and Pfhrp3 exon2.Additional file 3. Distribution of PfHRP2 and PfHRP3 amino acid repeat by study site.Additional file 4. BLASTP of Ethiopian PfHRP2 sequences for 4 Pattern and 25 distinct haplotype isolates.Additional file 5. BLASTP of Ethiopian PfHRP3 sequences for eight different Pattern.Additional file 6. Comparison of Ethiopia PfHRP2 novel repeat with other countries.Additional file 7. Comparisons of Ethiopian PfHRP3 novel variants with others.Additional file 8. Baker repeats types and P. falciparum infection diagnosis . PfHRP2 sequence isolates classified into four groups according to the frequency number of types 2 × type 7 repeats: group A , group B , group C  and group I  as describe previously .Additional file 9. PfHRP2 RDT and microscopy results in Pfhrp3 sequence isolates.Additional file 10. Effect of the number of PfHRP2 and PfHRP3 amino acid repeat on the performance of PfHRP2 RDT.


Plasmodium falciparum malaria highlights the need for an effective and deployable vaccine. RTS,S/AS01, the most effective malaria vaccine candidate to date, demonstrated 56% efficacy over 12 months in African children. We therefore assessed a new candidate vaccine for safety and efficacy.Stalled progress in controlling ClinicalTrials.gov, NCT03896724.In this double-blind, randomised, controlled, phase 2b trial, the low-dose circumsporozoite protein-based vaccine R21, with two different doses of adjuvant Matrix-M (MM), was given to children aged 5–17 months in Nanoro, Burkina Faso—a highly seasonal malaria transmission setting. Three vaccinations were administered at 4-week intervals before the malaria season, with a fourth dose 1 year later. All vaccines were administered intramuscularly into the thigh. Group 1 received 5 μg R21 plus 25 μg MM, group 2 received 5 μg R21 plus 50 μg MM, and group 3, the control group, received rabies vaccinations. Children were randomly assigned (1:1:1) to groups 1–3. An independent statistician generated a random allocation list, using block randomisation with variable block sizes, which was used to assign participants. Participants, their families, and the local study team were all masked to group allocation. Only the pharmacists preparing the vaccine were unmasked to group allocation. Vaccine safety, immunogenicity, and efficacy were evaluated over 1 year. The primary objective assessed protective efficacy of R21 plus MM (R21/MM) from 14 days after the third vaccination to 6 months. Primary analyses of vaccine efficacy were based on a modified intention-to-treat population, which included all participants who received three vaccinations, allowing for inclusion of participants who received the wrong vaccine at any timepoint. This trial is registered with From May 7 to June 13, 2019, 498 children aged 5–17 months were screened, and 48 were excluded. 450 children were enrolled and received at least one vaccination. 150 children were allocated to group 1, 150 children were allocated to group 2, and 150 children were allocated to group 3. The final vaccination of the primary series was administered on Aug 7, 2019. R21/MM had a favourable safety profile and was well tolerated. The majority of adverse events were mild, with the most common event being fever. None of the seven serious adverse events were attributed to the vaccine. At the 6-month primary efficacy analysis, 43 (29%) of 146 participants in group 1, 38 (26%) of 146 participants in group 2, and 105 (71%) of 147 participants in group 3 developed clinical malaria. Vaccine efficacy was 74% (95% CI 63–82) in group 1 and 77% (67–84) in group 2 at 6 months. At 1 year, vaccine efficacy remained high, at 77% (67–84) in group 1. Participants vaccinated with R21/MM showed high titres of malaria-specific anti-Asn-Ala-Asn-Pro (NANP) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. Titres waned but were boosted to levels similar to peak titres after the primary series of vaccinations after a fourth dose administered 1 year later.R21/MM appears safe and very immunogenic in African children, and shows promising high-level efficacy.The European & Developing Countries Clinical Trials Partnership, Wellcome Trust, and National Institute for Health Research Oxford Biomedical Research Centre. Plasmodium falciparum is a complex pathogen with numerous immune evasion mechanisms. Development of an efficacious vaccine against this parasite has remained elusive for many decades. The leading malaria vaccine candidate, RTS,S/AS01, induces partial efficacy through induction of antibodies against the central repeat (Asn-Ala-Asn-Pro [NANP]) of the circumsporozoite protein (CSP).Malaria remains one of the leading causes of morbidity and mortality worldwide. Evidence before this studyPlasmodium falciparum malaria. We searched PubMed from database inception to March 23, 2021, for published research articles using the terms “malaria vaccine”, “clinical trial”, “phase III”, AND “efficacy”. No language restrictions were applied. The search identified one published large phase 3 clinical trial describing a trial of the pre-erythrocytic malaria vaccine candidate, RTS,S/AS01 (Mosquirix), done at 11 sites in seven countries across sub-Saharan Africa. This vaccine candidate has now progressed to pilot implementation trials after showing efficacy of 36% after four doses, over a median of 48 months follow-up. Efficacy of 56% in children aged 5–17 months was observed over the first year.There are currently no licensed vaccines to protect against P falciparum, in the most recent update to the Malaria Vaccine Technology Roadmap, WHO called for the development of malaria vaccine candidates with a protective efficacy of at least 75% against clinical malaria by 2030, to address this unmet priority public health goal.Due to the high burden and wide geographical distribution of Added value of this studyThis study reports vaccine efficacy of a novel pre-erythrocytic candidate malaria vaccine in a phase 2 trial in children living in a malaria endemic area with high transmission in Burkina Faso. This new vaccine, R21 adjuvanted with 50 μg Matrix-M (R21/MM), administered before the malaria season, demonstrates high-level efficacy, reaching the WHO-specified efficacy goal of at least 75% in the target population of African children over 1 year. Furthermore, R21/MM demonstrates a favourable safety profile. It is well tolerated with the majority of local and systemic adverse events graded mild, and no serious adverse events related to vaccination in the trial. Importantly, although this vaccine immunogen is similar to RTS,S, it does not have the excess HBsAg found in RTS,S and provides a higher density of circumsporozoite protein epitopes on the particle surface, resulting in high levels of malaria-specific anti-Asn-Ala-Asn-Pro (NANP) antibodies. These antibodies were effectively boosted 1 year later to levels similar to those after the primary series of vaccinations.This phase 2 trial is currently continuing for a second malaria season, after the booster vaccine, to determine whether high vaccine efficacy can be maintained.Implications of all the available evidenceThese initial findings with the new R21/MM vaccine candidate appear to improve on the efficacy in children of all other malaria vaccines. These data support the further evaluation of this promising malaria vaccine candidate in a phase 3 trial that will embrace different malaria transmission settings, the coadministration of seasonal malaria chemoprevention, and encompass a wider age range with a high incidence of malaria. An important additional advantage of the new R21/MM malaria vaccine candidate is its potential for large-scale manufacturing and low-cost supply, to support global efforts to better control, sustainably eliminate, and finally eradicate malaria.There remains an urgent need to identify and develop improved vaccine candidates that could achieve the WHO goal of 75% efficacy against clinical malaria by 2030.11Following preclinical studies of R21 plus multiple adjuvants, Matrix-M (R21/MM) was selected for clinical development based on high immunogenicity.14Following an age de-escalation trial of R21/MM in Kenyan adults, children, and infants,This was a phase 2b, randomised, controlled, double-blind trial done at the Institut de Recherche en Sciences de la Santé, Nanoro, Burkina Faso. Participants aged 5–17 months were recruited from the Health and Demographic Surveillance System catchment area of Nanoro, which covers 24 villages, with an approximate population of 65 000 inhabitants. Nanoro is an area of high malaria transmission, with transmission occurring throughout the year, but with a marked peak during the rainy season (June to November).Eligible participants were recruited into three groups. Group 1 received 5 μg R21/25 μg MM, group 2 received 5 μg R21/50 μg MM, and group 3, the control group, received rabies vaccinations. Doses were administered before the seasonal peak of malaria transmission starting in July. Safety, immunogenicity, and vaccine efficacy are being assessed over 24 months, with the primary efficacy endpoint after 6 months, after the primary series of vaccinations (three doses). All participants also received a booster vaccination approximately 12 months after their third vaccination, before the start of the next malaria season. Field workers collected data on indoor residual spraying of households, insecticide treated net use , number of doses of seasonal malaria chemoprevention taken by the participant per month, and number of months seasonal malaria chemoprevention was taken during the malaria season.After community sensitisation, a list of eligible children was drawn from the Health and Demographic Surveillance System database, and parents or legally authorised guardians who expressed interest were invited to screening visits. During recruitment, parents or legally authorised guardians of participants provided written or thumb-printed consent, which was verbally checked at each study visit. Inclusion criteria specified that participants should be aged 5–17 months at enrolment, parents should provide written informed consent, and aim to be living in the study area for the trial duration. Exclusion criteria included significant comorbidities and participation in other malaria intervention studies and clinical trials. Further details are given in the protocol .The trial was approved by the Comité d'Ethique pour la Recherche en Santé, Burkina Faso (reference number 2019-01-012), and the national regulatory authority, Agence National de Régulation Pharmaceutique, Burkina Faso (reference number 5005420193EC0000). Ethical approval was also granted in the UK by the Oxford Tropical Research Ethics Committee (reference number 19-19).Children aged 5–17 months were randomly assigned (1:1:1) to groups 1–3. An independent statistician generated a random allocation list, using block randomisation with variable block sizes. A person independent of the trial prepared and sealed the envelopes using this list, which was then given to the pharmacist to assign to participants. Both malaria and control vaccines were prepared by the pharmacist using the same type of syringe, and the contents of the syringe were covered with an opaque label. The trial was double-blinded; participants, their families, and the local study team were all masked to group allocation. Only the pharmacists preparing the vaccine were unmasked to group allocation.Hansenula polymorpha, comprising the central repeat and the C-terminus of the CSP fused to the N-terminal end of HBsAgR21 was produced by expressing recombinant HBsAg virus-like particles in Plasmodium spp, they were treated for malaria in accordance with national guidelines before having a vaccination.On the day of vaccination, participants were tested for malaria if they had a fever of 37·5°C or higher. If their blood film was positive for After each vaccination, local and systemic solicited adverse events were collected for 7 days. Intensity of symptoms was evaluated following standardised methods. Unsolicited adverse events were collected for 28 days after vaccinations, and safety laboratory values were measured at 28 days after the first and third vaccinations to look for deviations from baseline. Serious adverse events are being recorded for the duration of the study. Clinical judgment by study clinicians was used to assess causality of adverse events and relationship to vaccine. All adverse events were followed up until resolution.Data safety monitoring board reviews were held after the vaccination of the first 30 participants, and after completion of the primary series of three vaccinations.Plasmodium spp. Two independent microscopists, who were masked to the vaccination status of all participants, analysed each blood film, with a third microscopist adjudicating in cases of discrepancy.Parents of participants were advised to attend the community health facility if their child had any illness, or a temperature of 37·5°C or higher or history of fever within the last 24 h, or both, for review and assessment for malaria. After the third vaccination, participants were visited by field workers every 30 days until 6 months after the third vaccination, when, if they had a temperature of 37·5°C or higher or history of fever within the last 24 h, or both, blood sampling was done for blood film microscopy to detect Anti-NANP antibodies were measured by ELISA before first vaccination, as previously described,P falciparum asexual parasite density of more than 5000 parasites per μL. The secondary case definition was presence of an axillary temperature of 37·5°C or higher or history of fever during the last 24 h, or both, and P falciparum parasite density of more than 0 parasites per μL. The secondary objective assessed protective efficacy of R21/MM from 14 days after the third vaccination to 12 months. In addition, cross-sectional asymptomatic P falciparum infection was analysed at months 6 and 12, defined as the presence of axillary temperature of less than 37·5°C, absence of history of fever within the last 24 h, and P falciparum parasite density of more than 0 parasites per μL. Safety, reactogenicity, and humoral immunogenicity of R21/MM were evaluated.The primary objective assessed protective efficacy of R21/MM against clinical malaria from 14 days after the third vaccination to 6 months. The primary case definition of clinical malaria was presence of an axillary temperature of 37·5°C or higher and The study was powered to provide an initial point estimate of the efficacy of the malaria vaccine in either group 1 or 2, assuming that the vaccine efficacy over 6 months was greater than 50%. Due to an unexpectedly high participant retention rate, we had power to detect efficacy greater than 37%.Cox regression models were used to analyse first episodes of clinical malaria from 14 days after the third vaccination to 6 months and 1 year. For participants without an episode of clinical malaria, their time was censored at the date of their withdrawal or the date of their 6-month or 12-month blood sampling. The primary comparisons were prespecified as being between groups 1 and 3 and groups 2 and 3, with comparison of groups 1 and 2 to 3 as a supplementary analysis. A secondary analysis adjusted for confounding factors of sex, age at randomisation  and bednet use (adequate or not). Vaccine efficacy was calculated as 1 minus the hazard ratio (HR).Primary analyses of vaccine efficacy were based on a modified intention-to-treat population, which included all participants who received three vaccinations, allowing for inclusion of participants who received the wrong vaccine at any timepoint. Because all vaccines were administered correctly, this is equivalent to a per-protocol analysis.The secondary outcomes of asymptomatic malaria infection at months 6 and 12 were analysed using a log binomial model, including randomised group as a covariate. Relative risks and 95% CIs were reported for comparisons of groups 1 and 3, and groups 2 and 3. This analysis was also done with adjustment for the confounding factors previously described.To search for an immunological correlate of protection, we divided participants by tertile on their antibody response 4 weeks after the third dose and searched for differences in risk of clinical malaria.To facilitate masking, analyses were done by statisticians external to the investigator teams.All statistical analyses were done using Stata, version 16.1.ClinicalTrials.gov, NCT03896724.This study is registered with The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.From May 7 to June 13, 2019, 498 children aged 5–17 months were screened . 48 chil186 participants had clinical malaria according to the primary case definition when assessing the primary objective of efficacy against clinical malaria of R21/MM from 14 days after the third vaccination to 6 months. These cases of clinical malaria occurred in 43 (29%) of 146 participants in group 1, 38 (26%) of 146 participants in group 2, and 105 (71%) of 147 participants in group 3. A Cox regression model comparing group 1 with group 3 resulted in vaccine efficacy of 74% . Comparing group 2 with group 3 resulted in 77% efficacy  after the third vaccination. 195 participants had clinical malaria—an increase of nine participants from the primary 6-month analysis . These cases occurred in 50 (34%) of 146 participants in group 1, 39 (27%) of 146 participants in group 2, and 106 (72%) of 147 participants in group 3. Cox regression showed vaccine efficacy of 71%  for group 1, and 77%  for group 2 . No sign224 participants had a first episode of clinical malaria by 12 months according to the secondary case definition, including clinical cases with parasitaemia of more than 0 parasites per μL. A Cox regression model comparing group 1 with group 3 showed vaccine efficacy of 70% , and 80%  when comparing group 2 with group 3 .Table 1TSecondary analyses of vaccine efficacy, according to the primary case definition, were done between 14 days after the third vaccination and 6 months or 12 months, adjusting for potentially confounding factors of sex, age at randomisation, and adequate bednet use. Using a Cox regression model, comparing group 1 with group 3 showed a vaccine efficacy of 75%  and comparing group 2 with group 3 showed a vaccine efficacy of 77%  at 12 months . FurtherCross-sectional blood films were done after 6 months and 12 months of follow-up. At 6 months, 28 (19%) of 147 participants in group 3 had asymptomatic parasitaemia, with fewer participants in groups 1 , and 2  with asymptomatic parasitaemia. At 12 months, this number was reduced to six (4%) of 142 participants in group 3, three (2%) of 132 participants (p=0·37) in group 1; and two (1%) of 141 participants (p=0·18) in group 2 .P falciparum. They were diagnosed and treated for severe malaria, and transferred to the referral hospital but died shortly thereafter.Seven serious adverse events were reported in participants  and all Local adverse events of redness, swelling, and pain were reported in a small proportion of participants. 25 (2%) episodes of pain were noted after 1159 R21/MM vaccinations . Fever, Unsolicited adverse events were collected for 28 days after each vaccination and were categorised according to the Medical Dictionary for Regulatory Activities preferred terms. 811 terms were assigned after three vaccinations, and there were no significant differences in number of events per group . LaboratAt baseline, no participant had detectable NANP IgG antibody levels. In group 1, titres reached a geometric mean of 6133 (95% CI 5161–7289) at 28 days after the third vaccination—about three times the level observed in vaccinated adults in the UK.The antibody levels 28 days after the third vaccination were assessed for correlation with vaccine efficacy. After dividing antibody response levels to NANP of the combined group 1 and 2 participants into tertiles—an approach used successfully to identify an immune correlate of vaccine efficacy for RTS,S/AS01We report malaria vaccine efficacy that reaches the WHO-specified efficacy goal of 75% or moreNCT03143218).R21/MM vaccinations were given before and during the start of the malaria season. It remains unclear whether, and by how much, vaccine efficacy might be increased by this approach in seasonal areas, but this is being studied with RTS,S/AS01 in Mali and Burkina Faso , compared with 44% (37–50) reported for RTS,S/AS01 at this site, without planned or implemented seasonal administration, over 12 months of follow-up.The most common local and systemic adverse events with RTS,S/AS01 across the phase 3 trial were pain at the injection site, after 12% (95% CI 11·4–13·4) of the primary series doses, and fever after 31% (30–33) of the primary series doses.15We also found that antibodies to the central repeat of the CSP correlate strongly with protection, and the antibody titres in group 2 with the higher adjuvant dose were about six-times higher than in adults vaccinated in the UK,A surprising finding of the kinetics of antibody response and efficacy with RTS,S/AS01 was that antibody immunogenicity after a fourth booster dose peaked at only about half the level observed after the third vaccination, and efficacy waned considerably over time.This study has some limitations, including the short period of follow-up, although we have now extended the follow-up of this phase 2 trial. The age range of participants was limited to 5–17 months and a wider age range will be investigated in the future. There is also a need to evaluate larger numbers of participants to assess more fully the safety of a relatively new adjuvant formulation and document efficacy at sites with more perennial malaria transmission using non-seasonal vaccine administration.An important advantage of R21/MM relates to its potential for large-scale manufacturing, which will be critical for the supply of hundreds of millions of doses of vaccine required annually for each birth cohort of children in malaria endemic regions of Africa. The R21 paediatric dose is just 5 μg.Follow-up of this phase 2 trial is currently continuing for a second malaria season, after a booster dose in June, 2020, to determine the durability of this high vaccine efficacy. A phase 3 trial across five African sites of differing malaria transmission and seasonality is underway, with the aim of licensure of a safe, low-cost, high efficacy vaccine, which can substantially reduce the malaria disease burden.The study protocol is provided in the appendix. Anonymised participant data will be made available when the trial is complete, upon requests directed to a corresponding author. Proposals will be reviewed and approved by the sponsor, investigators, and collaborators on the basis of scientific merit. After approval of a proposal, data can be shared through a secure online platform after signing a data access agreement. All data will be made available for a minimum of 5 years from the end of the trial.AVSH and KJE are named as coinventors on patent applications related to R21. GG, LF, and JR are employees of Novavax, developers of the MM adjuvant, and US is an employee of the Serum Institute of India, codeveloper of the R21/MM vaccine. The other authors declare no competing interests.


Plasmodium falciparum infection, clinical conditions such as anaemia, thrombocytopenia and leukocytosis are common. Mutation in haemoglobin sub-unit beta gene (HBB) may be a genetic factor responsible for these haematological changes during infection. However, the contributions of the carriage of different HBB genotypes on these changes remain largely unknown.In P. falciparum-infected children  with different haemoglobin sub-unit beta (HBB) genotypes . Children with acute febrile conditions were recruited at Jaramogi Oginga Odinga Teaching and Referral Hospital at the outpatient clinic. Haematological parameters were determined using Beckman Coulter counter ACTdiff2™ while HBB genotyping was done using TaqMan® SNP genotyping assay. Chi-square (χ2) was used to determine differences between proportions. Differences in haematological parameters were compared across groups using Kruskal Wallis test and between groups using Mann Whitney U test. Partial correlation test was used to determine correlation between haematological parameters and sickle cell genotypes while controlling for age and sex.In this cross-sectional study, we evaluated haematological abnormalities in P = 0.001], haematocrit (HCT), , red blood cells (RBC),  were markedly reduced in HbSS, however, red cell distribution with (RDW)  was increased in malaria infected children with HbSS. Severe anaemia was highest in HbSS (23.1%) followed by HbAA (8.6%) and HbAS (7.1%). There were no differences in platelet count (P = 0.399) hence no severe thrombocytopeania across the genotypes. Leukocytosis was highest in HbSS (69.2%), 42% in HbAS and 31% in HbAA. The RBC, HCT and Hb had negative correlation with RDW in HbSS in malarial-infected children ,  and , respectively.Haemoglobin (Hb), [median (IQR); 7.3 (1.3), HBB genotypes. Therefore, carriage of HbSS genotype is correlated with severity of haematological abnormalities.Our study reveals that anaemia is the most common abnormality in malaria-infected children with carriage of HbSS. The RBC, HCT and Hb concentration decrease with increase in RDW levels in infected children with carriage of HbSS compared to other The online version contains supplementary material available at 10.1186/s12879-021-06025-7. P. falciparum is the most deadly and most prevalent species  in the single nucleotide polymorphism rs334 by the following custom primer and probe sequences: Forward-TCAAACAGACACCATGGTGCAT, Reverse-CCCCACAGGGCAGTAACG, VIC-CTGACTCCTGAGGAGAA-MGB, 6FAM-CTGACTCCTGTGGAGAA-MGB, respectively and as per our previous work ,  and , respectively. Red cell distribution width , Mean Corpuscular Volume , Mean Corpuscular Heamoglobin , Mean Corpuscular Haemoglobin Concentration , white blood cells , lymphocytes (P = 0.103), monocytes (P = 0.084), and granulocytes (P = 0.354) counts were comparable between the two groups  were however, not different between the groups (P = 0.119). The children with malaria infection further had reduced plateletcrit (PCT), infected . Furthermore, red blood cell (RBC) count were equally significantly higher in HbAA  and HbAS  compared to HbSS . Nonetheless, Red Cell Distribution Width (RDW) was relatively lower in HbAA  and HbAS  compared to HbSS . In cases where significant differences were detected using the Kruskal-Wallis test, we performed a post-hoc pairwise analysis using Dunn’s multiple comparison test. The post-hoc test was run on haemoglobin, haematocrit red blood cell count and red cell distribution width. Our results revealed that haemoglobin was reduced in children with HbSS than those with HbAS (P = 0.001). Furthermore, haematocrit was markedly reduced in children with HbSS than those with HbAS (P = 0.002). Red blood cell count was reduced in children with HbSS than those with HbAS (P = 0.023). However, red cell distribution width was highly raised in children with HbSS than those with HbAS (P = 0.001). There were no significant differences in the levels of other hematological indices and carriage of different HBB genotypes in children with P. falciparum malaria. These results are summarized in Table Haematocrit levels were significantly higher in HbAA  and HbAS  relative to HbSS  and HP. falciparum infection and carriage of different sickle cell genotypes. Anaemia was defined according to the World Health Organisation’s criteria as a condition where haemoglobin level < 11 g/dl in children [To determine whether carriage of different sickle cell genotypes have effect on the severity of haematological abnormalities once an individual is infected, we evaluated their severity in children with children . These schildren . Severe 3 μL− 1 of blood [3 μL− 1), moderate (platelet count between 50 and 100 × 103 μL− 1) and mild (platelet count between 100 and 150 × 103 μL− 1). Otherwise, any platelet count above 150 × 103 μL− 1 was considered non-thrombocytopenic. None of the participants demonstrated severe thrombocytopenia in any of the HBB genotypes. Moderate thrombocytopenia was seen in 5.2 and 7.1% of HbAA and HbAS, respectively. However, none had thrombocytopenia in children with HbSS genotype. Mild thrombocytopenia was demonstrated in 15.5, 7.1 and 15.4% in individuals with HbAA, HbAS and HbSS, respectively. However, non-thrombocytopenia was demonstrated in 79.3% of children with HbAA, 85.7% of children with HbAS and 11% of children with HbSS.Thrombocytopenia was defined as a haematological condition in which platelet count is below 150 × 10of blood . For eva3 μL− 1 of blood. There was no leucocytopenia in any of the study participants. Leukocytosis was defined as a benign abnormality in which total leucocyte count is above 11 × 103 μL− 1) of blood [3 μL− 1) was seen in 70% of those with HbAA genotype, 57.1% in those with HbAS and in 30.8% in those with HbSS genotype.Leucocytopenia was defined as a condition in which total leucocyte count is below 4 × 10of blood . LeukocyMonocytosis was defined as a condition in which total monocyte count is below 12% of the total white blood cell count in peripheral blood , 25. Monr = − 0.750, P = 0.008 and r = − 0.634, P = 0.049, respectively). Furthermore, there was a negative correlation between RBC count and both MCV and MCH in malaria-infected children  and , respectively. Haematocrit was negatively correlated with RDW in both malaria-infected and non-infected children  and , respectively. Additional analysis in the carriage HbAS genotype, showed a negative correlation between haemoglobin with RDW in malaria-uninfected children . The RBC count showed a negative correlation with MCV  and with MCH  in malaria-infected children.Using partial correlation test while controlling for age and sex as factors that have previously been shown to affect haematological indices , the stur = − 0.324, P = 0.002), MCH , MCHC  and RDW  in children without malaria infection. Haematocrit showed a positive correlation with MCV , and a negative correlation with MCHC  and RDW  in children without malaria infection. Haemoglobin showed a positive correlation with MCV , MCH  and MCHC , however, it showed a negative correlation with RDW  in children without malaria infection. The RBC showed a negative correlation with MCV , MCH , MCHC  and RDW  in malaria-infected children. There was a negative correlation between haematocrit and PDW  in children infected with malaria. Haemoglobin showed positive correlation with MCH  and MCHC  in children infected with P. falciparum malaria. Further analysis revealed a negative correlation between haemoglobin and RDW  in children infected with P. falciparum malaria and  in children without P. falciparum malaria. It is therefore probable that the RBC count, haematocrit and haemoglobin concentration decrease with increase in RDW levels in P. falciparum-infected children with carriage of HbSS genotype relative to other HBB genotypes. Correlation between primary red cell measurements and red cell indices are summarized in Table In HbAA genotype, RBC count showed a negative correlation with MCV  was 0.21, which is consistent with previous studies in other malaria endemic parts of Sub-Saharan Africa [HBB rs334 allele distributions observed in the current study emphases that malaria infection is an important factor driving genetic drift towards sickle cell. In the current study, therefore, we determined severity of haematological abnormalities and correlation of haematological parameters with HBB genotypes in children with P. falciparum malaria in Kisumu County, western Kenya, a P. falciparum holoendemic region.Children from Sub-Saharan Africa continue to bear the largest burden of  malaria . Haemato malaria , 28. PreHBB gene , 27, 29.HBB gene  such as n Africa , 32. TheP. falciparum infection had increased red cell distribution width. The increased RDW in HbSS reported in the current study is explainable by pre-existing anisocytosis and poikilocytosis, which have been previously reported to be common biological conundrum in sickle cell disease [P. falciparum-infected children with HbSS genotype could be a modulated hereditary persistence of foetal haemoglobin (HPFH) [Our current study reveals the presence of severe anaemia in malaria-infected children with HbSS genotype. This observation could be due to chronic haemolysis observed in sickle cell anaemia  and acce disease , 26. Inc disease . The absn (HPFH) . It woulP. falciparum and had the carriage of HbAA genotype. Even though the mechanism was not explored in the current study, this phenomena is attributable to reduced haemolytic mechanisms and slowed removal of infected red blood cells by erythro-phagocytosis in non-severe malaria [P. falciparum infection had the lowest haemoglobin level as compared to HbAS and HbAA patients. This finding showed consistency with the findings of a similar study in hospitalized Cameroonian children with sickle cell anaemia and infected with malaria [The presence of mild and moderate anaemia was observed in children infected with  malaria . Our fin malaria . The min malaria , which t malaria , 42. Fur malaria .P. falciparum infection. Our study did not find thrombocytopenia as a haematogical abnormality in children with P. falciparum malaria irrespective of the HBB genotypes. However, this finding differed from earlier studies which demonstrated thrombocytopenia in HbAA genotype individuals infected with P. falciparum [P. falciparum infection and attributed this to peripheral destruction of platelets and their consumption by disseminated intravascular coagulation process, as well as splenic pooling [Even though lower platelet count was seen in the infected children relative to non-infected children in HbAA genotypes, it did not differ across other genotypes (HbSS vs. HbAS). This means that thrombocytopenia was not demonstrated in carriage of any haemoglobin type during lciparum , 9, 27.  pooling . On the  pooling . Our finP. falciparum malaria infection [P. falciparum malaria. This finding was not surprising since it has been previously established that leukocytosis is a common biological response in severe malaria anaemia to counter the infection [Our study further showed that children with HbSS genotype and infected with malaria had reduced monocytosis compared to those with HbAA and HbAS genotypes. A previous study showed a positive correlation between monocyte to lymphocyte ratio in the presence of malaria and the level of parasitaemia . Furthernfection . Monocytnfection , 36 and nfection . Monocytnfection –48. Perhnfection . In contnfection . Initialnfection , 50 whilnfection –53 in manfection , 54, 55,nfection . It is anfection .(HBB) have been found to primarily affect the red blood cells structurally resulting into sickle cell and physiologically affecting normal red cell functions [P. falciparum and presenting with different HBB (6GAG > 6GTG) genotypes. Erythrocyte counts revealed a strong negative correlation with RDW in children infected with malaria and had carriage of the HbSS genotype. Furthermore, this correlation trend was also seen in the malaria-infected children with the carriage of HbAS and HbAA. This implies that increasing the RDW reduces the erythrocyte counts once an individual is infected and this situation is aggravated by carriage of the HbSS genotype. These findings therefore demonstrate the effects of malaria infection on haematological parameters [Mutations in haemoglobin beta sub-unit gene unctions . Howeverrameters  and the Limitation in the current study included fewer number of subjects in HbAS and HbSS, lack of information on the nutritional status of the participants, possible presence of other co-infections e.g. HIV, bacteremia and intestinal infections such as hookworm and tapeworm which have been previously reported to worsen anaemia , 60, 61.We recommend that a similar longitudinal study with a larger sample size should be conducted in the same study area taking into account the level of HbF among other haematological parameters, as this may be more informative. We equally recommend inclusion of laboratory investigations of bacteremia and parasitic infections.P. falciparum-infected children with carriage of HbSS genotype compared to other HBB genotypes. Therefore, carriage of HbSS genotype contributes to the severity of haematological abnormalities once a child is infected P. falciparum.Our study reveals that anaemia is the most common haematological abnormality in malaria-infected children with carriage of HbSS genotype. The RBC count, haematocrit and haemoglobin concentration decrease with increase in RDW levels in Additional file 1.