Plasmodium falciparum while overlooking other Plasmodium species such as P. malariae. A better understanding of the role of Plasmodium species other than P. falciparum is needed to strengthen malaria elimination initiatives. The aim of the present study was to elucidate the contribution of P. malariae to malaria transmission in Cameroon.Malaria control efforts are highly skewed towards Plasmodium spp. was also determined by quantitative PCR, and dried blood spots were collected from 156 participants with the aim to detect different Plasmodium species by nested PCR.The study was conducted in the Ngatti Health District, a forest–savannah transition area in the Adamawa Region, Cameroon. A total of 497 individuals aged from 1 to 85 years were diagnosed with malaria in November 2020 using a rapid diagnostic test (RDT) and microscopy. Adult mosquitoes were collected between September 2019 and March 2020 by indoor aspiration and identified morphologically and molecularly. The infection status of Plasmodium prevalence was 50.3%, 51.8% and 64.7%, as detected by microscopy, the RDT and PCR, respectively. Based on the PCR results, P. falciparum was the most prevalent species (43%); followed by co-infections P. falciparum/P. malariae (17%), P. falciparum/P. ovale (1.3%), P. falciparum/P. ovale/P. malariae (1.3%); and then by P. malariae mono-infection (2.5%). The same trend was observed using microscopy, with 35% of participants infected with P. falciparum, 11% co-infected with P. falciparum/P. malariae and 4% infected with P. malariae. The prevalence and parasite density of malaria infection varied significantly with age group (P < 0.05), with the highest prevalence rate observed in children aged 6–10 years (P = 0.0001) while the density of Plasmodium infection increased significantly in children aged < 5 years compared to the other age groups (P = 10−3). Among the 757 Anopheles mosquitoes collected, 737 (97.35%) were An. funestus sensu stricto, 15 (1.9%) were An. gambiae and 5 (0.6%) were An. hancocki. The Plasmodium species recorded at the head/thorax level were P. falciparum and P. malariae, with a sporozoite infection rate of 8.4%; the highest sporozoite infection rate was recorded at Mibellon village (13.6%).The overall P. malariae, in addition to P. falciparum, to the high malaria transmission rate in this region. These findings highlight the need to deploy initiatives to also tackle this Plasmodium species to eliminate malaria in the region.The results of this study reveal the significant contribution of  The past decades have been witness to a substantial reduction in the number of malaria cases and deaths due to malaria worldwide by 37% and 60%, respectively, between 2000 and 2015 [Anopheles species can be considered to vectors of malaria disease to humans in Cameroon [Anopheles gambiae sensu stricto (An. gambiae s.s.), An. coluzzii, An. arabiensis, An. funestus, An. nili and An. moucheti [Plasmodium falciparum and, to a certain degree, Plasmodium vivax, making them the most studied Plasmodium species responsible for severe cases of malaria disease. However, Plasmodium malariae and Plasmodium ovale, which have been reported to be widely distributed in most malaria-endemic regions of the world, are less studied and difficult to identify by light microscopy because the low level of parasitaemia is often below detection thresholds of microscope readers. This failure is the result of sub-microscopic malaria, which accounts for 20–50% of all human-to-mosquito transmissions in sub-Saharan Africa [P. malariae infection is usually benign, but could cause morbidity such as severe anaemia, acute renal failure and splenomegaly [P. malariae and P. falciparum, it has been reported that P. malariae infection can reduce the burden of parasitaemia of P. falciparum infection by up to 30% and contribute to the reduction of the pathology due to P. falciparum as the people become even more asymptomatic [P. malariae prevalence when the prevalence of P. falciparum decreased. These results demonstrate that there is a need for more information regarding the epidemiology of non-falciparum species in the context of the distribution of malaria vaccines targeting only the P. falciparum antigen. Four Plasmodium species have been recorded to circulate in Cameroon: P. falciparum, P. ovale, P. malariae and P. vivax [Plasmodium falciparum remains the predominant Plasmodium species followed by P. ovale and P. malariae, with average prevalence rates of infection of 95%, 3% and 1%, respectively [P. falciparum malaria and especially on symptomatic malaria cases in patients aged < 15 years [Plasmodium in a patient with an axillary temperature of < 37.5 °C). In the study reported here, we first conducted an entomological survey to identify the vector competent to transmit P. malariae. Then we used molecular, microscopic and RDT techniques to evaluate the dynamics of P. falciparum and P. malariae infection transmission in all age groups of the study population.Despite past and ongoing efforts to eliminate malaria, the disease remains endemic in some parts of the world, with > 90% of cases recorded in the sub-Saharan region of Africa. This is largely due to the increasing resistance of mosquitoes to insecticides, which has diluted the effectiveness of malaria control measures . Hence, Cameroon –7, with moucheti , 8. To dnomegaly , 11. It nomegaly . In the ptomatic –15. A stptomatic  in TanzaP. vivax –19. PlasP. malariae/P. ovale or P. vivax infection rate of 4% in the mosquito population of Mibellon village, one of the villages in our study area.The entomological survey was performed by collecting mosquitoes in six locations between September 2019  and March 2020  /m2 [Z-score (a Z-score of 1.96 was used with a 95% confidence level), P = the percentage of population probability (assumed to be 50% = 0.5) and m = the margin of error (taken to be 5% = 0.05). All people from year 1 and thereafter were considered for blood collection.The sample size of the human population was calculated following the formula: s = z − P)/m2 , where sDuring the survey, malaria diagnosis was carried out using thick blood smears, RDTs  Combo, Access Bio Inc. 65 Clyde Road. Somerset, NJ, 08873). and PCR analyses. Body weight, temperature, gender and age of subjects sampled in the study were taken into account. The blood was collected on slides in all the localities and on Whatman FTA cards  only in Mibellon and Moinkoing villages.Plasmodium parasites. Parasite density was defined as the number of Plasmodium parasites per microlitre of blood, counted against 200 leukocytes and expressed according to the WHO guidelines as the number of asexual parasites per microlitre of blood, assuming a leukocyte count of 8000 cells/μl. Samples of blood drops were deposited on Whatman FTA cards and dried for 24 h at room temperature, following which they were stored in clean zippered bags until DNA extraction.Thick blood smears collected from the finger of each subject sampled in the study were air-dried and stained with 10% Giemsa solution for 20 min. The slides were screened by direct microscopy (100×) to visualise the different species of DNA was extracted from blood on Whatman paper using the Chelex protocol. Briefly, 400 µl of 5% chelex100 was added to an Eppendorf tube containing a blood sample on filter paper. The tubes were placed in a shaking dry heater and incubated at 56 °C for 1 h, following which it was incubated for 30 min at 95 °C in a shaking heater. The tube was then removed and centrifuged at a maximum of 13,500 rpm for 10 min. The supernatant was then transferred into a labelled tube and stored at − 20 °C .Plasmodium species infections were performed with all samples using the nested protocol with some minor modifications to differentiate the various species of Plasmodium [Plasmodium was amplified. In the first reaction, PCR amplifications were performed in a 15-μl volume containing 1.5 μl PCR buffer A, 0.75 μl 25 mM MgCl2, 0.12 μl 25 mM dNTPs, 0.51 μl forward and reverse primers (10 mM), 0.12 μl Kapa Taq, 10.49 μl ddH2O and 1 μl DNA. A 5-μl sample of the first reaction’s product was used as a template in a second nested reaction to specifically differentiate P. falciparum, P. vivax, P. ovale and P. malariae products. The temperature profile for the second PCR was: 5 min at 95 °C, followed by 25 cycles of 1 min at 94 °C, 2 min at 58 °C, 2 min at 72 °C; with a final cycle at 72 °C for 10 min. The cycles were repeated 30 times for the second reaction at the same cycling parameters. After electrophoresis in 1.5% agarose gel and staining with Midori green, the amplicon products were visualized under UV light. Plasmodium identification was made based on amplicon sizes.Real-time PCRs (RT-PCRs) for the identification of asmodium . A speci Anopheles mosquitoes collected were morphologically identified using morphological keys [Female mosquitoes were captured inside houses using an electric aspirator during the early morning hours of between 05.30 a.m. and 08.00 a.m. Femalecal keys . These mcal keys . Anopheles mosquitoes were dissected individually into between heads, thoraxes and abdomens. Genomic DNA (gDNA) was extracted from each body part (head + thorax and abdomen) using the LIVAK method [Plasmodium sporozoite infection rate was assessed using the TaqMan assay [P. ovale, P. vivax and P. malariae species [All femaleK method , and thean assay . As a la species .χ2). Parasitaemia was log transformed before analysis. Means were compared using an independent sample t-test and analysis of variance where appropriate; Kruskal–Wallis tests were used to make associations between P. falciparum, P. malariae and mixed infection and other variables of interest. P-values < 0.05 were considered to indicate significance, and a 95% confidence interval (CI) of geometric means that did not overlap was considered to indicate a significant difference.Data analysis was conducted using the Microsoft Excel sheet , GraphPad Prism V8.02R software . Data were summarised into means and standard deviations (SD), and percentages were used in the evaluation of the descriptive statistics. Proportions were compared using the Chi-square test  , Anopheles gambiae sensu lato (An. gambiae s.l.)  and Anopheles hancocki .A total of 757 Plasmodium sporozoites was 8.4%, recorded in An. funestus s.s. and An. gambiae s.l. , P. OVM  (2.5%) and mixed infections  (0.4%) were recorded in Mibellong village. Mosquitoes infected with P. OVM were found only in the Mibellong and Tibati localities, and two mosquitoes were found to be infected with a mixed infection  in Mibellong and Meng  was observed only in Mibellong (1.8%) and Mbougam (1.1%).The P.OVM were P. malariae for those with successful amplification .For the microscopy analyses, samples were collected from 497 people  enrolled in this study. Due to some amplification issues, only 156 samples were analysed by PCR, at two locations (Mibellon and Moinkoing), from 67 (43%) males and 89 (57%) females, respectively. The prevalence of malaria infection assessed by microscopy, RDTs and PCR on the same set of samples (156) was 47.4%, 43.7% and 64.7%, respectively. The general prevalence of infection taking into account the entire study population was 51% (252/494) for microscopy and 51.8% (252/484) for microscopy  of the individuals sampled were asymptomatic. Microscopy, RDTs and PCR showed that 44.8% (95% CI: 37.0–59.1), 43.7% (95% CI: 33.9–55.6) and 62.5% (95% CI: 37.0–59.1) of asymptomatic people, respectively, were infected with P. falciparum, four (2.5%) for P. malariae, 27 (17.1%) for P. falciparum/P. malariae, two (1.3%) for P. falciparum/P. ovale and two (1.3%) for P. falciparum/P. ovale/P. malariae  for P. malariae, 57 (11%) for P. falciparum/P. malariae and two (0.4%) for P. falciparum/P. ovale , Moinkoing (54.6%) and Ngatti (51%) villages was high, while the lowest prevalence of 12% was recorded in Mbougam village  was recorded in Mibellon (25.7%)  tested positive for .3% testeP. falciparum prevalence of 47.7% (95% CI: 34–64) was observed among older children aged 6–10 years, followed  by teenagers aged 11–15 years , infants aged 1–5 years , young adults aged 16–20 years  and adults, with the latter age group showing the lowest prevalence of 22% (95% CI 19–26%)  followed the same trend, with the highest prevalence of 27% observed in children aged 5–10 years, followed  by subjects in the age groups 1–5, 10–14, 16–20 and > 21 years, with 22%, 14.2%, 4.5% and 1% infection rates, respectively  was recorded in subjects aged 16–20 years, followed a prevalence of 7.1% in subjects aged 11–15 years. The prevalence of P. malariae obtained in subjects aged 6–10, 1–5 and 21+ years was 3.4%, 3.3% and 2%, respectively  was highest in children aged 6–10 years , while the younger adolescents (aged 10–14 year) were more infected with P. malariae with a prevalence of 9%.According to age groups, the highest  95% CI: 4–64 was  95% CI: 4–64 was  95% CI: 4–64 was P. falciparum and P. malariae parasitaemia was 4354.7 ± 7991.6 and 606.1 ± 572.9 parasites/μl, respectively. The highest parasitaemia was recorded in children aged 1–5 years, at 5873.9 ± 8910.2 and 854.4 ± 608.4 parasites/μl for P. falciparum and P. malariae,, respectively. The lowest parasitaemia was observed in adults (age group: 21–85 years), at 1901.8 ± 2389 and 215 ± 119.8 parasites/μl for P. falciparum and P. malariae, respectively .The overall geometric mean P. falciparum and P. malariae infections in mosquito and human populations. The study produced strong evidence that malaria remains highly prevalent in the study locations in Cameroon.In this study we assessed the transmission dynamics of An. funestus s.s., An. gambiae s.l. and An. hancocki, of which An. funestus s.s.(97.3%) was the most prevalent species. Our results are similar to those obtained by Tchouakui et al. in Mibellon [An. funestus prevalence. Our findings vary slightly from those of Tchouakui et al. [An. funestus s.s. in the same village and from those of Menze et al. [Three anopheline species were identified in the study area: Mibellon , where ti et al.  who recoe et al.  who obseAn. funestus is the dominant species in this locality and that the prevalence could vary according on the season since the different collections did not take place during the same season. To the contrary, in Tibati, we recorded 94.4% An. funestus prevalence compared to 55.2 %and 16% prevalence in collections from 2015 and 2017, respectively, by Feufack-Donfack et al. [An. gambiae development become scarce.The focus on the collection of indoor blood-fed mosquitoes may have prevented us from collecting the outdoor resting species of subfamily Anophelinae. Otherwise, our results showed that k et al. . This diAn. funestus and An. gambiae were infected with Plasmodium spp. A slight increase in the overall infection rate was observed in An. funestus in the present study compared to samples collected in 2017 in Mibellon by Menze et al. [X2 = 3.43, P = 0.06) and also to samples collected in Tibati in 2017 by Feufack-Donfack et al. [X2 = 3.14, P = 0.07). Although not significant, this increase in sporozoite infection rate could be due to the reduction in efficacy of LLINs (approx. 3 years) that were massively distributed in this part of the country in 2015. The prevalence of infection was also low in another location.Both e et al.   and, in particular, the infection rate by P. malariae  of mosquitoes collected in 2019 strongly support the role of physical barriers played by the mosquito midgut and immune genes in the process of oocyst migration to the salivary gland [P. falciparum and P. malariae could also be due to the lifespan of mosquitoes, which is generally no longer than 3 weeks in nature. In fact, P. malariae has a longer sporogonic cycle (21 days) [P. falciparum (14 days), which may explain the reduced likelihood of finding P. malariae sporozoites in the mosquitoes. The study site of Mibellon had a high infection rate with P. falciparum and P. malariae; this can possibly be explained by the perennial presence of the malaria vector, mainly An. funestus, during the year in Mibellon. The high infection rate of oocysts and sporozoites was recorded among the mosquito population of Mibellon village despite the human factors, vector immunity, midgut microbiota [P. malariae in the human population.The significantly higher overall infection rate found in the abdomen of the mosquitoes compared to the head + thorax   than P. crobiota , all of Plasmodium spp. infection rate recorded in the present study could be due to several factors, such as the sampling season and the molecular method used for species identification. The PCR test used in this study targeted the 18S rRNA gene [ cox III [ cox III method is able to detect an additional 10–50% more cases with high sensitivities for all four Plasmodium species due to the high number of parasite copies (20–150) found in cox III compared to 18S (4–8 copies) [The high prevalence of infection and high parasitaemia indicate that the Ngatti health district is a high-risk zone for malaria transmission. This is supported by earlier reports of the prevalence of malaria infection in the Adamawa Region varying from 8.1% to 10.6% in symptomatic children aged between 2 and 9 years , 20, 31.RNA gene  whereas  cox III  in the P copies) .P. falciparum and Plasmodium mixed) infection was recorded in children in the age group 6–10 years. Regarding this result, it should be noted that malaria control campaigns target children aged < 5 years of age and pregnant women, with parents advised to ensure that children aged < 5 years, who are considered at high risk of contracting malaria, sleep under a LLIN. This possibly explains the reduction in the prevalence of infection among children in this young age group. However, the good use of ITNs for children aged < 5 years may impede or delay the development of malaria protective immunity in children aged 6–10 years [P. falciparum decreased significantly with age from 10 to 85 years, which could be accounted for by the acquisition of protective immunity by adolescents and adults following multiple exposures to malaria infection [The results also revealed an association between the prevalence of malaria and age at infection. The highest prevalence of malaria  and, not surprisingly, the highest parasitaemia was recorded in children aged < 5 years. This result is explained by the low levels of immunity in young children and increasing naturally acquired immunity in older children through repeated exposure to the parasite [An estimation of malaria parasite density is necessary for patient management. Our study revealed that asymptomatic people coming from different villages in the Ngatti health district tolerated the high burden of asexual parasitaemia of up to 56,600 parasites/μl blood for parasite , 42.P. malariae obtained by microscopy was low, although higher than the general prevalence observed in Cameroon (3%) [P. falciparum in the lower age groups can be assumed to allow non-falciparum species, such as P. malariae, to become more important as P. falciparum is eliminated. In general, high transmission areas constitute a significant infectious reservoir of malaria parasites due to the potentially high percentage of asymptomatic malaria parasite carriers in this location [The prevalence of oon (3%) . The higlocation , 14, whiP. falciparum as well as P. malariae, in the asymptomatic population. The findings revealed that malaria endemicity in the Adamawa Region of Cameroon is driven by multiple Plasmodium species, although P. falciparum remains the most prevalent. In addition, P. malariae infection and P. falciparum/P. malariae co-infections are both transmitted to humans by An. funestus in the studied localities and play an important role in malaria transmission. We found that older children were the age group most infected with malaria. Thus, beyond the control of malaria targeting only the under-fives, pregnant women and P. falciparum, a comprehensive policy targeting all age groups of the population should be put in place to eliminate malaria and its reservoir species more effectively.The aim of this study was to examine the prevalence of malaria infection, targeting both


Plasmodium infection by intramuscular administration of a lipids-based formulation at a dose of decoquinate 200 mg/kg. The mice that were inoculated with Plasmodium berghei sporozoites 34 days after the administration of a one-time drug dose were continuously monitored for 60 days and shown to be free of Plasmodium parasites. The optimized formulation for the sustained release of decoquinate was prepared by hot melt extrusion, constructed by lipids including cholesterol and mono or diglycerides, and had a drug load of 20 to 40% and particle size of 30 to 50 μm. Decoquinate of the lipids-based formulation was slowly released in vitro at a constant rate for the duration of two months, and was examined and continuously exposed at a therapeutic level in the blood for as long as 4 to 6 months. Further evaluation showed that the lipids-based formulation at doses of decoquinate 100 to 150 mg/kg could protect mice from Plasmodium infection for a period of 120 days. It is the first time that cholesterol has been used for a controlled drug delivery system of decoquinate. The results may provide useful information, not only for preparing a formulation of long-acting decoquinate but also in general for developing a controlled drug release system. The one-time administration of pharmaceutical agents in such a slow-release system may serve patients with no concerns about compliance.Sustained-release formulations of decoquinate were evaluated for the long-term prophylaxis of malaria. In the initial experiment, mice were protected from liver-stage  The World Health Organization (WHO) reported that there were an estimated 241 million cases of malaria in 2020, and estimated malaria deaths of 627,000, about 80% of which were children under the age of 5. The incidence of malaria is increasing in some regions. In 2020, about half of the world’s population was at risk of malaria. Most cases and deaths occur in sub-Saharan Africa. However, the other WHO regions also report significant numbers of cases and deaths [Plasmodium sporozoites enter the hepatocytes of susceptible human beings when an infected mosquito bites. The sporozoites injected from the salivary glands of mosquitos are moved on into the blood circulation through which they travel to the liver and invade the hepatic parenchymal cells where sporozoites develop into liver-stage schizonts. Plasmodium (P) falciparum and P. vivax are the two most common parasites causing human malaria. In human primary hepatocytes, the development of P. falciparum takes 7 days, whereas P. vivax completes development after 9 days [P. vivax and P. oval can have their sporozoites differentiated into both liver-stage schizonts and hypnozoites. The schizonts then burst to release uninucleate merozoites. Hepatocyte-derived merozoites invade erythrocytes and enter a few-step cycle to develop into schizonts-containing asexual cells. Certain host erythrocytes develop into gametocytes associated with sexual cells. The hypnozoites are the dormant parasites in the liver that can cause a relapse of malaria months or even years later. The liver stage provides an ideal target for antimalarial intervention to block malarial progression before the manifestation of clinical symptoms caused by the rupture of schizonts from erythrocytes [r 9 days . Some pahrocytes ,4.Plasmodium liver stage and/or blood stage. Artemisinin, acting on blood-stage Plasmodium, is the first-line drug for treating severe malaria caused by P. falciparum. It must be combined with a different class of antimalarials to avoid drug resistance. Artemisinin has not been recommended for malarial prophylaxis. Chloroquine is prescribed for the treatment of P. vivax malaria and is no longer recommended for treating P. falciparum malaria. Chloroquine does not have activity against liver stage Plasmodium. It is prescribed for the intra-erythrocytic stages to prevent malaria only in a few areas with chloroquine-sensitive malaria [Antimalarial medications can target the  malaria . It is tPlasmodium liver stage, including atovaquone/proguanil , primaquine, and tafenoquine. Malarone is two combined drugs with synergistic actions that can be taken as a causal and suppressive prophylactic agent, but it is not effective in suppressing hypnozoites. Therefore, it cannot be used as a radical cure for malarial relapse caused by P. vivax or P. oval. It is, due to its cost, generally limited to travelers from industrialized countries. Primaquine and its derivative tafenoquine, a recently approved new drug, are the only drugs effective against hypnozoites. However, both primaquine and tafenoquine can cause fatal hemolysis in people with G6PD deficiency and should never be prescribed as prophylaxis to anyone with G6PD deficiency or unknown G6PD status [P. vivax in patients ≥ 16 years. Krintafel should only be used in combination with chloroquine, not other antimalarials, against acute P. vivax infection.There are several antimalarials that target the D status . TafenoqPlasmodium so that the activity of active pharmaceutical ingredients (API) can last for a longer period [As a measure of prophylaxis, vulnerable populations take antimalarial drugs at regular intervals to prevent acute malaria attacks. Currently, all drugs used for prophylaxis require frequent administration. There have been efforts to make controlled-release formulations of antimalarials to treat experimental cerebral malaria or blood-stage r period ,7,8. HowP. falciparum sporozoites 4 to 36 weeks after the administration of CIS43LS. The monoclonal antibody provided causal prophylaxis, which targets Plasmodium infection at the intrahepatic stage, and the results were encouraging. The only vaccine currently available is RTS, S/AS01, which prevents infection, maturation, and multiplication within the liver but has limited efficacy [In addition to chemoprophylaxis, another important aspect of controlling malaria involves monoclonal antibodies applicable to fight against malaria. Recently, a long-acting antimalarial monoclonal antibody CIS43LS was assessed in a two-part, phase 1 clinical trial . Particiefficacy . It has efficacy . AlthougOne alternative that has been in progress is the use of long-acting injectable formulations of API . Long-acA great deal of research aiming at application has been conducted by using biodegradable polymers. Poly (lactic-co-glycolic acid) (PLGA) is the most widely used polymeric biomaterial in controlled drug delivery systems including small chemical and large biopharmaceutical molecules ,14. HowePlasmodium parasites [P. falciparum as well as chloroquine- or multidrug-resistant P. falciparum in infected human erythrocytes and protect mice from severe malaria [Plasmodium infection. The nanoparticle formulations of DQ are very potent at inhibiting Plasmodium berghei in vitro at the liver stage (IC50 < 0.5 nM) and highly efficacious (1–3 mg/kg) in protecting mice from liver stage malaria [Plasmodium might also be associated with its pharmacokinetic feature in that there is high enrichment of DQ in the liver whether the nanoparticles are orally or intravenously administered [Decoquinate (DQ) is a potent antimalarial agent targeting multistage arasites ,16. As warasites . As an aarasites . DQ can  malaria ,20. To s malaria . The in nistered .Plasmodium infection [The lipophilic property may make DQ an appropriate candidate as a sustained-release formulation for the chemoprophylaxis of malaria. Recently, Li et al. created formulations of DQ in oil-based carriers to provide extended efficacy through continuous drug release over a prolonged period from a single injection of the depot to effectively protect mice from nfection . Interesnfection , but nannfection .Plasmodium infection [Plasmodium infection.Hot melt extrusion (HME) technology has been applied for pharmaceutical formulations to improve the dissolution rate and bioavailability of poorly water-soluble drugs and to make a sustained drug release of API over an extended period . HME hasnfection . In this® MCM) were from IMCD . Cholesterol was from Beijing Dingguo Changsheng Biotechnology Co., Ltd. . Pharma 11 HME machine was manufactured by ThermoFisher Scientific (Thermo Electron (Karlsruhe) GmbH, Germany). PLGA 75/25  and PLGA 50/50  were bought from Jinan Daigang Biomaterial Co., Ltd. . N-methyl-2-pyrrolidone (NMP) was from Sigma-Aldrich ((Shanghai) Trading Co., Ltd.). All other chemicals and solvents were of analytical grade. For DQ quantitation, HPLC grade acetonitrile, ethanol, and methanol were obtained from Tianjin Kemiou Chemical Reagent Company .Decoquinate  was purchased from Genebest Pharmaceutical Co. Ltd. . Decoquinate standard  was bought from USP Rockville, MD, USA. Mono or diglycerides of medium-chain fatty acids , and formulation toxicity. Animal studies were conducted in strict accordance with the relevant animal testing regulations . All experimental protocols involving animals were carefully examined and approved on 4 September 2017 by the Animal Care and Ethical Committee of CAS Lamvac Biotech Co., Guangzhou, China .Sustained-release formulation of DQ (SRFD) was prepared by different methods but the method eventually selected was HME. The percentage of each component was accounted for by weight. Cholesterol (40–90%) was fully mixed with DQ (10–40%). Then mono and diglycerides of medium-chain fatty acids (Capmul MCM) (5–20%) were added to the above mixture. The thoroughly mixed ingredients were loaded into the HME machine. The temperature for the HME process ranged from 70 °C to 100 °C, and the screw speed was 50 rpm. The extrudate was either made as a solid stick of a given length for subcutaneous (SQ) injection or pulverized into a powder and then suspended in normal saline (pH5.5) for intramuscular injection.The PLGA 75/25 or 50/50 (10–90%) was formulated into the SRFD either via HME or by being dissolved in NMP. For the HME process, the temperature ranged from 80 °C to 135 °C, and the screw speed ranged from 50 rpm to 100 rpm. For the non-HME process, PLGA 75/25 (50%) was first dissolved in NMP, and then DQ (50%) was added to the PLGA solution. The mixture was stirred for 2 h at 200 rpm at room temperature. The mixed composition was then solidified and evaluated for the in vitro release of DQ. PLGA 75/25 (50%) alone could not be formulated with DQ via the HME process. Therefore it was combined with cholesterol and processed via HME. As a result, it was incorporated into a formulation as a minor carrier of DQ  at the Test Center of Sun Yat-sen University in Guangzhou, China. XRD is used for observing the crystal structures of chemical compounds. The shining of an X-ray on a crystal generates a diffraction pattern characteristic of the chemical structure. A powder form of the material can be analyzed. Crystalline substance generates their characteristic sharp peaks. To obtain information on the physical property of SRFD, XRD patterns of HME product, the physical mixture of the formulation prior to the HME process, and pure DQ were observed by the standard operation rules. The targeting material used was copper with CuK α radiation, and settings were regularly used conditions.One gram of SRFD sample was placed in a 10 mL tube filled with 6 mL phosphate-buffered saline , with shaking speed at 100 rpm under 37 °C. DQ released from tested SRFDs was measured by HPLC 1260 analysis (Agilent) and compared to the total amount of the drug from each sample used in the tests. Samples were taken periodically and continuously for 52 days. The cumulative release rates of DQ against time duration were calculated by dividing the amount of the released drug measured at each time point over the total amount of the drug placed. The detected drug each time (day) is the amount of all drugs released and accumulated from preceding times.g . The ethanolic supernatants were taken for DQ quantification by a high-performance liquid chromatography (HPLC) system  (AGILENT TECHNOLOGIES (CHINA) CO. LTD, Chaoyang, Beijing) and the drug load of each SRFD was calculated. Experiments were performed in triplicate for all formulations.Depot formulations were suspended in saline or PBS. The suspensions were mixed with 3 times the volume of ethanol in Eppendorf tubes and vortexed for 2 min. After standing for half an hour, the extraction was centrifuged for 5 min at 5500× DQ was quantitated by HPLC using a Diamonsil C18 column . All the in vitro and in vivo experiments including drug concentrations measured in the blood, in vivo efficacy studies, drug load determination, and drug release tests, were based on HPLC quantitation to calculate the amount of drug used or determined. For instance, in an efficacy experiment for a mouse weighing 25 g at a dose of DQ 200 mg/kg, an SRFD used was determined by HPLC to contain 5 mg DQ/12.5 mg total weight (40% drug load).The wavelength of ultraviolet detection in HPLC was 260 nm. The isocratic mobile phase was absolute ethanol/H2O . The column temperature was set to 35 °C, flow rate 1 mL/min, and injection volume 20 μL. All chemicals and solvents were of analytical grade. HPLC grade acetonitrile, ethanol, and methanol were obtained from Tianjin Kemiou Chemical Reagent Company .Plasmodium infection. Animal studies were conducted in strict accordance with the relevant animal testing regulations .C57 mice were employed to assess the chemoprophylaxis efficacy of SRFD in P. berghei ANKA expressing firefly luciferase were isolated from the salivary glands of laboratory-reared female Anopheles stephensi mosquitoes. The mosquitoes were maintained at 26 °C for 17 to 22 days after feeding on Plasmodium parasites from infected Kunming mice. Salivary glands separated from malaria-infected mosquitoes were manually broken to obtain SPZ. For assessment of the prophylactic efficacy of SRFD targeting hepatic Plasmodium infection, 50,000 SPZ per mouse were intravenously injected via the tail vein.Seven-week-old C57 mice, one in each cage, were kept for at least 7 days after arrival before starting the experiments. Room temperature was 18–26 °C, the relative humidity was 34–68%, and the light and darkness alternately cycled for 12 h. Standard feeding food was provided before and during the study. Sporozoites (SPZ) of HME extrudates were dosed either in solid sticks given to mice by SC to each side of hind legs by using trocar needles or in liquid form administered to mice by IM after being pulverized into a powder and suspended in saline. The liquid form for administration by intramuscular injection was made by suspending the HME extrudates in normal saline with pH 5.5. The solid stick with a diameter of about 2 mm was shaped by HME output and the length of the stick was determined by weight based on the drug content equivalent to the amount of drug administered. The liquid suspension was given to both hind legs to cut the total volume in half to alleviate discomfort.P. berghei was monitored by using the in vivo imaging system (IVIS). The images were obtained at 24 h, 48 h, and 72 h post SPZ inoculation. P. berghei infecting mice reside in the liver for intrahepatic development in the first 48 h and then spread from the liver to the whole body after 48 h. This time course is reflected in images characteristic of bioluminescent signals in different regions of the body. The signals indicative of the location of parasites expressing firefly luciferase were generated by giving the mice 150 mg/kg luciferin  intraperitoneally. Meanwhile, fluorescent flux counts were also acquired to represent quantitative data. The counts measured were bioluminescent photons emitted from whole bodies or regions of intensity (ROI). The ROI settings of the Living Image® 4.0 software reflect the luminescent intensity. A 3-D bioluminescent imaging tomography was performed with the software to obtain sequential images with filters ranging from 580 to 660 nm.Liver stage infection of Plasmodium infection was monitored by microscopic examination of either a thin film or a thick film. Blood smears were stained with 3% Giemsa for 20 min. Parasite density was estimated by counting parasites against RBCs on the thin film:Blood-stage Parasite density per μL = Number of parasites counted × RBC count per μL ÷ Number of RBCs counted. Infected red blood cells per 10,000 red blood cells were counted in thin films. In the cases where parasitemia was undetectable on the thin film, the thick film was prepared to count parasites against WBCs:Plasmodium SPZ inoculation to the endpoint of the efficacy of each SRFD, to determine the time lengths of SRFD protection in various dosages.Parasite density per μL = Number of parasites counted × WBC count per μL ÷ Number of WBCs counted. Infected red blood cells per 100 WBCs were obtained. Survival rates were calculated from the date of g for 60 min at 4 °C. The supernatant 100 μL was transferred to each well of the 96-well plate and DQ was measured by liquid chromatography–mass spectrometry (LC-MS) system (Applied Biosystems-SCIEX model for API 3000 mass spectrometer).Propranolol 5% in purity, purchased from Sigma, St. Louis, MO. USA, was used as an internal standard. DQ standards were made by dissolving it in ethanol at 50 μg/mL as stock solution, which was then diluted to a series of 10 gradient concentrations from 0.5 ng/mL to 500 ng/mL. Each standard (20 μL) was mixed with blank blood 100 μL in a 1.5 mL tube by vortex for 3 min. Protein precipitation solution 400 μL (ethanol /acetonitrile (1:1) containing propranolol 1000 ng/mL) was added and then mixed again for 5 min. After standing at room temperature for 8 h, the tubes were vortexed for 5 min and centrifuged at 16,000× To measure DQ in the blood, the whole blood sample (100 μL) was mixed with 20 μL ethanol for 3 min. Precipitation solution (400 μL) was added and mixed for another 5 min. The rest of the procedure was the same as that for standard curve preparation. Data were analyzed by one-way analysis of variance (ANOVA) and expressed as mean ± SD.The same species of mice (C57) as those used in efficacy experiments was dosed with F2 at DQ 200 mg/kg by IM. After the drug administration, blood samples were collected from mice by cardiac puncture on different days  with a group of 5 mice for each time point. Due to the limited sample size, DQ was measured by LC-MS, only one point per animal sample. Original data points were plotted to represent the drug level in the blood on that day.DQ dissolutions of the SRFD are illustrated in Plasmodium SPZ 34 days after SRFD placement. The parasitemia was examined 4 days after the inoculation. F1, a cholesterol-based SRFD, and both F2 and F3, a cholesterol–MCM-based SRFD, were suspended in saline and administered to mice by IM. As shown in berghei malaria by day 60  in suppressing Plasmodium infection in mice but was not completely protective , F3 and F4 in the solid stick (implant) given to mice by SQ had only partial effectiveness in preventing mice from otective C.Plasmodium berghei. The experiment design is shown in berghei malaria by day 30. Thus, when PLGA was formulated for sustained-release, whether acting as a minor carrier (F4) in the solid form (Plasmodium infection in mice.Cholesterol–MCM-based SRFD (F3) and SRF (F3 excipients or F8) were suspended in saline for IM injection and compared in the efficacy experiments. The SRFD (F10), generated by dissolving PLGA (L/G 75/25) with an equal amount of DQ in NMP, was also assessed. Primaquine (PQ) phosphate was administered at 30 mg/kg to mice by IM. F3 and F10 were dosed at DQ 200 mg/kg. Six weeks later, the mice were inoculated with 50,000 SPZ of lid form B or as alid form C, did noPlasmodium infection than PLGA L/G 75/25. Therefore, the subtype L/G 75/25 was used. Polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL) were also assessed as excipients of DQ depot formulations, which did not turn out to be sufficient in suppressing Plasmodium infection (data not shown). There could be more validation work necessary for PLGA and these other polymers.PLGA with L/G 50/50 was evaluated in preliminary experiments but was less effective in suppressing Plasmodium infection  was only partially effective against otection F. The foThe X-ray diffractogram showed two peaks characteristic of pure DQ at the angles 3.77°2θ and 7.53°2θ A. SimilaPlasmodium infection. F2 was placed in six groups of mice for days ranging from 30 to 180. SPZ inoculation was given to each group of the mice only one time at different time lengths a month apart. IVIS images collected from the third month to the sixth month are shown in Plasmodium infection, suggesting that F2 could protect all animals for up to 4 months.F2 had a 40% drug load, higher than F3 (20%), but compositions were the same. Thus, F2 was suspended in saline and administered to mice by IM to further determine how long decoquinate could be effectively released to protect mice from berghei malaria. The longest duration for a decoquinate depot to have complete protection was estimated to be between 120 and 150 days. According to the report [Plasmodium infection.However, the animals inoculated with SPZ 150 days and 180 days after the decoquinate depot showed, in spite of the weak signals, the infection , indicate report , the minThe blood specimens were collected by cardiac puncture from C57 mice. The experiments paralleled the one with data shown in The mean particle sizes of HME-made SRFD were measured and shown to be between 31–52 µm. The SRFD (F2), used in the experiments for berghei malaria and the duration of the protection lasted 4 months. To determine the minimal effective dose, F2 was placed intramuscularly in mice at a dose of DQ 50, 100, 150, and 200 mg/kg. Mice were inoculated with the SPZ one month after the F2 injection. Parasitemia was examined 4 to 6 days after the SPZ inoculation. Mice with F2 placement at DQ 50 mg/kg and 100 mg/kg were shown to have parasitemia to some extent and were not sufficiently protected from the Plasmodium infection  chemoprophylaxis of malaria [The study conducted by Li et. al. used peanut oil as a vehicle to prepare a microparticle-sized depot formulation of DQ and high-pressure homogenization to reduce the particle size to less than 15 µM . SRFDs p malaria .Plasmodium infection much better than expected. The use of cholesterol in the sustainable formulation of drug release has not been reported previously and it is the first time that it has been used for forming antimalarial agents, specifically DQ, in the depot formulation.In pharmaceutical applications, cholesterol is commonly used in liposomal preparation ,31. CholCholesterol is a natural molecule that constitutes a structural component of about 30% of all animal cell membranes . The molPlasmodium parasites. These lipids could be absorbed by surrounding tissues faster than DQ, and then metabolized or excreted. Therefore, there was no need to remove any injected materials. The DQ of the SRFD residing in the muscle tissue could be diffused to local surrounding tissues or could enter the bloodstream and then spread to the sites where the killing event of Plasmodium parasites occurred. The target sites were the peripheral tissues, especially the liver and the skin. The dose of DQ ranging from 100 to 150 mg/kg in the SRFD was fully effective in preventing Plasmodium infection. To translate this dose of DQ for human beings, API 10 to 15 mg/kg or 1050 mg may be needed for injection.The implant made by melt exclusion traditionally needs to be placed surgically or by using a large-diameter needle . As for Primaquine and tafenoquine are used as primary and terminal prophylaxes for malaria . PotentiSustained-release formulations of DQ for the effective prophylaxis of malaria were prepared by using the hot melt extrusion method and cholesterol as a major drug carrier. An intramuscularly injected depot of decoquinate is one dose of administration for the long-term prophylaxis of malaria at the liver stage. It may play a role as a “vaccine”, but with a low cost and easy to produce and handle. The newly created formulation utilizes naturally available materials that can be metabolized or degraded or excreted by animals or humans with no need of removing the depot substances by surgery. Thus, decoquinate released slowly from the muscle depot was effective against liver stage malaria for a long period of time.Intramuscular depot of decoquinate compositions and method of prophylaxis and treatment thereof. Inventors: Hongxing Wang, Shuanghong Liang, Yinzhou Fan, Xiaoyi Huang, Li Qin, and Xiaoping Chen. International Application No.: PCT/CN2018/087307; Patent No.: ZL 2018 8 0001391.5. Granted by Chinese Patent Office. Patent issue No.: CN 109496152B; Issue Date of Patent: 18 June 2021.


Plasmodium falciparum infection outcomes. Several genetic polymorphisms were reported to be prevalent among populations living in tropical malaria-endemic regions and induce protection against malaria. The present study aims to investigate the prevalence of HBB (chr11) and G6PD (chrX) deficiencies polymorphisms among Senegalese populations and their associations with the risk for severe Plasmodium falciparum malaria occurrence.Host genetic factors contribute to the variability of malaria phenotypes and can allow a better understanding of mechanisms involved in susceptibility and/or resistance to HBB and G6PD. Using a multivariate regression and additive model, estimates of the impact of human HBB and G6PD polymorphisms on malaria incidence were performed.We performed a retrospective study with 437 samples, 323 patients recruited in hospitals located in three different endemic areas where malaria episodes were confirmed and 114 free malaria controls. The patients enrolled were classified into two groups: severe malaria (SM) (153 patients) and uncomplicated malaria (UM) (170 patients). PCR and DNA sequencing assessed host genetic polymorphisms in HBB gene  and rs713040) and two in the G6PD gene (rs762515 and rs1050828 (G6PD-202 G > A). Analysis of selected HbS polymorphism showed significant association with protective effect against severe malaria with a significant p-value = 0.033  for SM vs. UM comparison. Surprisingly, our study did not identify the protective effect of variant HbC polymorphism against severe malaria. Finally, we found some of the polymorphisms, like HbS (rs334), are associated with age and biological parameters like eosinophils, basophils, lymphocytes etc.Six frequent SNPs with minor allele frequencies (MAF) > 3% were detected in the HBB and G6PD polymorphisms in the Senegalese population and their correlation with severe/mild malaria and outcome. The G6PD and HBB deficiencies are widespread in West Africa endemic malaria regions such as The Gambia, Mali, and Burkina Faso. The study shows the critical role of genetic factors in malaria outcomes. Indeed, genetic markers could be good tools for malaria endemicity prognosis.Our data report  Plasmodium species infection and affects hundreds of millions of people per year. In 2020, an estimated 241 million malaria cases occurred in 85 malaria-endemic countries, increasing from 227 million in 2019, with most of this increase coming from countries in the African Region (95% of cases). Malaria disease remains a significant cause of death and is still the 4th leading cause of death from infectious diseases worldwide  and uncomplicated (UM) to severe clinical forms (SM)  . Reasonsrms (SM) . Understrms (SM) .Plasmodium species infection outcome (G6PD) and β-chain of haemoglobin (HBB) deficiencies are more prevalent in malaria-endemic countries and are two of the essential loci conferring resistance to severe malaria in humans , characterised by the 202G-376A allele. In sub-Saharan Africa, up to 40% of the population carries the G6PD A+ form (non-deficient type) characterised by 202G-376G allele with 85% enzyme activity  and Malawi (0.19)  .HBB and G6PD loci to identify potential variants associated with susceptibility to severe malaria and corresponding hematologic parameters.However, until now, there has been a lack of data about the frequencies of the general Senegalese population, from which full studies have not yet been performed. It is essential to perform additional screening of G6PD and HBB genes in the Senegalese population, (ii) secondly, we will check the correlation between main polymorphisms in HBB and G6PD genes and severe malaria outcome. In participants from three ecological zones, we characterised polymorphisms on HBB and G6PD genes where the malaria endemicity was different. More data on G6PD and HBB polymorphisms and their incidence on malaria outcome and corresponding haematological parameters are essential for different malaria eradication strategies in endemic West African countries.Senegal is a West African country where malaria risk varies from one region to another. Our present study has two aims: (i) first, to investigate the prevalence of key malaria-protective polymorphisms in The cohort included black Senegalese-born individuals recruited between 2003 and 2015, whose parents and grandparents were born in Senegal, a malaria-endemic country in the Sahelian zone of West Africa.The Senegalese population (SP) recorded in 2013 is 13.508.715 inhabitants, including near equality between men 49.9% and women 50.1% . SP is uSenegalese ethnic groups are located almost everywhere in Senegal, but the ‘Wolof Ethnic group’ is concentrated in the West and West-Center; the ‘Serere populations’ are focused on Peanut-Basin, specifically in Kaolack and Fatick regions. The Peulh ethnic groups are scattered throughout Senegal areas but are more established in Ferlo along the river and the Kolda region. The ‘Sarakholes’ live in Bakel. Other ethnic groups, such as Diolas, Baïnouks, Balantes and Madingues Ethnic groups, are located in Casamance. Bedik and Niokholonko populations are located in the country’s Southeastern part, such as Tambacounda and Kedougou regions.Clinical Malaria participants were enrolled from five “site-regions” in Senegalese regional hospitals in St Louis and Louga in the North, Dakar, Tambacounda and Kolda . These tFor convenience, these five regions were divided into three ecological areas according to the climatic gradient and malaria endemicity, translated from North to South. The northern part is Sahelian climates with unstable or seasonal malaria, including St Louis and Louga regions. These sites belong to the ecological area where malaria is hypoendemic and where the annual incidence in 2019 was less than five cases per 1,000 inhabitants. Some sites are under 1/1000 with very low anopheles vector density. This region is located in the malaria prelimination area context . The ‘ceClinical malaria participants corresponded to participants with Plasmodium-positive QBC (Quantitative Buffy Coat). The QBC is a qualitative test, conceptualised as far back as 1974, and is used to identify the malarial parasites in the peripheral blood faster than the conventional thick smear method. The technique involves staining the centrifuged and compressed red cell layer with acridine orange, which is later examined under a fluorescence microscope .P. falciparum infection. High parasitemia, Severe anaemia and cerebral form are the three most ‘severe forms’ currently explored. But other complications such as hypoglycaemia, thrombocytopenia, renal insufficiency, hepatic or even pulmonary oedema may appear alone or in combination. For uncomplicated forms, we considered patients who had fever with P. falciparum parasitaemia of <25,000 parasites/µL of blood with no evidence of impaired consciousness or seizures at the time of enrollment and no other past background of mental illness, meningitis or accidental head injury were included.According to the criteria defined by P. falciparum infection confirmed in diagnostic; (2) persons born in Senegal; whose parents and “grandparents” were born in Senegal. Exclusion criteria were: (1) other racial or ethnic groups living in Senegal; (2) participants with clinical signs of severity or any state that may interfere with the study, such as a recent pregnancy and childbirth. The control group (CTR) corresponded to unaffected participants, free of Plasmodium infection. These participants were recruited at Pasteur institute at the biomedical laboratory according to the same criteria used for clinical participants inclusion, and live in the same environmental and endemic area”.To ensure homogeneity of data, inclusion criteria were: (1) Only black Senegalese individuals with A blood sample (10 mL) was taken from venous blood in heparin-containing vacutainer tubes for each patient. Hemogram and Several blood cell indices, such as red blood cells (RBCs) count, Hematocrit, MCV (mean corpuscular volume) and MCHC (mean corpuscular volume concentration), were taken in the different groups and evaluated according to references normal values suggested elsewhere . In SM aAs previously described by HBB and G6PD by PCR. At all, PCR reactions were performed using a Gotaq®Green Master Mix  in a total volume of 25 µl containing 25 ng of genomic DNA (5 ng/µl) and 2.5 µl of each primer (10 µM). PCR conditions were initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 62 °C for 45 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min.Oligonucleotide primers were designed to amplify interest regions in HBB gene was performed using a reference sequence located at chromosome 11 (GRCh38:11:5226434-5227234). The sequence region was downloaded from the BLAST tools, and the primers positions of HbS—rs334 ([A/T]), HbC—rs33930165 ([C/T]) polymorphisms were designed. The reference sequence downloaded is orientated forward concerning the reference genome. The primers sequences designed for HBB amplicons were reversed concerning the reference, such as forward (5′-ACTCCTAAGCCAGTGCCAGA-3′) and reverse (5′-CGATCCTGAGACTTCCACAC-3′) primers. An amplicon of 801 bases was obtained, targeting mutations of deficiencies, including HbS (rs334) and HbC (rs33930165) and surrounding polymorphisms.PCR amplification for the Hemoglobin G6PD gene, the location of the amplicon was (GRCh38: X:154535674-154536374). PCR amplification was performed with forward (5′-GTCTTCTGGGTCAGGG AT-3′) and reverse (5′-GGAGAAAGCTCTCTCTCC-3′) primers, amplifying 701 bases long and targeting the G6PD-202 and their surrounding SNPs. Then the designed primers are reverse with respect to the human reference sequence (GRCh38: X:154535674-154536374) (http://www.ensembl.org/index.html). Then the amplicon left out SNP previously identified in the Senegalese population, such as G6PD +376, G6PD +376, G6PD +542, G6PD +680 and G6PD +968.For the via 2% agarose gel electrophoresis, and the amplicons were purified using BioGel P100 gels (Bio-Rad). Sequencing reactions (2 µl of PCR product) were performed using the Dye Terminator v3.1 method in an ABI PRISMs 3730 DNA Analyzer . Sequencing conditions were: 96 °C for 5 min, 25 cycles of 96 °C for 10 s; 60 °C for 4 min and 15 °C forever, and PCR products were purified with Sephadex G50 superfine columns . Alignment and SNP discovery were performed using NG_009015.2 and NG_059281. as reference sequences for G6PD and HBB, respectively. Analysis was performed with Genalys version 2.0 software  were performed to evaluate the association between malaria clinical status and escribed . The difescribed .p values < 0.05 were considered statistically significant.Associations between SNPs and different parameters such as age, biological and hematological parameters were performed using the Mann–Whitney test, and then associations with G6PD loci polymorphism to assess hemizygosity. First, a random inactivation was coded accordingly to genotype as  for males and  for females, accounting for the random inactivation and assuming an activation of one of the two X chromosomes. Second, an ‘SNP random inactivation escape’ was taken into account, and a test was performed in combined samples, given that a proportion of SNPs can escape from random inactivation used at first. Then gender was treated as a covariate, and the association tests were adjusted in the discovery sample. Third, the association test for males and females was performed separately to investigate sex-specific effects underlying X chromosome loci. An association test accounting for gender × SNP interaction was also performed. Meta-analyses were then conducted using the R package (version 1.9–8) for combined discovery analyses. For this model approach separating males and females would cause a lost statistical power according to the small size of our sampling. Finally, the heterogeneity among these datasets from the different models was evaluated by tests such as I2 and Q test > 50% or P < 0.05 were considered heterogeneous, and the random-effect model was applied; otherwise, the fixed-effect model would be used. Linkage disequilibrium (LD) was computed for each pair of polymorphisms within the HBB and G6PD genes using Haploview software  of Senegal and according to the procedures established by the Cheikh Anta Diop University of Dakar (UCAD) for the ethical approval of any research involving human participants. Written informed consent was obtained from adult participants and parents or legal representatives of children. In addition, based on the information provided, UCAD’s Committee on Research and Ethics (CER) considers that the research proposed respects the appropriate ethical standard and, as a result, approves its execution under “Protocole 0344/2018/CER-UCAD”. All patients enrolled in the cohort/or legal representative gave signed and informal written consent to provide a blood sample for further studies.We performed a retrospective study on 437 participants recruited between 2003 and 2015 in three ecological areas distinguished by their malaria endemicity , Table 1p < 0.01) with high-level parasitemia in the SM group (mean ± SD = 27220 ± 5458.3 P/µL). The haematological parameters of the three groups were compared, as shown in p-value < 0.0012). In contrast, the leucocytes count was significantly increased  in SM compared to UM patients. In addition, we found higher levels of eosinophils, basophils and eosinophils in UM participants than SM participants .The average values of selected haematological parameters were determined for three SM, UM and CTR groups and the association was calculated using ANOVA statistical tests. We found a difference in the level of parasitaemia between SM and UM groups, which was statistically significant  and HBB (Chr.11) genes and their flanking sequences were analysed in 437 Senegalese populations, including 153 SM, 170 UM and 117 CTR participants. To explore the prevalence of G6PD gene, PCR amplicon targeted G6PD-202 polymorphism amongst the commonly known SNPs in Western Africa and omitted polymorphisms such as G6PD +376, G6PD +542, G6PD +680 and G6PD +968. A set of 6 SNP were identified in an amplicon of 701pb , was observed at a high frequency of >3% in the overall population. In specific clinical phenotype populations, the MAF were 0.28, 0.37 and 0.32 in SM, UM, and control groups. There was no evidence of genotypic deviation from HWE except +10588_A > G (rs762515).For the of 701pb . Among tG6PD, the HBB gene showed more polymorphisms in the Senegalese population with 12 characterised SNPs. These identified SNPs were previously registered in the NCBI database, and rs numbers were already attributed. Then our study does not reveal any novel HBB polymorphisms. Among them, 6 SNP  and rs713040) were detected with high frequencies (with MAF > 3%), unlike 6 other SNPs , rs34598529, rs72561473, rs33944208) with MAF < 3% were observed  in each pair of polymorphisms. G6PDrs762515 A > G, rs1050828 G > A, and for HBBrs10768683 G > C, rs7480526 T > G, rs7946748 C > T polymorphisms, the values D’ > 0.97, showing a high linkage disequilibrium, and defining a block structure in HBB genes. A genetic segment appeared to be part of a haploblock structure in the HBB loci, composed of 3 ‘successive’ SNPs, for which the confidence interval D′ was 0.9–1 .The structure of G6PD and HBB polymorphisms were associated with malaria protective effect in our overall population, a statistical association analysis was performed using logistic regression by comparing the three phenotypes groups, SM, UM and underage, sex as covariates. For G6PD, the SNP +10588 A > G (rs762515) yielded a significant association with protection from severe malaria in Senegalese populations. For SM vs UM, the SNP +10588 A > G (rs762515) showed a borderline p-value = 0.047 . For G6PD-202 G > A, no association with protection against severe malaria was found in our overall population  yielded a significant p-value = 0.033  (rs33930165 (HbC) polymorphism is not a protective variant against severe forms in our population. There is no significant difference between SM vs UM, with a p-value = 0.26  . Surprise = 0.26 .rs334), HbC (rs33930165) and G6PD +10588A > G (rs762515) were associated with protection against severe malaria in the three regions by comparison of SM vs UM with higher frequency in UM group (HBB +9 C > T (rs713040) has variable protective effects depending on endemicity. In St-Louis, a low endemicity area, the SNP rs713040 and rs7946748 were associated with protecting the severe form, unlike Dakar and Tambacounda/Kolda/, which are more endemic areas. For G6PD-202 G > A, we found differences per the locality. The polymorphism was associated with protection against SM in the Dakar Capital centre but not in the other regions (St Louis-Louga and Tambacounda-Kolda). Indeed, the SNP is associated with the protection against SM form in Dakar but not in the other regions , HbS (rs334), HbC (rs33930165), HBB +9 C > T (rs713040), and G6PD-202 G > A with age, parasitaemia, and biological parameters  and survival/death outcome, were performed using the Mann–Whitney test.Analyses were conducted to test whether HBB +200_T > C (rs111851677), a variant responsible for β-thalassemia.Our results showed a significant association between age and rs7946748 with basophils levels p-value = 0.01 5  and lymphocytes p-value = 0.04   and p-value = 0.020  respectively. HbS (rs334) triggered a significant association with eosinophils p-value = 0.024 . Platelets were associated with G6PD-SNP +10707 G > A, G6PD-SNP +10983 G > C and HBB-rs72561473, with p = 0.009, 0.048, 0.006 respectively. Finally, G6PD- SNP +11000_C > T (rs782500951) was associated with parasitaemia with a p value = 0.016 (Then results showed significant association of  −0.44]) ; the rs1 = 0.016 .G6PD and HBB genes in the Senegalese population, two-loci previously associated with protection against severe malaria. A genetic analysis was performed to identify polymorphisms associated with clinical malaria and their prevalence, using three specific phenotypes of severity, including SM, UM and CTR groups living, from North to South, in three ecological malaria transmission areas.In our present study, we characterised the polymorphisms of G6PD genes and found that the minor allele frequency of G6PD-202 G > A was 0.022, 0.032, 0.018 in SM, UM, and CTR groups, respectively. The frequency of these polymorphisms was 0.026 in the overall population. In The Gambia, a country neighbouring Senegal, it was reported that the minor allele frequency of this polymorphism in the CTR sample was 0.03, considerably lower than reports from other east African countries such as Kenya (0.18) and Malawi (0.19) (We identified six polymorphisms on i (0.19) .G6PD-202 G > A in the Senegambian area compared to Kenya and Malawi populations. These results could ensure that G6PD polymorphisms prevalence is highly population-specific, and thus, attention should be paid to the study population from which future estimates may be drawn , age or immune status of participants, and study designs (case-control vs cohort)  yielded a significant association with protection from severe malaria in the Senegalese population.Our results confirm the weak prevalence of allele deficiency from be drawn . In our d Uganda . Howeverd Uganda , in maled Uganda , in bothd Uganda , or no pd Uganda . This di cohort) . G6PD deo gender . No such females . A case- anemia  . However results . In facte effect . Our stuHBB genes located on chromosome 11, we identified 12 polymorphisms and characterised their frequencies. The SNP HbS resulted in substituting a glutamate residue with a valine or lysine residue in the ß-globin chain at position 6. The Minor Allele Frequency of HbS and HbC polymorphisms was estimated to be 0.05 and 0.01, respectively, in the overall population. Several studies have shown a low frequency of the HbS allele in the control group (∼3.8%). The frequency is in keeping with other West African countries  and east African populations  . A higher frequency of the HbC allele has been observed in other West African populations  yielded a significant p-value = 0.033. The observed frequencies for HbS (rs334) are 0.026, 0.069 and 0.035 for SM, UM, and CTR groups, respectively  not showed significant; we found a p-value = 0.26 when comparing SM vs. UM. Previous studies showed that HbAS had been associated with 50% and 80% reduced risks of developing uncomplicated and severe malaria  variant and age. Moreover, this variant does not have a protective effect against severe malaria in our study. Age is an essential factor in the pathophysiology of malaria, and some reports from sub-Saharan Africa suggest that P. falciparum infections are more common among children aged 5–15 years than among younger children and adults .The results showed significant associations between several polymorphisms on P. falciparum infection is associated with a profound T-cell activation with the Th1-derived cytokine interferon-γ as a major pathogenic factor , +499G/C (rs2233860) and +577A/T (rs8019343) defined a haplotype risk (G-G-T) for malaria severity (rs334 (HbS) is a variant associated with eosinophils   . It woulP falciparum infection and rates of the clinical episodes of malaria are described in clinical studies in West Africa (The early and vigorous development of malaria immunity has been associated with a protective effect of sickle cell traits and thalassemia. Although, ethnic variations in the immunological response to t Africa .In conclusion, our study gives an overview of the importance of sickle cell polymorphism on severe malaria susceptibility in the Senegalese population.10.7717/peerj.13487/supp-1Supplemental Information 1Information about each participant: location, age, gender, malaria phenotype or control, haematological data, etc.Click here for additional data file.10.7717/peerj.13487/supp-2Supplemental Information 2These data were used for statistical analysis.Click here for additional data file.10.7717/peerj.13487/supp-3Supplemental Information 3These data were used for statistical analysis.Click here for additional data file.10.7717/peerj.13487/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.13487/supp-5Supplemental Information 5Click here for additional data file.


Both methods have low sensitivity and specificity. Loop-mediated isothermal amplification techniques have shown a sensitivity similar to PCR but with lower times of performance. This study aimed to assess a commercial LAMP for the diagnosis of malaria (AlethiaAlethia® Malaria, as it was easy to perform, minimal training requirement and 40 min run. Moreover, it includes an internal control to avoid false negatives. However, it also showed some limitations such as the need for a specific amplification and detection device, the detection of only Plasmodium spp. and a very high price.One hundred five samples out of 114 rendered valid results, obtaining 85 positive samples and 18 negative samples with an agreement of 98% compared to the reference method with a sensitivity, specificity and kappa coefficient of 98.84%, 94.74% and 0.94 respectively, with only two discrepant samples. The turnaround time was estimated in 1 h and 30 min, with a cost of 32.67€ per determination. The results showed several advantages of the  According to the last World Malaria Report, in 2020 an estimated 241 million cases of malaria occurred worldwide [In non-endemic countries, the majority of cases are imported  and locaP. falciparum; nevertheless they lack sensitivity at low parasite density, fail to detect non-falciparum malaria and do not allow quantification of parasites [Conventional methods for malaria diagnosis are microscopic examination of Giemsa-stained thick and thin blood smears, often combined with rapid diagnostic tests (RDTs) . Microscarasites , 8, 9.® Malaria LAMP assay (Meridian Bioscience), previously called illumigene® Malaria, which is a qualitative in vitro diagnostic LAMP test for detection of Plasmodium spp. that has been tested in endemic [Plasmodium spp. samples using Alethia® Malaria LAMP assay included none or very few samples of Plasmodium knowlesi parasites [Nucleic acid-based detection of malaria parasites (NAT) is now routine in reference laboratories of several non-malaria endemic countries, mostly based on polymerase chain reaction (PCR) . However endemic  and in n endemic , 18, 19.arasites , 19, 20.® Malaria LAMP assay for the five species of Plasmodium that infect humans commonly, including P. knowlesi, compared to the Nested-Multiplex-Malaria PCR (NM-PCR). This evaluation includes the performance of the tests, the analytical sensitivity and the operational characteristics.This study aimed to assess the diagnostic performance of the AlethiaPlasmodium knowlesi samples were collected in Malaysia under a project approved by the Medical Research Sub-Committee of the Malaysian Ministry of Health . One hundred fourteen blood samples, 93 malaria positive  and 21 negative samples were tested. Samples were tested retrospectively, and they were refrigerated at 4 ºC until the moment of performing the experiments.Samples used belonged to a project approved by the Medical and Health Research Ethics Committee (CEIC) of the Hospital Universitario 12 de Octubre (No. CEtm: 18/021). ® Malaria LAMP kit includes the material to perform the DNA extraction, using a simple filtration method, and the LAMP reaction. The assay was performed according to the manufacturer’s instructions using the Illumipro-10™ (Meridian Bioscience) for the amplification and reading of the results, obtaining a positive, negative or invalid result in 40 min. The LAMP reaction consists of two tubes with all the lyophilized components, one for the amplification of Plasmodium spp. and the second as internal reaction control.Alethia®, Inc.) according to the manufacturer’s instructions.DNA purification from 200 µl of whole EDTA blood was performed using the QIAamp DNA mini blood kit  according to the manufacturer and original authors’ recommendations , 22. TheThe parameters used for the amplification were an initial denaturation at 94 ºC for 7 min, followed by 40 cycles at 94 ºC for 20 s, 58 ºC for 20 s, and 72 ºC for 30 s. The last cycle was followed by an extension time at 72 ºC for 10 min. Conditions for the second PCR reaction were an initial denaturation at 94 ºC for 5 min, followed by 35 cycles at 94 ºC for 15 s, 53 ºC for 15 s, and 72 ºC for 20 s, finishing with an extension phase at 72 ºC for 10 min.P. falciparum positive blood sample belonging to a patient with a parasitaemia of 7500 parasites/µl determined by microscopy. Briefly, the sample was serially tenfold diluted with blood from a Spanish patient negative for malaria and no story of travelling to endemic malaria areas. DNA was extracted from each dilution by simple filtration method included in the kit and then tested by Alethia® Malaria assay twice. LoD was considered as the lowest parasite concentration in which both duplicates were positive.LoD was determined, by duplicate, using a ® Malaria assay and the NM-PCR method were referred to as the cost for reagents for one determination without including costs associated with labor or derived from duplicates or controls. Turnaround time was estimated as the time required from the initial moment the sample begins to be processed until obtaining the results, considering the possibility of more than one sample are included in the run; while hands-on work time was the time required for the staff to perform the assay.Estimated costs for the AlethiaSensitivity (S), specificity (E), positive (PPV) and negative predictive value (NPV) and Kappa coefficient (k) were analyzed with 95% confidence intervals using EPI Dat (3.1) software package .® Malaria assay was used, obtaining 85 positive samples and 18 negative samples with an agreement of 98% compared to the reference method , while for NM-PCR, it was estimated in 6 h 15 min .The turnaround time for the AlethiaThe cost for the commercial assay was calculated as 32.67€, higher than for the “in-house” assay of 5€, although in other countries or institutions, the costs of kits may be lower  were correctly characterized, meanwhile another was considered negative and the last gave an invalid result. Expanding the number of samples infected with P. malariae would be necessary to find the correct answer.There is a growing request for faster and more sensitive diagnostic methods for malaria. Conventional methods, such as microscopy and RDTs, lack sensitivity, while PCR-based methods, although very sensitive , requirechnology . Furtherchnology . In our chnology , 27 or echnology . LAMP cochnology , 12, 28,® Malaria kit has also been evaluated in Senegal obtaining high sensitivity (97.2%) and a good specificity [P. knowlesi specimens. In our study, the nine P. knowlesi-infected samples were detected, without any invalid or false-negative results, confirming the ability of the Alethia® Malaria LAMP to detect the main five Plasmodium human species.Similar good results in specificity and sensitivity have been shown in more studies in non-endemic countries , 18, 20.cificity . HoweverNine samples out of 114 provided invalid results. Several authors have described a similar situation where it was not possible to obtain a valid result with this LAMP assay , 19. Acc® Malaria kit showed a very good LoD value for P. falciparum, 0.075 parasites/µl, similar to reported for several malaria Nested PCRs [P. falciparum cultures [The Alethiated PCRs , 29. Othted PCRs  and 0.5 ted PCRs  with con® Malaria kit incorporates the components to purify DNA from whole blood and an internal reaction control to discriminate false negatives from inhibition reactions. Unfortunately, contrary to the best feature of LAMP technology, the reaction must be run on a specific device. Despite this, given our results, with excellent sensitivity and specificity and with a reduced time of diagnosis, Alethia® Malaria assay could be used as a good screening diagnosis method for malaria, as other authors have pointed out previously [Several malaria LAMP assays have been described previously –13, mosteviously , 19, 20.® Malaria for detection of the five most common Plasmodium human species, including P. knowlesi which was detected in 100% of samples assessed. The analytical sensitivity obtained was very good and our evaluation showed several advantages of the assay, as it was easy to perform, minimal training was needed, DNA purification was simple, fast and included in the kit, and the amplification run was completed in 40 min. Moreover, it includes an internal control to avoid false negatives. However, it also showed some limitations such as the need for a special amplification and detection device, the detection of only Plasmodium spp. with no information about species or the level of parasitaemia and the higher price of each test compared to non-commercial assays.In this study, we demonstrated the utility of the AlethiaOur study presented some limitations, as the retrospective analysis of the samples and the low number of samples evaluated, especially for non-falciparum species. In addition, mixed infections were not included in the assessment and we did not undertake a reproducibility analysis.


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of infected erythrocytes (IEs) are important virulence factors, which mediate vascular adhesion of IEs via endothelial host receptors and are targets of naturally acquired immunity. The PfEMP1 family can be divided into clinically relevant subgroups, of which some bind intercellular adhesion molecule 1 (ICAM-1). While the acquisition of IgG specific for ICAM-1-binding DBLβ domains is known to differ between PfEMP1 groups, its ability to induce antibody-dependent cellular phagocytosis (ADCP) is unclear. We therefore measured plasma levels of DBLβ-specific IgG, the ability of such IgG to inhibit PfEMP1-binding to ICAM-1, and its ability to opsonize IEs for ADCP, using plasma from Beninese children with severe (SM) or uncomplicated malaria (UM). IgG specific for DBLβ from group A and B ICAM-1-binding PfEMP1 were dominated by IgG1 and IgG3, and were similar in SM and UM. However, levels of plasma IgG inhibiting ICAM-1-binding of group A DBLβ of PFD1235w was significantly higher in children with UM than SM, and acute UM plasma induced a higher ADCP response than acute SM plasma.Members of the highly polymorphic  To help combat malaria, WHO in October 2021 recommended the widespread use of the RTS,S/AS01 vaccine in areas of moderate to high P. falciparum transmission2, and other vaccine candidates are in clinical trials including two (PAMVAC and PRIMVAC) based on P. falciparum erythrocyte membrane protein 1 (PfEMP1)3. Antibodies against variant surface antigens on the surface of infected erythrocytes (IEs) are known to protect individuals from severe and symptomatic malaria6, and members of the PfEMP1 family have been shown to be targets of naturally acquired antibodies10. The highly polymorphic PfEMP1 antigens are encoded by approximately 60 var genes per haploid parasite genome, mediate adhesion of IEs to vascular host receptors, and are associated with the pathogenesis of P. falciparum malaria11. Despite their extensive variation, the var genes can be divided into three major groups  based on chromosome location, DNA sequence, and promotor region13. Chimeric group B/A genes exist, and together with group A var genes, are transcribed in parasites isolated from children with severe disease, and in cytoadhering IEs linked to the pathogenesis of cerebral malaria (CM). A subgroup of group A and B/A PfEMP1 proteins can bind both to the intercellular adhesion molecule 1 (ICAM-1) and endothelial protein receptor C (EPCR), and this dual-binding phenotype has been linked to CM15 but not all studies16. Infected erythrocytes co-localize with ICAM-1 expression in the brain blood vessels suggesting that ICAM-1 mediates IE sequestration in CM17. Dual-receptor binding IEs have been shown in vitro to cause clustering of ICAM-1, to be taken up by brain endothelial cells in an ICAM-1-dependent manner, that results in the breakdown of the blood–brain barrier and swelling of endothelial cells, and antibodies directed to the PfEMP1 on their surface prevent sequestration of IEs18. Sequestration of large numbers of IEs in the microvasculature of specific organs is central to the pathogenesis of severe P. falciparum malaria19. CIDRα domains associated with CM21 are the focus of some research groups, while we focus on DBLβ domains. In this study, we hypothesized that children with uncomplicated malaria (UM) have higher levels of functional anti-PfEMP1 (anti-DBLβ domain) antibodies than severe malaria (SM) cases and because of this, they would have antibodies against PfEMP1 variants associated with severe disease. To test this, we measured plasma levels of anti-PfEMP1 (DBLβ)-IgG in a cohort of Beninese children with SM or UM and used ICAM-1-binding inhibition and ADCP as measures of antibody effector function.Malaria is one of the most important public health problems worldwide with an estimated global number of 241 million cases and 627 000 deaths (2020), the majority in children less than five years of ageTo investigate the anti-PfEMP1-IgG antibody reactivity against ICAM-1-binding DBLβ domains, 137 Beninese children  were recruited at three different hospital centers and divided into three clinical categories: cerebral malaria (CM), non-cerebral severe malaria (nCSM), and uncomplicated malaria (UM) (Table P = 0.04) compared to children with UM   and non-cytophilic  IgG in plasma samples from 84 (day 0) and 45 (day 30) of the children. Cytophilic IgG completely dominated the anti-DBLβ  antibody response in all children  showed varying adhesion inhibitory capacity against DBLβ domains of PFD1235w, HB3VAR03, HB3VAR21, and IT4VAR13. At day 0, the percentage of plasma samples showing ≥ 30% inhibition of the ICAM-1-binding of the DBLβ domains was 16% , 41% , 43% , and 78%   or CM (P < 0.001), while this was not the case for the DBLβ domain of HB3VAR03 (group A) or HB3VAR21 and IT4VAR13 (group B) , CD32+ (FcγRII) and CD16- (FcγRIII) THP-1 cells , the convalescent (day 30) Beninese plasma samples promoted a stronger phagocytosis of DBLβ-coated beads  than samples obtained on the day of hospitalization , suggesting that this type of PfEMP1-variants are major targets of IgG promoting ADCP , and against IT4VAR13 (group B), were positively correlated with the ADCP activity against the DBLβ domains . In malaria endemic areas with stable transmission of P. falciparum, protective immunity is acquired during childhood, first to severe complications and later to clinical disease28. This is believed to be a result of an ordered acquisition of antibodies, with antibodies to a relatively conserved set of PfEMP1 proteins associated with severe disease acquired prior to antibodies to a larger and more diverse set of PfEMP1 proteins associated with uncomplicated malaria and asymptomatic parasitemia29.30 and that of others31 did not observe differences in IgG reactivities to DBLβ domains between children of similar age with severe or uncomplicated malaria.In Beninese children, we found that IgG reactivity against ICAM-1-binding DBLβ did not differ among children of similar age with severe or uncomplicated malaria Fig. , and tha33. Here, we investigated the activity of naturally acquired functional antibodies from children who were exposed to P. falciparum infection, i.e., we looked at adhesion-inhibitory antibodies and antibodies mediating ADCP. Our findings show that antibodies from participants included in the study were able to not only react with the domains, but that the IgG antibodies have functional activity as they elicited inhibitory effects ranging from 16 to 78% upon the ICAM-1-binding of DBLβ domains encoded by dual-receptor binding PfEMP1 . Besides neutralization of pathogens by antibodies, phagocytosis is considered as one of the most important anti-pathogen activities44. ADCP involves receptor-mediated interactions between immune effector cells and the Fc domain of cytophilic IgG bound to pathogen45. Here, we measured opsonic phagocytosis activity using undifferentiated THP-1 monocytes and found that children with UM and SM had similar levels of IgG to the different DBLβ domains, but plasma from UM children (day 0) showed significantly higher percentage phagocytosis activity to group B IT4VAR13 DBLβ , No. 87/MS/DC/SGM/DRFMT/CNERS/SA Cotonou, République du Benin. All methods used in this study were performed in accordance with relevant guidelines and regulations.n = 137) were collected at three hospital centers in the cities of Cotonou, Southern Benin during the malaria transmission season of June–September 2019. After informed consent had been obtained from a parent or a legal guardian, children less than 5 years of age were subjected to clinical investigation and screened by malaria rapid diagnostic test . Venous blood samples were collected from the enrolled children and parasitemia quantified by microscopy. Blood samples were collected from each child on the day of hospitalization (disease presentation) and 30 days following discharge . Clinical manifestations were classified according to the definitions by the World Health Organization46. Patients were categorized as having cerebral malaria  if they had a positive blood smear of P. falciparum and unarousable coma (Blantyre coma score [BCS] ≤ 2) with exclusion of other causes of coma. Patients were categorized as having non-cerebral severe malaria  if they presented with hyperparasitemia , and/or severe anemia (hemoglobin < 5 g/dL), and no coma. Patients with uncomplicated malaria  had fewer parasites than 50,000 parasites per µL accompanied by fever, headache, or myalgia without signs of severity and evidence of vital organ dysfunction.Plasma samples  were maintained in long-term in vitro culture and selected for IE surface expression by repeated antibody selection as described50. The identity of isolates was routinely verified by genotyping as described51, and Mycoplasma infection was excluded by using the MycoAlert Mycoplasma detection kit (Lonza) according to the manufacturer´s instruction.The Escherichia coli Shuffle C3030 cells (New England BioLabs). The protein domains  and four group B ICAM-1-binding DBLβ protein domains , and three group A non-ICAM-binding DBLβ protein domains  were expressed as His-tagged proteins in ns Table  were pur32. Following a washing step in wash buffer  plates were blocked using blocking buffer . Plasma samples (diluted 1:200 in blocking buffer) were incubated  in duplicate wells. The plates were washed and bound IgG antibody was detected with horseradish peroxidase (HRP)-conjugated anti-human IgG  (Agilent). Following incubation (1 h) and washing as described above, bound detection antibody was visualized using TMB according to the manufacturer´s instructions (Agilent). The reaction was stopped by adding 0.2 M H2SO4 (50 µL/well) and the optical density (OD) values were read at 450 nm using a VERSAmax microplate reader (Molecular Devices). Antibody reactivity was expressed as ELISA units calculated as (ODsample − ODbackground)/(ODpositive control sample – ODbackground) × 100%53.Maxisorp microtiter plates (Sigma-Aldrich) were coated with recombinant DBLβ domains  in 0.1 M glycine/HCl buffer pH 2.75 overnight at 4ºC as described previouslyThe anti-DBLβ IgG subclass reactivity was measured in plasma samples collected from children with malaria. Maxisorp plates  were coated with different recombinant DBLβ domains  overnight at 4 °C. The plates were emptied and washed once with washing buffer and blocked with blocking buffer as described above. Plasma samples  were added duplicate wells. Following incubation (1 h) and washing as described above, bound antibody was detected with mouse HRP-conjugated anti-human IgG  . Following 1 h of incubation and subsequent three times washing, plates were developed, colour reactions stopped, OD values read, and results calculated as described above.Inhibition of recombinant DBLβ domain binding to ICAM-1 by participants’ plasma was measured by ELISA. Briefly, wells of Maxisorp plates were coated with recombinant Fc-tagged ICAM-1  by incubation overnight at 4ºC and blocked with blocking buffer . His-tagged recombinant DBLβ domains (0.5–16 µg/mL) were mixed with immune plasma  and subsequently added to duplicate wells  of ICAM-1 coated plates. Following washing, bound DBLβ protein was detected using HRP-conjugated anti-penta-His antibody  (Qiagen). Following 1 h incubation and three rounds of washing, bound antibody was visualized using TMB and plates read as described above.7 beads per mg DBLβ protein. Briefly, beads were resuspended by vortexing in 400 µL of ddH2O, biotinylated DBLβ protein (1 mg/ml) was added to the beads, mixed by vortexing and incubated at room temperature . Coupled beads were pelleted at 18,000 × g, washed twice in 1 mL of dilution buffer , and resuspended in 1000 µL dilution buffer and stored in the dark at 4ºC until used. On the day of assay, U-bottom plates were blocked  at room temperature for at least 30 min. For opsonization, this was followed by incubation of heat-inactivated plasma (40 µL of 1:200 dilution) with 105 DBLβ-coupled beads (10 µL). To prevent photobleaching, plates were covered with foil and placed on a shaker for 1 h at 100 rpm. Following two washes with 1 × PBS, beads were incubated 45 min with 5 × 104 THP-1 cells  in 50 µL of RPMI-1640 at 37ºC and 5% v/v CO2. To stop phagocytosis, 200 µL of chilled PBS was added to each well of the plates and immediately placed on ice or at 4 °C. Samples were immediately read on FACS BD Accuri C6-plus (BD Biosciences), and THP-1 cells were gated by size, and granularity on FSC and SSC plots and fluorescence in the FITC-channel. A minimum of 5,000 THP-1 cells were recorded for each sample. Data were analyzed using the FlowJo (ver 10.6.0) software. Percentage phagocytosis was the proportion of THP-1 cells that were positive for fluorescence in the FL-1 channel. THP-1 cells in two major fluorescence peaks were visualized by setting the FL-1 channel against the FL-4 channel.The DBLβ domains were biotinylated and coupled to Neutravidin-labelled fluorescent beads  in a ratio of 10P < 0.05. GraphPad Prism 9.0 was used for the analysis.Wilcoxon rank sum test was used to compare antibody levels in matched and paired data groups between the time of hospitalization and at convalescence. We used Kruskal-Walli’s test with Dunn´s multiple comparison test of functional data for children in different clinical categories i.e., CM, nCSM, and UM. Differences were considered significant when Supplementary Information.