Plasmodium infection are inconsistent. Therefore, we aimed to synthesise the overall evidence of the association between ovalocytosis and malaria infection using a meta-analysis approach. The systematic review protocol was registered with PROSPERO (CRD42023393778). A systematic literature search of the MEDLINE, Embase, Scopus, PubMed, Ovid, and ProQuest databases, from inception to 30 December 2022, was performed to retrieve studies documenting the association between ovalocytosis and Plasmodium infection. The quality of the included studies was assessed using the Newcastle–Ottawa Scale. Data synthesis included a narrative synthesis and a meta-analysis to calculate the pooled effect estimate (log odds ratios [ORs]) and 95% confidence intervals (CIs) using the random-effects model. Our database search retrieved 905 articles, 16 of which were included for data synthesis. Qualitative synthesis revealed that over half of the studies showed no association between ovalocytosis and malaria infections or severity. Furthermore, our meta-analysis demonstrated no association between ovalocytosis and Plasmodium infection . In conclusion, the meta-analysis results demonstrated no association between ovalocytosis and Plasmodium infection. Hence, the role of ovalocytosis in relation to protection against Plasmodium infection or disease severity should be further investigated in larger prospective studies.Reports of an association between ovalocytosis and protection against  Plasmodium species, including Plasmodium falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri, and P. knowlesi2. The parasite is transmitted between humans via the bite of the female Anopheles mosquito3. The World Health Organization estimated that there were 247 million malaria cases in 84 malaria-endemic countries in 2021, resulting in 619,000 deaths4. Plasmodium infection requires interaction with several protein receptors in the membrane of the red blood cell (RBC). The most important of these is P. falciparum erythrocyte membrane protein 1, which mediates the interaction of parasite ligands on the RBC surface5. Other important malaria parasite ligand/RBC receptors involved in RBC invasion are band 3, glycophorin A, glycophorin B, glycophorin C, Duffy antigen receptor for chemokines and semaphorin 7A6.Malaria is caused by at least six 8. Band 3 is a member of the Solute Carrier 4 family of bicarbonate transporters. It is the predominant glycoprotein of the RBC membrane, where it acts as an electroneutral anion exchanger10. A mutation in or decreased expression of the Band 3 gene has been associated with changes in RBC morphology, membrane instability, and blebbing11. In vitro studies have shown that SAO RBCs are relatively resistant to invasion by certain P. falciparum isolates13. Furthermore, an in vitro study recently revealed that P. vivax field isolates invaded SAO reticulocytes 67–71% less frequently than non-SAO reticulocytes14. Additionally, a clinical study demonstrated an association between SAO and protection against clinical infections of P. vivax and severe P. falciparum malaria15. Although ovalocytosis, particularly SAO, has been reported to confer a protective effect against malaria infection and severity, there are some inconsistencies in the association between ovalocytosis and malaria, with some studies reporting no such association17. Therefore, our systematic review and meta-analysis aimed to collate and pool the evidence of ovalocytosis-associated malaria infection and severity.Southeast Asian ovalocytosis (SAO) is an RBC disorder caused by a 27-basepair deletion in the band 3 protein gene and is characterised by the presence of macro-ovalocytes and ovalostomatocytes on a blood smear18. The systematic review protocol was registered with PROSPERO (CRD42023393778).This systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelinesPlasmodium OR ‘Remittent Fever’ OR ‘Marsh Fever’ OR Paludism). The searches were performed in the English language without limitations on the publication date. A search in Google Scholar was also performed on 30 December 2022 to expand the search and ensure that relevant studies were not missed in the search of the main databases. Table A systematic search of the MEDLINE, Embase, Scopus, PubMed, Ovid, and ProQuest databases was performed from database inception to 30 December 2022 to retrieve studies reporting cases of malaria in patients with ovalocytosis. The search strategy combined search terms with Boolean operators  as follows:  AND  P: participants with and without ovalocytosis, (ii) E: malaria and (iii) O: odds/risk of malarial infection. Microscopy, rapid diagnostic tests (RDTs), or molecular methods can be used to detect Plasmodium infection. The following articles were excluded with reasons: reviews, in vitro studies, case reports or series, conference abstracts without full reports, books or book chapters, letters or news articles, articles where the outcomes of interest could not be extracted and articles for which full texts were unavailable.The population, exposure, outcome approachPlasmodium species, age range (years) of patients, characteristics of ovalocytosis, method for investigating ovalocytosis  and method for Plasmodium detection. Study selection and data extraction were independently conducted by three authors  and any discrepancies or disagreements between two authors were resolved through consensus.The studies retrieved from the database searches were managed using EndNote version 20.0  reference management software. The study selection began by removing duplicate studies, and the titles and abstracts of the remaining studies were screened. Those with irrelevant titles and abstracts were removed, and then the full-text versions of potentially relevant studies were examined. Studies were included based on pre-specified eligibility criteria, and ineligible studies were excluded with specific reasons. Finally, the following data were extracted and input into a data sheet before synthesis: first author, year of publication, study design, country, participants’ characteristics, 20. This tool was designed to evaluate the quality of non-randomised studies from three broad perspectives: study group selection, study group comparability and identification of the exposure or outcome of interest for case–control or cohort studies, respectively. Each item received 1 star in the categories of selection and outcome. For each item in the comparability category, a maximum of 2 stars was awarded.Two authors (MK and KUK) independently assessed the risk of bias of the included studies using the Newcastle–Ottawa Scale (NOS)Both qualitative and quantitative data were synthesised. The qualitative synthesis involved a narrative synthesis of the outcomes of interest from the included studies. The quantitative synthesis involved a meta-analysis approach to pool the outcomes of interest from the included studies.21. The consistency index (I2 statistics) and Chi-square (Q) test were used to assess the significance and levels of heterogeneity among the included studies. I2 values of < 25%, 25%–75% and > 75% were interpreted as low, moderate and high levels of heterogeneity, respectively22. Meanwhile, a P value of 0.1 for the Q statistic was considered an indication of significant heterogeneity among the included studies, as described previously23. If the heterogeneity of effect estimates among the included studies was observed, with a P value < 0.1 for the Q statistic or an I2 value > 25%, the pooled effect estimate was computed using the random-effects model. However, if there was no significant heterogeneity of effect estimates among the included studies, with a P value > 0.1 for the Q statistic or an I2 value < 25%, the pooled effect estimate was computed using the fixed-effects model.To synthesise the pooled log odds ratio (OR) and 95% confidence interval (CI), data in 2 × 2 tables Table  were com24. The trim-and-fill method was used to correct for publication bias25, if any. Meta-regression analysis based on study design, country, participants’ characteristics, Plasmodium species, method used to investigate ovalocytosis, and method for malaria detection was performed to assess the impact of these parameters on the pooled effect estimate. If the meta-regression analysis showed a significant impact of covariates on the pooled effect estimate, a subgroup analysis was conducted to determine the difference in the pooled effect estimate between subgroups. The leave-one-out sensitivity analysis was performed to test whether a single study affected the overall pooled estimate. All analyses were performed using the meta command in Stata v17 software .The publication bias was assessed by visualisation of the funnel plot asymmetry and further validated using Egger’s test38 were included for qualitative and quantitative data synthesis , Embase n = 160), Scopus (n = 172), PubMed (n = 114), Ovid (n = 295), and ProQuest (n = 76). After removing duplicate records (n = 311), the titles and abstracts of the remaining records were screened (n = 594) to exclude non-relevant records (n = 443). Then, the remaining records (n = 151) were assessed for full-texts and eligibility. After applying the exclusion criteria, 133 records were excluded with specific reasons: reviews (n = 68), in vitro studies (n = 30), case reports (n = 10), gene mutation or polymorphism (n = 8), no abstract available (n = 4), conference abstracts (n = 2), books (n = 2), no information on ovalocytosis (n = 2), letters or news article (n = 2), inability to extract data (n = 2), unavailability of full text (n = 1), guideline (n = 1) and duplicate article (n = 1). The searches in Google Scholar identified 876 articles, none of which met the eligibility criteria. Finally, 16 studies60, Scopu37. Study designs included cross-sectional studies (50.0%)37, case–control studies (18.8%)29, cohort studies (25.0%)38 and both cohort and case–control studies (6.3%)15. Most studies were conducted in Papua New Guinea (68.8%)38, with the remainder in Indonesia (18.8%)37, Malaysia (6.3%)30 and Thailand (6.3%)27. Most studies enrolled patients infected with single or mixed infections of P. falciparum and P. vivax (56.3%)37, single or mixed infections of P. falciparum, P. vivax and P. malariae (18.8%)36, and infection with P. falciparum only (12.5%)35. The majority of study participants were children (50.0%)37, followed by pregnant women (18.8%)38. Most of the included studies tested for SAO using a polymerase chain reaction (PCR) assay to identify band 3 deletions (75.0%)38, and the remainder investigated ovalocytosis using microscopy (25.0%)36. Malaria parasites were mainly detected using microscopy (68.8%)36, followed by a combination of microscopy with PCR (18.8%)38, a combination of microscopy with RDT (6.3%)33 or PCR only (6.3%)37  published between 2000 and 201029 and three of the cohort studies33 scored the maximum of 9/9 stars. Two studies38 scored 8/9 stars: one did not demonstrate the outcome of interest at the start of the study35 and the other lacked selection of the non-exposed cohort38. 'The NOS scale for case–control studies was adapted to assess the risk of bias in cross-sectional studies. Six cross-sectional studies scored 8/8 stars36, and two37 scored 7/8 stars because they lacked a ‘definition of controls’ . Benet et al.28 reported that severe malaria infections in placental tissues were less common in pregnant women with SAO than in pregnant women in the control group. Cattani et al.29 reported that patients with ovalocytosis had a lower infection rate of P. falciparum (P = 0.044), P. vivax (P = 0.009) and all species of malaria combined (P = 0.013) compared with those without ovalocytosis. Foo et al.30 demonstrated fewer ever-positive ovalocytes for P. falciparum (P < 0.05) or any parasite species (P < 0.05) compared with the controls. Additionally, malaria parasitaemia was lower in individuals with ovalocytes compared with the controls. Rosanas-Urgell et al.15 reported that SAO was associated with a statistically significant reduction of 46% in the incidence of clinical P. vivax episodes and a 52% reduction in P. vivax blood-stage reinfection. Furthermore, in the case–control study, SAO was associated with protection against severe P. falciparum malaria  but not with protection against uncomplicated P. falciparum malaria. Allen et al.26 reported the absence of SAO band 3 in 68/68 (100%) of children with cerebral malaria compared with 6/68 (8.8%) matched community controls . Serjeantson et al.36 suggested that patients with ovalocytosis were more resistant to severe malaria than those with normocytes because the ratio of parasitaemia in 112 children with ovalocytes compared with 741 children with normocytes was 1.05 for P. falciparum; 0.90 for P. vivax; 0.54 for P. malariae, and 0.91 for infection with any Plasmodium species.Seven studies (43.8%) showed that ovalocytosis might protect against malaria infection38. Fowkes et al.16 found no association of SAO with P. falciparum prevalence (P = 0.29) or with mean parasite density (P = 0.66). Kimura et al.17 found no difference in the prevalence of SAO between patients with malaria and controls (P > 0.8) and no difference in the frequency of SAO between patients with P. falciparum and P. vivax malaria. Although Kimura et al.31 discovered a link between a higher rate of ovalocytes and a lower risk of malaria infection, they found that SAO did not protect against malaria infection. Lin et al.32 showed no significant associations between P. falciparum infection and SAO (P > 0.2). O’Donnell et al.34 demonstrated no difference in the proportion of SAO in acute malaria and the community controls . However, the degree of ovalocytosis was significantly lower in children with SAO during acute malaria compared with the community controls (P = 0.025). O’Donnell et al.35 showed that the SAO genotype was not associated with the frequency of placental P. falciparum infection . Shimizu et al.37 reported no significant difference in the prevalence of asymptomatic malaria infection between participants with or without SAO (P > 0.05), and Stanisic et al.38 reported no significant difference in the frequency of malaria infection among participants with or without SAO (P = 0.62). Manning et al.33 reported a frequently higher proportion of ovalocytosis in P. falciparum (5.7%) than P. vivax (3.8%) and mixed infections (4.3%).Eight studies  showed no association of SAO with malaria infection or severity38 were included in the meta-analysis of the risk of malaria among participants with ovalocytosis. Overall, the results demonstrated no difference in the log OR of malaria between patients with and without ovalocytosis . The results of both tests indicated a publication bias among the included studies. Although the trim-and-fill method was applied to correct for publication bias, the subsequent results showed no difference in the log OR of malaria among patients with ovalocytosis compared with those without ovalocytosis .Publication bias was assessed using a funnel plot and Egger’s test. The funnel plot showed the asymmetrical distribution of the log OR and indicated the standard error of the log OR Fig. . Egger’s38. Meanwhile, studies by Rosanas-Urgell et al.15 and Foo et al.30 both showed lower odds of malaria infection in SAO. Conversely, only the study by Cattani et al.29 reported higher odds of malaria infection in SAO. According to Rosanas-Urgell et al.15, SAO provides partial protection against P. vivax but not against P. falciparum infection or uncomplicated disease. Nevertheless, our meta-analysis contradicted these results, with no difference in the risk of P. vivax infection between patients with and without ovalocytosis. These findings suggest that ovalocytosis may not be a protective factor against P. vivax infection, at least for the limited studies included in this meta-analysis.SAO is a disorder of the RBC membrane caused by a mutation in the gene for band 3 protein, which leads to the formation of unique linear oligomers in the RBC membrane. Our meta-analysis results demonstrated no association between SAO and malaria infection, and almost all of the included studies demonstrated no association between SAO and malaria infection15. Secondly, alterations in RBC structures may impair the binding of malarial proteins from the parasite to the RBC membrane, thereby inhibiting parasite growth within RBCs39. Thirdly, SAO RBCs may provide protection against cerebral malaria due to the redistribution of sequestered infected RBCs away from the brain32. Similar to the protection against cerebral malaria, SAO RBCs may prevent parasitised RBCs from adhering to receptors in the placenta, thereby protecting against placental malaria. However, a study on placental samples of pregnant women demonstrated that SAO RBCs did not reduce the incidence of placental malaria and, therefore, did not protect against placental malaria35. Another study on pregnant women revealed that primigravidae with SAO had a lower prevalence of placental infection than primigravidae without SAO, whereas there was no difference in the prevalence and severity of placental infections between SAO and non-SAO multigravidae, indicating that the gravidae status in women with SAO confounds the association between SAO and placental infection. Thus, the mechanism by which SAO RBCs protect against placental malaria among primigravidae was suggested to be non-immune- rather than immune-based28. Since SAO RBCs protect against cerebral malaria or severe diseases but not uncomplicated diseases, this suggests that the protection involves post-invasion mechanisms, such as preventing sequestration of infected RBCs41.The protective mechanism of SAO against malaria is unknown, and several theories have been proposed. Firstly, modification of the RBC membrane may affect the ability of merozoites to invade RBCs and prevent the development of parasites within RBCs38. However, some showed that ovalocytosis might protect against malaria infection30 or severe disease36. Nevertheless, our meta-analysis results demonstrated no association between SAO and malaria infection. These results were stratified according to the study design, without or with minimal heterogeneity in cohort studies38 and in cross-sectional studies37. High or substantial heterogeneity was found in three case–control studies29. Besides differences in study design, the controversial association of SAO in the literature was suggested to be caused by the presence of molecularly heterogeneous SAO because SAO without B3∆27 was associated with decreased malaria infection rates31. Additionally, another study indicated that the inconsistency of SAO-related protection against malaria might be due to the detection of SAO with low sensitivity34.Our systematic review showed heterogeneous associations between SAO and malaria in individual studies. Most studies demonstrated no association of SAO with malaria infections or severityP. vivax malaria, some studies have suggested that SAO mainly protects against P. vivax malaria. Thus, it would be interesting for further studies to refine the investigation to that of P. vivax exclusively to assess the extent of this protection without any interference from a concomitant P. falciparum infection.Our systematic review and meta-analysis had several limitations. Firstly, the number of studies included in the meta-analysis was limited, which might limit the interpretation of the results. Nonetheless, because the results of the studies included in the meta-analysis were consistent, the findings were sufficiently robust to support the conclusion that there is no link between SAO and malaria infection. Secondly, a meta-analysis of the odds of severe disease between severe and non-severe diseases could not be performed due to the limited number of studies reporting an association between SAO and malaria severity. Third, there was heterogeneity in the association between ovalocytosis and malaria infection between studies. Although we conducted a meta-regression analysis, no probable source of heterogeneity was found. Fourth, although our meta-analysis revealed that ovalocytosis did not protect against Our systematic review revealed the heterogeneity of individual studies regarding the association between SAO and malaria. However, our meta-analysis results demonstrated no protection of SAO RBCs against malaria infection. Hence, the association of SAO with malaria infection or disease severity should be further investigated by larger studies.Supplementary Information 1.Supplementary Information 2.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.


In the study, a novel point-of-care Rapid Diagnostic Test designed for the detection of imported malaria infections was evaluated in the field. Methods: Suspected imported malaria cases reported from Guangxi and Anhui Provinces of China during 2018–2019 were enrolled to evaluate the novel RDTs. Diagnostic performance of the novel RDTs was evaluated based on its sensitivity, specificity, positive and negative predictive values, and Cohen’s kappa coefficient, using polymerase chain reaction as the gold standard. The Additive and absolute Net Reclassification Index were calculated to compare the diagnostic performance between the novel RDTs and Wondfo RDTs (control group). Results: A total of 602 samples were tested using the novel RDTs. Compared to the results of PCR, the novel RDTs presented sensitivity, specificity, PPV, NPV, and diagnostic accuracy rates of 78.37%, 95.05%, 94.70%, 79.59%, and 86.21%, respectively. Among the positive samples, the novel RDTs found 87.01%, 71.31%, 81.82%, and 61.54% of P. falciparum, P. ovale, P. vivax, and P. malariae, respectively. The ability to detect non-falciparum malaria did not differ significantly between the novel and Wondfo RDTs (control group). However, Wondfo RDTs can detect more P. falciparum cases than the novel RDTs . After the introduction of the novel RDTs, the value of the additive and absolute Net Reclassification Index is 1.83% and 1.33%, respectively. Conclusions: The novel RDTs demonstrated the ability to distinguish P. ovale and P. malariae from P. vivax which may help to improve the malaria post-elimination surveillance tools in China.Background: China was certified malaria-free by the World Health Organization on 30 June 2021. However, due to imported malaria, maintaining a malaria-free status in China is an ongoing challenge. There are critical gaps in the detection of imported malaria through the currently available tools, especially for non- Plasmodium parasites. It is reported that malaria cases were still on the rise between 2020 and 2021. However, the rate of increase is lower than that of 2019–2020; there were an estimated 247 million malaria cases in 2021 in 84 malaria endemic countries, this number was 245 million in 2020 and 232 million in 2019, and an estimated 619,000 malaria deaths [Malaria remains a serious public health problem worldwide and is caused by falciparum malaria, especially Plasmodium ovale , increased to levels higher than expected [With globalization and increased international movement, imported malaria cases continue to be reported in China, highlighting the challenges faced in preventing malaria re-establishment ,5. Priorexpected . Moreoveexpected .2 and a land area of 139,400 km2. It is a transitional region between warm temperate and subtropical climates, with a distinct monsoon climate. Anhui Province was once one of the key malaria endemic provinces in China, which seriously affected people’s physical health and socio-economic development. Anhui Province is an unstable malaria endemic area, and historically, the Huaibei Plain was an endemic area for P. vivax; in the hilly areas of the Jianghuai River, there are many cases of daily malaria, and some areas have the presence of P. falciparum; the mountainous areas in southern Anhui are mainly characterized by P. vivax. Prior to the 1970s, there were cases of P. falciparum and a small number of P. malariae. After nearly 70 years of prevention and control, the basic elimination of P. falciparum was achieved in 1996. The last local infection case in the province was reported in 2013. Since 2014, there have been no local cases of malaria reported in the province, and all cases have been imported cases. Among them, there were 68–190 reported cases from 2011 to 2019, mainly of P. falciparum, with reports of the other three species and mixed infections [Anhui Province is located in the southeastern part of China, in the middle and lower reaches of the Yangtze and Huai Rivers, with an area of 140,100 kmfections .P. vivax and P. falciparum. After the founding of New China, after more than 70 years of prevention and control, the incidence rate dropped from 296.7/10,000 in 1954 to less than 1/10,000 in 1987; The last local infection case was reported in 2012, and since 2013 there have been no local infection cases reported in the province. All cases occurred as imported cases, mainly from African and Southeast Asian countries and regions. Among them, a total of 3195 malaria cases were reported in Guangxi from 2010 to 2019, with the main species being P. falciparum, while the other three species and mixed infections were all reported as well [Guangxi is located in the southern part of China, bordering Guangdong and Hunan in the southeast and northeast, Yunnan and Vietnam in the west and southwest, and Guizhou Province in the north. The average annual temperature is 16.5–23.1 °C. There are 14 cities and 111 counties in the province, with a total area of 236,000 km and a total population of 56.95 million in 2019. Throughout history, Guangxi has been a severely prevalent area for malaria, with major outbreaks occurring in 1954, 1963, and 1971. Throughout history, malaria in Guangxi was mainly caused by P. ovale is easily and commonly misdiagnosed as an infection of P. vivax [P. malariae, P. ovale, and P. vivax infections. Thus, prompt and precise diagnostic tools that can detect and differentiate non-falciparum malaria species are needed.In China, when suspected malaria patients presenting with symptoms of malaria, especially combined with a history of travel to a malaria-endemic area, seek medical care, the physician provides a diagnostic test for malaria, commonly microscopy or rapid diagnostic test (RDTs) . HoweverP. vivax ,17, whicIn this study, a novel point-of-care RDT was designed for the detection of imported malaria infections and was evaluated using polymerase chain reaction (PCR) as the gold standard. We believe that the newly designed diagnostic tool can benefit the prevention of malaria re-establishment in the future.The provinces of Anhui and the Guangxi Autonomous Region were selected as study areas for the evaluation of the novel RDTs . HistoriIn China, each suspected malaria case should be mandatorily reported through the China Information System for Disease Control and Prevention (CISDCP) . This isPlasmodium species. The kits, targeting the 18s rRNA gene, were designed by referring to a previous study and provided internal control [In China, when a suspected case is reported through the network, the blood samples, including whole blood and smears that were collected from the patient before anti-malarial treatment , are sen control . Before P. vivax, T2 and T3 line were simultaneously positive, whereas, if the infections were P. ovale and/or P. malariae, only the “T3” line was positive, with a negative “T2” line. Using the combination of “T2” and “T3” test lines, the novel mRDT can distinguish P. vivax from P. ovale and/or P. malariae , and analysis was performed using SPSS 26.0 statistical software . The thematic map of geographic distribution was created by MapInfo 15.0 .The categorical data are presented as percentages. Values are presented as the mean ± standard deviation for data that were normally distributed. Differences in proportions were compared using McNemar’s χ2 test. Taking the results of PCR as the gold standard, the diagnostic performances of the novel and Wondfo RDTs were presented with the following parameters: sensitivity, specificity, positive (PPV) and negative predictive values (NPV), and Cohen’s kappa coefficient, with their respective 95% confidence intervals. The formula for calculating PPV and NPV is: PPV = (true positives)/, NPV = (true negatives)/, respectively. Additive Net Reclassification Index (NRI) and absolute NRI are calculated to compare diagnostic performance between the Novel and Wondfo RDTs . All staP. falciparum), 123 , 22 (P. vivax), 13 , and 7 (mixed infections) samples tested positive. The remaining 283 cases were confirmed negative by PCR. The mean age of the participants was 42.2 ± 9.1 years, and 578 participants (96.0%) were males.During the study period, a total of 602 blood samples collected from suspected malaria cases were tested to evaluate the performance of the novel and Wondfo RDTs. Cases came from 26 African and 2 Asian countries, with Africa  being the most common region of origin. The five countries of origin of infection were Ghana , Nigeria , Ivory Coast , Angola , and Mozambique . Of these, 154 , whereas the opposite is true for specificity (89.05% vs. 95.05%) .Plasmodium species from the blood samples which were collected. Compared to the PCR gold standard, the Wondfo RDTs detected 96.01% , 72.13% , 90.91%(P. vivax), and 92.31%, while the novel RDTs identified 87.01% , 71.31%, 81.82%(P. vivax), and 61.54%  of cases. Their ability to detect non-falciparum malaria did not differ significantly, but Wondfo RDTs detected more P. falciparum infections than the novel RDTs (96.10% vs. 87.01%)  .p > 0.05)  .The last indigenous malaria case in China was reported in 2016 and local transmission has been interrupted since 2017 . China wP. falciparum), parasite lactate dehydrogenase , and malaria parasite aldolase (Plasmodium genus). Therefore, distinguishing between P. falciparum and other species is not a simple task [P. falciparum, pan specificity, and malaria parasites, showing that differences in reactivity may be related to small differences on the surface of pLDH, with subtle amino acid changes being the cause of species specificity [For malaria detection, RDT that can distinguish between the types of malaria parasites, live and dead infections, and the sexual stage of parasitemia will be very helpful for diagnosis and guide intervention measures. The main challenge is in the field of low-level parasitemia. The examination of the life cycle stage of malaria parasite infection has identified many key targets, including the HRP2 protein  have been registered in the National Medical Products Administration that could be used in health facilities. Wondfo RDTs are used more in the market for price reasons, and presented a better performance for detecting P. ovale compared to CareStart pLDH PAN and SD BIOLINE Pf/Pan RDTs [P. falciparum from non-falciparum species. However, they could not further differentiate non-falciparum species. P. vivax, a common species in all malaria-endemic areas, is distributed nationwide and is the main species related to the risk of malaria reintroduction. Due to their morphological similarity, P. ovale is easily and commonly misdiagnosed as an infection of P. vivax in the field, further leading to inappropriate interventions. A novel RDT has been designed to fulfil the gap in field practice.In China, infections caused by Pan RDTs . AccordiP. falciparum and did not react with the samples of P. vivax (P. vi-vax); similarly, PvLDH antibodies only react with P. vivax samples and do not react with P. falciparum samples [Related studies have shown that, although the protein sequence of pLDH is very conservative within the same species, there are certain differences in the protein sequence of pLDH among the four human malaria parasites. Usually, the detection antibodies in RDT are monoclonal antibodies that are prepared based on a specific pLDH antigen of a certain insect species. Therefore, when detecting unknown samples, if the patient is infected with other insect species in the body, the detection antibodies in RDT cannot specifically bind to the antigen in the sample, resulting in false negative test results. The specificity experiment results showed that the monoclonal antibody of PfLDH only reacted with the samples of  samples .P. falciparum, 71.31% of P. ovale, 81.82% of P. vivax, and 61.54% of P. malariae infections. The diagnostic performance of the novel RDTs for non-falciparum species was similar to that of the Wondfo RDTs. Importantly, the novel RDTs had the ability to distinguish P. ovale and P. malariae infections from P. vivax. Although improvements are still needed to improve the novel RDTs, we believe that significant progress has been made in this initial development and through this diagnostic evaluation.In this study, the diagnostic performance of the novel RDTs was assessed using blood samples collected from the field. The novel RDTs found 87.01% of P. falciparum was lower than for Wondfo RDTs (87.01% vs. 96.10%), P. falciparum is the dominant species of imported malaria [Although the diagnostic sensitivity of the novel RDTs for  malaria . Novel RP. ovale and P. malariae were commonly considered as the ‘bashful’ malaria parasites, due to their low prevalence and limited geographic distribution [P. falciparum and P. vivax. Developing diagnostic tools that target the two main species of Plasmodium is challenging. One factor significantly impacting its success is that parasitaemia is typically very low in infections caused by P. ovale and P. malariae . According to a previous study, P. malariae only invades aged red blood cells (0.1% parasitaemia) and P. ovale preferentially invades youthful red blood cells (1% parasitaemia) [P. malariae.ribution . Howeverribution ,34. Furtitaemia) . This imP. malariae and P. vivax cases is small.Our study has two main limitations. First, limitations around clinical and patient-level RDTs occur frequently in non-endemic settings, especially in the presence of treatment delays . TherefoP. ovale and P. malariae from P. vivax. We believe that the newly designed RDTs can benefit POR practices within China and other malaria-eliminating and POR countries with further improvements.The diagnostic power of novel RDTs for non-falciparum species detection was comparable to the Wondfo RDTs, and demonstrated the ability to distinguish


N = 661) nested within the COSMIC clinical trial (NCT01941264) in Burkina Faso. Plasmodium falciparum infections during pregnancy and infants’ clinical malaria episodes detected during the first year of life were recorded. The levels of maternal IgG and IgG1-4 to 15 P. falciparum antigens were measured in cord blood by quantitative suspension array technology. Results showed a significant variation in the magnitude of maternal antibody levels in cord blood, depending on the PME category, with past placental malaria (PM) more frequently associated with significant increases of IgG and/or subclass levels across three groups of antigens defined as pre-erythrocytic, erythrocytic, and markers of PM, as compared to those from the cord of non-exposed control infants. High levels of antibodies to certain erythrocytic antigens  were independent predictors of protection from clinical malaria during the first year of life. By contrast, high levels of IgG, IgG1, and IgG2 to the VAR2CSA DBL1-2 and IgG4 to DBL3-4 were significantly associated with an increased risk of clinical malaria. These findings indicate that PME categories have different effects on the levels of maternal-derived antibodies to malaria antigens in children at birth, and this might drive heterogeneity to clinical malaria susceptibility in early childhood.In this study, we investigated how different categories of prenatal malaria exposure (PME) influence levels of maternal antibodies in cord blood samples and the subsequent risk of malaria in early childhood in a birth cohort study ( Infants are thought to be protected against malaria during the first months of life mainly due to the transplacental passage of maternal antibodies as well as additional factors such as fetal hemoglobin and behavioral practices reducing exposure. However, in high-transmission settings, malaria in early infancy is common, and susceptibility to the infection varies between individuals. Indeed, birth cohort studies across different African countries showed that up to 59% of infants can experience at least one clinical episode of malaria during the first year of life in certain transmission settings –4. In in utero and through breastfeeding  was demonstrated only in few studies –18. Main utero exposure to malaria parasites and/or antigens have a profound effect on fetal innate immunity, which affects malaria risk during the first year of life . In brief, COSMIC was a cluster-randomized controlled trial investigating the protective efficacy of adding community-scheduled screening and treatment (CSST) of malaria during pregnancy to the standard IPTp-SP  compared to IPTp-SP alone (control arm) in Burkina Faso, Benin, and The Gambia . The CSSIn Burkina Faso, from the 1,800 pregnant women participating in the COSMIC trial, 734 mother-child pairs were enrolled in a birth cohort study with a 12-month follow-up. Of the 734 mother-child pairs, 661 mothers and their offspring were included for the present study. Mother-child pairs not included were those for whom complete data on history of malaria infection during pregnancy and/or cord blood samples for immunological assays were not available. The study was conducted in the health district of Nanoro, a rural area located in Centre-West of Burkina Faso . MalariaThe recruitment and the follow-up procedures of the birth cohort study have been described elsewhere . BrieflyP. falciparum diagnosis by qPCR. Cord blood plasma samples were collected following a centrifugation at 3,000 rpm for 10 min, then frozen at −80°C. The plasma samples were subsequently shipped frozen to ISGlobal  for antibody quantification. For after birth malaria surveillance, peripheral blood was collected by finger-prick from each infant visiting the health facilities with presence of fever or history of fever in the previous 24 h, and used for RDT, blood smear, and spots on filter paper (Whatman 3 MM).Procedures have been described elsewhere . In brieP. falciparum 3D7/NF54 strain antigens. These included (i) pre-erythrocytic sporozoite antigens  (42) (1-4 subclasses (1/100). For all assays, 12 serial dilutions 1:2 of a positive control were used to perform antigen-isotype/subclass specific standard curves. The positive control consisted of a WHO Reference Reagent for anti-malaria P. falciparum human serum (NIBSC code: 10/198) at 1:50. A total of 10 different samples from malaria-naïve adult donors were used as negative controls. Two blanks were added to each plate for quality control purposes. In brief, antigen-coupled beads were added to a 384-well μClear flat bottom plate (Greiner Bio-One) in multiplex  resuspended in 90 µL of luminex buffer . Ten microliters of sample, negative or positive controls, and blanks were added to multiplex wells and incubated during 1 h at room temperature in a shaker at 900 rpm and protected from light. Plates were washed three times with 200 µL/well of wash buffer (PBS-Tween 20 0.05%) using an automatic plate washer machine (Biotech 405TS). Then, for IgG, 25 µL of goat anti-human IgG-phycoerythrin (PE)  were added diluted in luminex buffer (1/400). For IgG1 and IgG3 subclasses, 25 µL of biotinylated secondary antibody  were added diluted in luminex buffer . For IgG2 and IgG4 subclasses, 25 µL of mouse anti-human IgG2 and IgG4  were added diluted in luminex buffer (1/250 both), followed by 25 µL of biotinylated anti-mouse IgG (B7401) diluted in luminex buffer . All antibody incubations were performed for 30 min, at room temperature, at 900 rpm, and protected from light. Next, 25 µL of streptavidin-R-phycoerythrin  diluted 1/500 in Luminex buffer were added to all wells for all the antibodies and incubated 30 min, at room temperature, 900 rpm, and protected from light. Plates were washed as before and resuspended in 80 µL/well of Luminex buffer. Plates were read using a FLEXMAP 3D analyzer and at least 50 microspheres per analyte were acquired per sample. Data were captured using xPonent software. Antibody levels were reported as median fluorescence intensity (MFI).Quantitative suspension array technology (qSAT) applying the xMAP technology  was used to measure antibody levels to 15 P-NANP)] ; (ii) blP-NANP)] , C-termiP)] 42) , merozoi , merozoP)] (42) , merozoiP)] (42) , erythroP)] (42) , EBA175 P)] (42) , glutamiP)] (42) , reticulP)] (42) ]; and (iP)] (42) , 50. A gP)] (42) . The selP)] (42) , 52, 53.P. falciparum detection of Pf-varATS by qPCR, as previously described  acute infection , (ii) chronic infection , (iii) past infection (parasites absent but pigment present), and (iv) no infection . PME was categorized based on placental infection  and maternal peripheral infection either during monthly screening, ANC visits, unscheduled visits or at delivery. The “exposure No PM’’ status was defined as presence of a documented peripheral infection during pregnancy but with no evidence of PM. Congenital malaria infection was defined as the presence of P. falciparum parasites in the cord blood as detected by qPCR. Non-exposure of newborns to malaria during pregnancy was defined as absence of documented peripheral and placental malaria infection during the course of pregnancy and at delivery.SD-Bioline malaria antigen P.f test  detecting PfHRP2 was used for malaria RDT according to the manufacturer’s instructions. The microscopic examination of thick blood smears stained with Giemsa (10%) was performed according to standard procedures . DBS on escribed . Data onescribed . A clini10 scale for the subsequent analyses. The assay quality control for each antigen and plate was based on visual inspection of the performance of the standard curves and of boxplots [with medians and interquartile ranges (IQR)] of the different sample types .MFI measurements were transformed to log10 MFI) across antigens and between study groups were done through boxplots with medians and IQR, by Wilcoxon rank-sum test and P-values adjusted for multiple comparisons all together for IgG and subclasses by Benjamini-Hochberg correction. Radar plots were also used to visualize IgG and subclasses profiles across the different PME categories. Associations between antibodies were explored by Spearman correlations across antibodies levels .Analyses included either all subject sample or samples stratified by PME categories and other maternal and infant covariates including malaria in pregnancy preventive treatment strategy (MiP strategy), gravidity, insecticide treated net (ITN) use by the mother, prematurity , low birth weight , congenital malaria, infant sex, ethnicity, and transmission intensity at birth . Comparisons of crude levels of IgG and subclasses , P-value <0.05) and included in the Cox proportional-hazard regression models . P-value <0.05 was considered statistically significant. Ratios of protective antibodies relative to antibodies associated with increased risk of clinical malaria (the markers of PM) from the Cox proportional-hazard analyses were used to further assess the effect of maternal antibodies on malaria susceptibility during the first year of life. Values of the ratios above 1 were considered as high levels of protective antibodies as compared to that of the antibodies associated with PM. All analyses were performed using R statistical package version 3.2.3 (The associations between antibody levels (as continuous variables) at birth and the risk of clinical malaria during the first year of life were assessed in univariable and multivariable Cox proportional-hazard models. Proportionality of hazards assumption and functional form of each variable adjusted in the Cox models were tested using Schoenfeld residuals analysis and p-splines functions, respectively. Secondary variables  that showed significant associations with malaria during the first 12 months of life were determined in Kaplan-Meier survival analyses  and markers of PM  were higher compared to other antibodies. The Spearman correlation matrix showed a positive moderate correlation between antigens for IgG and all the subclasses . In addition, acute PM and exposed/No PM cases were, respectively, associated with increased levels of IgG1 and IgG3 against pre-erythrocytic antigens. All categories of PME were associated with significantly higher levels of IgG and/or subclasses against the markers of PM  compared with that among the non-exposed control group . However, past and chronic PM were more frequently associated with higher IgG levels against DBL1-2 and DBL3-4 than the other categories of PME .Variation of maternal antibody levels by PME category is shown using boxplots ; Fig. S242 as compared to those in the non-exposed control group. By contrast, chronic and past PM were associated with significantly lower levels of IgG subclasses against some erythrocytic antigens (IgG1 against MSP3 and IgG2 against MSP5 and GARP)  . The mulnd GARP) .42 and IgG3 against EBA140 and MSP5). Non-cytophilic antibodies (IgG2 and IgG4) against blood stage antigens were not significantly associated with malaria risk during the first year of life. By contrast, IgG and both cytophilic and non-cytophilic antibodies against markers of PM  were associated with an increased risk of malaria episodes during the first year of life  experienced at least one episode of clinical malaria with a median survival time of 9.9 months. Among the covariates from mothers and infants that were assessed for their effect on malaria risk, MiP preventive treatment , birth s of life .42/IgG1-DBL1-2). The proportion of individuals with a ratio >1 and the median ratio were, respectively, 26.6% (176/661) and 3.6 [IQR (1.6–7.9)] for IgG-EBA140/IgG-DBL1-2 ratio, 44.5% (294/661) and 2.4 [IQR (1.4–5.3)] for IgG-EBA175/IgG-DBL1-2 ratio, 59.3% (392/661) and 7.1 [IQR (2.1–34.8)] for IgG1-MSP142/IgG1-DBL1-2 ratio. We found that in all three cases, infants were at a reduced risk of developing a clinical malaria when levels of protective antibodies where higher than those of antibodies associated with an increased risk of malaria (ratios >1) as compared to infants in whom ratios where equal or below 1  were associated with an increased time to first clinical malaria episode during the first year of life. This is in agreement with previous studies that reported association between maternal antibodies against EBAs and MSPs with malaria protection in children  were significantly associated with an increased risk of clinical malaria during the first year of life. DBL1-2 and DBL3-4 are domains of the VAR2CSA (variant surface antigen 2-chondroitin sulfate A) protein, a variant antigen of the PfEMP1 family that is expressed on infected erythrocytes and mediates parasites sequestration in the placenta through binding to CSA on placental syncytiotrophoblast cells , 73. VARIn spite of the significant increase of antibodies against CSP antigens observed in PM, this study failed to find association between pre-erythrocytic antibodies and malaria protection while recent malaria vaccines studies demonstrated the relevance of these antibodies (in particular the CSP-NANP specific antibodies) in the protection against clinical malaria in children , 77, 78.42, IgG3 to MSP5, IgG4 to DBL3-4, and IgG, IgG1, and IgG2 to DBL1-2). To further assess how maternal antibodies influence malaria susceptibility in these infants exposed to malaria in utero, we examined the associations of ratios between protective antibodies and those associated with risk on malaria protection during the first year of life. Based on the Cox proportional hazard analyses results, the effect of two IgG ratios  and one IgG1 ratio  were evaluated, but none for IgG3 since IgG3 levels to both DBL1-2 and DBL3-4 were not significantly associated with malaria risk. Interestingly, we found that despite a prenatal malaria exposure, having higher levels of maternal antibodies against erythrocytic antigens than against DBLs protected children from clinical malaria. Humoral immunity to malaria is complex (Importantly, several antibodies whose levels were significantly modified by PME were found to be associated with differential malaria risk in infancy (e.g., IgG to EBA175, IgG1 to MSP1 complex  and thesin utero environment that probably influences maternal antibodies transfer. Past PM is characterized by persistent hemozoin deposits in fibrin in the placenta with no presence of infected erythrocytes, constituting a biomarker of early infection during pregnancy that has been cleared prior to delivery (P. falciparum specific antibodies with sufficient time to cross the placenta, also favored by the resolution of the placenta inflammation and the absence of sequestered parasites, and thus, could explain why past PM was found to be associated with significant increase of IgG and/or subclasses levels in cord blood. In chronic PM, there are infected erythrocytes and hemozoin, and it is interpreted as an infection of longer duration, or a recent placental infection in a pregnant woman with a pre-existing past PM. This could be the reason why chronic PM was associated with an increase of antibody levels against several antigens in bivariable (as illustrated by boxplots in Previous studies have reported a strong correlation between maternal antibodies in the peripheral blood and those in the cord blood at delivery and that these antibody profiles reflect the immunological experience of the mother in her living environment , 11, 13.delivery , 87. TheIt’s worth noting that maternal antibody levels decrease over time and, depending on their initial titers, they are expected to be low at 6 months and further decreased at 9 months after birth . Thus, tIn conclusion, these findings indicate that different PME categories have different effects on maternal-derived antibodies levels to malaria antigens in children at birth, and this might drive heterogeneity to clinical malaria susceptibility in early childhood.


This suggests that ALA may sensitize GSCs to BNCT by upregulating the expression of amino acid transporters, thereby enhancing the uptake of BPA and improving the effectiveness of BNCT. These findings have important implications for strategies to improve the sensitivity of malignant gliomas to BPA-BNCT.Boron neutron capture therapy (BNCT) is a high-LET particle radiotherapy clinically tested for treating malignant gliomas. Boronophenylalanine (BPA), a boron-containing phenylalanine derivative, is selectively transported into tumor cells by amino acid transporters, making it an ideal agent for BNCT. In this study, we investigated whether the amino acid 5-aminolevulinic acid (ALA) could sensitize glioma stem cells (GSCs) to BNCT by enhancing the uptake of BPA. Using human and mouse GSC lines, pre-incubation with ALA increased the intracellular accumulation of BPA dose-dependent. We also conducted in vivo experiments by intracerebrally implanting HGG13 cells in mice and administering ALA orally 24 h before BPA administration (ALA + BPA-BNCT). The ALA preloading group increased the tumor boron concentration and improved the tumor/blood boron concentration ratio, resulting in improved survival compared to the BPA-BNCT group. Furthermore, we found that the expression of amino acid transporters was upregulated following ALA treatment both in vitro and in vivo, particularly for ATB Presently, The standard treatment for malignant gliomas includes combined therapy with surgical resection, chemotherapy, and radiotherapy. Nevertheless, the median survival time (MST) of GBM patients is < 15 months because of tumor recurrence. Studies during the last decade suggest that malignant gliomas harbor self-renewing tumorigenic cancer stem cells, termed glioma stem cells (GSCs). GSCs are believed to be responsible for the therapeutic resistance of malignant gliomas and tumor recurrence2. Thus, treatment strategies targeting GSCs might help treat GBM3.Malignant gliomas are the most prevalent and lethal among intraparenchymal brain tumors, and glioblastomas (GBM) have the worst prognosis10B) is first administered to the patient, followed by irradiation with epithermal neutron beams focused on the tumor lesion. In the cells where the drug is taken up, a neutron capture reaction occurs specifically with 10B, generating alpha particles. Alpha particles release all their energy within a short range of less than the size of a cell (approximately 10 μm), leading to a high-density ionization reaction with cell organelles and various intracellular molecules. In particular, complex double-strand breaks, which are difficult to repair, occur in DNA, resulting in a powerful cytotoxic effect. The 10B drug for BNCT is designed to selectively accumulate in malignant tumor cells, allowing for the preservation of normal brain cells with low radiation exposure4. Thus, BNCT is a selective radiotherapy targeting tumor cells with high boron concentrations.BNCT (Boron Neutron Capture Therapy) is a high-linear energy transfer (LET) particle radiation therapy that allows for the selective targeting of tumor cells. In the actual treatment, a drug containing boron-10 , an analog of phenylalanine; BPA has already been approved for insurance coverage in Japan as the anti-neoplastic agent for BNCT, specifically for refractory head and neck cancer. BPA selectively accumulates in malignant tumor tissues via the amino acid transport system5. We have conducted clinical trials on BPA-mediated BNCT against malignant brain tumors, including gliomas, since 20026, and we are currently performing a multicenter phase II clinical trial for accelerator-based BNCT to treat patients with recurrent GBM in hospitals7. Unfortunately, even with BNCT, the recurrence of malignant gliomas cannot be prevented. Therefore, in BNCT using BPA, increasing BPA concentration in GSCs might help target these intractable tumor-propagating cells.The leading 8. As a result, excessive accumulation of protoporphyrin IX (PpIX), a photosensitive substance, occurs only in tumors. The fluorescence of this exogenous ALA-derived PpIX has been utilized for the PDD of tumors, including malignant gliomas9.5-Aminolevulinic acid (ALA) is a natural amino acid synthesized from glycine and succinyl-CoA in mammalian cells. Subsequently, via multiple enzymatic reactions, ALA is converted to porphyrin. Exogenous ALA, a drug approved by the FDA for photodynamic diagnosis (PDD), accumulates selectively in neoplastic cellsIn our preliminary study, we observed that ALA significantly increases the intracellular concentration of phenylalanine in human and mouse GSCs   (Table p = 0.02)  pretreatment significantly increased the BPA uptake in GSCs  Fig. a–c. We sors Fig. . In the 10B concentration  formed neurosphere-like spheroids  compared to corresponding GSCs (HGG13 and HGG30); however, the difference between GSCs and non-GSCs was more apparent in HGG13 cell line Fig. a–c. In cSLC3A2, LAT1 (SLC7A5), and ATB0,+ that are reportedly involved in BPA import in human GSCs (HGG13 and HGG30)  pretreatment. In HGG30, ATB0,+ expression was upregulated, whereas SLC7A5 expression was downregulated following ALA (300 μM) pretreatment.We investigated the effect of ALA treatment on the expression of neutral amino acid transporters Nrf2 and Nrf2-targeted ROS scavenger genes in GSCs . However, the blood 10B concentration ratio was lower in the ALA-pretreated group compared to the control group . Therefore, the T/Bl ratio was significantly higher in the ALA-pretreated group compared to the control group .Table SLC3A2, SLC7A5, and ATB0,+ in tumors obtained from HGG13 glioma-implanted mice. Results showed that ATB0,+ expression was significantly increased in tumors isolated from animals that received ALA before BPA administration . Comparing groups 5 and 6, the survival benefit of ALA pretreatment combined with BNCT was statistically significant, but only at an approximately 8.5% increase in life expectancy .We investigated whether ALA preloading could enhance the therapeutic efficacy of BNCT using the HGG13-implanted mice model. In this experiment, one animal each in groups 4 (neutron irradiation with ALA) and 6 (BNCT with ALA), died while receiving neutron irradiation. The survival data following BNCT are summarized in Table In vivo physical dose and equivalent dose in the tumors and the peripheral normal brain parenchyma estimated by the tissue boron concentrations are shown in Table 10B) to capture thermal or epithermal neutrons. Its nuclear capture reaction yields high-LET particles10. Theoretically, these high-LET particles can kill both proliferating and quiescent cells, even in hypoxic microenvironments. Therefore, when an adequate amount of 10B is delivered to GSCs, BNCT should be able to eliminate these cells. 18F-BPA-PET showed that, on average, approximately 30% of BPA is taken up into the normal brain compared to the target malignant glioma, suggesting that high-dose neutron irradiation causes considerable damage to the normal brain6. Hence, developing novel methods to increase BPA uptake by tumor cells is necessary further to enhance the therapeutic efficacy of BNCT against malignant gliomas while maintaining its safety.GSCs are believed to be responsible for tumor relapse following standard primary therapy and poor prognosis of malignant gliomas. BNCT is a high-dose tumor-selective radiotherapy that utilizes a non-radioactive isotope of boron (. reported that culturing GSCs (SU2 cells) and non-GSCs or DGCs (SHG-44 cells) in a medium containing 5 mM BPA leads to an increase in the intracellular BPA concentration only in non-GSCs, and accordingly, GSCs showed significantly higher resistance to BNCT than non-GSCs in the clonogenic assay12. In the present work, ALA (300 or 900 μΜ) preloading significantly increased the BPA uptake in the three GSC lines under study  and 2 (PEPT2)14. ALA is widely used in the fluorescence-guided diagnosis of various cancers, including malignant gliomas. The tumor-selective accumulation of ALA-derived PpIX, a fluorescent metabolite, well explains the tumor-directed nature of ALA. While PpIX fluorescence is rarely observed in tumor-adjacent normal tissues8.We showed that preincubation with ALA increases the intracellular concentration of BPA in all GSC lines examined . In contrast, preincubation with ALA did not promote BPA accumulation in non-GSCs 0,+ (SLC6A14) are involved in the transport of BPA into neoplastic cells. Wongthai et al. reported that among these amino acid transporters, ATB0,+ showed the highest rate of BPA transport in neoplastic cells16. Among SLC3A2, SLC7A5, and ATB0,+, the expression of ATB0,+ was significantly upregulated upon incubation with ALA in human GSCs (HGG13 and HGG30)  to the culture medium resulted in a significant increase in the cellular uptake of BPA Fig. a–c. Prev,+ SLC6A1 are invoATB0,+ expression might be one of the reasons for increased BPA uptake in GSCs in vitro  administration. Their results showed that the tumor PpIX concentration was approximately 10, 22, and 72 times higher than that in the peritumoral brain, normal gray matter and white matter, respectivelyHMOX1 (HO-1), SOD2, GPX3, NQO1, SLC1A2 (GLT1), SLC1A3 (GLAST), SLC1A5 (ATB0), SLC6A5 (GLYT2), and SLC7A11 (XCT), among others23. SLC6A14  is also a known target gene of Nrf224. We observed that the expression of ATB0,+ and HO-1 was significantly higher in ALA-exposed HGG13 and HGG30 cells -related factor (Nrf2) acts as a master switch for transcriptional upregulation of various genes coding antioxidant enzymes and transporters essential for the cellular defense against oxidative stress. These genes include 26. Under normal homeostatic conditions, Nrf2 is sequestered in the cytoplasm by the E3 ubiquitin ligase Kelch ECH-associating protein 1 (Keap1) and rapidly degraded in a ubiquitin–proteasome-dependent manner28. ROS are known to oxidize two reactive cysteine residues of Keap1, inhibiting Keap1-mediated ubiquitination of Nrf2, which finally results in cytoplasmic accumulation and nuclear translocation of Nrf228. Sugiyama et al. reported that ALA increases oxidative phosphorylation in human lung carcinoma cell line A549, increasing oxygen consumption and ROS production29. Furthermore, it has been reported that excess ALA can produce singlet oxygen through ALA dimerization, which leads to an increase in intracellular ROS stress30 and activation of Nrf231. Thus, ALA inactivates Keap1 by increasing ROS levels, allowing the Nrf2 protein to accumulate in the nucleus. Within the nucleus, Bach1 is known to act as a heme-responsive transcription factor and forms a heterodimer with small Maf proteins that represses Nrf2 transcriptional activity33. It has been reported that heme can bind to Bach1, inhibiting its DNA-binding activity and promoting its nuclear export. Consequently, Nrf2 forms a heterodimer with the small Maf nuclear protein (MafK) for effective binding to the ARE consensus sequence in the promoter region of Nrf2-target genes.Nrf2 is a basic leucine zipper (bZIP) transcription factor that targets the antioxidant response element (ARE) with a consensus sequence of 5′-A/GTGACNNNGC33.During porphyrin biosynthesis, the only rate-limiting step is ALA synthesis, catalyzed by ALA synthases (ALAS1 and ALAS2). Once the intracellular heme concentration reaches optimal levels, it exerts its regulatory effect on ALAS1 and ALAS2 via negative feedback inhibition. However, exogenously imported ALA bypasses this rate-limiting step and is promptly converted to PpIX and heme without any regulation. Therefore, we speculate that excess accumulation of heme produced from exogenously supplied ALA might inactivate the Bach1 protein and promote Nrf2 binding to ARE  of growth factors (EGF and bFGF) instead of FBS to preserve their stemness. However, these cytokines can activate porphyrin metabolism in GSCs, and consequently, GSCs might accumulate large amounts of PpIX, leading to a change in the native characteristics of these cells. Hence, there is no credible evidence that the in vitro response of GSCs to exogenous ALA observed in our study accurately represents ALA's effect on GSCs in vivo. Further studies are required to clarify whether exogenous ALA can increase the intracellular boron concentration in cancer stem cells within gliomas using in vivo glioma models.10B biodistribution study on day 14 after implantation, whereas in the BNCT experiment on day 7 after implantation. The brain tumor model used in this experiment dies from the tumor in a little over two weeks. Since removing the tumor to evaluate 10B biodistribution was necessary, we chose 14 days after transplantation, when the tumor should have grown to a size that could be identified by gross eye.We administered BPA in the On the other hand, since 14 days after transplantation is the time when some animals are already on the verge of brain herniation, we decided that it would be better to conduct the BNCT experiment at 7 days after transplantation, when the tumor is smaller, in order to examine the difference in therapeutic effect among the groups. Thus, the evaluation of biodistribution after implantation and the timing of the irradiation experiment were different, so the results may not reflect the actual biodistribution at the neutron irradiation.34. Further experiments will be devoted to fractionating GBM cells by trait to determine the amount of boron uptake.Finally, we performed in vivo experiments using only one GBM cell line. As GBM is highly heterogeneous, further studies using other cell lines are required to confirm whether ALA can enhance the therapeutic benefits of BNCTIn conclusion, the failure of current therapies to eliminate GSCs is considered to be one of the leading causes of the inevitable recurrence observed in patients with malignant gliomas. BNCT is a tumor-selective, high-LET radiotherapy. Therefore, it should be capable of eliminating GSCs if adequate amounts of BPA can be delivered into this low-dividing, tumor-propagating stem cells. Our data indicate that ALA could act as a drug delivery enhancer for BPA through the upregulation of the amino acid transport system in GSCs and might thus contribute to improving the therapeutic efficacy of BPA-based BNCT. Since, similarly to BPA, ALA can also selectively accumulate in neoplastic cells; it can be expected to exert a tumor-selective sensitizing effect when combined with BNCT.35. Thus, BPA used in the present study was used as a fructose complex.BPA (L-isomer) was kindly supplied by Stella Chemifa Corporation  and was converted to fructose complex before incubation40. In the present study, these human GSC lines were used after obtaining approval from the Research Ethics Committee of Osaka Medical College .In this study, we used two human GSC lines HGG13, HGG30) and a mouse GSC line (TS). /F12  supplemented with 20 ng/mL epidermal growth factor , 20 ng/mL basic fibroblast growth factor , B27 supplement without vitamin A (Thermo Fisher Scientific), 200 ng/mL heparan sulfate, GlutaMAX (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 ng/mL streptomycin (Thermo Fisher Scientific) at 37 °C in a 5% CO2 atmosphere41.These human GSCs were classified as mesenchymal-type GSCs based on their gene expression profiles42. TS cells were cultured in DMEM/F12  supplemented with 20 ng/mL EGF (PeproTech), 20 ng/mL bFGF (PeproTech), B27 supplement without vitamin A (Thermo Fisher Scientific), 200 ng/mL heparan sulfate, 100 U/mL penicillin, and 100 ng/mL streptomycin (Thermo Fisher Scientific) at 37 °C in a 5% CO2 atmosphere42.TS cells are murine GSCs established by overexpressing H-RAS V12 in normal neural stem/progenitor cells isolated from the subventricular zone of adult B6 mice harboring a homozygous deletion of the INK4a/Arf locusSLC3A2, LAT1 (SLC7A5), ATB0,+ (SLC6A14), Nrf2, HO-1 (HMOX1), and SOD2 were measured. Primer details are provided in Supplementary Table Total RNA was extracted from cells using the RNA extraction kit  and subjected to cDNA synthesis using the Transcriptor First-Strand cDNA Synthesis Kit . The reaction was run in the LightCycler  according to the TaqMan assay protocol. The reaction consisted of a denaturation step (95 °C for 15 min), and the amplification and quantification process was repeated 45 times (95 °C for 15 s and 60 °C for 40 s). Data were analyzed using the LightCycler 3 software. The mRNA levels of 10B was measured using inductively coupled plasma atomic emission spectroscopy . All the above experiments were performed under dark conditions to avoid the photodynamic effect on ALA-exposed tumor cells.HGG13, HGG30, and TS cells were incubated with 5-aminolevulinic acid (ALA) -containing medium  for 24 h. After incubation, the cells were centrifuged and washed with phosphate-buffered saline (PBS). Cells were then exposed to 1 mM BPA-containing medium for 6 h, following which the cells were centrifuged and washed twice with PBS. The cells were then digested for 24 h with 1 N nitric acid solution . The intracellular concentration of All of the animal experiments in this study were approved by the Ethical Committee on the Care and Use of Laboratory Animals of Osaka Medical College  and Kyoto University Research Reactor Institute  , respectively. Animal housing and the following animal methods and procedures were performed in accordance with the regulations and guidelines of our institutions (OMC and KURNS), both of which comply with the ARRIVE Guidelines 2.0. (PLoS Bio 8(6), e1000412,2010).Seven-week-old male BALB/c nude mice [19–23 g body weight (b.w.)] were used for the HGG13 cell transplantation . All animals were raised in Specific pathogen-free (SPF)-level animal rooms and fed a standard diet. Mice were anesthetized with an intraperitoneal injection of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) and placed in a stereotactic frame . A midline scalp incision was made, following which a small burr hole was drilled 2 mm right lateral to the bregma using an electric drill. A 25-μL Hamilton microsyringe with a 22-gauge needle  was inserted at 4 mm depth from the skull and withdrawn from 1 mm to the target in the brain (3 mm from the skull surface). Since HGG13 cells form spheroid, the cells were dispersed by pipetting and confirmed to be single-celled using a microscope. Ten thousand HGG13 cells suspended in 2 μL of Serum-free DMEM were injected at a rate of 1 μL/min using an automatic infusion pump. The needle was placed for 2 min after infusion and withdrawn slowly. The burr hole of the skull was sealed with bone wax, and the scalp was sutured with a sterilized clip.10B/kg b.w.) was administered intraperitoneally. After 2.5 h of BPA administration, the mice were euthanized, and the tumor and blood were collected to measure the tissue 10B concentration using ICP-AES as we described previously43.BPA biodistribution was evaluated 14 days after HGG13 cell implantation into the mouse brain. ALA or PBS (80 mg/kg b.w.) was orally administered to mice 24 h before BPA administration. BPA  seven days after tumor cell implantation. Forty-four glioma-bearing mice were randomly divided into six groups: group 1, untreated control (n = 5); group 2, ALA-only control (n = 5); group 3, neutron irradiation-only control (n = 7); group 4, neutron irradiation following ALA (n = 6); group 5, neutron irradiation following BPA (n = 10); and group 6, neutron irradiation following the combination of ALA and BPA (n = 9).10B/kg b.w.) was administered intraperitoneally to groups 5 and 6, and PBS was administered to animals in other groups. Mice in groups 3 to 6 were irradiated at a reactor power of 1 MW at the Heavy Water Neutron Irradiation Facility (HWNIF) for 50 min, 2.5 h after BPA administration. Sham irradiation was performed on mice in groups 1 (neither BPA nor ALA) and 2 . After implantation surgery, the mice’s body weight and neurological function were monitored daily. One day before death became imminent , the mice were euthanized in accordance with the ARRIVE guidelines 2.0.ALA was administered orally at 80 mg/kg b.w. to animals in groups 2, 4, and 6, 24 h before BPA administration. BPA  7Li and 14N  14C capture reactions, the 1H  1H scattering reaction, and gamma rays, which can be described by the following equation:DB (boron dose) = 7.43 × 10−14 (Gy cm2/μg 10B/g) × Boron concentration (μg 10B/g) × thermal neutron fluence (1/cm2), and DN (nitrogen dose) = 6.78 × 10−14 (Gy cm2/wt.%) × nitrogen concentration (weight%) × thermal neutron fluence (1/cm2). The above numerical values were taken from a previous study46. The thermal neutron fluence was measured by the radioactivity of a gold foil  that was set at the surface of the heads of the irradiated mice. DH is the physical dose due to the elastic scattering between epithermal or fast neutrons and the hydrogen nucleus47. Dγ is the measured dose of gamma rays mixed in the neutron beam48. To evaluate the therapeutic effect of BNCT, each radiation dose value was multiplied by the relative biological effectiveness (RBE) for each tissue to obtain the equivalent dose, as follows:Estimation of the physical dose and equivalent dose delivered to the mice was done as described previously49. RBE for nitrogen (RBEN) and hydrogen (RBEH) is 3.050.In BNCT, the relative biological effectiveness (RBE) is expressed as the compound biological effectiveness (CBE), which corresponds to the biological properties of the boron compounds. Its value for BPA is 3.8 for glioma and 0.9 for the brainSLC3A2, SLC7A5, and ATB0,+ in tumors derived from the HGG13 implanted glioma mice model. Fourteen days after HGG13 cell implantation, mice were administered ALA (80 mg/kg b.w.) or PBS (control). Twenty-four hours later, the mice were euthanized, and tumors were excised from the brains to extract RNA. Gene expression analysis was performed using RT-PCR.We examined the effect of ALA on the expression of p-value less than 0.05 was considered statistically significant. Survival of the GSC-implanted mice was calculated for each group using the Kaplan–Meier method, and the log-rank test was used to determine the differences between survival curves. A Cox proportional hazards regression model was used to analyze the survival data, with Bonferroni’s correction for multiple comparisons.In vitro data were generated from at least three independent experiments. All statistical analyses were performed using JMP Pro ver. 13 for Mac . Data are presented as mean ± SE. Comparison between two groups was performed using the Student’s t-test. A Supplementary Information.


Anopheles stephensi is a malaria-transmitting mosquito that has recently expanded from its primary range in Asia and the Middle East, to locations in Africa. This species is a competent vector of both Plasmodium falciparum and Plasmodium vivax malaria. Perhaps most alarming, the characteristics of An. stephensi, such as container breeding and anthropophily, make it particularly adept at exploiting built environments in areas with no prior history of malaria risk.An. stephensi were created, under current and future climate. Temperature-dependent transmission suitability thresholds derived from recently published species-specific thermal curves were used to threshold gridded, monthly mean temperatures under current and future climatic conditions. These temperature driven transmission models were coupled with gridded population data for 2020 and 2050, under climate-matched scenarios for future outcomes, to compare with baseline predictions for 2020 populations.In this paper, global maps of thermal transmission suitability and people at risk (PAR) for malaria transmission by An. stephensi presence. General patterns of poleward expansion for thermal suitability were seen for both P. falciparum and P. vivax transmission potential.Using the Global Burden of Disease regions approach revealed that heterogenous regional increases and decreases in risk did not mask the overall pattern of massive increases of PAR for malaria transmission suitability with An. stephensi transmission in a changing climate provides a key tool for planning, given an ongoing invasion and expansion of the vector. Anticipating the potential impact of onward expansion to transmission suitable areas, and the size of population at risk under future climate scenarios, and where they occur, can serve as a large-scale call for attention, planning, and monitoring.Understanding the potential suitability for The online version contains supplementary material available at 10.1186/s12936-023-04531-4. Anopheles gambiae species complex [Anopheles stephensi into the Horn of Africa, identifying this new vector species as a major potential threat to malaria control in the region [Malaria remains a critical global health challenge, with 241 million cases reported by the World Health Organization (WHO) in 2020 alone . Malariae region –8.An. stephensi represents a new critical threat, not only to communities in Africa, but also to global public health. A competent vector of both Plasmodium falciparum and Plasmodium vivax malaria, An. stephensi has been implicated in malaria transmission throughout much of its native range in Asia and the Middle East, including India, Iran, and Pakistan [An. stephensi is capable of exploiting containers of standing water for ovipositional habitat, similar to container-breeding mosquitoes in the genus Aedes, including Aedes aegypti and Aedes albopictus [An. stephensi into built environments, fueling urban outbreaks of malaria and facilitating invasions into new geographic areas. In the past decade, An. stephensi has successfully expanded its range into the African continent, with established populations in Djibouti, Ethiopia, and Sudan [An. stephensi, such as acquired insecticide resistance [An. gambiae [An. stephensi has been identified as a major risk to malaria elimination targets, with global public health organizations calling for increased entomological surveillance and vector control in areas at imminent risk of invasion [The expansion of Pakistan –13. In cbopictus . This nond Sudan . Alarminnd Sudan . This shnd Sudan , 18. Othsistance  and grea gambiae , raise f gambiae . Perhapsinvasion .An. stephensi invasions, and subsequent malaria transmission. A great deal of research has been conducted to delineate the extent of temperature-dependent malaria suitability in Africa [Anopheles spp. input parameters, primarily for P. falciparum malaria transmitted by An. gambiae [An. gambiae and An. stephensi, and the two main malarial parasites they transmit , were separately modelled to produce thermal suitability curves for transmission [An. gambiae is functionally limited by arid conditions, An. stephensi is a container breeder that is resilient to habitat extremes [An. stephensi is able to thrive in close association with people, and thus potentially able to establish itself everywhere that temperature is not limiting. Thus, understanding the potential areas for suitability for transmission by this invasive mosquito now, and in the future, is important for capacity building and planning control efforts.Mapping geographic estimates of transmission suitability can provide essential tools for assessing the current and future risk of n Africa , 23 and n Africa –29. In p gambiae , 31. In smission , 30. Whiextremes . TherefoAn. stephensi was mapped using modelled thermal limits under current and future climate scenarios. Unlike previous studies to map the distribution of malaria, the projected distributions are not limited to non-arid regions, given the life history of An. stephensi, and instead make similar assumptions to those for mapping Aedes spp. transmitted diseases. Additionally, An. stephensi thermal suitability maps were combined with projected human population density estimates, enabling us to assess not only the areas that are vulnerable to malaria transmission through An. stephensi expansions, but also the magnitude of threat in terms of people at risk (PAR).In this study, the global suitability of malaria transmission by P. falciparum and P. vivax by An. stephensi. Briefly, thermal response data for vector and parasite pairings were synthesized from published data. These data were used to parameterize a formulation for P. falciparum malaria transmission initially described in Mordecai et al. [EFD is mosquito fecundity expressed as the number of eggs per female per day; In a previous study , used mi et al. . The temA Bayesian approach was used in Villena et al. 2022 to fit unimodal thermal response curves of traits for each mosquito or parasite species [An. stephensi was greater than zero (S(T) > 0), with a posterior probability greater than 0.975 [An. stephensi are temperatures of 16.0–36.5 °C for P. falciparum and 16.6–31.7 °C for P. vivax [In this study, the thermal boundaries from Villena et al.  were usean 0.975 . The resP. vivax .An. stephensi transmitted P. falciparum and P. vivax suitability are described. Outputs are presented for a baseline climate scenario, and future potential climate driven outputs for four General Circulation Models (GCMs), following the methodology used in Ryan et al. [Aedes spp. transmitted diseases.In this paper, baseline and future scenarios for n et al. , 34 to dhttp://ccafs-climate.org/data_spatial_downscaling/), part of the Consultative Group for International Agricultural Research (CGIAR). The baseline climate model from which these are projected is the WorldClim v1.4 baseline [Baseline and future scenario climate model output data were acquired from the research programme on Climate Change, Agriculture, and Food Security (CCAFS) web portal  was used: the Hadley Centre Global Environment Model version 2, Earth-System configuration (HADGEM2-ES) under two scenarios for greenhouse gas emissions, or representative concentration pathways (RCPs): RCP 4.5 and RCP 8.5. The Intergovernmental Panel on Climate Change (IPCC) describes RCP 4.5 as a moderate scenario in which emissions peak around 2040 and then decline, while RCP 8.5 is the highest baseline emissions scenario in which emissions continue to rise throughout the twenty-first century. Mechanistic transmission models were projected onto climate data in R (v. 4.1.2) with the package ‘raster’ . MonthlyIn order to establish a population baseline, the 2020 Gridded Population of the World  was used . The decMonthly suitability maps were produced for baseline and future climate scenarios , using one GCM (HADGEM-ES) at a near-future time horizon , for illustration Fig. . FollowiP. falciparum and P. vivax by An. stephensi, under the two future RCP x SSP scenarios, averaged across the 4 GCMs  regions  were useAn. stephensi malaria transmission suitability for P. falciparum and P. vivax are shown in Fig. P. falciparum and P. vivax extends throughout the global tropics, throughout the known existing range of the Middle East, extending throughout Asia, Central America, South America, and marginally in North America. The predicted potential range for year-round transmission suitability of P. falciparum extends further North and South than P. vivax, with notable extension of the transmission season in northern Africa, the Middle East, India, and central Australia. The narrower thermal suitability bounds for P. vivax constrains the baseline potential extent, compared to that for P. falciparum. Seasonal transmission suitability at baseline climate conditions is projected to be globally widespread for both P. falciparum and P. vivax, extending well into temperate regions in North America, Europe, and Asia.Maps of months of P. falciparum is seen in both RCPs. Notably, the potential for any transmission  is predicted to expand at northern latitudes, where areas with no current malaria suitability will have the potential for transmission, at least for one month every year. This includes portions of Alaska in the US, northern Canada, Scandinavia, and Russia. The length of the P. falciparum transmission season is expected to increase in temperate regions of North America and Europe, southern Africa, and in central Australia. Yet, changing climate conditions will shorten the length of the P. falciparum transmission season in some areas, most notably in northern Africa, the Middle East, and northern India. The transmission suitability for P. vivax is also predicted to extend further North in the future, encroaching on areas that do not currently experience malaria transmission. The length of the P. vivax transmission season is expected to increase in southern Africa and in parts of North America, including Mexico and along the Gulf Coast in the US. There are also marked decreases in the length of the P. vivax transmission season, most notably throughout northern Africa, the Middle East, India, Asia, northern Australia, and South America. Raster output for additional scenarios of future climate change from this study is available on the Harvard Dataverse.Mapped transmission suitability in 2050, for RCP 4.5 and RCP 8.5 scenarios is shown in Fig. P. falciparum and P. vivax by AS for the two future scenarios of RCP 4.5 and RCP 8.5, in combination with matched population projections SSP2 and SSP5, respectively, revealed that in many regions of the world, increases in people at risk of transmission suitability will occur  transmission PAR increases, East and West Sub-Saharan Africa regions are the top two affected, under both the RCP 4.5 and RCP 8.5 scenario  and the pathogen , and how that will impact the vector microbiome [P. vivax [An. stephensi populations to reduce PAR in the future, particularly in countries neighboring current areas of expansion.This exploration of potential future climate impacts on a vector currently expanding its range is based on current vector-pathogen biology and thermal limits to the life-history of crobiome , potentiAnopheles quadrimaculatus in parts of N. America).A knock-on effect of the potential expansion of a novel, urban-adapted, malaria vector into, for example, the Americas, is that adding a competent malaria vector to areas with existing competent malaria vectors expands the competent vector community. This compounds the risk in a changing world for facilitating spillover from a novel invader experiencing perhaps only a shortened suitability season to established Anopheline species  for year-round transmission suitability of P. falciparum by An. stephensi in 2050, under RCP 4.5 (SSP2 population projection) and RCP 8.5 (SSP5 population projection) future climate scenarios, averaged across four general circulation models (GCMs) as described in the main methods. Global increase is the sum of all gains in PAR increases across all GBD regions. Table S2.Top 10 Global Burden of Disease defined regional increase in people at risk (PAR) for year-round transmission suitability of P. vivax by An. stephensi in 2050, under RCP 4.5 (SSP2 population projection) and RCP 8.5 (SSP5 population projection) future climate scenarios, averaged across four general circulation models (GCMs) as described in the main methods. Global increase is the sum of all gains in PAR increases across all GBD regions. Table S3.Top 10 Global Burden of Disease defined regional increase in people at risk (PAR) for one or months transmission suitability of P. falciparum by An. stephensi in 2050, under RCP 4.5 (SSP2 population projection) and RCP 8.5 (SSP5 population projection) future climate scenarios, averaged across four general circulation models (GCMs) as described in the main methods. Global increase is the sum of all gains in PAR increases across all GBD regions. Table S4.Top 10 Global Burden of Disease defined regional increase in people at risk (PAR) for one or months transmission suitability of P. vivax by An. stephensi in 2050, under RCP 4.5 (SSP2 population projection) and RCP 8.5 (SSP5 population projection) future climate scenarios, averaged across four general circulation models (GCMs) as described in the main methods. Global increase is the sum of all gains in PAR increases across all GBD regions.


Plasmodium falciparum is not only the most widespread but also the most deadly species. The share of Plasmodium infections caused by the other species  is clearly underestimated. The objective of the study was to determine the molecular epidemiology of plasmodial infection due to P. malariae and P. ovale in Côte d'Ivoire.Malaria is a major public health problem, particularly in the tropical regions of America, Africa and Asia. The study was cross-sectional. The study participants were recruited from Abengourou, San Pedro and Grand-Bassam. Sample collection took place from May 2015 to April 2016. Questionnaires were administered and filter paper blood samples were collected for parasite DNA extraction. The molecular analysis was carried out from February to March 2021. A nested PCR was used for species diagnosis. The data was presented in frequencies and proportions.Plasmodium positive rate was 72.5% (261/360). The specific index was 77.4% and 1.5% for P. falciparum and P. malariae in mono-infection, respectively. There was also 15% P. falciparum and P. malariae co-infection, 3.4% P. falciparum and P. ovale co-infection and 2.3% P. falciparum, P. malariae and P. ovale triple-infection. Typing of P. ovale subspecies showed a significant predominance of P. ovale curtisi (81.2% of cases).A total of 360 patients were recruited, including 179 men  for 181 women . The overall Plasmodium falciparum remains the most prevalent malaria species in Côte d'Ivoire, but P. malariae and P. ovale are also endemic mostly in co-infection. Malaria elimination requires a better understanding of the specific epidemiological characteristics of P. malariae and P. ovale with a particular emphasis on the identification of asymptomatic carriers. Plasmodium infecting humans, Plasmodium falciparum is the deadliest species as well as the most prevalent in Africa, whereas the incidence of Plasmodium vivax infection is higher outside Africa [Plasmodium ovale and Plasmodium malariae have long been widespread in tropical Africa and other major malaria-endemic areas worldwide [Malaria is a major public health concern, affecting the tropical regions of America, Africa, Eastern Mediterranean, South East Asia, and Western Pacific. Approximately 241 million cases and 619,000 deaths were reported in 2021 [orldwide .P. malariae and 4–10% for P. ovale. In these studies, thin blood smears were carefully examined by trained microscopists because parasitaemia is usually low [Anopheles breeding sites, most P. ovale and P. malariae infections are associated with P. falciparum [P. falciparum (95–99%), followed by P. malariae (3–4.2%) and P. ovale (0.3–0.7%) [Previous studies have reported that in the humid forest and savanna areas of West and Central Africa, high prevalence of both these species is common in children, often reaching 15–40% for lciparum , 7. In p.3–0.7%) , 9.P. malariae and P. ovale appear to be underestimated because of the low parasitaemia during the infections they induce and the presence of mixed infections with the main parasite species [Plasmodium pauci-infections [P. ovale into two distinct subspecies: P. ovale curtisi, the classical type, and P. ovale wallikeri, the alternative type [P. ovale may be responsible for distant relapses from dormant (hypnozoite) forms in the liver [These figures reported for  species , 10. Thefections . In addiive type . The perive type , 14. In he liver  and may he liver .P. malariae and P. ovale in Côte d’Ivoire.The objective of the study was to determine the molecular epidemiology of plasmodial infections caused by P. falciparum chemoresistance in the Côte d’Ivoire), and Grand Bassam .This cross-sectional study was performed in three localities, namely Abengourou, San Pedro  from samples of schoolchildren with or without malaria symptoms were randomly selected.. Each schoolchild was included after written informed consent was obtained from their parent or legal guardian. The children included in the study met the following criteria: (i) regularly enrolled in one of the randomly selected schools; (ii) from 4 to 16 years old, regardless of gender; (iii) living in the study area for at least 2 months; (iv) any patients were included  according to the manufacturer's instructions. Briefly, DNA was attached to a Zymo-Spin IIC-XL centrifugation column filter. The cells were then washed thrice; first wash with DNA Pre-Wash Buffer and then two washes with g-DNA Wash Buffer. Finally, pure DNA was eluted from the centrifugation column filter using DNA Elution Buffer.DNA was extracted using the Quick-DNAPlasmodium species [Plasmodium genus (rPLU5/rPLU6). For species typing, the primary PCR products of the positive samples were further subjected to four secondary amplifications with primer pairs specific for the four species that are likely to infect humans; rFAL1/2, rVIV1/2, rMAL1/2, and rOVA1/2 for P. falciparum, P. vivax, P. malariae, and P. ovale, respectively. However, this last primer pair has a limitation of preferentially binding or amplifying the P. ovale curtisi [The nested PCR technique has been used for molecular diagnosis of  species , 18. TheP. ovale isolates were typed to determine the subspecies of P. ovale using the nested PCR method described by Fuehrer [P. ovale curtisi and rOVA1v/rOVA2v specific for P. ovale wallikeri.After detection,  Fuehrer , with thThe collected data were coded and entered using Epi Data 3.1, Excel 2010, and Word 2010. Statistical analyses were performed using Statistical Package for Social Sciences version 21 . Descriptive statistics were used to assess the distribution of sociodemographic data and independent variables as well as the frequencies and proportions of malaria-positive samples.The study protocol was approved by the National Research Ethics Committee under Number 020/MSLS/CNER-dkn on 05 May 2015. The study was conducted in accordance with the text of the Declaration of Helsinki adopted by the 18th World Medical Assembly in 1964 and its amendments, the International Conference on Harmonisation recommendations. The study complied with Good Clinical Practice guidelines and all applicable regulatory requirements for clinical studies.DBS from 360 patients were used for molecular analysis. The distribution of samples collected was 89 (25%) in Grand Bassam, 129 (36%) in Abengourou and 142 (39%) in San Pedro.Among the 360 participants, 261 were diagnosed with malaria infection (72.5%). Out of the 261 malaria-infected cases, 59 were non-falciparum malaria infections. The demographic characteristics of the participants with non-falciparum malaria infection showed that 54.2% (27/59) of the participants with non-falciparum malaria were females. The study population was predominantly female, with a sex ratio of 0.84 (27/32). The mean age of the patients was 8.97 years (standard deviation = 2.75), with extremes of 5 and 16 years. The age group of 6–10 years was the most represented, with 64.4% (38/59) of the cases. Most cases were found in the towns of San Pedro and Abengourou with an identical percentage of 44.1% (26/59) for each. Majority of the samples 66.1% (39/59) were collected during the rainy season  schoolchildren selected were febrile; the majority of those selected had a normal temporal temperature. The mean temperature was 36.84 °C (standard deviation = 0.61) with extremes of 35.90 and 39.80 °C. More than half of the patients selected for this study were asymptomatic 64.4% (38/59). Clinical signs, when present, were diverse and varied and represented mainly by headache 20.3%) and abdominal pain 13.6% (8/59)  and non-falciparum infection constitutes 22.6% (59/261) of all malaria infections. 72.9% (43/59), 15.3% (9/59) and 11.9% (7/59) of the non-falciparum were P. malariae, P. ovale & P. malariae / P. ovale mixed infections, respectively. Of the P. malariae infections, 90.7% (39/43) were coinfected with P. falciparum; 100% (9/9) of the P. ovale infections were coinfected with P. falciparum 85.7% (6/7) of P. malariae & P.ovale mixed infection were triple infection with P. falciparum . 60. PlasmPlasmodium indices were 77.6% and 72.3% in the rainy and dry seasons, respectively. Depending on locality, the Plasmodium index was 59.6% (53/89) in Grand-Bassam, 79.8% (103/129) in Abengourou, and 81% (115/142) in San Pedro. In most cases, co-infection with P. falciparum was observed, and these cases mainly involved asymptomatic malaria regardless of the locality, while only 6.8% (4/59) of the P. malariae cases were monoinfected.The Plasmodium malariae was the most frequently encountered species in 84.7% (49/59) of the cases of co-infection with P. falciparum and P. ovale, Plasmodium ovale accounted for 27.1% (16/59) of cases, including co-infections . Both subspecies were found mainly in asymptomatic and female participants without any statistically significant association.The typing of The sex ratio of the study was 0.84 (27/32), similar to that found in studies performed in the Ivory Coast, which were 0.98 and 0.89 , 21. TheP. malariae and P. ovale had higher proportions at the end of the rainy part of the study period [The study observed high malaria prevalence in the rainy season compared to the dry season. The observations have been reported in all malaria infections. In the rainy seasons, the malaria vector prevalence increases due to increases in the breeding sites. Thus, increasing malaria transmission in the rainy seasons . In a sty period .. [P. malariae and P. ovale had fever at the time of examination (> 37.5 °C) and a history of fever of 2–5 days.The fever observed in 11.9% of the participants (mean axillary temperature 36.4 °C) could be explained by the fact that this study population was largely asymptomatic in 64.4% (38/59) of cases. These schoolchildren were apparently healthy; however, when present, their symptoms were dominated by headache 20.3% (12/59), abdominal pain 13.6% (8/59) and pale conjunctiva 8.5% (5/59). Similarly, several other studies have also reported various clinical signs at inclusion but in different proportions than those in this study, including chills, headache, myalgia, anorexia, and asthenia . Accordi.  in ChinaP. falciparum mono-infection, a significantly higher prevalence of P. malariae 66.1%(39/59) was observed compared to P. ovale 15.2%(9/59). However, both P. malariae and P. ovale were sometimes involved in co-infection with P. falciparum. In addition, a study in the Democratic Republic of Congo reported that most P. malariae and P. ovale infections were associated with P. falciparum and that the prevalence of mono-infections of these species was only 1.0 and 0.6%, respectively [P. malariae and P. ovale [P. malariae and P. ovale [In this study, after removing the cases of ectively . Similarectively , 28. In P. ovale , 30. In P. ovale , 28, whiP. ovale subspecies showed a predominance of P. o. curtisi compared to P. o. wallikeri (18.8%). This is the first study with epidemiological data of these two P. ovale subspecies in Côte d’Ivoire. Similar trends have been observed in Africa, China, and in places where clinical isolates have been reported to be more closely related to P. o. curtisi than P. o. wallikeri [P. o. wallikeri [P. o. curtisi. Moreover, P. o. curtisi was mainly implicated in malaria cases reported in travellers returning to France [P. o. curtisi and P. o. wallikeri have been reported in Bangladesh and Gabon [P. o. curtisi in this study could be explained by the use of the primer pair used for P. ovale, namely rOVA1/rOVA2. Indeed, this primer pair has a limitation of preferentially binding or amplifying the P. ovale curtisi [P. o. wallikeri and P. o. curtisi without binding to other human malaria parasites, is an alternative [Furthermore, typing of allikeri , 31, 32.allikeri . Howevero France , Italy [o France , and Chio France . Mixed iallikeri .8%. Thisernative .Plasmodium falciparum remains the most widespread malaria species in Côte d’Ivoire, but P. malariae and P. ovale are endemic with a low rate. However, no study has reported the presence of the subspecies Plasmodium ovale. Microscopy does not often reliably distinguish between P. falciparum, P. malariae, and P. ovale in Côte d’Ivoire, where all three species are frequently found both in mono-infection and more often in co-infection. Misdiagnosis of P. malariae as P. ovale, and vice versa, is common, which leads to inappropriate treatment or no treatment at all, given that many individuals are asymptomatic carriers of these species. Thus, P. ovale and P. malariae may remain important causes of morbidity in these areas and constitute reservoirs of the parasite. In addition, P. ovale, due to the presence of hypnozoites, may be responsible for distant relapses and even severe disease. Because P. malariae and P. ovale infections usually have low parasitaemia and occur as mixed infections with P. falciparum and P. vivax, molecular detection methods provide a useful tool for the diagnosis of the disease. Molecular detection methods provide an accurate estimate of malarial epidemiology.