The Lihir Islands of Papua New Guinea host a mining operation that has resulted in a mine-impacted zone (MIZ) with reduced malaria transmission and a substantial influx of mine employees, informal cross-country traders, returning locals, and visitors. Prevalence of malaria parasites was assessed in travellers arriving on the Lihir Group of Islands to evaluate the risk of parasite importation.Plasmodium parasite prevalence, employing logistic regression models to identify factors associated with qPCR positivity.In 2018, a cross-sectional study at the airport and main wharf was conducted, targeting asymptomatic travellers who had been away from Lihir for at least 12 days. Microscopy, rapid diagnostic tests (RDTs), and quantitative PCR (qPCR) were used to determine 398 travellers arriving by plane and 402 arriving by boat were included. Both cohorts were significantly different. Mean age among travellers arriving by plane was 40.1 years (SD ± 10.1), 93% were male and 96% were employed at the mine. In contrast, among travellers arriving by boat, the mean age was 31.7 years (SD ± 14.0), 68% were male and 36% were employed at the mine. The prevalence of malaria infection among travellers arriving by plane was 1% by RDT and microscopy, and increased to 5% by qPCR. In contrast, those arriving by boat showed a prevalence of 8% by RDT and microscopy, and 17% by qPCR. Risk factors for infection were arriving by boat , arriving from nearby provinces with high malaria incidence , and having been away from Lihir for 91 days or more . Being mine worker staying at the mine accommodation was related with less infection risk ; while Lihirian residents returning from a trip, VFRs, or people with trading unrelated to mining had higher risks (p = 0.0066).Travellers arriving by boat faced increased risk of malaria infection than those arriving by plane. This subpopulation poses an import risk to the MIZ and the rest of Lihir Islands. Screening of high-risk groups at wharfs, and collaboration with nearby Islands, could sustain reduced transmission and facilitate malaria elimination strategies.The online version contains supplementary material available at 10.1186/s12936-023-04804-y. Plasmodium species are Plasmodium falciparum and Plasmodium vivax with an overall prevalence by microscopy of 2.1% and 0.5%, respectively [Plasmodium malariae and Plasmodium ovale are also present [In 2020, Papua New Guinea (PNG) reported over 750,000 confirmed malaria cases , account present .2 area), and the airport, all located in the north-east of Aniolam. Similar to other public–private partnerships in regions with high endemicity of vector-borne diseases, efforts are also directed towards implementing vector control strategies [Lihir Islands, located in New Ireland province, host a gold mining operation of Newcrest Mining Ltd on their largest island, Aniolam. Due to the high malaria transmission rates in the area and the additional risks posed by deforestation and open-pit mining , the comrategies . Since 2rategies . The resrategies .In 2010, a cross-sectional study was conducted in Aniolam to evaluate malaria prevalence and the impact of the implemented vector control programmes . While tP. vivax isolates from different regions revealed patterns of transmission following major human migration routes, especially within the ‘Islands region’ of PNG [Travellers are common sources of imported malaria. For instance, a prevalence survey in the low-burden island of Bioko, Equatorial Guinea, showed that malaria infection was highly related with having a history of travel to mainland Equatorial Guinea . In PNG,’ of PNG . These f’ of PNG . Even af’ of PNG . Cabo Ve’ of PNG .Plasmodium parasites in travellers arriving to the Lihir Islands by plane or boat, and to evaluate the importation risk they pose to the ongoing transmission within the MIZ and in the rest of Lihir. Parasite prevalence was determined using microscopy, RDT and quantitative PCR (qPCR), and factors associated with qPCR positivity were identified. Estimating the burden of malaria among travellers to Lihir can provide valuable insights for refining current control strategies and guiding the sustainability of future interventions aimed at malaria elimination on these islands.Herewith, a cross-sectional study was conducted to assess the prevalence of This study obtained ethical clearance from the national ethical committee in PNG, the Medical Research Advisory Committee (PNG-MRAC), with MRAC No.18.07. Written informed consent was collected from all individuals in the study. Children under the age of 18 were verbally consented and parents or legal guardians signed the consent form on their behalf. Those participants unable to read and/or write were verbally consented and an impartial witness countersigned the consent form.2), and the smaller outer islands of Malie, Masahet, and Mahur. They are characterised by a tropical rainforest climate with extremely high precipitation figures all year round, a limited public health infrastructure, and a near-inaccessible geography in some areas. Londolovit is the main town and is the centre for most of the local business and those related with the mining activities. A population census, conducted between 2018 and 2020, estimated 26,528 inhabitants living in the Lihir Islands. Mining employees migrating from other parts of PNG, contribute to more than one third of the population on Aniolam [Lihir Islands are located 900 km northeast of Port Moresby in the New Ireland Province of PNG. Lihir consists of a group of four islands: Aniolam .After signing the informed consent, a questionnaire was obtained from all participants, which included demographic and clinical data, information on LLIN usage, and mobility/travel information. Data collection was conducted using a paper questionnaire in the field, and subsequently, two data clerks independently entered the data into a database upon their return from the field. For each participant, a health practitioner performed a finger prick to collect blood drops for a malaria RDT , a blood slide for light microscopy examination, and 2 dry blood spots in filter paper for qPCR. All tests and materials were labelled with the participant identification number before sample collection. RDT was performed according to the manufacturer instructions. For the slide, two drops of blood were used for a thick and thin blood smear, using a clean spreader slide for the latter. Slides were air-dried in horizontal position and stored in slide boxes to transport to the local laboratory for staining and reading. Two blood spots of approximately 2.5 cm of diameter were fulfilled on filter paper, air-dried, and placed into separate zip-lock bags with silica gel. Once in the local laboratory, they were stored at -20 ºC until shipped to the IMR Vector-borne Diseases Unit in Madang (PNG) for further processing. Results of the RDT were immediately interpreted by a clinician who provided antimalarial treatment according to PNG treatment guidelines in case of a positive result.Upon return from the field, only thin smear was fixed with methanol and blood slides were stained with 10% Giemsa during 10 min at the local laboratory following the MM-SOP-07A from the World Health Organization (WHO). Blood slides were examined under × 1000 magnification by two independent level 1–2 microscopists who had completed WHO quality assurance courses. A sample was considered negative after examining one hundred fields of view. When a parasite was observed, counts of white cells and parasites were conducted until 300 white cells were counted. The parasite density in parasites per μL was then calculated, assuming a white cell count of 8000 white cells/μL. The results for this first reading were cross-checked with the Institute of Medical Research (IMR) Vector-borne Diseases Unit at Madang (PNG). Any discrepancies were addressed at IMR with the involvement of a third WHO-certified level 1 microscopist.®) and performed according to the manufacturer’s instructions to obtain genomic DNA from blood. Following DNA extraction, a generic qPCR ‘QMAL’ assay that amplifies a conserved region of the 18S rRNA gene was run on all samples [P. falciparum, P. vivax, P. malariae and P. ovale were performed as previously described [P. falciparum and Pv-mtCOX1 for P. vivax were conducted [DNA extraction was conducted at the IMR Vector borne Diseases Unit in Madang (PNG) using FavorPrepTM 96-well Genomic DNA kit .The minimum sample size was 385 individuals arriving by plane and 385 individuals arriving by boat, considering a 5% precision and a 95% bilateral normal confidence interval (CI), and assuming an unknown prevalence of Data were analysed using the statistical software STATA  and descA total of 800 travellers arriving at the main points of entry in Lihir were recruited: the airport and the main wharf, located in Londolovit Town. Smaller wharfs outside the MIZ observe considerably fewer arrivals are were not included for sampling. The map in Fig. The study population comprised 398 participants arriving by plane and 402 participants arriving by boat. Table Travellers arriving by plane were predominantly male (93%) with a mean age of 40.1 (SD ± 10.1) years, who were employed at the mine (96%) and intended to stay at the mine accommodation facilities (99%). Given the mine's standard 14-day work roster , the majority of these fly-in fly-out employees (87%) had spent less than a month away from Lihir.In contrast, travellers arriving by boat displayed greater diversity. While males still formed the majority, females (32%) and children (12%) were present, with a mean age of 31.7 (SD ± 14.0) years old. Their travel purposes also varied widely, with only 36% being mine workers. Many travellers arriving by boat were either Lihir residents returning home or engaging in VFRs (30%), trading or business outside the mine (23%) or visiting Lihir for other reasons (11%). Consequently, only 35% of this cohort planned to stay at the mine’s accommodation facilities, with most opting for either one of the Lihirian villages (42%) or Londolovit Town (23%). They also had spent longer periods outside Lihir prior to travelling, with 41% of them being away for 3 months or more.There was a clear association between the place of stay on Lihir and the purpose of the visit, both in travellers arriving by plane (p = 0.0019) and in those arriving by boat (p < 0.0001). Conversely, despite variations in the place of stay and the intention of the visit, both cohorts shared a similar mean length of stay on Lihir (around 3-weeks).The origin of travellers arriving to Lihir differed significantly between those arriving by plane and those arriving by boat (p < 0.0001). Figure Despite the different origin of travellers in both cohorts, most reported having received a full course of antimalarial treatment during the preceding 12 months. However, this proportion was higher among travellers arriving by boat (95%) compared to those arriving by plane (80%) (p < 0.0001). The reported time of the last malaria episode was between 3 and 4 months prior to enrolment in the study for both cohorts. In addition, the use of LLIN was similar, with most travellers not utilizing them while being away from Lihir. No participant had fever, or any active symptom compatible with malaria upon recruitment.P. falciparum infection and two of P. vivax. DNA extraction was possible in 396 of the 398 blood spots collected in the travellers arriving by plane. When using qPCR to detect Plasmodium parasites, 18 (5%) of these travellers were positive, 16 (4%) were male and 2 (7%) females. Most of the positive cases arrived from provinces where malaria transmission is high or moderate, including East New Britain (6), East Sepik (3), Morobe (3), New Ireland (2), and Western Province (1). Unfortunately, the amount and/or quality of the extracted DNA from these samples was not sufficient to successfully determine Plasmodium species in the subsequent specification reaction, with 77% of the positive qPCR samples yielding a negative result.Regarding infection status of travellers arriving by plane, only 3 cases (1%) were positive by RDT and microscopy. Microscopy showed one case of P. falciparum infection, 5 (15%) of P. vivax, 1 (3%) of P. malariae, and 5 (15%) of mixed infections, which included P. falciparum and P. vivax. In contrast, RDT diagnosed 14 (42%) cases of P. falciparum infection, 10 (30%) of non-P. falciparum species, and 9 (27%) potential mixed infections. When using qPCR, 67 (17%) of travellers arriving by boat were positive; 43 (64%) were male and 24 (36%) were female. Most of them (98%) arrived from either New Ireland  or East New Britain , and 1 arrived from the Autonomous Region of Bougainville. In this cohort, DNA extraction was optimized and 63% of the positive qPCR samples yielded a positive result for the subsequent specification reaction. Among those, 21 were positive for P. falciparum, 6 for P. vivax, 2 for P. malariae, 11 for both P. falciparum and P. vivax, and 3 for P. falciparum and P. malariae. There were no P. ovale positive samples.Conversely, among travellers arriving by boat, 33 (8%) were positive by RDT and microscopy. Microscopy showed 22 (67%) cases of A flowchart of the samples tested in each cohort by each of the techniques, including their results and the tests’ accuracy using qPCR as the reference test, is available in Fig. The uni- and multivariate logistic regression models including both cohorts of travellers are shown in Table To further explore factors associated with malaria infection and their respective risk, separate analyses for each cohort were conducted. For travellers arriving by plane, no variable was found to be significantly associated with testing positive by qPCR or any of the utilized techniques  closely resembled the mean prevalence in the local Lihirian population (15%) found in a parallel study in 2019 . This high number of asymptomatic individuals  carrying malaria parasites likely sustain the ongoing transmission in Lihir Islands . In contAnopheles biting intensity and inoculation rates are highly reduced compared to the rest of Lihir .Furthermore, when looking for risk factors associated with malaria infection in the whole cohort and the cohort of travellers arriving by boat, the only independent risk factor identified was an extended absence from Lihir, specifically over 91 days. This further supports their increased exposure and subsequent elevated infection rates. Although arriving from high incidence PNG provinces emerged as a risk for infection, there was no independent association between point of origin and malaria infection, probably because most of the travellers arriving by boat arrived from high incidence provinces. Interestingly, the univariate logistic regression analysis in the whole cohort and in the cohort of travellers arriving by boat showed an association between infection and visit intent. Lihirian residents returning home, VFRs and traders had higher risk of infection compared to mine employees and contractors. These findings contrast with other mining settings, especially illegal operations, where mine workers presented higher malaria burden than the indigenous population , 24. ThiP. vivax, is likely to be carriers of hypnozoites, contributing to a high annual relapse burden [Anopheles when people are still engaged in outdoor activities [Despite differing malaria parasite prevalence and province origins, the large majority of both cohorts had experienced at least one malaria episode in the last 12 months. A potential explanation is that travellers from low transmission provinces could have been exposed during their work roster in Lihir; while those coming from nearby islands might have been infected locally or in Lihir. In addition, PNG’s population, having been exposed to e burden . Interese burden . On the e burden , 27, or tivities . Hence, P. falciparum typically manifest an incubation period of 9 to 14 days, other species can have longer incubation periods [P. vivax active cases are estimated to be relapses [This study included participants who had been away from Lihir for a minimum of 12 days, with the aim of capturing mine workers following the 14-day on-site and 12-day off-site work roster. Notably, while  periods . Additiorelapses , usuallyrelapses . Thus, drelapses , or by dIn the last decade, Newcrest Mining ltd has considered the possibility of malaria elimination in the Lihir Islands by implementing a pilot programme that integrates diverse strategies to reduce the malaria burden and ultimately halt local transmission. However, the amount of imported malaria parasites could challenge these efforts . For insIn islands regions like Lihir, where entry points are well identified, an effective approach for avoiding transmission of imported cases involves integrating passive and active case detection with travellers’ test and treat at borders , 37. In Finally, islands do not have a guaranteed success in control and elimination programs , and colTravellers arriving by boat to Lihir Islands exhibit a significantly higher risk of malaria infection compared to those arriving by plane, thus posing a higher risk for parasite importation. Lihirian residents returning home, VFRs, and traders face a heightened risk for malaria infection in contrast to mine employees. Implementing screenings among high-risk travellers arriving by boat could potentially prevent transmission from importation. In the long-term, particularly if malaria transmission decreases across the Lihir Islands, improved control endeavours could greatly benefit from integrating actions in the neighbouring islands. Failing to proactively address imported malaria cases among travellers to the island could pose challenges to ensuring the sustainability and impact of malaria control or elimination efforts.Additional file 1: Table S1. Variables and their associations with Plasmodium qPCR positive results and logistic regression models for the cohort of travellers arriving by plane.


Malaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared malaria-free and now without local transmission, imported cases remain a threat to re-introduction of the disease and a burden on the health system.Plasmodium falciparum infection. The 18S rRNA- PCR targeting the rPLU6-rPLU5 region was used to confirm the diagnosis. The next-generation sequencing (NGS) method was carried out according to the manufacturer’s instructions  Tagmentation kit (20060060), Illumina) to identify Plasmodium spp. Furthermore, NGS produced a whole-genome sequence of 22.8Mbp of the 14 chromosomes and 25Kbp of the apicoplast. A BLAST search of the apicoplast DNA and selected chromosomal DNA revealed that P. falciparum was the causative agent. The merozoite surface protein-1 (msp-1) was used to construct a phylogenetic tree of 26 P. falciparum, including the one isolated from the patient from Jericho, which clustered with the Sudanese isolate indicating genetic relatedness between the two.Three days after returning from a long trip to malaria- endemic countries; Abyei-Sudan, Chad and Uganda, a 41-year-old male resident from Jericho, Palestine, suffered paroxysms of fever, general fatigue, myalgia, arthralgia, headache, and a strong desire to vomit. Thin and thick Giemsa-stained blood smears were prepared and examined microscopically using oil immersion. Immature trophozoites (ring forms) were seen to parasitize approximately 10% of the erythrocytes revealing hyperparasitemia equivalent to > 100,000 parasites/ µl indicating severe malaria . The doumsp-1 were key means in tracking the place of origin of infection in the case of travel to multiple destination.The travel history together with signs and symptoms of malaria, followed by prompt diagnosis using conventional microscopic inspection of Giemsa-stained films together with molecular DNA tracking tools like  Plasmodium. It is a vector-born disease transmitted by female mosquitoes of the genus Anopheles. Of the four clinicallysignificant species in the genus, Plasmodium falciparum is the most widely distributed in Africa and having the most severe clinical consequences [P. falciparum and P. vivax was a major public health problem. The malaria infection rate ranged from 50, 20, 7, and 0.7% in the years 1905, 1913, 1922, and 1944, respectively [P. falciparum is characterized by fever, chills, headache, myalgia, arthralgia, weakness, nausea, anemia, jaundice, vomiting, or diarrhea. Other more severe manifestations include splenomegaly, hypoglycemia, pulmonary or renal dysfunction, and neurological changes such as mental confusion and seizures followed by coma and death. Here the geographical origin of a rare case of malaria imported into Palestine was tracked using next-generation sequencing and population genetics.Malaria is a potentially lethal infection caused by an apicomplexan parasite of the genus equences , 4. The equences . At the equences . Reportsectively , 10. Sevectively , 10, 11.Plasmodium falciparum DNA (Provided by Prof. Ron Dzikowski), 0.04ng/µl (1ng/25µl reaction) of DNA and PCR grade water was used as negative control. A PCR product of approximately 1200 bp was obtained from the DNA of the infecting parasites, which was equivalent to the positive control after running the samples on a 2% agarose gel and staining with ethidium bromide, using the Biorad gel doc XR documentation system  . Urinalysis, stool analysis, complete blood count, and examination of blood films were done, all of which were normal. Thick and thin blood films were prepared and examined in between the paroxysms. Blood films were stained for one hour with Giemsa’s stain made with 1:40 phosphate buffered water according to Garcia . Blood fmsp-1 (Accession number OP115966), msp-2 (Accession number OR198912), and any selected chromosomal DNA showed that P. falciparum caused the imported case of malaria. Based on the WHO recommendations and other studies, population genetics of the genus Plasmodium was based on the hypervariable region (556bp) of the merozoite surface protein I (msp-1) owing to its extensive polymorphism, stability throughout the life cycle, the availability of the widely used data and the easy distinguishable [msp-1 sequence was retrieved from the WGS resulting from this report. The phylogenetic tree based on the msp-1 of 26 isolates of P. falciparum from different countries was constructed. This include one from the resident of Jericho. The tree showed that this isolate clustered with the Sudanese isolate  of the antimalarial drug Coartem® (Novartis) as the drug of choice for acute malaria. The patient ingested four tablets every eight hours on day one, and four tablets every 12 h on days two and three. His condition improved with immediate resolution of all the acute symptoms, which was confirmed on examining thin blood films and employing PCR testing  and an apicoplast (25Kbp) DNA sequences [uishable . The Jer2.8Mbp anP. falciparum, the most severe form of malaria. This proved the efficacy of using NGS and msp-1 DNA in tracking the geographical origin of the parasites seen in the stained smears. The main limitation was the need of a high depth of sequencing coverage (6.2x) to cover the WGS of P. falciparum (22.8Mbp), which is within a much larger amount of human host DNA (3.2Gbp). Regarding this case, local transmission was ruled out owing to the lengthy incubation period of malaria caused by P. falciparum of at least two weeks. Also, in theory, local transmission could not have occurred, owing to the absence of the main vectors of P. falciparum, i. e., Anopheles gambiae and Anopheles funestus, as evidenced by the complete absence of anopheline species during insect trapping campaigns in Palestine in the last two decades [P. falciparum extracted from his infected blood with DNA from reference strains from other countries around the world. The phylogenetic tree based on the msp-1 of all the samples used clustered his causative agent with a Sudanese one, indicating, most likely, that the patient contracted his infection in the Abyei region of Sudan and not from any other region he travelled to. Imported cases of malaria from regions of malaria endemicity to those of non-endemicity are common. From 1990 and early 2021, 35 cases of malaria were recorded in Palestine but without mentioning whether the diagnosis was only symptom-based or also laboratory-confirmed, or if these cases were imported or autochthonous [This quite severe case of malaria was diagnosed in Jericho, Palestine, a zero-indigenous case area and declared by the WHO as a country free of malaria transmission , which m decades –25. A po decades –30. The  decades . In this decades , 33. In hthonous . In 2005hthonous . Ten yealciparum .8Mbp, whAnopheles in recently declared malaria free areas is crucial in preventing re-introduction of the disease.Cases of malaria imported into non-endemic countries, Palestine, is a challenge to their health system’s preparedness to diagnose and treat such cases. The careful taking of clinical histories, laboratory confirmation of cases including the identification of the infectious agents, using microscopy and molecular-based methods, and tracking the origin of the infection are recommended to enable timely life-saving treatment and to understand the disease epidemiology. Travelers to and from endemic countries should be dealt with according to WHO international health regulations and their amendments . Monitor


Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). Deepening our knowledge of parasite biology and its molecular components is key to develop new tools for malaria control and elimination. This study aims to investigate and characterize a P. vivax protein (PvVir14) for its role in parasite biology and its interactions with the immune system. We collected sera or plasma from P.vivax-infected subjects in Brazil (n = 121) and Cambodia (n = 55), and from P. falciparum-infected subjects in Mali (n = 28), to assess antibody recognition of PvVir14. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. IgG1 and IgG3 most frequently contributed to anti-PvVir14 responses. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21− CD27−) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. When analyzed at a single-cell level, the B cell receptor gene hIGHV3-23 was only seen in subjects with active P.vivax infection where it comprised 20% of V gene usage. Among T cells, CD4+ and CD8+ levels differed  between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. Specific B cell subsets, anti-PvVir14 circulating antibodies, and NKT cell levels declined after treatment of P. vivax. This study provides the immunological characterization of PvVir14, a unique P. vivax protein, and possible association with acute host’s immune responses, providing new information of specific host-parasite interaction.Trial registration: TrialClinicalTrials.gov Identifier: NCT00663546 & ClinicalTrials.gov NCT02334462. Plasmodium vivax is one of the main species causing malaria around the globe, thus the development of control tools are needed if progress is to be made towards malaria elimination. Among them, vaccines are a crucial one, since it’s been proven that a well-established immunization program can save millions of lives a year. Although the development of vaccines against malaria have had substantial progress in the last decade, most of the efforts are concentrated towards Plasmodium falciparum. In this study, the authors aimed to investigate the potential role of a recently P. vivax-protein (PvVir14) discovered in the urine of infected subjects from the Brazilian Amazon. Initial findings showed that, although the protein elicits a higher frequency of antibodies among the Brazilian population (where it was first discovered), antibody titers were also found within African populations exposed and/or infected by P. Vivax. Interestingly, populations exposed to P. falciparum only did not have any immune responses against the protein, indicating that it is exclusive to vivax. Discovering key antigens is one of the most important steps towards the development of control tools. These findings provide the first characterization of naturally acquired antibody responses from different malaria regions against a new and promising target. Plasmodium vivax is the most common human malaria species in Brazil, causing approximately 85% of the cases [P. vivax infection was considered to be benign and often asymptomatic, but in recent years, severe cases of P. vivax malaria have been reported more frequently, not only in Brazil but in several endemic regions worldwide [Despite numerous efforts to eradicate malaria, this disease is still a major cause of mortality and morbidity worldwide that is endemic in Sub-Saharan Africa, Southeast Asia, Middle East, Oceania, and Latin America. In 2021, an estimated 247 million cases of malaria occurred worldwide [orldwide –5. Amongorldwide .P. vivax strains [P. vivax malaria a public health issue of increasing importance. An efficacious vaccine targeting P. vivax would be a potent and cost-effective tool to reduce transmission, and thus an important measure to control malaria [P. vivax compared with those for P. falciparum.The recent increase in drug-resistant  strains , the evo strains , the ear strains , make P. malaria . HoweverP. vivax through targeting sites of vulnerability or mechanisms of parasite virulence [P. vivax encompasses numerous surface proteins that have been related to parasite virulence [P. vivax, named PvVir14 (from the Vir superfamily), was identified circulating in high levels during acute infection and recognized by P. vivax malaria patients [An efficient vaccine will protect from irulence . The Virirulence ,13, and irulence . Signifiirulence . In prioirulence . Recentlpatients .PvVir14 protein were present in a high proportion of sera/plasma from P. vivax-infected subjects in Brazil and in Cambodia. Considering the absence of previous immunological studies comparing different epidemiological regions, we characterized immune responses to PvVir14 at a serum and cellular levels to identify signatures of immunogenicity and assess the potential value of PvVir14 as a protein of interest.Here, we determined that specific antibodies targeting the ClinicalTrials.gov Identifier: NCT00663546). Written informed consent was obtained from each participant. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.The studies involving human participants in Brazil were reviewed and approved by the Tropical Medicine Research Center  and the Ethics Committee of the Federal University of Minas Gerais (CAAE: 27466214.0.0000.5149), Brazil. The human study in Cambodia was approved by the Institutional Review Board (IRB), National Institute of Allergy and Infectious Diseases (NIAD), National Institutes of Health (NIH), and National Ethics Committee for Human Research (NECHR), Cambodia  from Belo Horizonte, Minas Gerais State, Brazil, a non-endemic area for malaria, were also recruited as malaria-naïve controls (2019–2021). This study was approved by the Research Ethics Council of the Federal University of Minas Gerais, Brazil (CAAE 27466214.0.0000.5149). For some experiments, samples collected in Brazil were frozen and transported on dry ice to the U.S. National Institutes of Health (NIH), Bethesda MD, USA.The study population included 121 subjects living in the Brazilian Amazon and diagnosed with vivax malaria at the Research Center for Tropical Medicine (CEPEM) in Porto Velho, Rondônia, Brazil, in the years of 2014 (69 serum samples) and 2019 (52 serum samples and 22 PBMC samples) during the transmission season. The inclusion criteria in this study included acute illness at the time of blood sampling and presence of P. vivax infected subjects (3–55 years old) in Pursat, Cambodia, between 2008 and 2018, under a protocol approved by NIAID and the National Ethics Committee on Human Research , were used to perform IgG ELISA assays against PvVir14. Sera from 28 Malian adults (range of age: 18–55 years old) collected between 2015 and 2019 were used to investigate reactivity to PvVir14 and were approved for use under a protocol approved by the NIAID IRB and the University of Bamako (FMPOS) Ethical Review Committee, and the study registered at ClinicalTrials.gov (NCT02334462).Sera collected from 55 An additional malaria-naive control group included healthy adult subjects (N = 5) who donated samples during participation in clinical studies at the U.S in 2020. NIH .Each volunteer was required to sign a written informed consent specific to this study, and blood was obtained upon receiving the said document.PvVir14 protein was expressed in Escherichia coli. Briefly, the recombinant plasmid with the PUC/PvVir14 synthetic gene (GenScript) was resuspended and transformed with competent cells from E. coli XL-1 Blue . Positive clones were confirmed through digestion using Xho1 and Nhe1 enzymes. The PvVir14 insert was then cloned into a bacterial expression vector (pET28aTEV) and transformed with competent E. coli BL21-star  by electroporation . To confirm gene insertion, a colony PCR was performed using T7 primers . To produce at large scale, 1mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added following an incubation period of 3h at 37°C at 180rpm. Cells were ruptured by a high-pressure homogenizer and soluble fractions were obtained by centrifugation. The recombinant protein was purified using Ni2+ affinity chromatography with HisTrap HP 5 mL column  coupled to an ÄKTA Prime Plus system . The purified PvVir14 protein, having 325 amino acids and predicted molecular weight of 37kDa, was separated by SDS-PAGE. Pvs230D1M  and PvCSP  were expressed in Pichia pastoris following similar procedures previously described for Pfs230D1M and PfCSP [PvDBP-RII was expressed in E. coli BL-21 cells and refolded as previously described [The full-length (325 amino acids) predicted nd PfCSP  and the escribed ,21.PvVir14-IgG and IgM in serum was determined through a conventional enzyme-linked immunosorbent assay (ELISA) performed as follows: 96-well plates were coated with antigen (0.5μg/mL) in carbonate buffer overnight at 4°C. Wells were then blocked with 0.5M chloride, 1% Triton X-100 and 1% bovine serum albumin in PBS (1X) and incubated for 2 hours at room temperature (RT) followed by incubation with the primary antibody at a concentration of 1:500 for IgG and 1:100 for IgM (serum from infected subjects). After washing 4 times with PBS (1X) plus 0.05% Tween 20, a peroxidase HRP-conjugated secondary antibody was added at a concentration of 1:10000 (IgG) and 1:8000 (IgM) and incubated for 1 hour at RT with shaking. The plate was washed 4 more times with 1X PBS Tween (0.05%) before the developing reagent , SeraCare) was added to each well. The reaction was stopped after 15 minutes at RT by the addition of a TMB stop solution (SeraCare) and the optical density was measured at 450nm.The presence of cut-off value for detection was determined by testing 5 different negative control sera from subjects never exposed to malaria from Bethesda MD, United States of America. The mean optical density value at 450 nm ±3SD (VersaMax Microplate Reader) for duplicate determinations in negative sera was used as the cut-off value for different subclasses.ELISA assays to detect the IgG subclasses were performed as previously described . Sera wePvVir-14 seroreactivity among P. vivax-infected or exposed individuals, using flow cytometry and a panel of markers for the main B and T cell subtypes. The population was stratified by exposure, infection and seroreactivity status: PvVir14+ ; PvVir14- ; and HD . PMBCs were thawed in a 37°C water bath, resuspended in complete RPMI, and then in PBS. Cells were stained with the following antibodies: CD3 FITC (clone SK7), CD4 PerCP-Cy5.5 (clone SK3), CD8 APC-Cy7 (clone SK1), CD14 AlexaFluor700 (clone HCD14), CD16 PE-Cy7 (clone 3G8), CD19 BV605 (clone HIB19), CD21 BV711 (clone B-ly4), CD27 APC (clone LG3A10), and CD56 BV785 (clone 5.1H11). All antibodies are from BioLegend, except for CD21 BV711 (clone) which is from BD Biosciences. Cells were also stained with Zombie UV fluorescent dye (BioLegend) to assess live vs. dead cells. Cells were incubated at 4°C protected from light for 20 minutes while staining and then washed with PBS and resuspended in FACS Buffer. Flow cytometry was performed on a LSRII instrument (BD Biosciences) and analysis was done using FlowJo v.10. Cells were first gated for singlets by FSC-H and FSC-A, followed by live cells  and lymphocytes (SSC-A and FSC-A). Monocytes were gated out of total lymphocytes. B cells were thereafter gated as CD19+ followed by CD27 and CD21 to distinguish (i) classical memory B cells (CD27+ CD21+), (ii) activated memory B cells (CD27+ CD21-), (iii) atypical memory B cells (CD 27- CD21-), (iv) naïve B cells (CD27- CD21+). T cells (CD3+) were gated out of total lymphocytes and thereafter as CD4+, CD8+ (CD3+CD56-) and NKT subsets (CD19-CD3+CD56+) (We examined whether cellular immune responses were related to 3+CD56+) . Data anAmplification of BCR heavy and light chains from single sorted B cells was performed by iRepertoire Inc.  as previously described . BrieflyThe Reactivity Index (RI), used to define positivity in serology assessments, was calculated by dividing the mean OD of the assessed subjects by the mean OD plus 3 standard deviations (SD) of the malaria-naïve control subjects . Values above 1 were considered positive. Samples were submitted to a normality test to define statistics. For non-normal data or non-equal variances among groups (3 or more groups), a Kruskal-Wallis test was performed. For normal data with equal variances, we performed ANOVA or, for comparison between 2 groups, T test. To determine equality of variances among normal populations, a Fisher’s exact test was performed, and considered significant when p<0.05.P. vivax-infected adults from Porto Velho–Rondônia, Brazil and neighboring municipalities; 15 non-infected but exposed subjects from Porto Velho–Rondônia, Brazil; 5 non-infected and never exposed subjects from Belo Horizonte–Minas Gerais, Brazil; 55 P. vivax-infected adults and children from Pursat, Cambodia; 28 P. falciparum-infected adults from Bancoumana, Mali, West Africa; and 5 non-infected and never exposed subjects from Bethesda–Maryland, United States of America  and the age range was 29–49 years. The sex ratio was 1:3 . The non-infected but currently exposed had an age range of 20–47 with a sex ratio of 2.6:1 . The non-infected/never exposed had an age range of 26–32, and a sex ratio of 1:1.5 . For the Cambodian population, the range age was 3–55, and a sex ratio of 1:1.75 . As for the Malian population, the age range was 18–55 with a sex ratio of 1:3.4 . Finally, the American population was all males with age range 49–53 years, and the bias was merely due to sample availability. For the endemic regions, considering that the main factor to be enrolled was an acute infection, we observed a clear male bias within infected populations, which is a common feature for vertebrates [The study population is composed of 121  America . Concerntebrates . Among tP. vivax-infected subjects in Brazil, 74 (61%) had detectable IgG against PvVir14, compared to 19 out of 55 (34.5%) Cambodian subjects. To confirm that PvVir14 is a specific and exclusive target of P. vivax, sera from 28 P. falciparum infected subjects from Mali (Western Africa) were tested; P. vivax infections have not been reported at the study site in Mali. Of 28 sera tested, none were reactive to PvVir14, consistent with their lack of prior exposure to P. vivax and the absence of a P. falciparum ortholog for this protein. To assess whether PvVir14 seroreactivity is present even without an acute infection, we assayed sera collected from 15 uninfected subjects living in P. vivax endemic areas for anti PvVir14-IgG levels. Five subjects (33%) had detectable antibody titers three of the five seroreactive had a known history of prior clinical malaria, which might explain the presence of circulating antibodies, while the malaria history for the other two subjects is unknown  displayed PvVir14-IgM antibodies in their sera  and IgG3 levels (36.13%) were most frequent, while IgG2 (frequency of 9.24%) was less frequent and IgG4 (0.84%) was detected in a single individual , blood (PvDBP-RII) and mosquito-sexual stages (Pvs230D1). In ELISA assays to measure total IgG, the positivity rates were as follows: 61% for PvVir14, 7.6% for PvCSP, 42% for PvDBP-RII and 3.3% for Pvs230 domain 1 . In addition, a statistically significant but weak correlation between PvVir14-IgG levels and IgG levels to the sporozoite/liver and blood stage proteins was observed  (We compared IgG reactivity to domain 1 . The frenk test) , but notnk test) .PvVir14+ group . Among the B cell subpopulations, AtMBCs  were higher for the PvVir14+ subjects in comparison to the other two groups    , suggest4- group . These l(arrows) P. vivax-infected participants, total T cell levels  were lower than those of uninfected U.S. donors, but this difference was only statistically significant for the PvVir14+ group    . When me4+ group . Based o T test) . When us T cells  and a clPvVir14+ .-CD19-CD14-CD56+ and CD3+CD56+, respectively, and constituted 2 clusters   cells are known to be one of the first innate cells that respond to malaria parasites , helpingclusters . Measureclusters , whereas t test) . Of inteng CD16+ .PvVir14-IgG levels had significantly decreased among 8 individuals who returned for a follow up visit  .Upon the diagnosis of acute malaria, all subjects in this study received treatment consisting of Primaquine and Chloroquine . Forty dd-pairs) . Two sub+ expression decreased in naïve and classical B cell subsets  two subjects with acute infection and high titers against PvVir14 from the Brazilian Amazon; (ii) two non-infected subjects from the USA   were identified. Seven genes were shared between the two groups: hIGHV3-21, hIGHV3-30, hIGHV3-33, hIGHV3-66, hIGHV4-34, hIGHV4-61, hIGHV5-51 (To further characterize the B cell response in  the USA . All fouer (USA) . As for ple size . ConcernIGHV5-51 . One V gP. vivax has attracted increasing attention as the awareness of severe and lethal cases increased [P. vivax receives little attention when compared to P. falciparum. While vaccines could be a valuable tool for control of P. vivax [P. vivax candidates are in clinical and pre-clinical phases  [ncreased , and thincreased . NeverthP. vivax  when useP. vivax , only a d Pvs25) .P. vivax variant antigen family (Vir) was detected in patients’ urine and was assessed for seroreactivity and related immunological profiles. Pathogen proteins detected in body fluids during infection are widely used as biomarkers of different infections [In the present study, a member of a fections . Althougfections . The facfections  indicatePvVir14 IgG antibodies during acute infection was 61% in Brazil and 34.5% in Cambodia. The greater prevalence in the Brazilian amazon population is probably due the different transmission setting when compared to Cambodia. While Brazil is known to have a high API  in endemic regions such as Rondonia [P. falciparum-infected population in high-transmission Mali that is not exposed to P. vivax, none of the subjects recognized the protein, confirming that PvVir14 responses are exclusive to P. vivax. Besides IgG, a significant percentage (27%) of P. vivax-infected subjects from Brazil also displayed IgM antibodies against PvVir14. Although the IgM role in protective immunity against malaria is still unclear, IgM to the surface of merozoites inhibits binding in a complement-dependent manner and reduces the odds of clinical malaria [falciparum malaria have shown that IgM levels decline shortly after acute infection [Here, we observed that the prevalence of Rondonia , prevaleRondonia . AnotherRondonia . A serolRondonia , a lower malaria . Nonethe malaria ,41. Whilnfection , others nfection .In addition to IgM, cytophilic IgG subclasses can also work through complement fixation for some antigens and have been associated with protection against malaria ,45. In tP. vivax proteins from different phases of the parasite’s life cycle, PvVir14 showed more frequent seroreactivity. It has been described that vir genes from different families might be profusely expressed in different isolates at noun unspecified moments, unlike proteins such as PvDBP or PvCSP that are known to be expressed at a specific phase of the cycle [PvVIR14 positively correlated with that to PvCSP (liver stage) and PvDBP-RII (blood-stage). Although the functional role of anti-PvVir14 has yet to be defined, the prevalence of PvVir14-IgG is consistent with blood-stage parasite expression, which generally elicits a relatively greater antibody response [When compared to other well-known he cycle . The facresponse ,50.PvVir14 is a novel protein with features that suggest potential as a diagnostic or therapeutic target, we characterized immune cell subsets among acutely infected subjects with or without anti-PvVir14 titers. Our results demonstrated an expanded population of atypical memory B (AtMBCs) lacking CD21 and CD27 expression. The presence of this subset has been extensively described in subjects known to be chronically infected with malaria and living in varied transmission settings [PvVir14+ group have a higher median of previous episodes (9.2 episodes/lifetime) than the subjects within the PvVir14- group (median: 7.8 episodes/lifetime), and such profile corroborates previous studies correlating AtMBC levels with past malaria infections [Plasmodium, this subset is also found in many other diseases, especially chronic ones such as HIV, hepatitis C (HBC), and lupus [Since settings ,52. In ofections . Despitend lupus ,55.- CD27-, the presence/absence of several other expression factors distinguish AtMBC and its functions [PvVir14 antibodies are related to an increase of AtMBCs. However, other studies suggested that a reduced expression of CD21 can be associated with complicated malaria [Whereas in broad terms AtMBCs are mainly defined by CD21unctions . During unctions ,58. Howeunctions , and thi malaria ,60, and PvVir14+ subjects showed a higher intensity of CD21 whilst those from PvVir14- subjects showed a higher intensity of CD27. Although the frequency of these B cell subsets did not differ between the groups, the differential intensity of these markers may suggest that the cells are functioning differently. We speculate these differences may also contribute to inter-individual differences in PvVir14-IgG production. While B cells and antibody responses have a key role in protection, immunity to vivax malaria has recently been linked to the presence and functionality of T cells [PvVir14 antibodies. We observed that CD4+ cells are lower and CD8+ cells are higher in PvVir14-IgG+ subjects compared to malaria-naïve healthy donors. A CD4+ decrease is a common profile for P. vivax-infected individuals, potentially due to a propensity for apoptosis, loss of proliferative capacity, “exhaustion”, or redistribution to sites of inflammation [P. vivax has tropism) retain the protein translation machinery and surface-express these molecules [PvVir14- subjects also demonstrated an upregulation of CD27 on both CD4+ and CD8+ T cells, indicating perhaps a larger number of activated T cells within this group [When running a high dimensional analysis, classical and naïve B cells from the  T cells . Assuminammation –63. Wherolecules ,65. In tis group  albeit tPvVir14 antibodies were still found circulating in the peripheral blood for most individuals (5/7 subjects) despite the marked reduction observed among the patients, consistent with studies showing that Plasmodium infections induce a patent but short-lived antibody-responses to blood-stage antigens [Since treatment can alter the dynamics of the immune response, we explored immune response after Chloroquine/Primaquine therapy. IgG titers decreased for all assessed subjects after 40 days and anti-antigens –70.PvVir14 titers, but it is yet to be defined if the difference in expression has an impact on the disease progression [As for circulating immune cells, classical and naïve B, NK, and NKT cells presented a different profile when comparing acute and post-treatment scenarios. During acute infection, both naïve and classical clusters presented a higher CD27 intensity of expression when comparing groups with or without anti-gression . Althouggression –74. Nonegression ,76, therP. vivax-IgM BCR versus other isotypes, indicating that those cells are probably engaged in the immune response to infection. Although predominance of IgM B cells has already been demonstrated for acute P. falciparum malaria subjects [P. vivax. The IgM isotype has drawn attention lately since it can act as a protective factor through neutralization, opsonization, complement fixation, and functional inhibition of invasion with a higher and specific binding capacity [P.vivax-infected subjects, hIGHV3-23 was most common and accounted for 20% of all V gene usage within the heavy chain. The hIGHV3 family has already been related to high-affinity anti-Plasmodium antibodies against blood-stage antigens in naturally-infected subjects [P. falciparum for both Pf IgM+ and Pf IgG+, but with a lower percentage of use [PvVir14, the cell sorting was not performed using antigen to isolate Pv-specific B cells, and this is a limitation of our study, as well as the number of subjects selected for the experiments. However, by sorting all memory B cells from donors, we were able to fairly compare between malaria-immune and malaria-naïve donors.At the single B cell level, our study demonstrated that acute subjects present an unbalanced amount of subjects , little capacity ,78. At tsubjects . Of notee of use . Unlike P. vivax protein that we find is secreted in the urine of malaria patients. Further studies are warranted to understand the role of PvVir14 in infections, and its potential as a target of control tools to help reduce malaria around the globe.Our findings provide the first characterization of naturally acquired antibody responses from three different malaria endemic regions against a S1 Fig+ B cells, activated memory B cells CD19+CD27+CD21- (G1), Classical memory B cells CD19+CD27+CD21+ (G2), Naïve B cells CD19+CD27-CD21+ (G4), Atypical memory B cells CD19+CD27-CD21- (G4), Live CD3+, NK cells (CD19-CD3-CD14-), NKT cells (CD19-CD3+CD56+), CD4+ and CD8+ T cells (CD3+CD56-).Cells were pre-gated for live/dead and then identified as follows: CD19(TIF)Click here for additional data file.


Plasmodium vivax is a recognized cause of severe malaria including deaths. The exact burden and patterns of severe P. vivax monoinfections is however still not well quantified, especially in P. vivax endemic regions. We examined the magnitude and patterns of severe malaria caused by monoinfections of P. vivax and associated predictors among patients admitted to a tertiary care center for malaria in Vietnam.Infection with A retrospective cohort study was conducted based on the patients’ medical records at the Hospital for Tropical Diseases from January 2015 to December 2018. Extracted information included demographic, epidemiologic, clinical, laboratory and treatment characteristics.P. vivax were found in 153  patients of whom, uncomplicated and severe malaria were documented in 89.5%  and 10.5% , respectively. Patterns of severe malaria included jaundice (8 cases), hypoglycemia (3 cases), shock (2 cases), anemia (2 cases), and cerebral malaria (1 case). Among 153 patients, 73 (47.7%) had classic malaria paroxysm, 57 (37.3%) had >7 days of illness at the time of admission, and 40 (26.1%) were referred from other hospitals. A misdiagnosis as having other diseases from malaria cases coming from other hospitals was up to 32.5% (13/40). Being admitted to hospital after day 7th of illness  was a predictor of severe malaria. Severe malaria was statistically associated with longer hospital length of stay (p = 0.035). Early and late treatment failures and recrudescence were not recorded. All patients recovered completely.Monoinfections with P. vivax infection can be misdiagnosed which results in delayed treatment. To meet the goal of malaria elimination by 2030, it is crucial that the non-tertiary hospitals have the capacity to quickly and correctly diagnose malaria and then provide treatment for malaria including P. vivax infections. More robust studies need to be conducted to fully elucidate the magnitude of severe P. vivax in Vietnam.This study confirms the emergence of severe vivax malaria in Vietnam which is associated with delayed hospital admission and increased hospital length of stay. Clinical manifestations of  HTD is also a severe malaria referral hospital in southern Vietnam. As per HTD policy, patients’ information including medical history, epidemiology, diagnosis, and treatment must be correctly entered into the medical records. All malaria related laboratory tests including microscopy and rapid tests are performed at the HTD and in accordance with the national laboratory performance standards. All suspected cases are primarily diagnosed using microscopy, followed by screenings of all clinical and laboratory signs of severe malaria. Microscopy is subsequently performed at an interval of six or 12 h, depending on the disease severity until it turns into a negative result for at least two times. Rapid test is performed based on the physicians’ clinical judgement. According to the Vietnam Ministry of Health guidelines for the management of malaria infection, in order to be discharged from the hospital, patients must meet the following four criteria: receiving a full 3-day course of chloroquine of 25 mg (base)/kg or an oral artemisinin-based combination therapy, no fever, no severe malaria symptoms, and blood smear negative for parasites, excluding gametocytes stages, for at least two times . Patient2.2.P. vivax infected patients were used to extract information on the patients’ demographics, malaria related risk factors, pregnancy status , underlying health conditions, history of malaria infection, current course of malaria disease and treatments at previous hospitals and HTD. To ensure the validity of data, two authors (OP and TN) who are qualified infectious disease specialists at the HTD reviewed and extracted data from the medical records. Any discrepancies derived from this process were cross-checked until consensus was obtained.A 4-year retrospective cohort study was conducted based on the patients’ medical records at the HTD from January 2015 to December 2018. The study was approved by the HTD’s Ethics Committee (reference number 65/QD-BVBND) and UNSW Sydney’s Human Research Ethics Committee (reference number HC180340). Medical records of Demographic information included age, sex, job, residential address, and BMI. In addition, malaria related risk factors comprising of previous blood transfusions (transfusions within 1 month piror to the onset of disease), injecting drug use (IDU), living in or traveling to malaria endemic areas within the 7 days prior to the onset of disease, and previous history of malaria infection. Underlying health conditions included end stage renal disease, cirrhosis, and other chronic diseases. Information on the current course of malaria disease included admission time, number of days of illness at the time of admission, signs and symptoms , laboratory tests , aminotransaminases (AST and ALT), arterial blood gas, hemoglobinuria, and serum levels of electrolytes, creatinine, glucose, bilirubin, and lactate), abdominal sonography. Treatments included anti-malarial and other supportive treatments, response to treatment  and late treatment failure (LTF)), recrudescence, and outcomes (recovery and death). Diagnosis and treatment at previous hospitals were also documented.2.3.ETF and LTF were defined in line with the WHO recommendations . ETF inc2.4.P. vivax monoinfections were calculated based on the total number of patients with P. vivax monoinfection and the point estimate of the incidence of uncomplicated and severe P. vivax monoinfections. Descriptive statistics were performed to summarize characteristics of study participants. Categorical variables were presented as an absolute count and percentage, while continuous variables were presented as mean ± 1 standard deviation (SD). Inferential statistics including Fisher’s exact test and Student’s t-test were used to compare categorical and continuous data, respectively, that facilitate the identification of predictors of severe malaria. To test predictors of severe malaria, a multivariable logistic regression model was developed. Based on the purposeful selection process, covariates for the regression model were identified and included variables that have a p value <0.25 in the univariable analysis as well as those that are clinically important based on the authors’ judgement . For comparison purposes, 95% confidence intervals (CI) of the point incidence of uncomplicated and severe udgement , 31. In udgement , 31. Var3.3.1.P. vivax monoinfection. Of these 153 patients, males and females accounted for 80.4% (123/153) and 19.6% (30/153), respectively  of them resided in or traveled to malaria-endemic areas 7 days prior to the onset of malaria. These participants included 52 people worked in forest-related fields. Around one-third  of them reported having a previous history of a malaria infection. All these 50 patients previously acquired malaria infections more than 12 months prior to the current illness. Three patients reported having at least one previous blood transfusion. Among 30 female patients, three were pregnant. Chronic kidney disease, cirrhosis and injecting drug use were not documented. Among the 153 patients with P. vivax monoinfection, 57 (37.3%) of them had more than 7 days of illness at the time of admission. Forty patients (26.1%) were transferred from previous hospitals. Of these 40 patients, 13 had a wrong diagnosis of having other diseases. The remaining 27 patients were correctly diagnosed as having vivax malaria infection. Four (10%) of these transferred patients received antimalarial treatment at previous hospitals. Among four patients receiving antimalarials at previous hospitals, one received oral DHA/PPQ for 1 day, while another patient used chloroquine and DHA/PPQ for 1 day. The remaining two patients used chloroquine and primaquine for one and 2 days, respectively. Parasitemia still persisted in all of them at the time of admission.During the study period, the HTD received a total of 443 malaria infected patients including 153  with ectively . The mea3.2.All patients had fever on admission . The mosp > 0.05; p = 0.035, data not shown). All patients completely recovered.Regarding antimalarial treatment, among 137 patients with uncomplicated malaria, 112 were treated with chloroquine, while 25 were given DHA/PPQ . Among t3.3.p = 0.033), being diagnosed with malaria infection at previous hospitals  and being admitted to hospital after day 7th of illness .There was an association between severe malaria and age  had severe  to 4.7% . Our fin to 4.7% , 37. Alt.5–16.3%  country –40. SincP. vivax in Asia, and an increasing burden of morbidity and mortality associated with this malaria species has also been reported , hypoglycemia (three cases), shock (two cases), anemia (two cases), and cerebral malaria (one cases). These findings concurred with other studies  in the early 2000s  confirmed that chloroquine remained largely efficacious to treat this infection with an adequate clinical and parasitological response rate of 100% on day 28  receiving a dose below the recommended dose of 25 mg/kg similarly found that the risk of recurrence was 32.4% by day 42  and UNSW Sydney’s Human Research Ethics Committee in Australia (reference number HC180340). The patients/participants provided their written informed consent to participate in this study.MCD, OKNP, and HPN: conceptualization, formal analysis, and writing original draft. OKNP, MCD, TPN, VVCN, and HPN: data acquisition. MCD, OKNP, TPN, TTT, RL, VVCN, and HPN: reviewing and editing. All authors contributed to the article and approved the submitted version.MCD is funded by the Australian Government through the Australian Alumni Grants Fund. HPN is funded by OURCU Vietnam.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Historically, malaria has been the predominant cause of acute febrile illness (AFI) in sub-Saharan Africa. However, during the last two decades, malaria incidence has declined due to concerted public health control efforts, including the widespread use of rapid diagnostic tests leading to increased recognition of non-malarial AFI etiologies. Our understanding of non-malarial AFI is limited due to lack of laboratory diagnostic capacity. We aimed to determine the etiology of AFI in three distinct regions of Uganda.A prospective clinic-based study that enrolled participants from April 2011 to January 2013 using standard diagnostic tests. Participant recruitment was from St. Paul’s Health Centre (HC) IV, Ndejje HC IV, and Adumi HC IV in the western, central and northern regions, which differ by climate, environment, and population density. A Pearson's chi-square test was used to evaluate categorical variables, while a two-sample t-test and Krukalis-Wallis test were used for continuous variables.p = 0.001; p < 0.001) while SFGR in the northern region (p < 0.001).Of the 1281 participants, 450 (35.1%), 382 (29.8%), and 449 (35.1%) were recruited from the western, central, and northern regions, respectively. The median age (range) was 18 (2–93) years; 717 (56%) of the participants were female. At least one AFI pathogen was identified in 1054 (82.3%) participants; one or more non-malarial AFI pathogens were identified in 894 (69.8%) participants. The non-malarial AFI pathogens identified were chikungunya virus, 716 (55.9%); Spotted Fever Group rickettsia (SFGR), 336 (26.2%) and Typhus Group rickettsia (TGR), 97 (7.6%); typhoid fever (TF), 74 (5.8%); West Nile virus, 7 (0.5%); dengue virus, 10 (0.8%) and leptospirosis, 2 (0.2%) cases. No cases of brucellosis were identified. Malaria was diagnosed either concurrently or alone in 404 (31.5%) and 160 (12.5%) participants, respectively. In 227 (17.7%) participants, no cause of infection was identified. There were statistically significant differences in the occurrence and distribution of TF, TGR and SFGR, with TF and TGR observed more frequently in the western region (Malaria, arboviral infections, and rickettsioses are major causes of AFI in Uganda. Development of a Multiplexed Point-of-Care test would help identify the etiology of non-malarial AFI in regions with high AFI rates.The online version contains supplementary material available at 10.1186/s12879-023-08335-4. Fever without a localized cause is a common presenting symptom reported by persons seeking medical care in countries with resource-limited settings . Acute fIn tropical areas of SSA, the predominant cause of undifferentiated fever has historically been assumed to be malaria . During Plasmodium falciparum (99% of smears), P. malariae , P. vivax (< 2%), and P. ovale (< 2%) . Wa. Wa22]. 2. According to the 2014 Uganda National Population and Housing Census, the district had a population of 782,077, with Arua city  as the main urban center [This study clinic is located in Arua district, northern region Fig. , in a run center . Arua din center . Agriculn center .Study participants were recruited from each study clinic’s outpatient department (OPD), based on a recruitment strategy. The number of patients recruited per district during the study was proportionately based on the total number of malaria cases diagnosed by the district in 2007 as reported in the Health Management Information System (HMIS) of UMoH. Potential participants were systematically screened for AFI from the OPD registers using a pre-determined interval (every nth patient) and invited to participate in the study after signing informed consent  visit at the OPD, and a standardized questionnaire with patient’s demographic data, clinical presentation, and history , and reported vector previous past exposure  was completed by the study staff. To increase awareness, participants were informed of AFI etiologies and their risk factors during enrollment. Guidance for treatment was based on rapid diagnostic test (RDT) results provided to the participants’ healthcare providers (HCPs). A home or clinic follow-up visit was completed at least two weeks post-enrollment, during which an additional questionnaire assessed the patient’s follow-up clinical status and possible AFI risk factors, including housing characteristics, occupation, animal or livestock exposure, agricultural activities, use of mosquito nets, and water sources for consumption and other daily uses as well as handwashing practices.To minimize loss to follow-up, study staff provided appointment cards prior to discharge at the baseline visit, made reminder telephone calls a few days prior to the follow-up visits, reimbursed travel expenses of returning participants, and when necessary, performed in-home follow-up for those who did not return to the clinic for follow-up. The clinic staff were responsible for all aspects of patient management and were managed according to the existing standard of care guidelines from the UMoH .0C. Serum samples were screened at the time of patient’s visit for malaria, TF, brucellosis, and leptospirosis using their respective RDTs at study clinics. RDT results were provided immediately to clinic staff to inform patient management.Study staff drew venous blood (approximately 5 ml) from each eligible enrollee at baseline and follow-up visits. Samples were centrifuged and sera aliquoted on the day of collection, and aliquots were stored at 2-80C to the UVRI laboratory within one week to one month of collection for confirmatory testing as well as additional testing for diseases for which no RDT was utilized. Rickettsioses, chikungunya virus (CHIKV), dengue virus (DENV), and West Nile virus (WNV) testing were conducted at UVRI. Results of testing conducted at UVRI laboratory were provided to the clinic staff as they became available, supplementing RDT results to further guide patient diagnosis and treatment. For long-term storage at UVRI, sera were frozen at -800C. Additional quality assurance testing for leptospirosis and rickettsiosis was conducted at CDC in Atlanta, GA, USA.Aliquoted sera were transported at 2-8Plasmodium falciparum, P. malariae, and P. vivax on the baseline blood samples. Patients that were positive on the Rapid-1–2-3 malaria HEMA Express RDT were given diagnoses with malaria infection by the respective malaria parasite identified on the RDT cassette. A clinical laboratory evaluation of the Rapid 1–2-3 HEMA Pf  assay conducted by UVRI reported a sensitivity of 75.0% and a specificity of 87.0%. For a subset of patients from Ndejje HC IV in the central region, thick and thin blood smears were examined to evaluate the performance of malaria RDT. Details of malaria RDT evaluation will be reported elsewhere.Malaria testing in clinics was performed using Rapid 1–2-3 malaria HEMA Express Rapid Diagnostic Test (RDT) , which tested for S. enterica typhi). The kit insert reported a sensitivity of 95% and sspecificity of 80%.Patients with a positive test on Tubex serology assay  were considered having TF (Brucella micro-agglutination test (BMAT), with minor modifications including the use of U-bottom plates, overnight incubation at 28 °C (for about 16 hrs), and discontinued use of safranin. Patients who tested positive on the Brucella IgM or Brucella IgG lateral flow assay and confirmed with BMAT were considered to have brucellosis.Brucella IgM testing was performed at the baseline visit, while IgG testing was performed on convalescent serum at the follow-up visit, using the Brucella IgM and IgG Lateral Flow Assays  respectively and following the manufacturer’s instructions. The assay was reported to have a combined IgM and IgG sensitivity of 96% and a specificity of 99% in a study done in Spain . ConfirmSpecimens were screened for leptospirosis per manufacturer’s instructions using the Leptospira IgM Lateral Flow Assay . Patients with positive IgM lateral flow assay were considered to have acute infection. An evaluation study done in Andaman islands, off the coast of India, reported sensitivities of 52.9% during the first week of illness and 86% during weeks 2–4. The corresponding specificities were 93.6% and 89.4% . All samR.rickettsii) and typhus group Rickettsia . Because multiple spotted fever rickettsial species, as well as R. prowazekii (another typhus group species), can occur in Africa, we cannot be sure of the actual etiology for each patient utilizing the group-broad antigens. Reactivity in the assays can only be assigned to the group and not to that particular species. Confirmation of acute, recent, or past infection of spotted fever rickettsiosis and typhus fever was based on IgG endpoint titers at baseline and/or a comparison of titers from baseline and convalescent serum samples following the manufacturer’s instructions.Rickettsia Immunofluorescent assays  were used for detection and semi-quantitation of human IgG antibodies reactive with broad whole-cell antigens of representative members of the spotted fever group Rickettsia  tested in parallel or an IgG serum endpoint titer of 1:256 or greater with a single sample for the respective rickettsial sero group. Past infection was considered when a single IgG titer was between 1:64 and less than 256 or two paired samples whose titers do not give a fourfold increase in titer. Samples whose titration IgG end point titers were less than 1:64 were considered to be negative.Samples collected on the baseline visit were screened for WNV using West Nile Virus IgM Capture ELISA , while the convalescent samples were tested using West Nile Virus IgG ELISA  test. Samples that were West Nile virus IgM Capture ELISA positive are reported as acute WNV infection and those positive on West Nile Virus IgG ELISA as past infections. According to the manufacturer, the IgM kit had a sensitivity of 99% and specificity of 100%; and IgG kit had a sensitivity of 90.4% and specificity of 100%.Specimens were screened for CHIKV using the Chikungunya IgM ELISA kit  to detect IgM antibodies to CHIKV. The manufacturer, reported this test as highly sensitive and specific . Samples that tested positive on chikungunya IgM ELISA at baseline and/or follow-up visits are reported as acute CHIKV infection.Specimens collected from patients at the baseline visit were screened for DENV using the Dengue NS1 Ag ELISA  detecting the dengue virus NS1 antigen. Patients that were positive on the dengue NS1 Ag ELISA were considered to have DENV infection. According to the manufacturer, the RDT kit had sensitivity of 93.3% and specificity of 98.9%.n=200) from St. Paul’s HC IV in Kasese district where TF outbreaks had been previously reported [A subset of samples , rainfall (mm Hg), and humidity (%). Geographic Information System (GIS) coordinates (latitudes and longitudes) and ground elevation (feet) of the study participants’ residences were captured by hand-held Garmin GIS equipment. Details on data modelling investigating the relationship between climatic and environmental factors, and AFI disease occurrence and distribution will be presented elsewhere.p-value of < 0.05 was considered statistically significant.Data were entered in Epi Info™ (version 3.5.2)  and analOverall, 1327 patients were screened at OPD during the baseline visit; 1281 (96.5%) of the participants were seen with complete sociodemographic and clinical information at baseline, and follow-up visits were included in the analysis. Of the 1281, 450 (35.1%), 382 (29.8%), and 449 (35.1%) were recruited from western, central, and northern regions, respectively ,  years (Table –93 yearsp = 0.008),  had at least one AFI pathogen identified. Age-groups 2–10 and 11–20 years had over 50% of CHIKV, malaria, TF and SFGR infections (Supplementary Table S% had at Participants presenting with a fever had a mean temperature of 37.9 °C (sd = 1.05 °C) and a fever duration of 3.8 days (sd = 2.2 days). Other predominant symptoms included headache, chills, abdominal pain, arthralgia, nausea, and myalgia  reported having seen an ectoparasite in the last month; 705 (55.0%) had slept under mosquito nets the night prior to consultation at the study clinics, and 25 (2.0%) had the interior walls of their dwellings sprayed against mosquitoes in the last 12 months prior to this study Table .Table 4BOnly 51 (25.5%) samples were collected for blood culture, and all tested negative for TF.Because there was potential for the internal validity of the study to be compromised due to delays in sample collection from the study clinic to the hospital laboratory for blood culture; these results were therefore discarded.0C (sd = 0.6) Vs 36.5 0C (sd = 0.9), p-value = 0.003.Information on clinical outcomes is available from 1125 (87.8%) of the participants. Of 1125 participants, 1087 (96.6%) reported both improvement on treatment and fever resolution, during the convalescent visit  was relatively higher than in studies done in Tanzania , SenegalTo the best of our knowledge, this is the first study in SSA to investigate the etiology of AFI among outpatient and ambulatory patients recruited from all age-groups with excellent clinical outcomes, on such a broad range of pathogens in several distinct regions differing by climate, environment, and population density , 24, 26.CHIKV RDTs validation in studies done elsewhere demonstrated much lower sensitivity and specificity , likely due to cross-reactivity with other alphaviruses . EvidencThis study revealed that 69.7% of the participants had evidence of non-malarial AFI pathogen infection. Rickettsial infections and arboviral infections were the predominant etiologies. Limited data on CHIKV , 47, WNVIn our study, malaria was confirmed by laboratory testing in 44% of the participants. By contrast, a similar study among hospitalized AFI patients in northern Tanzania reported that malaria was diagnosed clinically in 60.7% but confirmed in only 1.6% . WHO recAnopheles funestus [A. funestus is also one of the principal vectors for P. falciparum in Uganda [A. funestus mosquitoes harboring both malaria parasites and an arbovirus, resulting in dual infection. Alternatively, consecutive bites from two infected mosquitoes or species  as postulated by Sow et al. could also have resulted in the dual infection [Our study found that concurrent infections were common, with up to 45.0% of the participants having evidence of infection with more than one AFI-associated pathogen. Similar findings have been observed elsewhere in Africa for malarial , 33, andfunestus , and A. n Uganda , 59. Thenfection .Xenopsylla cheopsis), a common vector of murine typhus [There were statistically significant variations in the occurrence and distribution of some AFI diseases studied between study clinics. TF and TGR were identified more frequently in Kasese district (western region). In contrast, SFGR was more frequent in the northern region in Arua district. Variations in climate and environment in the areas where these clinics are located may account for this finding. The southwestern region, especially Kasese district, is characterized by frequent TF outbreaks , 60. In e typhus . Risk fae typhus ; the morBrucellosis has been recognized as a significant cause of AFI in some regions of Uganda; for example, a prevalence of 14.9% was reported among pastoralists in a rural community hospital in Southwestern Uganda . However0C in refrigerators at the study clinics for several days before shipment to UVRI. The refrigerators were subject to occasional power outages without generator backup. Fluctuation in storage temperatures might have led to potential specimen degradation and affected the internal validity of the study findings.This study had several limitations. 1) Reporting of the ectoparasites' past exposure status during interviews by enrolled participants was a potential source of recall bias. This was minimized by providing charts with photographs of the ectoparasites and limiting the period of exposure not exceeding one month prior to their clinic visit. (2) Using a stringent case definition with inclusion criteria for body temperature of ≥ 38.50 C but not ≥ 38.00 C, a commonly used threshold, the current study was amenable to selection bias. In addition, the recruitment of only outpatient and ambulatory patients was also another potential source of selection bias as excluded ill and hospitalized patients with AFI etiologies. It could be that the spectrum of infections among inpatient patients was different from what we observed in our study. (3) The possible serologic cross-reactivity within the arboviruses families might have led to non-differential misclassification bias of outcomes (with bias of risk ratios towards the null) for some of the arboviruses (i.e. CHIKV) leading to overestimation of the true burden of arboviruses . We were Reportin. [This collaborative study shows that malaria and rickettsioses are major causes of AFI in selected clinics in the central, northern, and western regions of Uganda. Arboviral infections and TF in some regions may contribute a substantial burden to AFI as well. Further research and development of non-malarial AFI serological tests could improve the etiologic diagnosis of AFI, though, implementation of serological tests may be costly and complex in a limited-resource setting. The development of a cost-effective multiplex point-of-care testing is a future prospect and has been proposed by Yager P et al. . This coAdditional file 1: Table S1. Recruitment strategy of AFI study participants based on malaria cases in 2007.Additional file 2: Table S2. Age-group distribution of AFI study participants, April 2011 to January 2013.Additional file 3: Table S3. Effect of loss to follow-up on study validity.Additional file 4: Table S4. Diagnoses by age-group distribution, April 2011 to January 2013.


Antibody-mediated complement fixation has previously been associated with protection against malaria in naturally acquired immunity. However, the process of early-life development of complement-fixing antibodies in infants, both in comparison to their respective mothers and to other immune parameters, remains less clear.P. falciparum-positive atypical memory B cells.We measured complement-fixing antibodies in newborns and their mothers in a malaria endemic area over 5 years follow-up and found that infants’ complement-fixing antibody levels were highest at birth, decreased until six months, then increased progressively until they were similar to birth at five years. Infants with high levels at birth experienced a faster decay of complement-fixing antibodies but showed similar levels to the low response group of newborns thereafter. No difference was observed in antibody levels between infant cord blood and mothers at delivery. The same result was found when categorized into high and low response groups, indicating placental transfer of antibodies. Complement-fixing antibodies were positively correlated with total schizont-specific IgG and IgM levels in mothers and infants at several time points. At nine months, complement-fixing antibodies were negatively correlated with total B cell frequency and osteopontin concentrations in the infants, while positively correlated with atypical memory B cells and P. falciparum merozoites are produced in the mothers and placentally-transferred, and they are acquired in infants over time during the first years of life. Understanding early life immune responses is crucial for developing a functional, long lasting malaria vaccine.This study indicates that complement-fixing antibodies against  In 2021 there were 247 million cases and 619,000 deaths globally . MoreoveP. falciparum, and the humoral response plays a crucial role in this process  are thought to be relatively protected from clinical malaria, but the underlying immunological mechanisms are not yet fully known. Infant immunity has traditionally been attributed to the passive transfer of maternal antimalarial IgG antibodies through the placenta in the final trimester of pregnancy , 26. TheP. falciparum functions to mediate protection, including the rate of their development and their antigenic targets. Furthermore, it remains uncertain whether the levels and rate of decay of infant antimalarial antibodies rely on maternal antibody levels at birth, and which specific antibodies are transferred across the placenta.Currently, it remains unclear how the subsequent development of infant antibodies in response to natural exposure to P. falciparum-specific B cell subsets, as well as antibodies to P. falciparum schizont extract. Our study aims to provide insights into the development of functional antimalarial antibody responses in early childhood and the correlation with immune responses against malaria.Our study aims to address this gap in knowledge by examining functional infant antimalarial antibody responses during the development of naturally acquired immunity against malaria. Specifically, we have measured the levels of anti-merozoite C1q-fixing antibodies in cord blood and compared this to the mothers’ levels to determine the transplacental ratio. We also monitored the levels in the children until they reached 5-6 years of age and explored correlations between levels of C1q-fixing antibodies and different 22.1The first part of the study was conducted in Kasangati in Uganda, where malaria transmission is moderate . In brie2.2All samples underwent testing using Rapid Diagnostic Test (RDT) pLDH/HRP2 Combo . For all positive samples, thin and thick blood smears were examined by microscopy, and parasitemia was calculated in accordance with WHO guidelines .2.3P. falciparum was cultured at 37°C as described . To prevent batch/plate effects, longitudinal samples from the same mother-baby pairs were always analyzed on the same plates.The level of complement fixation to merozoite crude antigens was assessed as described  with som2.5P. falciparum+ IgG and IgM in plasma was conducted as previously described   were identified using carboxyl Quantum dots (Invitrogen) conjugated to extract of trophozoite/schizont-stage parasites (The immunophenotyping of ckinson) : IgG memarasites , 44.2.7Differences in antibody levels between groups were assessed by Wilcoxon matched-pairs signed rank and by Mann-Whitney test for paired/unpaired samples, respectively. To account for repeated measurements linear mixed model was used. Continuous variables were expressed as estimated means in AR  covarian33.1We analyzed complement-fixing antibodies in mother-infant pairs. Complement fixing activity was measured as the ability of antibodies to fix C1q, the first and essential step in the classical pathway of complement system activation. C1q fixation by antibodies to merozoites correlates with subsequent complement activation and formation of the C5-9 complex . We foll3.2The estimated mean levels of complement-fixing antibodies in infants at different time points were analyzed using linear mixed models, Figure 13.3Median complement-fixing antibody levels in the mothers remained unchanged from delivery to 9 months postpartum Figure 23.4There was no significant difference in median complement-fixing antibody levels between infant cord blood (0.36 (IQR: 0.31)) and median plasma levels in mothers at delivery (0.40 (IQR: 0.33))  and microscopy. However, upon analyzing the complement-fixing antibody levels, we did not observe any significant differences based on the degree of parasitemia . We concluded that our sample size was not large enough to detect any potential statistical differences.At delivery, among 131 mothers and 129 infants, only six mothers and one infant tested positive for 3.7Schizont specific IgG and IgM has been widely used as a broad marker of exposure to blood-stage malaria , 45, 46.3.8P. falciparum extract (Pf+ B cells), previously analyzed in this cohort   binding to s cohort . For inf3.9Both OPN and BAFF have important roles in the production of immunoglobulins and B cell differentiation. Our prior studies within the same cohort have shown interesting correlations between these factors in the context of malaria , 47. Res4Understanding the functional mechanisms that underlie naturally acquired immunity against malaria is of critical importance for the development of an effective vaccine. However, the exact process by which immunity is acquired is not yet fully understood especially among young children who are the primary target of malaria vaccines. In this study, we present, for the first time, the dynamics of complement-fixing antibodies in a longitudinal cohort of healthy mother-infant pairs residing in a malaria-endemic region of Uganda, from birth until five years of age. We show that complement-fixing antibodies in infants gradually decrease during the first six months of life, probably due to natural decay of antibodies transferred from the mothers. We further demonstrate that by five years of age, the antibody responses have nearly reached the same levels as those observed in both cord blood at birth and in the mothers.In malaria-endemic regions, it is widely acknowledged that children develop partial immunity against malaria during their first few years of life, while infants below 4-6 months of age seldom experience clinical malaria, despite the possibility of having sub-microscopic and asymptomatic infections –51. ThisP. falciparum parasitemia using more sensitive molecular methods, such as qPCR, was not performed; therefore, we may have missed infections. None of the participants in our cohort presented with malaria symptoms and those with parasitemia were most likely asymptomatic carriers of P. falciparum, or at least did not have fever or other obvious symptoms of infection. All 109 women had received intermittent preventive therapy and mosquito bed nets during their pregnancies, which could explain the low prevalence of parasitemia. Given that all women resided in close proximity to the clinic and needed to bring their children for multiple follow-up visits, we anticipate that the transmission of malaria would be similar among all individuals.When we correlated our data for complement-fixing antibodies to schizont-specific IgG and IgM, we were surprised to discover correlations in the mothers only at delivery, but not nine months later. The majority of the mothers have resided in the same area for several years, encountering malaria multiple times, suggesting a relatively long-developed immunity. Interestingly, a previous study on individuals with clinical malaria reported strong associations between complement-fixing antibodies and merozoite-specific IgM as well as subclasses IgG1 and IgG3 . Our resIn our assay, merozoite extract is bound to the plate and both IgG and IgM can bind to the merozoites, even though we only measure the levels of IgG. This could also mean that if there are very high levels of IgM, it could compete with binding of IgG. Nevertheless, considering the decline in IgG levels among infants in the months following birth, we do not consider this to be a concern for the overall interpretation of our results, even though it might have the potential to influence certain individual outcomes.P. falciparum-specific IgG from mothers to infants, providing early-life protection. Our findings align with previous studies on malaria-specific antibody responses in sub-Saharan African birth cohorts  compared to younger children, and had reached similar levels to those observed in adults , and  unclear , 68, 69.cal MBCs , 47, butn adults . Howevern adults . This inn adults , 15, 16.Upon dividing the infants into high and low complement-fixing antibody responders at birth, our findings revealed a faster decay rate of maternal complement-fixing antibodies in infants with initially high antibody levels. These results are in line with a study conducted on a birth cohort in Kenya, which demonstrated that the rate of decline of maternal total IgG levels against five recombinant merozoite antigens was inversely proportional to the starting levels in cord blood . MoreoveP. falciparum, as evidenced also by prior studies indicating that high levels of exposure result in high levels of antibodies , and Regionala Etikprövningsnämnden in Stockholm, Sweden (2014/478-32).SM: Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – review & editing, Data curation, Formal Analysis, Writing – original draft. AL: Data curation, Writing – review & editing. MN: Data curation, Formal Analysis, Investigation, Writing – review & editing. LD: Data curation, Formal Analysis, Methodology, Writing – review & editing. MT: Conceptualization, Investigation, Methodology, Writing – review & editing. JB: Conceptualization, Investigation, Methodology, Writing – review & editing. KP: Conceptualization, Investigation, Methodology, Writing – review & editing, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Formal Analysis.


Plasmodium spp. threatening millions of people in tropical and subtropical countries. The disease affected 247 million people globally and 619,000 deaths in 2021 increasing from 229 million cases and 409,000 deaths in 2019  as well as to different types of endothelial cells. The P. knowlesi-IRBCs displayed low affinity to brain (hCMEC/D3) as compared to the lungs (HPMEC) and kidneys (HRGEC) endothelial cell lines. Further, the IRBCs-endothelial cytoadherence affinity increased after priming with P. knowlesi culture supernatant in the lungs and kidneys, however not in cerebral endothelial cells. The research work is a novel finding that provides a framework for further studies to unravel P. knowlesi ligands and host receptors involved in the phenomenon.Rosetting and Cytoadherence leading to sequestration of infected RBCs (IRBCs) in deep vasculature are well known phenomenon associated with severity of Xie et al. revealed the gut microbiota changes in C57BL/6 mice after infection with P. berghei ANKA and in relation to the macrophage migration inhibitory factor (MIF). Human MIF is a pro-inflammatory factor and its increased level is life risk indicator in cerebral malaria (CM)  in P. vivax infections by screening different P. vivax patient isolates and primary human hepatocyte (PHH) lots. The findings suggested the involvement of both host hepatocytes parameters as well as P. vivax isolate characteristics as determinants of P. vivax infection progression in liver. Hepatocyte donor lot influenced hypnozoite formation rate and fating toward hypnozoites is more dependent on the P. vivax isolates. Further, researchers found that liver stage schizont growth was mainly influenced by the PHH donor lot. The sporozoite inoculum size was not found to be a regulator of sporozoite fating. The work underlines determinants of hypnozoite formation and need of future studies to better understand the liver stage parasite biology.After Djihinto et al. discussed the malaria-transmitting vectors microbiota as an innovative approach to target insect vectors based on “symbiotic control.” The review covered the viral, fungal, bacterial and bacteriophages diversity of Anopheles mosquitoes. Mosquito microbiota has been explored in search of new innovative tools to control the vector. The interplay between the vector microbiota and immune responses is one of the crucial aspects in developing mosquito resistance to the pathogen that could be exploited for effective management of the parasite  P. falciparum, Non-human primate spp., P. berghei followed by P. yoelii. Further, a detailed glimpse of reticulocyte tropism mechanisms, advantages, drawbacks and repercussions has been discussed. The review pins reticulocytes characteristics viz. nutrient rich environment, Glucose-6-phosphate dehydrogenase elevated level, membrane stability and zoonotic potential that might be favoring parasite propagation and thus underlines the need of interventions targeting reticulocytes.Erythrocyte tropism is an important phenomenon exhibited by the P. falciparum, the most deadly parasite, received all attention of researchers and policy makers to combat malaria whereas other non-falciparum Plasmodium spp. remain neglected. A comprehensive review by Brashear and Cui covered developments in population genomics of non-falciparum Plasmodium spp. vivax, malariae and ovale referred as “neglected malaria parasites”. These non-falciparum Plasmodium species set a challenge for malaria elimination efforts due to distinctive biological hallmarks favoring sustained transmission. Authors discussed challenges faced in whole-genome sequencing (WGS) of these non-culturable species and ways to overcome hurdles. Further, genome annotations of non falciparum malaria species need improvements and more focused inputs. Mapping SNP diversity of P. vivax population established differences among different populations. The authors elaborated applications of population genomics in understanding parasite biology , observing temporal changes in parasite populations, tracking parasite origins, and migrations, identification of natural selection signatures leading parasite evolution. At the end, authors emphasized significance of development of SNP barcodes for high-throughput genotyping and need of population genomic studies in zoonotic human infecting Plasmodium spp. The authors presented a thought evoking review about importance of population genomics and its role in designing innovative strategies thus channelizing malaria elimination efforts.Frontiers in malaria research” focussed on revealing cytoadherence properties of P. knowlesi, a detailed overview of malaria-transmitting vectors microbiota, factors for liver-stage fate determination in P. vivax, variations in host microbiota after Plasmodium infection and MIF knockdown deciphering microbial biomarkers, a detailed glimpse of erythrocyte tropism of different Plasmodium spp. and developments in population genomics of neglected malaria parasites. The Research Topic gave thoughtful insights about basic biology and interactions of all three players of malaria- parasite, vector, and human host. The concepts gained will add on to the efforts of curtailing malaria burden. Finally, tackling the malaria parasite requires innovative tools, concerted efforts and scientific collaborations to reach malaria free world milestone.The contributions to the Research Topic “RG prepared the first draft of the manuscript. RH, MA, AB, TB, and AS revised the MS. All authors approved the final submitted version.