Plasmodium vivax circumsporozoite (CS) protein in healthy malaria-naïve (phase IIa) and semi-immune (phase IIb) volunteers. Participants (n = 35) were randomly selected from a larger group (n = 121) and further divided into naïve (n = 17) and semi-immune (n = 18) groups and were immunized at months 0, 2, and 6 with PvCS formulated in Montanide ISA-51 adjuvant or placebo . Specific antibodies and IFN-γ responses to PvCS were determined as secondary outcome; all experimental volunteers developed specific IgG and IFN-γ. Three months after the last immunization, all participants were subjected to controlled human malaria infection. All naive controls became infected and drastic parasitemia reduction, including sterile protection, developed in several experimental volunteers in phase IIa (6/11)  and phase IIb (7/11) . However, no difference in parasitemia was observed between the phase IIb experimental and control subgroups. In conclusion, this study demonstrates significant protection in both naïve and semi-immune volunteers, encouraging further PvCS vaccine clinical development. Trial registration number NCT 02083068. This trial was funded by Colciencias (grant 529-2009), NHLBI (grant RHL086488 A), and MVDC/CIV Foundation (grant 2014-1206).A randomized, double-blind, controlled vaccine clinical trial was conducted to assess, as the primary outcome, the safety and protective efficacy of the  Plasmodium vivax circumsporozoite vaccine in naïve and semi-immune individuals from controlled human malaria infection as well as antibody and IFN-γ response to vaccination.In this phase 2 clinical trial, the authors assess protective efficacy of a  Plasmodium vivax is the second species of epidemiological importance with a wide geographical distribution in Asia, Oceania, and the American continents, where it coexists with P. falciparum, producing ~6.5 million cases annually1. Although it has been historically considered to cause benign disease, P. vivax infection results in a very algid febrile syndrome with headache and malaise and frequent pulmonary and hematological manifestations3. Moreover, severe illnesses, including cerebral malaria and death, have been recently documented5. Notably, the infection produces hypnozoites, dormant liver parasite forms that relapse periodically, generating clinical manifestations and contributing to an undetermined proportion of the P. vivax incidence worldwide7. Substantial evidence supports the feasibility of developing malaria vaccines, which are considered valuable tools to complement classical malaria control strategies8. P. falciparum RTS.S/AS01E, based on the circumsporozoite (CS) protein, is currently the most advanced malaria vaccine candidate with a mean efficacy of ~34% in phase III trials9. Recently the World Health Organization (WHO) recommended widespread use of the RTS,S/AS01  malaria vaccine among children in sub-Saharan Africa and other regions with moderate to high P. falciparum malaria transmission10. The recommendation is based on a pilot implementation program ongoing since 2019 in Ghana, Kenya, and Malawi in more than 800,000 children11; in addition, several other candidates are in advanced clinical development13. Although P. vivax vaccines have received significantly less attention, a systematic analysis of the P. vivax CS antigen (PvCS) has been performed over the last more than 20 years14, leading to promising results for its development.An estimated ~229 million clinical cases and >409,000 deaths occurred worldwide in 2019 due to malaria, with a substantial economic impact on populations living in developing regions16, three long synthetic peptides (LSP) covering the amino-terminal (N), the central repeats (R), and C-terminal regions were designed and synthesized. Studies in BALB/c mice and Aotus monkeys showed high LSP immunogenicity17. Phase I vaccine clinical studies were subsequently conducted in healthy malaria-naive volunteers (n = 69) to individually evaluate the three LSP formulated in Montanide ISA-720 . A vaccine dose-escalating protocol using 10, 30, and 100 μg/dose18 indicated good safety, tolerability, and immunogenicity in a phase Ia trial. A second phase I study was conducted in 40 volunteers who were vaccinated three times with different combinations of the peptides formulated in either Montanide ISA-720 or Montanide ISA-51 at 50 and 100 μg/dose19. Because of the known immunodominance of the R fragment, it was only included in two of the immunization doses to equilibrate the response to the three protein fragments. Vaccine formulations were well-tolerated, and no serious adverse events (SAE) were observed. All immunized individuals seroconverted and developed comparable ELISA titers of antibodies to the three protein fragments, which also recognized the native protein on P. vivax sporozoites as determined by indirect immunofluorescence test (IFAT). Antibodies to the three fragments inhibited sporozoite invasion (ISI) to liver-cell lines in vitro, in the same proportion20.After identification of multiple B- and T-cell epitopesP. vivax sporozoite controlled human malaria infection (CHMI) in healthy and semi-immune volunteers23. PvCS recombinant products were also developed and tested in phase I trials in studies by other groups. In those studies, the R region of the VK210 P. vivax variant was expressed in E. coli24, formulated in Alum, and tested in 13 volunteers (doses ranging from 10 to 1000 μg/dose), displaying a good safety profile but low and no boostable ELISA antibody response. A recombinant protein expressing 70% of the PvCS protein sequence was produced in yeast25, formulated in Alum and tested in doses ranging from 50 to 400 μg/dose. Volunteers exposed (n = 30) to the higher doses (200–400 μg/dose) generated minimal humoral and cellular responses. Then, a hybrid E. coli recombinant construct (VMP001) encompassing VK210 and VK247 repeats alleles26 was expressed, formulated in AS01B adjuvant, and evaluated in a phase I/IIa vaccine trial. Volunteers (n = 30) developed robust humoral and T-cell responses, and a slight delay in patency (1–2 days) was observed in 59% of the volunteers.Further studies allowed the standardization of a PvCS LSP phase I trials and the establishment of P. vivax sporozoite CHMI protocols, a comparative phase IIa/IIb clinical trial was designed and conducted in a malaria-free region of Colombia to evaluate the safety and protective efficacy of PvCS LSP formulated in Montanide ISA-51 adjuvant in healthy malaria-naive (phase IIa) individuals and the feasibility of the same study protocol in semi-immune volunteers (phase IIb).Based on the reproducible results of the 27. From a larger group of interviewed subjects, 121 accepted to be screened to participate in the trial   was conducted as in previous clinical studies28. Pain occurred more frequently in the Exp (16 subjects) than in the Ctrl (8 subjects) groups. Fever, nausea, chills, diarrhea, and abdominal pain occurred at low frequencies during the vaccination period  reported adverse event (AE) during vaccination 24/32), followed by headache in 31.25% (10/32) and malaise in 31.25% (10/32), which resolved in all cases the next day. These AEs were scored as mild (grade 1) to moderate (grade 2) according to the FDA guidelines4/32, folMild to moderate biochemical or hematological laboratory-related AE were observed. Mild anemia (10.7–11.5 g/dL) occurred in two naive females and two semi-immune  volunteers after the first immunization ; however, all volunteers normalized before the CHMI. Transient low-level proteinuria (Grade 1–2) was observed after the second immunization in the naive group but reached normal values in the following month. Two semi-immune volunteers presented prolonged thromboplastin time . After the third immunization, a volunteer showed glycosuria of 500 mg/dL and glycosylated hemoglobin of 8.1%  and was diagnosed with diabetes mellitus unrelated to vaccination ; all control volunteers remained seronegative during the immunization and infection phases  and enzyme-linked immunosorbent assay (ELISA) response to parasite blood-forms antigens, although all volunteers were negative to the PvCS.Positive ELISA using the N- and C-peptides indicated seroconversion of all 22 naive and semi-immune Exp volunteers after the first immunization. However, despite their previous parasite exposure, lower reactivity was observed in the semi-immune group than in the naive group. The second immunization induced a moderate boosting of antibodies to the amino (N-) and carboxyl (C-) fragments, as well as priming of the response to the R region. After the third immunization, a slight but significant boosting of antibodies to the three fragments was observed in some volunteers, higher in the naive than in the semi-immune groups  nor in the semi-immune group  titers presented association with infection (parasitemia levels).A regression analysis of the antibody titers to the protein fragments  determined by ELISA on months 7 and 10 indicated that neither in the naive group . IFN-γ production developed in all volunteers upon cell stimulation with the protein fragments in an unstable manner throughout the immunization phase, indicating no clear boosting trend through the immunizations period. However, a slight increase of IFN-γ levels developed at time 2 in the naive group against N (p = 0.0188) and R (p = 0.0060) peptides  nor the semi-immune IFN-γ levels  determined at month 7 before CHMI, were associated with parasitemia. Additionally, there was no boosting of the IFN-γ response after sporozoites CHMI; instead, there were decreased cytokine levels in both groups after CHMI. This decrease was highly significant in the naive group  whereas it was non-significant in the semi-immune group   was composed of 36.36% men and 63.64% women, while the Ctrl group (n = 5) had 20% men and 80% women, aged 19–43 years. In these groups, patent parasitemia developed between days 14 and 17 (mean 15.7 days), with no difference between the Ctrl (15.8) and Exp (15.7) volunteers. Furthermore, all naive-Ctrl volunteers (5/5) (100%) were infected, whereas, in the Exp group, a total of 6/11  individuals displayed a reduction of the parasite load <100 parasites/μL. This parasitemia level was considered the threshold as it was below the minimal parasitemia in naive Ctrl (100 parasites/μL) and corresponded to quartile 1 . Notably, 4/6  of the protected volunteers did not develop patent parasitemia over the 60 days follow-up, indicating overall sterile protection of 4/11  in the phase IIa trial; and general vaccine efficacy of 55% (1- RR = 0.45)  and Exp (7/11) individuals in the naive group (phase IIa) and from days 12 to 19 by Ctrl (4/5), and Exp (8/11) in semi-immune volunteers (phase IIb), which lasted until malaria treatment. The naive Exp group (/5 and Exn = 11), 45.45% were men and 54.55% women, while the Ctrl group (n = 5) had 20% men and 80% women, all volunteers aged between 19 and 28 years. In this phase, patent parasitemia developed between days 12 and 19 (mean 16.1 days), with no difference between the mean patency of the semi-immune Ctrl (16.7) and Exp (17.6) volunteers. Using the protection cutoff of the Phase IIa study, three volunteers of the semi-immune Ctrl group   were considered protected as parasitemia levels were ≤100 parasites/μL, including the volunteer (CSI 572) that did not develop patent parasitemia. Likewise, 3/7 semi-immune Exp volunteers  did not develop patent parasitemia over the 60 days follow-up. The reduction in infection intensity and frequency in the Ctrl  and Exp  groups indicated no difference. However, when the prepatent periods of the Exp naive (15.8) and semi-immune (17.6) groups were compared, we found that there was a significant difference (p = 0.0034) . Furthermore, the phase IIb component analysis indicated an even greater reduction of parasitemia frequency and intensity in the Exp group ; however, the reduction observed in 3/5 volunteers of the corresponding Ctrl group , makes it difficult to establish protection in this phase independently. Nevertheless, when compared with the phase IIa (naive) groups, the Phase IIb group (semi-immune) displayed a 2 days delay (17.6) (p = 0.0034) in the prepatent period, which is similar to that induced by the recombinant VMP001 PvCS vaccine formulation in 59% of the naive vaccinees29. In addition, the number of volunteers with lower parasitemia and sterile immunity indicate a significant protective efficacy of the PvCS, both in naive (Phase IIa) and semi-immune volunteers (Phase IIb).This study confirmed the safety, tolerability, and immunogenicity observed in the previous phase I vaccine studies with similar Although the outcome of the phase IIb component is less conclusive, the result is encouraging and has provided valuable bases for further Phase IIb trials with larger groups, subjected to CHMI, directly in endemic areas.30 recombinant PvCS VMP00126 and phase I PvCS LSP formulated Montanide ISA-5131. This tolerability is in contrast with unacceptable reactogenicity previously described in HIV- and malaria Pfs-25 vaccines formulated in Montanide adjuvants32. Regarding the infection experienced by one of the semi-immune Ctrl participants after returning to the endemic area, it may have corresponded to a reinfection. Unfortunately, the volunteer was in a rural setting, and the parasites were not available for genotyping; this patient was one of the not protected volunteers.Importantly, AEs related to vaccination occurred with similar frequency in all groups. AEs were limited to local, transient pain at the vaccine injection site, similar to those reported before with commercially available vaccines19. The lower antibody levels of the semi-immune groups  as well as the IFN-γ had also been reported23. Although the antibody response was higher in the naive group, we did not find any association between antibody titers and specificity to the N, R, or C at the time of the CHMI, and protection. The lack of significance between protection and immune response may be due to the small size of the final groups, as well as to the highly variable immune response among participants, likely due to the population heterogeneity in terms of ethnicity, sex, and nutrition status among other potential factors. Moreover, the results are in agreement with a previous study26 where no correlation was found when we analyzed antibody titers and IFN-γ responses but continues to be intriguing as both effector mechanisms have been experimentally shown to be associated with protection against Plasmodium pre-erythrocytic infection34.In terms of immunogenicity, these studies confirmed the results of previous phase I trials where all volunteers displayed the induction of antibody and IFN-γ specific responses to the three LSP in a fragment-balanced fashion; both the antibody patterns and the unstable behavior of IFN-γ resembled those of the previous trials35.The hypo-responsiveness of the semi-immune Exp groups in terms of specific IgG and IFN-γ to the immunogens is also in contrast with the delay in patency displayed by this group of volunteers. However, the identification of surrogate markers or signatures of immune protection remains elusive even by using systems biologyγ decrease in the naive group   Fig.  might ben = 4), which reduced the power of the study, (2) the volunteers’ ethnic heterogeneity in both Ctrl and Exp groups, (3) a possible dysregulation of the semi-immune volunteers; and potentially other factors35, generating a great heterogeneity, and (4) the semi-immune status of phase IIb volunteers, where protection mechanisms may be different from the ones studied here, which warrants further studies with larger groups.The contrast between the outcomes of phase IIa and IIb appears to be related to (1) the reduced number of volunteers remaining in the Ctrl group  both phase IIa and phase IIb were simultaneously conducted, and (2) we deliberately avoided inclusion of the PvCS R-fragment in the first vaccine dose to diminish its immunodominance. The rationale behind simultaneously conducting phase IIa/phase IIb trial is based on Cali being a malaria-free city in Colombia with conditions to explore the feasibility of comparative vaccine trials under similar conditions, and Buenaventura a malaria-endemic town, located at ~70 miles from Cali. In addition, the response of naive and semi-immune volunteers to P. vivax sporozoite CHMI had been already evaluated23. Furthermore, the geographic and epidemiological characteristics provided privileged conditions for this proof-of-principle on the feasibility of closely controlled phase IIb P. vivax pre-erythrocytic vaccine trials.Two main features make the design of these 35.The overall data are valuable in different ways, (1) they indicate an important degree of protection (54.5%) in the naive vaccinated group, with remarkable sterile immunity (36%), and potentially greater efficacy (≥60%) in semi-immune populations, (2) they highlight the importance of CHMI to ensure volunteers’ exposure to parasites in a controlled manner, (3) they provide a foundation to further cost-effective phase II trials, and (4) they warrant efficient comparison of the immune response, i.e., using systems biology and system-level data analysis for comparing populations exposed to different environmental conditionsPf-RTS,S39 showed a similar hypo-responsiveness, suggesting that individuals from malaria-endemic regions, either actively infected or not, display an altered basal immune status with a paucity of regulatory mechanisms and altered memory cell function leading to lower responsiveness to vaccines41. It has been hypothesized that this corresponds to an immunological imbalance caused by permanent exposure to malaria parasites, mosquito bites, and other host and environmental factors such as sex, ethnicity, nutrition, malaria endemicity, and others that may influence the host’s immune response and immunity to malaria in endemic areas. Although these studies do not provide further light on the correlates of protection, they generated reagents for further identifying or confirming potential critical PvCS epitopes.Recent studies on the response of individuals from malaria-endemic and non-endemic areas to P. vivax infections, and generated preliminary data for further and larger Phase IIb trials.One of the limitations of this study was volunteer retention. Although semi-immune volunteers were requested to be outside the endemic area for the parasitemia follow-up, some decided to return to their homes about a month after CHMI, making follow-up more challenging. Nevertheless, it provided a unique opportunity to directly assess the vaccine candidate’s protective efficacy in volunteers previously exposed to natural P. vivax malaria in endemic areas contributed to the vaccine-elicited immunity and therefore it has to be evaluated differently. Because the study sample was not representative of the whole population of individuals living in the country’s malaria-endemic areas, a comparative trial with larger volunteer groups is being prepared (NCT 04739917) in malaria-endemic and non-endemic regions and will address ethnicity; that trial will include more powerful system biology and system data analyses. A highly efficacious P. vivax pre-erythrocytic vaccine is of utmost importance due to the high relapsing rate of this parasite species in some regions of the world7.The differential protective efficacy achieved here may have been influenced by ethnic factors and the final limited sample size. While all naive Exp and Ctrl volunteers were mestizo, the semi-immune group was heterogeneous . Alternatively, one could hypothesize that the levels of premunition to The study protocol was reviewed and approved by the Institutional Review Boards of the Malaria Vaccine and Drug Development Center (MVDC-CECIV) and Centro Médico Imbanaco (CMI # 0992304-493-26202)  for P. vivax.All study participants complied with the following general criteria. (1) Healthy men and women aged 18–45 years; (2) participants freely and voluntarily providing signed IC, accompanied by two witnesses signatures; (3) for women, adequate contraception from the time of enrollment; (4) accept not to travel to areas considered endemic for malaria during the infection follow-up period (1 month); (5) be reachable by phone throughout the study period; (6) being Duffy blood group positive (Fy+); (7) harboring Hb levels >11 g/dL; (8) availability to attend all visits during the study period; (9) not participating in other clinical studies. Naive volunteers had no history of malaria infection, whereas semi-immune volunteers had a history of previous malaria infection(s) and positive serological tests for P. vivax infected blood for sporozoite production had to comply with the following criteria: (1) Be healthy men and women aged 18–45 years; (2) harbor P. vivax infection with parasitemia ≥0.1% as determined by thick blood smear examination; (3) not having P. falciparum or P. malariae circulating malaria parasites; (4) Hb levels ≥9 g/dL at the time of malaria diagnosis; (5) have the capacity to provide IC freely and voluntarily. Should he/she be illiterate, accept to assert the decision to participate by a fingerprint in IC form; (6) if the potential donors were minors aged 15 to 17 years, sign the IC, and one of his/her parents must sign the IC, accompanied by two additional witness signatures. Potential parasite donors were excluded from enrolment if (1) they had negative IFAT (<1:20) for P. vivax in screening tests, (2) if patients had chronic or acute disease, different from malaria by P. vivax; (3) hemoglobin levels <9 g/dL at the time of recruitment, (4) having received antimalarial treatment before blood draw; (5) having a history of disease or clinical conditions that according to medical criteria might significantly increase their risk by participating in the study.Malaria patients accepting to provide PvCS formulated in Montanide ISA-51. Thirty-five healthy, Fy+ men and non-pregnant women (19–44 years of age) were recruited from a larger group (n = 121) and allocated into two groups: phase IIa with healthy malaria-naive (n = 17), all mestizo  with age range of 20–43 years; and phase IIb with malaria semi-immune (n = 18) divided into afrodescendants (n = 3), mestizo (n = 13), and indigenous (n = 2), with age range of 19–44 years of age. Volunteers were recruited as before27 using several communication strategies, including mass media approaches. Participants were recruited from October 3, 2014 (first patient) to December 22, 2014 (last patient) and were randomly (simple) assigned in a 2:1 ratio based on the pre-specified (study protocol- Supplement Note n = 25) or placebo   was further divided into Exp (n = 12) and Ctrl (n = 5), as was the semi-immune group [n = 18 into Exp (n = 13) and Ctrl (n = 5)]. Naive volunteers were from Cali , a malaria-free area, located at the southwest of Colombia at ~1.000 m.a.s.l. Volunteers were eligible based on no history of malaria and negative P. vivax serology. The semi-immune volunteers were recruited in Buenaventura, the main port on the Pacific coast of Colombia, at 80 km from Cali with an altitude of 7 m.a.s.l. The region is a low to moderate malaria-endemic area were both P. vivax and P. falciparum parasites are transmitted. Selected semi-immune volunteers had a history of malaria and antibodies against P. vivax blood stages with titers by immunofluorescence (IFAT ≥ 1:20) or enzyme-linked immunosorbent assay (ELISA ≥ 1:200) against a P. vivax recombinant MSP-1 protein42.The naive group  20–96 (N-term), and the C peptide to C-terminal aa 301–372 (C-term). In contrast, the R peptide VK210 (type I) corresponded to a construct based on the first central repeat (aa 96–104) in tandem three times and collinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin43  in the same proportion according to manufacturer recommendations on the same day of subject immunizations. Saline solution  was emulsified with the same adjuvant and used as a placebo.The three LSP  synthesized under good laboratory practices (GLP) conditions at the Biochemistry Institute, University of Lausanne, Switzerland, were packaged, lyophilized, and tested for sterility and apyrogenicity . As previously describedP. vivax CS LSP vaccine’s protective efficacy against the P. vivax CHMI in malaria-naive and semi-immune volunteers as determined by the parasite reduction in terms of frequency and density, and the secondary outcome was the B and T-cell immune response associated with protection. During the immunization period, blood samples were collected before the first immunization and at several time points thereafter to study the immune response and the blood chemistry. Enrolled participants received three vaccine doses (Exp group) or placebo (Ctrl group) at months 0, 2, and 6 by i.m. injection in the deltoid muscle with a volume of 0.5 mL. Vaccines were prepared by staff researchers not involved with patient care.The primary outcome was the P. vivax blood infection45, and normal G6PD status to prevent the risk of hemolytic anemia upon P. vivax treatment with primaquine47. Complete blood count (CBC), prothrombin time (PT), partial thromboplastin time (PTT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, alkaline phosphatase, blood urea nitrogen (BUN), creatinine, and pregnancy were performed. During the immunizations period, volunteers were under direct medical supervision during the hour following immunization to detect any adverse reaction to the vaccine injection, after which a physical examination was performed. Eight hours post-injection, volunteers’ physical status was assessed by a telephone call. Also, a personal follow-up was conducted 1 week before the following immunizations. Clinical laboratory tests evaluated the vaccine tolerability and safety at months 0, 1, 2, 3, 6, 7, 9, and 10. Volunteers were also under observation for 1 h after the CHMI and then by phone monitoring 8 h after and once a day until day 423. Volunteers were then evaluated daily for clinical manifestations and microscopic patent parasitemia from days 5 to 30 after the challenge and every second day until day 60. Two experienced, independent microscopists evaluated parasitemia by counting the number of asexual P. vivax parasites per 400 white blood cells (WBC), assuming normal WBC counts (8000 cell/μL). Samples were considered negative after observing 200 microscopic fields, and qPCR was performed subsequently for retrospective analysis. Adverse events (AE) were recorded, graded, and classified according to FDA recommendations28.At enrolment, blood samples were collected to determine Fy blood group and G6PD deficiency. Fy+ was confirmed to ensure the volunteer’s susceptibility to P. vivax in Leticia, Colombia, and used to infect colonized Anopheles albimanus mosquitoes. Fed mosquito batches were dissected and microscopically examined for the presence of oocysts in the midgut (day 7) and sporozoites in salivary glands (day 14) as previously described17. For CHMI, only batches with ≥60% sporozoite infection rates were considered acceptable. CHMI was performed 3 months after the last immunization by volunteers’ exposure to 2–4 P. vivax-infected mosquito bites22. After biting, individual mosquito dissection confirmed the presence of blood in midguts and sporozoites in salivary glands22.Whole blood (15 mL) was collected by venipuncture  from patients diagnosed with VK210 18. Controls were selected from a pool of sera from semi-immune blood donors (positive ctrl) and a pool of sera from malaria-naive donors (negative ctrl). In addition, parasite recognition by anti-LSP antibodies was determined by IFAT using P. vivax fixed sporozoites18. Previous malaria contact in the group of semi-immune volunteers was confirmed by IFAT using parasite blood forms and ELISA test using a Pv-MSP-1 recombinant protein42.Antibody response was assessed using blood samples collected on months 0, 1, 3, 7, and 10 before immunization and CHMI and measured by ELISA with N, R, or C peptides (1 μg/mL) antigensγ, as previously described19. Briefly, fresh PBMCs (4 × log 105/well) were mixed with 10 μg/mL of each LSP, and after 40 h culture, the number of IFN-γ spot-forming cells (sfc) was counted using an ELIspot reader ; results were expressed as the mean number of IFN-γ sfc per log106 PBMC. In each assay, PBMC were also incubated in the absence of peptides to determine the IFN-γ background production level. The background level of each test was subtracted from the specific counts obtained from peptide stimulated cells. Volunteers were considered responders if the number of sfc in their samples increased from their baseline level; any increase ≥5 sfc was considered positive19.Peripheral blood mononuclear cells (PBMC) were separated immediately after whole blood was collected by venipuncture, at months 0, 1, 3, 7, and 10, using Ficoll-Histopaque  density gradients. PBMC was used to determine the ex vivo production of IFN-P. vivax infection in volunteers vaccinated with PvCS LSP formulated in Montanide ISA-51. The study sample size was calculated with a confidence level of 95%, taking into account a previous census of ~5603 subjects with recent malaria infections in Buenaventura48, where the estimated prevalence of the Fy+ genotype (30%) and G6PD deficiency (12%) in the target population indicated that only 1479 subjects would be suitable for participation. Likewise, we estimated that only 3–10% of the subjects (44–147) would volunteer for the trial based on previous studies in the same area.Data were collected and managed using REDCap 5.10.1  electronic data capture tools, analyzed using SPSS version 16.0 software , and plotted using Graph Pad Prism version 6.0 . The primary outcome evaluated the frequency and density of p values ≤0.05 considered significant. A T-student test was used to compare differences between the response to N, R, C fragments in naive and semi-immune groups. Regression analyses were performed both as separate models per peptide  as well as with grouped (N + R + C) fragments. In addition, Fisher’s exact test was used to compare proportions of protective efficacy between Exp and Ctrl in naive and semi-immune groups. The association between immune response and protection was assessed using paired sample T-test. A volunteer was considered protected if the parasitemia reached levels significantly lower than that of the control groups. Parasitemia was treated as a continuous variable.For the secondary outcome, antibody titers and IFN-γ elicited by immunization were compared among groups at several time points in the study using regression analysis with Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Standard dosage regimens of quinine formulated for adult patients with uncomplicated and complicated malaria have been applied for clinical uses in children, pregnant women, and elderly. Since these populations have anatomical and physiological differences from adults, dosage regimens formulated for adults may not be appropriate. The study aimed to (i) review existing information on the pharmacokinetics of quinine in children, pregnant women, and elderly populations, (ii) identify factors that influence quinine pharmacokinetics, and (iii) analyse the relationship between the pharmacokinetics and treatment outcomes (therapeutic and safety) of various dosage regimens of quinine.Web of Sciences, Cochrane Library, Scopus, and PubMed were the databases applied in this systematic search for relevant research articles published up to October 2020 using the predefined search terms. The retrieved articles were initially screened by titles and abstracts to exclude any irrelevant articles and were further evaluated based on full-texts, applying the predefined eligibility criteria. Excel spreadsheet  was used for data collection and management. Qualitative data are presented as numbers and percentages, and where appropriate, mean + SD or median (range) or range values.Twenty-eight articles fulfilled the eligibility criteria, 19 in children, 7 in pregnant women, and 2 in elderly . Severity of infection, routes of administration, and nutritional status were shown to be the key factors impacting quinine pharmacokinetics in these vulnerable groups.The recommended dosages for both uncomplicated and complicated malaria are, in general, adequate for elderly and children with uncomplicated malaria. Dose adjustment may be required in pregnant women with both uncomplicated and complicated malaria, and in children with complicated malaria. Pharmacokinetics studies relevant to clinical efficacy in these vulnerable groups of patients with large sample size and reassessment of MIC (minimum inhibitory concentration) should be considered.The online version contains supplementary material available at 10.1186/s12936-022-04065-1. Plasmodium falciparum infections in pregnant women during the first-trimester, as well as for chloroquine-resistant Plasmodium vivax infections [Parenteral artesunate, followed by artemisinin-based combination therapy (ACT), is the recommended treatment for severe malaria, when available. Quinine is usually used if artesunate is not available, and is always used in combination with an additional agent, such as doxycycline . Quininefections . A systefections . A recenfections .The current dosage regimens of quinine for children, elderly, and pregnant women, based on adults’ regimens, may be suboptimal due to altered physiology and pharmacokinetics . None ofThe Web of Science, Cochrane Library, Scopus, and PubMed were used by two independent researchers as search databases up to October 2020. The search terms included ‘Quinine’ AND ‘Pharmacokinetics’ AND ‘Pregnancy’ OR ‘children’ OR ‘Pediatrics’ OR ‘Neonates’ OR ‘Newborns’ OR ‘Infants’ OR ‘Geriatrics’ OR ‘elderly’ (> 65 years).The inclusion criteria were: the article published in English, and the article involved clinical pharmacokinetic research on quinine in either children, elderly, or pregnant women.max), time to reach maximum plasma concentration (tmax), trough plasma concentration (Ctrough), elimination or terminal half-life (t1/2 or t1/2β), apparent volume of distribution , total clearance (CL or CL/F), efficacy, and toxicity parameters. The AUC, Cmax, and Ctrough were normalized with body weight. As there are different values of half-life, volume of distribution, and clearance parameters reported in various studies, the full descriptions of parameter terms are used in the articles. Qualitative data are presented as number and percentage (%). Quantitative data are summarized as mean, mean ± SD, median, median (range), or range values. The volume of distribution and clearance for the intramuscular (im), intrarectal (ir), and oral (po) doses are reported by adjusting to an absolute bioavailability (F) of 0.95, 0.60, and 0.76, respectively.TS and PK independently retrieved and reviewed articles, and any argument or inconsistency was resolved by the third person (KN). The information extracted were: area under the plasma drug concentration–time curve (AUC), maximum plasma concentration  reported in children, pregnant women, and elderly with uncomplicated and complicated malaria.Three pharmacokinetic analysis approaches were applied i.e., non-compartmental analysis NCA, n = 13) –32, 34,  –32, 34,max, and Ctrough were reported in 20 (71.4%) [Three out of 28 articles 10.71%) reported the pharmacokinetic parameters of both quinine and the active metabolite 3-hydroxyquinine  .71% repo, 28. For (71.4%) , 31, 34, (71.4%) , 31–35,  (71.4%) , 29–31 a (71.4%) , measure0-8 h (mg/l/h) data were reported in 7 articles following different routes and dosage regimens; 5 in uncomplicated and 2 in complicated malaria . The ranges of body-weight adjusted AUC0-8 h in uncomplicated and complicated malaria were 0.97–2.47 [AUC.97–2.47  and 3.10.97–2.47 –23, 25 m0-8 h following 4.74 mg base/kg in uncomplicated and complicated malaria were 1.2–2.47 [Plasmodium chabaudi-infected mice [0-8 h following the im and ir routes were contradictory, being either higher [0-8 h [0-8 h comparable to the iv route [0-8 h following both the im and iv, but not ir route, increased linearly with dosage.Malaria severity, nutritional status, administration route, and body weight are the key factors influencing quinine exposure and treatment outcomes. With iv dose administration, AUC1.2–2.47 , 13 and 1.2–2.47 , 23–25 c1.2–2.47  and healted mice . Kwashioted mice , but notted mice , 23, hadr higher  or compar higher –20 with r [0-8 h  may be rmax in complicated malaria (0.4–1.22 µg/ml/kg) [max  [max values in nourished and malnourished children were 0.5 and 0.67 µg/ml/kg, respectively [max showed a linear relationship with dose size following ir and im administration. Simulated Cmax in children weighing 5 kg was 15.9% lower than those weighing 20 kg. However, this difference was unlikely to significantly affect quinine efficacy since plasma quinine concentrations in 95% of the patients remained within the therapeutic range [Following 7–16 mg base/kg, the Cg/ml/kg) , 15, 16,g/ml/kg) –23, 25. µg/l/kg) , 11. Theectively . Cmax sh0-inf for both groups were comparable (8.24 ± 2.43 vs. 9.90 ± 2.50 µmol/l/h) [AUC data was reported in pregnancy with uncomplicated malaria in only 4 articles –29, 31. mol/l/h) . Physiolmax was reported in all articles, 5 and 2 articles in uncomplicated and complicated malaria, respectively (0.07–0.22 vs. 0.19 µg/ml/kg) [max [max following iv and po routes were 0.20 ± 0.028 [Cg/ml/kg) –29, 31. kg) [max . The Cma ± 0.028 , 28 and max values were comparable [A study in healthy elderly following a single oral dose of 600 mg quinine salt (497 mg base)  showed amparable .Quinine clearance was reported in 8 studies , 2 in uncomplicated , 14, andThe volume of distribution of quinine was reported in 8 articles, 6 in complicated , 23–25 aThree out of 19 studies reported models with covariates , 24, 25.The half-life values of quinine reported in healthy, uncomplicated, and complicated malaria were 6.1 ± 1.5 , 7.2 ± 3max in uncomplicated and complicated malaria ranged from 1.7 to 4.0 h [max  [Quinine tto 4.0 h –11, 13 ato 4.0 h –21, resp 2.7–4.7 , 20, 23  2.7–4.7 –19, 21 v 2.7–4.7 , 15 h). ± 2.9 h) .Quinine clearance during pregnancy was reported in 2 articles in uncomplicated –31 and 1Quinine half-life was reported in 3 articles, 2 in uncomplicated –29, 31 amax. The tmax was reported only in uncomplicated malaria in 3 articles, i.e., pregnant [Physiological changes during both pregnancy and malaria infection are unlikely to have affected quinine tpregnant –29, 31 apregnant , 28 womeQuinine clearance was reported in elderly subjects  and eldeThere was no effect of diabetic status on quinine volume of distribution (1.29 ± 0.42 vs. 1.29 ± 0.45 l/kg for non-diabetic vs. diabetic subjects) . The volThe prolongation of quinine half-life in the elderly compared with young adults (19.2 ± 1.1 vs. 10.5 ± 1.6 h) was consistent with the decrease of quinine clearance .max was unaffected by age (2.3 ± 1.2 vs. 2.5 ± 0.7 h in elderly vs. young adults) [Quinine t adults) , which w adults)  or uncha adults) , are disEthnicity can influence quinine clearance due to associated polymorphisms in drug-metabolizing enzymes. Twenty-three studies were carried out in Africans; the other 5 studies were carried out in Cambodians, New Zealanders, and Australian. There were no clinical studies that reported across different ethnic groups on the same malaria severity and vulnerable populations. The conclusion of the influence of ethnicity on quinine exposure is, therefore, limited. However, the distribution of genetic polymorphisms, including those in genes coding for drug-metabolizing enzymes, varies with ethnicity, suggesting the possibility that ethnicity could influence quinine clearance. The allele frequencies of CYP3A4*3, CYP3A4*13, CYP3A4*18, and CYP3A4*19 were 0.021 in European-Americans, 0.011 in Han-Chinese, 0.01 in Japanese, and 0 in Africans . The proP. falciparum treatment. Three out of the 9 articles reported 100% cure rates in complicated malaria [P. falciparum to quinine has been decreasing over time [P. falciparum isolates in Thailand (Thai-Cambodian border) was 3.04 µmol/l (0.98 µg/ml) [50 (concentration that inhibits parasite growth by 50%) values in adults with uncomplicated malaria in Western and Eastern Cambodia gradually increased from 2001 to 2007  of quinine reported in 2009 in adult patients with uncomplicated malaria in Northwestern Thailand was 36.5 µmol/l (11.84 µg/ml) [The clinical efficacy of different dosage regimens and routes of administration in uncomplicated malaria was excellent with a 100% cure rate , 11–16.  malaria , 21, 23.ver time –55. Quinver time , but by ver time . During 8 µg/ml) . QuinineSixteen out of 19 articles reported clinical efficacy of quinine, 8 each in uncomplicated and complicated malaria. Quinine doses in a 3-day or 5-day regimen for uncomplicated malaria during the period of 1993–2005 varied from 4.74 to 11.85 mg base/kg . Since 2max following im administration compared with ir routes. Time to regain consciousness was 36–39 h [Quinine doses used in complicated malaria treatment varied from 4.74 to 16.35 mg base/kg (with or without a loading dose). Four out of 8 articles reported both FCT , 21–23 a 36–39 h . A recen 36–39 h , but nottrough at different time points, 8 articles in uncomplicated [P. falciparum to quinine over time, the current analysis suggests that total Ctrough in most studies in both complicated and uncomplicated malaria were maintained above the MIC for 7 days with a clinical efficacy of 100%. Total quinine Ctrough during 1983–2010 in uncomplicated malaria ranged from 1.42 to 10.43 µg/ml (0.1–0.73 µg/ml free quinine) [trough of at least 3.4 µg/ml  [trough in children  [trough of quinine in children with uncomplicated malaria is therefore, 9.5–11.50 µg/ml. The dosage regimens that provided the required total Ctrough in children with uncomplicated malaria were a 7-day course of multiple doses of 8 [Fifteen out of the 19 articles reported Cplicated , 15, 16 plicated , 23, 25 plicated . Therapeplicated , and 0.2plicated , respectquinine) , 15, 16. = 0.109 ) for cur = 0.109 . The conquinine) , which itrough during 1982–2010 in complicated malaria ranged from 2.7 to 12 µg/ml (0.15–0.66 µg/ml free quinine) [trough based on data analysis from uncomplicated malaria during 1948–1995 was 8–15 µg/ml (0.72–1.05 µg/ml unbound quinine) for curative treatment of complicated malaria [trough in children with complicated malaria is therefore 14.4–19.09 µg/ml   or 10–15quinine) , 23, 25.trough, 2 in uncomplicated [trough values for uncomplicated [trough values for uncomplicated and complicated malaria during pregnancy  were 2.re 2–3.9 , 31 and re 2–3.9  µg/ml, rtrough in elderly, estimated from non-elderly adult patients with uncomplicated malaria (4.5 µg/ml) [trough concentration of 0.4 µg/ml in elderly is, thus, considered sufficient for uncomplicated malaria therapy. Plasma quinine concentration at steady-state in adult patients with severe malaria following standard regimen was 14 (10–20) µg/ml [trough is within the therapeutic range (10–20 µg/ml), and quinine dose adjustment may not be necessary for elderly  , is 4 µg5 µg/ml) . An unbo0) µg/ml , which cThe effects of anatomical and physiological differences in children and elderly, but not pregnant women, do not appear to influence the pharmacokinetics and clinical efficacy of quinine. Standard dose regimens of quinine are likely to be sufficient for effective malaria treatment in children  and elderly . Due to the expansion of plasma volume which may result in inadequate plasma quinine exposure, dose adjustment is likely to be required in pregnant women.max ranging from 4.43 to 9.86 µg/ml (0.31–0.69 µg/ml unbound Cmax) [max of over 20 µg/ml (1.4 µg/ml free Cmax) was reported in adult patients with cerebral malaria without toxicity [Eight out of 19 articles reported adverse reactions after quinine dosing, 3 in uncomplicated –13 and 5nd Cmax) –13. A Cmtoxicity . The estmax of 25.9 µg/ml at the time of hypoglycaemia [Hypoglycaemia is a commonly reported adverse reaction to quinine in complicated malaria treatment (1–15%) , 24, 25.lycaemia . Two artmax of one of them was 16.9 µg/ml [QRS prolongation is one of the most serious concerns of quinine toxicity. One study reported a 10% incidence of QRS prolongation in children with complicated malaria receiving quinine treatment ; in this.9 µg/ml . However.9 µg/ml . Althougmax in fatal  and nonfatal  cases were similar, although the Cmax in one fatal case was markedly high (25.9 µg/ml) [Local irritation or pain at the injection site was commonly reported (12%) following im quinine administration but these symptoms resolved within 4 weeks . The inc9 µg/ml) . About 5dly high .9 µg/ml 9 µg/ml) .Three out of 7 articles reported adverse reactions to quinine, 1 in uncomplicated  and 2 inHypoglycaemia was the most commonly reported (50–100%) adverse reaction to quinine in complicated malaria , 33. TheAdverse reactions to quinine were reported in two subjects after a single oral dose of 600 mg, one in an elderly patient with dizziness (4 µg/ml) and another in a young subject with tinnitus (4.3 µg/ml) . QuinineThe average half-life based on NCA, CA, and pop-PK approaches in uncomplicated malaria were 10 , and 9.1P. falciparum malaria has not been reported since 2007 [P. falciparum to quinine changes over time, inaccurate estimation of MIC and MCOC values may lead to inappropriate dose optimization in populations. The sample sizes used in most studies are small, which may not provide adequate power to detect small differences in the parameters under investigation, and thus, lead to incorrect data interpretation and conclusions. The study designs applied in most studies are not double-blind, or randomized controlled trials (RCT), which could result in bias influencing data interpretation and conclusions. Only a few studies applied statistical analysis to draw conclusions on the significant difference between the observed parameters. The heterogeneity of blood sampling frequency for pharmacokinetic investigations makes comparison among various groups or populations difficult. A conclusive understanding of the effect of a single factor  on quinine pharmacokinetics is obscured by the intertwined nature of these factors in the studied populations. Further, pregnancy status (trimester periods) and age groups are not well defined in some studies. As quinine pharmacokinetics varies as a result of the physiological differences between children, elderly, and pregnant women, data obtained from such ill-defined subpopulations could lead to misguided conclusions. Conducting large clinical trials with a sufficient number of participants may have several limitations and ethical concerns in these vulnerable groups of malaria patients. In recent years, PBPK has emerged as a promising pharmacokinetic analysis tool which could be applied for accurate dose optimization for various drug classes with no requirement of large study sample sizes. PBPK is a mathematical model describing drug disposition in the human body based on prior knowledge from both in vitro and in vivo studies [The MIC or MCOC of quinine for nce 2007 . As the  studies . This mo studies   without adjustment, even though these special populations have differences in their physiology and anatomy. Current standard dose regimens for malaria treatment for both uncomplicated and complicated malaria are sufficient for the elderly, but not for pregnant women. In addition to children, only the standard dosage regimen of quinine for uncomplicated malaria treatment is sufficient for malaria treatment. However, no standard dosage regimen of quinine for complicated malaria has been applied in children.Additional file 1: Table S1. Summary of quinine pharmacokinetic studies in children with malaria . Pharmacokinetic parameters (Cmax and systemic exposure) are presented as mean + SD or mean or median (range) or median values. Table S2. Summary of quinine pharmacokinetic studies in pregnant women with malaria . Pharmacokinetic parameters (Cmax and systemic exposure) are presented as mean + SD or mean or median (range) or median values. Table S3. Summary of quinine pharmacokinetic studies in the elderly. Pharmacokinetic parameters (Cmax and systemic exposure) are presented as mean+SD or mean or median (range) or median values. Table S4. In vitro quinine susceptibility (IC50) for Plasmodium falciparum isolates collected from Cambodia (2001-2007) and Thailand border (1998-2003).


Malaria elimination by 2030 is an aim of many countries in the Greater Mekong Sub-region, including Vietnam. However, to achieve this goal and accelerate towards malaria elimination, countries need to determine the extent and prevalence of asymptomatic malaria as a potential reservoir for malaria transmission and the intensity of malaria transmission. The purpose of this study was to determine the prevalence of asymptomatic malaria and seropositivity rate in several districts of Gia Lai province in the Central Highlands of Vietnam.Plasmodium falciparum and 4 Plasmodium vivax antigens by indirect enzyme-linked immunosorbent assay (ELISA). Age-seroprevalence curves were fitted using reverse catalytic models with maximum likelihood.A cross-sectional survey of asymptomatic malaria and serological testing was conducted in 3283 people living at 14 communes across seven districts in Gia Lai province in December 2016 to January 2017. Finger prick capillary blood samples were tested for malaria using rapid diagnostic testing and polymerase chain reaction (PCR), as well as detecting antibodies against 3 P. falciparum 1.07%, P. vivax 0.40%, Plasmodium malariae 0.15% and mixed infections 0.12%). In contrast, the annual malaria prevalence rates for clinical malaria in the communities where the participants lived were 0.12%  in 2016 and 0.22%  in 2017. Seropositivity for at least one P. falciparum or one P. vivax antigen was 38.5% (1257/3262) and 31.1% (1022/3282), respectively. Age-dependent trends in the proportion of seropositive individuals in five of the districts discriminated the three districts with sustained low malaria prevalence from the two districts with higher transmission.The study population was predominantly male , adults  and of a minority ethnicity , with most participants being farmers and outdoor government workers . Using a small volume of blood (≈ 10 µL) the PCR assay revealed that 1.74% (57/3283) of the participants had asymptomatic malaria (Plasmodium carriers were found to be substantially more prevalent than clinical cases in seven districts of Gia Lai province, and a third of the population had serological evidence of previous malaria exposure. The findings add knowledge on the extent of asymptomatic malaria and transmission for developing malaria elimination strategies for Vietnam.Asymptomatic The online version contains supplementary material available at 10.1186/s12936-022-04060-6. Plasmodium species of malaria by 2030 [Despite continued decreases in the number of malaria cases outside of Africa, there are still more than 200,000 cases reported annually across Asia and the Pacific, primarily in 11 endemic countries  [ by 2030 .P. falciparum parasites resistant to the current artemisinin-based first-line treatments described in six provinces  [P. falciparum. While P. falciparum is the focus of current elimination efforts, malaria caused by Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi may require different strategies due to either dormant liver stages (hypnozoites), importation from areas outside of Vietnam or presence of animal reservoirs [Significant reductions in malaria mortality and morbidity were observed in Vietnam between 1991 and 2013 with continued decreases reported through 2015 [h Phuoc) , (2) ideservoirs .It is widely accepted that asymptomatic malaria parasite carriers present a unique challenge for elimination programmes as they provide a transmission reservoir capable of sustaining malaria endemicity –10. As Vi.e. hotspots of transmission surrounded by areas of no transmission), where geographical stratification in transmission levels becomes essential to target NMCP interventions efficiently [As transmission decreases, parasitological surveys designed to identify ongoing infections will require large sample sizes, becoming logistically and economically unfeasible for NMCPs. Other malaria transmission metrics such as entomological inoculation rate also have inherent limitations in low transmission areas due to very low numbers of infected mosquitoes –13. Alteiciently .P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi in an asymptomatic population working and residing in Gia Lai province, Vietnam. Three endpoints were assessed to characterize malaria burden in the study population: (1) prevalence of parasite positive individuals determined by PCR, (2) prevalence of individuals with a history of malaria exposure measured by detection of antibodies to P. falciparum or P. vivax, and (3) proportion of P. falciparum parasites with molecular markers associated with drug resistance.The primary objective of this cross-sectional study was to characterize the parasite reservoir of 2 distributed in 17 districts and city divisions  was reported in Gia Lai province in 2010 and 2017 [The study was conducted between December 2016 and January 2017 at located in seven different districts of Gia Lai province. Gia Lai is located in the Central Highlands of Vietnam on the border with Cambodia, has a population of approximately 1.3 million people residing in an area of 15,500 kmand 2017 , 20.Fig.Cross-sectional malaria surveys were done in 14 military-civilian health facilities across the seven districts: Chu Prong (Ia Mo and Ia Puch communes), Chu Puh , Chu Se (Ia Hlop and Ia Ko communes), Duc Co , Ia Grai (Ia Chia and Ia O communes), Kong Chro (So Ro commune), and Krong Pa (Chu RCam commune) . It was determined that a minimum of 3,052 participants would be required to detect a PCR positive rate of ≥ 1.25% with a two-sided 95% confidence interval no wider than 1% with alpha and beta errors of 0.05 and 0.20, respectively. Allowing for 7% to 8% of subjects withdrawing their consent to participate in the study, it was decided to recruit at least 3,265 to 3,296 participants.e.g. occupation, recent travel history, health check) and a finger prick blood sample. The exclusion criteria were: (1) being unwilling to provide consent, information or finger prick capillary blood sample, (2) inability to communicate well with the study staff, and (3) presenting with tympanic body temperature > 38 °C or reporting other malaria symptoms such as headache; these individuals were referred to the nearest health care facility. Following informed consent, study staff collected finger prick capillary blood samples for RDT  and four filter paper blood spot samples were collected on 3MM filter paper  for PCR analysis and antibody detection. Blood spots were stored individually in zip-closure plastic bags with desiccant (10 silica gel beads). Body temperature was measured, and demographic information was collected.Both asymptomatic civilians and government workers from the Vietnam People’s Army eligible for medical care in the area covered by the selected military medical facilities were non-randomly invited to participate in the study. The inclusion criteria for the study were: (1) working or residing in Gia Lai province during the 14 days prior to the study visit, (2) 5 to 65 years of age, and (3) willingness to provide information . Briefly, highly synchronous ring-stage parasite cultures at 5% parasitaemia were centrifuged, washed twice in 100% plasma and resuspended at 50% haematocrit. These suspensions were dispensed into 50 μL aliquots in LoBind Eppendorf tubes and stored at -80℃. The parasite DNA was extracted from 50 μL aliquots and serially diluted. Using these standards, the lower limit of detection (LOD) for PCR was ≥ 0.25 parasites/µL. The PCR performance acceptance threshold was set tenfold greater than LOD at ≥ 2.5 parasites/µL  with respective positive DNA controls included into every batch of PCR analysis. As DNA extraction efficiency control, filter paper spots containing 50 parasites per spot were prepared using the synchronous ring-stage D6 parasites and included in every batch of DNA extraction and subsequent PCR. When testing field samples, positive PCR results were accepted (i.e. bands on the gel of the size corresponding to the respective positive controls) if they were reproduced in at least 2 out of 3 PCR conducted. In addition, detection of P. knowlesi was carried out as previously described by Imwong et al. [DNA was extracted from filter paper blood spots (approximately 10 µL of whole blood) using the QIAamp DNA mini kit  as per manufacturer’s protocol and eluted in 150 μL of AE buffer. Five µL was used as template in a single round multiplex PCR targeting small subunit (SSU) ribosomal RNA for simultaneous detection of escribed . PCR prog et al. .P. falciparum kelch-13 gene  were evaluated by sequencing of the 850 bp Pfkelch-13 gene region covering amino acid 427 to 687, using a previously described method [exo-E415G (exonuclease gene PF3D7_1362500) and copy number of plasmepsin 2 and 3 were analysed as previously described [P. falciparum chloroquine resistance transporter (Pfcrt) gene at codons 72 to 76 associated with chloroquine resistance were analysed by PCR amplification and sequencing, as previously described [P. falciparum multidrug resistance protein 1 (Pfmdr1) gene, which is associated with lumefantrine and mefloquine resistance, was determined by qPCR using SYBR Green chemistry [Prevalence of artemisinin-resistance validated mutations in the st 2018, ; i.e. F4d method . For marescribed . In addihemistry . Copy nuP. falciparum and P. vivax were detected by indirect enzyme-linked immunosorbent assay (ELISA). For antibody elution, two 5 mm discs were punched from dried blood spots, placed in a 96-well plate, and mixed with 1,120 μL of elution buffer containing PBS/0.05% Tween20/0.05% NaN3. Plates were incubated overnight at ambient temperature (~ 23 °C) and with shaking (200 rpm), and pink/red colored eluates were stored at 4 ºC. The following recombinant proteins were produced: PfAMA-1 (3D7 strain), PfMSP1 , PfCSP (3D7), PvAMA-1 , PvMSP1 , PvCSP allelic variant 210  and PvCSP allelic variant 247 (PNG_M69059.1). All proteins were produced at Burnet Institute , except PfMSP1 and PvMSP1 produced at Dalian Institute of Biotechnology .Antibodies against 2SO4, and optical density (OD) values read at 405 nm in a Multiskan™ FC Microplate Photometer . Duplicates with > 25% variation were repeated once, and those with > 25% variation after repetition were excluded from analysis. The mean OD from blank wells was subtracted from samples, controls and IgG standards at each plate, and total IgG standards were then used to normalize mean sample OD between plates. Fifteen percent of the study samples (n = 575) were retested for all seven antigens to assess reproducibility of serological assays, resulting in pairwise Pearson’s correlation coefficients ranging 0.877–0.952  were coated with 25 ng/well of antigen in PBS or carbonate buffer, sealed and incubated overnight at 4 °C. Total IgG from human serum  were used as plate standards and directly coated in control wells at three different dilutions . Plates were washed and blocked with 1% skimmed milk powder in PBS/0.05% Tween20 for 2 h. Fifty μL of antibody eluates were tested in duplicate at 1/200 dilution together with positive  and a negative control (unexposed Vietnamese). After incubation at room temperature for 2 h, peroxidase-conjugated goat anti-human IgG secondary antibody (Sigma-Aldrich) was added for 1 h, washed and mixed with 50 µL of 2,2’-azino-di-(3-ethylbenzthiazoline sulfonic acid (Sigma-Aldrich). Reaction was stopped after 15 min with 50 μL of 2 M HThe main dependent variable used this study was malaria infection based on PCR analysis. Independent demographic variables included age (stratified in seven age groups), ethnicity (Kinh versus ethnic minorities), sex, occupation  and district.P. falciparum or P. vivax if they tested seropositive for at least one of the species antigens. Age-seroprevalence curves were fitted using reverse catalytic conversion models with maximum likelihood methods in R , as previously described [i.e. the rate at which seronegative individuals become seropositive per year) and one seroreversion rate . Seroconversion rate can thus be used as a measure of malaria force of infection or transmission intensity, whereas seroreversion rates can be interpreted as a measure of persistence of the antibody response [2) tests with Bonferroni corrections, or Fisher’s exact test when sample size was small. Risk factor analysis for P. falciparum and P. vivax seropositivity was conducted by running univariate logistic regression models for age, sex, ethnic group, occupation, district of residence and PCR positivity. Statistical analyses were performed in IBM SPSS 22.0 or Stata Version 12.0 , and graphs were created in Prism v8.0 . A significance level of p < 0.05 was defined for all tests.Individuals were considered exposed to escribed . The modresponse . DiffereA total of 3,283 individuals consented to participate and were enrolled in the study from health facilities in Chu Prong (n = 330), Chu Puh (n = 600), Chu Se (n = 400), Duc Co n = 530), Ia Grai (n = 220), Kong Chro (n = 600) and Krong Pa (n = 603) districts. Overall, the participants were predominantly adults , male , and belonging to an ethnic minority group , with most participants engaged in farming and outdoor work activities  , whereas P. vivax accounted for 39.7% (24.9–59.2%).The annual number of clinical malaria cases reported by the 14 communal health stations by microscopy and/or RDT between 2014 and 2017 are presented in Table P. falciparum 1.07%, P. vivax 0.40%, P. malariae 0.15%, and mixed infections 0.12%; Table P. falciparum was the predominant species  followed by P. vivax . The difference in total and individual Plasmodium species malaria distribution was significantly different between the seven districts surveyed. P. ovale and P. knowlesi malaria were not detected in the participants’ blood samples. Out of the 57 PCR positive cases, three presented with clinical malaria within a period of two years after recruitment, including one case of acute vivax malaria seven months after recruitment .Two individuals tested positive for malaria by RDT 0.06%, 2/3283). The prevalence of asymptomatic malaria detected by PCR was 1.74% . No significant differences in PCR malaria prevalence were seen by age group , sex  or ethnicity .By occupation, PCR positive samples were detected in 2.0% 35/1737) of farmers, 3.1% (10/323) of students and 1.0% (12/1223) of government workers . Among markers associated with piperaquine resistance, none of the samples had the exo-E415G mutation (0/11), but 13.3% (2/15) had increased copy numbers of plasmepsin 2/3. Chloroquine resistant haplotype CVIET at positions 72–76 of Pfcrt gene was observed in 27.3% (6/22) samples. Four out of 15 samples (26.7%) had multiple copies of Pfmdr1. The two isolates with multiple plasmepsin 2/3 copies also had multiple copies of Pfmdr1 gene .Molecular markers of drug resistance were analysed in samples with sufficient amount of DNA for PCR amplification and sequencing. No samples had mutations associated with artemisinin resistance in P. falciparum antigen, and 31.1% (1022/3282) responded to at least one P. vivax antigen  and were highest in Krong Pa and Krong Chro districts and lowest in Chu Puh and Chu Se. Noted, that participants from these latter two districts were predominately male government workers with low clinical malaria rates as well as evidence of low malaria exposure: seroprevalence in Chu Puh and Chu Se was 1% (6/600) and 4.8% (19/400) for PfAMA1, and 0.5% (3/600 and 2/400) for PvAMA1  of participants had antibodies to at least one 57/3262 oP. falciparum and P. vivax exposure (defined as being seropositive to any antigen for each species) were determined using logistic regression in five of the study districts  and 42% (962/2282) to P. vivax. There was no association between PCR positivity  and antibody responses. Female sex, being from an ethnic minority group, farming or residing in Krong Pa and Kong Chro districts indicated an increased risk of both P. falciparum and P. vivax exposure (p < 0.001). Overall, there was an increased risk of seropositivity for older age groups as compared to young children, especially for P. falciparum.Factors associated with ts Table . In thesP. falciparum and P. vivax exposure at each district . Seroconversion rates were highest in Krong Pa and Kong Chro , the districts with the highest prevalence of clinical malaria in 2014–2015  below the RDT detection threshold. This prevalence was markedly higher than the mean clinical malaria prevalence rate of 0.12%  reported in 2016 among residents from the 14 communes where the participants were recruited. Although the survey included a subset of the province population, other studies in similar epidemiological settings have indicated that asymptomatic Importantly, the PCR assay in the present study using a finger prick capillary blood of approximately 10 µL and based on the PCR performance acceptance threshold was designed to detect sub-microscopic malaria infections with a sensitivity of at least 1.6 to eightfold greater than that of microscopy (the LOD in thick blood films for expert microscopists is estimated at 4 to 20 parasites/µL ). MoreovP. ovale and P. knowlesi infections in the study population. In the present study, the PCR assay sensitivity was only tested for P. falciparum. Although rare, P. ovale infection detected by PCR has been reported in several southern Vietnam provinces  [P. knowlesi has been reported in south-central Ninh Thuan province [P. malariae  supports other reports of cryptic or asymptomatic P. malariae infections in Southeast Asia [The results suggest no presence of anh Hoa)  and in sanh Hoa) , but theprovince  and in cprovince , but notast Asia . The cliP. falciparum no Pfkelch-13 mutations that confer artemisinin resistance were detected (0/13). This finding contrasts with other recent studies that reported a prevalence of Pfkelch-13 validated mutations of 74% (20/27) in Ia Grai district in 2015, and up to 89% (39/45) in Krong Pa district in 2017, in both cases C580Y mutations were predominant [PfKelch-13 mutations in samples collected from asymptomatic individuals in the present study could be due to the small sample size of the subset of P. falciparum positive samples (13/35) for which the sequence data were obtained and/or heterogeneity in the local parasite populations. Another possible explanation is that in the absence of drug pressure resistant parasites appear to be less fit in comparison with the wild type parasites [plasmepsin 2/3 genes and pfmdr1 gene were found in 13.3%, (2/15) and 26.7% (4/15) of infections, respectively, including two double mutants. Amplification of plasmepsin 2/3 confer resistance to piperaquine, the partner drug in the first-line artemisinin-combination therapy DHA-PPQ used in Vietnam up until 2020. The rate of plasmepsin 2/3 amplification coincides with that reported in a clinical trial conducted Krong Pa district during the same period in Vietnamese subjects  [pfmdr1 copy number are associated with resistance to mefloquine, which has not been used in the study area since the 1990s . This suggests the possibility of importation of resistant malaria parasite from neighboring Cambodia, where artesunate-mefloquine has been used to treat P. falciparum malaria in early 2000s [Among asymptomatic infections caused by dominant , 20. Unldominant , 20. Thearasites , and thup to 89% /45 in KrP. falciparum or P. vivax antigen, respectively. Although no other serological surveys had been conducted previously in Gia Lai, these rates compare to those found in similar epidemiological settings, such as a population survey in Quang Nam, Vietnam, in 2015 , or a study among malaria-negative adults in Oddar Meanchey, Cambodia, in 2011  [i.e. > 6 months) of responses against Pf/PvAMA1 or Pf/PvMSP1 antigens generated after exposure events [i.e. 297 days for PfMSP1, 569 for PfCSP, 367 for PvAMA1, 312 for PvMSP1, 885 for PvCSP210 and 725 for PvCSP247) [P. vivax antigens, may partly explain the high seroprevalence found for CSP antigens in some of the districts included in the present study.This study also aimed to explore serology as an alternative malaria transmission metric for surveillance purposes. The antigens AMA-1, MSP1 and CSP, were selected as they are present in both species, represent both pre-erythrocytic and erythrocytic stage parasites and have been extensively used as markers of exposure in the past. The analysis of antibody responses of study participants found that 38.5% (1257/3262) and 31.1% (1022/3282) of individuals responded to at least one ax-MSP1) , 40. Thee events , 42 and,P. falciparum and P. vivax antigens, what can be interpreted as ongoing transmission of both species. Although the higher number of individuals < 20 years of age in Krong Pa and Kong Chro might have contributed to these differences, retrospective data showed these two districts had the highest clinical malaria rate among study districts in the two years preceding the survey, in line with serological results. The decrease in malaria cases reported between 2014 and 2016 in all districts by an average of 6.4-fold was not captured in the age-seroprevalence curves conducted at the end of 2016, probably due to the high immunogenicity and long duration of responses to selected antigens.Age-seroprevalence curves allowed to identify two types of districts. For Chu Prong, Duc Co, and Ia Grai districts a linear association between age and seroprevalence rate was seen, suggestive of a sustained decrease in malaria prevalence in the past years. In contrast, Krong Pa and Kong Chro districts had the highest seroconversion rates for all antigens and a rapid acquisition of antibodies at early ages for both i.e. Chu Puh and Chu Se). Even so, district of residence significantly increased risk of seropositivity for both Plasmodium species, in agreement with the higher number of malaria cases that detected in Krong Pa and Kong Chro communal health stations in recent years  together with their history of exposure to infection during their lifetime. Similarly, the higher seroprevalence in females than males may be due to a higher proportion of females being farmers by occupation  compared with males  in these communities. Farming activities often include going far into forests where risk of malaria is higher. On the other hand, no association was seen between PCR positivity in the cross-sectional survey and P. falciparum or P. vivax seroprevalence. Although studies in low endemic areas have shown that even low-density asymptomatic infections can induce strong long-lived IgG responses against P. vivax [Risk factors for seropositivity were assessed after excluding the two districts with only military personnel and negligible seropositivity rates . Table S1. Malaria prevalence for blood film and/or rapid diagnostic testing positive people in the seven districts of Gia Lai province, Vietnam. Table S2. PCR results by Plasmodium species and self-reported occupation of study subjects from Gia Lai province, Vietnam. Table S3. PCR result and Plasmodium species by age-group of study subjects from Gia Lai province, Vietnam. Table S4. PCR result and Plasmodium species by sex of study subjects from Gia Lai province, Vietnam. Table S5. PCR result and Plasmodium species by ethnicity of study subjects from Gia Lai province, Vietnam. Table S6. Seroprevalence against P. falciparum and P. vivax by districts of study subjects from Gia Lai province, Vietnam. Table S7. Seroconversion rates by districts of study subjects from Gia Lai province, Vietnam.Figure S2. Age-seroprevalence curves for individual P. falciparum antigens by districts of study subjects from Gia Lai province, Vietnam. Reversible catalytic conversion models allowing one seroconversion rate (λ) were fit to the data (dashed line shows 95% CI). Figure S3. Age-seroprevalence curves for individual P. vivax antigens by districts of study subjects from Gia Lai province, Vietnam. Reversible catalytic conversion models allowing one seroconversion rate (λ) were fit to the data (dashed line shows 95% CI).


ABSTRACT IMPACT: This study aims to provide insight into naturally acquired immunity against severe malaria, thereby laying the foundation for the design of novel vaccine candidates to prevent severe disease as well as monoclonal antibody therapies to treat severe malaria. OBJECTIVES/GOALS: Severe malaria is caused by parasite surface antigens that contain high sequence diversity. Nevertheless, P. falciparum-exposed individuals develop antibody responses against these antigens. Our goal is to isolate antibodies with broad reactivity to understand how disease protection is acquired. METHODS/STUDY POPULATION: Our study cohort consists of Ugandan adults living in a malaria-endemic region with high transmission intensity, who are protected against severe malaria. Using fluorescently labeled probes of parasite surface antigens, we have isolated antigen-specific B cells from these donors. We then expressed the corresponding monoclonal antibodies in vitro. These antibodies were screened against a library of variant surface antigens to determine antibody breadth and potential to inhibit interaction of the parasite surface antigen with host receptors, a critical step in pathogenesis. Additionally, using a panel of variant surface antigen mutants, we have predicted the epitopes targeted by the broadest monoclonal antibodies. RESULTS/ANTICIPATED RESULTS: We have identified three monoclonal antibodies with exceptionally broad reactivity and inhibitory activity against our panel of severe disease-inducing variant surface antigens. We have identified two major sites targeted by these broadly reactive antibodies. The first site was associated with the largest breadth, but limited inhibitory potential, while the second site showed high-affinity antibody binding and inhibition of receptor binding. Interestingly, two of these three antibodies were very similar in structure, even though they were isolated from different donors. Isolation of antigen-specific B cells from additional donors will enable us to identify how common such broadly reactive antibodies are and allow the identification of additional epitopes DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is the first to isolate broadly reactive antibodies that are likely to protect against severe malaria in naturally immune individuals. Further characterization of antibody-antigen interactions will inform the development of this surface antigen as a vaccine candidate for malaria.


P. falciparum and P. vivax. Data was gathered on demography and a priori risk factors of participants. Data was analysed nationally and within each of four domains defined by API data. Prevalence and seroprevalence of malaria were 0.74% and 16.01% nationwide, respectively. Prevalent infection was primarily asymptomatic P. vivax, while P. falciparum was predominant in serology. There was large heterogeneity between villages and by domain. At the township level, API showed moderate correlation with P. falciparum seroprevalence. Risk factors for infection included socioeconomic status, domain, and household ownership of nets. Three K13 P. falciparum mutants were found in highly prevalent villages. There results highlight high heterogeneity of both P. falciparum and P. vivax transmission between villages, accentuated by a large hidden reservoir of asymptomatic P. vivax infection not captured by incidence data, and representing challenges for malaria elimination. Village-level surveillance and stratification to guide interventions to suit local context and targeting of transmission foci with evidence of drug resistance would aid elimination efforts.Malaria incidence in Myanmar has significantly reduced over recent years, however, completeness and timeliness of incidence data remain a challenge. The first ever nationwide malaria infection and seroprevalence survey was conducted in Myanmar in 2015 to better understand malaria epidemiology and highlight gaps in Annual Parasite Index (API) data. The survey was a cross-sectional two-stage stratified cluster-randomised household survey conducted from July-October 2015. Blood samples were collected from household members for ultra-sensitive PCR and serology testing for  The incidence of malaria in Myanmar has reduced significantly in recent years, falling by over 80% from a reported 1341.8 cases per 100,000 population in 2005 to 253.3 cases per 100,000 population in 2014 . CompletPrevalence surveys can highlight gaps in epidemiological understanding and if conducted regularly can measure the rate of impact of interventions on decreasing transmission. There has been increasing interest in the use of highly sensitive infection tests and serological methods in monitoring malaria transmission intensity in low transmission settings where prevalence is low since a large proportion of infections are asymptomatic and low density and thus missed by conventional diagnostic methods , 10. TheHere, we present results from this survey in relation to geography, incidence data and associated risk factors to better understand the epidemiology of malaria in Myanmar and the challenges faced by drug resistance and ultimately malaria elimination.The MIS protocol, questionnaire and tools were submitted to the Ethics Review Committee on Medical Research Involving Human Subjects, Department of Medical Research. Following defence of the submission full ethical approval was granted by the committee on 21/07/2015 ."Myanmar (Burma) sits within the GMS, sharing borders with Thailand, China, India and Bangladesh. In 2015, Myanmar had an estimated population of 53,900,000, and an estimated malaria at-risk population of 32,120,000 residing in 15 malaria endemic states/regions .The Myanmar Malaria Indicator Survey (MIS) was a national cross-sectional household survey conducted from July to October 2015. The survey used a two-stage stratified cluster-randomised design, with a cluster defined as a ‘village’ and a ‘household’ as the sample unit. All malaria endemic townships were included and were categorised into four non-contiguous strata, termed domains, three based on intensity of malaria transmission using API data from the township level  and non-state areas included in a separate fourth domain due to limited incidence data and variable administrative structures .Townships were randomly selected with probability proportionate to size (PPS) and with replacement to ensure the required number of clusters within each domain was reached. One village tract/ward (VT/W) per selected township was randomly selected with uniform probability. In each selected VT/W, field teams updated village level population and one village per VT/W was randomly selected by PPS. In each selected village, field teams updated household listings to include all registered and non-registered households at the time of the survey and then randomly selected 31 households for participation. In areas covered by Domain 4 (non-state areas), administrative systems were independent and unique. In each area, the survey team interposed the government of Myanmar township boundaries and made random selection of townships with PPS. Available population data supplied by non-state actors was then used to make random selection with PPS of villages per township. Households were selected in the same manner as in the other domains. Certain areas that were absolutely inaccessible at the time of surveying due to active conflict or flooding were excluded from the sampling frame.Sample size was calculated based on assumed prevalence levels detectable by PCR in each of the strata and using the standard formula for proportions, assuming a minimum precision of 0.01, a non-response rate of 10%, a design effect of 2.0, average household size of 4.5 and selection of 31 households per cluster. Based on this, a total 20,138 individuals from 4,495 households and 145 clusters across the four domains were required.Prior to data collection, fully informed consent was gathered from the head of the household on behalf of all household members and for children, where applicable, for both questionnaire information and blood collection. Study information was provided in written and/or verbal form in a language understandable to the participant and written consent was provided by signature or thumb print.In each selected household, a standard questionnaire was administered to the head of the household or his/her representative. The questionnaire was designed to gather data on factors related to demographic background, including age, sex and wealth of the household; malaria risk factors, including fever, travel and forest-going habits; net ownership and usage, including type of net; case management practices, including healthcare seeking behaviour; and knowledge of malaria. These data were gathered on all household members, including temporary visitors staying in the household at the time of the survey. A traveller was defined as a household member who has travelled and stayed overnight away from home in the previous six months. A forest-goer was defined as a household member who sometimes goes to the forest and stays there overnight.P. falciparum/P. vivax) and asked to provide four dried blood spots on filter paper for subsequent PCR, K13 molecular analysis and serological testing. Blood spots from each individual from a finger prick using a sterile lancet and necessary measures to ensure sterile blood collection . The survey team revisited households up to three times to get blood samples from all household members. GPS coordinates were recorded in every sampled household and from a central point in each cluster to assess for geographical clustering of infection, except in domain 4 where no geolocation data were provided due to local sensitivities.Upon obtaining informed consent, all household members aged over six months present in the households were tested for malaria infection by rapid diagnostic test (RDT)  were estimated using ultrasensitive PCR with a detection limit of 20 parasites per millilitre, as previously described by Adams et al. , but was significantly higher in Domain 4 at 10.96%, 95% CI [5.63–20.25], followed consecutively by Domain 1, Domain 2 and finally Domain 3  followed by Domain 1 . Prevalence of P. falciparum was low at only 0.17%, 95% CI [0.07–0.41] and did not differ significantly by domain. No P. falciparum cases were detected in Domain 3. There were 28 mixed infections identified, giving an overall prevalence of 0.05%, 95% CI 0.01–0.21, but the majority (n = 20) were detected in Domain 1.Of 13,648 RDTs conducted, only 10 positive cases were identified including eight Domain 3 . P. vivaP. falciparum seroprevalence was over two-times higher than P. vivax. P. falciparum seroprevalence was significantly lower in Domain 3 and appeared highest in Domains 1 and 4 though CIs overlapped with Domain 2. P. vivax seroprevalence was highest in Domain 4, followed consecutively by Domain 1, 2 and finally 3 (though CIs overlap between each level). Antibody responses split by age showed there were individuals with marked antibody responses in all age groups, including children aged 1–10  and significantly lowest in Domain 3  had a mutation in the F446I gene, each from three different clusters in Domain 1, two of which were in Pinlebu township, north-western Myanmar.Of the total 80 P. falciparum and P. vivax . Two of these clusters were from the same township, Pinlebu, with prevalences of 18.33% and 9.49%, one of which also contained a K13 mutant. These were followed by lower prevalences in another township of Sagaing (4.05%) and two clusters in Ann township of Rakhine State, south-west Myanmar . The highest prevalence clusters for P. vivax were in Domain 4, including 60.34% from a cluster in Yephyu township, Thanintharyi State; 42.40% and 29.91% from two clusters in Yay township, Mon State; and 19.79% from a cluster in Myawaddy township, Kayin State .Clusters (villages) were plotted based on prevalence and seroprevalence of both P. vivax . ClusterP. falciparum. Although there were many clusters without any evidence of infections by PCR (coloured in black), there were only few clusters without any seropositive individuals.Seroprevalence was higher and more widespread throughout the country particularly for P. vivax infections were asymptomatic, defined as not having current fever nor reported fever in the previous two weeks  and in Domains 2 and 3, all P. vivax infections were asymptomatic. P. falciparum had a higher proportion of symptomatic infection compared to P. vivax .The vast majority of PCR-positive Plasmodium species and per domain  and P. vivax (ranging from 2.2 to 3.7% across domains) the majority of these had antibody levels close to the seropositivity threshold. Seroprevalence of P. falciparum was also associated with wealth quintile, staying in the forest and ownership of nets, as well as domain, and being PCR positive , current fever (domain 3) and living in a rural area (domain 4). Interestingly, higher odds of infection and exposure indices were associated with higher ITN/LLIN ownership and/or knowledge of ITN as a prevention tool for both plasmodia species and in most domains.Analysis of the whole population sample, however, hides interesting differences in risk factors between domains for both prevalence and seroprevalence of each species Tables –5. For eDomain 4 . P. vivaDomain 2 . In Domadomain 4 . P. falcP. vivax and P. falciparum PCR prevalence and township API. A positive relationship was observed between increasing API and increasing seroprevalence to P. falciparum. However, the association between API and P. vivax was less clear with a positive association observed only until seroprevalence estimates of 25%.Township API data acquired during sampling was plotted against prevalence and seroprevalence figures aggregated by township . No cleaP. falciparum and P. vivax infection, respectively. However, these overall estimates mask huge variation between the domains which ranged from an all-species prevalence of 10.96% in Domain 4 to 0.19% in Domain 3. Species-specific prevalence figures show that P. falciparum infections were relatively low and uniform across the country whereas P. vivax, made up the vast majority of infection in all domains and accounted for the observed heterogeneity. Given that serology captures both current and past infection, seroprevalence was expectedly higher at 16.01%, but showed similar variation between domains. In contrast to PCR data, the dominant Plasmodium species detected by serology was P. falciparum, including in young children. In comparing API data with prevalence and seroprevalence, where API was <25 per 1000 population, serology was able to capture a wider range of API variability than PCR, particularly for P. falciparum which showed a strong positive correlation between API with seroprevalence, although there was significant variation. Although there was an initial upward trend in API with P. vivax seroprevalence, this correlation was no longer apparent at prevalences greater than 25%.The results of the first ever MIS in Myanmar reveal programmatically important epidemiological findings not apparent in routine surveillance data. Use of RDTs, now a common tool for identifying malaria infection in the field, only detected a tiny fraction of infections identified by PCR. RDTs can be useful for diagnosis of clinical disease but, as demonstrated here, are not a good surveillance tool, particularly in a context where asymptomatic and sub-patent infection may be highly prevalent . Use of P. vivax prevalence is in contrast to routine surveillance data, which detects incident symptomatic clinical cases only and places P. falciparum as the major malaria species (75%) in Myanmar [P. falciparum is associated with higher parasite densities and more serious clinical outcomes than P. vivax, and as such leads to the poor correlation of P. vivax prevalence with API data we have shown [P. vivax parasitaemia is >10 parasites/ul, and that this may be the optimal diagnostic threshold since mosquito infection is rare at levels below this threshold [P.vivax infections tend to reach peak gametocytaemia prior to symptoms and are infectious before treatment this coupled with the relapse of infection due to their dormant liver stage hypnozoites, means transmissibility is difficult to predict [P. vivax infection reservoir. In practice this will be a challenge given that sub-clinical infections are poorly detected by RDTs and the only currently available radical cure for P. vivax, Primaquine, can be fatal in individuals with G6PD deficiency [P. vivax also doesn’t respond as well to the mainstays of vector control—ITNs/LLINs and IRS—largely due to its hypnozoite stage and association with outdoor and early biting vectors [The dominance of  Myanmar . This isve shown , 24. Wheve shown –27. A rehreshold . Moreove predict . Programficiency , 31. P.  vectors , 33. PerP. falciparum may be due to the timing of data collection, much of which was after the transmission season, or because the ELISA assay used was originally developed to detect responses to P. falciparum antigens and thus may be more sensitive in detecting those. P. falciparum also circulates at higher parasite densities that induce higher antibody levels than P. vivax [P. vivax antibody detection [The higher seropositivity from P. vivax  and thisetection .The difference exhibited in prevalence between domains (API strata) supports the stratification design of the survey and is important to consider in the design of future surveys to capture important heterogeneities between regions. It also highlights the need for the Myanmar NMCP to be able to stratify its surveillance and approaches to interventions to suit the different transmission intensities in the country, including in the types of risk factors used to target high-risk population groups . MoreoveP. falciparum (2.33%) and P. vivax (1.41%) was low some had notable antibody responses, which, since young children are less likely to have travelled, indicates possible local and recent transmission  of potential high transmission that would have been missed by looking at infection prevalence results alone. In addition by comparing areas of high seropositivity (suggesting areas of past transmission) with areas of PCR positivity (current transmission) it is also possible to explore areas of potential high receptivity to complement surveillance efforts and guide programmes on optimal implementation of malaria control interventions. These higher transmission areas appear to be largely concentrated in north-west Myanmar, in the region of Sagaing, south-east in Rakhine and in the south-western tip of Myanmar in Mon, Kayin and Thanintharyi. Interpreting these areas as sites of high transmission requires the assumption that the infections were transmitted in the same location as that in which the diagnosis took place, which may not always be the case, particularly in areas with high population mobility. Although not presented here, basic travel history information was collected as part of the MIS questionnaire and only seven individuals had travelled outside the same state/region as their household in the previous six months. All other identified travellers had stayed away from home overnight but remained within the same state/region. Furthermore, whilst seroprevalence in children aged less than five years for both smission . Future Secondly, serology provided an increased sample size of infections with which to analyse risk factors, improving uncertainty around OR estimates. This is useful programmatically to guide best targeting of resources, particularly as resources become more finite following reduction in caseloads. The recent National Strategic Plan has rightly specified activities for these risk groups such as, introducing quarterly malaria screening and additional LLINs for pregnant women through antenatal care services; emphasising case detection and treatment for forest-goers and mobile groups and engaging NGO networks in distribution of ‘forest-packages’ for forest-goers; and monitoring arrival of mobile population groups into communities by VHVs .The findings offer important insights into malaria epidemiology in Myanmar, though the survey does have certain limitations. The proportion of non-response from household members was higher than anticipated and disproportionately consisted of males, travellers and children aged less than five years across all domains with the exception of D4 that had a younger population structure than the other three domains. Males and travellers were identified as risk groups and thus overall prevalence and seroprevalence may be slightly underestimated. Although attempts to revisit households were made, missing a certain proportion of household members is unavoidable and is expected to miss more mobile population groups. In contrast, pregnant women were overrepresented since, assumedly, they are more likely to be at home during day time. Despite not reaching the desired sample size, the loss in power to calculate the prevalence estimates was negligible. Finally, comparison of API and prevalence data is limited because API was only available at township level and did not include infections that might have been treated outside the formal health system. For comparison, we aggregated prevalence to township but townships are thus only represented by one or two villages, which with high amount of transmission heterogeneity may not be accurate. Work is ongoing in the country to measure and report API at village level; once this is achieved better understanding of the association between these two measures could be elucidated.Despite these limitations, the survey presents the first nationwide malaria prevalence baseline findings for the Myanmar NMCP and partners as they move further toward malaria pre-elimination and elimination goals. The utility of these findings would be maximised if future surveys were to be regularly conducted from which to measure progress toward elimination of malaria. Use of PCR and particularly serology would enable monitoring of changes in transmission intensities to inform targeting of future interventions and ultimately certify elimination status . IdentifS1 Table(DOCX)Click here for additional data file.