Malaria remains an important cause of morbidity and mortality in Africa. Previous studies that assessed C-reactive protein (CRP) have centered on the conventional method. This study evaluated the usefulness of high-sensitivity CRP (hs-CRP) in malaria diagnosis and morbidity in a pediatric population in Ghana.  A total of 267 subjects , between the ages of 7 months and 18 years, were recruited for this case-control study. Blood samples were collected for malaria parasite density by microscopic examination; full blood count, electrolytes, and liver function tests using an automated analyzer; and hs-CRP levels by sandwich ELISA method.  The median hs-CRP concentration was lowest in the control group and increased significantly from low to high parasitaemia. The median hs-CRP level was significantly higher in high malaria parasitaemia compared to moderate and low malaria parasitaemia. Increasing hs-CRP cutoff (3.12-4.64 mg/L) presented with increasing specificity (79.3-93.1%) and sensitivity (96.4%-97.4%), except for moderate parasitaemia where a decline in sensitivity (80.9%) was observed. However, hs-CRP had relatively lower PPV but high NPV at low parasitaemia while both the PPV and NPV were moderate in moderate parasitaemia.  hs-CRP yielded a high sensitivity, specificity, and accuracy for low, moderate, and high-grade malaria, respectively, and thus may serve as an effective supplementary diagnostic and prognostic biomarker for Plasmodium parasite infection. However, hs-CRP might not be readily useful yet for diagnostic purposes in hospitals due to the relatively low PPV and NPV for low and moderate parasitaemia and thus necessitates further studies in larger cohorts. Malaria is a widespread disease in the tropical and subtropical regions, affecting mostly sub-Saharan Africa, Asia, and Latin America . As an iIn endemic areas, there may be afebrile presentation of malaria even in patients with high parasite density . ConfounAs a result, some blood tests, including leukocyte count and liver function tests, have previously been used to assess the malaria severity but have shown to have a rather low sensitivity. Other markers related to immune activation in malaria have also been explored. C-reactive protein (CRP), an acute phase reactant, whose plasma concentration increases during inflammatory disorders , has gaiDespite the reported usefulness of traditional CRP tests in malaria, a more sensitive approach could potentially enhance early detection and monitoring of malaria severity. High-sensitivity CRP (hs-CRP) allows the detection of low-range differences in C-reactive protein levels (1-10 mg/L) as opposed to the conventional method (10-1000 mg/L). Previous studies have focused on its use in cardiovascular diseases –21, seps2 in size and is bounded by four districts and is located in the rainforest zone of West Africa with a population of about 2 million inhabitants [This case-control study was conducted at Komfo Anokye Teaching Hospital (KATH) located in Kumasi, the capital of the Ashanti Region in Ghana, between August 2015 and April 2016. Kumasi is Ghana's second largest city located about 300 km from the national capital, Accra. The city of Kumasi lies between latitude 6.35 °N and 6.40 °N and longitude 1.3 °W and 1.35 °W, and it is 150 kmabitants . The cliabitants .Participants between the ages of 7 months to 18 years were recruited for the study. Participants positive for malaria were recruited as cases  while those negative for malaria were considered as controls. The study participants were obtained through random sampling technique. Patients' corresponding clinical data was retrieved from the hospital's archive. Subjects with evidence of recent chronic illnesses, chronic liver disease, inflammatory diseases, and viral exanthema as well as those receiving anti-inflammatory drugs were excluded.Ethical approval for this study was obtained from the Committee on Human Research Publication and Ethics (CHRPE) of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology (CHRPE/AP/184/16), and also from the Research and Development Department of KATH. Written informed consent was obtained from parents or guardians after the aim and objectives of the study had been explained to them.3 EDTA tubes and mixed to prevent clotting. The whole blood in the EDTA tube was used for plasmodium parasite identification and parasite density as well as full blood count (FBC). The remaining 2 mL was dispensed into gel separator tubes, placed in a centrifuge, and spun at 3000 rpm for 10 minutes to obtain the serum. The serum was stored at -20°C until analysis. Serum was used for the estimation of hs-CRP, liver function tests, and electrolyte.Five (5) milliliters of blood was aseptically collected from the antecubital vein of each respondent at the hospital. Blood samples were collected immediately after written informed consent was received. Three (3) milliliters of whole blood was dispensed into Kμl of participant. Blood films were declared negative if no parasites were seen in 200 oil-immersion fields as described by Squire et al. [P. falciparumcounts ≥ 100 parasites at a high-power field in thick smear, parasite counts were confirmed in thin films  and recalculated with 200 WBCs. Malaria parasitaemia was graded as low parasitaemia , moderate parasitaemia , and high parasitaemia  according to WHO criteria [Standard thick and thin films were prepared on clean grease free slides for each participant and the thin films fixed with absolute methanol, followed by staining with 10% Giemsa and subsequent microscopic examination for malaria parasite . Using te et al. . For P. criteria .μL of hs-CRP standards, diluted specimens, and controls (100-fold dilution) was dispensed into appropriate precoated microtitre wells followed by 100 μL of hs-CRP enzyme conjugate reagent, mixed thoroughly for 30 seconds, and incubated at room temperature for 45 minutes. After, the incubation mixture was removed from the wells by flicking plate contents into a waste container, rinsed, and flicked again with deionized water. Residual water droplets were removed by sharply striking the wells onto absorbent paper. 100 μL of tetramethylbenzidine (TMB) solution was then dispensed into each well and mixed gently for 5 seconds, followed by 20 minutes' incubation at room temperature. The reaction was stopped by adding 100 μL of stop solution to each well and gently mixed for 30 seconds. The absorbance of the final colored product was measured spectrophotometrically at 450 nm using the Thermo Electron Multiskan EX plate reader . The mean absorbance value (OD450) for each set of reference standards, controls, and samples were calculated. The calculated mean OD450 obtained for each reference standard was used to construct a standard curve and the concentrations of samples and controls determined from the standard curve. The obtained values of the patient samples and control sera were then multiplied by the dilution factor of 100 to obtain actual hs-CRP concentration. Patient samples with hs-CRP concentrations greater than 10 mg/L were further diluted 10-fold after the initial 100-fold dilution , and the final CRP values were multiplied by 1,000 to obtain the actual concentration of hs-CRP.Serum bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, sodium, potassium, chloride, and calcium estimation were assayed using the Cobas Integra automated chemistry analyzer . The reagent for the determination of hs-CRP was purchased from DRG International Inc.  and analyzed based on the principle of solid phase sandwich enzyme-linked immunosorbent assay according to the manufacturer's instructions (standardized with an intra-assay CVs = 2.5%-4.1% and interassay CVs = 2.3%-7.5%). Briefly, 10 Daily calibration and maintenance of the analyzer was performed according to the manufacturer's instructions as previously described . Briefly3/μL and WBC > 4.5 × 103/μL of whole blood, respectively.Anaemia was defined as haemoglobin level < 13.5 g/dL for males and < 12.0 g/dL for females. Thrombocytopaenia and leukocytosis was defined by platelet count < 150.0 × 10b to estimate the effect size. Continuous variables were presented as mean ± standard deviation for parametric variables and median (interquartile range) for nonparametric variables. The Kruskal-Wallis test was used to assess significance of differences for nonparametric variables between controls and different grades of malaria parasitaemia, followed by the Bonferroni post hoc multiple comparison test. One-way ANOVA was used to test for significance of differences for parametric variables between controls and different grades of malaria parasitaemia, followed by the Tukey post hoc multiple comparison test. Partial correlation was used to test for associations between various parameters upon adjusting for age and gender. The ROC analysis was used to ascertain the diagnostic performance of hs-CRP for malaria. Confidence was set at 95%, and p value less than 0.05 was considered statistically significant. All statistical analyses were performed using IBM SPSS 20.0 Statistics and GraphPad Prism 7 version 7.04.All categorical data were presented as frequency (percentages), and the Chi-square test statistics was used to test for association between variables. The Mantel-Haenszel test of trend was used to examine rates across levels of parasitaemia. The trend across age ranges was evaluated by the Jonckheere trend test followed by Kendall's tau-A total of 267 participants comprising 167 confirmed malaria positive cases  and 100 malaria-negative controls  were recruited for the study.The prevalence of low, moderate, and high-grade malaria parasitaemia were 16.8%, 37.1%, and 46.1%, respectively. A higher proportion of the participants with low, moderate, and high parasitaemia were 1-5 years old, in elementary school, used ITN, were anaemic, and had normal platelet and WBC counts. Most of the participants with moderate and high parasitaemia were females, and there was an equal proportion of males and females with low parasitaemia .As shown in Our results show that the median hs-CRP concentration is lowest in the control group and increases significantly from low to high parasitaemia .As shown in Malaria is a disease of global health importance requiring urgent interventions to reduce the disease burden. The WHO has proposed appropriate and early diagnosis as a way to decrease morbidity and mortality. Some biomarkers have been assessed in this effect. A significant one is C-reactive protein. Previous studies on usefulness of C-reactive protein in malaria have only centered on the conventional CRP , 30–34 wpfhrp 2 gene [This study reports increasing level of hs-CRP across increasing parasite density among children with malaria. Coupled with the high sensitivity and specificity, this finding suggests that the cutoff values of hs-CRP obtained in this study may serve as a potential marker for malaria morbidity and help to identify patients with a chronic low-grade proinflammatory state, a critical contributor to the clinical manifestations of malaria , 36. Addp 2 gene , making Nevertheless, the relatively low PPV for low parasitaemia and the moderate PPV and NPV in moderate parasitaemia suggest that, though hs-CRP may assist in making some informed clinical decisions, especially in highly malaria-endemic areas, where fever is frequently equivalent to malaria, it might not be readily useful yet for diagnostic purposes in hospitals and thus necessitates further studies in larger cohorts.Additionally, studies have shown that, in malaria-endemic regions, children acquire passive immunity from their mothers. However, this immunity is limited to the first 6 months of life, after which the immunity is lost , 40, andThis study is somewhat limited by the large age range of the cohort and the fact that only microscopy was utilized for malaria diagnosis and not tests such as RDTs or the more sensitive diagnostic tools such as PCR. Additionally, the increased hs-CRP level could be due to other inflammatory conditions such as viral and bacterial infections which were not screened for.hs-CRP yielded a high sensitivity, specificity, and accuracy for low, moderate, and high grade malaria, respectively, and thus may serve as an effective supplementary diagnostic and prognostic biomarker for Plasmodium parasite infection. However, hs-CRP might not be readily useful yet for diagnostic purposes in hospitals due to the relatively low PPV and NPV for low and moderate parasitaemia and thus requires further studies in larger cohorts.


Plasmodium vivax rhoptry proteins have been shown to induce humoral immunity during natural infections. Therefore, these proteins may be potential novel vaccine candidates. However, there is a lack of data on the duration of antibody and memory B cell (MBC) responses. Here, the longitudinal analysis of antibody and MBC responses to the P. vivax rhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence.Rhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. P. vivax-infected subjects (age 18–60 years) were recruited to explore the frequency and persistence of antibody and MBC responses against rhoptry proteins (PvRALP1-Ecto and PvRhopH2) using both cross-sectional and longitudinal cohort study designs. Antibody levels were determined by ELISA during clinical malaria, and at 3, 9 and 12 months post-infection. The frequency of MBC sub-sets and presence of rhoptry-specific MBCs in subjects 18 months after treatment were detected by flow cytometry and ELISPOT assay.Thirty-nine samples from The seroprevalence of antibodies against PvRALP1-Ecto and PvRhopH2 proteins was found to be high during acute infection, with IgG1, IgG2 and IgG3 sub-classes predominant. However, these anti-rhoptry responses were short-lived and significantly decreased at 9 months post-infection. To relate the durability of these antibody responses to MBC persistence at post-infection, 18-month post-infection peripheral blood mononuclear cells (PBMCs) samples were taken to detect rhoptry-specific MBCs and frequency of MBC sub-sets, and correlate with antibody responses. These late post-infection samples revealed that rhoptry-specific MBCs were present in about 70% of total subjects. However, the persistence of specific MBCs was not correlated with antibody responses as the majority of malaria subjects who were positive for PvRALP1-Ecto- or PvRhopH2-specific MBCs were seronegative for the rhoptry antigens. The frequencies of classical MBCs were increased after infection, whereas those of activated and atypical MBCs were decreased, indicating that MBC responses could switch from activated or atypical MBCs to classical MBCs after parasite clearance, and were maintained in blood circulating at post-infection.P. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.The study showed that rhoptry antigens induced the development and persistence of MBC responses in  Plasmodium vivax is the most prevalent cause of recurring malaria and has a wide geographic distribution, mainly in Asia–Pacific and Latin American regions [P. vivax biology, making it difficult to control because the parasite can be hidden for a long time in the patient’s liver and then emerge to cause relapsing P. vivax [Malaria remains an important global health issue causing illness and death worldwide, especially in tropical and sub-tropical areas. There were an estimated 219 million malaria cases and more than 400,000 deaths in 2017 [P. vivax , 4. GivePlasmodium falciparum have moved into clinical trials. In contrast, P. vivax vaccine development has lagged far behind, as only three P. vivax vaccine candidates  have even reached phase I clinical trials [P. vivax, technical challenges such as maintaining P. vivax in culture, and limited animal models of infection. For blood-stage malaria vaccines, the aim is to block parasite invasion and subsequently reduce the clinical burden. At present, P. vivax Duffy-binding protein region II (PvDBPII) is a leading vaccine candidate because P. vivax invasion of erythrocytes is largely dependent upon its interaction with the Duffy blood-group antigen [P. vivax and protects against high-density P. vivax infection by inhibiting parasite invasion into red blood cells (RBCs) [P. vivax isolates [P. vivax invasion [There is currently no licensed vaccine available for malaria. Several antigen candidates against l trials . This ma antigen . It indus (RBCs) . Howeverisolates . Moreoveinvasion . Thus, aP. falciparum infections can induce antibody responses to blood-stage antigens but that these responses were relatively short-lived [P. vivax were short-lived and had returned to near baseline by day 145 [Plasmodium-specific MBCs without frequent boosting [P. falciparum-specific MBCs despite repeated infections annually [Antibody responses to blood-stage malaria are required for inhibition of parasite invasion –15. Longrt-lived . Likewis day 145 . The preboosting , 19. Howannually , 21. Thiannually –24. TherP. falciparum rhoptry neck protein (RON) 2–4, are involved in tight junction formation between the parasite and its target cells by interacting with the micronemal protein apical membrane antigen (AMA) 1 [P. falciparum infection [P. falciparum merozoites [Plasmodium spp. [The rhoptry is a large secretory organelle of the merozoite that contains a wide range of proteins which are involved in invasion of RBCs , 26. It  (AMA) 1 , 28. How (AMA) 1 . The rhonfection , 31. Therozoites , 32. RALrozoites  and Rhoprozoites . Furtherium spp. , suggestP. vivax-exposed Korean individuals, and purified serum anti-PfRALP1 from Malian P. falciparum-infected patients inhibits parasite invasion [P. vivax proteins at the rhoptry bulb, rhoptry-associated membrane protein (RAMA) and RhopH2 antigens, show an ability to induce the acquisition of humoral immunity in both mouse models and patients [P. vivax patients living in a low-malaria-endemicity region of southern Thailand. Longitudinal sampling of participants in a cohort study was conducted to determine whether individuals exposed to P. vivax developed and were able to maintain antibody and MBC responses to these rhoptry proteins.High antigenicity of rhoptry proteins has often been reported in malaria patients , 35–37. invasion , 35. Twopatients , 38. IntP. falciparum and P. vivax were common. Both a cross-sectional survey and a cohort study were set up between 2014 and 2017 to explore the antigenicity of rhoptry proteins and monitor the longevity of antibody and MBC responses. First, serological responses to rhoptry proteins were determined from 39 patients with acute P. vivax infection  where both P. vivax infection, the kinetics of B cell sub-sets in the acute and recovery phases, and the persistence of rhoptry-specific MBCs were investigated in the sub-cohort 2 study (May 2016 to May 2018). Peripheral blood mononuclear cells (PBMCs) from 10 individuals were isolated to perform for flow-cytometric analysis and ELISPOT assays. Plasma was collected for detection of total IgG and IgG subclasses. Peripheral blood samples (10 mL) from participants were obtained by venipuncture into heparinized tubes before administration of treatment, and transported to the laboratory for processing within 6 h of collection. The PBMCs were separated by Ficoll-Hypaque density-gradient centrifugation and used for MBC experiments. Plasma was stored at − 20 °C for antibody analyses.To explore the basis of persistence of humoral immunity against rhoptry antigens in P. vivax infection was diagnosed by microscopic examination of both thin and thick blood films, and confirmed by nested PCR. Data on occurrence and number of prior P. vivax infections in each individual were obtained from the records of the local Vector-borne Disease Unit 11.4.2. The malaria staff collected post-treatment blood samples from patients every 3 months to check for sub-clinical malaria by nested PCR. To estimate the incidence of clinical malaria over the study period, malaria staff had carried out weekly house-to-house visits from May 2014 to May 2017, and in May 2018, with confirmation of infection by nested PCR. Malaria-unexposed persons living in Bangkok, Thailand, were recruited as healthy controls . Ethical approvals were obtained from the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand (MUIR 2012/079.2408).The inclusion criteria for subject recruitment were the following: (1) systolic blood pressure not less than 90 mmHg; (2) body temperature not higher than 40 °C; (3) haematocrit not less than 25%; (4) no prior treatment with corticoids or non-steroidal anti-inflammatory drugs (NSAIDs); and, (5) age of 18 years or above. Those who did not meet the criteria were excluded. Written informed consent was obtained from all participants before enrollment. The pvralp1-ecto, comprising amino acids 31 to 675 without the signal peptide sequence, and the first exon of the pvrhoph2 sequence, amino acid position 67 to 1161, were amplified with the following primers: PvRALP1-Ecto F: 5′-atcactagttctcgagATGGCGTACCGCCTAAAGAGG-3′ and the reverse primer PvRALP1-Ecto R: 5′-ccctatatatggatccTCACTAGAACATGTCGTAGAGCAGGC-3′, RhopH2 F:PvRhopH2 F: 5′-GGGCGGATATCTCGAGGAGCTGAGCCACAGCTTGT-3′ and PvRhopH2 R: 5′-GCGGTACCCGGGATCCTCACTTCTCCACATCCTCGTGGT-3′. The purified PCR products were then cloned into the XhoI and BamHI sites of the pEU-E01-His-Tev-N2 plasmid vector, which is an expression plasmid with an N-terminal hexa-histidine (His)-tag followed by a tobacco etch virus protease cleavage site. Plasmid DNA was prepared, purified and eluted in 0.1× TE buffer , and used for in vitro transcription. The recombinant proteins were synthesized with a wheat germ cell-free protein expression system , using the bilayer translation reaction method as described previously [To express the recombinant PvRALP1-Ecto and PvRhopH2 proteins, the protocol followed that of previous studies , 35. In eviously . The HisP. vivax plasma that showed seropositive to rDBPII antigen from previous study [Naturally acquired antibodies against recombinant PvRALP1-Ecto and PvRhopH2 were detected by ELISA. In brief, 96-well, flat-bottom microplates  were pre-coated with 0.5 μg recombinant PvRALP1-Ecto or PvRhopH2 per well and incubated overnight at 4 °C. After blocking with PBS containing 4% BSA for 2 h, 100 μL of each plasma sample at a dilution of 1:200 was added to duplicate wells and incubated at room temperature for 1 h. The plates were washed with PBS containing 0.05% Tween 20 (PBS-T) five times and incubated with peroxidase-conjugated, goat anti-human IgG  for 1 h. Subsequently, washing was repeated, and the plates were developed with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) substrate  for 45 min. The optical density (OD) was determined at 405 nm using a plate reader. The positive samples (n = 2) were IgG isotype responses to rhoptry proteins were detected following a protocol similar to that for total IgG detection. In the step when each plasma sample was added at a dilution of 1:100, followed by addition of conjugated antibodies, horseradish peroxidase (HRP)-conjugated anti-human IgG1, IgG2, IgG3, and IgG4 antibodies at a dilution of 1:1000 were used for detection. The OD of the reaction was measured at 450 nm after the addition of the tetramethylbenzidine (TMB) enzyme–substrate. The positive controls were plasma (n = 2) of the vivax malaria patient who had the highest OD value for total IgG. A baseline OD was established using plasma samples from 20 healthy individuals living in non-malaria endemic areas (HC).The levels of total IgG or IgG subclasses specific to PvRALP1-Ecto or PvRhopH2 were expressed as reactivity indexes (RI), which were obtained by dividing the mean sample OD by the cut-off value. The cut-off value (mean OD + 2 standard deviations (SDs)) was calculated from a set of negative controls obtained from plasma samples from HC. A sample was considered to be positive for a specific antibody if the RI was ≥ 1.PBMCs collected during acute malaria and 18 months post-infection were used for B cell sub-set phenotyping. Fluorochrome-conjugated, mouse anti-human monoclonal antibodies were used to stain 1 million PBMCs/100 μL FACS buffer. A cocktail consisting of the following mouse monoclonal antibodies was used: FITC-CD19, PerCP-CD20, PE/Cy7-CD10, PE-CD27, and APC-CD21 . After staining for 15 min, cells were washed with FACS buffer. Finally, cells were suspended in 250 μL FACS buffer. The analyses were done by a flow cytometer . Data were processed using FlowJo software .6 PBMCs from P. vivax subjects were prepared for cell stimulation along with negative controls. First, 1 × 106 cells per mL were stimulated with polyclonal activator R848 and recombinant human IL-2  at 37 °C in a humidified 5% CO2 incubator for 72 h. Then, the ELISPOT assay was performed by coating with 15 μg/mL of monoclonal antibodies of anti-human IgG , 5 μg/mL of recombinant PvRALP1-Ecto or PvRhopH2 rhoptry antigens, or 1 μg/mL of tetanus toxoid (TT) antigen  onto MultiScreen Filter PVDF Immobilon plates (Merck Millipore). Cells were harvested and seeded in duplicate to yield 5 × 104 cells per anti-human IgG-coated well, and 1 × 106 cells per specific antigen-coated well. After 24 h of incubation at 37 °C under 5% CO2, 1 μg/mL of detection monoclonal antibodies MT78/145 (Mabtech) diluted in PBS-0.5% FBS was added and incubated for 2 h at room temperature. Following a thorough washing with PBS, the diluted (1:1000) streptavidin-HRP-conjugated, polyclonal goat anti-human IgG (Mabtech) and TMB substrate for ELISPOT (Mabtech) were added. The filtered plates were rinsed with deionized water after distinct spots emerged. Antigen-specific MBCs were expressed as spot-forming cells (SFCs) in the wells. The plates were analysed with a Bioreader 5000 Pro-F gamma ELISPOT Reader . PBMCs from P. vivax-infected subjects that were cultured without stimulation and incubated overnight with rhoptry antigens were used as negative controls. A positive ELISPOT response was defined as detectable spots in duplicate wells with the total spots in the specific antigen-coated wells being at least twice the number of spots detected with the negative control samples.The presence of rhoptry-specific MBCs was determined by ELISPOT assay as performed in a previous study . In brieP-values < 0.05 were considered significant. The statistical analysis was performed and graphs prepared using GraphPad Prism software .The Mann–Whitney U test was used to compare data of 2 groups of samples (unpaired) from different subjects, while the Wilcoxon signed rank test was used to compare data of 2 groups of paired data from the same subjects at different time points. Kruskal–Wallis one-way ANOVA was used to compare data from more than 2 groups. The association between the responses of rhoptry-specific MBCs by ELISPOT and antibodies by ELISA was done using Fisher’s exact tests. In all analyses, 2-tailed P. vivax-infected patients, using plasma samples collected from May 2014 to May 2017 . Subject positivity to PvRALP1-Ecto and PvRhopH2 was 66.67% (26 of 39) and 29 74.36% (29 of 39), respectively . Both anti-PvRALP1-Ecto and anti-PvRhopH2 titres were significantly reduced at 9 months post-infection , or anti-RAP2 antibodies that inhibit P. falciparum parasite invasion [Aotus primate model by showing the capacity to reduce parasitaemia after rPvRAP2 immunization [P. vivax parasite invasion, as well as identification of target anti-rhoptry antibodies, will be useful for consideration of these two proteins as potential vaccine candidates.Rhoptry proteins are among the blood-stage antigens that have been characterized and shown to induce antibody responses , 25. HowP. vivax –46. Thesand Mali , 35. Furinvasion , 47, 48.Plasmodium spp infection through complement-mediated lysis [P. falciparum exposure [P. vivax and P. falciparum have the ability to trigger B cells to produce cytophilic IgG1 and IgG3 [P. vivax-infected patients in killing the parasite. This conclusion comes from previous reports that cytophilic IgG1 has the high affinity to Fcγ receptors [P. vivax.Analysis of IgG sub-class responses to PvRALP1-Ecto and PvRhopH2 antigens is important for evaluating protective activity since different IgG sub-class responses mediate distinct immune effector functions , 43. Alsed lysis  and celled lysis –52 and aed lysis . In conted lysis , 55 wherexposure –58. The exposure . Consistand IgG3 –62 and nand IgG3 , 60, 63.eceptors , thus, weceptors , 52, 53.eceptors , 65. HowP. vivax infections was reported. The specific MBC responses to PvRALP1-Ecto and PvRhopH2 antigens were detected at high frequency at 18 months post-infection (approximately 70 and 90% of subjects). These findings suggest the ability of PvRALP1-Ecto and PvRhopH2 antigens to induce MBC development during malaria infection, and their persistence in the absence of additional malaria antigen stimulation. The literature on MBCs to malaria is based on populations with various intensities of transmission. The P. vivax subjects who live in low malaria transmission areas of northern Thailand and northwestern Brazil appear to maintain MBC responses against malaria-specific antigens for many years after their last infection [P. vivax infections. One explanation is that the alterations of circulating MBC sub-sets, including the expansion of atypical MBC sub-sets, could interfere with the development of long-lived antibody secreting cells and thus antibody production as this MBC sub-set in P. falciparum exposure expressed lower levels of surface IgG, possessed reduced B cell receptor signalling, and impaired antibody production in vitro [MBCs are major contributors to antibody production, but it is unclear what factors interfere with or promote maintenance of long-lived antibodies. Here, the presence of rhoptry-specific MBCs in individuals with natural nfection . In contnfection , 21. Howin vitro , 67. How+ classical MBCs were triggered via parasite infection, and class-switched into CD27+ activated MBCs or CD27− atypical MBCs, helping in parasite clearance. After infection these two MBC sub-classes were reduced and class-switched back to CD27+ classical MBCs. However, the relationship these MBC sub-sets to plasmablast/plasma cell responses, as well as that between long-lived plasma cells and antibody longevity in recovered subjects (18 months post-infection) was not demonstrated in this study. A full understanding of these relationships would be useful for the context of immunity to malaria infection and vaccine development, since generation of pre-existing antibody from long-lived plasma cells could help to kill parasites. In accordance with the previous studies, the temporary changes in MBC sub-sets was previously reported from an area with seasonal malaria transmission [+CD21−CD27−  or CD19+CD27+ , without any malaria specificity [An alteration of circulating MBC populations was detected during malaria infection , 68, 69.smission , 68, 69.smission . Most ofcificity , 68, 69.P. vivax and P. falciparum exposure that would be involved in the persistence of antibody and MBC responses. The study was also limited by the inability to recruit a larger group of P. vivax subjects into the cohort study.  The observation in this study that showed persistence of long-lived PvRALP1-Ecto- and PvRhopH2-specific MBCs in the absence of re-infection should be confirmed by future studies. In addition, a larger sample size would allow correlation analyses among anti-PvRALP1-Ecto and -PvRhopH2 levels, specific MBCs and frequency of MBC sub-sets.The limitations of this study include the lack of an independent measure of prior The results in this study provide additional information to the available data on the characteristics of PvRALP1-Ecto and PvRhopH2 proteins, and their ability to induce humoral immunity during acute malaria and after infection. The anti-PvRALP1-Ecto and -PvRhopH2 responses in the majority of individuals were short-lasting after infection. However, the specific memory B cells in a sub-group of individuals was stably detected (through 18 months). This evidence supports previous reports that effective long-lived MBCs can develop and persist in regions with low rates of malaria transmission.Additional file 1. PvRALP1-Ecto and PvRhopH2 specific MBC response by ELISPOT assay.