Hematology analyzers display abnormal parameters during malaria infection providing insightful information for suspecting and assessing malaria infection. The goal of this study is to demonstrate the potential of a three-part differential hematology analyzer to assess malaria, provide information about the parasitemia, and discuss the importance of combining C-reactive protein (CRP) with hematology parameters to obtain further information about the malaria infection.P. vivax and 31 P. falciparum positive malaria samples, five cases of mixed P. vivax and P. falciparum infection, and three co-infection cases of P. vivax and dengue. Raw data from the three-part analyzer LC-667G CRP (HORIBA) and the corresponding microscopic findings  were obtained for each sample.The present study shows the results of a case–control study during the monsoon season of years 2018 and 2019 in Mumbai, India. The study considers 1008 non-malaria febrile cases, 209 3/µL, while CRP medians were 67.4, 81.4 and 10.4 mg/L in P. vivax, P. falciparum and control groups respectively . Compared with negative samples, platelets counting less than 161.5 × 103/µL were observed on malaria patients . Especially in P. vivax cases, an abnormal peak was frequently observed in the white blood cells (WBC) histogram around the 37fL channel. The events counted around that channel showed a linear correlation with the counting of red blood cells infected predominantly with larger parasitic forms. Parameters like CRP , WBC  and PLT  were correlated with the parasitemia of P. vivax samples. Between the malaria and dengue groups, the highest area under the receiver operating characteristic curve was observed on CRP .The medians of platelet counts (PLT) were 102.5, 109.0, and 223.0 × 10A three-part differential hematology analyzer has the potential to not only trigger malaria diagnosis confirmation but also assess the severity of the infection when CRP is considered.The online version contains supplementary material available at 10.1186/s12879-021-06704-5. According to the WHO an estimated 228 million cases of malaria occurred in 2018 worldwide and 85% of this burden was located in 19 sub-Saharan Africa countries and India. For rapid disease management and surveillance of malaria, the WHO recommends two main strategies to eradicate malaria: closing the gaps of healthcare access between urban and rural areas, and diagnosis  by microscopy or malaria rapid diagnostic tests (RDTs) in all patients with malaria suspicion. Fever is the main manifestation for suspicion of malaria infection. In malaria endemic areas, it acts as a trigger for parasitology testing [RDTs are currently replacing microscopy, the golden standard for diagnosing malaria, especially in endemic countries. Thus, complementary information should be considered to ensure correct interpretation and correct disease management particularly in surveillance programs . The twoP. falciparum in comparison with detecting P. vivax . This is most probably due to the difficulty of detecting early stage parasites and their low nucleic acid content [P. falciparum infections where trophozoites and schizonts are barely seen in peripheral blood.The potential to diagnose clinically unsuspected cases of malaria for patients subjected to complete blood count (CBC) is one of the main benefits of flagging malaria in hematology analyzers . A comprP. vivax infected samples [In spite of all promising results from ACCs to identify malaria, fewer studies have been conducted for three-part differential ACCs. One study combined hemoglobin (HGB), total lymphocyte count (TLC), platelets (PLTs) and red cell distribution width (RDW) data in a KX-21 (Sysmex) achieving a 26.1% sensitivity and 96.3% specificity in a study mainly consisting of P. vivax malaria infection with inflammatory activation, including CRP, and cytokine imbalance [CRP is a member of the pentraxin family of proteins, an acute-phase reactant and a biomarker for inflammation. Although CRP is not a specific biomarker for detecting malaria infection, several studies have identified CRP to be a potential marker to assess the severity of malaria infections –13. A stmbalance .Dengue and malaria infections, which frequently coexist in endemic areas, present with similar symptomatology and produce similar abnormalities in CBC parameters. CRP has been observed to be a useful biomarker to discriminate between malaria and dengue  using CRP > 5 mg/L as a cut off to differentiate malaria from dengue, obtaining sensitivity of 99% and specificity of 35% . In addiThe goal of this study is to demonstrate the potential of a three-part differential hematology analyzer to assess malaria, provide information about the parasitemia, and discuss the importance of combining CRP with hematology parameters to obtain further information about the malaria infection.The present study shows the results of a case–control study involving comparison of raw instrument data from a three-part differential hematology analyzer with CRP measurement  and corresponding microscopic findings of samples obtained from patients visiting Dr. Dharap’s Diagnostic Centre, Mumbai, India during the monsoon season of years 2018 and 2019.During monsoon season, the laboratory routinely caters to patients for diagnostic testing in cases of acute febrile illness which comprises of a CBC + CRP, peripheral examination of blood smear for malarial parasites and RDTs for malaria and dengue NS1, being an endemic area with coexistent infections. All groups considered for the current study were evaluated as patients with acute febrile illness. Documented historical data of known malaria cases, as well as dengue positive and negative cases were used for the study.P. vivax RDTs but negative in blood smear, and one sample was positive with P. falciparum RDT but negative in blood smear. For the current study, the historical documented data was further anonymized in coded fashion in order to protect patient confidentiality in accordance with local ethical or IRB guidelines, according to national and international standards for the conduct of clinical studies including 21 CFR Parts 50 and 56 and International Conference on Harmonization (ICH) E6-Good Clinical Practice Consolidated Guideline. Ethical clearance was not obtained as the study was carried as a retrospective review of the available raw instrument and historical patient data, without any collection of extra specimen or monetary charges to patients and did not involve any communication of results of the study to the clinician so as to affect the diagnostic and therapeutic management of the patients.As being the golden standard for diagnosis, malaria positivity was determined solely by the examination of blood smears. We found four discordant cases between the blood smear inspection and the RDTs: one case was confirmed by blood smear but negative in both species RDTs, two samples were positive with Testing for ‘Acute Febrile Illness’ in the laboratory comprises of a full blood count, CRP value, peripheral examination of blood smear for malarial parasites, and RDTs for malaria and dengue NS1.A full blood count is performed using a three-part differential hematology analyzer  which additionally provides CRP level estimations. For peripheral blood smear examination the following technique is routinely used. Anticoagulated (K2-EDTA) blood of the patient is used to prepare thick & thin blood smears for peripheral blood smear examination (PBSE). Both types of smear preparations are air dried. The thin preparation is fixed with methanol, whereas the thick blood smear is subjected to ‘process of de-hemoglobinisation’ with deionized water. Both smears are then stained by ‘Field staining technique’ using an automated slide stainer. Thin smears are examined for presence of malarial parasites and species identification, presence of platelet aggregates, macroplatelets and nucleated red blood cells, if any.Malaria cases are further assessed for parasite density by counting parasite life cycle forms against 100 white blood cells by an experienced pathologist. The life cycle forms are grouped as small forms being ring and early ameboids, and large forms including late ameboids with ample parasitic material, schizonts and gametocytes.P. falciparum HRP-II (0.75 ± 0.15 µg) with monoclonal anti-P. vivax pLDH (0.75 ± 0.15 µg) respectively to detect presence of P. falciparum & P. vivax related antigens are used.For additional screening and confirmation of malaria cases, Rapid Malaria Antigen detection test kits manufactured by SD Biosensor Healthcare Pvt. Ltd., Gurugram, India using monoclonal anti-For screening and diagnosis of dengue, RecombiLISA NS1 Antigen tests are performed on separate serum samples, utilizing pairs of specific polyclonal & monoclonal anti-dengue antibodies of all four serotypes  and analytical sensitivity of 0.3 ng/mL for type 2 NS1 antigen, manufactured by CTK Biotech, Inc., United States of America.P. vivax & P. falciparum samples respectively, as well as malaria & dengue positive samples, Mann–Whitney U and Krustal–Wallis tests were applied due to their non-parametric distribution.To determine the significance of the differences between the hematology parameters extracted from LC-667G CRP of positive & negative samples of P. vivax positive samples and to understand if CRP is correlated with the age of patients in a group stratification of malaria species and patient sex. Moreover, to evaluate the hypothesis of CRP values being different between P. vivax and P. falciparum groups, Mann–Whitney U test was used to evaluate the difference in medians.The Spearman’s rank correlation coefficients were calculated for understanding the independent association of the inspected parameters with the parasites load of A receiver operating characteristic (ROC) curve, the area under the curve (AUC), sensitivity, specificity, and the odds ratios were calculated for evaluating the clinical significance to detect malaria positivity among fever patients and to differentiate malaria from dengue samples.Due to the confounding factors in all the parameters from the analyzer, particularly CRP, and the limited distribution of samples, a matching method for reducing the risk of selection bias caused by the age and sex of patients was performed only in individual parameter comparisons . Please refer to the Additional file All the whisker-plots and statistical analyses were computed with OriginPro 2021 9.8.0200 and Excel 2016. The histogram graphs were created using R software 3.5.1.P. vivax positive cases, 31  P. falciparum samples, five cases of mixed P. vivax and P. falciparum malaria infection , and three co-infections of P. vivax and dengue  were considered for the present study. Nonetheless, the mixed malarial infections, and the three P. vivax and dengue co-infection samples were excluded from all statistical tests. In addition, 1008 non-malaria febrile cases were examined including 197  dengue positive cases. The median and range of ages were 28 (1–91), 35 (5–87) and 36 years (15–77) for non-malarial, P. vivax and P. falciparum cases, respectively.From the monsoon seasons of 2018 and 2019, based on selection criteria and availability of corresponding data, 209  Using the Krustal–Wallis test to compare the four groups  were found. Table P. falciparum  and P. vivax . Table P. vivax and P. falciparum, and each patient sex group. In addition, the Spearman’s rank correlation coefficients between age and CRP value in each subgroup of malaria species were calculated. The null hypotheses of equality of medians were not rejected when comparing the CRP values between malaria species , nor in patient sex of each malaria specie group. CRP correlates positively with patient age in the P. falciparum group (p = 0.008), but not in the P. vivax group (p = 0.825).The CRP measurement value has many confounding factors including patient’s age and sex. In order to evaluate the association and possible confounding factors of the available clinical variables with CRP value, a stratification of groups was created by matching patient age and sex between positive groups of P. vivax samples with high density of large forms of parasites . A multiple linear regression model was calculated obtaining the following equation y represents the events per µL from 19.3fL to 43.9fL in the WBC histogram , rather than P. vivax (76.44%), which is similar to the observations reported in [P. vivax parasite density with PLT (p < 0.001). To analyze the thrombocytopenia dependency on malaria species, the analysis must considers the parasite load and patient condition, as pointed out in a study where parasitemia was significantly higher in individuals with thrombocytopenia only in P. vivax, but not in P. falciparum infections [The most commonly reported affections on hematology parameters observed in malaria infection are thrombocytopenia, anemia, and leucopenia , 17. Throrted in , 22. Autorted in  have confections .Among the hematology parameters with significant statistical difference in medians between malaria positive and negative samples, platelets related parameters, especially MPV, should be further analyzed due to the strong affection with the elapsed time between collection and measurement of sample.P. vivax large forms, supporting the same observation of moderate correlation (r = 0.707) in [P. falciparum infections as the larger forms are sequestered by the spleen. Mature trophozoites and schizonts in P. falciparum infections are barely present in peripheral blood samples. According to [P. falciparum infections, can be used as a marker for disease severity. Therefore a semi-quantitative estimation of schizontaemia can be inferred from the events located before the 37fL channel of the LC-667G WBC histogram and might help to assess the severity of malaria infections.In both three and five part differential ACCs a peak around the RBC ghosting area in the WBC histogram has been identified as one of the main abnormalities to predict malaria infection –29. In t.707) in . The abnrding to , the preThe abnormal peak in the WBC histogram is not exclusively due to malaria iRBCs. Common interferences include RBC lysed debris, PLT aggregations, macro-PLTs, nRBCs, and others. Researchers used this abnormal peak along with two other parameters of the LH780 (Beckman Coulter) to identify malaria infections having an 83.1% specificity but failed to discriminate from samples with nRBCs . The disP. vivax infections. The positive and negative correlations of the P. vivax parasitemia with %LYM (p = 0.042) and %MON (p < 0.001) respectively, could be related to the interference in the WBC histogram, which suggests an incorrect counting of WBC subpopulations when iRBCs are present.Moreover, considering the parameters that are correlated with the total percentage of parasitemia such as WBC, PLT, and CRP, it might be possible to assess the level of parasitemia in combination with the WBC histogram analysis, especially in In low cost ACCs where the Coulter principle is the only technology responsible for differentiating the WBC population into three parts based on volume, the identification of malaria or dengue infected samples is rather difficult due to the impossibility of differentiating granulocytes populations. Addition of CRP increases the specificity of detecting malaria due to the inflammatory response secondary to parasite infection.P. vivax and P. falciparum groups (p = 0.526). The non-statistical significance in age and CRP correlation in the P. vivax group might be affected by the wider distribution of parasite density . The parasitemia was not included in the current study’s matching method due to the limited variation on P. falciparum positive samples. However, for future analyses on CRP, parasitemia should be also considered and treated as a confounding variable.Supporting observations of –35, in tP. vivax samples, the difference between medians was statistically significant. Nonetheless, both variables have a poor clinical relevance for differentiating malaria from dengue samples using the Youden’s index (AUC of WBC = 0.580 and AUC of MCHC 0.620). In contrast, PLT medians of both species of malaria infections were lower than dengue cases (p < 0.001) and the odds ratio of malaria positive patients having less than 160 × 103 platelets/μL was 10.83. Discrepancy of results with [Dengue and malaria in many countries are co-endemic diseases that present similar clinical presentations. The importance of distinguishing them has been reported by , who devlts with  might beThe CRP parameter showed the most significant difference between malaria and dengue samples. From our current data set, patients with malaria are 15.78 times more likely to present CRP greater than 26.85 mg/L, compared to dengue patients. In comparison with negative patients with fever, patients with malaria have a smaller odds ratio  to present CRP greater than 27.65 mg/L. Although CRP is not a specific biomarker for malaria, the response of CRP to virus infections is rather small, making it a good parameter to understand the etiology of the infection and to help differentiate malaria and dengue from similar hematology parameters. Nevertheless, dengue and other non-viral infections can co-exist in a single sample, thus relying only on CRP should not be an exclusive criteria in differentiating both diseases.P. falciparum), the single location of the study, and the bias selection from retrospective results.In general, hematology parameters and CRP depend on clinical characteristics and might vary upon demographics. Additionally, the immune response against malaria, dengue or other infections is related to the condition of the patient and the disease’s stage. Caution to generalize the current results must be taken considering the limited distribution of parasites in the inspected samples .P. falciparum samples. The discussion and observations provided were focused on P. vivax positive cases. Further research following this study should be oriented to measure P. falciparum samples, to evaluate the potential of using hematology analyzers to provide additional information to the RDTs, and to monitor anti-malaria drugs treatments.Our current data set is limited by the number of Additional file 1: Annex 1. Annex 1 QUADAS-2 analysis.Additional file 2: Annex 2. Raw data.


Plasmodium falciparum malaria could contribute to the design of effective malaria vaccines. Using a recently developed multiplexed FluoroSpot assay, we assessed cross-sectional pre-existing memory B-cells (MBCs) and antibody responses against six well known P. falciparum antigens , MSP-2 (FC27), MSP-3, AMA-1 and CSP) and measured their associations with previous infections and time to clinical malaria in the ensuing malaria season in Kenyan children. These children were under active weekly surveillance for malaria as part of a long-term longitudinal malaria immunology cohort study, where they are recruited from birth. After performing Cox regression analysis, we found that children with a breadth of three or more antigen-specific MBC or antibody responses at the baseline had a reduced risk for malaria in the ensuing P. falciparum transmission season. Specifically, MBC responses against AMA-1, MSP-2 (3D7) and MSP-3, as well as antibody responses to MSP-2 (3D7) and MSP-3 were prospectively associated with a reduced risk for malaria. The magnitude or breadth of MBC responses were however not correlated with the cumulative number of malaria episodes since birth. We conclude that increased breadth for merozoite antigen-specific MBC and antibody responses is associated with protection against malaria.Identifying the mechanism of naturally acquired immunity against  Plasmodium falciparum malaria is a leading cause of death in Sub-Saharan Africa, especially in children. Globally, over 241 million malaria cases and 627,000 related deaths were reported in 2020 . Although MBCs by themselves are unlikely to neutralize infectious agents, they are critical for maintaining anamnestic antibody driven immunity, whereby they rapidly proliferate and differentiate into antibody secreting cells in response to antigen re-stimulation . In humaCirculating malaria specific MBCs can be long-lived even in the absence of re-exposure to parasites, whilst antibodies may decay to below detectable levels over time , 29. ThuP. falciparum antigens  in children living in an endemic region of Kenya. Circulating antibody responses to these antigens have previously been identified as possible markers of immunity against malaria  and blood smears for microscopy. All children with RDT positive tests are treated with artemether/lumefantrine . All positive RDT results are confirmed by microscopy. For the purpose of this study, the definition of a clinical malaria episode was set as having an axillary body temperature of ≥37.5°C combined with a P. falciparum parasite density of ≥2500 parasites per microliter of blood , MSP-2 (FC27), MSP-3 and AMA-1 were expressed recombinantly in a mammalian expression system for use in the FluoroSpot and peptide tag ELISA assays, respectively, as described elsewhere  with GAL; MSP-2 (FC27) with Twin-Strep-tag® ; MSP-3 with WASP, AMA-1 with Twin-Strep-tag®; and the newly expressed CSP was tagged with the peptide tag WASP.lsewhere . CSP waslsewhere : MSP-11919, MSP-3 and AMA-1 in one well, and MSP-2 (3D7), MSP-2 (FC27) and CSP in another (2 in the presence of 1 μg/mL R848 and 10 ng/ml of recombinant IL-2 (both from Mabtech), were added to the wells used for the multiplex MBC antigen-specific analysis; and 25,000 cells to wells used for MBC total IgG analysis. Cells were then cultivated in these wells for 20-24 hours in 37°C and 5% CO2. Plates were washed, followed by addition of combinations of supernatants containing the respective tagged parasite antigens diluted in PBS and 0.1% BSA  (PBS/BSA) and incubated at room temperature (RT) for 1 hr. Irrelevant control antigens  were added in separate wells. After another wash, fluorophore-conjugated anti-tag antibodies or fluorophore-conjugated Strep-Tactin®XT (IBA LifeSciences) were added to wells an incubated for 1 hr at RT in PBS/BSA supplemented with filtered human plasma diluted 1:100. For the total IgG analysis, a biotinylated anti-human IgG (mAb MT78/145-biotin from Mabtech) followed by fluorescently conjugated streptavidin (SA) were added instead. After a last wash, Fluorescent enhancer (Mabtech) was added to the plates and incubated for 10 minutes at RT before plate underdrains were removed, and plates were dried in the dark at RT. The FluoroSpot plates were analysed using the Mabtech IRIS™ reader system equipped with Apex™ software version 1.1.7. Definition of assay disqualification was applied to samples with <1000 total IgG spot forming units (SFU) per 250,000 cells. Magnitudes of MBC responses were expressed as proportions of antigen-specific IgG spots per total IgG spots  and SFU per 106 PBMCs after subtracting reactivity to negative control antigens . The frequency of MBCs to the expression tag control antigen was low for all antigens , MSP-2 (FC27), MSP-3, AMA-1 and CSP as well as a negative control antigen (horse IFN-γ) as previously described (®XT (IBA LifeScience) at a concentration of 1 μg/mL in PBS and incubated overnight. The following day, additional binding sites were blocked by the addition of 100 µL of PBS supplemented with 0.1% Tween 20, 0.1% BSA (both from Sigma-Aldrich) (incubation buffer), and incubated for 1 hr at RT. Plates were then washed in PBS supplemented with 0.1% Tween 20 (Sigma) followed by addition of Twin-Strep® tagged variants of the P. falciparum antigen diluted 1:5 in incubation buffer. Plates were then incubated for 1 hr at RT. After another wash, human plasma samples diluted to 1:2000 in incubation buffer was added to plates and incubated for 1 hr in RT. Plasma antibodies having bound the respective antigen were detected by addition of horse-radish peroxidase conjugated anti-human IgG mAb (Mabtech AB) at a concentration of 0.5 µg/mL followed by subsequent addition of TMB substrate to develop signal. The reaction was stopped by addition of ELISA Stop solution (Mabtech AB) and the plates were read in a Synergy HTX Absorbance reader and analysed using Gen5 software . Mean OD values are presented as antibody levels.Plasma samples were analysed by ELISA for detection of circulating IgG antibodies against escribed . Briefly6 PBMCs and antibody levels between two age groups (1-6 and 7-12 years old) within the children in Junju  and children in Ngerenya  aged 1-6 years. Spearman correlation was used to determine the association between logarithmically transformed MBC and antibody responses, as well as to determine the association of MBC and antibody responses with age, number of clinical malaria episodes since birth and parasite density at baseline in separate multivariate analysis. A Cox-regression model was used to investigate the risk of subsequent clinical malaria after baseline (date of sample collection in March 2016) until one year later, and similarly for time since last malaria episode until baseline. The models were performed by either subgrouping individuals considered positive or negative for MBC and antibody responses against an antigen or performed with number of SFU/106 PBMC. In addition, the risk analyses were also performed with the levels of MBC responses as continuous values. Proportional hazards were tested using Schoenfeld’s residuals. The p-values below 0.05 and hazard ratio (HR) with 95% CI not crossing 1 were considered significant.Statistical analyses were performed using STATA MP version (16.0), and GraphPad Prism (version 8.3) . Mann-Whitney test was used to compare IgG spots , spot forming units (SFU)/10P. falciparum malaria since birth. None of them had symptoms of clinical malaria either at the time, or within 15 days before and after sampling. However, 16 of the 96 (16%) children had asymptomatic P. falciparum infection detected by PCR at sampling. In addition, 20 children from the Ngerenya cohort, aged 1-6 years, who had no recorded malaria episode during their lifetime, or detection of asymptomatic parasites in either the current, or in any of the preceding annual surveys (since birth) were included as a malaria naïve comparison group , median 4 (range 0-240) for MSP-2 (3D7), median 0 (range 0-96) for MSP-2 (FC27), median 4 (range 0-204) for MSP-3, median 4 (range 0-344) for AMA-1 and median 0 (range 0-28) for CSP  was not observed between the two allelic forms of MSP-2 (3D7 and FC27), when assessing spots counted using different wavelength-specific filters. Data from six malaria-exposed children were excluded from the analysis due to assay disqualification i.e., <1000 total IgG spot-forming units (SFU) per 250,000 cells in the FluoroSpot assay. Furthermore, MSP-119, and 3/62 (5%) for CSP. None of the responding children had MBC responses to all antigens tested, 3 children (3.3%) responded to four antigens, 9 children (10%) responded to three antigens, 7 children responded to two antigens (7.8%), 16 children (17.7%) responded to one antigen, and 55 children (61.1%) were negative to all the antigens. All the malaria-naïve children were negative against all the antigens tested.The proportions of malaria-exposed children qualifying as MBC positive were 19/90 (21%) for AMA-1, 17/90 (19%) for MSP-3, 13/90 (14%) for MSP-2 (3D7), 7/90 (8%) for MSP-2 (FC27), 10/62 (16%) for MSP-1p= 0.007, 0.022 and 0.022, respectively) , rs 0.298 p=0.004 for MSP-2 (FC27), rs 0.213 p=0.044 for MSP-3 and rs 0.284 p=0.006 for AMA-1). Furthermore, the breadth of MBC responses was also weakly correlated with age (rs 0.258 p=0.014) (The median frequencies of MBCs for MSP-2 (3D7), MSP-3 and AMA-1 were higher in children aged 7-12 years compared to 1–6-year old’s (Mann-Whitney test p=0.014) Table 2.p=0.001), MSP-3 (Mann-Whitney test p=0.006) and for AMA-1 (Mann-Whitney test p=0.009) in children aged 7-12 compared to 1-6 year old’s among malaria-exposed children  and MSP-3   (p=0.011) (Median antibody OD levels were higher for MSP-2 (3D7) (Mann-Whitney test p=0.031) Table 2.s 0.221 p=0.038), MSP-2 (FC27) (rs 0.347 p ≤ 0.001), MSP-3 (rs 0.274 p=0.09) and AMA-1 (rs 0.420 p= p ≤ 0.001). However, this was not the case for MSP-119, and neither was it for CSP (Frequencies of MBCs were positively but weakly correlated with antibody levels for MSP-2 (3D7) . The point estimates for antibody levels to the other antigens as well as their breadth showed a negative but non-significant correlation with the number of clinical episodes since birth   Table 2.P. falciparum parasitemia detected by microscopy and PCR. Parasite positivity at baseline was positively correlated with frequencies of MBCs and levels of antibodies against MSP-2, MSP-3 and AMA-1, as well as breadth of MBC responses  of the 90 malaria-exposed children  with MBC data had asymptomatic esponses Table 2.p=0.019), MSP-3  and AMA-1  (p=0.021) and MSP-3  were associated with a reduced risk of malaria (p=0.017), and MSP-3 . Also, after adjusting for parasite positivity the risk of malaria was only reduced for individuals that were MBC positive for MSP-3 . Finally, after adjusting for both age and parasite positivity, the risk of malaria was reduced for individuals that were MBC positive for MSP-2 (3D7) , and MSP-3 .Since the MBC and antibody levels were measured from samples collected in March, towards the end of a 4-month dry season when malaria transmission is very low, we assessed the prospective risk of subsequent clinical malaria associated with MBC and antibody positivity during the ensuing malaria transmission season starting from May. Time to a clinical episode of malaria from baseline in children with and without MBC and antibody responses, respectively, is visualized in p=0.044) Table 3.p=0.017) (p=0.035) and MSP-3 (HR 0.33 95% CI 0.13-0.84 p=0.021) , with significant associations for MSP-3 (HR 0.08 95% CI 0.01-0.64 p=0.034) for children aged 7-12 and with a breadth of MBC responses exceeding 3 antigens (p=0.009) (p=0.018)  instead of MBC positivity, and the results were similar with the outcomes of the models based on MBC positivity. Similar to the Cox analysis based on MBC positivity  individually, and sometimes by both age and asymptomatic parasitemia. Importantly, a breadth of three or more antigens for either MBCs or antibodies remained protective even after controlling for age. Similarly, MBC positivity towards MSP-2 and MSP-3 remained protective after controlling for age and parasite positivity.We assessed MBC and antibody responses specific for six well characterized P. falciparum antigens have been rigorously investigated  and MSP-3, as well as breadth of responses, with reduced risk of clinical 19 and AMA-1 , the Swedish Research Council (Development Research project grant 2018-04468) and Karolinska Institutet Travel Grant (2018-02525). FMN is supported by an MRC/UKRI African Research Leadership Award (MR/P020321/1), and Senior Fellowship from EDCTP (TMA2016SF).PJ and NA are employed by Mabtech AB, Sweden.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Several of the reagents and the FluoroSpot reader systems used in this study are produced by Mabtech. Mabtech has no influence on the content of this study or interpretation of results.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Major challenges have been encountered regarding the development of highly efficient subunit malaria vaccines, and so whole-parasite vaccines have regained attention in recent years. The whole-killed blood-stage vaccine (WKV) is advantageous as it can be easily manufactured and efficiently induced protective immunity against a blood-stage challenge, as well as inducing cross-stage protection against both the liver and sexual-stages. However, it necessitates a high dose of parasitized red blood cell (pRBC) lysate for immunization, and this raises concerns regarding its safety and low immunogenicity. Knowledge of the major components of WKV that can induce or evade the host immune response, and the development of appropriate human-compatible adjuvants will greatly help to optimize the WKV. Therefore, we argue that the further development of the WKV is worthwhile to control and potentially eradicate malaria worldwide. Malaria remains a potentially fatal public health problem, resulting in high morbidity and mortality in tropical and subtropical regions. Recently, malaria control interventions, such as artemisinin-based combination therapy (ACT), insecticide-treated bed nets (ITNs), and other mosquito vector control strategies, have greatly reduced the incidence of malaria all over the world . HoweverPlasmodium genus. There are four human malaria parasites, namely, Plasmodium vivax (P. vivax), P. falciparum, P. ovale, and P. malariae. Of these, P. falciparum is the deadliest. A Plasmodium infection is initiated by sporozoite inoculation into the host skin by an infected female Anopheles mosquito. The sporozoites travel to the liver and infect a small number of hepatocytes, with a single sporozoite giving rise to tens of thousands of merozoites. Next, merozoites are released into the bloodstream when merosomes bud from an infected hepatocyte, and they invade red blood cells (RBCs), initiating blood-stage development. A merozoite can multiply up to 20-fold every 1–3 days in cycles of invasion, replication, and RBC rupture, which releases many infectious merozoites. Some of the asexual blood stages transform into sexual forms called as gametocytes, which can eventually move into the midgut of a blood-feeding mosquito and then develop into sporozoites in the salivary gland. The blood-feeding mosquito then injects these sporozoites into another human, thereby initiating a new human infection  and blood stage in mammals and the sexual-stage in its vector. The blood stage is the main cause of the clinical manifestations , and a blood-stage vaccine aims to reduce mortality and morbidity in malaria patients. In contrast, the liver stage is clinically silent, and a liver-stage vaccine is promising for preventing malaria infection , which plays an important role in sporozoite invasion of hepatocytes . The mosP. falciparum reticulocyte-binding protein homolog 5 (PfRH5), which interacts with basigin (CD147) on the RBC surface during merozoite invasion  and MSP3, apical membrane antigen 1 (AMA1), and erythrocyte-binding antigen 175 (EBA-175). These blood stage malaria vaccine candidates seek to induce high titers of plasmodium specific antibody that inhibit erythrocyte invasion by merozoites or limit parasite replication in red blood cells. However, most of the vaccine candidates exhibit extensive polymorphism between malaria strains from different geographical regions, and immunization with these vaccines only led to partial protection against vaccine-like strains . In addiinvasion . PfRH5 einvasion . Pre-cliinvasion  and conf monkeys . Recent  monkeys . However monkeys . This maP. falciparum Pfs230 and Pfs48/45, which are expressed by gametocytes, and Pfs25, which is exclusively expressed by zygotes and ookinetes. In phase 1 clinical trials, vaccines based on Pfs25 and its P. vivax ortholog Pvs25 have induced antibodies that block mosquito infection  with purine nucleoside phosphorylase (PNP), nucleoside transporter 1 (NT1), or histamine-releasing factor (HRF) deficiency confer complete sterile protection against both homologous and heterologous blood-stage challenge . However, a recent study showed that P. vivax-infected reticulocytes express HLA I and circulating CD8+ T cells in malaria patients can recognize the P. vivax-infected reticulocytes in a HLA-dependent manner and kill the intracellular parasites -B cell interactions in germinal centers via secretion of CTLA-4 (+ (TH1-like) TFH cells  malaria parasite components that positively regulate the host immune responses and (2) the mechanisms and parasite components that suppress or evade the host immune responses, which would affect the magnitude and duration of the protective immune response induced by WKV. With this knowledge, negative regulatory components could be removed and positively components could be strengthened from the parasite by genetic manipulation to greatly improve the immunogenicity and protective efficacy of the WKV.WX and TL: conceptualization, writing-review and editing, and funding acquisition. JC, SC, XL, and FZ: writing-original draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Outcome of falciparum malaria is largely influenced by the standard of care provided, which in turn depends on the available medical resources. Worldwide, the COVID-19 pandemic has had a major impact on the availability of these resources, even in resource-rich healthcare systems such as Germany’s. The present study aimed to determine the under-explored factors associated with hospital length of stay (LOS) in imported falciparum malaria to identify potential targets for improving management. This retrospective observational study used multivariate Cox proportional hazard regression with time to discharge as an endpoint for adults hospitalized between 2001 and 2015 with imported falciparum malaria in the Charité University Hospital, Berlin. The median LOS of the 535 cases enrolled was 3 days . The likelihood of being discharged by day 3 strongly decreased with severe malaria  and by 40% with each additional presenting complication . The 55 (10.3%) severe cases required a median LOS of 7 days . In multivariate analysis, occurrence of shock , acute pulmonary oedema or acute respiratory distress syndrome , and the need for renal replacement therapy  were independently associated with LOS. All patients survived to discharge. This study illustrates that favourable outcomes can be achieved with high-standard care in imported falciparum malaria. Early recognition of disease severity together with targeted supportive care can lead to avoidance of manifest organ failure, thereby potentially decreasing LOS and alleviating pressure on bed capacities. Falciparum malaria has a unique pathophysiology with exponential growth of parasite biomass every 48 h, reduced deformability and sequestration of infected (IE) as well as non-infected erythrocytes in the microcirculation, and systemic inflammation and endothelial dysfunction ,2. This In Europe, falciparum malaria occurs as a rather rare imported disease. In contrast to regions where the disease is endemic, the affected patient population is more heterogeneous, consisting of malaria-naïve travellers, immigrants with waning semi-immunity visiting friends and relatives (VFR-immigrants) in their home countries, and travellers residing in regions of endemicity. As severe and fatal courses are more likely to occur among these patients, hospitalization is generally recommended. In the European countries mostly affected, namely, France, the United Kingdom, and Germany, outcome of imported falciparum malaria is considerably better compared to many high- and low-transmission areas, which is primarily the result of a higher standard of care. When treatment takes place in specialized centers, mortality rates less than 5% can be achieved ,9,10.Standard of care depends on the entirety of the available medical resources, such as pharmaceutics, diagnostic interventions, hospital and intensive care unit (ICU) beds, technical organ support, and, in particular, trained critical care staff including respiratory therapists, dialysis specialists, and ICU nurses . ThroughThe aim of the present study was to identify factors associated with prolonged LOS among patients with imported falciparum malaria treated in a specialized center in Berlin, Germany, in the pre-COVID-19 era.All adult patients (≥18 years) hospitalized with imported falciparum malaria between 1 January 2001 and 31 December 2015 in the Department of Infectious Diseases and Pulmonolgy of the Charité, Berlin, a tertiary care university hospital, were enrolled . During Diagnosis of falciparum malaria relied on thin and thick blood smears. Parasitaemia was expressed as percentage of parasitized erythrocytes . Severe malaria was defined according to the World Health Organization (WHO) 2014 definition  with minThe study’s only endpoint  was LOS, which was calculated in days by subtracting the day of admission from the day of discharge. Selection of covariates  was based on a literature search using the Cochrane Library, PubMed, and Google Scholar databases that had recently identified factors associated with malaria LOS .2 test, while the Mann–Whitney U-test was used for continuous data. Length of hospital stay was markedly right-skewed  had been treated with artemisinin-based regimens. In 480 cases (89.7%), the disease was uncomplicated, while 55 (10.3%) patients suffered from severe malaria. Admission to an intensive care unit (ICU) was required in 68 (12.7%) cases. Among these, 13 (2.4%) needed vasopressors for the treatment of shock. Thirteen patients (2.4%) had radiologically confirmed acute pulmonary oedema (APO) or ARDS. In four individuals (0.8%), oxygen supplementation was sufficient. Nine patients (1.7%) required mechanical ventilation for a total of 950 ventilation hours . Four patients were non-invasively ventilated, five invasively. Indications for invasive mechanical ventilation were moderate to severe ARDS, deep coma , advanced shock, multi-organ dysfunction, and cardiac arrest. In 7 (1.3%) of the 13 patients with renal impairment, RRT was required for acute kidney injury stage 3 (AKI3). Median time to initiation of RRT was 20 h. These patients were on RRT for a total of 51 days .A total of 558 adult patients with imported falciparum malaria were treated in the Department of Infectious Diseases and Pulmonology of the Charité Universtiy Hospital, Berlin, between 2001 and 2015, corresponding to 7.1% of all 7.866 imported falciparum malaria cases notified in Germany during the study period . Twenty-The overall outcome was favourable: weaning from mechanical ventilation was successful in all patients, renal function normalized and RRT could de discontinued in all but one individual. This patient, however, had pre-existing chronic renal failure with a baseline creatinine >10 mg/dL and progressed to end-stage renal disease as a result of the malaria. Individuals with coma did not suffer from long-term neurologic deficits, including a patient who had to be resuscitated due to cardiac arrest in the context of severe metabolic acidosis. All patients survived to discharge.p < 0.0001). Presence of a ≥1 criterion for severe malaria was strongly associated with prolonged hospitalization , an autocrine regulator of endothelial inflammation promoting an increase in capillary permeability, correlate with disease severity. Significantly higher Ang-2 levels have been detected in patients with four severity criteria compared to individuals with only two . ElevateMortality in patients with shock was 16% in the original SEAQUAMAT trial . Shock nImportantly, the risk of death sharply increases with multi-organ involvement in falciparum malaria: while mortality is 9.5% in patients with a single complication, it reaches 50% in patients with >5 complications .The results of the present study are in line with the above observations. Similar to a recent nationwide study from Sweden, the proportion of patients with severe imported falciparum malaria was around 10% . Due to These results have two implications. To achieve optimal outcomes, reliable identification of patients at the highest risk of death that will require the most resources is mandatory. Yet, externally validated scoring systems for the assessment of disease severity are unavailable for imported malaria. Therefore, the earliest recognition of severe disease, and particularly of multi-organ involvement, appears to be crucial in patients with imported falciparum malaria.Successful management of critically ill falciparum malaria patients has two cornerstones: effective antimalarial therapy and best supportive care. The exponential disease dynamics call for swift actions. Initiation of an effective antimalarial as soon as the diagnosis is established is the most important intervention. The artemisinins achieve the fastest parasite clearance of all antimalarials. Accordingly, their use has demonstrated a strong impact on survival in both children and adults in high- and low-transmission areas. In the present analysis, ACTs were associated with a shorter LOS in uncomplicated cases. The prognostic benefit of intravenous artesunate is greatest in patients with a high parasite burden. Yet, in non-immune travellers, a survival benefit could not be found ,9, a facIn addition to the rapid initiation of an effective antimalarial, early recognition of (incipient) complications that immediately results in targeted interventions is needed to achieve optimal outcomes. Without RRT, malaria-associated AKI carries a high mortality of up to 75% ,40. If iIn summary, it appears, that hospitals caring for patients with imported falciparum malaria can calculate with roughly 10% of severe cases and that these patients will have a median LOS of about 7 days. The above outlined treatment strategies may contribute to prevent manifest organ failure and thereby possibly aid in shortening LOS.The present study has several limitations, the main being its retrospective, single-center design and a long observation period. This limits the generalizability of the results. Yet, the study is intended to analyze an “ideal-world scenario” for the treatment of falciparum malaria in order to identify the factors that require most resources under the best possible conditions. Generalizability is, therefore, a priori limited. Importantly, the identified factors are all well recognized prognostic factors of falciparum malaria. In addition, with a caseload of >500, the analysis covered at least 7% of the total cases notified in Germany during the study period. The availability of standardized electronic files for all patients ensured a high level of data capture. Discharge after 3 days, i.e., the median LOS of the cohort, was arbitrarily chosen to define the primary outcome parameter. However, performing sensitivity analyses using other thresholds ensured the robustness of this approach. Most importantly, data on factors influencing LOS in imported falciparum malaria are scarce, and resources for critically ill patients may continue to be limited. As standardized prognostic scoring systems for the assessment of disease severity are currently unavailable, more data supporting resource allocation are needed for the best management of this complex illness.Falciparum malaria is a protean illness with rapid disease dynamics. Micro-vascular obstruction plays a central role in its unique pathophysiology. Immediate initiation of an effective antimalarial is therefore of paramount importance for successful management. However, rapid parasite clearance is not the only significant task to be achieved. Adverse effects from more indirect disease mechanisms such as generalized endothelial dysfunction, systemic inflammation with increased capillary permeability, renal tubular damage, or muscle weakness may require considerably more time to resolve. The results of the present study illustrate that with a high standard of care, mortality from the disease can be kept to a minimum. Yet, due to the COVID-19 pandemic, the medical resources required to provide a high standard of care have become more scarce in many settings. Optimal supportive care can achieve two important tasks simultaneously: improve outcomes and save valuable resources. Correct assessment of disease severity at the earliest possible stage, use of a restrictive fluid management strategy, timely initiation of RRT if indicated, and avoidance of invasive mechanical ventilation if non-invasive ventilation is appropriate are key measures that will likely reduce the LOS of patients with imported falciparum malaria and thereby relieve pressure on the need for hospital and, in particular, ICU beds.