Plasmodium falciparum Malaria and Epstein-Barr Virus (EBV) infection are risk factors in the development of Burkitt’s lymphoma. In Indonesia, 100% of the population is persistently infected with EBV early in life and at risk of developing EBV-linked cancers. Currently, 10.7 million people in Indonesia are living in Malaria-endemic areas. This cross-sectional study was initiated to investigate how acute Malaria dysregulates immune control over latent EBV infection. Using blood and plasma samples of 68 patients with acute Malaria and 27 healthy controls, we measured the level of parasitemia for each plasmodium type  by microscopy and rapid test. The level of 4 regulatory cytokines was determined by quantitative ELISA and the level of circulating EBV genome by real-time PCR targeting the single copy EBNA-1 sequence. All Plasmodium-infected cases had high-level parasitemia (>1000 parasites/ul blood) except for one case. EBV-DNA levels were significantly more elevated in P. falciparum and P. vivax infections (P<0.05) compared to controls. EBV-DNA levels were not related to age, gender, Malaria symptoms, or plasmodium type. TNF-α and IL-10 levels were increased in Malaria cases versus controls, but IFN-γ and TGF- β levels were comparable between the groups. Only TNF-α levels in P. falciparum cases showed a clear correlation with elevated EBV DNA levels (R2 = 0.8915). This is the first study addressing the relation between EBV (re)activation and cytokine responses during acute Malaria, revealing a clear correlation between pro-inflammatory cytokine TNF-α and EBV-DNA levels, specifically in P. falciparum cases, suggesting this cytokine to be key in dysregulating EBV homeostasis during acute P. falciparum Malaria. P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), causing unregulated activation of EBV+ B-cells, lymphoproliferations and potentially leading to BL . All cases with a positive RDT were followed-up and confirmed in the regional health center(s) on Sumba Island by further blood examination using thick smear microscopy by expert parasitologists to confirm the parasite species and to quantify the proportion of infected red blood cells in relation to a predetermined number of white blood cells (WBC), according to WHO-2010 guidelines Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: No**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Overall Impression: Although the study did not find a clear link between inflammation-associated EBV viral control and severity of malaria infection, the laboratory methods used were robust, and the study offers a contribution to the literature on co-infections in the context of EBV-associated cancer risk.Introduction: The introduction as written is clear but quite long. At least half of the material in the inflammatory cytokines paragraph could be omitted or transferred to the Discussion. The length of the Background detracts a bit from getting to the hypothesis / objective of the study.Methods: All of the analyses as presented are not adjusted for age or sex. These two factors should be included, even if in simple linear regression models, to validate that any reported correlations are not driven by a differential distribution of age and / or sex in patients with elevated EBV replication.Results:(1) As the reported association between EBV and TNF-alpha is specific to P. falciparum, this needs to be clearly stated everywhere in the Abstract, Introduction, etc…. where it is stated that EBV and malaria-related inflammatory cytokine changes are associated.(2) Please include the correlations between EBV and TNF-alpha and the other malaria groupings in the text on page 9.(3) Per Figure 5, it appears that the reported correlation specific to P. falciparum is entirely do to a somewhat linear association at very low levels of the EBV genome number . This should be at least mentioned in the Discussion text.Discussion: Again, the text here, particularly pages 11-14, is far too much. Please select which paragraphs to highlight based on your findings. An extensive literature review of EBV in different populations and various cytokines that showed no association with EBV are not needed.Reviewer #2: This manuscript describes a cross-sectional study comparing pro-inflammatory plasma cytokine levels with EBV loads in adults with acute malaria compared to controls residing in Indonesia. It is novel to compared Pf and Pv single and mixed infections within this context since the question often arises as to the specificity of the Pf-EBV interaction. However, without independent confirmation that all the viral loads measured where remarkably high (with none being negative), it is difficult to interpret these findings and their comparisons with cytokine levels.Major comments:1. It is extremely unlikely that all your study participants had viral loads above 2.5 log copies and that none of them were negative, not even in your healthy control group. There are many published studies on EBV detection in healthy sero-positive adults that strongly refute your data. Therefore, it would be helpful if your results could be independently validated to demonstrate that there was no cross-contamination with the positive control DNA used for the RT-PCR assay. In addition, the DNA isolated from plasma could be treated with DNAse to determine the fraction of encapsidated virus  to confirm lytic cycle reactivation in your study populations.2. The authors state that their study is the first to “measure EBV loads in adults during acute malaria” however they overlooked earlier studies published in Kenya where there was a malaria-exposed adult control group  and other studies by Rochford that measured EBV loads in pregnant women. Therefore, this statement should be tempered. Replications studies done in another study population or at another time that either confirm or refute earlier findings are valid study designs and contribute knowledge to the field.3. The authors appear to be citing more recent publications or secondary analyzes, as opposed to seminal studies. A more in-depth literature review should be done so this can be remedied.4. Clinical symptoms listed by individual in Table 1 could be summarized in aggregate across groups, with associated p-values to highlight significant differences, if they exist.5. The authors interpret higher EBV loads overall in their study groups as being due to stress. Is there any published findings to support this level of stress in people living in Indonesia compared to other study populations residing in malaria endemic regions to support this claim?**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  8 Sep 2021Reply to the reviewer’s comments on manuscript PONE-D-21-19672We are grateful to the positive and supportive comments of the reviewers.Our answers to the reviewer’s questions are detailed here below and we have adjusted the revised manuscript accordingly.Reviewer #1: We thank the reviewer for indicating the positive overall impression.As suggested, we shortened the introduction by reducing (too many) details on inflammatory cytokines and focus better on the hypothesis / objectives of the study.We made a statement in the results section that the distribution of EBV-DNA load and 4 individual cytokine levels was analysed for age/sex influences in patients and controls. Data on EBV-DNA male/female comparison are shown in Supplemental. Figure 2 .Data on linear regression analysis for each parameter with age is shown in Suppl. Figure 3  and Supplemental Figure 4 . These analyses did not influence the original results or conclusions. #1: We thank the reviewer for this good suggestion and have emphasized the association between EBV and TNF-alpha as being specific to P. falciparum everywhere in the manuscript .#2: We have included the correlations between EBV and TNF-alpha for the individual groups in the text of result section as suggested.#3: The reported correlation specific to P. falciparum is linear and significant (P=0.8915) and holds in both male and female Malaria cases. Due to a systematic error in our DNA load calculation .# Discussion: We have significantly (500 words) reduced the discussion section by removing detailed background information on role(s) of cytokines in anti-Malaria defenses and likewise reduced the number of references. Reviewer #2: We thank the reviewer for the positive remarks on the novelty of our findings. We have addressed the concerns on EBV-load measurement.#1. The EBV-DNA PCR used in this study is an UNG-controlled quantitative PCR with internal calibration and H2O control for each PCR run. Erroneous amplification or PCR cross-contamination is eliminated by the UNG-procedure. Therefore, we consider it highly unlikely that the data are false-positive. This is indicated in the method section.Unfortunately, due to the Coronavirus epidemic, the Indonesian government has officially called all research PCR instruments in the country to be used for Covid-19 testing only, and we cannot independently re-test our samples. Despite several requests and due to government blocking of new PCR kit import, we are unable to do independent PCR analysis. By re-analysing our raw data, we found a systematic error in the EBV-DNA load calculation for all samples . This has resulted in a 6-fold reduction of the reported EBV-DNA levels. All data and figures were corrected for this systematic error.Yet the resulting EBV-DNA levels remain rather high for healthy individuals, but now they are in line with previously reported data for healthy adults and children in malaria-risk regions in Kenia . One healthy donor had near negative values and her husband had approx. 1100 copies/ml plasma. He has a known IM history > 10 years ago, with persistent high EBV-DNA load in multiple independent PCR analyses but remains without any symptoms. Indeed, we were also surprised by elevated EBV-DNA loads found in the nine healthy controls. However, we consider this to be a reflection of the local living conditions, which may be similar to sub-Saharan Africa where healthy children and adults also have higher EBV-DNA loads (ref 35 added). Similar strenuous life conditions in Surabaya, East-Java may affect EBV homeostasis . We also noted a recent publication on frequent (9.4%) subclinical Malaria infections (detected by PCR) in the Surabaya urban population , where we collected our apparent healthy controls and added this as ref.26. Subclinical malaria may explain -in part- the elevated EBV-DNA levels, similar to malaria-endemic regions in Africa.We present these arguments (see discussion) about environmental and population stressors which are especially abundant in Surabaya, East-Java compared to other parts of Indonesia , and suggest that these may be responsible for the high EBV-DNA loads in this East-Java population, compared to our prior findings in Jakarta and Yogyakarta controls . In African healthy controls  the reported EBV-DNA loads are higher in a significant part of the controls compared to US healthy controls (ref. 35) and similar life-stress factors may be due to this.Although we did not pursue the DNAse treatment, we consider that “overall” the EBV-DNA load in Surabaya EBV carriers is high, whatever the origin  and is comparable with EBV-DNA loads in healthy African children in the Moormann 2005 study. The environmental factors that may lead to higher EBV-DNA loads (as indicated above), may influence both latent B-cell proliferation  and lytic reactivation . It is not the aim of this study to analyse the origin of EBV-DNA in detail.#2. We appreciate the reviewer’s comment and studied the Moormann et al., JID2005 paper. We noticed that this paper deals with children with Malaria and used local non-Malaria children and adult as controls, all being healthy and living locally in a high malaria risk region (Kisumu). Of relevance is their finding that the healthy African children displayed a wide range of EBV-DNA loads and healthy African adults in Kisumu also have elevated EBV-DNA loads compared to US-based individuals. (See our reply here above).We eliminated the remark that “our study is ‘first’ to measure EBV loads in adults with acute malaria”. #3. We appreciate the reviewer’s point of view. Because recent papers cite and discuss earlier seminal findings, we chose to focus on recent publications relevant to our study.#4. We have aggregated the clinical symptoms in a graphic display  to accommodate the reviewers suggestion. #5. Unfortunately, no prior study has addressed this “stress” issue in Indonesia, but it is well known that food and lifestyle  as well as stress hormones (glucocorticoids) are inducers of EBV reactivation and contribute to elevated EBV-DNA loads . These factors are abundant in the East-Indonesian region, as mentioned in the discussion with related references .AttachmentReply to reviewers comments PONE-D-21-19672 .docxSubmitted filename: Click here for additional data file. 18 Oct 2021PONE-D-21-19672R1Quantitative cytokine level of TNF-α, IFN-γ, IL-10, TGF-β and circulating Epstein-Barr Virus DNA load in individuals with acute Malaria due to P. falciparum, P. vivax or double infection in a Malaria endemic region in Indonesia.PLOS ONEDear Dr.  Middeldorp,Thank you for submitting your manuscript to PLoS ONE. After careful consideration, we feel that your manuscript will likely be suitable for publication if the authors revise it to address specific points raised now by the reviewers.According to the reviewers, there are some specific areas where further improvements would be of substantial benefit to the readers (please see reviewer #3).   For your guidance, a copy of the reviewers' comments was included below.  plosone@plos.org. 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Luzia Helena Carvalho, Ph.D.Academic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #3: (No Response)Reviewer #4: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #3: PartlyReviewer #4: Partly**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: NoReviewer #4: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #3: YesReviewer #4: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #3: YesReviewer #4: No**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors’ responses have adequately addressed all of my concerns, and I recommend publication of the current version of the manuscript revision.Reviewer #3: Quantitative cytokine level of TNF-α, IFN-γ, IL-10, TGF-β and circulating Epstein-Barr Virus DNA load in individuals with acute Malaria due to P. falciparum, P. vivax or double infection in a Malaria endemic region in Indonesia by Budiningsih et alThe authors report their findings of correlations between EBV level and parasitemia in in subjects in Indonesia. This works sheds light on the interaction between malaria parasites and EBV in a population with low incidence of Burkitt lymphoma. The authors are to be congratulated for designing and implementing this study. It is timely and represents an important step forward for understanding the pathobiology of EBV and malaria. Their manuscript would benefit from some improvements as suggested below:General comments:a) The authors report the study as a case-control study, but it is not clear whether this is not a cross-sectional study. This difference is important and needs further clarification. A cross-sectional study is one where all assessments (exposure and outcome) are made at the same point in time. In this case, if classification as either a case or control is made based on responses to a questionnaire and blood test made at the same time on the subject, the study should be classified as a cross-sectional study, with subjects enrolled from two different locations. A case control study is defined as enrollment of subjects with or without the condition of interest . Thus, cases are evaluated for the presence of the condition and excluded if they don’t have the condition, while controls are similar to the cases in all respects except in having the outcome of interest. The requirement for comparability of cases and controls imposes on the author the need to describe the source of cases, the population which they represent, and to describe the controls so readers can conclude whether the controls are from a population where cases originated. These details are lacking or do not seem to be met for a case-control design. I suggest that the authors report this as a cross-sectional study. Regardless, the authors need to report the subjects who were assessed and if any were excluded and the reasons for exclusion.b) The authors need to clarify what they refer to as malaria. Malaria is the disease ie, infection PLUS symptoms. Individuals with malaria have a range of symptoms categorized as severe, moderate, mild, or asymptomatic. These are defined in a standard way by the WHO and should be used as such. For example, hyper-parasitemia is defined as parasitemia of 250,000/uL. It is doubtful that the subjects included here meet that definition, but if they do they should us the standard WHO definition and show the results in a primary table.c) The analytic methods need to specify and distinguish the primary hypotheses being tested from the secondary explorative hypotheses being tested. The methods should also specify how multiple comparisons are being handled.d) The authors need to provide some epidemiological context for their work – for example what is the malaria pressure in heir population - this can be provided in the form of malaria prevalence, entomology inoculation rates, and the age when severe malaria is observed in that population or the annual burden of malaria. This background information would be helpful for readers to understand how the cases being reported compared to the malaria patterns observed in the area.e) The authors need to provide more information about the sites for the enrollment – what are these malaria hospitals?f) Please add page numbers!Specific commentsg) The sentence “Enhanced EBV-DNA levels were significantly more frequent in P. falciparum and P. vivax infections (P<0.05) compared to controls.” In the abstract and elsewhere in the manuscript is confusing. Levels are higher, positivity is more frequent. The authors need to read the manuscript carefully and distinguish instances where they are talking about EBV DNA positivity as a binary variable versus when they are talking about the quantity.h) 2nd paragraph of the introduction – please add some malaria contextual information as suggested above. Some of this information is I the first 2-3 paragraphs of the discussion.i) Last paragraph of the introduction – please “in Indonesia” where applicable and “in East Africa” or “Kenya’ as applicable, eg, “The aim of this research was to investigate how acute Malaria dysregulates EBV homeostasis and what cytokines would be involved “in Indonesia”j) In the material and methods revise “Malaria samples”… to “venous blood samples”k) Please provide more information about the sites where the subjects were enrolled – hospitals, communities etc etc using the STROBE guidelines.l) Please clarify whether all subjects were assessed for malaria – this would include a screening for temperature, symptoms, and parasites. A standard definition of malaria should be used, denoting cases as either severe, moderate, mild, or asymptomatic.m) For healthy controls, the test used to exclude infection should be stated as well as its limit of detection.n) The word “Clinical” in the sentence “Clinical diagnosis of Malaria was confirmed by a laboratory test for Malaria antigen detection, i.c. “Rapid Diagnostic Test” (RDT) .” should be replaced with “presence of plasmodial parasite infection”. Malaria by definition is clinical, ie, associated with symptoms. Thus, the authors could say “the diagnosis of malaria was made based on demonstration of presence of parasites, based on …tests, in patients with a fever and XX symptoms”o) The sentence “… followed-up with thick smear microscopy by expert parasitologists to confirm the parasite species and to quantify the proportion of infected red blood” needs to be revised for clarity and correctness. It is impossible to determine the number of red blood cells infected from a thick film.p) Please provide some additional details of the giemsa stain q) In the statistics section – this needs to be revised for clarity “ The correlation between EBV genome and Malaria parasites levels in a population was defined by student t-test” this test is normally used to test for quality of means – is this what is meant here? The authors need to describe what is being evaluate – means, correlation, association and what statistics are being used. This section requires careful attention.r) In the results section - the definition of hyper-parasitemia is unclear here. Do these patients really have severe malaria? Could other measures of malaria severity be provided? Eg, hemoglobin, platelet count, WBCs?s) If the authors believe that the EBV levels might differ by type plasmodium – this should be articulated. Are there differences in the severity of malaria? Given the limitations of sample size, this segmentation of data should be considered carefully.t) The results presented as “Overall, the mean EBV genome level was significantly higher in cases of P. falciparum  and P. vivax  infection compared to the mean level in the healthy controls  .” are interesting, but I would suggest re-vising this such that the different statistical results can be presented, ie, “Compared to healthy controls , EBV levels were significantly elevated in those with P. falciparum  and those with P. vivax .” The authors could also compare the different groups according to log levels, i.e., 2 log higher …u) I think the authors need to present the significant cytokine results and report those that are not significant as not significant without going into details.v) The discussion should be shortened and focused on the key findings, what they mean, and the strengths and weaknesses of this study, and brief suggestion of the next steps.Reviewer #4: Reviewers commentsManuscript Number: PONE-D-21-19672R1Manuscript Title: Quantitative cytokine level of TNF-α, IFN-γ, IL-10, TGF-β and circulating Epstein-Barr Virus DNA load in individuals with acute Malaria due to P. falciparum, P. vivax ordouble infection in a Malaria endemic region in Indonesia.Summary of articleThe work is very interesting, novel and is well suited for publication in PLOS-ONE.I have reviewed the author’s response to the previous reviewers comments and am happy to see that all relevant points were taken on board to improve the manuscript.IntroductionLine – “EBV was first identified in cells of Burkitt Lymphoma (BL), an endemic cancer among sub-Saharan children, that is triggered by co-infection with Malaria parasites . BL is the most common cancer in children living in Malaria endemic regions in sub-Sahara Africa and Papua New Guinea ,” - recent publications have been published highlighting new perspectives such as Ellis et al 2021 (see below) which can be referencedhttps://doi.org/10.1186/s13027-021-00370-7Ellis, T., Eze, E. & Raimi-Abraham, B.T. Malaria and Cancer: a critical review on the established associations and new perspectives. Infect Agents Cancer 16, 33 (2021). Line – “There appeared to be a direct correlation between increases in plasma EBV viral load and progression of endemic Burkitt Lymphoma” abbreviate to BLInformation in the introduction on the four cytokines chosen to quantify in the study should be provided in greater detail beyond the summary paragraph provided.. Further details on the selection/choice of specific cytokines used in this study is important. For example “ in this study the amount of x,x,x, and x plasma cytokines was evaluated. X has a role in xx etc repeat.Materials and Methods/ Results and DiscussionSample collection and Malaria parasite analysis – because of the age association with relationship between malaria and EBV it would have been useful for the field to have full malaria patient data i.e. positive parasites of P. falciparum (n=26), P. vivax (n=28), and mixed  (n=14), or healthy controls (n=27) data presented in a table with age information to know for example how many patients with P. falciparum infection were under 5 etc. This should be reflected in the results and discussion. Whilst the gender could be of interest especially in any of the samples were from pregnant women, the age is of greater interest and should be included.The authors should highlight any sample data from pregnant women.Line – use of Kenya and Kenia in the manuscript change all “Kenia” to Kenya for continuity.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  23 Nov 2021PONE-D-21-19672R16. Authors Reply to: Review Comments to the AuthorReviewer #1: The authors’ responses have adequately addressed all of my concerns, and I recommend publication of the current version of the manuscript revision.Au reply: We thank the reviewer for the positive consideration.Reviewer #2: No further comments upon the first reply and revised manuscript.Au reply: The Reviewer #4 has taken notice of these corrections and consider them adequate (see comments and reply for reviewer #4).Reviewer #3: The authors are to be congratulated for designing and implementing this study. It is timely and represents an important step forward for understanding the pathobiology of EBV and malaria. Their manuscript would benefit from some improvements as suggested below:Au reply: We thank reviewer #3 for the congratulations and positive response and the proposed adjustments on which we reply here below:General comments:a) The authors report the study as a case-control study, but it is not clear whether this is not a cross-sectional study. This difference is important and needs further clarification. I suggest that the authors report this as a cross-sectional study. Regardless, the authors need to report the subjects who were assessed and if any were excluded and the reasons for exclusion.Au reply: The authors agree that this research is a cross-sectional study . This study has two specific goals: (1) to compare EBV genome and cytokine levels in acute Malaria cases infected with different malaria parasites  versus regional healthy controls and (2) to study the relation between defined cytokine levels and EBV-DNA loads. Sampling was done based on inclusion and exclusion criteria as was mentioned in the original sections on Sample collection and Malaria parasite analysis: - Inclusion criteria of clinical samples: 1) People who live in high endemic malaria region, i.c. Sumba Island, Nusa Tenggara province, Indonesia2) Suffering from clinical malaria  plus having a positive malaria rapid test result3) Age and gender were not limiting - Inclusion criteria of healthy control samples:1) Healthy residents who live in Surabaya city, a regional low Malaria region2) Not suffering from malaria or other acute or chronic diseases for 1 year3. Age: adulthood - Exclusion criteria of patient and control samples: 1) Donors suffering from HIV and/or sexually transmitted diseases2) Being non-coöperative (not filling-out questionaire or not giving consent)b) The authors need to clarify what they refer to as malaria. Malaria is the disease ie, infection PLUS symptoms. Individuals with malaria have a range of symptoms categorized as severe, moderate, mild, or asymptomatic. These are defined in a standard way by the WHO and should be used as such. For example, hyper-parasitemia is defined as parasitemia of 250,000/uL It is doubtful that the subjects included here meet that definition, but if they do, they should us the standard WHO definition and show the results in a primary table.Au reply: All patients had clinical symptoms characteristic of acute Malaria at intake  and had a positive on-site score in the Rapid Antigen test (RDT defined in Methods). All patients subsequently visited the local hospital for more detailed examination by an expert parasitologist . Parasitemia levels in blood were categorized as 4 groups according to WHO-2010, as specified in the Methods section (ref. 24).All parasitemia levels of P.falciparum, P. vivax and mixed were >50 parasites per single oil-immersion thick film field (therefore categorized as high parasitemia or Group 4), except one patient with P.vivax. The final number of parasites per μL of blood was calculated as the formula: [(counted parasites/500WBC) x counted or assumed WBC/μL(8000)] as prescribed by the WHO-2010 guidelines. These details are mentioned in the Methods and shown in Figure 1 (Parasitemia levels) and Suppl. Figure 1 (Symptoms).We changed hyper-parasitemia into high parasitemia (2x) in the manuscript.c) The analytic methods need to specify and distinguish the primary hypotheses being tested from the secondary explorative hypotheses being tested. The methods should also specify how multiple comparisons are being handled.Au reply: The comparison of EBV DNA levels between Malaria cases and controls was done by unpaired t-test, Pearsons R test was used for correlation analysis between EBV-DNA levels and parasite or cytokine levels. Linear regression analysis was used to determine the relationship between age, gender and one or more independent variables (i.c. EBV genome and cytokine levels), as already mentioned in the statistical analysis.d) The authors need to provide some epidemiological context for their work – for example what is the malaria pressure in their population - this can be provided in the form of malaria prevalence, entomology inoculation rates, and the age when severe malaria is observed in that population or the annual burden of malaria. This background information would be helpful for readers to understand how the cases being reported compared to the malaria patterns observed in the area.Au reply: The background on Malaria-incidence and spread in this study population (Sumba Island) was deduced from the ongoing Indonesian Malaria reduction campaign, summarized briefly in Lancet Global Health (2018), indicated here as ref. 15. The prevalence of Malaria in the control population  was described by Arwati et al., 2018, named here as ref. 26. We indicated some background info on EBV infection in Indonesia on lines 65-70. Although many studies on malaria epidemiology, immunology, and drug resistance have been conducted at many sites in Indonesia, there is little published literature describing malaria prevalence at the district, provincial, or national level (ref. 15). For extra info on local Malaria-situation in Sumba Island to the reviewer, not included in the manuscript because of ref. 15: Prior research  investigated two stages of cluster sampling malaria prevalence surveys in the wet season and dry season across West Sumba, Nusa Tenggara Province, Indonesia. The overall prevalence of malaria infection in the West Sumba District was 6.83%  in the wet season and 4.95%  in the dry. In the wet season Plasmodium falciparum accounted for 70% of infections; in the dry season P. falciparum and Plasmodium vivax were present in equal proportion. Malaria prevalence varied substantially across the district; prevalence rates in individual sub-villages ranged from 0-34%. The greatest malaria prevalence was in children and teenagers; the geometric mean parasitemia in infected individuals decreased with age. Malaria infection was clearly associated with decreased hemoglobin concentration in children under 10 years of age, but it is not clear whether this association is causal. Syafruddin D. et al. Seasonal prevalence of malaria in West Sumba district, Indonesia. Malar J. 2009;9,8:8. doi: 10.1186/1475-2875-8-8. PMID: 19134197.e) The authors need to provide more information about the sites for the enrollment – what are these malaria hospitals?Au reply: As part of Sumba-Island health care system, experienced health workers visited each of the suspect malaria patients at their homes in different villages and then examined the patients on site with RDT. If the RDT result is positive, the patient was referred to the nearest public health center in the district  to follow-up with clinical and microscopic examination of the malaria status and to obtain their questionnaire and plasma. The plasma of malaria patients was placed in a cool box with dry ice and shipped to the Institute of Tropical Disease, University of Airlangga, Surabaya. Upon arrival, the plasma was immediately aliquoted and frozen at –80°C. When being used, plasma samples were thawed and stored on melting ice or in a refrigerator at +2°-8°C. The manuscript is adjusted with these details (see lines 96-106). f) Please add page numbers!Au reply: We added page numbers and line numbering throughout the manuscript.Specific commentsg) The sentence “Enhanced EBV-DNA levels were significantly more frequent in P. falciparum and P. vivax infections (P<0.05) compared to controls.” In the abstract and elsewhere in the manuscript is confusing. Levels are higher, positivity is more frequent. The authors need to read the manuscript carefully and distinguish instances where they are talking about EBV DNA positivity as a binary variable versus when they are talking about the quantity.Au reply: We screened and adjusted the manuscript as suggestedh) 2nd paragraph of the introduction – please add some malaria contextual information as suggested above. Some of this information is in the first 2-3 paragraphs of the discussion.Au reply: See Au reply under d). In Indonesia, with around 270 million population that is 100% positive for EBV, about 10,7 million people are still living in Malaria endemic areas [15]. Children in Indonesia are exposed to EBV at early age with high dose of EBV via saliva (pre-chewed food). Later in life chronic exposure to EBV carcinogens, such as formalin, tobacco additives, herbal drugs/oils, butyrate acid (dried meat) and nitrosamine  are common in Indonesia, which can trigger aberrant and pathogenic EBV activity and malignancy [16-18].More detailed background was excluded from the introduction to reduce the manuscript size upon earlier reviewer’s suggestion (R1) and is now summarized in the discussion only. Because we found a rather high EBV-DNA load in Surabaya controls compared to our previous studies in Yogyakarta and Jakarta (= new result), we placed information on local life-conditions and malaria incidence in the discussion section .i) Last paragraph of the introduction – please “in Indonesia” where applicable and “in East Africa” or “Kenya’ as applicable, eg, “The aim of this research was to investigate how acute Malaria dysregulates EBV homeostasis and what cytokines would be involved “in Indonesia”.Au reply: We adjusted this issue in the manuscript, where applicable.j) In the material and methods revise “Malaria samples” to “venous blood samples”.Au reply: We adjusted this in the manuscriptk) Please provide more information about the sites where the subjects were enrolled – hospitals, communities etc etc using the STROBE guidelinesAu reply: We adjusted this issue: see also Au reply to e)l) Please clarify whether all subjects were assessed for malaria – this would include a screening for temperature, symptoms, and parasites. A standard definition of malaria should be used, denoting cases as either severe, moderate, mild, or asymptomatic.Au reply: These details are mentioned in the manuscript and follow the WHO-2010 guidelines. Clinical samples obtained were from symptomatic malaria patients, who often appear with a spectrum of symptoms such as fever, headaches, nausea, pale, and conjunctival pallor . Clinical findings were confirmed on-site by a “Rapid Diagnostic Test” (RDT) for malaria antigen detection (specified in Methods). RDTs are available in the remote areas in all Indonesia provinces, as an alternate way of quickly establishing the diagnosis of malaria infection. All positive RDTs were followed-up in the nearest regional hospital on Sumba Island (see above) with thick smear microscopy to confirm the parasite species and to quantify the proportion of infected erythrocytes in relation to a predetermined number of WBCs. After the examinations were concluded and prior to the start of treatment, fresh venous blood was collected for plasma preparation with informed consent from the patients who were confirmed malaria positive.m) For healthy controls, the test used to exclude infection should be stated as well as its limit of detection.Au reply: We did not specifically test the health status of the controls. We performed only physical examinations and personal interview to establish their condition at the time of sampling: all were healthy with no underlying disease in the recent year.n) The word “Clinical” in the sentence “Clinical diagnosis of Malaria was confirmed by a laboratory test for Malaria antigen detection should be replaced with “presence of plasmodial parasite infection”. Malaria by definition is clinical, ie. associated with symptoms. Thus, the authors could say “the diagnosis of malaria was made based on demonstration of presence of parasites, based on …tests, in patients with a fever and XX symptoms”AU reply: We adjusted the text as suggested (Lines 114-117). “The diagnosis of Malaria was confirmed in symptomatic patients by demonstrating the presence of plasmodial parasite infection in a fresh drop of finger prick blood using a Rapid test for Malaria antigen detection, i.c. “Rapid Diagnostic Test” (RDT), …o) The sentence “… followed-up with thick smear microscopy by expert parasitologists to confirm the parasite species and to quantify the proportion of infected red blood” needs to be revised for clarity and correctness. It is impossible to determine the number of red blood cells infected from a thick film.AU reply: We modified the text as suggested, by referring to the WHO-2010 guidelines (ref. 24). See lines 118-122: “All cases with a positive RDT were followed-up in the regional health center(s) on Sumba Island by further examination using fresh venous blood and thick smear microscopy by an expert parasitologists to confirm the parasite species and to quantify the proportion of infected red blood cells, according to WHO guidelines [24]. Briefly,…etc”.p) Please provide some additional details of the Giemsa stain .AU reply: These details are compliant with the WHO-2010 guidelines (24) and are not mentioned in such detail in the manuscript. We gladly will specify our procedures for the reviewer here: Using a clean lancet the tip of a finger was punctured and the first drop of blood was wiped away with clean gauze. The next drop of blood was touched with a clean slide and a small drop of blood was placed in the center of the pre-cleaned, labeled slide and was spread in a circular pattern until the size was of a dime , the corner of another slide was used to spread the blood drop. The slides were laid flattered and the smears were allowed to air-dry thoroughly in 20-30 minutes with a fresh air fan being protected from heat, dust and insects. Fresh working Giemsa stain (2.5% w/v in PBS) was prepared in a staining jar with 40ml fills and 2 drops of Triton X-100 were added. Fixed thick smear slides were placed into Giemsa stain for 45-60 minutes. Thereafter slides were removed and left in PBS for 5 minutes and dried upright in a rack. Staining Procedure for Quality Control = a positive smear  was included with each new batch of working Giemsa stain. q) In the statistics section – this needs to be revised for clarity “The correlation between EBV genome and Malaria parasites levels in a population was defined by student t-test” this test is normally used to test for quality of means – is this what is meant here? The authors need to describe what is being evaluate – means, correlation, association and what statistics are being used. This section requires careful attention. AU reply: The statistics section has been modified in detail as suggested by the reviewer #3 (Lines 162-167). Detailed calculation results for the reviewer #3 are shown here below.EBV Genome = P. falciparum vs Healthy controlEBV Genome = P. vivax vs Healthy controlEBV Genome = Mixed  vs Healthy controlP. falciparum Parasitemia vs EBV GenomeP. vivax Parasitemia vs EBV GenomeMixed  Parasitemia vs EBV Genomer) In the results section - the definition of hyper-parasitemia is unclear here. Do these patients really have severe malaria? Could other measures of malaria severity be provided? Eg, hemoglobin, platelet count, WBCs?AU reply: see Au reply to reviewer’s questions under b). At intake, all patients had multiple symptoms characteristic of acute Malaria , plus a positive “on site” RDT and >50 parasites per thick blood smear (category 4+) by subsequent expert parasitologist examination (WHO-2010 guidelines) prior to treatment. We did not make any statement on the clinical severity of Malaria, other than being acute symptomatic and RDT confirmed Malaria cases. To avoid confusion, we changed hyper-parasitemia into high (4+) parasitemia (2x) in the manuscript and show all parasitemia numbers in Figure 1.s) If the authors believe that the EBV levels might differ by type plasmodium – this should be articulated. Are there differences in the severity of malaria? Given the limitations of sample size, this segmentation of data should be considered carefully.AU reply: We indeed found significantly elevated mean levels of EBV-DNA in plasma in both P. falciparum and P.vivax Malaria cases versusthe healthy controls , as stated in the results, but the mean EBV-DNA levels were not different among both malaria groups. We did not address case-to-case differences in Malaria severity other than having multiple symptoms and Group-4+ level of parasitemia in all but 1 case of Mixed parasite infection. Details are shown in Figure 1 (parasitemia level) and in Supplemental Figure 1 (distribution of symptoms).Sample size for this cross-sectional study was defined using the following formula :N=[(Zα+Zβ)//(1-r)) )]^2+3 = [//) )]^2+3=15,872 ≈ 16If the estimated degree of relationship is a moderate degree with a correlation coefficient (r) = 0,653; standard deviation (type I error rate) = 5%, α = 0,05 , then Zα = 1,96 (from Z table); standard deviation (type II error rate) = 20% and the standard normal deviate for β = 0,2 (research power 80%), then Zβ = 0,842, thus the minimum sample size is 16 samples. If a 10% drop out is expected , the sample size with drop out correction is: n_do= n/(1-do)^(2 ) = 16/^(2 ) =19.75≈20Therefore, a minimum sample size of 20 samples is needed for this research. We did not specify this calculation in the methods, but it was part of the initial planning and study approval protocol.Note: due to the decreasing incidence of Malaria in Sumba Island it was difficult to find equal numbers of mixed infection cases in this cross-sectional survey. t) The results presented as “Overall, the mean EBV genome level was significantly higher in cases of P. falciparum  and P. vivax  infection compared to the mean level in the healthy controls  .” are interesting, but I would suggest re-vising this such that the different statistical results can be presented, ie, “Compared to healthy controls , EBV levels were significantly elevated in those with P. falciparum  and those with P. vivax .” The authors could also compare the different groups according to log levels, i.e., 2 log higher …Au reply: We modified the text as suggested (lines 190-195). “Compared to the healthy controls , cases of P. falciparum  and P. vivax infection  had significantly higher mean EBV DNA levels .”u) I think the authors need to present the significant cytokine results and report those that are not significant as not significant without going into details.Au reply: Because -in our opinion- this is a first study detailing 4 cytokine levels versus EBV-DNA load in acute symptomatic Malaria cases, we consider it relevant to mention all result details. In particular, the high levels of pro-inflammatory TNF-α found in this research may reflect how malaria P. falciparum infection causes dysregulation of endogenous EBV co-infection by impairment of the basic immune regulation. The elevated levels of IL-10, IFN-γ and TGF-β, though not significantly different from regional controls indicate a (pre-existing) dysregulation of normal immune balance , which may also affect EBV balanced latency. This may be similar to observations in healthy controls living in malaria-endemic regions in Africa, as indicated in the discussion (ref. 35).v) The discussion should be shortened and focused on the key findings, what they mean, and the strengths and weaknesses of this study, and brief suggestion of the next steps.Au reply: Because our study is first in quantitatively analyzing the levels of 4 different cytokines versus the EBV-DNA loads in acute malaria cases, in particular in Indonesia where such details are not described before, we consider a detailed description and discussion (lines 263-281) of the underlying regional life-style/environmental factors in Indonesia that may affect “normal” cytokine and EBV balances is relevant here and has not been considered as such in previous studies (ref. 35). Such underlying dysregulated EBV latency may be more profoundly altered upon repeated episodes of severe malaria, and especially in young children in endemic areas may increase the risk of lymphoma formation . The strength of our study is the combined marker analysis (4 cytokines and EBV-DNA) in 3 groups of Malaria cases, being Pf, Pv and Mixed. A weakness is the relatively low number of cases overall, particularly in the mixed group.The key finding in our work is the high level of pro-inflammatory TNF-α correlating with elevated EBV-DNA levels particularly in P. falciparum malaria cases, thereby supporting the previously described special interaction of P. falciparum parasites with EBV-infected B-cells involving the CIDR1α domain of P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), (ref 11-13). This is highlighted in the abstract, the discussion and conclusion. Further cancer-registry and clinical-immunological studies are needed to analyse this suggested interaction and its consequences in the Indonesian situation.Reviewer #4 comments:1. Summary of articleThe work is very interesting, novel and is well suited for publication in PLOS-ONE.I have reviewed the author’s response to the previous reviewers’ comments and am happy to see that all relevant points were taken on board to improve the manuscript.Au reply: We thank the reviewer for these positive remarks.2. Introductionhttps://doi.org/10.1186/s13027-021-00370-7), which can be referenced.Line – “EBV was first identified in cells of Burkitt Lymphoma (BL), an endemic cancer among sub-Saharan children, that is triggered by co-infection with Malaria parasites . BL is the most common cancer in children living in Malaria endemic regions in sub-Sahara Africa and Papua New Guinea ,” - recent publications have been published highlighting new perspectives such as Ellis et al 2021  on clinical-epidemiological and pathogenic-therapeutic aspects of Malaria and Cancer including BL. In our manuscript we named 6 references from recent 10 years on the pathogenesis and clinical aspects of BL in Malaria endemic regions  and 1 from 2005 (ref. 10), and we mentioned 2 recent reviews on Malaria and Cancer risk from 2017 (ref.13) and 2020 (ref. 31), which we feel cover all relevant aspects. Therefore, we prefer not to include any further references. 3. Line – “There appeared to be a direct correlation between increases in plasma EBV viral load and progression of endemic Burkitt Lymphoma” abbreviate to BL.Au reply: We checked and corrected all subsequent BL abbreviations.4. In the introduction information on the four cytokines chosen to quantify in the study should be provided in greater detail beyond the summary paragraph provided. Further details on the selection/choice of specific cytokines used in this study is important. For example “in this study the amount of x,x,x, and x plasma cytokines was evaluated. X has a role in xx etc repeat.Au reply: In the first round of reviewer’s comments and suggested revisions it was suggested to shorten the introduction regarding the role(s) of cytokines in acute Malaria, which we did. The suggested details and arguments for selecting the 4 cytokines and their proposed role in Malaria is now given in the discussion.5. Materials and Methods/ Results and DiscussionSample collection and Malaria parasite analysis – because of the age association with relationship between malaria and EBV it would have been useful for the field to have full malaria patient data i.e. positive parasites of P. falciparum (n=26), P. vivax (n=28), and mixed  (n=14), or healthy controls (n=27) data presented in a table with age information to know for example how many patients with P. falciparum infection were under 5 etc. This should be reflected in the results and discussion. Whilst the gender could be of interest especially in any of the samples were from pregnant women, the age is of greater interest and should be included.Au reply: These detailed patient data were also requested by reviewer #1 and are provided in the revised manuscript as supplemental information. This also includes the correlation analysis of patient’s age and gender against EBV and cytokine levels for each Plasmodium type infection and for the controls . 6. The authors should highlight any sample data from pregnant women.Au reply: We did not use any sample from pregnant women.7. Use of Kenya and Kenia in the manuscript change all “Kenia” to Kenya for continuity.Au reply: This was checked and corrected throughout the manuscript.AttachmentPLOS ONE Reviewers Comments-Revisions (JM).docxSubmitted filename: Click here for additional data file. 14 Dec 2021Quantitative cytokine level of TNF-α, IFN-γ, IL-10, TGF-β and circulating Epstein-Barr Virus DNA load in individuals with acute Malaria due to P. falciparum, P. vivax or double infection in a Malaria endemic region in Indonesia.PONE-D-21-19672R2Dear Dr. Middeldorp,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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Plasmodium falciparum elicits antibody responses against many of its protein components, but there is also formation of antibodies against different parts of the red blood cells, in which the parasites spend most of their time. In the absence of a decisive intervention such as a vaccine, people living in malaria endemic regions largely depend on naturally acquired antibodies for protection. However, these antibodies do not confer sterile immunity and the mechanisms of action are still unclear. Most studies have focused on the inhibitory effect of antibodies, but here, we review both the beneficial as well as the potentially harmful roles of naturally acquired antibodies, as well as autoantibodies formed in malaria. We discuss different studies that have sought to understand acquired antibody responses against P. falciparum antigens, and potential problems when different antibodies are combined, such as in naturally acquired immunity.Antibodies are central to acquired immunity against malaria.  Plasmodium species and is transmitted in human populations by bites of infective female Anopheline mosquitoes. The success of Plasmodium species as parasitic organisms is based on their ability to evade immune attacks directed against them by the human host, as well as the mosquito vector. During evolution, the genetic background of both humans and mosquitoes has been of importance in forming which parasites can multiply successfully.Malaria still inflicts an extreme level of burden on people living in endemic regions . Africa P. falciparum and highlight antibody interactions with important antigens expressed as the parasite goes through different stages in the human host as well as the mosquito.Antibodies are considered an efficient product of the immune system and they are generally produced by B cells/plasma cells, but there is an increasing body of evidence to support antibody production by cancerous and normal non-B cells, such as in proximal tubuli cells and epithelial cells ,4,5,6. NAnopheles mosquito injects about 10–150 sporozoites into human skin [Plasmodium, both in vitro and in vivo [Plasmodium studies, there is evidence to show that about half of the inoculated sporozoites could remain in the skin where they form extrahepatic exoerythrocytic forms. P. falciparum is not known to induce a significant dermal immune response, but a recent study based on a Plasmodium berghei animal model showed that anti-sporozoite antibodies targeting mainly the circumsporozoite protein (CSP) have protective functions by inhibiting sporozoite motility through the skin [Plasmodium species.The complex and somewhat treacherous interaction of malaria parasites with the human host begins when a parasite-infected female man skin . Recent man skin ,20 in a  in vivo ,22. Morethe skin . AlthougP. falciparum sporozoites have been demonstrated in endemic areas [The few sporozoites that are able to make it to the bloodstream quickly invade the hepatocytes and are exposed to the immune system for a relatively short period of time. However, naturally acquired antibodies against whole ic areas ,25. Circic areas ,28,29,30ic areas . Naturalic areas .P. falciparum is responsible for all known symptoms of malaria; thus, functional antibodies against parasite antigens at this stage should be able to reduce or prevent clinical symptoms of malaria. Merozoite-filled vesicles, merozomes, from parasite-infected hepatocytes release merozoites into the blood stream where they invade erythrocytes to begin a 48-h life cycle that will lead to an exponential increase in production of merozoites. Merozoites in the bloodstream are more exposed to the immune system and studies have identified naturally produced antibodies against a majority of the known merozoite surface proteins as well as against merozoite organelles such as rhoptries and micronemes [The asexual stage of cronemes .P. falciparum is used for the experiments [P. falciparum lines, but when W2mef was used, the growth was actually enhanced, sometimes as much as 25–50%. It is well established that in endemic areas, many different parasites circulate [P. falciparum parasites in vitro [Leishmania or viruses like Zika and Dengue, it has been shown that antibody-mediated responses involving the complement or FcR pathways do not always lead to protection or reduced infection, but can sometimes be exploited by these pathogens for enhanced invasion of host cells [P. falciparum could also use this mechanism to enhance hepatocyte or erythrocyte invasion. A study suggested that this could definitely be possible, since a monoclonal antibody obtained from MSP142 vaccines enhanced parasite invasion and polyclonal IgG enhanced invasion in a complement-dependent manner [Merozoite surface protein 1 (MSP1) is the most abundant protein on the merozoites and plays an important role in erythrocyte binding and invasion . Some steriments . When puirculate ,44 and iirculate  and therin vitro . For othst cells . This pht manner .P. falciparum [Apical membrane antigen 1 (AMA1) is a micronemal protein that is transferred to the merozoite surface just prior to merozoite egress . Like MSlciparum . Protectlciparum .P. falciparum.Interethnic differences in antibody responses to some blood-stage antigens such as ring-infected erythrocyte surface antigen (RESA) and Pf322 have been used as indirect evidence for the existence of an association between naturally acquired immunity and human genetic factors . A genomP. falciparum on the surface of infected red blood cells (iRBC) to human endothelial cells. This process is central for the development of severe forms of malaria mainly in children. One of the main iRBC proteins is P. falciparum erythrocyte membrane protein 1(PfEMP1), and antibodies targeting PfEMP1 have been shown to be associated with a reduced risk of severe malaria in children in endemic areas [P. falciparum-iRBC in the placenta by binding to chondroitin sulphate A (CSA) [Obstructions of small blood vessels are caused by binding of proteins expressed by ic areas . A studyic areas . VAR2CSA A (CSA) . The seq A (CSA) . Natural A (CSA) ,75,76,77 A (CSA) . The mai A (CSA) ,80. Howe A (CSA)  could no A (CSA) ,83.P. falciparum infections in endemic areas. Autoantibodies can be directed against membrane phospholipids [Autoantibodies targeting different human cellular components have been associated with holipids ,85,86, eholipids ,88, or Dholipids . During holipids ,91. Anotholipids , indicatNot much is known about the mechanisms through which autoantibodies are generated in malaria infection, but it has been suggested that the expression of parasite proteins such as PfEMP1 on erythrocyte membranes could be an initiation step. For instance, a genome-wide study  found thP. falciparum infections. On the contrary, they could instead do more harm than good. Autoantibodies have been shown to aggravate malaria pathology by inducing cell lysis, microthrombosis, and inflammation [P. falciparum malaria [P. falciparum [Autoantibodies targeting DNA and other cytoplasmic components could be a form of homeostatic response to mop up cellular fragments, or debris produced during erythrocyte rupture that accompany blood-stage infection, or are generated by oxidative damage associated with antimalarial drug use . If thisammation . A recen malaria . Anemia, malaria ,89,97. H malaria , and ser malaria . Anti-Ba malaria ,100, suglciparum .Plasmodium that are responsible for parasite transmission from the human host to the Anopheles mosquito. Mosquitoes pick up gametocytes when they take blood meals from infected humans. In response to the environment in the mosquito lumen, gametocytes transform and after fertilization, they form a zygote. Pfs230 and Pfs48/45 belong to the 6-cystein protein family that is expressed on the surface of gametocytes and play important roles in fertilization [Anopheles mosquitoes can limit Plasmodium infection, but Pfs47 can be used by the parasite to subvert the complement-like immune mechanism of the mosquito in order to establish an infection [P. falciparum parasites in their transmission cycle. It could be challenging to produce effective antibodies against Pfs47 as the majority of monoclonal antibodies produced against full-length recombinant Pfs47 lacked transmission blocking activities, and some antibodies even seemed to increase the transmission [Gametocytes are specialized and permanently differentiated forms of lization . Studieslization . A more lization . Mosquitlization . The immnfection . The intnfection . The midnfection . This efsmission .P. falciparum in the human host as antibodies accompany the parasites through all the stages of development, and human anti-parasite antibodies are even carried on into the mosquitoes. Although the functional roles of autoantibodies are still enigmatic, their contribution to malaria pathogenesis cannot be disregarded. The ability of malaria parasites to complete their life cycle despite the abundance of antibody attacks is probably a high level of evolutionary success with a win–win situation between the human host and the parasites. Antibody responses are able to protect the host enough for the host to survive, but not enough to kill the parasites; thus, parasites can survive in low numbers and be transmitted to a new host. The field of malaria research where combinations of antibodies with different specificities are used together needs more focus in future studies to elucidate what happens in real life in a human being during the development of natural immunity against malaria.There is no lack of antibody attacks against


Plasmodium species in donors with low-density parasites were identified using PCR. Nine (75.0%) were identified as Plasmodium falciparum, two (16.7%) were identified as Plasmodium vivax, and one (8.3%) was identified as Plasmodium ovale. All were imported from malaria-endemic areas. New action plans designed to meet the challenges of TTM are necessary to ensure the elimination of malaria in China. Paying more attention to the frequency of TTM could help to enhance blood safety in China.Although China is moving toward the eradication of malaria and no indigenous malaria has been reported in most Chinese provinces for several years, recent evaluations have revealed that imported cases remain a major challenge to eliminating malaria, with the number of transfusion-transmitted malaria (TTM) cases increasing over time. Here, we review several TTM case reports published after the implementation of the National Malaria Elimination Program in 2010. A total of 12 TTM cases were reported in China between 2013 and 2018. All recipients and donors were diagnosed using rapid diagnosis test and peripheral blood smears.  Plasmodium, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Falciparum malaria is the most prevalent and severe. Plasmodium species are always transmitted by the bite of Anopheles mosquitoes from an infected person to healthy subjects [Plasmodium spp. presenting with low parasitaemia. These infections may result in transfusion-transmitted malaria (TTM) which may be fatal if not diagnosed timely and treated effectively [Malaria is an important parasitic disease worldwide, which threatens the health and safety of various populations and whose infections slow both economic and social development. According to the WHO malaria report, there were 229 million malaria cases and more than 400,000 deaths globally in 2019 with the majority of these distributed in Sub-Saharan Africa and Southeast Asia [subjects . This isectively . In anotectively . None ofP. vivax as the causative agent. When the National Malaria Control Program was launched, the number of malaria cases declined dramatically. However, in the late 1980s and early 1990s, China experienced several blood donor-based malaria outbreaks in various parts of China, especially in those areas where natural transmission had been eliminated for several years [Plasmodium spp. were reported between 1993 and 1995 with the majority of cases in Hebei, Jiangsu, Henan, and Sichuan [P. falciparum among paid plasmapheresis blood donors was significantly higher than that of residents and whole blood donors [P. vivax and P. ovale in Guangdong, Guangxi, Yunnan, Heilongjiang, and other places [In the past, China suffered seriously from malaria epidemics, which makes it difficult to determine accurate statistics concerning total malaria cases, including TTM, till the foundation of the People's Republic of China in 1949. In addition, the lack of a complete information reporting system before the appearance of severe acute respiratory syndrome (SARS) in 2003 has also inhibited these evaluations . Therefoal years . Some la Sichuan , 12. Ther places . Under tr places , and no r places .Plasmodium spp. reservoirs present a unique challenge for blood banks, and infections of this kind can evolve into severe cases. To address these new challenges to blood safety in light of the malaria elimination goals, the main objective of this paper is to comprehensively review any TTM cases reported between 2011 and 2020 and characterize the epidemiology of TTM and provide some suggestions for reducing TTM and supporting the Chinese malaria elimination goals.The incidence of imported malaria is increasing and poses a serious threat to total malaria elimination in China. Due to global economic integration, people travel to malaria-endemic countries for trade, tourism, labour, and other purposes. The number of imported malaria cases has continued to rise on an almost daily basis in almost all provinces in China, with more than 2000 cases of imported malaria diagnosed every year for the past decade or more . These ihttp://www.cnki.net), Wanfang Data (http://www.wanfangdata.com), and the China Science and Technology Journal Database . The keywords for the literature search included “transfusion transmitted malaria OR Blood transfusion AND malaria OR Blood transfusion AND plasmodium.” All pertinent case reports were downloaded and analysed in the context of the epidemiology of the disease in China. English papers were also identified using the PubMed database. The following data were extracted from each study: province, sex, and age of blood donor and recipient, occupation of blood donors, diagnostic method employed, blood donor screening method, blood component transfused, Plasmodium species, Plasmodium spp.'s original country, incubation period , and delayed diagnosis .We examined all publicly available TTM case reports produced between January 2011 and March 2021 in the Chinese literature databases, including CNKI ) [Plasmodium spp.A total of 21 papers reported cases associated with TTM in Chinese patients, only five of which seem to be duplicated. In one report, the recipient had not developed malaria after being transfused with 200 ml of blood contaminated with  (King)) . In tota (King)) –28. All Plasmodium parasites were all imported from endemic countries, such as those in Africa and Southeast Asia. One older patient died (Case 6) due to his illness, and the others recovered successfully.Of the 12 TTM cases, seven were female and five were male. The ages of the TTM cases ranged from 19 to 85 years, and they shared several common characteristics. First, none of the patients had a history of travel to a malaria-endemic country, and none had a history of infection. Second, all patients were transfused with red cell suspension prepared from infected blood donors. No corresponding plasma transfused patient was found to infect malaria. Third, the patients were not diagnosed in time with several patients experiencing a delay between fever onset and diagnosis, with the shortest period 3 days and the longest over a month. Fourth, the sources of the The female-to-male ratio was 1 : 6 for blood donors. All were first-time donors. They were all young people, with a mean age of 27 years , and all had a history of malaria or were living in an endemic area. The mean gap between their return to China and their donation was not more than 1 year . These donors included 8 employees dispatched abroad and 4 overseas students. A total of five of the donors  had no malaria symptoms, and no parasite could be observed using microscopy  had no symptoms and remained parasite free under the microscope. All cases, with the exception of Case 4, were confirmed to be malaria DNA positive by PCR. These PCRs included normal PCR, nested-PCR (nPCR), and real-time fluorescence quantitative PCR (RT-fqPCR). In addition, the donors in Cases 2 and 12, which had no detectable parasites under the microscope, produced positive results using more sensitive nested-Multi PCR. A total of 9 (75%) of the cases were shown to be P. falciparum, 2 (16.7%) were P. vivax, and 1 (8.3%) was P. ovale . A global TTM review showed that these results were largely consistent throughout the world and that the mean incubation time for P. falciparum TTM was 25.7 days [P. falciparum TTM was 12.2 ± 4.1 days (range: 7–16 d) [P. falciparum TTM. This may be because most of the donors presented with more robust subclinical infections which allowed the parasites to be observed using microscopy and meant that the parasite inoculums were sufficient to establish clinical symptoms in a shorter period of time.In theory, if the infected blood product is able to directly transmit the erythrocytic stage of the parasite to the recipient during TTM, the incubation period should be shorter than that in natural mosquito-transmitted malaria (MTM). In MTM, parasites experience a liver stage, where they mature over 5–16 days depending on the parasite species, and then infect red blood cells over 2-3 days leading to the development of erythrocytic schizonts, eventually destroying the erythrocyte . In a re5.7 days . In anot 7–16 d) , which iAs reviewed, 30.6% malaria cases were delayed in their diagnoses, often exceeding the more common 3-day timeframe in clinical interventions .Blood banks usually evaluate the donor candidates' health before donation, including screening for current infections, and a detailed history of previous disease and travel. In China, donor candidates should meet the Blood Donor Physical Examination Requirements (GB18467-2011) which include weight, blood pressure, and haemoglobin evaluation using the copper sulphate method, alanine aminotransferase (ALT) activity, and HBV surface antigen testing by colloidal gold strip. However, before these evaluations, donors are required to complete the Health Check Questionnaire, which itemizes disease and travel history, and sign informed consent. For malaria, it rules that those who have had a diagnosis of malaria or lived in an endemic country for 12 months are deferred to donate for 3 years after becoming asymptomatic. Therefore, the donors in this review should have been prevented from donating for between one and three years following their return to being asymptomatic. In practice, these guidelines were not properly applied. These donors intentionally or unintentionally concealed or forgot their malaria history. Some foreign donors may not fully understand the questionnaire due to the language barrier. In most areas of China, indigenous malaria has been eliminated for more than 20 years. The public's perception of risk for malaria is very low, and many younger donors have not even heard of this disease . Most ofµL [Effective screening of the donations can also significantly decrease the risk of TTM. Microscopy, detection of plasmodia antibodies or antigens by immunology assay, or plasmodial DNA by PCR can all be applied to screen donations reducing the risk of contamination. In a systematic review and meta-analysis , the medµL , but notµL . That saµL . DespiteµL . In our µL  is used P. vivax. Indigenous vivax malaria has been effectively controlled through the comprehensive implementation of effective interventions [Anopheles mosquitoes, has not been eradicated, Jiangsu Province continues to implement stringent criteria for malaria infection reporting. It is the first province to implement the 1-3-7 surveillance and response strategy  [Plasmodium species negative blood components be used in the clinic.Jiangsu, located in eastern China, was once an endemic home for ventions . No indiventions , 40. In ventions . Given ten days) , which ten days) . This haen days) . This isen days)  and realen days)  to screeChina launched its malaria elimination program in July 2010, with a plan to achieve total eradication by 2020. Although many malaria research papers have been published in China, TTM has been largely neglected. The challenges faced in TTM mirror the considerations in reducing national malaria risk and improving general blood safety in China. To mitigate the risk of TTM as much as possible, knowledge gaps in TTM need to be highlighted and addressed in the pursuit of malaria elimination, and public health authorities should consider how to deploy the best blood safety surveillance strategies. Currently, there are no mandatory malarial agent screening methods available to Chinese blood banks. The first and only defence against TTM is strengthening our blood donor management and using questionnaire-based risk assessment. In areas where the number of imported malaria cases is increasing, it is advised that high-risk blood should be selectively screened by in-house PCR.


Recent findings suggest that changes in human odors caused by malaria infection have significant potential as diagnostic biomarkers. However, uncertainty remains regarding the specificity of such biomarkers, particularly in populations where many different pathological conditions may elicit similar symptoms. We explored the ability of volatile biomarkers to predict malaria infection status in Kenyan schoolchildren exhibiting a range of malaria-like symptoms. Using genetic algorithm models to explore data from skin volatile collections, we were able to identify malaria infection with 100% accuracy among children with fever and 75% accuracy among children with other symptoms. While we observed characteristic changes in volatile patterns driven by symptomatology, our models also identified malaria-specific biomarkers with robust predictive capability even in the presence of other pathogens that elicit similar symptoms. However, a major challenge for the development of volatile disease biomarkers is posed by the inherent variability of human volatile emissions, which are highly labile and can be influenced by a wide range of genetic, physiological, and environmental factors3. In light of this variability, there is reason to speculate that volatile biomarkers might be of particular value in diagnosing diseases caused by insect-borne pathogens, which frequently manipulate the odors of their hosts in ways that influence vector behavior and might therefore be highly conserved6. Indeed, a number of recent studies on human malaria have identified characteristic changes in the volatile emissions from the skin and breath of infected individuals12. Yet, uncertainty remains about the physiological bases of these volatile changes and the extent to which they are uniquely caused by malaria or products of pathological processes that might be shared with other disease states. Addressing this uncertainty has important implications for understanding the diagnostic value of such biomarkers, particularly for use in human populations where numerous pathological conditions are widespread and may give rise to similar symptomatology, as is often the case for malaria endemic regions.The presence of disease can alter human odors, including volatile emissions from skin and breath. The potential diagnostic value of such changes in volatile chemistry has long been recognized, and volatile-based diagnostics are being actively explored for a number of diseases, including several types of cancer9 and breath12 volatiles, as well as the volatile emissions of rodent malaria hosts13, while others have characterized the emissions of Plasmodium cells grown in vitro16. Effects of infection on host odors have also been reported to influence vector behavior, with several studies reporting increased mosquito attraction to hosts harboring transmissible stages of Plasmodium parasite18 and at least one showing enhanced attraction to host odor profiles in which individual compounds were manipulated to mimic the relative up- or downregulation caused by infection13.A number of previous studies have documented malaria-induced changes in human skin19, suggesting that the pathogen may benefit from actively manipulating host volatiles20. To the extent that Plasmodium parasites, or other vector-borne pathogens, alter host odors in ways that consistently influence vector attraction, they may also generate unique patterns of effects on host volatile profiles—possibly tailored to the olfactory responses of particular vector species—distinct from more general changes in volatile emissions that arise as mere byproducts of pathology. The presence of such unique signatures of infection might, in turn, facilitate the identification of pathogen-specific biomarkers capable of reliably predicting infection status even in populations where numerous pathological conditions elicit similar symptoms.Enhanced vector recruitment during the transmissible stage of infection is hypothesized to facilitate malaria transmission7. Yet, while these characteristic changes were highly predictive of malaria infection, the specific processes by which the presence of malaria parasites alter human volatiles remain almost entirely unknown. One recent study reported that a malaria-derived isoprenoid increases the production of several monoterpenes and aldehydes in vitro21, but this does not account for the majority of volatile alterations observed by our and other studies12 suggesting that the underlying mechanisms are likely to be complex.The identification of such robust biomarkers is of particular interest for malaria, which frequently occurs in populations affected by numerous other diseases and ailments that elicit similar symptoms, including fever, headaches, diarrhea, vomiting, etc., and might therefore plausibly be expected to have somewhat similar effects on underlying physiological processes that also influence volatile emissions. In the case of malaria, disease progression often entails alternating asymptomatic and symptomatic phases that may recur indefinitely if not treated. In a previous study, we reported differential up and down regulation of specific volatile organic compounds in both symptomatic and asymptomatic schoolchildren in Kenya7. Specifically, we compared the skin volatile profiles of symptomatic schoolchildren who tested positive for malaria to those of children presenting similar  symptoms, but who tested negative for malaria. We used a genetic algorithm framework to predict malaria infection and identify volatile compounds associated with disease status. Our goal was thus to discover which volatile changes might be specific to malaria, and which might be caused by infection with other illnesses or conditions.To explore the extent to which changes in the odors of malaria infected humans are associated with the presence of symptoms, we analyzed the volatile profiles of symptomatic children, using volatile data previously collected from a large field trial in Western Kenya-F1,48 = 2.96, p = 0.022) and feet . These results indicate that the effect of malaria infection on volatile profiles is apparent even when directly compared to other conditions that produce similar symptoms.We first explored whether there was a clear signature of malaria infection among symptomatic children. In addition to examining differences in the volatile profiles of malaria-infected and uninfected children presenting any symptom , we made similar comparisons for non-exclusive subsets of children exhibiting the two most commonly observed symptoms in our dataset: fever and diarrhea. For each of these three symptom categories, discriminant analysis of principal components (DAPC) revealed clear separation in the overall volatile composition between individuals with and without malaria infection Fig. , S1, witWe next used a genetic algorithm predictive model trained to recognize malaria infection based on a subset of the data (70%) to predict malaria infection status in the remaining test set (30%). For children exhibiting any symptom, this model was able to predict malaria status with 75% accuracy Tables and 2. GiPlasmodium completes its lifecycle within the host, fever is typically associated with the rupture of mature schizont cells and the release of merozoites that then reinvade red blood cells22. The resulting intermittent cyclic fever may cause unique physiological effects compared to other ailments, and may also play a role in the downregulation of volatiles, including the compounds that drive the predictive accuracy of our model  and using either foot or arm volatiles Table . Several7. The overlap in compounds identified as important predictors of malaria infection and of symptoms in malaria-free children (but with different patterns of up and down regulation) is intriguing and may reflect the influences of different pathological conditions on the same underlying physiological processes.Comparing the volatile emissions of malaria-free children exhibiting any symptom to those without symptoms revealed significant downregulation of several compounds Fig. , with et25. In the case of lung cancer, a large-scale trial for diagnostic detection using VOCs is currently underway26. For many other diseases, however, significant challenges remain to be overcome prior to the implementation of large-scale trials, including the identification of a robust and reproducible set of candidate biomarkers3, which can be complicated by the absence of standardized methods for volatile collection and analysis27. In the case of malaria, several previous studies have reported successful prediction of infection status based on analysis of VOCs; however, there is considerable variation in the predictive compounds identified, likely owing to divergent methodologies across studies. Two compounds, hexanal and nonanal, have consistently been found informative with respect to with malaria status, each reported in three separate studies from skin and breath volatiles as well as in the prediction here of malaria status in symptomatic children , and the Kenya Medical Research Institute (SERU 391) and all experiments were performed in accordance with relevant guidelines and regulations. Before sample collection, the study and consent form were explained to parents/guardians and their written informed consent was obtained.This study was approved by The Pennsylvania State University , ETH Zürich (EK2015-Participant exclusion criteria included (1) receipt of antimalarial medication during the previous 2 wk; (2) chronic disease, such as HIV; (3) not signing (or having a parent sign) the consent form; and (4) refusal of malaria treatment.7. Symptoms were self-reported in an initial interview using a standardized questionnaire; symptoms indicative of malaria included fever, abdominal pain, rash, diarrhea, vomiting and body aches. For our analysis, we sorted children into the categories (1) any symptom (2) fever, (3) diarrhea and (4) asymptomatic. The any symptom category comprised children with fever, diarrhea, abdominal pain or vomiting; the fever and diarrhea categories were non-exclusive subsets of the any symptom category. Abdominal pain and vomiting were not analyzed independently due to low numbers of children with these symptoms. Malaria infection status was initially assessed via rapid diagnostic testing (SD Bioline), then confirmed by light microscopy and PCR. For the current study, children in the malaria infected category tested positive by both microscopy and PCR.Our analyses were performed on data derived from skin volatile samples (1 h collections from arms and feet) collected from students at 41 primary schools near Mbita Point, Kenya between 2013 and 2016The dataset used for this study comprised volatile profiles for 114 children. Once categorized into symptoms for the below analyses, numbers of children in each category were as follows: malaria infected children with any symptom = 29, fever = 18, diarrhea = 11. Malaria-free children with any symptom = 22, fever = 11, diarrhea = 9, asymptomatic = 16.7).Arm and foot volatiles were collected simultaneously for 1 h by enclosing the arm (from wrist to above the elbow) or foot (to below the knee) in a teflon bag, pushing filtered air through an entry port  and pulling it through an exit port  where it was collected on an adsorbent HaySepQ filter. Compounds trapped on filters were then eluted with 150ul dichloromethane and analyzed by GC–MS  with the Euclidean similarity on the scaled data43.Discriminant Analysis of Principal Components (DAPC)44. They have been used as a promising multivariate approach in the analysis of metabolomics datasets46. The algorithm works by evolving initial sets of variables (chromosomes) from a random population via cycles of replication, recombination and mutation of the fittest chromosomes. The iterations were repeated for 100 generations, with a population size of 50 candidate solutions for each model and crossover and mutation probabilities of 0.8 and 0.1, respectively. The performance of the fittest GA model was used to predict the samples in the validation set. To complement the predictive models, a differential analysis between infection statuses was performed using the limma R package47. The GA models were formulated with the caret R package48.We used a Genetic Algorithm (GA) to predict disease condition and to select one or more sets of compounds for each of the symptom subsets to be assessed as a potential biomarker for malaria. Each subset was randomly split into 70% for model training and 30% for validation. Using the training set, each model was trained five times using a repeated tenfold cross-validation. Genetic Algorithms are feature selection procedures that are conceptually based on the principle of evolution by natural selectionSupplementary information.