Background: Gastrointestinal symptoms are commonly associated with acutePlasmodiumspp infection. Malaria-associated enteritis may provide an opportunity for enteric pathogens to breach the intestinal mucosa, resulting in life-threatening systemic infections.Methods: To investigate whether intestinal pathology also occurs during infection with a murine model of mild and resolving malaria, C57BL/6J mice were inoculated with recently mosquito-transmittedPlasmodium chabaudi AS. At schizogony, intestinal tissues were collected for quantification and localisation of immune mediators and malaria parasites, by PCR and immunohistochemistry. Inflammatory proteins were measured in plasma and faeces and intestinal permeability was assessed by FITC-dextran translocation after oral administration.Results: Parasitaemia peaked at approx. 1.5% at day 9 and resolved by day 14, with mice experiencing significant and transient anaemia but no weight loss. Plasma IFNγ, TNFα and IL10 were significantly elevated during peak infection and quantitative RT-PCR of the intestine revealed a significant increase in transcripts forifng andcxcl10. Histological analysis revealed parasites within blood vessels of both the submucosa and intestinal villi and evidence of mild crypt hyperplasia. In faeces, concentrations of the inflammatory marker lactoferrin were significantly raised on days 9 and 11 and FITC-dextran was detected in plasma on days 7 to 14. At day 11, plasma FITC-dextran concentration was significantly positively correlated with peripheral parasitemia and faecal lactoferrin concentration.Conclusions: In summary, using a relevant, attenuated model of malaria, we have found that acute infection is associated with intestinal inflammation and increased intestinal permeability. This model can now be used to explore the mechanisms of parasite-induced intestinal inflammation and to assess the impact of increased intestinal permeability on translocation of enteropathogens. Plasmodium spp-infected red blood cells (RBCs) detectable in the peripheral circulation. Gastrointestinal symptoms are commonly noted in malaria patients, with diarrhoea reported in both travelers and those residing in malaria-endemic areas  or exudative . Gastrointestinal pathogens,  are highly prevalent in malaria-endemic areas and episodes of diarrhoea are common, especially among children , a particularly frequent cause of invasive bacterial disease  in sub-Saharan Africa resulting in considerable morbidity and mortality  , along with autoclaved water. Animals were monitored according to institutional guidelines, with routine daily health checks and increased monitoring duringP. chabaudi infection. Euthanasia was performed by cervical dislocation at the end of phenotypic experiments, or by exsanguination under anaesthesia . This specific method of anaesthesia reduces animal suffering whilst maximising blood volume obtained.This study was reviewed and approved by the Ethical Review Body of the University of Edinburgh. All procedures were carried out in accordance with the UK Home Office regulations  under Project Licence number P04ABDCAA. Throughout this study, all efforts were made to reduce animal usage and ameliorate harm to animals. Mice were housed in the University of Edinburgh Licenced Animal Facilities 60/2605), and all animal procedures were performed in laboratories within the animal facilities. Six to eight weeks old female C57BL/6J mice were purchased from Charles River . All animals were maintained with at least one companion, randomly housed in individually ventilated cages furnished with autoclaved woodchip, fun tunnel and tissue paper at 21 ± 2°C, under a reverse light-dark cycle  at a relative humidity of 55 ± 10% in a specified pathogen free facility. Mice were housed under these light-dark cycle conditions to allow collection ofmus musculus-Plasmodium chabaudi chabaudi AS animal model of malaria was chosen to minimize host genetic variability and to obtain robust infections with a very low incidence of severe disease. Animals were inoculated i.p. with 1×105 PcAS-GFP or PcAS-mCherry-infected RBCs (iRBC) that had been blood passaged 3–6 times since primary infection by mosquito; deemed ‘recently mosquito-transmitted PcAS infection’, as previously described , mCherry-expressing PcAS , or were uninfected controls (n=58). For each experiment, 2 mice were used to expand frozen stocks of stabilate parasites, . Four mice were excluded; three inoculated mice which were uninfected and one with an unexpectedly high parasitemia. For each experimental readout per time-point, two independent experiments were performed.-1 heparin sodium , and then diluting a further 1:5 prior to acquisition on a BD Fortessa . At least 100,000 events were analysed per sample; gates were set using uninfected control blood using FlowJo V10 (Tree Star), as previously shown , centrifuged at 10,000g for 5 min, and plasma stored at -70°C for subsequent analysis. For multiplex analysis, a magnetic Luminex assay  was performed according to the manufacturer’s instructions for IFNγ (BR33), TNFα (BR14), and IL-10 (BR28), using undiluted samples and analysed on a Bio-Plex 200 . For IFNγ analysis by enzyme-linked immunosorbent assay (ELISA), plasma was diluted 1:2 and assayed with the mouse IFNγ ELISA MAX deluxe  according to the manufacturer’s instructions. Samples which gave values below the detectable range were reported at the limit of detection for each analyte.Cardiac blood was collected into 5µL of heparin sodium  containing 10µL Brilliant III Sybrgreen Ultrafast Mastermix , 6.4µL ultrapure water (Gibco), and 0.8µL of both forward and reverse primers (diluted to 10µM) for each gene target (sequences listed inT) method. Target gene transcription of each sample was normalized to the respective levels of β-actin mRNA and represented as fold change over gene expression in control animals, as described previously (ΔΔCt).At necropsy, the intestines were divided into five equal lengths , and cleaned of contents by flushing with dPBS. Tissue was then immersed in 1mL RNAlater  and stored at -70°C after chilling according to the manufacturer‘s instructions. For isolation of RNA, tissue was transferred to 2mL FastPrep Lysing Matrix D tubes  containing 1mL of TRIzol (Invitrogen). Tissues were then homogenized using a Precellys 24 tissue homogenizer (Bertin instruments) at 30sec on high speed, followed by phenol/chloroform extraction with TRIzol according to the manufacturer’s instructions. Residual DNA was then removed . Purified RNA was measured using a NanoDrop spectrophotometer and diluted to 100 ng/mL prior to cDNA synthesis using the AffinityScript Multiple Temperature cDNA synthesis kit , according to the ‘1Plasmodium parasites, immunohistochemistry was performed targeting GFP in MT-PcAS-GFP-infected mice. Antigen retrieval was achieved by autoclaving  in TRS , followed by washing in PBS/0.1% Tween20. Slides were blocked with 3% hydrogen peroxide for 10min, followed by non-specific horse serum matched to the secondary antibody for 15min, followed by blocking with avidin and biotin for 15min . Slides were incubated with goat anti-GFP  diluted 1:500 in PBS/0.1%Tween+1%FBS) in a humified chamber at 4°C overnight. After 3 x 15min washes in PBS/0.1%Tween, slides were incubated with biotinylated horse anti-goat IgG H+L  diluted 1:500 for 1h at room temperature. Normal goat IgG  or no primary antibody were used as controls. Finally, slides were stained with DAB  using an ABC reagent kit , according to the manufacturers’ instructions with substrate development for 10min, and counterstained with hematoxylin  using an autostainer XL (Leica).Rolls of cleaned intestinal tissue were fixed immediately in 10% PBS-buffered formalin, followed by embedding into paraffin prior to sectioning into 4–5µm slices onto charged slides. Slides were deparaffinised and rehydrated using an AutoStainer XL (Leica) prior to staining. For visualisation ofQuPath Software (v0.2.3)  and analysed withIntestinal permeability was assessed as described previously . 100µL oLarge intestines were excised from anus to caecum, split open with scissors and the contents collected with a blunt metal edge into a 2mL eppendorf tube. Samples were placed at -70°C until processing. Contents were weighed and 0.5mL of ‘faecal buffer’  in dPBS) added. Samples were allowed to rest for 30min at 4°C, then placed on a vortex adapter for 30min with continuous shaking, as described previously  and parasite load or fecal lactoferrin were assessed using Pearson’s correlation coefficient. All statistical analyses were performed, and graphs made, using GraphPad Prism (v 8.2.1 or v 9.1.0). A p value of <0.05 was considered statistically significant.P. chabaudi AS parasites, expressing GFP ( rMT-PcAS-GFP), parasitaemia peaked 9 days post infection (p.i.) at a low to moderate density   , in lineifng andcxcl10 were raised between 7 and 11 days p.i., and were significantly higher than controls in all sections of the intestine on day 7 p.i. Raised inflammatory markers were particularly evident in the proximal colon with significant elevations ofil10 andtnfa on day 7 p.i.; withlcn2,cxcl1, andmip2 at 11 days p.i. Whilst plasma IFNɣ peaks at 7 days p.i., intestinal transcripts ofifng remain elevated in the colon through to day 11 p.i., and show a significant positive correlation to the circulating protein levels in the proximal colon, but not the distal colon. Thus,ifng transcripts can remain in the intestine whilst circulating protein levels have become undetectable. These data are indicative of a generalised, low-grade enteritis which coincides with the period of peak parasitaemia and systemic inflammatory response.To determine whether rMT-PcAS-GFP infection and the associated systemic inflammatory response has any intestinal consequences, inflammatory markers were analysed in samples of duodenum, jejunum, ileum, caecum, proximal colon and distal colon by qRT-PCR . IntestiTo determine whether parasite localization in the intestine may be driving the enteritis, qPCR for PcAS ribosomal 18S (r18s) was conducted on the same tissue samples . PcAS r1P. chabaudi AS via binding to the cell-surface receptor ICAM-1 has been reported in the spleen and liver  (in order not to interfere with measurement of FITC-dextran). To determine whether any changes in permeability might be linked to intestinal inflammation, faecal inflammatory protein concentrations were also assessed.Given the evidence of a generalized enteritis in PcAS-infected mice, we considered whether there might also be changes in intestinal permeability. For these experiments, mice were infected with recently mosquito-transmittedParasitaemia of rMT-PcAS-mCh peaked at a similar density to rMT-PcAS-GFP and between days 8–11 p.i.  . Mice inP. chabaudi AS) that more closely reflects mild to moderate human malaria infections, with rapidly resolving parasitaemia peaking below 2% and mild to moderate anaemia that resolves upon parasite clearance, we have confirmed previous reports of malaria-associated intestinal inflammation  have reported moderate intestinal inflammation, with infiltration of the intestinal mucosa by monocytes, mast cells, and T cells , suggests that the enteritis may be driven by systemic inflammation rather than localization of parasitised RBCs in the intestine.Previous studies using murine malaria models that lead to high parasitaemia, severe anaemia and significant weight loss  into the plasma. Moreover, this increased permeability was highly correlated with both parasitaemia and intestinal inflammation , suggesting a causal pathway in which parasitaemia drives systemic inflammation, systemic inflammation drives enteric inflammation and enteric inflammation drives increased intestinal permeability.P. falciparum  , as well as their location within the tissue and their contribution to intestinal permeability. In summary, we have established a clinically relevant murine model of malaria-associated enteritis characterized by systemic and local inflammation and increased intestinal permeability. This model can be exploited to better understand the pathophysiology of enteric disease during malaria infections and to understand the mechanisms by which current or recent malaria infections substantially increase the risk of invasive enteric bacterial infections. I thank the authors for having comprehensively addressed my comments and suggestions. The revised manuscript represents a solid addition to this field and I am happy to approve version 2.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Mucosal ImmunologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This reviewer does not have any additional comments to the manuscript.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunology, parasitologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. P. chabaudi AS, they show the presence of low-grade intestinal inflammation and increased intestinal permeability that is concomitant with peak parasitemia. While parasite sequestration in the intestine is not observed, infected RBCs were seen circulating in the intestinal vasculature.In this study the authors report that gastrointestinal symptoms noted during malaria are indeed associated with the malarial disease and are not just coincidental. Using blood stage recently mosquito-transmitted  As noted by the authors similar observations have been made in other mouse models of malaria but these observations, they claim, seem to better represent what is normally observed during human malaria in endemic regions.il10 along with the inflammatory markers. As the authors may be very well aware, IL10 is considered anti-inflammatory cytokine and hence they need to make that clear in the text. It is also recommended that they give a brief description as to the implications of both pro and anti-inflammatory cytokines upregulated simultaneously.While assessing transcripts levels of various cytokines in the intestine, the authors have grouped Minor points:The study is merely observational without any mechanistic experimentation. Despite this, the authors have attempted to draw parallels between intestinal complications arising from human malarial disease and those that they observed in this study , thus validating why their study more representative and useful for experimentation to learn that particular aspect during malaria. Major points:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Immunology, parasitologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. P. chabaudi AS, they show the presence of low-grade intestinal inflammation and increased intestinal permeability that is concomitant with peak parasitemia. While parasite sequestration in the intestine is not observed, infected RBCs were seen circulating in the intestinal vasculature.In this study the authors report that gastrointestinal symptoms noted during malaria are indeed associated with the malarial disease and are not just coincidental. Using blood stage recently mosquito-transmitted As noted by the authors similar observations have been made in other mouse models of malaria but these observations, they claim, seem to better represent what is normally observed during human malaria in endemic regions. Major points: While assessing transcripts levels of various cytokines in the intestine, the authors have grouped il10 along with the inflammatory markers. As the authors may be very well aware, IL10 is considered anti-inflammatory cytokine and hence they need to make that clear in the text. It is also recommended that they give a brief description as to the implications of both pro and anti-inflammatory cytokines upregulated simultaneously.---- RESPONSE:P. yoelii malaria model, Th1 cells are a major source of both IL-10 and IFNγ+ . This work was then extended by Rosario et al. (2012) (DOI: 10.4049/jimmunol.1102755) during virulentP. chabaudi infection using IL-10 reporter mice. Therefore, we have now included additional clarification in the results section. Thank you for the opportunity for us to provide additional context to this phenomenon for the reader. Thank you for your comments and for this observation. The simultaneous production of both pro- and anti-inflammatory cytokines is well noted, and its purpose is to protect the host from immune-mediated pathology. In fact, previous work from co-author Eleanor Riley and others have shown that, in a highly virulentUpdated text: Results (paragraph 1): “These kinetics are typical of acute malaria parasite infections, denoting a switch from a pro- to anti-inflammatory systemic response to protect the host from immunopathology, driven in part by the co-production of IFNɣ+ and IL-10+ in activated T cells ).”---- Minor points: The study is merely observational without any mechanistic experimentation. Despite this, the authors have attempted to draw parallels between intestinal complications arising from human malarial disease and those that they observed in this study , thus validating why their study more representative and useful for experimentation to learn that particular aspect during malaria.---- RESPONSE: Thank you for your point and we agree this is entirely observational work. We hope to utilize this mouse model, along with extending to human malaria, to address both the clinical relevance and underlying mechanism(s). We have clearly defined this work as observational and further commented on future mechanistic work in the discussion.Updated text: Discussion (paragraph 6): “This study reveals novel observational changes in the intestine during a mild, avirulent murine malaria parasite infection. Future work is needed to unpick the mechanism by which intestinal inflammation is induced, and how this relates to intestinal permeability. This would include phenotypical analysis of the cellular source of the inflammatory mediators observed , as well as their location within the tissue and their contribution to intestinal permeability.”---- Plasmodium chabaudi AS (PcAS) and assessed intestinal pathology.This study characterizes intestinal pathology in a well characterized murine model of malaria infection. This is an important topic because human malaria patients often exhibit gastrointestinal symptoms, especially diarrhoea, and they are also more prone to develop invasive disease associated with non-typhoidal Salmonella infection. Determining the contribution of malaria infection versus coincidental enteric infections to intestinal pathology is difficult, so mouse models of malaria are useful in this context. There have been reports of intestinal pathology in mouse models of severe malaria, but the extent of intestinal alterations in milder rodent malaria infections (that mimic the majority of infections in endemic areas) had not been investigated. Hence, this study employed infection with ‘recently mosquito-transmitted’ Figure 1 presents data showing that the PcAS infection elicited a consistent pattern of low-to-moderate parasitaemia that peaked around day 9 post-infection (pi). Further analyses of one experiment indicated that there was a transient systemic inflammatory cytokine response on day 7 pi. It would be helpful to know if this pattern of systemic cytokine release was reproducible across multiple experiments.ifng andcxcl10 along the entire intestinal tract and that these peaked again at day 7 pi. Figure 2C extended the analyses to additional markers for the proximal colon samples. Again, it would be helpful to know that these changes were reproducible across multiple experiments. In addition, whether the changes in gene expression corresponded to increased mediator secretion could have been tested using intestinal explants followed by Luminex and/or ELISA assays of the supernatants. Figure 2A and B demonstrate that PcAS infection also led to increased expression of Figure 4 presents histological analyses of changes in small intestinal morphology in PcAS-infected mice. They observed a modest, transient reduction in villus/crypt ratio on day 7 pi and infer that this reflects ‘mild villous atrophy and or crypt hyperplasia’. It should be possible to discern these by separately plotting the villous and crypt lengths. In addition, it would be of interest to know whether the composition and/or activation status of leukocyte populations in the lamina propria was affected by PcAs infection, although this is perhaps a question for future studies. Figure 5 presents data showing that PcAS infection also led to increased intestinal permeability, as evidenced by increased levels of plasma FITC-dextran, as well as elevated lactoferrin in the colon contents. Figure 6 extends these analyses by showing that these increases correlated with the levels of parasitaemia, suggesting that they are a consequence of the systemic infection. In this respect, it would be of interest to determine whether plasma IFNg and TNF levels also correlated positively with the measures of intestinal permeability. Overall, the data are clear, well controlled and very nicely presented. The conclusions made by the investigators are balanced and reasonable and the discussion places them into the wider context and identifies areas for further study. The key strengths of the study are that it represents a good model of milder malaria infection and presents clear data and detailed kinetic analyses. The model offers scope for further development to better understand the consequences of altered intestinal permeability on the ongoing malaria infection, as well as predisposition to enteric pathogens. The limitations are that the data are largely descriptive and offer limited mechanistic insight. For example, which cytokines are driving the increased intestinal permeability and what are the key cellular sources of these inflammatory mediators? Are they all systemic or are some local to the intestine? The model provides a solid basis for interrogating these and other questions.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Mucosal ImmunologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This study characterizes intestinal pathology in a well characterized murine model of malaria infection. This is an important topic because human malaria patients often exhibit gastrointestinal symptoms, especially diarrhoea, and they are also more prone to develop invasive disease associated with non-typhoidal Salmonella infection. Determining the contribution of malaria infection versus coincidental enteric infections to intestinal pathology is difficult, so mouse models of malaria are useful in this context. There have been reports of intestinal pathology in mouse models of severe malaria, but the extent of intestinal alterations in milder rodent malaria infections (that mimic the majority of infections in endemic areas) had not been investigated. Hence, this study employed infection with ‘recently mosquito-transmitted’ Plasmodium chabaudi AS (PcAS) and assessed intestinal pathology. Figure 1 presents data showing that the PcAS infection elicited a consistent pattern of low-to-moderate parasitaemia that peaked around day 9 post-infection (pi). Further analyses of one experiment indicated that there was a transient systemic inflammatory cytokine response on day 7 pi. It would be helpful to know if this pattern of systemic cytokine release was reproducible across multiple experiments.----RESPONSE:including Fig 2A-C, Fig 3A, and Fig 4).  As with all WOR publications, raw data is open and accessible for full scrutiny, and version 1, with the representative data, remains available online. Thank you. This is a very valid point. Previously, we showed data from a representative experiment for clarity in figures 1-4, as the data showed similar conclusions across independent experiments. In response to your comment, and for uniformity, all graphs in this updated manuscript version now show pooled data from at least three independent experiments  As outlined above, pooled data from two independent experiments is now shown.Ifnγ, in both the proximal and distal colon, against circulating plasma levels in new Figure 2B. At 7 d.p.i, when circulating IFNγ is highest, there is a strong positive correlation. As Th1 cells are a major source of IFNγ in PcAS infection, this isn’t unexpected. However, at 11 d.p.i, when circulating IFNγ has significantly diminished,Ifnγ transcripts remain in the colon. This data suggests that IFNγ may come from both local and systemic sources, depending on the infection dynamics and we have discussed this conclusion further in paragraph 1 of the results. Indeed, dissecting out the cellular sources, along with their location within the tissue (i.e. in the blood vessels and/or lamina propria) would be important in future. Thank you for the thoughtful comment. (Comment 2) We agree that directly testing for a correlation between mRNA and inflammatory protein expression within tissues, either by explant culture and/or FISH, would be definitive experiments. Here, we have asked whether circulating IFNγ correlates to intestinal transcripts, and have graphed the transcript level ofUpdated text for ‘Comment 2’: Results (paragraph 2): “Whilst plasma IFNɣ peaks at 7 days p.i., intestinal transcripts of ifng remain elevated in the colon through to day 11 p.i., and show a significant positive correlation to the circulating protein levels in the proximal colon, but not the distal colon. Thus, ifng transcripts can remain in the intestine whilst circulating protein levels have become undetectable. These data are indicative of a generalised, low-grade enteritis which coincides with the period of peak parasitaemia and systemic inflammatory response.”---- Figure 4 presents histological analyses of changes in small intestinal morphology in PcAS-infected mice. They observed a modest, transient reduction in villus/crypt ratio on day 7 pi and infer that this reflects ‘mild villous atrophy and or crypt hyperplasia’. It should be possible to discern these by separately plotting the villous and crypt lengths. In addition, it would be of interest to know whether the composition and/or activation status of leukocyte populations in the lamina propria was affected by PcAs infection, although this is perhaps a question for future studies.----RESPONSE: (Comment 1) We originally graphed villous height to crypt depth ratio, as this is a common histomorphic measurement used on neighbouring villi and crypts. As the reviewer rightly points out, this measurement cannot differentiate between villous atrophy and crypt hyperplasia. In the new Fig 4, we have now graphed solely villous height and crypt depth. While there is a trend for both villous atrophy and crypt hyperplasia, the data is not significant. In our analysis, differences are only observed when neighbouring villi and crypts are compared against one another. This calculation may be more sensitive, as the Vh:Cd ratio takes into account the proliferation of intestinal progenitor/stem cells within the crypt against the blunting of the neighbouring villous rather than 10/30 randomised single measurements. Pathological changes may not be uniform across the intestinal surface and thus larger sampling may be warranted in future studies. Regardless, the data here suggests that neither villous atrophy nor crypt hyperplasia alone is a dominant phenotype. In all, we would conclude that changes in intestinal pathology during PcAS are mild.Updated text for ‘Comment 1’: Results (paragraph 5): “However, neither villous height nor crypt depth alone was significantly different in the small intestine.” (Comment 2) We agree that understanding the cellular infiltrate would be important. We are actively exploring this avenue in order to fully characterise the source of faecal lactoferrin, which is presumably via an influx of intestinal neutrophils. These points have been expanded on in a new paragraph in the discussion.Updated text for ‘Comment 2’: Discussion (paragraph 6): “This study reveals novel observational changes in the intestine during a mild, avirulent murine malaria parasite infection. Future work is needed to unpick the mechanism by which intestinal inflammation is induced, and how this relates to intestinal permeability. This would include phenotypical analysis of the cellular source of the inflammatory mediators observed , as well as their location within the tissue and their contribution to intestinal permeability.”---- Figure 5 presents data showing that PcAS infection also led to increased intestinal permeability, as evidenced by increased levels of plasma FITC-dextran, as well as elevated lactoferrin in the colon contents. Figure 6 extends these analyses by showing that these increases correlated with the levels of parasitaemia, suggesting that they are a consequence of the systemic infection. In this respect, it would be of interest to determine whether plasma IFNg and TNF levels also correlated positively with the measures of intestinal permeability.----RESPONSE: We agree, and appreciate that it would be useful to show all parameters in a single figure for related animals. Therefore, we now show circulating IFNγ (measured by ELISA) in Figure 5 and, in Figure 6B, we have plotted this plasma IFNγ against permeability (FITC-Dextran). The data shows that a positive correlation is found only at 9 d.p.i., when IFNγ has diminished. Nevertheless, it is important to recognise that the link between IFNγ and intestinal permeability may not be linear and as the reviewer suggests, it may be important to measure additional inflammatory mediators, such as TNF. We have included additional comments in the discussion , highlighting that a more robust characterisation of proteins pertinent to intestinal permeability, rather than indicative of a generalised inflammatory response such as IFNγ, would be of great interest. We thank the reviewer for his comment and look forward to future research on this topic.---- Overall, the data are clear, well controlled and very nicely presented. The conclusions made by the investigators are balanced and reasonable and the discussion places them into the wider context and identifies areas for further study. The key strengths of the study are that it represents a good model of milder malaria infection and presents clear data and detailed kinetic analyses. The model offers scope for further development to better understand the consequences of altered intestinal permeability on the ongoing malaria infection, as well as predisposition to enteric pathogens. The limitations are that the data are largely descriptive and offer limited mechanistic insight. For example, which cytokines are driving the increased intestinal permeability and what are the key cellular sources of these inflammatory mediators? Are they all systemic or are some local to the intestine? The model provides a solid basis for interrogating these and other questions.----RESPONSE: Thank you for this very constructive and thoughtful review. Whilst this data does investigate malaria-related inflammation in a new model, we agree that more mechanistic work is now needed to identify the drivers of increased permeability and what, if any, contribution this has on promoting dissemination of secondary enteropathogens.----


Plasmodium falciparum histidine-rich protein 2 (HRP2) antigen are widely used for detection of active infection with this parasite and are the only practical malaria diagnostic test in some endemic settings. External validation of RDT results from field surveys can confirm appropriate RDT performance.Malaria rapid diagnostic tests (RDTs) based on the detection of the A community-based cross-sectional survey was conducted between July and November 2017 enrolling participants of all ages in households from 15 villages in four border regions of Tanzania: Geita, Kigoma, Mtwara and Ruvuma. All participants had an RDT performed in the field and provided a blood sample for later laboratory multiplex antigen detection of HRP2. In assessing the continuous HRP2 levels in participant blood versus RDT result, dose–response logistic regression provided quantitative estimates for HRP2 limit of detection (LOD).From the 15 study villages, 6941 persons were enrolled that had a RDT at time of enrollment and provided a DBS for later laboratory antigen detection. RDT positive prevalence for the HRP2 band by village ranged from 20.0 to 43.6%, but the magnitude of this prevalence did not have an effect on the estimated LOD of RDTs utilized in different villages. Overall, HRP2 single-target tests had a lower LOD at the 95% probability of positive RDT  when compared to pLDH/HRP2 dual target tests , though this difference was not significant. With the exception of one village, all other 14 villages (93.3%) showed RDT LOD estimates at 90% probability of positive RDT between 0.5 and 12.0 ng/mL.Both HRP2-only and pLDH/HRP2 combo RDTs utilized in a 2017 Tanzania cross-sectional survey of border regions generally performed well, and reliably detected HRP2 antigen in the low ng/mL range. Though single target tests had lower levels of HRP2 detection, both tests were within similar ranges among the 15 villages. Comparison of quantitative HRP2 detection limits among study sites can help interpret RDT testing results when generating population prevalence estimates for malaria infection.The online version contains supplementary material available at 10.1186/s12936-022-04383-4. Plasmodium falciparum accounts for 96% of malaria infections in Tanzania [Malaria continues to be a significant public health threat worldwide, with sub-Saharan Africa remaining disproportionately affected by the disease. The World Health Organization (WHO) reported an estimated 241 million cases of malaria and 627,000 deaths globally in 2020, of which > 90% of deaths occurred in sub-Saharan Africa [Tanzania –5. Due tTanzania , the NatPlasmodium species. However, microscopy-based diagnosis in malaria-endemic countries can many times present challenges given the need for trained microscopists, quality microscopes, reliable electricity and quality reagents for accurate diagnosis. Malaria rapid diagnostic tests detecting parasite antigen in the blood of infected persons have overcome many of these limitations and are widely used in Tanzania for diagnosis of P. falciparum infection [Plasmodium blood stage antigens: aldolase, lactate dehydrogenase (LDH), and histidine-rich protein 2 (HRP2) [P. falciparum is the predominant species responsible for symptomatic malaria in sub-Saharan Africa, many countries use RDTs detecting only the HRP2 antigen as this provides a sensitive and species-specific marker for infection with this parasite.[P. falciparum parasites can sequester in host tissue and vasculature, detection of parasite antigen (such as HRP2) may provide a more accurate representation of true parasite density during infection and has been linked to pyrogenic threshold and disease severity [In adherence to the WHO 2010 recommendation that all suspected cases of malaria have parasitological confirmation before treatment , Tanzaninfection . RDTs arnfection . Antigen2 (HRP2) , 9. Howeparasite., 10. As severity , 12. TanP. falciparum parasites which have been diluted to 200 p/µL blood [P. falciparum strains and individual infections lead to a weak correlation between parasite and antigen levels [In order to assess ability of RDTs to detect lower-density infections, the WHO recommends that malaria RDTs have a panel detection score of ≥ 75% against a panel of µL blood . Howevern levels –15. The n levels . For a pn levels .P. falciparum transmission intensities.In this study, the analytical sensitivity of RDTs was assessed across 15 villages in Tanzania from a 2017 survey of border regions with persistently-high malaria burden in the country [As described previously , study aAs described previously , samplesIn each of the sampled villages, a random sample of ≥ 120 households were selected, and all members (regardless of age) were asked to enroll in the survey . Blood sEscherichia coli extract. Elution from filter paper diluted the samples to a 1:20 whole blood.A single 6 mm punch of each sample was taken and eluted in blocking buffer containing: phosphate buffered saline , 0.5% polyvinyl alcohol , 0.8% polyvinylpyrrolidine (Sigma), 0.1% casein , 0.5% BSA (Sigma), 0.3% Tween-20, 0.05% sodium azide, and 3 µg/mL Plasmodium antigens, DBS samples were screened by a bead-based multiplex antigen assay for the simultaneous detection of P. falciparum HRP2 (HRP2), pan-Plasmodium aldolase , and pan-Plasmodium lactate dehydrogenase (pLDH) based on previously described protocol [For detection of protocol , 17. To protocol .Local regression (LOESS) and logistic regression dose–response curves comparing RDT result to DBS HRP2 antigen concentration were created in R  as described previously . BrieflyIn total, 6941 persons had valid RDT results and provided a DBS for later antigen detection by laboratory assay. Of these persons, 2512 (36.2%) were tested with a HRP2-only RDT, and 4429 (63.8%) were tested with a pLDH/HRP2 combo RDT  from the study would have provided a positive RDT result. This was previously done for a study of febrile patients in Tanzania and showed that the study’s conventional HRP2-based RDT had a reliable sensitivity of approximately 3.7 ng/mL [Plasmodium targets can allow for detection of other Plasmodium species , but this augmented detection capacity inherently leads to a slight reduction in the efficiency of HRP2 binding and detection. Though this current study found HRP2 detection significantly reduced in these combo tests when compared to HRP2-only tests, with such overall low LOD estimates and the low magnitude of percent difference, there is no indication from this data indicating that combo tests are deficient for HRP2 detection.In order to assess concordance of malaria diagnosis among different tests, many previous studies have compared RDT results to microscopy or nucleic acid-based tests –23. Howey assays , 24–26. y assays . Logisti.7 ng/mL . From th.7 ng/mL , and 10 .7 ng/mL . Additio.7 ng/mL , 30. ModP. falciparum prevalence. In this study, RDT prevalence rates ranged from 20.0% to 43.6%, and these results from this community population shows evidence for the high P. falciparum transmission in these northwestern and southeastern border regions of Tanzania. These high-transmission regions have been confirmed by recent reports [P. falciparum at the time of enrollment. This finding provides further evidence that the RDTs utilized in this survey performed well in ability to detect persons with HRP2 antigenaemia. Low-density P. falciparum infections can still produce nanogram/mL levels of HRP2 in host blood [P. falciparum infection by the RDT prevalence rates. This endeavour is an important analysis for a study to investigate if there are inherent reasons why study site may have higher (or lower) RDT prevalence estimates, or if significant differences may exist in RDT performance among sites.When comparing LOD estimates by village of enrollment, two prominent findings were observed: more sensitive HRP2 detection in villages only utilizing HRP2-only RDTs, and LOD estimates did not differ by  reports , 31, 32, reports . No corrst blood , and thest blood . For theThis study was also subject to multiple limitations. DBS were prepared in the middle to late 2017, and not processed at CDC until the first half of 2019. This age of specimens combined with storage at ambient temperature would have led to some degree of HRP2 protein degradation, so even though very similar to previous estimates, the LOD estimates presented here are almost certainly an underestimate of the RDTs’ ability to detect the antigen in fresh blood at POC. Additionally, DBS sample storage factors such as humidity and degree of air conditioning could have differed among health facility storage sites and led to varying levels of protein degradation, which would ultimately affect laboratory assay performance and LOD estimates by village. Only two types of RDTs were used in this study, and both from the same manufacturer, so detection of HRP2 by other manufacturers’ products was not able to be assessed here. Unfortunately, lot numbers for the RDTs used in this study were not available, so potential lot-to-lot variation could not be assessed as a potential factor leading to variations in RDT performance through modelled LOD estimates.pfhrp2 and pfhrp3 gene deletions are largely absent in Tanzania [P. falciparum infections in the country. As Tanzania works towards malaria control and elimination, continuous evaluation of the performance of this tool  will be critical to ensure the tests are truly capturing infections with this parasite. Moreover, as RDT estimates generated from surveys of non-healthcare settings are increasingly being used for evaluating P. falciparum prevalence in many countries, similar data collection and analyses as performed here can provide quantitative estimates for RDT performance among mostly low-density infections.The ability of HRP2-based RDTs to detect low levels of target antigen coupled with the previous finding that Tanzania  provide Additional file 1: Dose-response logistic modeling for limits of HRP2 antigen detection by RDT utilized for different study populations. Logistic (red curves) and LOESS (blue curves) regression of probability of RDT positivity by antigen concentration in study participants by village. Shading indicates 95% confidence limits for regression curves. Regression outputs shown in Table 2.Additional file 2: Comparison of village specific RDT prevalence to HRP2 antigen concentration at different probabilities of RDT positivity. Plots shown for estimates for HRP2 concentrations by 50, 75, 90, and 95% probability of positive RDT result with dashed line the linear line of best fit and model estimates. The 95% probability plot does not include Kitunguli village as that point estimate was unable to be calculated.Additional file 3: Antigen concentration versus RDT result in comparison with estimated pyrogenic threshold. Histograms of log-transformed HRP2 concentration shown for each of the village enrolment sites with green bars indicating specimens from RDT-positive persons and blue bars from RDT-negative persons. Grey shading on each plot indicates HRP2 concentrations previously estimated as the pyrogenic threshold for this antigen at >3000 pg/mL (3 ng/mL) (11), and hashed vertical lines for each plot indicate 50% reliability estimates for HRP2 limit of detection (LOD) for each study site.


Malaria is a mosquito-borne disease that is one of the most serious public health issues globally and a leading cause of mortality in many developing countries worldwide. Knowing the prevalence of both symptomatic and asymptomatic malaria on a subnational scale allows for the estimation of the burden of parasitaemia present in the transmission system, enabling targeting and tailoring of resources towards greater impact and better use of available capacity. This study aimed to determine the PCR-based point prevalence of malaria infection, by parasite species, among three high-risk populations in Mondulkiri province, Cambodia: forest rangers, forest dwellers, and forest goers.Plasmodium spp using PCR.A cross-sectional survey was performed during the transmission season in November and December 2021. Blood samples collected on filter paper from participants (n = 1301) from all target groups were screened for Plasmodium species. Malaria prevalence in the forest ranger group was 8.1%, was 6.8% in forest goers, and 6.4% in forest dwellers; all infections were asymptomatic. Plasmodium vivax was detected in all participant groups, while the few Plasmodium falciparum infections were found in goers and dwellers. 81% of all infections were due to P. vivax, 9% were due to P. falciparum, 3% due to Plasmodium cynomolgi, and the rest (7%) remained undefined. Gender was associated with malaria infection prevalence, with male participants having higher odds of malaria infection than female participants . Passively collected malaria incidence data from the Cambodian government were also investigated. Health facility-reported malaria cases, based on rapid diagnostic tests, for the period Jan-Dec 2021 were 521 Plasmodium vivax , 34 P. falciparum (0.06%) and four P. falciparum + mixed (0.01%)—a total of 559 cases (0.95%) for all of Mondulkiri.Malaria prevalence among all study participants was 6.7% for any This reservoir of asymptomatic parasitaemia may be perpetuating low levels of transmission, and thus, new strategies are required to realize the goal of eliminating malaria in Cambodia by 2025.The online version contains supplementary material available at 10.1186/s12936-023-04630-2. Anopheles mosquito bites [The Greater Mekong Subregion (GMS) in Southeast Asia has experienced dramatic declines in malaria transmission, with 65,297 cases reported in 2021, representing a 16% decrease from 2020 . Accordito bites .Plasmodium carriage is below the threshold of detection of the diagnostic method used [Plasmodium detection by PCR is much more sensitive compared to RDT or microscopy, particularly in low density or mixed infection cases, and it is valuable for the accurate collection of malaria epidemiological data [Knowing the point prevalence of both symptomatic and asymptomatic infections on a subnational scale allows for the estimation of the parasite reservoir in the transmission system , enablinhod used , 13. Placal data , 15. As 1) Forest rangers and forest patrol teams, who spend at least 21 days/month in forested areas,2) Forest dwellers, who live and work inside forests or within 1 km of forest edge, and.3) Forest goers, who live more than 1 km from forest edge, but typically visit forested areas at least 1 day per week.This study was conducted in Mondulkiri Province, Cambodia. Mondulkiri was the location for this study given its higher burden status relative to other provinces in Cambodia, and because Mondulkiri was the site for other related research activities under a broader vector control research program in collaboration with CNM. Villages were chosen based on ongoing local malaria passive case reporting (ongoing vector-based transmission) and consultations with local health authorities, with the 21 villages with the highest reported number of cases included in the study, with the addition of forest ranger stations which are associated with rangers who work in high-risk areas . Where active malaria transmission remains in Cambodia, the foci are largely related to forested areas, and forest exposure has been documented elsewhere as a risk factor for malaria in the country , 16. TheThese locations and populations were specifically chosen for this study as a preliminary investigation into current levels of malaria in advance of a then-planned randomized controlled trial. The trial was intended to be run in the same locations and with the same at-risk populations as were selected in the present study. While the villages selected were chosen based on the number of malaria cases reported through the government tracking system, the province, operating district, and the particular at-risk populations were selected to conform with the planned trial.Three sample size estimates were determined for each of the three target populations  based on precision estimates of malaria infection, with a coefficient of variation of 0.5, a significance of 5%, and 80% power. All sample size estimates were powered to provide a baseline estimate of the prevalence of malaria by PCR for each population using the Hayes and Bennett (1999) approach. Based on sample size calculations for the three target groups combined, a total samples size of 1450 was determined.This study used a cross-sectional design targeting clusters with high rates of malaria, as reported to the district health offices and the national Malaria Information Service. The primary clusters for rangers were ranger stations and villages for forest goers and forest dwellers. Villages and ranger stations were randomly selected for inclusion in the study. Within selected ranger stations, all rangers who were willing to participate in the study were enrolled. In selected villages, household were selected using a simple random sample, and all eligible individuals living in that household were invited to participate. Inclusion criteria for study participants comprised of being in one of the target groups, weight of > 20 kg , ability to provide informed consent, and speaking Khmer or Bunong . Study sPlasmodium spp infection by the COX-III direct PCR method [Blood samples were collected (~ 50 ul blood via finger prick), for a total of four dried blood spots (DBSs) per individual on filter paper (Whatman Filter Paper #3), from each participant at a single time point. All blood samples were screened for R method  using AcThe outcome measure was the malaria infection prevalence (by parasite species) per target population and was determined using the number of positive individuals divided by the total sample size. Univariable logistic regression analyses were used to identify potential risk factors for malaria infection against data collected via the questionnaire. All factors were included in the univariable analysis and variables that were related to malaria infection prevalence with a p-value of < 0.25 in the univariable analyses were subsequently combined in a multivariable model. A p-value of < 0.05 was considered statistically significant, and the corresponding odds ratios with 95% confidence interval were also estimated. STATA version 14.0 was used for data analysis.Passive malaria incidence data is generated by village malaria workers (VMWs). VMWs are trained to diagnose malaria using RDTs and treat uncomplicated malaria cases . SymptomThis study was approved by the National Ethics Committee for Health Research of the Ministry of Health of Cambodia (ref. no. 241 NECHR) and the University of California, San Francisco (ref. no 21-34947). The purpose of the study was explained to all study participants and written informed consent was obtained. Participants understood that they were free to remove themselves from the study at any time without repercussion. All the data and samples were de-identified and coded towards analysis following IRB guidelines.A total of 1301 participants were recruited into the study, of which 770 (59.2%) were from the forest goer group, 469 (36.1%) from the forest dweller group, and 62 (4.7%) from the forest ranger group. Individuals were recruited from 21 different villages  and dweller groups (0.4%). Infections with a simian malaria species, P. cynomolgi, were also found in both the forest goer group (n = 2) and the dweller group (n = 1). Six Plasmodium species were not determined, with four from forest goers and two from the dweller group.Overall, 87/1301 (6.7%) of participants were positive for any .In both the univariable and multivariable analyses, only gender was found to be associated with malaria infection prevalence, where female participants had lower odds of being malaria positive compared to male participants   were P. vivax, and four (0.01%) were mixed infections , which collects malaria case data from all villages in Mondulkiri. These health facilities reported the highest malaria caseloads in Mondulkiri in 2019–2020, which was the reason they were selected for this study. As part of the reporting, a total of 58,584 symptomatic (temperature > 98°F) patients from Mondulkiri were screened for malaria in 2021, of which 559 (0.95%) were RDT positive. Thirty-four (0.06%) were Anopheles bites, to explore the reservoir of asymptomatic parasitaemia as the country accelerates towards malaria elimination. Data on asymptomatic parasitaemia among specific populations can support programmatic decision-making on appropriate malaria control measures to clear those reservoirs, including more targeted prevention interventions, such as bite prevention tools and chemoprevention approaches.In this study, a malaria prevalence survey was conducted among high-risk populations, including forest-goers, forest dwellers, and forest rangers living in, traveling to, and/or working in and around the forests who are exposed to P. vivax, 9.2% were P. falciparum, 3.5% were P. cynomolgi, and 6.9% were of an undefined Plasmodium species due to inadequate genetic material or sample degradation—for sequencing. By comparison, a previous study in Mondulkiri conducted from 2017–2018 among the high risk population living outside the forest, at the forest fringe, and inside the forest, found an 8.3% point prevalence (349 infections/4200 population) [All malaria PCR-positive individuals in the survey were asymptomatic (temperature < 98°F). The overall PCR-based prevalence of malaria in this sample was 6.7%—of which, 80.5% of the cases were ulation) . The sigulation) . Previouulation) , 24–27.P. vivax infections predominate over P. falciparum in Cambodia in high-incidence provinces [P. vivax infections, which may be regularly observed as relapses (though this was not investigated in this study), can lead to higher immunity levels and, thus, overall lower parasite density [P. vivax reservoirs and relapses, radical cure with primaquine (currently the only WHO-approved drug that clears P. vivax hypnozoites) is necessary. Administration of primaquine has significant limitations, including serious haemolysis risks in individuals with glucose‐6‐phosphate‐dehydrogenase enzyme (G6PD) deficiency [This study supports prior molecular data which demonstrated that rovinces , 28–31.  density , 33. To ficiency .P. vivax reservoirs and accelerate progress toward malaria elimination.There are access and delivery challenges associated with both G6PD testing and primaquine administration, including availability, non-compliance with treatment regiments, and/or side effects, among others. Despite these challenges, countries across the GMS, including Cambodia, are actively working to scale up implementation of G6PD testing and primaquine use to address Individuals with asymptomatic infections generally remain undiagnosed and untreated as they do not present to health facilities, and consequently may remain infective to mosquitoes over long periods . IndividP. cynomolgi human infections, similar to another report in Cambodia from 2018, which was the first to report asymptomatic infections with this malaria parasite in the country [P. cynomolgi is not new, but it is highly uncommon in Cambodia [P. cynomolgi and it was concluded that Anopheles balabacensis (now Anopheles dirus) probably was the vector of both human and monkey malaria and that the risk of cross infection was considerable if monkey malarias infective to humans exist in the Pailin area [PCR analyses detected three simian  country . Human iCambodia –44. Thislin area .P. cynomolgi, as observed by microscopy, are almost identical to that of P. vivax, and P. cynomolgi also presents hypnozoites which can initiate relapses [P. cynomolgi is included in the WHO definition of elimination as a human parasite. As human malaria becomes more controlled and prevalence falls, primate malaria parasites may comprise an increasing proportion of cases in areas where monkeys live close to people [The morphological features of relapses . The pero people .A)The main focus of health workers during this time was on COVID-19 screening, and not on malaria;B)With lockdowns imposed, people who would normally be travelling to and spending significant amounts of time in the forest, where they were at higher risk of malaria infection, were no longer doing so;C)VMWs were less active during this time, owing to the restrictions on movement and of supplies;D)Due to heightened awareness of disease transmission, people were potentially less likely to visit others in the community, including VMWs. It is unknown how the COVID-19 pandemic may have influenced the main finding of this paper, that active and targeted surveillance identifies more malaria cases than the existing passive surveillance system. However, if it is indeed true that there were fewer malaria cases than if the COVID-19 pandemic had not occurred, then the findings of this study could potentially be underestimated.It is of interest to note that this study was carried out during the COVID-19 pandemic, during which time many of the villages in Mondulkiri were on self-imposed lock down, before, during, and/or after the research was complete. The study team worked closely with local health officials and village leaders and were granted access to villages that may otherwise have been off limits to outsiders. While a thorough analysis and report has not been completed by the Cambodian government, there is a possibility that the COVID-19 pandemic may have impacted how malaria cases were detected, and ultimately the number of reported cases, both globally and in Cambodia . The majP. vivax predominating, and gaps in passive surveillance. Targeting high-risk populations with more sensitive diagnostics and prevention and treatment interventions could substantially reduce the infectious reservoir [These findings highlight challenges that are faced by many malaria elimination programmes, including asymptomatic parasite reservoirs, eservoir , 48.P. vivax radical cure, with tailored bite prevention or vector control tools will help clear these reservoirs and support near-term elimination goals in Cambodia and across the GMS.Compared with standard, passive malaria case detection using RDTs on symptomatic individuals, active and targeted malaria detection using more sensitive PCR testing confirms that there is more malaria circulating in specific high-risk groups than previously assumed. This asymptomatic and untreated reservoir may be perpetuating low levels of malaria transmission and could slow progress towards elimination. New strategies targeted to high-risk populations that combine drug-based approaches, including chemoprevention and Additional file 1: Appendix 1. Cross-sectional Survey.


In 2021, India contributed for ~79% of malaria cases and ~ 83% of deaths in the South East Asia region. Here, we systematically and critically analyzed data published on malaria in pregnancy (MiP) in India.ClinicalTrials.gov were used to identify articles dealing with MiP in India. The archives of local scientific associations/journals and website of national programs were also consulted.Epidemiological, clinical, parasitological, preventive and therapeutic aspects of MiP and its consequences on both mother and child were reviewed and critically analyzed. Knowledge gaps and solution ways are also presented and discussed. Several electronic databases including Google scholar, Google, PubMed, Scopus, Wiley Online library, the Malaria in Pregnancy Consortium library, the World Malaria Report, The WHO regional websites, and Plasmodium falciparum (Pf) and P. vivax (Pv), and on rare occasions to P. ovale spp. and P. malariae too. The overall prevalence of MiP is ~0.1–57.7% for peripheral malaria and ~ 0–29.3% for placental malaria. Peripheral Pf infection at antenatal care (ANC) visits decreased from ~13% in 1991 to ~7% in 1995–1996 in Madhya Pradesh, while placental Pf infection at delivery unit slightly decreased from ~1.5% in 2006–2007 to ~1% in 2012–2015 in Jharkhand. In contrast, the prevalence of peripheral Pv infection at ANC increased from ~1% in 2006–2007 to ~5% in 2015 in Jharkhand, and from ~0.5% in 1984–1985 to ~1.5% in 2007–2008 in Chhattisgarh. Clinical presentation of MiP is diverse ranging from asymptomatic carriage of parasites to severe malaria, and associated with comorbidities and concurrent infections such as malnutrition, COVID-19, dengue, and cardiovascular disorders. Severe anemia, cerebral malaria, severe thrombocytopenia, and hypoglycemia are commonly seen in severe MiP, and are strongly associated with tragic consequences such as abortion and stillbirth. Congenital malaria is seen at prevalence of ~0–12.9%. Infected babies are generally small-for-gestational age, premature with low birthweight, and suffer mainly from anemia, thrombocytopenia, leucopenia and clinical jaundice. Main challenges and knowledge gaps to MiP control included diagnosis, relapsing malaria, mixed Plasmodium infection treatment, self-medication, low density infections and utility of artemisinin-based combination therapies.Malaria in pregnancy is mainly due to All taken together, the findings could be immensely helpful to control MiP in malaria endemic areas. MalariaIndia accounted for ~79% of malaria cases and ~ 83% of deaths seen in SEA in 2021  and websites of national programs were also consulted. We used the search terms “malaria,” “pregnancy,” “pregnant woman,” “burden,” “prevalence,” “epidemiology,” “outcome,” “placental infection,” “congenital malaria,” “neonatal malaria,” “diagnostic,” “prevention,” “control,” “management,” “India,” and Indian regions . Boolean operators including “AND” and “OR” were used in combination with the above mentioned search terms to identify relevant papers through databases such as PubMed. The search strategy was tailored to each of the search databases using search terms and Boolean operators . We also included all publications on neonatal and congenital malaria (NCM). To do so, the same search strategy used for MiP papers was used with some differences. For example search terms for NCM papers were “malaria,” “placental infection,” “congenital malaria,” “neonatal malaria,” “India,” and Indian regions .The strategy used to identify relevant studies was inspired from MiP reviews reported previously , 9. Brie2.2.Titles and abstracts of studies retrieved from databases were independently reviewed by the authors in order to identify those relevant to the study. The full texts were retrieved and scrutinized to extract data of interest. Principal investigators were kindly contacted to request full length paper and/or more details on studies. We also contacted editors-in-chief of national journals to request full length papers in case of a negative reply or no reply at all from principal investigators. Additionally, we reviewed relevant articles cited in references of identified literature and included them as primary sources.2.3.Only papers published in English and Hindi were included. Publications were considered of interest if they addressed any aspect of MiP and NCM in India including prevalence, clinical presentation, determinants, maternal and fetal/neonatal outcomes, diagnostic, prevention, and treatment. The list of studies is presented in 2.4.Data of interest were independently extracted from eligible publications, and these consisted of (i) characteristics of studies ; (ii) demographical, obstetrical, and gynecological data ; (iii) clinical characteristics of MiP and NCM ; (iv) parasitological information , (v) factors associated with malaria infection in pregnant women and babies; (vi) MiP and NCM outcomes, (vii) malaria preventive methods used, and (viii) efficacy of treatments to control MiP. Setting urbanization was categorized as urban, semi-urban, rural, and not specified. Timing of screening consisted of women screened for malaria parasites in community or at health facility for delivery (DU) and antenatal care visit (ANC). Type of malaria was defined either as asymptomatic malaria, uncomplicated malaria (UM) or severe malaria (SM). In the early 90s, the WHO defined a set of criteria used for diagnosing severe malaria (SM) in children and adults  6, 10, . The lat2.5.Data were keyed into an Excel spreadsheet  by reviewer authors and then coded and verified for consistency, and removed any duplicates. Any discrepancies between the authors were resolved through discussion and consensus. Data were analyzed using StatView 5.0 for Windows  and GraphPad v8.01 for Windows , and summarized as percentages and mean in tables and charts where appropriate. Overall aggregation of data using sophisticated approaches such as meta-analysis was not possible due to the heterogeneity of studies related to study design, diagnostic methods, timing of screening, blood origin, analysis conducted, and effect measures presented. Thus, only summaries of study findings stratified by variables such as diagnostic methods, timing of screening and blood origin are presented in this review. In addition, findings from studies with minimum sample size of 30 were extracted to generate charts . We thin3.3.1.https://nvbdcp.gov.in; Malaria burden profoundly decreased in last two decades in India, and this is due to diverse strategies implemented and/or scaled up over the country  . The recPf and Pv species are major species in the country, with ratio close to one at the country level but varied between the different regions , P. malariae (Pm), and P. knowlesi (Pk) are also found in India but much fewer in extent than that of Pf and Pv ..Pf and Pf and Pv . The Pm t states , 23. Tya Islands . Recentlnd Delhi .3.2.viz. Madhya Pradesh, Rajasthan, and Chhattisgarh , timing of screening , and blood origin . Again, results are more documented from forested, tribal, and rural areas of three states ttisgarh . Informattisgarh . In Chhattisgarh , 49. Comttisgarh , 40, andttisgarh . In Madhttisgarh , 47, thettisgarh , 54.3.3.Plasmodium species-wise MiP proportion is mainly originated from hospital based studies, in a limited number of states such as Madhya Pradesh, Jharkhand, Maharashtra, Rajasthan, and Chhattisgarh  both at ANC and DU while Pf was dominant in Bastar ..PlasmodiPf infection at ANC decreased from ~13% in 1991 to ~7% in 1995–1996, while prevalence of peripheral Pv infection at ANC levels off at ~5% between 1991 and 1997–1998. In Jharkhand, the prevalence of Pv infection at ANC increased from ~1% in 2006–2007 to ~5% in 2015. The same trend was observed for peripheral Pf infection among women attending ANCs in Chhattisgarh where the prevalence of this species increased from ~0.5% in 1984–1985 to ~1.5% in 2007–2008 (Pv infection at DU was noted in Jharkhand (~0.5% in 2006–2007 to ~4% in 2015). Regarding placental infection, the burden of Pf infection slightly decreased from ~1.5% in 2006–2007 to ~1% in 2012–2015 in women living in Jharkhand  in Ghana   . Trackinic areas , especia3.4.2.Pf and Pv UM-related symptoms reported in Indian pregnant women attending ANC and/or DU included fever, weakness/fatigue, body/joint pain, headache, loss of appetite, diarrhea, dizziness, hepato-splenomegaly, and nausea/vomiting , host and parasite factors , 65. Uncvomiting , 52, 54.Plasmodium-infected and uninfected red blood cells (RBCs) and impaired/suppressed hematopoiesis  and Uttarakhand (57.8%), respectively (Pv was the main contributor of SM cases (56.6%)  . Most of Pradesh . No seve3.4.3.1.Plasmodium-infected pregnant women  vka state , and , 74Pv-re3.4.3.4.Pf-induced ARF proportion of 20%, but this estimate was obtained on only 45 pregnant women  is multifactorial in pregnancy, and studies outline that malaria contributes toward a small fraction as reported from Gujarat state where ARF proportion is ~0–8% in pregnant women . A studynt women  80)..Pf-induc3.4.3.5.Pv-infected preeclamptic pregnant woman  compared to the above mentioned clinical manifestations  compared to those living in Rajnandgaon (low endemicity area)  .3.6.4.Plasmodium parasitemia with molecular tools which have higher sensitivity than LM and RDTs  and RDTs . Using P Pradesh , 7 47)..Plasmodi3.7.Plasmodium infections are associated with adverse maternal outcomes such as miscarriage, stillbirth, abortion, and mortality. In India, Pf and Pv have been associated with these maternal outcomes which are frequently seen in primigravidae mothers  , prematurity, intrauterine growth retardation (IUGR), respiratory distress, and deaths in the worst case  . Such Plrst case . LBW, IUPf- and Pv-infected women gave birth babies with LBW, respectively. In Madhya Pradesh, it was reported that almost all babies (95.2%), born to malaria infected women, had LBW  for radical cure of Pv infection is not recommended to pregnant women and her fetus. The treatment of uncomplicated Pf MiP is CQ for first trimester women while ACTs artemether + lumefantrine (AL) or artesunate + sulfadoxine/pyrimethamine (AS + SP) are used for second and third trimesters. Due to high level of SP resistance in Northeastern states, AL is recommended for treatment while AS + SP is used in other states. Parenteral quinine, artesunate, or artemether are given for SM  in Arunachal Pradesh and West Bengal , Bihar and Uttarakhand where malaria endemicity is still high. Also, more studies on MiP and NCM in tribal and forested settings are also greatly needed , 123.Po and Pm are also associated with MiP in India  especially Pv parasitemia are problematic given efficient transmission of Pv LDI to Anopheles vectors  deficiency  which encodes a Pf-specific protein antigen  are also involved. The epidemiology of these species in MiP is greatly varied with important role of mother’s demographic and obstetrical characteristics, geography, and ecoclimatic features of the area. Plasmodium infections in pregnant women, often associated with comorbidities and concurrent infections, may progress from asymptomatic carriage of parasites to SM, which is mostly represented by SMA, CM, and hypoglycemia, and more frequently seen in Pf infections. MiP has deleterious effects on mother and her child that can often end with death. This review provided a comprehensive overview on epidemiological situation and identified important missing links in MiP and NCM to inform population, clinicians, and researchers. There is urgent need for further studies on the different above mentioned points addressed in the present review. If adequately addressed, the future findings could be greatly helpful for efficiently controlling MiP in India through development, implementation, and scale up of control strategies and policy makers, and thus achieve malaria control and elimination objectives in the country.Malaria in pregnancy is still a serious public health concern in India. Its epidemiological burden is high in Indian pregnant women, with LPKF and VS conceptualized the paper. LPKF conducted literature review and extracted and analyzed data from papers, conceived the figures and maps, performed extraction and analysis data, and drafted the first version of the final manuscript. VS revised the manuscript for important intellectual content and supervised the work at all stages. All authors contributed to the article and approved the submitted version.


Water resource development projects such as dams and irrigation schemes have a positive impact on food security and poverty reduction but might result in increased prevalence of malaria.Two cross-sectional surveys were conducted in the dry and wet seasons in irrigated and non-irrigated clusters of Arjo sugarcane and Gambella rice development areas of Ethiopia in 2019. A total of 4464 and 2176 blood samples were collected from Arjo and Gambella. A subset of 2244 microscopy negative blood samples were analyzed by PCR.p < 0.001), but no difference was found in Arjo . Level of education was an individual risk factors associated with infection in Arjo  and in Gambella . While duration of stay in the area for < 6 months  and being a migrant worker  were risk factors in Gambella. Season , no ITN utilization  were risk factors in Arjo, and irrigation  and family size  risk factors in Gambella. Of the 1713 and 531 randomly selected smear negative samples from Arjo and Gambella and analyzed by PCR the presence of Plasmodium infection was 1.2% and 12.8%, respectively. P. falciparum, P. vivax, and P. ovale were identified by PCR in both sites.Prevalence by microscopy was 2.0% (88/4464) in Arjo and 6.1% (133/2176) in Gambella. In Gambella, prevalence was significantly higher in irrigated clusters (10.4% vs 3.6%) than in non-irrigated clusters (Strengthening malaria surveillance and control in project development areas and proper health education for at-risk groups residing or working in such development corridors is needed. Water resource development projects such as dams and irrigation schemes have a positive impact on food security and poverty reduction . HoweverAn. gambiae, the major malaria vector in Africa, prefers environments with direct sunlight for breeding, thus the dense vegetation cover by sugarcane development would make the environment undesirable  and rice (Gambella) development area of Ethiopia. The Arjo Didessa Sugarcane development irrigation is located in Jimma Arjo, Dabo Hanna and Bedele districts of East Wellega and Buno Bedele Zones of Oromia regional state. It is 540 km Southwest of Addis Ababa and is located at latitude 8.6° N, longitude 36.4°E, and; an altitude with a ranges of 1300 to 2280 m. The mean annual rainfall is 1477 millimeters, with bimodal rainfall (long rainy season June to September and short rainy season February to March) and with a relatively short dry season between December to January. The Saudi Star Agricultural Development PLC is located in Abobo district of Gambella region in West Ethiopia. It is 811km west of Addis Ababa located at latitude 7.9° N and longitude 34.5°E, and; an altitude with a ranges of 400–500m. The annual rainfall in Abobo is ranges between 900 to 1200 mm, with unimodal rainfall (long rainy season May to October) and long dry and hot season occurs between November-April .Kebeles  were randomly selected from twenty six Kebeles surrounding Arjo Didessa Sugar cane irrigation scheme in the three districts with a catchment population of 51,407. These Kebeles were Abote-Didessa (AD), Ambelta (AMB), Bekelicha-Biftu (BEB), Beyima (BEM), Bildima-Daru (BLD), Chilalo-Bildima (CBL), Chefe-Jalala (CHJ), Hunde-Gudina (HNG), Kerka (KER), Sefra-Tabia (SFT), Command 2 (CO2), Command 5 (CO5), and Command 8 (CO8). From the above 13 Kebeles, fifteen , the others were KER, CHJ, BEM, CO2, CO5, and CO8. Non-irrigated cluster were AMB, BEB, BLD, CBL, HNG, HG1and SFT , Saudi Star GRC (GRC) and Village-17 (V17). Non-irrigated clusters were in Terkudi (TER), Village-12 (V12) and Village-13 (V13)  . The ricAnopheles mosquito flight range (Demographic data and blood samples were collected simultaneously in eight irrigated and seven non-irrigated clusters of Arjo and in three irrigated and three non-irrigated clusters of Gambella rice irrigation .. Irrigaht range . The spaCapillary blood samples (300μL) were collected from all consenting participants for diagnosis by Rapid Diagnosis Tests (RDT), microscopy, and qPCR. For qPCR, dried blood spot (DBS) were prepared. The dried blood smear slides and the four dots of DBS samples were packed in slide box and plastic bag with desiccant, respectively. All samples were stored at room temperature then transported to Jimma University diagnostic and parasite culture laboratory for microscopic examination, DNA extraction and qPCR parasite determination.The sample size (n) calculation was done using the formula for estimating single proportion at 95% confidence interval (CI), (Z α/2 = 1.96). To attained maximum sample size 50% was assumed for prevalence (P). A total of 384 household (n) were generated using 5% marginal error (d). Then by considering 4.9 household size in Oromia and 3.8 in Gambella region  the finaThick and thin blood films were prepared and stained with 10% Giemsa solution and examined under light microscope at Jimma University. Blood films were considered negative if no parasites were detected in 200 fields of the thick blood film. Thick blood films were used for detection and quantification of the parasites. Thin blood films were used for species identification.Gametocytes and asexual stage parasites were counted against 200 and 500 white blood cells (WBCs), respectively, and densities (parasite/μl) was estimated using a factor of 8000 leukocytes/μl , 29. AllChelex-100 resin was used for DNA extraction following an established protocol . One dotP. falciparum, P. vivax and P. ovale each  then 0.5μl Pf-fam, 0.5μl Pv-vic, and 0.5 μl Po-Ned for P. falciparum, P. vivax and P. ovale TaqMan probe in 0.1 double distilled water , 0.4 μl forward and reverse primers of ed water , 32 sincPlasmodium positivity. Multivariate logistic regression run following stepwise backward selection of independent variables and removal of non-significant variables with p > 0.05. All significant variables remain in the final model and the model selection was based on the Akaike’s information criterion (AIC). The R statistical software package version (4.2.2) was used to compute W Mann−Whitney test, bar graph and box plot. Model assumption was tested for each models.Data was analyzed in JMP Pro . Univariate analysis was done on the associations between malaria prevalence and independent variables by regressing a single independent variable against Demographic information was obtained from 6640 individuals including 4464 (67.2%) from Arjo and 2176 (32.8%) from Gambella and can be found in Plasmodium species by microscopy was 76.1% (67/88) P. falciparum and 23.9% (21/88) P. vivax.Of 4464 blood samples collected in Arjo, the overall malaria prevalence by microscopy was 2.0% (88/4464). The proportion of Plasmodium species in submicroscopic malaria infection was 12 P. falciparum, 6 P. vivax, and 3 P. ovale (One thousand seven hundred thirteen (1713) smear-negative samples were randomly selected for PCR analysis. Of the total samples analyzed by PCR, 1.2% (21/1713) carried submicroscopic infections. 90.5% (19/21) were afebrile and 9.5% (2/21) were febrile during or 48 h prior to the survey. The number of P. ovale .Malaria prevalence by microscopy showed no significant difference between sugarcane irrigated 2.0% (44/2230) and in non-irrigated clusters 2.0% (44/2234) (p = 0.993).p = 0.0001) and non-irrigated clusters (wet: 2.9% (40/1372); dry: 0.5% (4/862); p = 0.0001). The prevalence of infection in the dry season in both sugarcane-irrigated and non-irrigated clusters was very low, 0.2% (2/977) and 0.5% (4/862) respectively ; dry: 0.2% (2/977); ectively ..Plasmodium infection by microscopy. The median parasite density/μl for symptomatic and asymptomatic infection in Arjo was: 112, IQR: 88–672 and 80, IQR: 64–96, respectively with Mann–WhitneyW  = 197.5, n1 = 12, n2 = 76, p = 1.51e-03) (There was significant difference in parasitemia load between symptomatic and asymptomatic .51e-03) ..Plasmodium species by microscopy was 75.9% (101/133) P. falciparum and 24.1% (32/133) P. vivax.The overall malaria prevalence by microscopy was 6.1% (133/2176) Gambella. The proportion of P. falciparum, 16 P. vivax, 3 mixed , and 1 P. ovale  were submicroscopic malaria infection. 92.6% (63/68) were afebrile and 7.4% (5/68) were febrile. Among submicroscopic infections, 48 were P. ovale ..p = 0.0001, p = 0.0046). In non-irrigated clusters, marginally significant difference in malaria prevalence was observed between the wet season (4.9% (25/511)) and dry season (2.9% (25/865), p = 0.055, The prevalence of malaria was 10.4% (83/800) and 3.6% (50/1376) in rice irrigated and non-irrigated clusters, respectively  (There was significant difference in parasitemia load between symptomatic and asymptomatic .11e-06) ..p = 0.010]. Also individuals who never attended school had higher malaria infection than those with higher education  , = 0.014] .. Stepwi = 863.5 ..p = 0.0001] and not utilizing LLIN  were significantly associated with infection , nfection .. StepwiIC = 300 ..p = 0.0026], age group < 15 , primary educational status , being a preschool child , < 6 months , being a migrant worker  were associated with Plasmodium infection , nfection .. Sex wa = 920.5 .Plasmodium infection in the univariate analysis. Whereas, irrigation , mud/earth floor material , corrugated iron wall material  and no LLIN utilization  were significantly associated with infection  whereas, 12.8% in Gambella (high transmission setting). Studies in Ethiopia showed different prevalence of sub-microscopic malaria 2.4%  than high transmission season (6.7%) in rice irrigation clusters. Similar findings were also documented in two studies from east-central Tanzania .9% than . On the Also, studies conducted in our study sites demonstrated that anopheline mosquito abundance was higher in rice-irrigated clusters than non-irrigated clusters and the sporozoite rate in rice-irrigated clusters was 10-fold higher than non-irrigated clusters in Gambella . WhereasIn the present study, prevalence of infection was 16-fold higher in the wet season compared to dry season. This finding was complemented by an entomological study that showed higher mosquito abundance in wet season than the dry season in Arjo . In addiVector control is one of the most cost effective tool in reducing malaria transmission. This includes spraying IRS and ITN utilization. Previously high coverage of IRS was done by the African IRS programme funded by USAID-PMI until 2010. At the moment, IRS is conducted by the national malarial elimination program of the Ethiopian government and the coverage was limited and targeted to malaria hotspot areas. However, IRS coverage was higher 37.6% in Arjo area compared to the 2015 regional coverage 29.8% . SimilarPlasmodium infection in both irrigated and non-irrigated clusters in Arjo. The risk of Plasmodium infection was 10-fold increase in households who had a habit of poorly utilization of LLIN than households who used persistently. Additionally, households that did not utilize LLIN were 22 times at higher risk of Plasmodium infection than persistently utilizing in Arjo. This result is in line with the results of different studies  p = 0.0016] in Gambella study. Our findings indicates that poor LLIN utilization could be correlated with the level of education.Although, LLINs utilization was 32.2% in 2017  and 40.3 studies , 55–57.  studies –60. LeveNewly migrant population is more prone to malaria infection due to different reasons. In our study, being a migrant worker were 4.7 times more prone to malaria infection than the non- migrant residents. In addition, migrant workers who stayed in the area for < 6 months and 7 to 12 months were 4.7 and 5.6 times at risk of malaria infection than those who stayed for more than three years, respectively. Our finding is in line with findings from several studies on migrant workers involved in commercial agricultural activities , 58–61. P. vivax, P. ovale and P. falciparum coupled with the high malaria prevalence recorded in irrigated areas indicated the significant impact of irrigation schemes on malaria transmission in the study areas. Poor ITN utilization being illiterate and population movement were the main malaria risk factors in the area. In addition, staying in irrigated less than 12 months and having a family size more than 5 were malaria risk factors. Therefore, strengthening malaria control and elimination; including proper utilization of vector control tools and health education for migrant population, should be implemented in water resource development projects such as irrigation schemes.The co-existence of


The complex interaction between malaria and undernutrition leads to increased mortality and morbidity rate among young children in malaria-endemic regions. Results from previous interventions suggest that improving nutritional status of young children may reduce the burden of malaria. This study tested a hypothesis that provision of lipid-based nutrient supplements (LNS) or corn-soy blend (CSB) supplementation to 6–18-month-old children in Malawi would reduce the prevalence of asymptomatic malaria among them.Plasmodium falciparum was determined by real-time PCR from the participant’s dried blood spots (DBS) collected at the baseline and every 3 months. The global null hypothesis was tested using modified Poisson regression to estimate the prevalence ratio (PR) between the control group and three intervention groups at all ages combined. All the models were adjusted for malaria at baseline, season of DBS sample collection, site of enrolment, and household asset Z-score.A total of 840 6-month-old children were enrolled in a randomized trial. The participants received 12-month supplementation with three different daily dietary supplementations: CSB, soy-LNS, or milk-LNS, and one control group without supplementation. The prevalence rate of asymptomatic P. falciparum was 14.1% at enrollment, 8.7% at 9 months, 11.2% at 12 months, 13.0% at 15 months and 22.4% at 18 months of age. Among all samples that were taken after enrolment, the prevalence was 12.1% in control group, 12.2% in milk-LNS, 14.0% in soy-LNS, and 17.2% in CSB group. Compared to children in the control group the prevalence ratio of positive malaria tests was 1.19  in the milk-LNS group, 1.32  in the soy-LNS group and 1.72  in the CSB group.All children combined, the prevalence of The study findings do not support a hypothesis that LNS or CSB supplementation would reduce the prevalence of asymptomatic malaria among Malawian children. In contrast, there was a signal of a possible increase in malaria prevalence among children supplemented with CSB.The online version contains supplementary material available at 10.1186/s12936-023-04701-4. Plasmodium falciparum ), weight-for-age Z score [WAZ] and weight-for-length Z score [WLZ]) were calculated using the STATA macro developed by the World Health Organization (WHO) using the WHO 2006 multicentre growth standards .With the sample size of 210 participants allocated to each group, the original trial possessed an 85% likelihood of detecting significant contrasts in severe stunting rates. This was achieved while maintaining a two-sided Type I error of 5%, even with a potential 10% drop in participants due to attrition during the follow-up period. Additionally, the exclusion of individuals who were already severely stunted at the outset  accounted for 5% of the sample. The proportion of positive malaria parasitaemia DBS samples by intervention group at enrollment, at 9, 12, 15, and 18 months was calculated separately, and for all intervention time points combined (9–18 months). All randomized participants were eligible to be included in the analyses, with the exception that subjects with missing data on an outcome variable were excluded from the analysis of that outcome. The modified Poisson regression with a robust variance estimator was used to estimate the prevalence ratio (PR)  between Null hypothesis of no difference between groups was rejected if P < 0.05. Wald’s test was used to test the global null hypothesis of no differences between the control group and the intervention groups, and the pairwise comparisons. For pairwise comparisons with P < 0.05, the hypothesis of no difference between groups was rejected only if the global null-hypothesis was also rejected.The main analysis was conducted with covariate adjustments. Covariates used in the adjusted models were derived from a predefined list of variables that could modify the prevalence of malaria parasitaemia. The covariate selection with modified Poisson regression models was performed. All the models were adjusted for the same set of covariates, which were malaria parasitaemia at enrolment, season of DBS sample collection, study site, and household asset Z-score.A sensitivity analysis was performed by combining children in the soy- and milk-LNS groups and compared it to the control group at any time of study visit to validate the association between LNS and the prevalence of asymptomatic malaria.A total of 1385 infants were screened, out of whom 840 were enrolled in the trial. The recruitment, group allocation, reasons for exclusions at enrollment, and success of their follow-up including available laboratory measurements at different visits are shown in Fig. There were no differences among intervention groups in terms of baseline characteristics Table . The proAmong all the collected blood samples, the proportion of positive malaria tests was 13.9%. After enrollment, the prevalence of asymptomatic malaria was 8.7% at 9 months, 11.2% at 12 months, 13.0% at 15 months, and 22.4% at 18 months of age.The prevalence of asymptomatic malaria by visit and intervention group is shown in Table A sensitivity analysis combining children from the soy-LNS and milk-LNS groups into one group confirmed that there was no association between LNS provision and asymptomatic malaria at any of the study visits  reduced the probability of random error or bias in the current study. Differences in laboratory methods, excluded participants or those with missing data  could have affected internal validity. Nevertheless, standard procedures for laboratory sample collection, storage, and methods were used across the study. Furthermore, included and excluded participants had similar baseline characteristics. Therefore, the findings of this study are valid and representative and not supportive of a hypothesis that the tested dietary supplements would reduce the prevalence of asymptomatic malaria prevalence in this target population of 6–18-month-old children in Malawi.There are a limited number of clinical trials that have evaluated the potential effects of micronutrient supplements on children living in malaria-endemic areas which lead to different outcomes either protective, adverse, or no effects , 41. In P. falciparum [In this study, CSB supplementation was associated with a higher malaria prevalence among 6–18-month-old children. Among the three intervention used in this study, CSB has a lower nutrient density and it was associated with relatively poor linear growth outcomes in a prior study . Giving lciparum –54. ThesThe study findings did not support the hypothesis that children between 6 to 18 months who receive daily dietary supplementation with LNS or CSB would have a lower risk of asymptomatic malaria in rural Malawi. In contrast, there was a signal of a possible increase in malaria prevalence among children supplemented with CSB. It would be important to monitor malaria parasitaemia with adequate control measures when further studies will be done with micronutrients fortified CSB.Additional file 1: Table S1. Prevalence of malaria at each study visit by the intervention group .