Plasmodium vivax antigen PvMSP-119 to evaluate the prevalence of antibody positive subjects. A reversible catalytic model was used to assess the risk of exposure. The prevalence of anti-PvMSP-119 IgG was 12.84% [95% confidence interval (CI): 9.22%–16.47%], 13.93% (95% CI: 10.11%–17.74%), and 3.57% (95% CI: 1.40%–5.75%) in three different line-of-defense areas, which differed significantly (P < 0.0001). The prevalence of anti-PvMSP-119 IgG increased with age and no statistically significant difference was detected between the sexes. The reversible catalytic model indicated that the seropositive conversion rate and seronegative reversion rate were 0.0042, 0.0034, 0.0032 and 0.0024, 0.0004, 0.0065 in the first-, second-line-of-defense area and total areas, respectively, and the fitted value did not differ significantly from the observed value (P > 0.1). Although this study found the prevalence of antibody-positive subjects and the seroconversion rate in this post-elimination setting were lower than that in transmission setting, the population still had an exposure risk. Serological surveillance should be considered in post-elimination settings to provide valuable information with which to evaluate the risk of malaria re-establishment. China was declared malaria free in June of 2021. In the post-elimination setting, vigilant surveillance is essential to sustain malaria free status. Serological surveillance has been recognized as an efficient tool for assessing the immunity levels and exposure risk in a population. In this study, a cross-sectional serological survey was conducted in Yingjiang County, China, in August–September, 2021. The study sites were villages along the borders with Myanmar, which have no local transmission since the last indigenous case registered in 2016. A total of 923 participants from six villages were enrolled. The majority was aged > 36 years (56.12%) and 12.46% (115/923) participants had experienced malaria infection at least once. A magnetic- bead-based assay was used to test antibodies against  China launched the “National Action Plan for Malaria Elimination in China (2010–2020)” in 2010  was widely used in China during the 1980s–2000s  and the The key areas in the national malaria elimination program, which were surveyed in the last step of the program, were the border areas between China and Myanmar, mainly in Yingjiang County. The last indigenous case of malaria in China was registered in this county in April 2016. In the last stage of the elimination program, a joint cross-border malaria prevention and control project termed “3+1 Strategy”, which was initiated for establishing a buffer zone of border malaria in these areas in 2017. Here, the “+ 1” was a catchment area in Myanmar side with a length of 20.5 km and a width of 2.5 km along the boundary, where the joint work and integrated measures were implemented, including funding and technical assistance, training on early case detection and management, focus response, vector control and surveillance, and health education, etc. The “3” referred to establishing three lines of defense in Yingjiang County where undertook national 1–3–7 approach to accelerate the process of malaria elimination . In this22.1Anopheles minimus is the dominant species of mosquito  was used as an indicator of the risk of malaria exposure in the population and sample sizes calculated for the different transmission settings were based on the value of SCR. In this study, as China was malaria free, the local parasite prevalence was defined as 0. Sample size was estimated based on previous study on size determination for estimating antibody seroconversion rate , for a r2.3After informed consent was obtained from all subjects, their demographic characteristics and behavioral and travel-related information were collected with a standardized questionnaire, using an E-data system. All data were uploaded and stored in a secure database at National Institute of Parasitic Diseases, China CDC.Finger-prick blood was collected from all subjects for the diagnosis of malaria with a rapid diagnosis test (RDT) . The finger-prick blood was also spotted onto filter papers , air-dried, and stored in individual sealed plastic bags with desiccant in a temperature-controlled facility until molecular and serological analysis.2.42.4.1Plasmodium DNA was extracted from the dried blood samples on 3MM filter paper with the QIAamp 96 DNA Blood Kit , according to the manufacturer’s instructions. A real-time reverse transcription ultrasensitive polymerase chain reaction (PCR) analysis was performed following published methods, with an estimated limit of detection of 16 parasites/mL , diluted in Phosphate Buffered Saline (PBS) to a concentration of 3.32 mg/mL, was used in the serological test. Carboxylated Luminex beads with different fluorescent moieties  were covalently coupled with bovine serum albumin (BSA) and the PvMSP-119 antigen, using the modified protocol of Bio-Rad, as follows. The beads (5 × 106 per assay) were resuspended in 80 μL of activation buffer  and activated with 10 μL of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride  and N-hydroxysulfosuccinimide . The activated beads were washed and resuspended in 100 μL of PBS (pH 7.4). The beads coupled beads with 8.0 μg of PvMSP-119 antigen were resuspended in storage buffer and stored at 4°C in the dark.The recombinant blood-stage 2.6The tested sera were isolated from blood spots eluted from 903 filter paper and diluted 1:4 before testing. Antigen-coated beads were thoroughly resuspended and diluted to a final concentration of 80 beads/μL. The secondary antibody , diluted 1:500 (1 μg/mL), was added and the beads were resuspended in 100 μL of PBS (pH 7.4) before analysis on Bio-Plex 200 system. The results were expressed as median fluorescence intensities (MFI). The cut-off value was defined as the MFI of the antibody in the negative control plus three standard deviations. Samples with an MFI value exceeding the cut-off value were determined as antibody positive.2.72 test and Fisher’s exact test were used to evaluate the differences in seroprevalence among the different groups. A reversible catalytic model (a/(a+b)] × [1 − e−(a+b)t], where a refers to the SCR from negative to positive; b refers to the SCR from positive to negative; and t is time. The seropositivity rates in the different defense areas were fitted according to subject age. The parameters of the seroconversion rate were estimated with Muench’s nomogram for the reversible catalytic model (2 test (α =0.05) in GraphPad Prism 8.4.2 . According to the parameters estimated with the best model, the curve-fitting equation for the seropositive rates in the three different line-of defense area was obtained, and the fitting curves for the different age groups and areas were plotted. Maps were created with ArcGIS 10.1 . A P value < 0.05 was deemed to indicate statistically significant differences.All statistical analysis was performed with the statistical software SAS 9.4 . A χic model  was usedic model . The fit33.1The field work was conducted in August–September, 2021. The total number of participants enrolled was 923, and the first-, second-, and third-line-of-defense areas contributed 327, 316, and 280 enrollees, respectively Table 1.P. vivax, and was deemed to be an imported case. All the samples were tested with ultrasensitive PCR, and only one was P. vivax positive, the RDT-positive subject mentioned above.The ratio of females to males was 0.97:1. The majority of participants  were aged > 36 years. The age distribution differed slightly among the different areas, and the highest percentages were aged 6–15, 46–55, and 36–45 years in the first-, second-, and third-line-of-defense areas, respectively. Of the participants, 12.46% (115/923) had been exposed to malaria infection at least once in the past, and this value increased as subjects lived closer to the border. One participant who had a history of travel to Myanmar was RDT-positive for 3.2Plasmodium antigen PvMSP-119 IgG positive were 42, 44, and 10 in the first-, second-, and third-line-of-defense areas, respectively. The prevalence of anti-PvMSP-119 IgG was 12.84% [95% confidence interval (CI): 9.22%–16.47%], 13.93% (95% CI: 10.11%–17.74%) and 3.57% (95% CI: 1.40%–5.75%), respectively. The prevalence of anti-PvMSP-119 IgG differed significantly among three line-of-defense areas on a χ2 test and Fisher’s exact test (P < 0.0001). A paired analysis was used to determine the difference between each two pairs. There was no significant difference between the first- and second-line-of-defense areas (P > 0.05). When the first- and second-line-of-defense areas were combined and then compared with the third-line-of-defense area, the difference was significant (P < 0.0001).The numbers of participants showing 3.32 test (P > 0.05). When the differences between each line-of-defense area were analyzed separately, there was also no statistically significant difference (P > 0.05). The age-specific seropositive rates were calculated in the different line-of-defense areas  and 9.67% (44/455), respectively, which did not differ significantly on a χse areas Figure 23.42 = 0.8582, P < 0.05). There was no significant difference between the fitted value using the reversible catalytic model and the observed value when the goodness-of-fit was evaluated with the χ2 test (P > 0.1).In terms of the conditions of the reversible catalytic model and the increasing trend in the age-specific seropositivity rates, the reversible catalytic model was fitted using the prevalence of IgG antibody positivity. The model indicated that the rate of conversion to seropositive were 0.0042, 0.0034, and 0.0032 in the first- and second-line-of-defense area and total areas, respectively, whereas the rate of reversion from seropositive to negative were 0.0024, 0.0004, and 0.0065, respectively Table 2.The curve fitting equation for the antibody positive rate was obtained using the parameters estimated with the reversible catalytic model Figure 34Plasmodium-species-specific antibodies as biomarkers, and has significant potential utility in enhancing the effectiveness of malaria control and elimination programs , the erythrocyte binding protein (EBP), the merozoite surface proteins (MSPs), apical membrane antigen 1 (AMA1), glutamate-rich protein-R0 (GLURP-R0), and liver-stage antigen 1 (LSA-1). PvMSP-119 is the most immunogenic antigen of P. vivax evaluate the cumulative exposure risk in an investigated population  among three line-of-defense areas. These results may be attributable to differences in the exposure risk and geographic locations of these areas, because the villages from the first- and second-line-of-defense areas were in Nabang Township, adjacent to Laiza City, Kachin State, Myanmar, which reported thousands of cases of malaria annually , whereas no children aged 1–5 years were antibody positive. The seroprevalence in adults reflected the exposure to infection over a longer time period (P. vivax with residual dormant stages in the liver and periodic relapses (P. vivax infection might lead to relapse and seropositivity over a prolonged period. While, the seropositivity in younger children could reflect the recent malaria exposure rates and transmission intensities. The prevalence of antibodies in children aged 1–5 years in three line-of-defense areas were all zero, which strongly supported the indigenous malaria transmission has been suppressed in recent years in the study areas. Moreover, the fitting curve based on the age-specific antibody positivity rates also predicted that the exposure risk of malaria in the 1–5 year age group was 0, which proved the absence of local transmission in the malaria elimination phase, and provided a serological basis for the local confirmation and consolidation of malaria elimination. A similar study in Sri Lanka based on a serological survey after the declaration of malaria elimination also showed that seropositivity in the child group was 0 (In this study, the prevalence of PvMSP-1e period  and sevee period . Populate period , which wrelapses , populatup was 0 .19 antibody positivity increased with age in all the study areas. The risk of malarial exposure predicted with the reversible catalytic model showed that the seroconversion rates in the first- and second-line-of-defense areas were 0.0042 and 0.0034, respectively. The predicted rate was much lower than in other studies that used the same serological marker, PvMSP-119. Two studies undertaken in Cambodia in 2005 and 2011 found seroconversion rates of 0.184 in 2005 and 0.011 in 2012 (In this study, the results showed that the prevalence of PvMSP-1 in 2012 . Another in 2012 , which wAlthough this serological survey showed that seroconversion rates were lower in a post-elimination setting, there was still a risk of exposure to malaria among the populations in the first- and second-line-of-defense areas. When moving into the elimination or post-elimination phase, local transmission is zero, but the potential risk of malaria re-establishment does not disappear, especially in border areas where malaria is still endemic on the other side of the border. Migrant population crossing the border and the lack of barriers to malarial vectors are also challenges to the maintenance of a malaria-free status. Although great achievements have been made in eliminating indigenous cases of malaria in China, the elimination campaign still faces challenges in regions close to international borders . It is d5P. vivax, no standardized serological marker is currently available. In this study, only one antigen, P. vivax antigen PvMSP-119, was used in the serological test. Second, the catalytic model was used at the population level, so individual information and other related factors were ignored. Third, the study sites were only selected in border areas with a potentially high risk of re-establishment.This study had several limitations. First, although multiple serological assays have been used to measure the transmission of 6This is the first serological report to estimate the exposure risk of a population in the post-elimination phase of malaria in China. It indicated that the prevalence of anti-malaria antibody positivity was relatively low in the post-elimination setting, although an exposure risk persisted. Serological surveillance should be considered in the post-elimination setting to provide valuable information about the potential re-transmission risk in post-elimination settings.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.FH and Z-GX conceived and designed the study; FH and YC carried out the data analysis. YC and SW conducted the laboratory work. SL and XRG collected the field samples. FH drafted the initial manuscript. ZH and XG contributed to data review and manuscript revision. All authors contributed to the article and approved the submitted version.


Plasmodium falciparum prevalence by ownership status of common domesticated animals in DR Congo, where 12% of the world's malaria cases occur and anthropophilic Anopheles gambiae vectors predominate.Domesticated animal ownership is an understudied aspect of the human environment that influences mosquito biting behaviour and malaria transmission, and is a key part of national economies and livelihoods in malaria-endemic regions. In this study, we aimed to understand differences in Plasmodium quantitative real-time PCR (qPCR) to estimate P falciparum prevalence differences by household ownership of cattle; chickens; donkeys, horses, or mules; ducks; goats; sheep; and pigs. We used directed acyclic graphs to consider confounding by age, gender, wealth, modern housing, treated bednet use, agricultural land ownership, province, and rural location.In this cross-sectional study, we used survey data from individuals aged 15–59 years in the most recent (2013–14) DR Congo Demographic and Health Survey and previously performed P falciparum infections per 100 people, whereas cattle ownership was associated with 9·6 (–15·8 to –3·5) fewer P falciparum infections per 100 people, even after accounting for bednet use, wealth, and housing structure.Of 17 701 participants who had qPCR results and covariate data, 8917 (50·4%) of whom owned a domesticated animal, we observed large differences in malaria prevalence across types of animals owned in both crude and adjusted models. Household chicken ownership was associated with 3·9 (95% CI 0·6 to 7·1) more An gambiae feeding away from humans. Studies of animal husbandry practices and associated mosquito behaviours could reveal opportunities for new malaria interventions.Our finding of a protective association conferred by cattle ownership suggests that zooprophylaxis interventions might have a role in DR Congo, possibly by drawing The National Institutes of Health and the Bill & Melinda Gates Foundation.For the French and Lingala translations of the abstract see Supplementary Materials section. Plasmodium falciparum, predominates.P falciparum transmission.Despite substantial research investment and attention, malaria cases and deaths are increasing in high-burden countries, with an estimated 247 million cases and 619 000 deaths worldwide in 2021.Anopheles gambiae predominate, with An gambiae sensu stricto most commonly found, followed by Anopheles coluzzii.P falciparum prevalence, such as wealth, modern housing, and low agricultural cover,P falciparum infection remains undefined.DR Congo accounts for the second highest percentage of malaria cases worldwide (12%) and 54% of the cases in central Africa.Anopheles arabiensis dominates,An gambiae sensu stricto, An melas, and An arabiensis predominate.Human–animal interactions influence local disease ecology, and the effect of livestock on malaria is an important question given that the introduction of livestock as zooprohylatic decoys  or as a means of using veterinary endectocides has been proposed as an intervention  to reduce malaria infections.Evidence before this studyAnopheles arabiensis predominates, cattle ownership was protective against malaria infection, but had no effect in anthropophilic Anopheles gambiae sensu stricto regions of The Gambia or zoophilic An arabiensis regions of Ethiopia. Chicken ownership appears protective in Ethiopia, but chicken DNA is not frequently found in bloodmeals across contexts. In a feeding analysis of 1886 An gambiae sensu lato in Senegal, 37·1% of An gambiae sensu lato feedings involved single-animal blood meals and a similar proportion were observed to be single human origin. A 2015 systematic review of zooprophylaxis for malaria control concluded that zooprophylaxis has potential as an effective strategy—particularly in contexts of zoophilic vectors, large distances between where animals are kept at night and where humans sleep, and frequent bednet use—but more evidence is needed for specific environmental conditions. Further, increasing use of insecticide-treated bednets is predicted to increase feeding on cattle by anthropophilic An gambiae sensu stricto vectors.We searched PubMed using search terms  AND  for any articles in any language published from database inception to May 24, 2022. None of the studies identified investigated this question in DR Congo. Several studies have evaluated aspects of zooprophylaxis and zoopotentiation for malaria control in Ethiopia, The Gambia, Ghana, Mozambique, Senegal, Tanzania, and Zambia, or included specific animals owned in malaria risk factor analyses . The evidence is mixed across these settings: in Macha, Zambia and southern Tanzania, where the moderately zoophilic Added value of this studyPlasmodium falciparum remains highly prevalent despite ongoing control efforts and where the anthropophilic An gambiae sensu stricto vector is widespread and the anthropophilic Anopheles coluzzi is also common. In the largest analysis of malaria zooprophylaxis in Africa conducted to date, we found a protective effect of cattle ownership against P falciparum infection, even after accounting for household wealth, treated bednet use, agricultural land, and modern housing. This finding raises the possibility that cattle kept near household sleeping quarters could draw mosquitos away from humans and reduce household P falciparum transmission risk. We also found increased P falciparum infection prevalence with chicken ownership. Although rare in this population, ownership of horses, donkeys, or mules was also associated with an increased P falciparum prevalence; however, this result was not statistically significant in adjusted models. Goat, sheep, pig, and duck ownership did not result in a difference in infection prevalence.Our study investigates whether zooprophylaxis could offer protection against malaria in DR Congo, where Implications of all the available evidenceP falciparum infection and that chicken ownership confers risk of P falciparum infection in DR Congo. There might be a role for zooprophylaxis in DR Congo and other settings where P falciparum transmission via An gambiae vectors is high. Future studies of animal ownership and husbandry practices could reveal opportunities for novel malaria control interventions, such as optimising distance between humans and household animals combined with the use of veterinary endectocides.These findings suggest that cattle ownership in the household compound offers protection against An arabiensis and Anopheles funestus sensu lato vectors where livestock are present in the household compared with households without livestock, whereas human blood meals in An gambiae sensu stricto appeared unaffected by the presence of household livestock.An gambiae sensu stricto and zoophilic tendencies of An arabiensis and An funestus,An arabiensis ranges from 0% to 80% across surveys, suggesting adaptability of the zoophilic vector to feed across hosts, including an increased proportion from humans.An gambiae sensu stricto might be driven towards a preference for cattle feeding with increasing use of insecticide-treated bednets.Feeding preferences of the mosquito species are an important determinant of an animal's zooprophylaxis or zoopotentiation effect on human malaria infection. These effects are evaluated through analysis of mosquito blood meal content to establish the proportion from humans . In Tanzania, human blood meals have been found to be less frequent in P falciparum prevalence and ownership status of common domesticated animals in DR Congo.This adaptability of mosquito vectors underscores the need to move beyond human-centred malaria prevention, and consider the broader environment, including non-human hosts. Improved understanding of the relationship between household animals and malaria prevalence in DR Congo is needed to identify One Health strategies that might reduce DR Congo's high malaria burden. In this study, we aimed to investigate the association between P falciparum infection using the most recent (2013–14) DR Congo Demographic and Health Survey (DHS) and previously collected dried blood spots.In this population-based, nationally representative, cross-sectional study with a two-stage cluster design, we investigated the association between animal ownership and In the first stage, 540 sampling clusters across 26 DR Congo provinces were sampled, and in the second stage, households were randomly sampled within each cluster. Men (aged 15–59 years) and women (aged 15–49 years) from the selected households were offered enrolment, including collection of a dried blood spot specimen. Consent for participation, specimen collection, and specimen analysis for other biomarkers was more than 95%.Additional ethics approval was not required for this analysis of publicly available, de-identified data. The parent studies from which data used in this analysis were generated were approved by Kinshasa School of Public Health (ESP/CE/015/14) and the University of North Carolina at Chapel Hill Institutional Review Board (number 14-0077).P falciparum infection identified using participant dried blood spot samples. As described elsewhere,P falciparum lactate dehydrogenase with a limit of detection of 5–10 parasites per μL.We defined the exposure as a binary variable of each type of livestock, herd, or farm animal owned versus not owned by the household, including cattle; chickens; ducks; goats; horses, donkeys, or mules; pigs; and sheep. We defined the outcome as a binary variable reflecting quantitative real-time PCR (qPCR)-confirmed We used a directed acyclic graph  to identP falciparum prevalence. We also conducted a post-hoc analysis of different flock sizes for chickens.The survey did not include information on where animals were kept overnight, but we used past qualitative research to inform a subanalysis with cattle ownership—ie, households with fewer cattle might be more likely to keep animals within the household compound, as they are less likely to be stolen when kept near the household.P falciparum infections between participants from households that owned each animal compared with participants from households that did not own each respective animal. We applied survey sample weights to provide estimates of the target population based on the sampling design. For subanalyses, we used the log and logit links to calculate prevalence ratios and odds ratios to enhance model convergence given small sample sizes. We calculated profile likelihood 95% CIs.We used linear binomial regression models with generalised estimating equations to account for the clustered data structure to estimate crude and adjusted prevalence differences in We conducted all analyses in R (version 4.0.3).The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.P falciparum malaria  had non-missing values for all covariates , represe malaria .Table 1CP falciparum infection tended to be slightly younger than those who were P falciparum negative, and more participants with P falciparum infection were men than women , whereas of P falciparum-negative participants, the largest proportion were in the highest wealth quintile  did not show a significant protective or harmful association with P falciparum infection (Participants from households that owned chickens had 3·9 (95% CI 0·6 to 7·1) more rurality . Particinfection .Figure 2P falciparum prevalence among individuals living in households with chickens and cattle was 20% . All chicken flock sizes  were associated with increased P falciparum prevalence compared with households that owned no chickens in unadjusted models, with an increasing prevalence ratio with more chickens owned  nfection . When thns owned . All excd models .Table 2CP falciparum infection remained significant after assessing agricultural land ownership as a modifier  did not provide a zooprophylactic effect;An Arabiensis.An gambiae sensu stricto landings on humans.The protective association of cattle ownership was strongest when the herd size was small. Although the DHS did not collect information about where animals were kept overnight, we used herd size as a proxy for where cattle might be kept overnight, based on past research in DR Congo indicating that households with a small number of cattle were more likely to keep animals within the household compound.P falciparum prevalence, and that prevalence increased with flock size. An gambiae sensu lato bloodmeals from chickens are found least frequently,An arabiensis vectors in Ethiopia.An arabiensis, volatile compounds secreted by chickens acted as medium-to-long range deterrents for female An arabiensis.An gambiae sensu stricto species, a common vector in DR Congo. Second, living conditions in DR Congo for chickens might provide additional mosquito breeding habitats, amplifying vector abundance. This amplification could be particularly plausible given the increasing prevalence ratios with larger flock sizes.We further observed that household chicken ownership was associated with increased P falciparum prevalence in households with horses, donkeys, or mules compared with households without these animals, ownership of these animals was rare and this estimate was imprecise. In contexts with An gambiae, donkeys have been associated with reduced malaria in univariate analyses,P falciparum infection with each of the remaining animals . In contexts with anthropophilic An gambiae sensu stricto and An funestus, pig ownership has been found to be associated with increased odds of positivity on malaria rapid diagnostic test among children (aged 1–15 years).An arabiensis regions of Ethiopia, sheep and goats have not been associated with an increased risk of P falciparum infection.Although we did not observed a significant increase in This study has a few notable limitations. First, the cross-sectional design limits investigation of temporal trends between the exposure and outcome, malaria seasonality, and migratory patterns of some animals, particularly cattle. Second, although multiple iterations of the models resulted in estimates of similar magnitude and direction as the models presented, unmeasured confounding or measurement error of the self-reported exposure could nonetheless induce bias. Third, refusal to participate or provide a blood sample could result in selection bias; however, refusal rates were very low (<5%) across this nationally representative study and it is unlikely to substantially bias results. Finally, absence of information about household-specific animal husbandry restricts our ability to identify the behaviours or practices most amenable to intervention.An gambiae sensu stricto vectors such as DR Congo.These findings suggest that interventions focused on household animals, particularly cattle and chickens kept in designated pens on the household compound, could have a role in DR Congo as a complement to current malaria control interventions. Future studies are needed to evaluate where animals are kept in relation to human sleeping quarters, as well as environmental and behavioural conditions in DR Congo that might affect the interplay between the vector, livestock, and human host. Promotion of husbandry practices found to be protective against malaria could complement ongoing malaria control activities in high-burden countries with dominant https://dhsprogram.com/methodology/survey/survey-display-421.cfm), including malaria PCR results. R code used for the analysis is available at https://github.com/IDEELResearch/animalaria.Data are available in a public, open access repository and upon request from the DHS programme (JBP reports research support from Gilead Sciences, non-financial support from Abbott Laboratories, consulting for Zymeron Corporation, and honoraria from Virology Education, all outside the scope of this work. JBP also reports malaria research support from WHO, unrelated to this work. All other authors declare no competing interests.


Malaria diagnosis has been improved by the introduction of rapid diagnostic tests (RDTs), recommended by the World Health Organization (WHO) for areas where microscopy is not available. To be used, these RDTs must meet performance criteria defined by the WHO. This study was conducted to evaluate the diagnostic performance of two RDTs: Advantage P.f. Malaria CardThis was a cross-sectional analytical study conducted from December 2019 to February 2020 on malaria-suspected cases received in three sentinel sites in Togo and from whom capillary blood was collected to perform the two RDTs according to the manufacturer's instructions. Sensitivity and specificity were estimated by comparing to thick/thin blood smear, the gold standard, and to PCR, which is a more sensitive.® compared to thick/thin blood smear was 91.8% and 91.3%, respectively, and for both the specificity was 94.7%. Compared to PCR, the sensitivity was 84.2% and 83.8%, respectively, and the specificity 96.5%.A total of 390 participants  with a median age of 18 (± 0.8) years were included in the study. The sensitivity of both Advantage P.f. Malaria Card® and Advantage Malaria Pan + Pf Card® and Advantage Malaria PAN + Pf Card® compared to microscopy, considered the gold standard, were acceptable under the field conditions found in Togo. They can therefore be used for the biological diagnosis of malaria.The performances of the Advantage P.f. Malaria Card Plasmodium and transmitted to humans by infected female Anopheles mosquitoes. In 2021, the World Health Organization (WHO) estimated the worldwide number of malaria cases to be 247 million with 619000 deaths attributable to malaria, 95% of which occurred in the African region  and 94.7% [CI = 90.0–97.4], respectively; PPV and NPV values were 95.7 [CI = 92.2–97.7] and 90.0 [CI = 85.2–93.3]  were P. falciparum was inconclusive for seven samples; therefore, the estimation of the performance of the two RDTs evaluated compared to PCR was done on a total of 383 patients divided into 143 for the PCR-negative group and 240 for the P. falciparum PCR-positive group . The PPV and NPV values are shown in Table For the PCR analysis, detection of plasmodial DNA of ® and Advantage Malaria Pan + Pf Card® are WHO pre-qualified RDTs whose performance has been evaluated against clinical wild-type samples containing P. falciparum and P. vivax at low (200) and high (2000) parasite densities (plasmodia/μl) as well as negative samples for any pathology [The World Health Organization recommends that malaria-endemic countries and their partners procure pre-qualified RDTs for the biological confirmation of suspected malaria cases. As such, the FIND group conducts regular evaluations and makes the results available to countries. The Advantage P.f. Malaria Cardathology . J. MitrP. falciparum compared to microscopy were 91.8% and 91.3% for sensitivity, respectively, and 94.7% for specificity. Our results are similar to other evaluations done that also considered microscopy as a gold standard method. For example, a study in northwest Ethiopia evaluating CareStart™ showed a sensitivity of 92.9 and specificity of 95.4 [®(HRP2) RDT [®.The performance levels of the Advantage P.f. Malaria Card® and Advantage Malaria Pan+Pf Card® for the detection of  of 95.4 ; anotherRP2) RDT . A studyRP2) RDT  noted a P. falciparum of 100 to 200 parasites/µl were not reliably detected by the RDTs used.In our study, density-dependent sensitivity was observed with microscopy as the reference: indeed, it was higher for high parasitemia for both evaluated RDTs . This trend corroborates the finding of Wanja et al.  who, in fhrp2 and pfhrp3) genes in field isolates of P. falciparum [This limitation of RDT detection for low parasitemia  would exlciparum .In our study, the RDTs evaluated showed a relatively high specificity (94.7%) compared to microscopy. Indeed, WHO recommends that RDTs used for biological confirmation of malaria should have at least a specificity > 90% . The low® and Advantage Malaria Pan+Pf Card®, 90.0 and 89.5%, respectively, are probably related to the time of our study, which started at the end of the rainy season, while the evaluations of the other authors were done during the period of high transmission. Indeed, the predictive values (PPV and NPV) are a function of the prevalence of the disease in the study area [Significant positive and negative predictive values were found for these RDTs compared to TTBS in this study. Thus, the high PPV for both RDTs (95.7%) reinforces the sensitivity and specificity data found for these RDTs to be used for biological confirmation of malaria cases in malaria-endemic areas. The NPVs of the Advantage P.f. Malaria Cardudy area .®, which were 91.8% and 91.3%, respectively, compared to microscopy, fell below 90% when PCR was used as a reference method . Indeed, PCR can detect parasites at levels as low as 0.002 P/µl, implying that the lower sensitivity range observed could be due to sub-microscopic infections in the population. Also, Matangila et al. [Although microscopy is the gold standard detection technique for malaria, the performance of these RDTs was also estimated with PCR, a technique with better sensitivity than TTBS. The performance of the RDTs was slightly lower than that obtained when considering TTBS. The sensitivity of the Advantage P.f. Malaria Card® and Advantage Malaria Pan+Pf Carda et al.  showed tThe opposite was observed for specificity whereby the levels were higher compared to PCR (96.5%) than microscopy (94.7%). This indicates that compared with PCR, they have a higher ability to actually detect a malaria-negative patient. False-positive results could be explained by the persistence of HRP2 circulation in the blood for > 2 weeks even after antimalarial treatment . The difP. falciparum, the performance of this RDT to detect other plasmodial species has not been assessed because of the low number of positive cases for these other species. Among the included subjects whose malaria test result was positive according to the results of the microscopic examination, 2.2% had a positive result for P. malariae and 0.9% for P. ovale.Our study has some limitations. Indeed, although the Advantage Malaria Pan+Pf Card® RDT detects species other than ® and Advantage Malaria Pan + PF Card® RDTs for the parasitological confirmation of malaria cases compared to the microscopy as the gold standard, although varying according to the parasite density, was acceptable under the field conditions found in Togo; even if considering PCR as the reference method, this performance has slightly declined. As sub-microscopic infections are expected to be more frequent, an impact of the control strategies undertaken by Togo, the determination of the performance of the new RDTs should perhaps consider PCR in the evaluation procedure as part of the continuous improvement of these diagnostic tools to achieve the elimination of malaria in the countries where it remains endemic.The diagnostic performance of the Advantage P.F Malaria Card


Water resource development projects, such as dams and irrigation schemes, have a positive impact on food security and poverty reduction. However, such projects could increase prevalence of vector borne disease, such as malaria. This study investigate the impact of different agroecosystems and prevalence of malaria infection in Southwest Ethiopia.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 study participants from 1449 households in Arjo and 546 households in Gambella enrolled in the study and blood samples were collected, respectively. All blood samples were microscopically examined and a subset of microscopy negative blood samples (n = 2244) were analysed by qPCR. Mixed effect logistic regression and generalized estimating equation were used to determine microscopic and submicroscopic malaria infection and the associated risk factors, respectively.p < 0.001), but no difference was found in Arjo . On the other hand, of the 1713 and 531 samples analysed by qPCR from Arjo and Gambella the presence of submicroscopic infection was 1.2% and 12.8%, respectively. Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale were identified by qPCR in both sites. Irrigation was a risk factor for submicroscopic infection in both Arjo and Gambella. Irrigation, being a migrant worker, outdoor job, < 6 months length of stay in the area were risk factors for microscopic infection in Gambella. Moreover, school-age children and length of stay in the area for 1–3 years were significant predictors for submicroscopic malaria in Gambella. However, no ITN utilization was a predictor for both submicroscopic and microscopic infection in Arjo. Season was also a risk factor for microscopic infection in Arjo.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 (The study highlighted the potential importance of different irrigation practices impacting on submicroscopic malaria transmission. Moreover, microscopic and submicroscopic infections coupled with population movement may contribute to residual malaria transmission and could hinder malaria control and elimination programmes in the country. Therefore, strengthening malaria surveillance and control by using highly sensitive diagnostic tools to detect low-density parasites, screening migrant workers upon arrival and departure, ensuring adequate coverage and proper utilization of vector control tools, and health education for at-risk groups residing or working in such development corridors is needed.The online version contains supplementary material available at 10.1186/s12936-023-04762-5. Water resource development projects such as dams and irrigation schemes have a positive impact on food security and poverty reduction . HoweverAnopheles 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 . In non-irrigated clusters, marginally significant difference in malaria prevalence was observed between the wet season [4.9% (25/511)] and dry season   and 3.6% (50/1376) in rice irrigated and non-irrigated clusters, respectively  .There was significant difference in parasitemia load between symptomatic and asymptomatic 01) Fig. . This alP. falciparum, 16 P. vivax, three mixed , and one P. ovale  were submicroscopic malaria infection. Nine-tenth 92.6% (63/68) of the positive individuals were asymptomatic and 7.4% (5/68) were symptomatic. Among submicroscopic infections, 48 were Plasmodium infection by microscopy. Survey seasons was strong predictor of microscopically detected malaria infection . Similarly individuals who did not use ITN had higher microscopically detected malaria infection than those who use ITN daily  , the multilevel logistic regression of the fixed effect analysis indicated, survey season and ITN utilization were associated with Plasmodium infection by microscopy analysis. Being a migrant worker increased microscopically detected malaria infection by 14.7 times . Microscopically detected malaria risk also increased with the length of stay in the area, individual who stayed < 6 months was 7 times higher risk than those stayed longer than 3 years . Similarly, individuals who stayed seven to 12 months more likely to be infected with Plasmodium than those who stayed longer than 3 years . In addition, irrigation , individual involved in outdoor jobs were 3 times risk for microscopically detected malaria infection , however, homes constructed with corrugated iron walls  were significant malaria predictors  being a migrant worker, length of stay in the area, irrigated cluster, type of occupation and household construction material were significantly associated with p < 0.001]. While, the individual level, cluster level and full model variance were higher than the null model and remained significant ,  and , respectively . In the full model, 40.88 in the odds of Plasmodium infection variation was explained after adding individual and cluster level risk factors. MOR 4.07 was observed in the null model, individual-level predictors 4.18, cluster level predictors 4.43 and full 4.21 models were greater than one. This shows the presence of unexplained variation among the cluster. Akaike information criteria (AIC) identified the full model as the best fit.In sugarcane growing (Arjo), the null model estimates of random effects variance revealed there was significant variation in microscopic infection between clusters . The individual, cluster level and full model variance was lower than the null model  respectively. The VPC showed 49.98% variability in the odds of Plasmodium infection was due to cluster level risk factors after controlling individual level risk factor (model I). In the full model, 39.23% in the odds of Plasmodium infection variation was explained after adding individual and cluster level risk factors. MOR 5.64 was observed in the null model, individual-level predictors 4.22, and 4.01 in cluster level and full models were greater than one. This shows the presence of unobserved variation among the cluster. Akaike information criteria (AIC) identified the full model as the best fit (Table In rice growing areas (Gambella), the cluster variance of the null model showed significant difference in P. ovale was detected in irrigated clusters of both sugarcane growing (Arjo) and rice growing (Gambella), southwest Ethiopia.In the present study, submicroscopic malaria transmission was influenced by irrigation and ITN utilization in sugarcane growing (Arjo) area. While in rice growing (Gambella) area, submicroscopic malaria was associated with irrigation, age, survey season, number of rooms in the house and length of stay in the area. However, microscopically detected malaria infection had shown seasonal pattern and affected by ITN utilization in sugarcane growing (Arjo). Irrigation, being a migrant worker, outdoor jobs, length of stay in the area, and house construction material were significant predictors of microscopically detected malaria. Interestingly Plasmodium infection was 12.8% in rice growing areas (Gambella), a high transmission setting whereas, 1.2% in sugarcane growing areas (Arjo), low transmission setting. Several studies showed a range of submicroscopic prevalence in low transmission setting from 0 to 16.8 and 16.3 to 82.5 in high transmission setting respectively [P. falciparum and P. vivax caused submicroscopic malaria was reported in the aforementioned studies. It would be expected that in lower transmission areas the prevalence of both microscopic and sub-microscopic infections would be lower, as shown in your study . In Arjo area, since 2018 primaquine (PQ) was extensively used targeting malaria elimination in low transmission setting. The use of PQ could lower the overall transmission, but it is not clear why that would only impact submicroscopic infections. Asymptomatic submicroscopic malaria is a major obstacle to malaria elimination due to its being a hidden reservoir and facilitate onward transmission. Therefore, malaria elimination requires targeting submicroscopic carriers [In this study, the prevalence of submicroscopic ectively –44. Studectively , 45, 46.carriers .Plasmodium ovale was reported in both study sites for the first time. All P. ovale infections were submicroscopic. Species identification and parasite distribution are important epidemiological parameter to understand a country’s parasite population that eventually informs the malaria control and elimination which interventions and diagnostic method to use. Previously P. ovale was reported in Ethiopia in 1969, but it was not reported until 2013 and 2015 from northwestern [P. ovale and Plasmodium malariae in different parts of the country [hwestern  and southwestern . Two rec country , 50. ThiPlasmodium infection among 5–15 years of children were 3.7 higher than individuals who were over the age of 15. This result was in line with research from other sources [The finding of this study indicated submicroscopic malaria was disproportionately higher in school-age children in rice growing, Gambella area and the odds of submicroscopic  sources , 51–53. This study also showed 4.9 and 3.1 fold increased submicroscopic infection by irrigation in both sugarcane growing areas (Arjo) and rice growing areas (Gambella), respectively. In Arjo, no significant increment of microscopically detected malaria was observed between sugarcane-irrigated and non-irrigated clusters however, rice irrigation showed fourfold increase in microscopically detected malaria infection than non-irrigated clusters. Moreover, microscopically detected malaria infection doubled in dry season (12.9%) than wet (6.7%) in rice irrigation clusters. Similar findings were also documented in two studies from east-central Tanzania , 16 and Also, studies conducted in these 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 tenfold higher than non-irrigated clusters in rice-irrigated (Gambella) . WhereasIn the present study, microscopically detected malaria infection was 21-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 addiNewly migrant population migrated in are more prone to malaria infection due to different reason. In rice growing (Gambella) study, being a migrant worker were 14.7 times more prone to microscopic malaria infection than the non-migrant residents. Half (50%) of this migrant population did not owned ITN during the study period while, only one fourth (22.9%) of the local residents did not owned ITN. In addition, individuals who stayed in the area for < 6 months and 7 to 12 months were seven and 5 times at risk of microscopic malaria infection than those who stayed for more than three years, respectively. Similarly, submicroscopic infection was higher in individual who stayed one to 3 years. This finding is in line with findings from several studies on migrant workers involved in commercial agricultural activities , 58–60. Plasmodium infection in sugarcane growing (Arjo). The risk of submicroscopic and microscopic Plasmodium infection was 7.4-fold and fivefold increase in individuals who had no habit of utilization of ITN than households who used persistently, respectively. This result is in line with the results of different studies [This study demonstrated a lower than expected protective effect of IRS. Reasons for this could be due to insecticide resistance, the insecticide dosage concentration, duration of insecticide on the wall, and other factors could affect the protective efficacy of IRS. Studies documented multiple insecticide resistance particularly to dichloro diphenyl trichloroethane (DDT) and pyrethroids at nationwide level and at the study site, Arjo and Gambella in Ethiopia –63. Howe studies , 58, 64.Plasmodium infection than those who had indoor jobs in rice growing (Gambella). This could be explained by malaria transmission occur at outdoor. However, wall construction material such as corrugated iron wall and number of sleeping rooms were 90% and 68% less likely to acquire microscopically detected and submicroscopic Plasmodium infection than their counterparts respectively in Gambella. Different studies showed poorly constructed house such as a wall or a roof, which had a gap or an open eaves may serve to a mosquito enter to a house than well-constructed houses [In this study, socio-economic factors, such as occupation and wall construction material, were also significantly associated with malaria. Study participants who had outdoor jobs were 2.9 times at higher risk of d houses –67.There are various limitation in this study that have not been included. These limitation includes lagged rainfall, enhanced vegetation index (EVI) and land surface temperature (LST) as a cluster level variable were not included in the data collection. Therefore, this calls for the need for further investigations in these areas.P. falciparum and P. vivax were identified by microscopy however, P. ovale was detected by qPCR. This indicates there is a need of a highly sensitive diagnostic tool to detect the low-grade parasite in a resource-limited setting and to accelerate the progress of malaria elimination by detecting submicroscopic malaria reservoirs. Population movement such as migrant workers are also a challenge for malaria control and elimination efforts. Since their frequent mobility of this group, they have limited access to malaria diagnosis, treatment, and vector control measures. Therefore, it is crucial to specifically screen and treat this population upon arrival and departure in order to minimize residual malaria transmission. Length of stay in the area, and sociodemographic factor such as outdoor jobs and school-age children were shown risk factors for malaria infection. In addition, in sugarcane growing areas, poor ITN utilization and season were significant predictors. Therefore, strengthening malaria control and elimination efforts by ensuring adequate coverage and proper utilization of vector control tools, prompt diagnosis and treatment coupled with health education should be implemented in water resource development projects such as irrigation schemes.The study highlighted the potential importance of different irrigation practices impacting on submicroscopic malaria. Moreover, asymptomatic and submicroscopic malaria also adds a hidden burden to malaria control and elimination thus continues the ongoing transmission pattern. In this study, only Additional file 1: Table S1. The clusters name and abbreviation of the sugarcane growing (Arjo) and rice growing (Gambella), Ethiopia: March 2019 and October2019. Table S2. Univariate mixed effect logistic regression analysis of individual level risk factors and malaria infection by microscopy in Arjo and Gambella, Ethiopia: March 2019 and October2019. Table S3. Univariate mixed effect logistic regression analysis of cluster level risk factors and malaria infection by microscopy in Arjo and Gambella, Ethiopia: March 2019 and October 2019. Table S4. Univariate analysis of GEE model submicroscopic malaria infection and risk factors in Arjo and Gambella, Ethiopia: March 2019 and October 2019


Plasmodium falciparum parasites in Senegal. The analysis is based on 1097 samples collected at health facilities during passive malaria case detection in 2019; it provides a baseline for analyzing parasite genetic metrics as they vary over time and geographic space. The study’s goal was to identify genetic metrics that were informative about transmission intensity and other aspects of transmission dynamics, focusing on measures of genetic relatedness between parasites. We found the best genetic proxy for local malaria incidence to be the proportion of polygenomic infections , although this relationship broke down at low incidence. The proportion of related parasites was less correlated with incidence while local genetic diversity was uninformative. The type of relatedness could discriminate local transmission patterns: two nearby areas had similarly high fractions of relatives, but one was dominated by clones and the other by outcrossed relatives. Throughout Senegal, 58% of related parasites belonged to a single network of relatives, within which parasites were enriched for shared haplotypes at known and suspected drug resistance loci and at one novel locus, reflective of ongoing selection pressure.We here analyze data from the first year of an ongoing nationwide program of genetic surveillance of  Senegal has initiated a national sentinel surveillance program for malaria parasite genetics. Here, the authors report data from the first year of the program and use it to investigate local malaria incidence, patterns of transmission, and genetic loci under selection. Plasmodium falciparum malaria remains a major global cause of human morbidity and mortality. As countries seek to improve the targeting and effectiveness of malaria interventions, they require detailed information about the prevalence of drug resistance, about local epidemiology, and about how the malaria burden is changing in response to interventions and other factors. Parasite genetic surveillance shows great promise as a source of information to support decision-making in all of these areas4. It already has a well-established role in tracking the spread of known drug resistance markers and detecting the appearance of new resistance alleles6. In low transmission settings, genetic data can determine whether new cases arise from ongoing local transmission or from importation, and it can potentially identify sources of imported parasites9.Despite progress over the past several decades toward malaria control and elimination, 10. However, the relationship between malaria burden and genetics is complex, in part because the parasite life cycle includes both a haploid asexual stage in humans and a diploid sexual stage in the mosquito. Both self-fertilization and outcrossing can occur during the sexual cycle, with the latter requiring the presence of multiple genetically distinct parasites in the mosquito, something that occurs more frequently when transmission is higher. Thus, as malaria transmission drops, we can expect a decrease in the typical complexity of infection  and an increasingly clonal parasite population, both of which have been observed11. The exact relationships between these aspects of parasite population genetics and transmission, and whether they are able to provide useful information for malaria control, are still being investigated. Even less is known about the utility of partially related parasites (close relatives that are not clones) in understanding transmission; these should become less common with declining transmission (and hence declining outcrossing) but also easier to detect in a smaller parasite population.Detecting changes in malaria burden is challenging, both because directly surveying parasite prevalence is difficult and because indirect estimates can be skewed by varying care-seeking behavior and access to health resources. Here again, genetic surveys have the potential to provide important information since parasite genetics has already been observed to reflect changes in transmission ratesP. falciparum genetic surveillance across Senegal. The project is intended as a testbed for learning how to apply parasite genetic surveys to inform malaria control strategy across the range of malaria transmission intensity seen in Senegal, which is stratified into three zones  sentinel surveillance network. All samples were subjected to barcode genotyping based on 24 single nucleotide polymorphisms (SNPs), which were used to identify clonal parasites and polygenomic infections (those with COI > 1). A subset of primarily monogenomic samples was chosen for more detailed analysis using whole genome sequencing (WGS) and subsequent genome-wide SNP genotyping, which gave us the ability to detect partially related parasites and identify signatures of selection. Our goal was broadly to explore ways in which this genetic data could be informative for malaria control decision-making, and in particular to investigate the relationship between parasite genetics and malaria burden.The dataset consists of genetic data from 1097 12. The final dataset used for analysis comprised 1097 samples: 310 with both barcode and genome-wide SNP data, 757 with barcode data only, and 30 with genome-wide SNP data only . Samples chosen for genome-wide analysis were subjected to P. falciparum selective whole genome amplification and Illumina-based short-read whole genome sequencing. Monogenomic samples were analyzed at ~40,000 polymorphic genome-wide SNPs filtered to remove highly heterozygous regions and SNPs  from febrile individuals with uncomplicated malaria infections at 23 health facility sites that serve as national sentinel sites across the country . We found no evidence for systematic differences in allele frequencies and thus no evidence for parasite population structure within Senegal. Mean parasite diversity between study sites was independent of the distance between sites and was indistinguishable from diversity within sites Fig. .Fig. 2Ge13). Pairs of relatives were identified either as clones, based on barcode data, or as partial relatives, based on the fraction of their sequenced genomes that were identical by descent (IBD), that is, the fraction that consisted of chromosome segments with essentially identical alleles throughout17 (see “Methods”). If the IBD fraction exceeded a threshold  movement between sites. It has the highest human population density within Senegal as well as the most well-developed road network. In addition, many of our study sites in this region have specific characteristics that increase connectedness with the rest of the country, whether because they are in or near the capital , a major destination for religious education or pilgrimage , or located at important road crossings . While the high degree of parasite relatedness between these sites must reflect parasite movement, it should also be noted that all sites that have a high pairwise relatedness with other sites also display high levels of within-site relatedness  and partial relatedness  within each site  and the relationship was not at all linear. Partial relatedness and clonality calculated separately were even less correlated with incidence . Primarily, the observed pattern suggests that at higher incidences (with a threshold in the range of 10–50 per 1000 per year), relatedness is always low, while at lower incidences it can lie in a wide range.We next addressed one of our core goals, which was to investigate how well genetic measures can be used to estimate the changing malaria burden. Previous work has shown that relatedness increases with decreasing transmissionnce Fig. . However18 is COI, which has been proposed to offer both more accurate and more current information about transmission changes. The measure we investigated for this purpose was the frequency of polygenomic infections at each study site (termed the polygenomic fraction hereafter), based on the large number of samples for which we had barcode data. Given the small number of SNPs in the barcode, we treated COI as a dichotomous trait, with samples classified as “probable monogenomic” (<2 heterozygous SNPs) or “probable polygenomic” . The breakdown of the correlation at very low incidence is consistent with the previously reported behavior of COI19. The relationship was little changed when we adjusted the raw incidence estimates for differing levels of care seeking, testing of suspected cases, and reporting completeness at our study sites, using a previously described approach20  on the polygenomic fraction and on relatedness, the former was the much better single predictor , as did replacing relatedness with its partial and clonal components (R2adj = 0.507).The polygenomic fraction was more informative about incidence than relatedness, whether the latter was measured as total relatedness or separately as partial and clonal relatedness. When we regressed logWhen we grouped sequenced parasites from throughout the country into networks of partial relatives, we found a strikingly skewed distribution of network sizes. Omitting clones, there were 110 parasites with a relative in the dataset or 32% of the total. These formed a total of 22 clusters of relatives. Twenty-one clusters contained two or three parasites and together accounted for 46 of the 110. The remaining single cluster contained the other 64 parasites  and included parasites from eight study sites across central and southern Senegal Fig. . This obp = 0.033, two-tailed Fisher’s exact test)—although the marked variation in clonality and partial relatedness between sites makes interpretation difficult. To address this possibility, we looked for genome segments that were more widely shared  between cluster parasites than between non-cluster parasites. While we found no specific segments shared among the majority of cluster parasites, we did find multiple regions of increased sharing within the cluster  locus (PF3D7_0709000) on chromosome 721 is known to modulate drug resistance, the amino acid transporter (aat1) gene (PF3D7_0629500) on chromosome 6 has been implicated in drug resistance22, and the GTP cyclohydrolase 1 (gch1) locus (3D7_1224000) on chromosome 12 has been implicated in antifolate resistance24. In all of these cases, enhanced sharing was evident in both cluster and non-cluster-associated parasites but was more pronounced among those in the cluster. The same was true of a region on chromosome 9 that has been reported previously to be under selection in Senegal and Gambia25 but that has not been associated with drug resistance. A ~ 200 kb region, containing dozens of genes, on chromosome 11 showed excess sharing only among parasites in the cluster, raising the possibility that it is an early signal of an emerging sweep .One process that could contribute to this kind of clustering of relatives is ongoing positive selection, with increased relatedness driven by the sharing of genomic segments under selection. This possibility would also be consistent with the observation that parasites within the large cluster were twice as likely to have a clone in our dataset as those from the same study sites but outside the cluster—26.7% (16/60) vs. 12.5% (14/112)  between parasites and the frequency of complex (polygenomic) infections in the population. We drew two main conclusions from the comparison. First, while both measures were correlated with local incidence, the polygenomic fraction was a much better predictor than relatedness, confirming modeling studies that identified COI-based statistics as superior to relatedness- or diversity-based ones19. In the far north of Senegal, incidence is so low (~1/1000) that the same individual should rarely be infected twice simultaneously, and yet 40% of infections in the region are polygenomic. Based on the known epidemiology and a previous study of parasite genomics of this region, it seems likely that many parasites there represent recent importation from areas with higher burden27 and that polygenomic infections are either acquired elsewhere or are the result of local co-transmission of multiple parasite strains from a single imported polygenomic infection. It is thus not surprising that local parasite genetics in this part of Senegal reflect the characteristics of the source region more than the local transmission rate28. Similarly, relatedness should be low where the bulk of infections represent recent importation from more diverse regions, and where it does appear  it signals ongoing local transmission. A better understanding of the transition of genetics into an importation-dominated regime will likely require both more in-depth study of particular regions and detailed modeling.We also found that, while the correlation between either genetic measure and local incidence holds true for moderate and higher transmission regions, it breaks down where transmission is low . In our low-incidence sites, genetic measures suggest a much higher incidence than is actually observed; similar behavior has been previously reported specifically for COI29 and mix with more than 1 million local inhabitants; in 2019, this took place on October 16–17, roughly in the middle of the sample collection period. Diourbel, on the other hand, is a smaller urban setting  that contains numerous large  religious boarding schools called daaras, where up to several hundred school-aged boys, many from elsewhere in Senegal, live communally. Previous work has shown evidence of distinct, genetically identical parasites among infections from individual daaras elsewhere in Senegal, consistent with local and focal transmission9. The findings here have helped lead Senegal’s NMCP to start developing new strategies to address the infection risks associated with large population movements like the Grand Magal and have motivated a PMI-funded operational research study that will evaluate new interventions specifically targeting these at-risk students and increased community case management targeting these schools.Despite its limitations as a predictor of transmission intensity, parasite relatedness proved to be illuminating about the heterogeneity of malaria transmission in Senegal. An unexpected finding was that two geographically close (~50 km apart) cities, Touba and Diourbel, with similarly high overall parasite relatedness, differ markedly in the patterns of that relatedness, with partially related parasites common in Touba while clones dominate in Diourbel. Touba does have a higher reported incidence than the main study site in Diourbel (15/1000 vs. 8/1000), but the different transmission dynamics in the two cities may also reflect their differences as settings for malaria transmission. Touba, the second most populous city in Senegal, is located at the crossing of major roadways and is the site of the annual Grand Magal pilgrimage, during which more than 4 million individuals travel to the areaSubsequent surveillance data should provide more fine-grained temporal data about transmission in Touba and the role that the Grand Magal pilgrimage plays in local transmission. This pilgrimage may serve as a natural experiment for studying the effect of malaria importation at different points in the transmission season; it draws millions of people from throughout Senegal and the broader region to a single city for a brief period, and that period shifts by ~11 days each year. Further data will also show whether the large number of clones and clonal clusters in Diourbel is a persistent feature and, if so, whether the same clones persist across the dry season or are replenished by new imported infections each year. Modeling and epidemiological investigation could both provide insights into the cause of the pattern in Diourbel, in particular investigating the possibility that it is driven by highly localized hot spots of transmission. Such hot spots could then be addressed with targeted, vector-based interventions. More broadly, these observations illustrate both the potential for genetic surveillance to provide actionable information about local transmission patterns and the need to better understand the implications of specific patterns.15), and in particular, was more common among related parasites. This revealed multiple signals of selection. Most of these were at loci known  or suspected (chromosome 12) to be under selection for drug resistance, while one was at a previously identified locus on chromosome 9 that is likely not associated with drug resistance. The excess IBD sharing on chromosome 6 was consistent with a recent report30 from Gambia of the rapid rise of nonsynonymous mutation S258L in the gene aat1: this allele was present in 100% of common shared haplotypes in our data, while its frequency was 79% in the dataset as a whole. No single selected allele was responsible for all relatedness in the network of relatives, but several loci had markedly higher sharing within the network, and a signal at one candidate locus on chromosome 11 was seen only among samples in the network.Finally, another striking observation from the country-wide dataset was that clusters of partially related parasites had a highly skewed size distribution, consisting of 21 small clusters (2–3 parasites apiece) and a single large cluster connecting multiple sites and containing 64 parasites. Since this kind of pattern could signal the rapid spread of one or more positively selected alleles, we looked for genomic loci where IBD sharing was unusually common  and approved by the Institutional Review Board of the Harvard T.H. Chan School of Public Health (IRB protocol 16330). The CDC Human Research Protection Office reviewed the protocol and determined the CDC to be non-engaged.This work represents a collaboration between Senegal research (CIGASS at UCAD) and the National Malaria Control Program (NMCP), with Harvard T.H. Chan School of Public Health and The Broad Institute, and other partners. All authors, which include local  researchers, participated in the work represented here from study design and implementation through data generation and analysis. All data are owned by the country of Senegal under the Ministry of Health and Social Action. The capacity to carry out the genotyping and sequencing data generation is held at CIGASS within the University Cheikh Anta Diop in Senegal and the data are analyzed jointly across the collaboration.Blood samples were collected from clinics and health posts across Senegal from uncomplicated malaria infections detected by microscopy (clinics) or rapid diagnostic testing . These samples are biologically unique and were fully utilized for the data generation described. Collection sites were across the range of transmission intensity in Senegal, from very low reported annual incidence (<1 per 1000) in the northwest to high reported annual incidence (>500 per 1000) in the southeast, including three main clinic sites, located in Thies, Diourbel, and Kedougou, that were augmented by health posts as part of the sentinel survey system of the National Malaria Control Program, with additional sites located in areas of interest. Blood was collected on Whatman 903 ProteinSaver filter paper material.12. We estimated the incidence from nearby sites for two sites (SLP and CSS) for which we did not have incidence data. The ROB site was used for CSS in Richard Toll, and a combination of incidence data from Medina Fall 1 and 2 was used as a proxy for the SLAP clinic in Thies.Incidence per thousand individuals in 2019 was calculated for each health facility using data provided by the Senegal NMCPNucleic acid material was extracted from 1353 blood spots collected on filter paper, with 1066 of these samples subjected to pre-amplification, before molecular barcode genotyping of all samples. A total of 1034 of the 1353 samples passed initial barcode genotyping, with 636 samples classified as monogenomic infections. Of the 1353 samples, 648 were subjected to whole genome sequencing (WGS).Nucleic acid material was extracted from dried blood spots collected on filter paper obtained from all microscopy- and/or RDT-positive individuals presenting at clinics or health posts with fever according to routine Senegal protocols. Briefly, dried blood spots collected on ProteinSaver cards were extracted for genomic DNA from 2 to 3, 6-mm punches using the manufacturer protocol from the Promega Maxwell DNA IQ Casework Sample kit .31 and molecular barcode analysis32. The genotypes were called by their base designation  with missing alleles identified by “X” and working alleles where both the major and minor alleles were designated by “N”. Only samples missing zero or one of the twenty-four assays were used for analysis, and monogenomic infections were called if there were zero or one of these assays called as “N”.Extracted nucleic acid material was subjected to pre-amplification33 involving multiple displacement amplification using Phi29 polymerase followed by magnetic bead cleanup and quantification. The resultant material was subjected to fragmentation and NEBNext Ultra II FS DNA library construction according to manufacturer instructions  and then sequenced using Illumina Hi-Seq X machines.Genotyped samples with barcode calls of up to four missing positions and no more than two “N” calls were then used for WGS. Even though the criterion for monogenomic infections was no more than one “N” call, we included samples with two “N” calls as well to increase the likelihood of including all samples that could be analyzed for relatedness (described below). Samples were subjected to sWGAPlasmodium falciparum 3D7v.3 reference assembly for read alignment with bwa-mem34. Picard toolkit was used to mark and remove duplicates and to assess quality control metrics. For genome sequence data, individual genotypes were discarded if the supporting read was <5 reads. The downstream analysis was limited to a set of preferred SNP sites all located within the callable loci of P. falciparum35.Variant calling was performed in accordance with the best practices established as a part of the Pf3K project using GATK3.5.0 and P. falciparum genome, we restricted our sequence analysis to a highly filtered set of 149,582 single nucleotide polymorphism (SNP) sites, where the filters  removed sites and regions with anomalously high heterozygosity.To avoid artifacts caused by the high AT content and extensive low-complexity regions of the 34. Polygenomic samples were identified with DEploid36, and removed, along with duplicate samples from Malawi and a small number of samples from Nigeria , leaving a total of 1328 samples.To develop these filters, we downloaded the complete set of VCF files for Pf3k release 535. (2) Remaining regions were divided into non-overlapping 2 kb windows, which were filtered on the basis of mean heterozygosity in the window, with a threshold at 0.03 . To study shared IBD segments in possible selective sweeps, a modified version of hmmIBD was used with the same parameters. In this version, the fraction of sites that are IBD, which is normally a free parameter in the model, was kept fixed at 50% while identifying IBD segments. This was done to remove a bias in the hidden Markov model, which is more likely to identify segments as IBD when the overall relatedness of the two samples is high. hmmIBD was modified by deleting line 949 of hmmIBD.c, version v2.0.4 (‘pi[0] = count_ibd_fb / (count_ibd_fb + count_dbd_fb);’). Pileup of IBD segments across sample pairs was then calculated in 1 kb windows; a sample pair was considered IBD within that window if an IBD segment overlapped the window at all.IBD for estimating pairwise relatedness was calculated with hmmIBDTo identify a minimum IBD fraction that would reliably signal close relatedness between parasites, we generated IBD distributions for two sets of sample pairs, one enriched for related pairs, based on time and location of sampling, and the other similarly depleted. For this purpose, we used an expanded dataset that included sequence data from Thiès (SLP) from prior years. The enriched sample set contained pairs of parasites where each parasite was from 2019 and from one of three sites with a high degree of relatedness: SLP, SES, and TBA. For the depleted set, the parasites in each pair had to have been sampled at least ten years apart and one of them had to come from a site other than the three listed above. We compared the distribution of IBD fractions between the two sample sets, making the comparison separately for pairs with high sequencing coverage (>50% of SNPs with at least 5x coverage) and for those with at least one parasite with lower coverage. Based on the comparison between the distributions  +  * , where clonal relatedness is the fraction of all pairs that are clones and partial relatedness is the fraction of non-clone pairs that are partially related. When calculating diversity and relatedness within sites and as a function of binned distance Fig. , sites wWe empirically evaluated methods for calculating confidence intervals for pairwise partial relatedness within study sites. To do so, we took the expanded dataset mentioned above (this dataset plus earlier sequence data from Thiès), restricting sites to TBA, SLP, SES, and SMS in order to approximate the relatedness seen within one site, and treated the dataset as a population with measured relatedness. From it, we repeatedly drew sample sets of specified size to simulate the sample set for one of our sites and evaluated how often a particular method for calculating confidence intervals contained the true value. A standard bootstrap approach was anticonservative and biased, the latter because sampling with replacement introduces spurious relatedness. The method we identified as the most accurate was repeated downsampling of the test set without replacement, specifically, downsampling to 55% of the initial sample. Performing a similar test with the full set of barcodes, we identified 60% as the appropriate downsampled size for estimating CIs for pairwise clonality. When determining the final confidence intervals, downsampling was repeated 1000 times for each CI. Confidence intervals for relatedness between sites were determined by simple bootstrapping.Network diagrams of related parasites were created with the software package Gephi, version 0.9.2.R2adj, which corrects for the number of explanatory variables in the regression.Ordinary least squares regression was performed with the Python package statsmodel.api. The incidence was log-transformed because of the wide range in incidence values, because fold changes at all incidence levels are relevant to malaria control efforts, and because of the highly nonlinear relationship between incidence and genetic measures. The goodness of fit was assessed by Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Malaria and tuberculosis remain highly prevalent infectious diseases and continue to cause significant burden worldwide. Endemic regions largely overlap, and co-infections are expected to occur frequently. Surprisingly, malaria-tuberculosis co-infection is relatively understudied. Malaria has long been known to have immunomodulatory effects, for example resulting in reduced vaccination responses against some pathogens, and it is conceivable that this also plays a role if co-infection occurs. Data from animal studies indeed suggest clinically important effects of malaria-tuberculosis co-infection on the immune responses with potential consequences for the pathophysiology and clinical course of both infections. Specifically, rodent studies consistently show reduced control of mycobacteria during malaria infection. Although the underlying immunological mechanisms largely remain unclear, an altered balance between pro- and anti-inflammatory responses may play a role. Some observations in humans also support the hypothesis that malaria infection skews the immune responses against tuberculosis, but data are limited. Further research is needed to unravel the underlying immunological mechanisms and delineate possible implications of malaria-tuberculosis co-infection for clinical practice. Malaria and tuberculosis (TB) remain among the leading infectious diseases causing morbidity and mortality worldwide. About one-quarter of the world’s population is infected with TB, and in 2021 an estimated 10.6 million people developed TB disease, with approximately 1.6 million deaths . MoreovePlasmodium parasites are transmitted to humans through the bites of infected female Anopheles mosquitoes. The injected sporozoites move to the liver, invade hepatocytes, and mature and multiply. Once the infected hepatocytes burst open, merosomes containing merozoites are released into the bloodstream with exponentially increasing parasitaemia due to repeated invasion and multiplication in erythrocytes. Symptoms may occur as soon as parasites occur in the blood [P. falciparum infection may result in severe anaemia, cerebral malaria, acute respiratory distress syndrome, organ failure, or death in non-immune individuals [Plasmodium parasite [Malaria has a complex life cycle. The protozoan he blood . If not ividuals . Some blparasite .Mycobacterium tuberculosis (MTB) bacilli are transmitted through aerosols from an infected individual to another person. In most immunocompetent individuals, the mycobacteria are phagocytosed by macrophages and contained after inhalation, resulting in latent infection [nfection . Acute pnfection , 13. Abonfection .TB and malaria are ancient infections, with co-infections demonstrated using molecular techniques in mummified remains from lower Egypt dating back to circa 800 BC . Some auP. falciparum parasitaemia [It is difficult to ascertain the prevalence of co-infections, with a considerable variation in reported rates. In a cross-sectional study in the early 2000s in Mwanza, Tanzania, newly diagnosed patients with TB were screened for co-infections. Out of 655 patients with TB, 4.1% had malaria parasites in their blood. This did not differ significantly between the microbiologically confirmed and clinically diagnosed TB groups, nor did the geometric mean parasitaemia, with 378 and 928 parasites/µl, respectively . In a sisitaemia . In Limbsitaemia . Co-infesitaemia . The larIt is conceivable that malaria/TB co-infection is a common scenario in endemic regions, but there is limited data on its clinical implications. The clinical presentation of malaria varies with age in endemic areas. Young children often experience severe symptoms, including high fever and anaemia, and are at a higher risk of mortality. In contrast, older individuals may exhibit asymptomatic parasitaemia, due to acquired immunity over time.M. tuberculosis, it supports the notion that malaria co-infection could be a clinically important phenomenon.A study in Guinea-Bissau demonstrated that malaria prevention through bed nets, cotrimoxazole prophylaxis during the wet season and insecticide spraying resulted in a reduction of mortality from 26 to 19% in patients admitted with TB . Even thA study in Cameroon showed a trend toward lower haemoglobin (Hb) levels in malaria/TB co-infected patients compared to mono-infections. While the average Hb was 13.4 g/dl (SD 2.6) for patients with TB and 11.4 g/dl (SD 2.3) for malaria, the average Hb was lower with 10.4 g/dl (SD 2.6) in co-infected patients . This stPlasmodium falciparum and TB in a 34-day-old child, indicating that clinicians should be aware of this possibility in all age groups [Another study showed a reduction in total white blood cell count, neutrophils and lymphocytes in patients with malaria/TB co-infection compared to TB mono-infection , althouge groups .An important clinical consideration when treating co-infections involves the side effects and potential drug interactions. Since endemic areas largely overlap and both diseases are highly prevalent, concomitant treatment may occur. One important issue is QT-prolongation in the case of fluoroquinolones and many anti-malarials . FluoroqNaturally-acquired immunity against malaria develops gradually after repeated exposure occurring over the years. This is evidenced by the observation that young children suffer the largest disease burden in endemic countries. Antibodies that prevent the sequestration of infected erythrocytes and inhibit the inflammatory cytokine response probably play an important role here. With increasing age, presumably due to repeated infections, children and adults become resistant to severe disease . On a poPlasmodium have not been elucidated entirely yet but include splenic removal of infected erythrocytes, opsonin-independent phagocytosis by macrophages and interferon-gamma (IFN-γ) production by NK cells [Immunity to malaria is complex, inherent to the different intra- and extracellular lifecycle stages of the parasite. Innate responses against NK cells . MoreoveNK cells .Adaptive immunity develops gradually due to repeated infections and involves both humoral and cellular immunity. Antibodies are important in the protection against malaria, as demonstrated elegantly in the 1960s through the adoptive transfer of serum from immune to naïve individuals . AntibodAlthough much is still unclear about the role of various T-cell subsets in anti-malaria immunity, they appear to play a critical role against both pre-erythrocytic (sporozoites/liver stages) and erythrocytic parasites . In animThere is also a role for the host immune response in malaria pathology, as demonstrated by the correlation between the severity of the disease and levels of pro-inflammatory cytokines such as TNF-α , 37. IndThe immune response to MTB varies according to age and involves both innate and adaptive immune system components. Macrophages, neutrophils, natural killer cells, and dendritic cells, along with airway epithelial cells, are the first to respond to the inhaled tubercle bacilli . Once myTNF-α facilitates macrophages to phagocytose and kill mycobacteria and is important in containing infection, reflected by the increased risk of TB in individuals receiving anti-TNF-α treatments . GranuloM. tuberculosis [About one-quarter to a third of the world’s population is estimated to be infected with rculosis . In apprrculosis . The incrculosis , 46.Salmonella typhi, they generated significantly fewer specific antibodies than healthy controls [Malaria has long been known to have immunomodulatory effects. This is illustrated by observational data on vaccination responses and viral, bacterial and parasitic co-infections. In the 1970s, it was shown that if children with acute malaria infection were vaccinated against meningococcus or controls . Even ifcontrols .The presumed role of Epstein Barr Virus (EBV) and malaria co-infection in the pathogenesis of Burkitt lymphoma prompted a study that demonstrated significantly higher EBV loads in young children in a region with holoendemic malaria transmission compared to children of the same age from an area with sporadic malaria transmission . AlthougPlasmodium in their blood than in those who had not [An association between non-typhoid salmonella septicaemia and recent malaria infection was found in children from the Gambia . Moreove had not .Trichuris muris is suppressed if they are simultaneously infected with the murine malaria parasite Plasmodium berghei [In mice, the expulsion of the nematode  berghei .Plasmodium falciparum sporozoites while on chloroquine prophylaxis can induce sterilizing protection against subsequent controlled challenge [Immunization of healthy subjects with viable hallenge . Howeverhallenge .et al. investigated co-infection dynamics in a non-lethal C57BL/6 mouse model of disseminated M. bovis BCG and Plasmodium chabaudi chabaudi AS [in vitro follow-up experiment, MTB proliferated in macrophages approximately two-fold higher when co-cultivated with P. falciparum-infected red blood cells compared to uninfected red blood cells [Animal models have contributed significantly to understanding the immunological effects of malaria/TB co-infection, consistently showing reduced control of MTB during malaria infection. Hawkes baudi AS . A higheod cells .et al. established a rodent model of MTB and P. berghei co-infection [P. berghei NK65-infected mosquito bites. MTB load in the lungs of co-infected mice was significantly higher after 12 days compared to control mice. Moreover, co-infected mice had more severe lung pathology with increased inflammation [et al., who used a P. yoellii-MTB co-infection model, showed that co-infected mice had increased MTB load in their lungs, spleen and liver and had increased mortality, with a concurrent increased number of IFN-γ producing CD4 T cells [P. yoelii, these findings were confirmed. When sacrificed after fifty-one days, co-infected mice showed a slight increase in MTB burden in the lungs, spleen, and liver, suggesting that malaria infection limited the control of MTB. It has been difficult to disentangle the underlying immunological factors, but it probably involves an altered balance between pro- and anti-inflammatory responses since co-infected mice showed an increased number of immune cells and levels of both pro- and anti-inflammatory cytokines such as IFN-γ and IL-10 [Plasmodium appears to influence granuloma formation, delaying this process in a P. yoelii-BCG co-infection model in BALB/c mice [Mueller nfection . They chammation . Also, S T cells . In anotnd IL-10 . MoreoveB/c mice .Reduced CD4+ T cell counts and TNF-α blockage can trigger TB reactivation, but much remains unknown. Interestingly, in mice with latent BCG infection, malaria infection resulted in the reactivation of mycobacterial infection with inflammation in the spleen and liver, a rise in mycobacterial counts and disorganization of granulomas .Plasmodium berghei nor Plasmodium chabaudi infection abrogated BCG-induced protection against TB, even though the malaria infections significantly affected both B- and T-cell immunity such as central memory T lymphocyte apoptosis and reduction of marginal zone and follicular B-cell populations [P. yoelii infection in C57BL/6 mice in another study [The effect of malaria on BCG-induced protection against TB appears to be limited. In a C56BL/6 mouse model, neither ulations . Moreoveer study .M. tuberculosis and Plasmodium yoelii 17XL resulted in a lower peak parasitaemia and increased survival compared to P. yoelii 17XL mono-infection [P. berghei ANKA in C57BL/6 mice, nor were there any significant alterations in cytokine and T-cell responses [The effect of TB co-infection on the course and pathophysiology of malaria is less clear. Co-infection of C57BL/6 mice with nfection . Enhancenfection . In anotesponses . These oIn humans, data on immunological aspects of malaria/TB co-infection are limited. A cross-sectional study from Nigeria showed a significant reduction of IL-10 and IL-6 in co-infected patients compared to TB mono-infection. TFN-α levels were not different, and IFN-γ was not measured . The autex vivo data, however, suggest an effect of malaria on TB-specific immune responses. For example, the lymphoproliferative response to purified protein derivate (PPD) was lower in the majority of healthy adults during the transmission (wet) season compared to the dry season in a study in Sudan [Plasmodium falciparum-infected individuals suppressed the in vitro lymphoproliferative response to Plasmodium antigens and PPD [Some in Sudan . Why thi and PPD . This wa and PPD .An evaluation of data from refugee health assessments in Perth, Australia, showed that having malaria was associated with a three-times higher chance of having an indeterminate QuantiFERON-TB Gold IFN-γ release assay (IGRA) result . This waStimulation of PBMCs isolated from Nigerian adolescents and adults with PPD showed similar IFN-γ production but increased IL-4 and IL-10 secretion in malaria/TB co-infected subjects compared to individuals with TB mono-infection . This fiPlasmodium parasites [Results on the effect of placental malaria on PPD-specific responses are conflicting. A study in Gambian infants suggested a reduced proportion of PPD-specific IFN-γ producing CD4+ T cells in infants with histological evidence of placental malaria compared to healthy controls . In anotarasites .Plasmodium blood stage infection, Treg cell populations expand, and their frequencies are associated with improved outcomes and lower parasitaemia [Regulatory T (Treg) cells are a subset of CD4+ T cells defined by expression of the transcription factor FOXP3 and have been recognized to have an important role in regulating immune homeostasis . By limisitaemia . Due to sitaemia , 79. Simsitaemia . And agaBabesia microti and P.berghei yoelii 17X malaria [P. berghei challenge [P. yoelii 17XL challenge in A/J mice concurrent with a shift towards Th1 response with higher levels of IFN-γ and lower levels of IL-4 in BCG-vaccinated mice compared to controls and enhanced the production of Plasmodium-specific IgG2a antibodies. Neutralization of IFN-γ abrogated protection [P. yoelii rodent model, parasitaemia was significantly lower in BCG-vaccinated animals. Subsequent depletion experiments showed a critical role for both CD4+ and CD8+ T cells [There is ample experimental evidence for the relative protection of BCG vaccination against malaria, most likely through a non-specific modulation of the immune response. Animal studies from the 1970s demonstrated a nonspecific protective effect of BCG vaccination against  malaria . This wahallenge . A follootection . In anot T cells .P. berghei ANKA-induced experiment cerebral malaria [Plasmodium species. C57Bl/6 mice had higher levels of parasitaemia and increased mortality from P. c. chabaudi AS infection if they had received BCG four weeks earlier, associated with an increased proportion of CD4 + CD25 + Foxp3 + regulatory T cells and reduced levels of IFN-γ and TNF-α [BCG also protected C57Bl/6 mice from  malaria . Interesnd TNF-α .P. falciparum in a controlled human malaria infection model, although BCG-vaccinated individuals developed earlier symptoms and more robust inflammatory responses, suggesting some extent of trained immunity [Some observational data support the hypothesis that BCG induces non-specific protection against malaria in humans. For example, in Guinea-Bissau, analysis of mortality patterns showed that having a BCG scar significantly reduced the risk of death from malaria . Similarimmunity .Malaria/TB co-infection is a surprisingly underexplored area, given the high prevalence of both infections and the large geographical overlap between endemic regions. A substantial amount of animal data suggests clinically important effects of co-infection on the immune response with potential consequences for diagnosis, pathophysiology and clinical course of both infections. Some observations in humans support these hypotheses, but data are limited, possible implications for clinical practice remain unclear, and many questions remain. Does malaria infection trigger the reactivation of TB in humans? Does malaria influence TB disease severity and vice versa? Knowledge evolution on both infections has suffered from a relative lack of funding compared to diseases with increased impact in high-income countries. Immunity to either infection is highly complex and incompletely understood, making it challenging to disentangle the crosstalk. Still, a systematic investigation of the pathophysiology of malaria/TB co-infection could provide important insights. This could include prospective studies evaluating the effects of co-infection and treatment on clinical and immunological outcomes, the effect of malaria on TB disease progression, and the interaction of infections with vaccination. Improved understanding may lead to more targeted interventions that could have a major impact on both diseases.iqad008_Supplementary_DataClick here for additional data file.