Iron deficiency (ID), the leading cause of anemia and the most common nutritional deficiency globally, is not well reported among children in malaria-endemic settings, and little is known about its contribution to anemia in these settings. We aimed to assess the prevalence of anemia, the role of ID using multiple parameters, and the factors associated with anemia in a malaria-endemic rural area. We conducted a community-based cross-sectional study of 432 children aged 1–5 years from the Popokabaka Health Zone, Democratic Republic of Congo. Sociodemographic characteristics, medical history, anthropometric parameters, and biochemical parameters were considered. Hemoglobin and malaria prevalence were assessed using rapid finger-prick capillary blood testing in the field. Venous blood samples were analyzed for serum ferritin, serum iron, total iron-binding capacity, and C-reactive protein (CRP) in a laboratory. Anemia was found in 294 out of 432 (68%) patients. Malaria was found in 375 out of 432 (87%), and ID in 1.8% according to diagnosis by adjusted ferritin only and in 12.9% according to transferrin saturation. ID indicators were not significantly correlated with low hemoglobin levels. Malaria, fever, and CRP > 5 mg/L were major factors associated with anemia in Popokabaka. Anemia control should focus on treating inflammatory conditions and infectious diseases among children in such settings. Anemia, which is characterized by a hemoglobin level below 11.0 g/dL, continues to be a serious global public health problem that particularly affects young children and pregnant women . AccordiWhile multiple factors are known to cause anemia, the literature ,6,7,8 shHowever, according to the WHO , half ofPlasmodium falciparum is the most prevalent malaria-causing species found in DRC [We conducted a community-based cross-sectional study in the Popokabaka Health Zone, DRC, between May and June 2019. This region is an entirely rural area located in Kwango Province, where the population is facing food insecurity and poverty . The head in DRC . Availabd in DRC . The divA total of 432 children aged 12–59 months were included in this study, based on a proportional sample size calculation for anemia prevalence = 0.59, a precision d of 0.075, a design effect of two, and an expected response rate of 0.80. Children were selected using a three-stage cluster sampling technique. First, we selected five clusters  among the nine accessible areas through a probability-proportion-to-size technique. The term “accessible” considered the time constraints for collecting venous blood samples from the field and being able to process them within 3 h at the Popokabaka Hospital. Only 9 of the 25 health areas fitted this technical constraint. Second, we randomly selected 3 villages in each cluster. In the last stage, an equal number of 30 households having children aged 12–59 months old were systematically selected from a detailed list pre-established by community workers in each village. Only one child from each household was selected and assessed. A parent had to provide written consent for their child to participate in the study. Children for whom the parents refused blood drawing or children who were hospitalized for any diseases in the two previous weeks were excluded.Data were collected using a questionnaire completed on tablet computers using the Survey CTO collect application. The questionnaire consisted of eight modules: household characteristics; water, hygiene, and sanitation (WASH); household food security ; child health history; IYCF; anthropometric measures; dietary practices ; and biochemical sampling.Data collection took two consecutive days in each cluster, three teams working in parallel for each of the three selected villages of the cluster. A team comprising three data collectors and two phlebotomists was formed. On the first day, the data collectors visited the households, obtained consent forms, and completed the surveys for each child selected in the village. The biological mother or caretaker responded to the questionnaire while anthropometric measurements were taken for the child. When finished, the personnel provided a card to the mother with the correct personal identifier for blood collection the next day. On the second day, the phlebotomists went to the same villages at a specific identified location  from 7 to 9 am for blood collection and hemoglobin and malaria rapid testing. The mothers were then invited to bring the children and the cards for blood collection.The data collectors were trained by the investigator on the survey questionnaire and interview techniques, and the phlebotomists were trained by a lab expert on appropriate standard operating procedures (SOPs) and blood sample management.The phlebotomist first performed a capillary finger-prick test for hemoglobin (Hgb) assessment (Hemocue 301) and a rapid test for malaria in the field. The information was recorded using tablet computers and the Survey CTO application. Additionally, the phlebotomist collected up to 6 mL of venous blood from the child using trace-element-free serum BD vacutainers (BD-368380) and powder-free sterile disposable gloves. Tourniquet application took a maximum of one minute. The collected blood was allowed to clot for at least 30 min in the field at room temperature and was then transported to the Popokabaka Hospital within 3 h. There, it was centrifuged, and the supernatant was separated at 2300 rpm for 10 min using a Hettich Rotor 32A centrifuge . The serum was aliquoted into two polypropylene vials: 0.5 mL tricoded FluidX, Brooks Life Science vials and 2 mL Sarstedt vials.All the vials were immediately stored in a −40 °C freezer that worked nonstop on solar energy during the day and on a generator overnight in Popokabaka. When the survey was complete, the samples were transported from Popokabaka to Kinshasa (a 12 h vehicle trip with self-transported fuel) stored in liquid nitrogen. Then, every sample was stored at −80 °C in an ultra-low freezer at the Kinshasa School of Public Health for a week before being shipped on dry ice to Norway.All the 0.5 mL vials were sent to Haukeland University Hospital  for the analysis of serum ferritin (using electrochemiluminescence immunoassay (ECLIA)), C-reactive protein (S-CRP) (using the immunoturbimetry method), and total iron-binding capacity . Two-milliliter vials were sent to the Norwegian University of Life Sciences  for the analysis of serum iron and other minerals (using the Agilent 8900 Triple Quadrupole inductively coupled plasma mass spectrometer (ICP-MS)).Transferrin saturation (TSAT), expressed as a percentage, was then calculated as the value of serum iron divided by the TIBC.Anthropometric indices including the weight for height, height for age, weight for age, and mid-upper-arm circumference for age and their Z-scores were calculated using the WHO Anthro software. Wasting was defined as a weight-for-height Z-score (WHZ) < −2, stunting was defined as a height-for-age Z-score < −2, and underweight was defined as a weight-for-age Z-score < −2. Wealth index quintiles were generated using principal component analysis on ownership variables in the household. The food consumption score was calculated using data from the Food Frequency Questionnaire (FFQ) and categorized as adequate, borderline, and inadequate consumption. The dietary diversity variable was generated from 24 h food recall, capturing the intake of common food items in the area. The Household Food Insecurity Access Score (HFIAS) and its categories were also calculated.Anemia was defined as Hgb levels < 11 g/dL . Accordi3+) levels by the TIBC. A child is said to have ID when TSAT is <20% [We also estimated ID by calculating TSAT by dividing the serum iron  < 0.20. Multicollinearity was checked for all the covariates included in the regression model. All the statistical analyses were performed at the 0.05 level of confidence.All the data were merged and analyzed using STATA 14.0. We performed a descriptive analysis. The median and 25th–75th percentiles are reported for continuous variables that were not normally distributed, while the mean (SD) is used for continuous variables that were normally distributed. Frequencies are used to describe categorical data. The confidence intervals (CIs) for prevalence were calculated using a normal Z-test. Moreover, an independent-sample t-test or the Wilcoxon rank-sum test were used to compare, respectively, mean or median values. A Pearson chi-squared test was used to test for association, with anemia as the outcome. The Spearman correlation coefficients were used to describe the relationships between the iron biomarkers and hemoglobin concentrations. Linear regression was used to adjust ferritin for inflammation (CRP). Bivariable and multiple logistic regression were used to identify factors associated with anemia, and the crude and adjusted odds ratios (ORs) were reported. The regression model was constructed by a forward stepwise selection approach with covariate inclusion probability  affected, and every tenth (11%) child exhibited wasting. Only one in three had a diversified diet, and approximately 3% of the children were defined as being obese.Plasmodium falciparum rapid testing. We found that anemia was also common, at 68.1% , with severe anemia seen among 3.3%  of the children. The prevalence of malaria in Popokabaka was 86.8%  according to p value = 0.001).Half of the children (49.4%) had signs of inflammation, with CRP > 5 mg/L. This proportion was significantly higher among anemic children than nonanemic children . In another approach, accounting for inflammation as proposed by the BRINDA project , we founWe estimated the IDA prevalence and the contribution of ID to anemia, and the former 7.5% according to transferrin saturation and 1.5% according to adjusted ferritin. Thus, the remaining anemia cases were not iron-deficiency-related.Hgb was not correlated with transferrin saturation (TSAT) but showed a weak, negative correlation with ferritin (r = −0.17) and serum iron (r = 0.07) . In addin = 8280) according to the latest Demographic and Health Survey in 2014 [In the present study, we demonstrated the burden of ID and anemia using multiple biomarkers for a representative community-based sample of children in a poor part of the DRC, which, to the best of our knowledge, had not been assessed before. Our study found that anemia was highly prevalent (68.1%) among children aged < 5 years, while ID was remarkably low . This anemia prevalence seemed much higher than the previously reported national prevalence  administered at home to anemic children by parents in Popokabaka, even though only 35% of those who took part in our study reported that their children had received iron supplementation in the previous three months .In a recent review, Camaschella et al.  explaineWe estimated the association between anemia and other factors in a multivariate logistic regression analysis, and we found that fever in the previous two weeks, malaria status, and inflammation status (CRP > 5 mg/L) were factors associated with anemia. Taking into account the weak correlation of hemoglobin with iron biomarkers, we hypothesized from these results that the remaining cases of anemia could be explained or have been caused by malaria or other diseases that we did not explore in our study. Unfortunately, we did not assess other illnesses such as helminthic infections ,27. ChapPlasmodium falciparum. This could have underestimated the true prevalence of malaria in terms of parasite count. In a review of how malaria was associated with anemia, White [We used rapid malaria tests to determine the prevalence of parasitemia based only on the presence of a, White  stated tIn our study, anemia prevalence was indifferently distributed within nutritional parameters—food insecurity, dietary diversity, and anthropometric indicators—which did not explain any of the variation in anemia. This finding is in contrast to the summary findings reported by the WHO , indicatThe present study had several strengths. This was a community-based biochemical study for a hard-to-reach rural settlement with many sample-conservation constraints. We obtained a representative study population from Popokabaka through a probabilistic sampling scheme with a large sample size. We demonstrated that iron deficiency might not be the main cause of anemia in such malaria-endemic areas, that malaria itself or other inflammatory conditions may be leading factors. We examined multivariate regression models and checked for multicollinearity to validate the associated factors found.However, the study had several limitations. The study population was restricted to technically accessible areas in the study area, as explained in Despite the fact that the interpretation of Hgb concentrations requires some adjustments  as recommended by the literature ,30, for Finally, anemia has complex pathways, and there are likely to be other important factors involved that were not measured.Iron deficiency should be considered prevalent in populations when its prevalence is more than 20%. In this study, we demonstrated a low iron deficiency prevalence within a context of high anemia and malaria prevalence in Popokabaka. ID was not a risk factor for anemia in this study. However, malaria was a major contributor to anemia. Further research with more hematological biomarkers is needed to characterize this anemia, which was not explained by ID. Due to the findings from this study, we suggest that anemia control strategies in Popokababa focus on malaria prevention and other childhood infectious-disease controls for effective impact.


Low birthweight (LBW) is a worldwide problem that particularly affects developing countries. However, limited information is available on its magnitude in rural area of Burkina Faso. This study aimed to estimate the prevalence of low birthweight and to identify its associated factors in Nanoro health district. A secondary analysis of data collected during a cross-sectional survey was conducted to assess the prevalence of low birthweight in Nanoro health and demographic surveillance system area (HDSS). Maternal characteristics extracted from antenatal care books or by interview, completed by malaria diagnosis were examined through a multi-level logistic regression to estimate odd-ratios of association with low birthweight. Significance level was set at 5%. Of the 291 neonates examined, the prevalence of low birthweight was 12%. After adjustment for socio-demographic, obstetric and malaria prevention variables, being primigravid , or multigravid with history of stillbirth , as well as the lack of long-lasting insecticide treated bed net use by the mother the night preceding the admission for delivery  were significantly associated with neonate low birthweight. The number of antenatal visits however did not confer any direct benefit on birthweight status within this study area. The prevalence of low birthweight was high in the study area and represents an important public health problem in Burkina Faso. In light of these results, a redefinition of the content of the antenatal care package is needed. Indeed, low birthweight—a birthweight below 2500 g—is an important risk factor for neonatal deaths3, which particularly affects developing countries where malaria transmission is endemic4. Sub-Saharan Africa is particularly affected due to the high risk of maternal infections during pregnancy6. Malaria, an example of these infections, causes up to 20% of all low birthweights in this region and 6% of neonatal deaths7. Thus, potentially, 100,000 infant lives could be saved each year if adequate malaria prevention measures were implemented. In Burkina Faso, malaria is the primary reason for seeking health care, and pregnant women are particularly affected.Despite halving the global neonatal mortality rate from 36.6 to 18.0 per 1000 live births between 1990 and 20178. IPTp-SP consists of monthly administration of sulfadoxine-pyrimethamine (SP) in pregnant women from the second trimester until delivery8. The strategy is effective in reducing malaria incidence and its consequences9. Burkina Faso adopted the strategy in 2005, with a minimum of three doses of sulfadoxine-pyrimethamine administered under the supervision of a healthcare worker10.To reduce malaria-related low birthweights in Sub-Saharan Africa, the World health organization (WHO) recommends a set of measures for pregnant women during antenatal care visits, which include the intermittent preventive treatment of malaria using sulfadoxine-pyrimethamine (IPTp-SP)3, little information is available on its prevalence in developing countries such as Burkina Faso. Indeed, at the start of the IPTp-SP policy in Burkina Faso, the prevalence of low birthweight was 15.8%11. Five years later, an evaluation reported a prevalence of 13.4%12. Since then, no other evaluation was conducted despite the numerous concerns raised by the low coverage of IPTp-SP strategy13 and the spread of Plasmodiumfalciparum resistance to sulfadoxine-pyrimethamine throughout the African continent14. Thus, a re-evaluation of the prevalence of adverse birth outcomes is necessary to timely seek alternative approaches. Therefore, this study aimed to assess the prevalence of low birthweight in rural Burkina Faso ten years after the IPTp-SP policy was adopted.Although low birthweight adverse effects on neonatal survival have been well established15. Nanoro is situated in the centre-west region of Burkina Faso at about 90 km from Ouagadougou, the capital city, and has a population of 63,000 inhabitants15. Malaria transmission is holo-endemic, with peak transmission overlapping with the rainy season (June-November). The commonest malaria vectors are Anopheles gambiae, A. funestus, A. arabiensis and P. falciparum is the predominant malaria parasite. Malaria is the main reason for visiting health centres with a case-fatality rate between 5 and 30%16.The study was conducted in all eight peripheral health centers of the Nanoro health and demographic surveillance system (HDSS) area17. All eight peripheral health centers of the HDSS were included. The study site’s map is described elsewhere15. For each enrolled pregnant woman, their babies were assessed within 24 h of delivery. Demographic, gyneco-obstetric, and relevant medical history data were collected from antenatal care books or face-to-face interviews with the mother during both high and low malaria transmission seasons.We conducted a secondary analysis of data collected among pregnant women attending health centers for delivery in the Nanoro HDSS area from September 2013 to April 2014. The primary study of this analysis was an ancillary topic annexed to a main project (NCT01703884) entitled ‘ANC & Malaria Diagnostic in Pregnancy” and aimed to improve the quality of antenatal care and diagnostic services for malaria in pregnancyPregnant women aged 15–45 years, attending the health center for delivery with a gestational age of at least 37 weeks, living in the Nanoro HDSS area, and that provided written informed consent were eligible for inclusion. Those who delivered non-singleton babies, not willing to participate, or whose neonate was transferred before documentation of birthweight were not included. Of the 323 women included in the primary study, 9.9% (32/323) were excluded  and 291 (90.1%) were included.11. We hypothesized that the new policy would reduce the low birthweight proportion to 10% after ten-year of widespread implementation. The Cochran formula n = Z2 *p*(1-p)/i2 was used to calculate the sample size where p = 10% is the expected proportion, i = 3.5%, represents the margin of error, Z the z-score that corresponds to the 95% confidence interval (1.96), n is the minimal sample size. The minimum required sample size was 282. This was a secondary analysis of database collected on 291 participants.At the time of policy change, term LBW proportion was 15.8% of all live birthsPf® test strips (sensitivity 99.7% and specificity of 99.5%). Table 19.Data were extracted from antenatal care books and by face-to-face interviews. Maternal age, educational level, occupation, and sulfadoxine-pyrimethamine uptake were collected from the antenatal care books. The use of insecticide-treated net (ITN) the night before visiting the health facility for delivery or any other variables not available in the antenatal care book were obtained by interview of the mother. Gestational age was estimated using the knowledge of the last menstrual period (LMP), or the Ballard score whenever LMP was unknown. Neonate examination was conducted within 24 h of delivery either in the health facility or at home by trained midwives. Birthweight was measured using a calibrated electronic scale with 10 g resolution and a precision less than 10 g . In addition, a malaria test using peripheral blood was performed by rapid diagnostic tests SD-Bioline Malaria Antigen Data were collected on a paper-based questionnaire by trained midwives and double-entered onto Redcap (Research—Electronic Data Capture) data collection tool, then imported into Stata version 15  for cleaning and analysis. Categorical variables were summarized on frequency tables. Mean or median with respective measures of spread, including standard deviations and quartiles, were used to summarize numerical variables. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated according to each maternal factor using univariate logistic regression. Adjusted odds ratios (aOR) and 95% CI were derived by a backward multivariate logistic regression analysis of variables with statistically significant p-values at the univariate analysis, except for malaria. The final model retained variables which p-values were statistically significant. Variable age was not included due to its strong correlation with gravidity. A p-value < 0.05 was considered statistically significant.All data were anonymized before publication, thus consent for publication was not applicable.Ethical clearance for this study was obtained from the National Ethics Committee of Burkina Faso. All data were anonymized before analysis. All participants or their authorized legally representatives provided a written informed consent prior to their participation. All methods were performed in accordance with the relevant guidelines and regulations.Table Of the 291 neonates, the mean birth weight (± standard deviation) was 2933 ± 390 g and 12.0% (35/291) live births were low birthweight. Table Multi-level logistic regression analysis included variable gravidity, the use of insecticide-treated bed nets the night before hospital admission for delivery, and malaria test results. Being primigravid , multigravid with an obstetrical history of stillbirth  and the lack of bed net use  were significantly associated with higher proportion of low birthweight among neonates.11. Several factors, including the mother's socioeconomic status, obstetrical characteristics, and malaria prevention methods, influence low birthweight prevalence, and those factors differed from each setting. Indeed, lower low birthweight rates were observed in Ethiopia (10%)20 and in Nigeria (7%)21. However, these estimates were understated as the Nigerian’s study used data collected retrospectively from the routine surveillance system, which is not adapted to SSA as half of neonate birthweights are not recorded due to weaknesses of the reporting system4. Also, the study in Ethiopia was conducted in urban areas where women's economic level tends to be higher than those from the rural areas, and the impact of socioeconomic status on the prevalence of low birthweight is well established23.Low birthweight prevalence was high in Nanoro given that those from preterm births were not included in this analysis. However, the report showed a substantial decrease (3.8 percentage points) from its level at the start of the IPTp-SP strategy24 and in a study conducted in Zimbabwe (16.7%)25. These could be related to the higher prevalence of human immuno-deficiency virus (HIV) infection26. Indeed, HIV is considered an important risk factor of LBW, although the mechanism is not yet fully elucidated27.In contrast, rates in excess were reported in South Africa (38.5%)28.It is reported that maternal obstetrical history and events occurring during pregnancy impact the outcome of pregnancies, although those factors are not always identified29 and increasing risk of low birthweight, or because some physiologic changes are less efficient during first pregnancies thus increasing the risk of low birthweight30. Indeed, uteroplacental blood flow, responsible for delivering oxygen and nutrients to the fetus, is greater during subsequent pregnancies than in the first pregnancy32. Also, we have noted that mothers with multiple pregnancies were also at higher risk if there was an obstetrical history of stillbirth. Chen et al. in 2018 and Rozi et al. in 2016 made such observations in their respective studies34, while Ahrens et al. went further to notice that the risk was highest after a history of three stillbirths35. Particular attention should be paid to pregnant women with such background characteristics during their antenatal care visits.We found that the order of pregnancy was significantly associated with low birthweight and first pregnancy infants were at higher risk than others. The biological ground for first pregnancy neonates to be at higher risk of low birthweight may be related to the susceptibility of first pregnancy placentas to parasite-infected red blood cells sequestration, disrupting fetus nutrients supply36. Bed net usage has long been recommended in areas with sustained malaria transmission as an effective tool to prevent malaria-related adverse birth outcomes, including low birthweight37. Although bed net is freely provided to pregnant women during the first antenatal care visit in Burkina Faso, a high proportion of women (up to 20%) were not covered, and actions toward improving bed net usage are required.As expected, the lack of long-lasting insecticide-treated bed net usage was associated with an increased risk of low birthweightPlasmodium falciparum resistance to sulfadoxine pyrimethamine reported in many parts of sub-Saharan Africa and Burkina Faso could be a limiting factor of the efficacy of IPTp-SP strategy41. Indeed, the high number of triple mutants, and the presence of quintuple mutants (triple dhfr and double dhps mutations) reported in Burkina Faso may have decreased the efficacy of sulfadoxine-pyrimethamine on P. falciparum in the study settings, and thus the effects of the IPTp-SP strategy on low birthweight42.The number of antenatal care visits performed and the intermittent preventive treatment of malaria in pregnancy did not directly influence the prevalence of low birthweight in this study. Although the limited sample size did not allow an accurate evaluation of all low birthweight factors, 43. Studies building on the most accurate diagnosis methods are needed in this study setting.Contrary to what is reported in the literature, malaria was not associated with low birthweight in the current study. The use of malaria rapid diagnosis test could have understated the actual extent of malaria infection in the study population and thus, underestimated the extent of malaria negative effects on the fetusA number of limitations in this study are worth noting and taken into consideration. The study was conducted on term pregnancies excluding low birthweight from preterm deliveries, which could understate the problem's actual extent. It remains to bear in mind that gestational age was dated from parental knowledge of the last menstrual period (LMP) or the Ballard score, which could be prone to some errors. The use of malaria RDT for the diagnosis in peripheral blood sample could have understated the extent of malaria infection in the study population and thus undermine the strength of association with low birthweight. Although the analysis had limitations, it depicted the overall picture of the magnitude of low birthweight in rural Burkina Faso.Low birthweight prevalence was high in Nanoro and required more effective policies. Exploring novel approaches or improving current approaches is needed. But first, quality data building on larger sample size are necessary to update maternal risk factors of low birthweight.


Plasmodium falciparum (Pf) malaria induced by circulating asexual parasites. Although clinical manifestations of inflammation are associated with parasite density, this relationship is influenced by a complex network of immune-modulating factors of both human and parasite origin.Fever and inflammation are a hallmark of clinical Pf-infected mosquito bites  or intravenous administration of Pf-infected red blood cells .In the Controlled Human Malaria infection (CHMI) model, we compared clinical inflammation in healthy malaria-naïve volunteers infected by either in vitro response of subsets of peripheral blood mononuclear cells showed attenuated Pf-specific IFNγ production by γδ T-cells in the BS-arm.All volunteers developed patent parasitaemia, but both the incidence and duration of severe adverse events were significantly higher after MB infection. Similarly, clinical laboratory markers of inflammation were significantly increased in the MB-group, as well as serum pro-inflammatory cytokine concentrations including IFN-γ, IL-6, MCP1 and IL-8. Parasite load, as reflected by maximum parasite density and area under the curve, was similar, but median duration of parasitaemia until treatment was longer in the BS-group compared to the MB-group (8 days [range 8 – 8 days] versus 5·5 days [range 3·5 – 12·5 days]). The Pf-infections by MB induce stronger signs and symptoms of inflammation compared to CHMI by BS infection. The pathophysiological basis remains speculative but may relate to induced immune tolerance.In conclusion, irrespective the parasite load, The trial was supported by PATH's Malaria Vaccine Initiative; the current analyses were supported by the AMMODO Science Award 2019 (TB). We conducted an online literature search on inflammation or severity of symptomatology following human malaria infection using PubMed for articles published up to October 1st, 2021. Articles were searched using the terms "Malaria"[Mesh] AND ("Virulence"[Mesh] OR "Inflammation"[Mesh] OR "disease severity" OR "tolerability") AND  and screened on title and abstract. Of the 397 screened articles, 15 contained information on inflammation or disease severity following mosquito bite or blood-stage infection in humans specifically. None of the articles included a side-by-side analysis of symptoms or signs of inflammation in humans following infection by mosquito bite and blood-stage infection. Some articles referenced in the screened publications were also included, but contained mostly information on non-falciparum strains in animal models. These studies were mutually contradictive and provide no clear leads for pathophysiologic mechanisms underlying a difference in disease severity resulting from different infection routes.Pf stages; sporozoite and blood-stage. This study adds new insights into the pathophysiology of malaria. Our combined findings suggest that exposure to initially low densities of blood-stage parasites induces a level of tolerance towards Pf parasites, leading to significantly milder symptomatology and laboratory abnormalities following blood-stage infection compared to infection by mosquito bites.Here, we directly compare clinical inflammatory reactions to controlled human malaria infection (CHMI) commencing at two distinct Recognising and understanding this phenomenon is of significant importance for the understanding of general malaria pathophysiology and parasite-host interaction. Clinical trials using the controlled human malaria infection model should carefully consider the implications of the chosen infection route on the clinical symptoms and inflammation parameters.Alt-text: Unlabelled boxPlasmodium falciparum (Pf) infection is characterised clinically by marked systemic inflammation that is generally associated with circulating asexual parasite density. However, even in malaria-naïve individuals without pre-existing immunity, presentation and severity of clinical symptoms following Pf infection can be heterogeneous due to modulation of host immunity.Pf gametocytes, the life-cycle stage responsible for malaria transmission from the human host to the mosquito vector. In order to achieve a predefined density of asexual parasitaemia acting as source of these gametocytes, volunteers were subjected to distinct infection regimens; volunteers received either mosquito bites from laboratory-reared Pf-infected mosquitoes (MB-group) or intravenous inoculation of asexual blood-stage parasites (BS-group) from an established master cell bank.,We recently conducted a controlled human malaria infection (CHMI) in malaria-naïve Dutch volunteers with the aim of generating in vitro stimulation.Here we report on the contrasting clinical parameters and signs of systemic inflammation in these two groups, which were monitored by number, severity and duration of adverse events, as well as markers of inflammation in the circulation and in peripheral blood mononuclear cells (PBMC) after An open-label, single centre, randomised, trial was conducted at the Radboud university medical center  as described before.Pf 3D7 isolate, or by intravenous injection of ∼2,800 Pf 3D7 infected red blood cells. Volunteers in the MB-group received a sub-curative treatment of piperaquine (480mg) when parasitaemia reached 5,000 parasites/mL as defined by 18S qPCR, or at an earlier timepoint if pre-defined safety criteria were met. All volunteers of the BS-group received sub-curative treatment of piperaquine (480mg) on day 8 post-infection based on earlier findings In short, twenty-four healthy, malaria naïve volunteers (18 – 35 years) provided informed consent and were screened and enrolled to receive infection by either bites of 5 mosquitoes infected with the Original primary and secondary study outcomes focussed on safety and transmissibility to mosquitoes as previously described in detail.Adverse events were recorded from inclusion until day 51 post-infection, and graded for severity; grade 1 (mild) defined as: awareness of symptoms that are easily tolerated and do not interfere with usual daily activity; grade 2 (moderate) defined as discomfort that interferes with or limits usual daily activity; and grade 3 (severe) defined as disabling, with subsequent inability to perform usual daily activity, resulting in absenteeism or requiring bed rest. For each AE a start and stop date and time was recorded to calculate its duration. Relatedness to the trial procedures (CHMI and drug treatment) was categorised for each adverse event as not related, possibly related or probably related. Only adverse events that were possibly or probably related to the trial were included in the current analysis. Local adverse events at the site of injection or mosquito bite were recorded but not included in these analyses.Clinical laboratory parameters including complete blood count, C-reactive protein, liver enzymes  and troponin-T were determined at predefined timepoints during the trial. Laboratory normal values are represented in supplemental table 1. Parasitaemia was quantified by 18S qPCR as described before.PBMCs and sera were collected and stored as previously described.Inflammatory cytokines  were determined in serum prior to challenge infection (C-1), 7 or 9 days after infection (C+7 for BS and C+9 for MB), at time of treatment (T), as well as two (T+2) and four (T+4) days thereafter, using a flow-based multiplex kit  measured on a Beckman-Coulter Gallios flow cytometer.+ human serum  and plated at a concentration of 0·5 × 106 PBMCs/well in 96-well U-bottom plates. PBMCs from each volunteer were stimulated in duplicate with 1 × 106 NF54 parasitised red blood cells (PfRBC) per well for a ratio of 2 PfRBC:PBMC for 24h at 37°C in 5% CO2. PBMCs were also stimulated with 1 × 106 uninfected RBCs (uRBCs) per well as a control. CD107a-Pacific Blue  monoclonal antibody was added for the duration of the stimulation period. For the last 4 h of stimulation, 10µg/mL Brefeldin A  and 2µM monensin  were added to each well. PBMCs were stained with fixable viability dye eFluor 780  and subsequently with: CD14-FITC , pan-γδ T cell receptor-PE , CD69-PerCP-Cy5.5 , HLA-DR-PECy7 , CD86-APC , CD56-AF700 , CD3-PB  and CD16-BrilliantViolet510 . Cells were permeabilised using FoxP3 Fix/Perm kit  and then stained with IFN-γ- PE-DazzleCF594  and resuspended in 1% paraformaldehyde in PBS. All samples were measured on a 10-colour BC Gallios (Beckman Coulter) using Kaluza 4.0 (Beckman Coulter). Uninfected RBC background measurements for each volunteer were subtracted from the corresponding PfRBC measurement.Cryopreserved PBMCs collected at baseline and prior to treatment  were thawed, counted using a haemocytometer and 0·1% Trypan Blue, and resuspended in culture media consisting of Dutch modified Roswell Park Memorial Institute (RPMI) 1640  media supplemented with 2mM Glutamax , 0·05M gentamycin, 1mM pyruvate, and 10% AAnalyses were performed using SPSS, Flowjo and Graphpad Prism 5. The Mann-Whitney U test was used for comparison of continuous variables between study arms; for dichotomous variables the Fisher's exact test was used. For comparison of variables at different timepoints within groups, the Wilcoxon signed-rank test was used. The area under the curve of asexual parasite density over time (AUC) was computed with the (ΔX)*(Y1 +Y2)/2 formula in GraphPad Prism 5. Cytokine concentrations were calculated using Graphpad Prism 5 using a 5 parameter logistic curve with log-transformed data and 12-point standard curves for each cytokine standard. Sample sizes were based on the primary objectives of the trial and allowed the detection of a proportion of infectious individuals of 34% or higher.PATH's Malaria Vaccine Initiative supported the original trial, was involved in the trial design and approved the final manuscript. AMMODO science award had no role in the design, conduct or analysis of the trial.Randomisation resulted in balanced groups as demonstrated by comparable baseline characteristics as shown previously.9/L). All other subjects in the MB-group received treatment based on the predefined treatment threshold of >5,000 parasites/mL. Median peak parasitaemia, generally observed half a day after start of treatment, was 32,807 parasites/mL  for the MB-group and 27,700  for the BS-group. Median area under the parasitaemia curve (AUC) for MB was 37,654  and for BS 38,735 . Neither the peak nor the AUC of the asexual parasitaemia were statistically significantly different between groups  while all BS-infected participants were treated on day 8 post-infection. The duration of detectable parasitaemia was shorter in the MB-group  compared to the BS-group (8·0 days)  and lymphocyte count . In particular grade 3 liver enzyme abnormalities, considered a marker of systemic inflammation in uncomplicated malaria ,All volunteers experienced adverse events (AEs), but none were serious adverse events. The median number of AEs per subject  did not differ significantly between groups, with 13·5 AE/subject (range 9 – 25) in the MB-group and 11 AE/subject (range 5 – 35) in the BS-group . However, the number of severe (grade 3) adverse events was significantly lower in the BS-group . In addition, the median duration per AE  was significantly shorter in the BS-group . Moreover, the median duration per severe AE was also significantly lower in the BS-group . Fever, defined as a temperature ≥38·0°C, occurred slightly more frequently in the subjects infected by MB (12/12) compared to BS (9/12), though this was not statistically significant . At time of treatment, temperature was significantly different between the two groups  in MB-infected subjects than in BS subjects . MCP1, Iin vitro with Pf-infected RBC at different study time points . The proportion of Pf-specific IFN-γ+ cells were assessed by flow cytometry, focusing in particular on the innate(-like) γδ T-cell, NK-cell and monocyte populations in both study groups. While total numbers of γδ T-cells, NK-cells and monocytes remained stable over the two respective time points, there was a decrease in the percentage of responding IFN-γ+ cells in the BS-group at day 7 post-infection  parasitaemia), most pronounced and reaching significance in the γδ T-cell population (Pf-responding IFN-γ+ cells in the MB-group did not differ at C+9 (i.e. after only 2 days of detectable parasitaemia) compared to baseline. We did not detect significant differences in monocyte or lymphocyte activation parameters including HLA-DR and CD86 or CD69, respectively (not shown). The combined data show an in vitro Pf-hypo-responsiveness over time in particularly γδ T-cells in the BS-subjects.To further dissect the nature of the inflammatory response, BS and MB subjects’ PBMC were stimulated P. falciparum 3D7 parasite line in malaria-naïve volunteers, despite similar asexual parasite densities. This comprises the more frequent occurrence and longer duration of grade 3 adverse events in the MB-group. Objective measures including temperature and clinical parameters of inflammation such as CRP, platelet and lymphocyte counts and liver enzymes also suggest more severe systemic inflammation in the MB-infected subjects. The difference in inflammatory status between the two groups is further confirmed by significant differences in a number of circulating cytokines . The mechanism behind these observations remains speculative, but the host response to parasite kinetics may play an important role. In the BS-group, with a gradual increase of parasite densities over 8 days starting at unphysiologically low parasite densities -innate PBMC responsiveness in the BS-group may play a role, thereby modulating the severity of clinical symptoms. The decreased IFN-γ production after stimulation with PfRBCs in vitro may be due to exposure to relatively low numbers of blood-stage parasites over a sustained time period. This observation is most pronounced in the γδ T-cell subset previously identified as one of the main IFNγ-producers in response to PfRBC.in vivo observations of milder symptomatology, lower levels of inflammatory parameters and cytokines (including IFN-γ) in the BS-group. Yet, attenuation of induced inflammatory responses is not reflected by an increase of circulating IL-10 in the BS-group , suggesting that this cytokine does not represent the immunological mechanism that prevents inflammation. The kinetics of parasite exposure in BS may induce a tolerogenic or immunoregulatory effect through (an) alternate immunological pathway(s), that in turn limit(s) the development of inflammation. As we considered the elapsed time from first exposure to parasites to the sampled time points too short to expect significant adaptive responses, our current flow analysis focused on (semi-)innate immunity. It would however be interesting to include regulatory cellular immune responses in future investigations.Our ,It should be noted that a combination of factors might be responsible for the observed difference in clinical inflammatory signals. In addition to human host factors, differences in parasite characteristics may play a role including parasite multiplication rate or virulence as a result of mosquito- or liver passage. For instance, changes do occur in variant surface antigen (VSA) expression profile after mosquito passage.Plasmodium species, increased disease severity is observed after  blood passage of asexual blood-stages.,P. chabaudi model by mosquito passage of the parasites.th century, blood passage of P. vivax parasites through the human host was frequently used to treat patients suffering from neurosyphilis. Given the large numbers of patients receiving this therapy, it is remarkable that only few literature reports describe increased pathogenicity after blood passage through the human host.,Interestingly and contrary to our findings, in rodent In this trial transmissible gametocytaemia was only observed in BS-volunteers.A number of possible limitations can be considered. The post-hoc analyses were not part of the original study objectives, sample sizes were small and the trial was conducted in a single centre. Although the observed inflammatory and symptomatic differences between the study groups were confirmed by multiple parameters, for unequivocal evidence of induced tolerogenic responses in γδ T-cells, this should be confirmed by a second dataset.Pf-infections by MB induce stronger signs and symptoms of inflammation compared to CHMI by BS-infection at similar parasite densities. The pathophysiological basis remains speculative but may relate to immune tolerance induced by prolonged low density parasitaemia following BS-infection. These results are of importance for our understanding of parasite-host interaction. Finally, these results highlight that caution is needed when interpreting and comparing safety and efficacy results obtained from CHMI trials with different infection routes.In conclusion, Isaie J Reuling, Christian F Ockenhouse, Katharine A Collins, Teun Bousema and Robert W Sauerwein designed the study. Manon Alkema, Gerdie M de Jong, Isaie J Reuling, Quirijn de Mast, Reinout van Crevel, Teun Bousema and Robert W Sauerwein conducted the clinical trial.X Zen Yap, Matthew BB McCall, Robert W Sauerwein and Manon Alkema designed, performed and analysed the laboratory experiments. X Zen Yap, Matthew BB McCall, Robert W Sauerwein and Manon Alkema wrote the first draft of the manuscript, had access to and verified the data. All authors contributed to the final manuscript, had full access to the data and accept responsibility to submit for publication.RS received consulting fees from Biomedical Primate Research Centre, Rijswijk, The Netherlands and has stocks in TropIQ Health Sciences, TropIQ, the Netherlands. MA was supported by PATH's Malaria Vaccine Initiative. ZY was supported by the AMMODO science award 2019 awarded to TB. All other authors: No reported conflicts.


Plasmodium, has several known etiologies of hemolytic anemia as a possible complication in cases such as concurrent G6PD deficiency, severe parasitemia, or use of parenteral antimalarials. Although artemisinin-based antimalarial therapies are generally well-tolerated, several cases of severe post-artemisinin delayed hemolysis (PADH) have been recently reported, which present a diagnostic challenge, and affect morbidity and mortality in patients with malarial infection. We highlight the case of a young lady with Plasmodium falciparum severe parasitemia who developed hemolytic anemia after parenteral artesunate therapy.Malaria infection, which results due to the parasitic protozoan  Plasmodium. The Plasmodium falciparum and Plasmodium vivax cause the majority of human malarial infections, with P. falciparum being the more virulent [Malaria is a major health problem worldwide. It results from infection by parasitic protozoans belonging to the genus virulent .Malaria affects many countries, especially those with poor public health resources, and it has been reported that the incidence of the disease in 2004 was between 350 and 500 million cases. Over two billion people, representing more than 40 % of the world's population, are at risk of contracting malaria, and the number of deaths due to malaria worldwide has been estimated at 1.1–1.3 million annually in reports by World Health Organization (WHO) between 1999–2004 [Malaria typically presents with high fever, which may be accompanied by chills and headache. Symptoms and signs may be generally be more subtle, and may also include anemia and hepatosplenomegaly. Patients with malaria may also experience respiratory distress and/or rapidly progressing cerebral malaria, manifesting as altered sensorium and, infrequently, as seizures. For initial diagnosis, thick blood smears are performed; however, a single smear showing no evidence of parasites is not sufficient to exclude malarial infection. To identify the type of parasitic species, thin blood smears are used . ManagemPlasmodium falciparum infection worldwide, and has resulted in saving more patients with severe malaria than the previous reference drug quinine. Use of artesunate is associated with fatality rates consistently lower than 5%; however, a considerable proportion of patients are affected by hemolytic episodes occurring after parasitological cure. Such episodes are referred to as post-artemisinin delayed hemolysis (PADH) [The parenteral artemisinin drug, artesunate, is a first-line treatment for severe s (PADH) .PADH is linked to the destruction of circulating infected red blood cells (i-RBCs) from which, after artesunate therapy parasites have been removed by “pitting” in the spleen, thereby deemed as once-infected RBCs (O-iRBCs) .Here, we present this rare case of post-artesunate delayed hemolysis to highlight diagnostic challenges, to improve patient care, management plan and result in better treatment outcomes.We present the case of a previously healthy 35-year-old female, who presented to our emergency department (ED) complaining of high-grade fever of waxing-and-waning nature for five days, associated with vomiting, body aches and sleepiness. She works at a local bank and developed fever during her mandated hotel quarantine upon return from a holiday in Sudan seven days ago, during which she was not on malaria prophylaxis. COVID-19 PCR from nasopharyngeal swab was negative. She did not improve despite anti-pyretics and anti-emetics during the hotel quarantine, and sought medical advice at a private clinic where she tested positive for malaria and was referred to our hospital for inpatient management.Patient reported that she had previous history of malaria 10 years ago but not as severe. She had no other sick contact in her family or friends.Plasmodium falciparum with a parasitemia level of 27 %. She was admitted to medical intensive care unit (MICU) for further management under close monitoring due to severe malaria with overlapping features of sepsis and acute respiratory distress syndrome (ARDS). In line with guidelines for malaria management, she was started on parenteral artesunate, which was administered until the parasite level decreased to less than 1%. She was then started on oral artemether-lumefantrine for five days thereafter, and the malaria parasite level at the end of the treatment was less than 0.1 %. In the meantime, she received other supportive treatment including intravenous fluids, antiemetics, antipyretics and antibiotics.Her initial labs showed hemoglobin of 12.1 gm/dl, normal white blood cell count of and platelets of 150 with elevated AST and ALT of 58 and 57U/L, respectively and high total bilirubin of 26 μmol/L. Malarial smears were positive for She was transferred to medical ward after MICU care, and had significant improvement in her condition, except for persistent fever and a gradual decline in her hemoglobin.3/μL, but improved without intervention. Liver transaminases also increased up to 150sU/L and total bilirubin was 35 μmol/L. Labs revealed increased lactate dehydrogenase (LDH) of 595 U/L (reference range: 135–214 U/L) and low haptoglobin reported as less than 10 mg/dl (30−200 mg/dl). Coombs’ test was positive with low complement 4 (C4). Subsequently, all liver function tests improved within one week.Labs later in the hospital course revealed continuous gradual drop in hemoglobin from 12 g/dL to 6.5 g/dL and she received multiple packed red blood cell transfusions. Her hemoglobin would initially increase post-transfusions, followed by a decrease again within one to two days. She continued to spike fever at least once per day and temperatures ranged from 38.5 to 40C and she was on as-needed paracetamol infusion. Sepsis workup including blood cultures and urine culture were negative. She had bilateral pleural effusion, more prominent on the right side associated with dyspnea and increased requirement for oxygen supplementation, thus piperacillin-tazobactam was initiated empirically and she underwent therapeutic and diagnostic thoracentesis on the left side – as the right-sided effusion was complicated with consolidation and collapse. Pleural fluid analysis revealed exudative effusion with predominantly neutrophilic leukocytosis.In the next few days, she continued to spike fever and had persistent drop in hemoglobin, due to which antimicrobial coverage was escalated to tigecycline to treat suspected empyema, for which right-sided effusion was drained by interventional radiologist, and pleural fluid analysis showed the same results as those from left pleural effusion. Once the results of all cultures from pleural fluid and peripheral blood were negative, the antibiotics were discontinued.Despite all measures, there was no further improvement in her fever. Further workup to investigate possible fever of unknown origin included ultrasound of the abdomen to evaluate for any intrabdominal collections and it showed an ill-defined hypoechogenicity in the pancreatic head  and multAs no significant improvement was noted in her clinical condition, further workup was performed to exclude connective tissue disease, involving ANA which was positive. Direct antiglobulin test was also positive, along with high LDH and low haptoglobin, confirming autoimmune hemolytic anemia. Bone marrow aspiration (BMA) was not performed as the patient declined the procedure after discussion of benefits of BMA in diagnosis and potential risks of the procedure including hematoma and bleeding, especially considering thrombocytopenia. After ruling out all other causes including hemolytic anemia due to autoimmune etiologies or related to sepsis, along with resolution of the fever, the delayed autoimmune hemolytic anemia was deemed most likely attributable to parenteral artesunate in our case.After one-week follow-up, she had stable hemoglobin and her fever chart showed no more spikes with good over all condition as per the patient.As per our conclusions, with accumulating available information, it appears our patient had a rare case of artesunate-induced delayed hemolysis by exclusion of all other possible diagnoses and we highlight the challenges of diagnosis of PADH due to its rare occurrence and as the concept is not yet well-recognized.Plasmodium falciparum. Intravenous artesunate is the recommended parenteral therapy by WHO for severe malaria [Anemia is a common complication of severe malaria and it can be caused by the parasite or the antimalaria medications ,6. Malar malaria  as studi malaria . Althoug malaria .The mechanism of artesunate-induced hemolysis, referred to as post-artemisinin delayed hemolysis (PADH), is not fully understood but it is speculated to be secondary to the destruction of infected RBCs after killing the parasite and usually occurs anytime between few days to four weeks after receiving the medication . Thus, iWhile the etiology of anemia in malaria is multifactorial , autoimmPlasmodium falciparum malaria with parasitemia level of 23 %. She received three doses of IV artesunate and showed good clinical response along with her reduction in parasitemia level to less than 0.1 % after 3 days. Despite this improvement, she continued to produce dark urine, her hemoglobin level declined, LDH increased and haptoglobin decreased, and moreover, Coombs’ test was positive with low complement 4 (C4). She improved after packed red blood cell transfusion. After few days, her hemoglobin dropped again and responded to blood transfusion once more. Coombs’ test repeated after 1 week was positive again. Further workup to exclude other underlying causes such as connective tissue disease and lymphoma were all negative. Her case was thoroughly discussed with the hematologist, who recommended supportive management as she had completed her antimalarial therapy course and no other clear causes of hemolytic anemia were identified.Our patient was admitted as a case of severe With supportive measures, her hemoglobin level improved, and she was thus discharged with outpatient clinic follow-up after four weeks, upon which she had improved hemoglobin and stable but low C4.We can conclude that this uncommon presentation and its diagnosis is a challenging feat for physicians to suspect clinically and exclude other possible etiologies, along with prolonged hospital course and sometimes, extensive investigations. The authors would like to emphasize the important key points in this case for physicians to be aware of this subtype of hemolytic anemia associated with intravenous artesunate therapy in patients managed for malaria, which thereby requires close observation and careful supportive management.Plasmodium falciparum malaria. Blood transfusions, supportive measures and close follow-ups are helpful in appropriate management; however, more studies are required to ascertain the demographics, possible predictors and associated factors of PADH for a better understanding of the concept to facilitate diagnosis and curb morbidity and mortality.Post-artemisinin delayed hemolysis (PADH) is a rare cause of prolonged autoimmune hemolysis in patients with severe Qatar National library funded the open access publication fees of this case.Obtained for publication by local medical research committee.Written informed consent was obtained from the patient for publication of this case report.Writing the initial draft of the manuscript: Sundus Sardar, Mohammed Abdurabu.Conceptualization and supervision: Anand Kartha, Khalid Farooqui, Sundus Sardar.Medical management of the case: Anand Kartha, Mohammed Abdurabu, Sardar Sundus, Ahmed Abdelhadi, Muhammad Bilal Jamshaid, Khalid Farooqui.Additions to manuscript: Adnan Humam Hajjar, Munir Abu Ageila, Tasnim Abdalla, Mhd Baraa Habib.Revising the manuscript critically and literature review: Sundus Sardar, Khalid Farooqui.The first authors (SS and MA) contributed equally to the writing and preparation of this article. SS, MA, AA have written the initial draft of the manuscript and performed the literature review. The draft was revised and updated by SS with supervision from AK and KF. The authors AK, SS, MA, AA, MBJ, AHH, MAA, TA, MBH, KF were all part of the medical treating team and submitted additions to the manuscripts. All the authors critically reviewed the initial and the final draft of the manuscript and approved it for submission.The authors report no declarations of interest.


Despite being preventable and treatable, malaria still puts almost half of the world’s population at risk. Thus, prompt, accurate and sensitive malaria diagnosis is crucial for disease control and elimination. Optical microscopy and immuno-rapid tests are the standard malaria diagnostic methods in the field. However, these are time-consuming and fail to detect low-level parasitemia. Biosensors and lab-on-a-chip devices, as reported to different applications, usually offer high sensitivity, specificity, and ease of use at the point of care. Thus, these can be explored as an alternative for malaria diagnosis. Alongside malaria infection inside the human red blood cells, parasites consume host hemoglobin generating the hemozoin crystal as a by-product. Hemozoin is produced in all parasite species either in symptomatic and asymptomatic individuals. Furthermore, hemozoin crystals are produced as the parasites invade the red blood cells and their content relates to disease progression. Hemozoin is, therefore, a unique indicator of infection, being used as a malaria biomarker. Herein, the so-far developed biosensors and lab-on-a-chip devices aiming for malaria detection by targeting hemozoin as a biomarker are reviewed and discussed to fulfil all the medical demands for malaria management towards elimination. Plasmodium parasites, is one of the most life-threatening infectious diseases worldwide, with a significant impact on human lives. Most malaria cases occur in tropical and sub-tropical developing countries, where poverty limits access to proper healthcare conditions and infrastructures . T. T91]. Tnfection . In thisnfection . Neverthnfection .Myrand-Lapierre and colleagues developed a multiplexed fluidic plunger to evaluate the deformability of RBCs through microscale funnels within a microchannel . Later, P. falciparum-infected RBCs and, in less than 2 min, were able to distinguish 1% of infected RBCs from non-infected RBCs [6/µL of blood, the detection limit  is not competitive with microscopy and RDTs yet, nor do the authors specify whether this sensor operates based on hemozoin detection. Nevertheless, the sensitivity and specificity of SAW sensors might be improved by their coating with absorptive materials [Recently Wang et al.  designedted RBCs . The autted RBCs . Despiteaterials . Despiteaterials .Furthermore, Graham et al.  proposedCatarino and colleagues, after demonstrating that the absorbance spectra of synthetic hemozoin and hemoglobin is different, developed a first prototype of portable optical microdevice for hemozoin-based malaria detection and quantification ,100. TheP. falciparum and 35 parasites/µL of P. vivax patients samples with 95% and 100% accuracy, respectively [P. vivax-predominant regions [Recently, Kumar et al. developed the magneto-optic Gazelle device D for hemectively . Gazelleectively ,104. Theectively . Similar regions ,106. Nev regions ,104.Current malaria diagnostic methods are time-consuming and fail to detect low-level parasitemia in both asymptomatic and symptomatic patients. Nevertheless, diagnosis at the point-of-care is of vital concern since it provides information regarding parasitemia, allowing disease control, and might contribute to better patient care and treatment. Several methods have been targeting prompt and accurate malaria diagnosis, and despite quite promising approaches that have been developing, there is still room for improvement ,12,13,14Particularly, the heme detoxification pathway, which goes from hemoglobin degradation to hemozoin formation, presents several players that could be exploited for biosensors-mediated malaria diagnosis. Hemozoin is formed as parasites invade the host RBCs, its concentration correlates with disease progression, and it is present in the malaria transmitters gametocytes. Therefore, it is a unique biomarker for infection identification. Moreover, the hemozoin crystal itself presents specific features that, coupled with the technology of biosensors, could result in the development of an indispensable device that would fulfil all the medical demands for malaria management and elimination. Consequently, in recent years, hemozoin has been actively explored for diagnosis ,103,107.Therefore, the current biosensors based on this biomarker still hinder the success of biosensors for the aimed simple, sensitive, and low-cost malaria detection. Nevertheless, an instrument might be expensive at first (when buying) but low-cost over time, bringing the potential for point-of-care diagnosis. For example, Kong et al. report that their device might cost several thousand USD, but then the cost per assay would be less than USD 0.50 . This isThe currently proposed biosensors and lab-on-a-chip devices for hemozoin detection show interesting results , despite requiring significant performance improvement for field applicability. Thus, improving the current sensing strategies and unveiling the hemozoin formation pathway might render targets for the development of both antimalarial drugs and new biosensors and lab-on-a-chip devices for diagnosis. Therefore, by considering the currently available literature, we believe that biosensors and lab-on-a-chip devices based on hemozoin detection might be, indeed, the future in malaria control and elimination.


Plasmodium falciparum cases. Although there are historical reports of Plasmodium vivax and Plasmodium malariae, today, non-falciparum infections would remain undetected because of extensive use of falciparum-specific histidine-rich protein 2 (HRP2) rapid diagnostic tests (RDT) at health facilities. A recent case–control study was conducted in Grand’Anse to identify risk factors for P. falciparum infection using HRP2-based RDTs . Post hoc multiplex Plasmodium antigenemia and antibody (IgG) detection by multiplex bead assay revealed one blood sample positive for pan-Plasmodium aldolase, negative for P. falciparum HRP2, and positive for IgG antibodies to P. malariae. Based on this finding, we selected 52 samples with possible P. malariae infection using IgG and antigenemia data and confirmed infection status by species-specific PCR. We confirmed one P. malariae infection in a 6-month-old infant without travel history. Congenital P. malariae could not be excluded. However, our finding—in combination with historical reports of P. malariae—warrants further investigation into the presence and possible extent of non-falciparum malaria in Haiti. Furthermore, we showed the use of multiplex Plasmodium antigen and IgG detection in selecting samples of interest for subsequent PCR analysis, thereby reducing costs as opposed to testing all available samples by PCR. This is of specific use in low-transmission or eliminating settings where infections are rare.Haiti is targeting malaria elimination by 2025. The Grand’Anse department in southwestern Haiti experiences one-third to half of all nationally reported  However, affordable methods for identifying these infections in routine surveillance are currently not available. PCR remains impractical at large scale owing to costs, processing time, and the lack of appropriate laboratories in many endemic settings. Multiplex bead assays (MBAs) can be used to rapidly collect antibody and antigen data at scale, thereby selecting samples of interest for confirmation by PCR.14 Because of the limited incremental costs of adding non-falciparum and/or pan-Plasmodium targets to a P. falciparum antigen and antibody detection panel on an MBA, this is a highly cost-effective approach to identify the presence of possible non-falciparum infections.In some cases, P. falciparum, although there are historical reports of P. malariae and Plasmodium vivax.16 Evidence for P. malariae was found in Haitian refugees arriving in Jamaica in 2004.17 Haiti and the Dominican Republic, sharing the island of Hispaniola, aim to eliminate malaria by 2025 . Although malaria elimination activities target P. falciparum and P. vivax, the elimination goal includes all Plasmodium species. As P. malariae has caused outbreaks decades after apparent successful elimination in other regional settings , it is important to know whether this parasite is endemic. Following the devastating 2010 earthquake in Haiti, rapid diagnostic tests (RDTs) were deployed for malaria diagnosis to supplement microscopy, which was available at some locations in-country. Based on the historical high rates of P. falciparum,16 RDTs detecting P. falciparum–specific histidine-rich protein 2 (HRP2) were deployed; thus, non-falciparum infections would be undetected by the primary diagnostic test being used in the majority of Haitian health facilities. Before the earthquake, national malaria prevalence was considered low, though highly focal,21 but there were concerns about underreporting due to a lack of access to diagnosis and a weak surveillance system.23Today, malaria transmission in Haiti is primarily due to P. falciparum malaria cases in recent years . A case–control study that was conducted as part of operational research efforts to identify risk factors for P. falciparum offered an opportunity to test for the presence of non-falciparum malaria species.24 Post hoc laboratory quantification of HRP2 and pan-Plasmodium aldolase (pAldolase) antigens allowed for selection of a subset of samples with possible P. malariae infection for species-specific testing of parasite nucleic acids. We aimed to determine the presence or absence of P. malariae infection in the selected subset of samples collected in Grand’Anse.Recent efforts have improved national reporting and access to malaria diagnosis by RDTs. The Grand’Anse department in southwestern Haiti has experienced one-third to half of all the nationally reported 24  and Plasmodium falciparum 19-kDa fragment of merozoite surface protein 1 (PfMSP-119) for all study participants  using the MAGPIX platform . Unique bead regions  were coated by the EDC/Sulfo-NHS intermediate reaction with anti-HRP2  or anti-pAldolase .13 Biotinylated detection antibodies  were prepared at a final concentration of 1 mg/mL using the EZ-Link Micro Sulfo-NHS-Biotinylation Kit . Beads and detection antibodies were transported to LNSP and stored at 4°C.A MBA was performed to collect HRP2 and pAldolase antigenemia and IgG data to 25 Dried blood spots were punched (6 mm discs) and eluted overnight in buffer B  to a final sample dilution of 1:20 whole blood. Bead mix was prepared in buffer A , beads added to plates  aiming for 800 beads/region/well, and washed ×2 with 100 µL wash buffer . The samples (50 µL) were incubated with the beads for 90 minutes  and subsequently washed ×3. Beads were incubated with detection antibodies  for 45 minutes. The plates were washed ×3 and then incubated with streptavidin–phycoerythrin  for 30 minutes. The plates were washed ×3 and incubated with a final 30-minute wash step with buffer A. The plates were then washed ×1, resuspended in 100 µL PBS, shaken briefly, and read on a MAGPIX instrument (Millipore Sigma), generating median fluorescence intensity (MFI) with a target of 50 beads/region/well. Median fluorescence intensity values were corrected for responses of a blank well (containing buffer B), providing an MFI-bg signal for analyses. The threshold of positivity was set at the mean +3 SD of the MFI-bg signal of a panel of known negative DBS samples from U.S. residents without history of travel in the last 6 months.25 Data were successfully collected for 1,081 participants for PfHRP2 (98%) and 1,059 for pAldolase (96%). Data were excluded for either target if providing a bead count of < 20 within an assay well.At LNSP, an MBA was used to collect antigenemia data, as previously described.191928 with bead preparation, wash, and incubation methods, as described previously. Proteins were covalently coupled to beads at 20 µg per 12.5 × 106 beads. Dried blood spots were punched (3-mm disc) and eluted in buffer B at 1:100 approximate serum concentration. Bead mixture was prepared, added to plates aiming for 600 beads/region/well, and washed ×2. Sample (50 µL) and anti-IgG  were incubated simultaneously overnight, and the plates were read the next day after washing ×3 and resuspending in PBS. Median fluorescence intensity was corrected for buffer B responses  and log10-transformed. A two-Gaussian mixture model of log-transformed data was used to determine the threshold for seropositivity, set at the mean +3 SD of the lower distribution. Sufficient bead counts were available for 1,071 participants for PfMSP-119 and 1,070 for PmMSP-119 (97%).IgG to PmMSP-1Plasmodium antigenemia and IgG results (n = 52) were stored at −20°C until shipment at ambient temperature to the CDC in Atlanta, GA, where they were stored at −20°C until processing.Samples for species specification by PCR were selected according to two sets of criteria using  results . Dried b29 DNA was extracted from DBSs using the Qiagen Mini Kit (Qiagen). PCRs for Plasmodium genus, P. falciparum, and P. malariae was performed in 20 μL reactions containing ×2 TaqMan Environmental buffer 2.0 . For the genus reaction, 0.5 μL of both forward and reverse (which was labeled with FAM dye) primers at 250 nM were included in each reaction. For the P. falciparum primers, 0.5 μL forward primer at 250 nM and 0.25 μL reverse primer at 125 nM (which was labeled with HEX dye) were included. For the P. malariae primers, 0.5 μL of both forward and reverse (which was labeled with FAM dye) primers at 250 nM were included in each reaction and the sequences were forward (5′-AAGGCAGTAACACCAGCAGTA-3′) and reverse (5′-agg cgc ata gcg cct ggTCCCATGAAGTTATATTCCCGCTC-3′) primers. PCR occurred with the following cycling parameters: initial hot-start at 95°C for 15 minutes, followed by 45 cycles of denaturation at 95°C for 20 seconds, annealing at 63°C for 40 seconds, and final elongation at 72°C for 30 seconds. The cycle threshold (CT) values were recorded at the end of the annealing step. All assays were performed using an Agilent M ×3005pro qPCR system (Agilent Technologies). Positivity of DNA amplification was set at CT < 40.0. The sensitivity and specificity of the PET-PCR assay compared with nested PCR is 92% and 100%, respectively, with an estimated lower limit of detection of two parasites/µL blood.29At the CDC, a PET-PCR assay was performed, as previously described.The study protocol was approved by the National Bioethics Committee of the Haitian Ministry of Public Health and Population (1718-20), Tulane University (2017-366), and the London School of Hygiene and Tropical Medicine (14556). The study protocol and institutional ethical determinations were reviewed and approved by the U.S. CDC, Office of the Associate Director of Science at the Center for Global Health, and CDC investigators were determined not to be engaged in human subject research.Consent from all participants was recorded electronically on tablets during recruitment at health facilities and confirmed at follow-up interview. Consent for individuals younger than 18 years was given by a parent or guardian. Mature minors  were able to provide consent directly.Plasmodium antigenemia and IgG data were successfully collected for 1,048 case–control study participants (95%). Data were excluded if insufficient bead counts were available for any of the targets . In total, 841 participants were negative for HRP2 and pAldolase (HRP2−/pAldolase−); 116 were positive for HRP2 and pAldolase (HRP2+/pAldolase+); 90 were HRP2+/pAldolase−; and one was HRP2−/pAldolase+. In addition, high IgG levels to PmMSP-119 were seen in the study population , 6- to 10-year olds: 9% (15/163), 11- to 20-year olds: 18% (35/195), 21- to 40-year olds: 28% (71/253), and > 40-year olds: 38% (61/160).Using an MBA, pulation . Age-spe19  but might also be mixed P. falciparum/P. malariae infections or single P. falciparum infections as 98% were HRP2+ (42/43) and 95% were seropositive to PfMSP-119 . Alternatively, these could represent a mixed P. falciparum/P. malariae infection below the lower limit of detection for pAldolase and/or HRP2 for which seroconversion to PfMSP-119 has not taken place (yet). Lastly, these could represent a single P. falciparum infection  for which seroconversion to PfMSP-119 has not taken place (yet).Selection 2 comprised children ( aged < 15 years) seropositive to PmMSP-19 n = 9; , removinP. malariae infection , he would have remained undiagnosed and untreated for malaria. Plasmodium malariae infections can lead to hospitalization and have had fatal outcomes even in well-resourced settings.1 The fact that no travel history was reported suggests that the infection was locally acquired and thus that P. malariae is circulating in this part of Haiti. However, the possibility of congenital malaria could not be excluded as we did not have a sample available from the infant’s mother. It may be possible that the infant’s PmMSP-119–seropositive status was due to maternally derived IgG. Nearly one in five participants in the overall case–control study was seropositive to PmMSP-119. The health facility that the infant attended was located in Les Irois, in the southwestern part of Grand’Anse, which was identified as a focus of P. falciparum transmission by Ashton et al.24The confirmed P. malariae infections, though rare . Before this report, the most recent published evidence suggesting the presence of P. malariae in Haiti was identification of P. malariae infections in Haitian refugees arriving in Jamaica in 2004.17 The current surveillance system in Haiti does not support reporting by Plasmodium species. Historical reports from Haiti showed that during localized outbreaks in mountainous areas in 1966, about 15% of the malaria infections were due to P. malariae, although nationally, they made up 3%.15 Overall, in 1964–1965, the monthly incidence of P. malariae was found to roughly parallel P. falciparum incidence in nationwide surveys in Haiti.15Data collections in the Artibonite department in 2017 also found evidence of Plasmodium malariae parasites are believed to be sensitive to chloroquine treatment, the national first-line malaria treatment in Haiti, although data are limited.1 When P. malariae presents as a mixed infection with P. falciparum, treatment could still occur in Haiti, unless it is a low-density P. falciparum infection below the limit of detection of RDTs. However, in the case–control study, 13% of participants seropositive to PmMSP-119 were seronegative to PfMSP-119 (27/202), and therefore, their infections may have escaped detection, and they might not have received antimalarial treatment during their infection. Moreover, P. malariae and mixed P. falciparum/P. malariae infections are frequently of low density and/or asymptomatic; thus, they will likely be missed by RDTs or microscopy.5 In the case described here, the infection appears to be a single P. malariae infection, though the possibility of a mixed infection with P. falciparum below the lower limit of detection of the PET-PCR protocol used (< 2 parasites/µL blood) cannot be excluded. Regardless, the identified P. malariae infection was not diagnosed by routine practices at the health facility because of the lack of appropriate diagnostics to detect non-falciparum infections.P. malariae has caused outbreaks decades after successful elimination . These outbreaks were hypothesized to be due to recrudescent infections. Plasmodium malariae is known to cause extended periods of latency; one case in Greece had a latency of more than 40 years (and possibly as long as 70 years).11 In addition, there is evidence from some settings that P. malariae transmission remained or increased while P. falciparum decreased.12 Combined, these reports emphasize the importance of knowing whether P. malariae is still endemic in Haiti.In other countries in the Caribbean, Plasmodium antigenemia and IgG at the Haitian National Laboratory. This enabled us to select relevant samples with possible P. malariae infection from the case–control study. As antigenemia data were collected at < 1 United States Dollar per sample13 and PmMSP-119 was added to a panel of P. falciparum antigens at limited additional costs, this was a highly cost-effective method of selecting possible non-falciparum infections for subsequent confirmation by PCR as compared with testing all available samples by PCR.A MBA was used to screen for P. malariae case’s family, a further limitation of our study is the fact that the original study protocol was not designed to determine the presence of non-falciparum malaria in this setting. A history of febrile illness was the inclusion criterion for the case–control study, although P. malariae is often asymptomatic.5 Future efforts to assess the burden of non-falciparum malaria in Haiti should therefore  focus on asymptomatic populations.In addition to the inability to exclude congenital malaria or gather additional information from the P. malariae case. However, this may have been a locally acquired infection, and there is currently a lack of diagnostic capacity for non-falciparum infections in routine care. Considering Haiti’s elimination aims, this preliminary evidence warrants further exploration of the extent of non-falciparum transmission, surveillance approaches, and efficacy of first-line treatment against non-falciparum species present.Whether this finding should have policy implications for Haiti is difficult to conclude based on a single identified Supplemental materials