Despite malaria, malnutrition and anaemia being major public-health challenges in Cameroon, very little has been reported on the interaction between these interconnected health determinants. This study therefore sought to investigate the relationship between malaria, anaemia, nutritional and socio-economic status amongst under—ten children living in six localities within two health districts in the North Region of Cameroon.Accordingly, a cross- sectional survey was conducted during the peak malaria season in November 2014, in Pitoa and Mayo-Oulo Health Districts. Three hundred and sixty eight children aged 6months—10 years were enrolled. Structured questionnaires were used to assess socio-economic status. Anthropometric indices were taken using standard methods and nutritional status assessed by calculating Height for Age (HA), Weight for Age (WA) and Weight for Height (WH) z-scores to determine stunting, underweight and wasting respectively. Finger-prick blood samples were used to prepare thin and thick blood smears for microscopy. Whole blood was collected to determine the PCV and blood spots on filter paper were used to extract plasmodium DNA for speciation by PCR.Overall prevalence rates of malaria, malnutrition and anaemia were 32.9%, 54.1% and 20.6% respectively. Stunting, underweight and wasting were detected in 56.9%, 63.5% and 34.8% of the children respectively. There was a significant association between malaria and malnutrition . Malnutrition was also strongly associated with malaria status . The prevalence rates of mild, moderate and severe anaemia were 8.1%, 9.2% and 3.3% respectively. Both malaria status and anaemia correlated with development index  and  respectively.Our findings show a synergistic relationship between malaria and malnutrition. Effective collaboration between malaria control and nutrition intervention programmes is essential for proper case management and improved socio-economic status. Malaria, anaemia, and malnutrition are public-health challenges in paediatric populations in sub-Saharan Africa with malaria and undernutrition being major contributors to childhood mortality . MalariaOn the other hand, undernutrition is known to cause more than 50% of deaths among under-five children . It is aP. falciparum infection . Generally, based on the age groups, the prevalence was higher amongst children less than 5 years old compared to the older group. Comparing results from Microscopy, RDT and PCR amongst the age groups, only the prevalence by microscopy showed a significant difference (p = 0.023) between the two age groups. There was no observed significant differences between RDT and PCR (p>0.05) between the ˂5 and ≥5 age groups.Parasite densities ranged from 40parasites/μL to 129600parasites/μL of blood with a geometric mean (GMPD) of 1783 ±19118parasites/μL of blood. The difference between the two age groups was not significant (P = 0.096). Of the 185 analysed by PCR, 123 were positive. PCR Analysis also confirmed 11 cases that were missed by microscopy .A total of 196 malnourished children were identified giving an overall prevalence of malnutrition of 54.1% (95% CI: 48.9–59.2). The rate of malnutrition did not differ significantly between the two age groups (P>0.05) with 101 (51.5%) being children aged ˂5 years and 95 (48.5%) aged ≥5 years.The overall prevalence rates of stunting, underweight and wasting were 56.9%, 63.5% and 34.8% respectively, showing that most of the children were underweight. Although not significantly different (p = 0.223), the prevalence of malnutrition was higher in the Pitoa Health District 109(55.6%) than Mayo-Oulo 87(44.4%), with the highest prevalence recorded in Dourbeye (60.7%) and lowest in Mayo-Oulo (40.3%).Malnutrition was significantly higher in males (60.6%) than in females (48.1%). Stunting was also significantly higher in males (66.9%) than females (47.6%). Underweight was more in males (65.7%) than females (61.5%) although the difference was not significant while wasting was more in females (37.9%) than males (31.4%).The prevalence rates of severe stunting, severe underweight and severe wasting were 29.1%, 20.9% and 9.3% in the ˂5 age group and 18.3%, 10.0% and 7.8% in the ≥5 age group respectively, with males being generally more severely malnourished than females (p˂0.05) as well. Comparisons among age groups in males revealed that the prevalence for severe underweight was significantly higher (p = 0.002) in ˂5 than the ≥5. Although not significantly different (p = 0.296), severe stunting was higher in the ˂5 age group than the ≥5 while severe wasting was more in the ≥5’s than the ˂5 age group (p = 0.963). In females, severe stunting and severe underweight were significantly higher (p = 0.028) and (p = 0.003) respectively in the ˂5 age group compared to the ≥5 while severe wasting was higher in the ≥5 age group than the ˂5, but not statistically significant (p = 0.185).Out of the 119 children who had malaria 70(58.8%) were ˂5years old while 49(41.2%) were ≥5years. For the children infected with malaria parasite, the mean±SD z-scores for HAZ, WAZ and WHZ in the ˂5 age group were -1.580±2.100, -1.504±2.121 and -0.231±2.729 respectively while for the ≥5 age group, -1.245±1.925, -1.732±1.224 and -1.355±1.566 respectively.Overall, malaria prevalence in malnourished children was 37.8% (n = 196), of which 67.2% were underweight, 56.3% were stunted and 41.2% were wasted. Among age groups, although not significantly different (p = 0.331), malaria prevalence in malnourished children was higher 33(67.3%) in the ≥5 age group than the ˂5 age group 41(58.6%).Anaemia was assessed by Hb levels. Children with haemoglobin levels less than 11 g/dL were considered to be anaemic in accordance with the WHO classification system . Out of Overall, the categorization of the development index into quintiles revealed that the majority of children in the study population belonged to the poor SES quintile, followed by the very poor. Indicators for socio-economic status included; the number of people living in a household, the level of education of caregiver and family head, employment status of family head, type of housing, type of toilet, source of lighting and cooking energy, availability of potable water and possession of household assets.Children belonging to the average SES quintile were observed to have the highest prevalence of malaria (42.4%) as opposed to those of the rich SES quintile who had the lowest prevalence (18.0%). The overall prevalence of malnutrition was 54.1% with children from the poor SES quintile observed to have the highest prevalence of malnutrition (64.9%) while children in the very rich SES quintile had the lowest prevalence (48.0%). With regards to anaemia, overall prevalence was 20.6%, with children belonging to the poor SES quintile having the highest prevalence of anaemia (26.8%) while those in the rich level had the lowest prevalence (7.1%).For Malaria, the model demonstrated that location  and nutritional status  were significant predictors of malaria, with nutritional status having 2 times greater risk of malaria . Other rThe LRM also demonstrated sex of child , level of education of care giver , and malaria status  as significant predictors of malnutrition as shown above . The levAnaemia had family head relationship with child  and socio-economic development index  as significant risk factors .Malaria represents a major public health problem. The overall prevalence of malaria in the study sites was 32.9%. The prevalence of malaria was also observed to be significantly higher (p<0.01) in Mayo- Oulo health district (38.5%) than Pitoa health district (27.6%), though both were peri-urban settings, probably because our study covered only 6 villages as opposed to earlier studies which covered a wider range of villages. This result was lower than 44.3% observed by Kimbi et al., in the Mount Cameroon region , and couAlso, our findings revealed that the prevalence of malaria was inversely related to age. Such increased susceptibility of younger children to infections has been attributed to their poorly developed immune systems .The logistic regression analysis demonstrated that location  and nutritional status  were significant risk factors for malaria. Thus there are 1.62 times more chances that a child will become infected with malaria in Bala than in Mayo-Oulo. The key to effective management of malaria is prompt and accurate diagnosis. The WHO recommends that malaria case management where possible should be based on parasitological diagnosis, except when considering young children in endemic areas where lack of resources or urgency of response temporarily limits its application , 33. MalWith regards to malnutrition, the overall prevalence was 54.1% with an overall prevalence of 56.9% for stunting. Although this is higher than is reported previously in the same region with 40.2% for malnutrition and 37.7% for stunting,  in the MWith regards to malnutrition, the high prevalence is indicative of poor feeding habits, absence of health/ nutritional education and lack of proper knowledge on nutrition and balanced diets as well as the low level of education of the caregivers in the studied area. The observed results on the prevalences of stunting, underweight and wasting in both males and females, was consistent with the observations of Sumbele et al., in the MA higher prevalence in malnutrition was observed among the ˂5years (55.5%). This is unlike reports from South Western Ethiopia where older children were more likely to suffer from undernutrition. This difference is probably because younger children are more vulnerable to infection due to their poorly developed immune systems . Poor feLogistic regression analysis was run and it identified malaria status, sex of child and level of education of caregiver as significant predicting risk factors for malnutrition. As revealed by the odd ratios, the level of education of the care giver was associated with 2 fold elevated risk of being malnourished. This implied that children of illiterate mothers were 2.40 times more likely to become malnourished compared to those that had basic education.Unlike Snow et al.,  who repo–The foremost aim of nutritional assessment studies is to determine the actual magnitude of under nutrition and thereby introduce appropriate nutritional intervention programmes to improve the existing nutrition situation –42.In order to meet up with the World Health Assembly’s Global Nutrition Targets 1 and 2 for 2025 on a 40% reduction in the number of children under-5 who are stunted as well as reduce and maintain childhood wasting to less than 5%, then effective strategies need to be sought .Therefore, appropriate measures such as improved nutritional practices could be recommended in areas with high malnutrition . AnotherAnaemia is widely distributed and often found in developing countries . ChronicAnaemia is a key public health challenge in Cameroon. Its prevalence as revealed by our study was 20.6%. This is unexpected in a peri-urban community where malaria is hyper endemic. Earlier studies by Jourdan et al., reported prevalence as high as 82% for children attending a clinic in Northern Cameroon . The preIn our study, the lower prevalence of anaemia observed may probably be due to the low malaria prevalence in the studied area coupled to the fact that ours was a community-based household survey and most of the children examined were healthy children and not necessarily sick children in a hospital. It also revealed mild moderate and severe anaemia to be 8.1%, 9.2% and 3.3% respectively. This indicates that severe anaemia was rare in the area. Meanwhile the WHO prevalence rates for mild, moderate and severe anaemia stood at 25%, 33% and 3% in 2017.Anthropometry has become a practical tool for evaluating the nutritional status of populations, particularly of children in developing countries and nutritional status is the best indicator of the global well-being of children . DespiteThe results equally showed that there was no statistically significant association between anaemia and malnutrition (p = 0.599). Contrary to our findings, malnutrition was reported in Ghana , to be aThe LR analysis significantly identified development index and family head relationship with child as significant risk factors for anaemia. Meaning, anaemia correlated with socio economic status (p = 0.023). Children with a low SES were 1.45 times more likely to become anaemic  compared to children in the high socio-economic level.All in all the study highlighted the fact that, malaria and malnutrition may not have been the causes of anaemia in the study population. Our findings show no linear relationship between anaemia and malaria or malnutrition, suggesting the possible involvement of other factors in the development of anaemia in children. Therefore, the results are suggestive of the fact that the anaemia observed in the children may have been due to other presumptive causes like helminthes among others. The prevention as well as timely management of anaemia is essential to attain Sustainable Development Goal- 3 (SDG) on ensuring healthy lives and promoting well-being for all at all ages. Further actions are required to reach the World Health Assembly target of a 50% reduction of anaemia in women of reproductive age by 2025 .Socio-economic status has been defined as a position that an individual or family occupies with reference to prevailing standards of cultural and material possessions, income and education . Our finControl and intervention programmes will have limited effects if we fail to recognize and consequently target the underlying causes of the diseases. Nutritional counselling and health education interventions of mothers followed by prompt feeding programs have to specifically focus on improving the health of the malnourished and anaemic. These, alongside continuous proper malaria-control measures of proven efficacy, may be apt to reduce the burden of malaria, anaemia, malnutrition and poverty on a large scale. This study therefore elucidates the interplay between the various health determinants and provides preliminary baseline data needed by policy makers, programme planners and implementers, non-governmental organizations, for strategic planning and implementation of the rapid scale-up of nutritional interventions as well as scaled-up malaria parasite control measures in the north region of Cameroon.Children screened for malaria should also be treated for anaemia and helminthes, especially in the poor communities. Children with pale mucosae should be provided with iron supplementation. There is need for appropriate nutritional and deworming interventions so as to ameliorate the anaemic status of the children. Further research needs to be done to ascertain the exact cause of the anaemia in the children.


Malaria can be transmitted by blood transfusion from human to human and it is responsible for the majority of transfusion-transmitted infectious diseases worldwide. In sub-Saharan Africa, it had been estimated that almost a quarter of blood donations contain malaria parasites. Since rapid diagnostic tests and thick blood smear microscopy lack sensitivity for low density parasitaemia, particularly in asymptomatic adults, the most reliable method to assess the problem of transfusion-transmitted malaria are nucleic acid-based molecular approaches such as quantitative polymerase chain reaction. The study was undertaken to determine the prevalence of sub-microscopic malaria parasite infection among blood donors in Malabo, Equatorial Guinea.Between July and August 2017, a total of 200 individual blood samples from blood donors at the Malabo Blood Bank were collected and screened by rapid diagnostic tests and thick blood smear microscopy. Retrospectively, the same samples were analysed for the presence of undetected, low-density malaria parasites using quantitative polymerase chain reaction.Plasmodium falciparum positive blood donations analysed by quantitative polymerase chain reaction was significantly higher . Densities of P. falciparum positive blood donations were ranging from 0.06 to 3707.0 parasites/µL with 79.6% below 100 parasites/µL and therefore not detectable by non-molecular malaria diagnostic tests. qPCR based species identification revealed that P. falciparum was the dominating species responsible for 88.1% (52/59) of positive blood donations, followed by Plasmodium malariae  and Plasmodium ovale .In comparison to 6.5% (13/200) by rapid diagnostic test and 2.0% (4/200) by microscopy, the proportion of P. falciparum, P. malariae and P. ovale parasites were identified. Currently widely used malaria diagnostic tools have missed more than 75% of P. falciparum containing blood donations, demonstrating the value of quantitative polymerase chain reaction to reliably detect low density P. falciparum infections. Since the availability of molecular diagnostic methods in malaria endemic countries is still limited, the blood recipients living in malaria endemic countries should be treated following WHO recommendations.This study confirms that in malaria endemic settings, sub-patent malaria infections among blood donors are prevalent. In blood collected from healthy donors living in Malabo, The online version of this article (10.1186/s12936-019-2639-8) contains supplementary material, which is available to authorized users. Plasmodium species are known to infect humans including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri and Plasmodium knowlesi . The mean Cq of the two replicates was reported and in case of one qPCR replicate interpreted as malaria positive and the other replicate negative, then the assay had to be repeated to arrive at an unequivocal result.Amplification and qPCR measurements were performed using the Bio-Rad CFX96 Real-Time PCR System . The thermal profile used for PlasQ qPCR is as follows: 60 s at 95 °C; 45 cycles of 15 s at 95 °C and 45 s at 57 °C. Each reaction contained 2 µL DNA and 8 µL reaction master mix containing 1× Luna Universal Probe qPCR Master Mix  and 1× PlasQ Primer Mix , a serial dilution ranging from 0.05 to 10,000 parasites/µL was prepared and used for quantification of P. falciparum. The WHO standards were run as duplicates in two out of the total 13 qPCR runs conducted.Using the first WHO International Standard for P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi were evaluated and the best performing primer and probes for each Plasmodium species were combined into a new multiplex assay (PlasID). The primers and probe combinations used for this novel pentaplex malaria qPCR assay is provided in Additional file Several published , 33–37 a® Probe qPCR Mix Plus  and 1× PlasID Primer Mix . Samples with a Cq value less than 45 for any of the five Plasmodium targets were considered positive for the corresponding Plasmodium species.Amplification and qPCR measurements were performed using the Bio-Rad CFX96 Real-Time PCR System . The thermal profile used for the PlasID qPCR is as follows: 15 min at 95 °C; 45 cycles of 15 s at 95 °C and 60 s at 55 °C. Each reaction contained 2 µL DNA and 8 µL reaction master mix containing 1× HOT FIREPolGeneious version 8.1.9  was used for sequence alignments and oligonucleotide designs. Cq values were obtained from the Bio-Rad CFX96 Manager 3.1 software  and transferred to a Microsoft Access based in-house database designed for storage and analysis of qPCR data. Cq values were transformed to parasite densities and linked to patient’s metadata collected at the blood bank. Categorical variables were compared by Fisher’s exact test and continuous variables by Mann–Whitney using GraphPad Prism version 7.00 for Windows . P values < 0.05 were considered to be significant.P. falciparum and/or non-falciparum species. Additionally, the internal control serves as a general control for quality and performance of the DNA extraction and qPCR reaction. The performance of the PlasQ assay is shown in Fig. Plasmodium spp. positive samples, was designed for rapid identification of five different malaria causing Plasmodium species. Performance of the PlasID assay was assessed using well-defined clinical samples as references and this novel assay’s ability of identifying P. falciparum, P. malariae, P. ovale curtisi, P. ovale wallikeri, P. vivax and P. knowlesi is demonstrated in Fig. Two independent qPCR assays were systematically used for the screening and identification of malaria parasites. The use of two consecutive qPCR assays maximizes the amount of information generated by the analysis. The first assay (PlasQ), applied to all samples, was designed to identify and quantify Blood donor characteristics are given in Table P. falciparum positive donations, while RDTs detected 13 malaria positive donations. Three donations were positive for both antigens, the P. falciparum specific HRP2 and the Plasmodium spp. LDH antigens. The remaining ten donations were positive for HRP2 only. Microscopy and RDT positive blood donations were not considered for donation and were included blinded into this study sample collection on purpose to test the performance of the PlasQ assay.The flow diagram depicting the sequential malaria diagnostic tools applied to the blood donations is summarized in Fig. P. falciparum (PfvarATS), Plasmodium spp. (Pspp18S) and the human RNAseP gene . All 200 samples amplified the internal control with Cq < 28 and were therefore eligible for further analysis. Over 70% (n = 141) of blood donations were negative for any malaria species. Forty-six donations were positive for both Plasmodium targets, while six and seven donations were positive only for P. falciparum or Plasmodium spp., respectively. All samples (n = 53) with a positive amplification of the Pspp18S target were analysed with the species identification assay (PlasID). Apart from confirming P. falciparum (n = 33) cases, we also found cases of P. malariae (n = 9) and P. ovale (n = 2). Amongst the 200 blood samples analysed, no P. vivax or P. knowlesi was detected.Upon arrival at the EGMVI laboratory all samples were screened with the first multiplex qPCR assay (PlasQ), targeting P. malariae or P. ovale in all seven samples which were positive for Plasmodium spp. and negative for P. falciparum, highlighting the assays ability to identify non-falciparum species. Compared to the PlasQ assay, a reduced sensitivity  for P. falciparum detection using the PlasID assay was observed. The 13 P. falciparum positive samples missed by the PlasID assay had moderate parasitaemia (median of 36.9 with IQR of 1.3–380.0 parasites/µL) and the five samples with the highest parasitaemia were positive by RDT. None of these samples were positive for any other non-P. falciparum species.The assay identified Plasmodium spp. cases were detected, resulting in 100% specificity.An additional 29 malaria negative blood samples were also run with the PlasID assay to test specificity of the assay. No false positive falciparum species had been identified by microscopy or RDT. Pan-positive RDTs, which detected the Plasmodium spp. LDH, were rather associated with higher P. falciparum density than with the detection of non-falciparum malaria parasite species. The three HRP2 and LDH positive RDTs had a geometric mean of 916.3 parasites/µL (range 244.1–3707.0) while the nine HRP2 only positive RDTs had a geometric mean of 106.0 parasites/µL (range 11.2–543.3)  and P. ovale (3.4%). Mixed species infections were found in 6.8% (4/59) of the malaria positive blood donations. One blood donation carried a P. malariae and P. ovale co-infection and three donations contained a combination of P. falciparum and P. malariae. The observed co-infection proportion between P. falciparum and P. malariae was slightly higher than expected .In summary, 59 blood donations were positive for at least one malaria species (29.5%). P. falciparum positive blood donations. Parasite densities calculated based on both targets show a high correlation  and the two positive P. ovale samples had Cq values of 37.9 and 39.8.Data obtained from the PlasQ assay was used to quantify P. falciparum positive blood donations was observed. Non-cumulative parasite density data grouped by diagnostic test is shown in Fig. A strong association between the diagnostic tests and the parasitaemia in P. falciparum positive blood donations ranged from 0.06 to 3707.0 parasites/µL with a median of 4.6 parasites/µL (IQR 0.8–49.0), which is lower than the LOD for conventional diagnostic tests. Only samples with the highest parasitaemia were detectable by microscopy (381.1 [145.4–2909.0] parasites/µL) and RDTs (134.6 [65.1–536.5] parasites/µL), while the two qPCR assays detected additional infections with lower parasite densities. The median parasitaemia was 7.8 parasites/µL (IQR 1.3–65.1) for the Pspp18S target and 4.6 parasites/µL (IQR 0.8–49.0) for the PfvarATS target , RDT (23.1%) and microscopy (7.7%) have all reduced sensitivities. Specificities were for all tests above 95%. One RDT positive sample could not be confirmed by qPCR, while the specificity for Pspp18S was reduced because of non-falciparum malaria parasite species, which are also detected  reported to be in good health at the time of blood donations. Apart from self-reported malaria and/or fever episodes within the last 3 weeks, no other risk factor had been identified based on the provided questionnaire. Since only about 3% (6/198) had reported to have malaria and/or fever episodes, the use of this risk factor to pre-select blood donors would not be very efficient. Deferral of about a quarter of blood donors that are sub-patent malaria carriers in a region where blood supply shortage is eminent could be even more damaging to public health.Plasmodium spp. infections with a LOD between 100 and 1000 parasites/µL which is significantly higher when compared to NATs [P. malariae [P. ovale [P. vivax [Conventional diagnostic tests are important tools, since they are affordable and can be applied immediately at blood banks, but lack in sensitivity limits their usefulness. Microscopy is still an important tool for malaria detection since it allows for species identification and quantification. The detection limit of an expert microscopy is close to 20–50 parasites/µL, but for non-experienced microscopists, it is up to tenfold higher (i.e. 500 parasites/µL) [ to NATs . In addi to NATs , 45. Infmalariae , P. ovalP. ovale  and P. vP. vivax  are usuaP. vivax .P. falciparum and all non-falciparum infections were missed by the conventional diagnostic tools. A suitable screening method for malaria detection in blood units must have high sensitivity, high negative predictive value, must include all Plasmodium species, and should be cost efficient. Such a test must enable a reduction of TTM risk, as well as that of falsely deferred blood donors found to have false positive results.In this study, more than three-quarter of P. falciparum and includes all other relevant Plasmodium species. This assay was extensively used during controlled human malaria infections in Tanzanian and Equatorial Guinean volunteers and no issues with specificity were observed (unpublished). As a limitation, qPCR analysis requires an advanced laboratory infrastructure, trained personnel and the costs for reagents and consumables for this assay is about USD 5, which is higher than the costs for RDT or microscopy-based malaria diagnosis.The PlasQ malaria screening assay applied in this study meets some of these requirements. With a LOD of 0.05 parasites/µL, PlasQ has high sensitivity for falciparum malaria species circulating in Malabo. Therefore a novel multiplex qPCR assay for parallel malaria parasite identification was developed. Using the PlasID assay, the main five human infectious malaria species can be detected qualitatively in a multiplex assay rendering it highly cost efficient since only one DNA extraction and qPCR reaction needs to be run. This novel malaria species identification assay should be used in combination with the PlasQ assay, based on the finding that in the PlasID assay, a reduced sensitivity for P. falciparum is observed. Further development of diagnostic assays for the detection of P. malariae and P. ovale, should include the ability of species-specific quantification. Estimations based on Pspp18S target of the PlasQ assay, indicate that P. malariae and P. ovale single infections had a geometric mean density of about 0.6 parasites/µL, which is more than 14 times lower than P. falciparum single infections (8.8 parasites/µL).This study was used to obtain more information about non-Educating the general public in malaria endemic regions via TV, radio, and pamphlet about the existence of TTM and its potential health impact might help to raise the awareness of this malaria transmission route, both in potential blood donors and medical and nursing staff involved in blood transfusions . This stP. falciparum, P. malariae and P. ovale were detected using two different qPCR assays. The majority of these infections were not identified by the currently widely used malaria diagnostic tools such as RDTs and microscopy. Since the availability of molecular diagnostic methods in malaria endemic countries is still limited, the blood recipients living in malaria endemic countries should be treated following WHO recommendations.More than one quarter of blood donations from healthy donors living in Malabo were infected with malaria parasites. Three species, including Additional file 1. Oligos used in this study.Additional file 2. Identification of risk factors associated with a malaria positive blood donation.


As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens—mainly vaccine candidates—have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance. Plasmodium falciparum are specific biomarkers that can be used to monitor parasite exposure over broader time frames than microscopy, rapid diagnostic tests, or molecular assays. Consequently, seroprevalence surveys can assist with monitoring the impact of malaria control interventions, particularly in the final stages of elimination, when parasite incidence is low. The protein array format to measure antibodies to diverse P. falciparum antigens requires only small sample volumes and is high throughput, permitting the monitoring of malaria transmission on large spatial and temporal scales. We expanded the use of a protein microarray to assess malaria transmission in settings beyond those with a low malaria incidence. Antibody responses in children and adults were profiled, using a P. falciparum protein microarray, through community-based surveys in three areas in Zambia and Zimbabwe at different stages of malaria control and elimination. These three epidemiological settings had distinct serological profiles reflective of their malaria transmission histories. While there was little correlation between transmission intensity and antibody signals (magnitude or breadth) in adults, there was a clear correlation in children younger than 5 years of age. Antibodies in adults appeared to be durable even in the absence of significant recent transmission, whereas antibodies in children provided a more accurate picture of recent levels of transmission intensity. Seroprevalence studies in children could provide a valuable marker of progress toward malaria elimination.Antibodies to IMPORTANCE As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens—mainly vaccine candidates—have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance. Plasmodium falciparum infection , or molecular methods through passive or active case detection . Althoug4–nfection –16. Howefection –.P. falciparum antigens in samples collected in community-based surveys in three transmission settings in Zambia and Zimbabwe: (i) Choma District in Southern Province, Zambia P. falcipA total of 429 samples were collected in serial, cross-sectional, community-based surveys from 290 individuals from 3 sites in 2015. Data from 213 individuals were analyzed . In the P. falciparum antigens on the array, the number of antigens recognized in individual participants (breadth of the antibody profile) was highly variable, ranging from less than 10 to more than 300 antigens, depending on the age of the participants and the sites where the samples were collected. Overall, 129 antigens were above the seropositivity threshold  based on the mean signal for the whole study population. Shown in P value [P_BH] < 0.05) by children and adults was 97, 100, and 1 in the Choma District, Mutasa District, and Nchelenge District, respectively (data not shown).Of the 500 Unsupervised principal-component analyses (PCA) of the profiles in children separated the three sites from each other and from U.S. naive controls , consist10.1128/mSphereDirect.00061-19.1FIG S1P. falciparum exon products on the array are represented and ranked from top to bottom by the average signal across the three sample populations. Average linkage hierarchical clustering of serum samples (left to right) was generated with multiexperiment software (http://mev.tm4.org) using Pearson correlations as the distance metric. The results are for Choma District, Zambia (Ch) samples from June 2015 (n = 28); Mutasa District, Zimbabwe (Mu), samples from August 2015 (n = 15); Nchelenge District, Zambia (Nc), samples from August 2015 (n = 19). Download FIG S1, TIF file, 0.5 MB.Hierarchical clustering dendrogram and heat map of protein microarray data for children 0 to 5 years of age from 3 sites of differing malaria transmission intensity. All 500 Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum antigens on the array by the average fluorescence signal in descending order. The top 30 dominant antigens  exon products mapping to the conserved intracellular domain, five merozoite surface proteins , three early transcribed membrane proteins , and two liver-stage antigens (LSA1 and -2).We then examined the responses to the dominant antigens, defined by ranking all 500 10.1128/mSphereDirect.00061-19.7TABLE S1Plasmodium falciparum-reactive exon products with the highest antibody signal intensities. Antigens are ranked by descending average signal intensity across the S. African study population. Shown is a comparison of the top 30 seroreactive P. falciparum antigens ranked using the fold over control (FOC) and two-component mixture model (MM) methods. The asterisk indicates a marker of recent exposure in Uganda, as described by Helb et al.  and adults from Papua New Guinea  , revealeIn the resurgent malaria transmission setting in Mutasa District, Zimbabwe, antibody signals for the highest-intensity 30 antigens also increased with age  but wereIn the high-transmission setting in Nchelenge District, Zambia, antibody signals were high across all ages, with all participants, including children younger than 5 years of age, being seropositive for the highest-intensity 30 antigens . These sP. falciparum antigens  and individuals from an additional high-transmission setting in Papua New Guinea . Individual antigens are represented by different symbols. The number of seropositive antigens out of 500 on the Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum on the magnitude of antibody signals and the breadth of antibody profiles. Therefore, we compared RDT-positive and -negative samples for the 3 sites  and 1.44 for RDT-positive individuals (n = 12). In this age group there was also an increase in the breadth of the antibody profiles associated with parasitemia, ranging from 9 to 57 seropositive antigens (P_BH < 0.05) between those who were RDT positive and those who were RDT negative  owing to the elevated signals found in RDT-negative individuals.It was of interest to determine the effect of infection with antigens . Of the 10.1128/mSphereDirect.00061-19.3FIG S3Plasmodium falciparum-specific seropositive antigen responses in RDT-negative and RDT-positive children  in Choma District, Zambia. Antibody responses in RDT-negative  and RDT-positive  individuals aged 10 years or younger, arranged from left to right according to average signal intensity. P values with Benjamini-Hochberg correction for false discovery (P_BH) for t tests comparing RDT-positive and -negative individuals are shown in green (secondary y axis), and the cutoff value for significance (P < 0.05) is indicated by a hashed purple line. Antigens are segregated into those that are differentially recognized (P_BH < 0.05), shown on the left (n = 19), and nondifferentially recognized (P_BH ≥ 0.05), shown on the right. Antigens are considered seropositive if the log2 FOC is ≥1. The 19 differentially reactive/seropositive antigens are listed in FIG S3, TIF file, 0.1 MB.Comparison of Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The utility of children younger than 5 years of age as a sentinel surveillance population for serological monitoring of ongoing malaria transmission and documentation of elimination was tested by comparing the signals to the top 30 antigens in children and adults across the three transmission settings and in the U.S. and PNG controls . The sig10.1128/mSphereDirect.00061-19.8TABLE S2P values for all pairwise comparisons shown in Table S2, DOCX file, 0.2 MB.Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The signals from children from Choma and Mutasa Districts were highly significantly different from those from children in Nchelenge District, consistent with the higher transmission in the latter setting. Unlike the signals from children, signals from adults were less discriminatory, with the distribution of the average signals for the top 30 antigens being indistinguishable between Choma and Mutasa Districts. The signals from adults in both districts were significantly lower than those from adults in Nchelenge District. Antigen-specific antibody signal intensities among adults in the high-transmission setting in Nchelenge District, Zambia, were not significantly different from those in a high-transmission reference sample from PNG. Antigen-specific antibody signal intensities among adults in the low-transmission setting in Choma District, Zambia, and the intermediate-transmission setting in Mutasa District, Zimbabwe, were lower than those in the high-transmission setting but higher than those in U.S. adults.t tests and receiver operating characteristic (ROC) analyses , signals for individual top 30 antigens were compared using both analyses . A numbe10.1128/mSphereDirect.00061-19.4FIG S4t test comparisons of top 30 antigens identify antigens associated with different transmission intensities. Shown are AUC and P values for 3 different site comparisons . In each ROC comparison, the analysis was performed twice, with the status for each site defined as 0 or 1, although only AUC values of >0.5 are shown. For Mutasa District versus Choma District, the AUC values apply to a Mutasa District status of 1, while those marked with an asterisk are applies to a Choma District status of 1. Antigens with an AUC of >0.8 in a column for a site with a status of 1 were considered discriminatory for that site. P values with the Benjamini-Hochberg correction for false discovery (P_BH) that are <0.05 are colored red. Download FIG S4, TIF file, 0.1 MB.ROC analysis and Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum in adults in Choma District, Zambia, following a dramatic decline in malaria transmission over the past decade suggests long-term antibody persistence. This was confirmed by measuring antibody responses to the 30 antigens with the highest signal intensity in a cohort of 13 adults from Choma District, Zambia, from whom samples were assayed at five time points between June 2013 and June 2015 -expressed exon products were also represented by Escherichia coli-derived purified proteins on the same array (R2 = 0.89) and EBA175 (R2 = 0.75), and modest correlations were seen for CSP (R2 = 0.6), LSA1 (R2 = 0.57), and MSP1 (R2 = 0.58 for IVTT segment 2 and R2 = 0.47 for IVTT segment 1). The highest correlation was seen for MSP2, where both the IVTT and purified versions were full-length proteins. Full-length PfCSP was also represented as an IVTT protein and a purified protein, although the reactivity to the purified protein was considerably higher than that to the IVTT protein, with a correspondingly lower correlation. This may reflect the low expression levels of this particular protein in the IVTT system. EBA175 also gave relatively good correlations, despite the different lengths of proteins used. Thus, for IVTT expression, exon 1  was amplified and expressed as two half-size segments (segments S1 and S2) and printed separately. Shown in S-transferase fusion protein. Segment S1 and MSP1-19 do not overlap, which presumably accounts for the weakest correlation seen in Analysis of the data using the two-component mixture model generated a list of 30 dominant antigens , of whicme array , namely,me array . Good coexon 1 4,3 bp was 10.1128/mSphereDirect.00061-19.5FIG S5FIG S5, TIF file, 0.1 MB.Scatter plots for protein microarray data for the IVTT-expressed exon product and the corresponding purified protein. Download Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphereDirect.00061-19.9TABLE S3Table S3, DOCX file, 0.02 MB.Comparison between purified proteins and corresponding IVTT-expressed exon products. Correlations are shown in Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Additional validation using purified proteins, preferably of equivalent length, is needed. Nevertheless, these arrayed purified proteins also reflected the same age- and transmission intensity-dependent patterns seen with the average top 30 IVTT expression exon products . Thus, a10.1128/mSphereDirect.00061-19.6FIG S6FIG S6, TIF file, 0.2 MB.Dot plots of array data from five purified protein antigens with serum samples from sites with three different transmission intensities. (A) Children ages 0 to 5 years; (B) adults ages >20 years. Download Copyright © 2019 Kobayashi et al.2019Kobayashi et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. falciparum antigens in three transmission settings in Zambia and Zimbabwe. Based on the 30 antigens with the highest signal intensity, distinct age-specific patterns that reflected recent and past P. falciparum transmission levels were identified. Thus, young children in the low-transmission setting of Choma District, Zambia, had antibodies to the fewest P. falciparum antigens. This reflected the recent decline in malaria transmission and the sustained low malaria transmission and could lead to methods to document progress toward elimination. By comparison, adults in the low-transmission setting had high antibody signals, which persisted for the 2 years of a longitudinal study, highlighting their durability. We speculate that these are a legacy of the hyperendemic malaria that existed in this area over a decade ago but could reflect periodic exposure to P. falciparum in the absence of clinical disease. Asymptomatic and low-parasitemic infection occurs even in the setting of declining malaria transmission in Choma District, Zambia. Community-based epidemiological surveillance studies reported a low but persistent parasite prevalence in the population  and merozoite surface protein 1 (MSP1) blood-stage antigen, were used to identify the spatial heterogeneity in malaria transmission that could be used to target malaria interventions  was performed using blood samples collected by finger prick. Artemether-lumefantrine was offered to treat RDT-positive male and nonpregnant female participants. For serological testing, blood was collected by finger prick using a capillary tube  and stored on ice packs until it was transported to a local laboratory. The blood sample was centrifuged at 14,000 rpm for 1 min to separate the plasma. Plasma samples were stored at −80°C until they were shipped to the University of California, Irvine, on dry ice, where they were again stored at −80°C until probing.P. falciparum as described previously  were amplified from genomic DNA and cloned into a T7 expression vector using homologous recombination. Each was expressed in individual E. coli-derived in vitro transcription/translation (IVTT) reactions  and printed directly onto nitrocellulose-coated glass slides . In addition to the 500 exon products, each array also contained 24 IVTT control (IVTTc) reactions that lacked expression plasmids. Also printed on the array were several purified proteins designed to provide a useful bridge between the array platform and other assay formats that use purified antigens. These antigens included recombinant PfCSP   , 53, PfLt, PATH) , and PfEt, PATH) . Each IVArray probing and data acquisition were performed as described previously . To mini2[IVTT − (SM − GM)]/GM, where IVTT is the exon product signal, SM is the median of the sample-specific IVTTc signals, and GM is the global median of the IVTTc signals. Fold over control (FOC) normalization provides relative measures of the specific antibody binding to the nonspecific antibody binding to the IVTT controls. Thus, with the normalized data, a value of 0.0 means that the intensity is not different from the background intensity and a value of 1.0 indicates a doubling with respect to the background. A threshold of a log2 FOC of 1 was used to define seropositivity.Raw data were collected as the median pixel signal intensity for each spot subtracted automatically from the local background. Data were normalized using the formula log2 FOC for each antigen, in which antigens were ranked by the mean signal across the whole study population and the mean for the top 30 was used for comparisons between different age groups and settings, and (ii) the breadth of the antibody profile, defined as the number of seropositive antigens (log2 FOC > 1) recognized by an individual or for a population using the mean signal intensity for the population. Differentially reactive proteins between groups were determined using signal intensities and a Bayes regularized t test for protein arrays  (P_BH of <0.05 was considered significant. Receiver operating characteristic (ROC) analyses and the area under the ROC curve (AUC) were determined using the R statistical environment and the ROCR tool. Graphical outputs were produced in JMP  and Excel  software. Principal-component analyses (PCA) on seroreactive antigens were conducted using JMP software to visually summarize signal intensity variance among the subject groups.To compare the antibody responses between groups, two metrics were used: (i) magnitude, defined as the logn arrays . To accos (P_BH) ; a P_BH In parallel with the FOC method described above, a two-component mixture model was also used to rank antigens. Reactivity against all 500 exon products at all time points for each subject was applied to the mixture model using an R package (mixtools) for the R statistical environment . A subjeThe study was approved by the Institutional Review Board at the Johns Hopkins Bloomberg School of Public Health, the Tropical Diseases Research Center Ethics Review Committee in Ndola, Zambia, for studies in Choma District and Nchelenge District, Zambia, and the Biomedical Research and Training Institute Institutional Review Board and the Medical Research Council of Zimbabwe for studies in Mutasa District, Zimbabwe. Participants provided written informed consent for themselves and their children.www.ncbi.nlm.nih.gov/geo/) under accession number GPL18316, and the array data obtained in this study are publicly available at the PlasmoDB Plasmodium Genomics Resource (plasmodb.org).The full content of the array (which is designated PfPv500) was deposited with the Gene Expression Omnibus database (


Plasmodium infection, offer a unique opportunity to investigate the kinetics and composition of the antibody response after natural infection.Antibodies against merozoite antigens are key components of malaria immunity. The naturally acquired antibody response to these antigens is generally considered short-lived; however, the underlying mechanisms remain unclear. Prospective studies of travellers with different levels of prior exposure, returning to malaria-free countries with P. falciparum malaria in Stockholm, Sweden  were sampled at diagnosis and 10 days and 1, 3, 6, and 12 months after treatment. Total and subclass-specific IgG responses to P. falciparum merozoite antigens  and tetanus toxoid were measured by multiplex bead-based immunoassays and ELISA. Mathematical modelling was used to estimate the exposure-dependent longevity of antibodies and antibody-secreting cells (ASCs).Adults diagnosed with P. falciparum merozoite antigens at diagnosis; however, the magnitude and breadth were greater in individuals with prior exposure. In both exposure groups, antibody levels increased rapidly for 2 weeks and decayed thereafter. Previously exposed individuals maintained two- to ninefold greater antibody levels throughout the 1-year follow-up. The half-lives of malaria-specific long-lived ASCs, responsible for maintaining circulating antibodies, ranged from 1.8 to 3.7 years for merozoite antigens and were considerably short compared to tetanus-specific ASCs. Primary infected individuals did acquire a long-lived component of the antibody response; however, the total proportion of long-lived ASCs generated in response to infection was estimated not to exceed 10%. In contrast, previously exposed individuals maintained substantially larger numbers of long-lived ASCs .A majority of individuals mounted detectable antibody responses towards The short-lived nature of the naturally acquired antibody response, to all tested merozoite antigens, following primary malaria infection can be attributed to a combination of a poor acquisition and short half-life of long-lived ASCs. Greater longevity is acquired with repeated infections and can be explained by the maintenance of larger numbers of long-lived ASCs. These insights advance our understanding of naturally acquired malaria immunity and will guide strategies for further development of both vaccines and serological tools to monitor exposure.The online version of this article (10.1186/s12916-019-1255-3) contains supplementary material, which is available to authorized users. Antibodies are critical components of naturally acquired immunity to malaria and are particularly important during the blood stage of the infection where targets include antigens expressed on the surface of the merozoite and the infected red blood cell –3. An unThe longevity of an antibody response is primarily determined by the generation and survival of long-lived antibody-secreting cells (ASCs) –13. For Plasmodium chaubaudi [1–4), each with different sets of effector functions and rates of turnover due to the inherent differences in their biochemical properties. The underlying IgG subclass profile may therefore influence the half-life of the antigen-specific total IgG response [The longevity of long-lived malaria-specific ASCs is difficult to study as they reside in the bone marrow and the secondary lymphoid tissues and are only transiently detectable in peripheral blood following acute infection . Howeverhaubaudi  and HIV-haubaudi . In addihaubaudi . IgG conresponse , 24.Reliable estimates of decay rates of antimalarial antibody responses require detailed study of the kinetics of the response after infection. However, studies of antibody kinetics in malaria-endemic areas are hampered by difficulties in determining the timing of the latest exposure due to frequent asymptomatic carriage of low-density infections  and the Plasmodium falciparum infection in absence of re-exposure [Studying returning travellers, diagnosed with malaria in malaria-free countries, provides a unique opportunity to investigate the kinetics of the antimalarial immune response after a naturally acquired exposure , 30. Furexposure –31. HoweP. falciparum schizont extract and eight recombinant vaccine candidate antigens after a naturally acquired infection in travellers followed prospectively after treatment in Sweden. We use mathematical models to compare how the kinetics and the longevity differ with regard to prior malaria exposure and provide quantitative estimates of the decay of the antibody response as well as the relative contribution from short- and long-lived ASCs.Here, we investigate the kinetics of the total IgG, and IgG subclass-specific, antibody response to n = 64) treated for P. falciparum malaria at Karolinska University Hospital in Stockholm, Sweden, were enrolled in the study upon hospitalisation and followed prospectively for up to 1 year. Venous blood samples were collected at the time of diagnosis, and participants were invited to contribute follow-up samples 10 days and 1, 3, 6, and 12 months after the first sample. A questionnaire detailing country of birth, previous countries of residence, travel history, use of antimalarial prophylaxis, previous malaria episodes, and co-morbidities was administered to each study participant upon diagnosis as well as at the end of follow-up. Additional clinical data were extracted from hospital records. All participants were treated with a full course of artemether-lumefantrine. Sixteen participants, who were hyperparasitaemic (i.e. > 5% parasitaemia), showing signs of severe malaria according to the WHO classification [Adults . These 20 individuals were considered likely primary infected and are referred to as being “previously naïve”. Forty-one participants (39 born in sub-Saharan Africa) reported multiple prior malaria episodes and a cumulative time of residency of more than 13 years in a highly malaria-endemic area . They were considered to have had repeated prior exposure and are referred to as being “previously exposed”. Three European natives had a documented single previous P. falciparum infection and were excluded from analyses.To examine the effect of exposure on the kinetics of the antibody response, and maximise exposure-related differences, we included individuals with either a likely primary malaria infection or with multiple previous infections but excluded individuals with a documented single previous 1–4), to eight recombinant P. falciparum antigens as previously described [19) [1–4. In addition, a singleplex bead-based immunoassay was used to quantify total IgG antibodies to tetanus toxoid (TTd) according to a previously described protocol [n = 82), collected at the peak of the antibody response and towards the end of the follow-up period, was analysed by a validated ELISA previously used for quantification of RH5-specific IgG [An ELISA was used to quantify total IgG levels to schizont extract (PfSE) (3D7 clone) according to a previously described protocol . Multiplescribed , 36, 37.escribed  with a Cescribed   , and twobed [19) , MSP-3 (bed [19) , and apibed [19) . The assprotocol . To examific IgG . PositivWe used a previously described mathematical model  to estimτ0 days before the individual presents to the hospital  and that A(t) is the antibody level at time t > τ0 and is given by the following equation:We assume that the infection causes antibody levels to rise ra is the rate of decay of IgG molecules; rs and rl are the rates of decay of short- and long-lived ASCs, respectively; β is the boost in ASCs following infection at time τ0; and ρ is the proportion of ASCs that are long-lived. A0 is the pre-existing levels of antibodies. For previously naïve participants A0 = 0. The models were fitted separately for P. falciparum antigen-specific total IgG and all subclasses (IgG1–4) in a Bayesian framework, and mixed-effects methods were used to capture the natural variation in antibody kinetics between individuals while estimating the average value and variance of the parameters across the entire cohort. To provide a validation of the estimated half-lives of the response to P. falciparum antigens, the model was also fitted to the data on total IgG levels to TTd, an unrelated antigen for which the half-life of the response is well characterised [where cterised , 46. A dasses IgG–4 in a BP. falciparum antigens were positively correlated, in particular, for the two allelic variants of AMA-1 and MSP-3 (range r 0.93–0.98). However, antibody levels to P. falciparum antigens were not correlated with antibody levels to TTd  while smallest for MSP-3_3D7 . The individual tetanus vaccination status (i.e. number and timing of doses received) was not known, but all study participants were highly seroreactive towards TTd  for any exposure group  nor with time since residency in a malaria-endemic area (years) within the previously exposed group . Late treatment failures were observed in five previously naïve individuals  for the antibody molecules, short-lived ASCs, and long-lived ASCs, respectively  for antibody molecules, short-lived ASCs, and long-lived ASCs, respectively  in the previously exposed group ranged from 0.11 to 19.92 years compared to 0.03 to 5.67 years in the previously naïve  reached its maximum within a month following malaria diagnosis and was greater among individuals with previous exposure Fig. . FollowiP. falciparum infection by studying a cohort of differentially exposed travellers followed for 1 year after treatment in complete absence of re-exposure. We observed greater magnitude, breadth, and longevity of the response to all tested P. falciparum antigens  in individuals with previous repeated malaria exposure. Through mathematical modelling of longitudinal data on antigen-specific total IgG and IgG subclass responses, we provided quantitative estimates of the kinetics of antibodies and short- and long-lived ASCs. The half-lives of the antimalarial long-lived ASCs were notably short in comparison with tetanus-specific long-lived ASCs, and although long-lived ASCs were acquired following primary malaria infection, they represented only a small fraction of the total ASCs generated. We observed a greater longevity of the antibody response in previously exposed individuals and show that this can be explained by the maintenance of a greater number of long-lived ASCs.We investigated the kinetics of the antibody response after a naturally acquired 1 and IgG3 subclasses [1 or IgG3. High-level IgG1 and IgG3 responses to several merozoite surface antigens have been associated with clinical protection [1 and IgG3 responses to AMA-1 and MSP-2. Furthermore, a large proportion of individuals with previous exposure, but almost none of the previously naïve, mounted detectable IgG4 responses irrespective of antigen specificity. The role of IgG4 in malaria is to date incompletely resolved but increased levels have been associated with prolonged or chronic antigen exposure [During the year following infection, geometric mean antimalarial antibody levels of all antigen specificities  were consistently higher in individuals with prior malaria exposure. The kinetics of the antigen-specific total IgG response, which is reported to consist predominantly of the IgGbclasses –50, was otection –50, and exposure , 48, 51.P. falciparum-specific short-lived ASCs ranged from a few days to a few weeks whereas the half-life of long-lived ASCs range from approximately 2 to 6 years depending on IgG subclass and antigen specificity . There was a tendency for the estimated half-life of long-lived IgG2 and IgG4 secreting ASCs to be longer than for IgG1, IgG3, and total IgG. However, this could be due to biases related to the small number of samples with seroreactivity exceeding the lower limit of detection of the assay, particularly among the previously naïve. The estimated decay rates of antibody molecules as well as both short- and long-lived ASCs are highly consistent with previous estimates for antibodies and ASCs specific for MSP-1, MSP-2, and AMA-1 in African children [Although the half-lives of antibodies and ASCs have previously been estimated from longitudinal antibody data in children living in endemic areas , no quanchildren , 19, 20.The greater boosting of antibody levels in previously exposed individuals is indicative of a greater number of short-lived ASCs, likely due to the presence of a memory B cell response that upon reinfection rapidly proliferates and differentiates into short-lived ASCs –54. It hP. falciparum infection. However, this component was small, and in previously naïve individuals, only minor proportions of the ASCs generated in response to the infection were estimated to be long-lived . Individuals with prior exposure were estimated to maintain a greater proportion of long-lived ASCs , supported also by the maintenance of two- to ninefold higher antibody levels at the end of follow-up approximately a year after the acute infection. Because of the rapid decay in short-lived ASCs, the antibodies they produce will disappear completely from the circulation within a few months after the infection has been cleared. Any circulating antibodies remaining at the end of follow-up will be maintained entirely by long-lived ASCs. The magnitude of the antibody response at the end of the follow-up will therefore be proportional to the number of long-lived ASC [Interestingly, the previously naïve individuals were found to acquire a long-lived component of the antibody response during this primary ived ASC , 23. Theived ASC , 60, 61.The mathematical model used represents necessary simplifications of the underlying processes that regulate the generation and survival of ASCs . DiffereBecause the exposure-dependent differences in antibody kinetics can be described by the differences in the size of the antibody boost and the acquired proportion of long-lived ASCs, we suggest that a more long-lived antibody response to malaria is acquired slowly over time by small consecutive additions to the pool of long-lived ASCs with each repeated infection. The relatively small number of long-lived ASCs generated by each new infection, as indicated by the low proportion of long-lived ASCs in the previously naïve group, contributes to the slow acquisition of a long-lived high titre antibody responses. This is supported by the data from Ghana and the Gambia, where the proportion of long-lived ASCs in children was estimated to increase with age , 19, andTo date, most successful vaccines induce protection mediated through antibodies that are maintained following just a few immunisations . The mosSerology has proven to be a promising tool to monitor medium- and long-term trends in malaria transmission intensity, particularly in low transmission settings . AvailabP. falciparum infection gives new insight into the acquisition and maintenance of the antibody response to eight malaria vaccine candidate antigens. Through mathematical modelling of longitudinal antibody data, using an approach that is widely applicable for the study of the humoral response to both infection and vaccination, we provide quantitative estimates of the exposure-dependent kinetics of antibodies and ASCs. The data suggest that the overall short-lived nature of the antibody response to natural infection can be attributed to a combination of a short half-life and an inefficient acquisition of long-lived antimalarial ASCs. The quantitative estimates of the total and IgG subclass specific kinetics of the response may help guide strategies for further vaccine development and improve serological tools for malaria transmission surveillance.This prospective study of differentially exposed travellers treated for Additional file 1:Figure S1-S6. (PDF 7872 kb)Supplementary information. Supplementary methods including technical details of antibody assays and model fitting procedures. Additional file 2:Table S1. Antibody kinetics model estimates of the population level parameters with corresponding variance parameters. (XLSX 39 kb)Additional file 3:Table S2. Antibody kinetics model estimates of the geometric mean antibody levels at the end of follow-up. (XLSX 27 kb)Additional file 4:Table S3. Antibody kinetics model estimates of the time-to-seroreversion. (XLSX 22 kb)


With an overall decline of malaria incidence, elimination of malaria is gradually becoming the next target for many of countries affected by the disease. In Kenya the national malaria control strategy is aiming to reach pre-elimination for most parts of the country. However, considerable heterogeneity in prevalence of the disease within the country and especially the remaining high prevalent region of the Lake endemic region is likely to slow progress towards this target. To achieve a sustained control and an eventual elimination, a clear understanding of drivers of ongoing malaria transmission in remaining hotspots is needed.Data from the 2015 Malaria Indicator Survey (MIS) were analysed for prevalence of malaria parasitaemia in children (6 months to 14 years) of different countries within the highly endemic Lake region. Univariate and multivariate logistic regression analysis were preformed to explore associations between selected risk factors and being parasitaemic. A predictive model was built for the association between malaria and the risk factors with the aim of identifying heterogeneities of the disease at the lower administrative levels.Overall, 604/2253  children were parasitaemic. The highest prevalence was observed in Busia County (37%) and lowest in Bungoma County (18%). Multivariate logistic regression analysis showed that the 10–14 years age group , households in the poorest socio-economic class , farming  and residence in Busia , Kakamega , and Migori counties (OR = 4.6 95% CI 2.1–10.3) were associated with higher risk of parasitaemia. Having slept under a long-lasting insecticide-treated bed net (LLIN) was associated with a lower risk . No association were found between malaria infection and the gender of the child, the household head, and the education status of the household head.Detailed analysis of malaria prevalence data in a hotspot area can identify new threats and avail opportunities for directing intervention. In the Lake endemic region of Kenya, interventions should be focused more on counties with the highest prevalence, and should target older children as well as children from the lower socio-economic strata. Precisely targeting interventions in remaining hotspots and high-risk populations will likely make impact and accelerate progress towards pre-elimination targets. Kenya has witnessed a significant decrease in the prevalence of malaria over the past years. Parasitaemia declined from 11% in 2010 to 8% in 2015 [However, further progress in reducing or eliminating malaria will likely be challenging. This is related to a number of factors, including reduced funding for control activities as authorities lower priority on malaria following reducing prevalence and the emergence of resistance to insecticides used in indoor residual spraying (IRS), which has resulted in the discontinuation of indoor residual spraying (IRS) in the majority of Kenyan counties in the highest endemic region . Furthervs urban), and sources of water for domestic use are risk factors associated with malaria parasitaemia [In order to implement targeted strategies to control malaria in this region, there is need to assess malaria risks at local levels to identify vulnerable populations and implement prevention strategies that are context-specific and tailored to address observed disparities in the prevalence of the disease. This requires understanding of the modifiable risk factors that increase the risk of disease in this region so that steps can be taken to mitigate them. Previous studies in settings similar to the Kenya has shown that factors such as age of child, socio-economic status of households, level of education of household head, housing conditions, land use, residence  within the Lake region of Kenya was performed. In addition, the associations between malaria parasitaemia in children and selected risk factors in this high endemic region was explored.The Lake endemic region is administratively made up of eight counties of Kisumu, Siaya, Migori, Homabay, Kakamega, Busia, Bungoma, and Vihiga Fig. . With thCross-sectional data collected during the 2015 Malaria Indicator Survey (MIS)—a household survey was analysed. Eligibility of households was determined through a representative probability sample designed to get independent estimates of the prevalence of malaria parasitaemia nationally, by endemicity zone and by urban/rural residence. Sampling involved a two-stage stratified cluster sampling. In the first stage, 246 clusters including 131 rural and 115 urban clusters were selected with equal probability from the country’s national sample survey and sampling programme [As a dependent variable in the logistic regression analysis, Parasitaemia was defined as the detection of any malaria parasite in a peripheral blood smear examined by microscopy. For most of the independent variables tested in our logistic regression, the definitions of the MIS 2015 without any alteration was used . HoweverMIS used rapid diagnostic tests (RDT) and microscopy to test for malaria in the selected children. For RDT, SD BIOLINE Malaria Ag P.f/Pan  was used. Briefly, blood was obtained by a finger prick, collected with a sterile loop applicator and placed in the RDT device. A buffer was added to the cassette so that the blood sample flows through the reaction area of the kit by capillary action. After 15 min, the results were read and recorded as either positive, negative or indeterminate. Indeterminate results were repeated until the test met the validity criteria.Peripheral blood smears were collected and sent to a malaria diagnostic centre for staining and examination by an expert microscopist. At the malaria diagnostic centre, the thin smear was fixed with methanol and both the thick and thin blood smears were then stained in 20% Giemsa stained for 15 min. The smears were dried and examined microscopically with a high power objective. For quality assurance purposes, every slide was read by two trained microscopists at different times. A third reader was called to settle any discrepancy between the two readers.As microscopy is considered the gold standard technique for the diagnosis of malaria, all analysis presented in this report are based on malaria microscopy results alone and not on RDT.This study involved the secondary analysis of data collected in 2015 and archived for further explorative analysis. No ethical approvals were necessary. Kenyatta National Hospital and University of Nairobi institutional review boards approved the primary protocol for MIS Kenya.vs traditional), sources of water for domestic use , occupation of household head, and residence .Analysis was done with Stata version 12.0 software . Prevalence rates were assessed by counties and for children of different age groups. Univariate and multivariate logistic regressions were done to test for the association between malaria parasitaemia and selected risk factors, taking into account survey design effects. Microscopy confirmed malaria parasitaemia was used as dependent variable. Selected independent variables included the child’s age and gender, gender of the household head, socio-economic status of households defined by wealth index, house characteristics  were included in a multivariate logistic regression model. A stepwise backwards-elimination was used to identify final covariates in the model. Wald test was used to check for the effect of removing factors from the model. Factors maintained in the final model are presented as odds ratio plot with 95% confidence interval.Nationally, MIS selected 7380 households but included 6667 (90.3%) in the final survey. A total of 10,721 equal to 93% of eligible children in the households participated [In general, of the 2253 children that were tested for malaria by microscopy, 604 (27.1%) tested positive compared to 8.0% positivity at national level. However, prevalence in the different counties was variable, ranging from highest 37% in Busia county to lowest 18% in Bungoma county , and county of residence. In the multivariable analysis, only age, socio-economic status, agricultural farming and county of residence remained significantly associated with parasitaemia Table .Table 3UCompared to children living in Bungoma County, which had the lowest observed prevalence, the adjusted odds of having malaria parasitaemia was significantly higher for children living in Migori (aOR = 4.64), Kakamega (aOR = 2.65) and Busia (aOR = 4.12).To identify the main risk factors that drive malaria prevalence in the Lake endemic region, a predictive logistics regression model was built. Factors predicting increased risk for parasitemia included age group, wealth index, the county of residence, materials used in making roofs and floors of houses, and farming see Fig. . SleepinUsing data from MIS 2015, prevalence and risk factors associated with parasitaemia in children from the Lake endemic region of Kenya was assessed. MIS is a highly standardized survey and is considered a gold standard surveillance tool to measure both the burden of malaria and the uptake of control strategies .Anopheles funestus and Anopheles gambiae breed on vegetation and in lagoons along the shores of the Lake [Analysis of MIS data has brought forth a number of important insights into the epidemiology of malaria in the Lake endemic region of Kenya. Firstly, despite the average malaria prevalence in this region standing at 27%, significant variations were evident in the levels of parasitaemia in children from different counties in the same region. Expectedly, the counties of Migori and Busia that border Lake Victoria had the highest prevalence relative to other counties in this region. This may be because environmental conditions around the Lake create conducive environments to sustain the mosquito vector and increase parasite transmission. A previous study from Mbita, a district on the shores of Lake Victoria has shown that important malaria vectors, such as the Lake . The avaSurprisingly, Kakamega County, which is geographically considered a highland area but classified under the Lake endemic regions for operational convenience by National Malaria Control Programme (NMCP), had a malaria parasitaemia prevalence of 33%, the second highest in the region. This observation was unexpected as highlands areas are generally considered to have unsuitable environmental conditions for mosquito breeding and survival. Despite this, malaria in the highlands is increasingly becoming a health problem not only in Kenya but also in other sub-Saharan Africa countries such as Ethiopia, Rwanda and Burundi . Such neThe differences in the levels of parasitaemia between the counties in the same endemic region require practical and policy considerations. In counties such as Migori, Kakamega and Busia, more than 30% of children tested were parasitaemic indicating a high level of exposure. Identifying these high-risk counties is important step towards implementation of targeted interventions. Considering the halted application of IRS in this region due to reported resistance to pyrethroids, the provision of universal access to bed nets alone, even though beneficial, may not be sufficient .Anopheles mosquito bites occur during early and late hours of the day as the reason for higher levels of parasitaemia in the older children who are likely to be playing outside [The shift in the burden of the disease to older age groups reported here agrees with similar findings in Tanzania, Uganda and The Gambia , 8, 12.  outside .The shift in the age burden to the older age groups requires strategies that address malaria in older children, as older children and adults may become the primary reservoirs of the parasite . Given tWealth index and the nature of materials used for the roof, the wall and the floor of houses, which are both considered proxy measures of socio-economic status of households, have been shown to have an association with being parasitaemic. The richer households enjoy a purchasing power which allows them to not only dwell in better conditioned houses but also enables them to afford a LLIN so that exposure of their household members to mosquitoes is lowered and risk of getting malaria reduced. These findings collaborate with the widely held view that malaria is a disease of poverty at the household levels. Similar findings relating to the associations between household socio-economic status and malaria have been reported in other studies in sub-Saharan Africa , 16, 17.Children whose households reported that they slept under LLIN had 30% lower likelihood of having parasitaemia after adjustment for age, socio-economic status and other factors. Like other studies of similar findings, this result adds to the current evidence of the efficacy of LLINs which are the mainstay of malaria prevention. The modest protective effect of LLINs reported here against parasitaemia is similar to many other findings in different studies , 18, 20.Malaria parasitaemia was also associated with households that practised agricultural farming. People living in the Lake endemic region of Kenya generally practice large-scale farming of cane and rice and engage in small-scale farming of corn and other vegetations, which all create the right breeding environment for the mosquito vector. This increased occupational risk for farmers is in agreement with many other findings reported previously , 21, 22.Even though the design of MIS is generally suitable for the analysis carried out in this study, potential limitations within the data need to be noted. Firstly, like all studies based on a cross-sectional survey, the associations observed may be unsuitable to draw causal relationship as they may be confounded by unmeasured factors . SecondlDespite the positive progress made by NMCP in the control of malaria in Kenya in the last decades, there are still some regions of the country where prevalence of parasitaemia is very high and heterogenous. It is therefore imperative to ensure that the current malaria control tools are deployed to get a maximum impact by targeting the areas and individuals who are at greater risk of the disease. Designing control strategies aimed at protecting school-age children and adults and implementing targeted interventions in counties with higher burden may be a useful strategy to make an impact on the levels of transmission of the parasite, to try to achieve nationwide pre-elimination.


Plasmodium falciparum is now widespread, MDA has been proposed as an elimination accelerator, but the contribution of asymptomatic infections to malaria transmission has been questioned. The impact of MDA on entomological indices has not been characterized previously.The objective of mass antimalarial drug administration (MDA) is to eliminate malaria rapidly by eliminating the asymptomatic malaria parasite reservoirs and interrupting transmission. In the Greater Mekong Subregion, where artemisinin-resistant Plasmodium infections were detected by polymerase chain reaction (PCR) analysis. The relationship between the entomological inoculation rate, the malaria prevalence in humans determined by ultrasensitive PCR, and MDA was characterized by generalized estimating equation regression.MDA was conducted in 4 villages in Kayin State (Myanmar). Malaria mosquito vectors were captured 3 months before, during, and 3 months after MDA, and their P. falciparum and Plasmodium vivax infections were cleared by MDA. The P. vivax entomological inoculation rate was reduced by 12.5-fold , but the reservoir of asymptomatic P. vivax infections was reconstituted within 3 months, presumably because of relapses. This was coincident with a 5.3-fold  increase in the vector infection rate.Asymptomatic Asymptomatic infections are a major source of malaria transmission in Southeast Asia. Plasmodium infections to the transmission of malaria in Southeast Asian settings. Mass drug administration with dihydroartemisinin-piperaquine can interrupt malaria transmission rapidly in areas where the prevalence of asymptomatic infection is high.This study demonstrates the major contribution of asymptomatic low-density  Plasmodium falciparum has emerged and spread in the Greater Mekong Subregion  falciparum or [Plasmodium] vivax infections to onward transmission to human populations. Intervention trials to directly assess the effect of identifying and treating low-density infections are warranted” [21pCommunity-wide access to early diagnosis and treatment with an effective ACT is the most effective strategy to reduce the transmission of falciparum malaria . In thisubregion . Thus, iubregion . Over tiubregion . Numeroudemicity . The prodemicity . Althougdemicity . In addidemicity , 20. Thented” 21p.P. falciparum parasites [P. vivax infections, the effect on prevalence was only transient: the asymptomatic reservoir reconstituted within 3 months of the intervention, presumably because of relapses from persistent liver stages of the parasite  [We have shown previously that mass drug administration (MDA) with dihydroartemisinin-piperaquine is effective in clearing the malaria parasite reservoirs in asymptomatic individuals, even in areas with low-level piperaquine resistance among arasites , and thaarasites , 23. Howozoites) . PrimaquEntomological data characterizing the contribution of asymptomatic carriers to malaria transmission in Southeast Asia are lacking, and there have been no assessments of the impact of MDA on entomological indices. These uncertainties may have contributed to unclear policies for malaria elimination in the Greater Mekong Subregion . The objThe study was conducted in Kayin State, Myanmar, from 2013 to 2015. Four villages, namely A1-KNH, A2-TOT, B1-TPN, and B2-HKT, were selected because they had high prevalences of malaria parasite infection , as determined by high-volume ultrasensitive polymerase chain reaction (uPCR) analysis .A malaria post was set up in each village to provide community-wide access to early diagnosis and treatment, and long-lasting insecticide-treated bed nets were provided to all villagers. Three rounds of MDA (1 dose of dihydroartemisinin-piperaquine given on 3 consecutive days and 1 low dose of primaquine given on day 1 or day 3) conducted at 1-month intervals eliminated asymptomatic malaria parasite carriage. MDA was administered sequentially, from months 0 to 2 in group A villages (A1-KNH and A2-TOT) and from months 9 to 11 in group B villages (B1-TPN and B2-HKT).Plasmodium sporogony in the anopheline vector [Plasmodium parasites was determined by uPCR  [P. falciparum infections were treated with artemether-lumefantrine and infections with other malaria species were treated with chloroquine, according to standard protocols and Myanmar national policy [Anopheles organisms were identified on the basis of morphologic characteristics, and malaria vectors from the Funestus, Maculatus, and Leucosphyrus groups were analyzed by real-time quantitative PCR, to determine Plasmodium infection rates  [The study period started 80 days before the beginning of MDA campaigns and ended 130 days after they ended . This took into account the approximately 30-day posttreatment prophylactic effect of piperaquine and the approximately 10-day duration of e vector , 25  . Clinical policy . Detailsl policy . Brieflyctively) . The datP. falciparum and P. vivax sporozoite indices (SIs) were defined as the number of mosquitoes testing positive for P. falciparum and P. vivax, respectively, by PCR, divided by the total number of specimens analyzed. The entomological inoculation rates (EIRs) for P. falciparum and P. vivax were defined as the number of mosquitoes testing positive for P. falciparum and P. vivax, respectively, by PCR, divided by the corresponding number of person-nights of collection [The human-biting rate (HBR) was defined as the number of mosquitoes collected, divided by the corresponding number of person-nights of collection. llection .Plasmodium sporogony in the mosquito vector and the posttreatment prophylactic effects of piperaquine. Therefore, entomological surveys conducted ≥10 days after the beginning of a MDA campaign and ≤40 days after the end of a MDA campaign were assigned to the during-MDA category. Entomological surveys conducted <10 days after the beginning of a MDA campaign or >40 days after the end of a MDA campaign were assigned to the before-MDA and after-MDA categories, respectively  reflect the impact of MDA on malaria prevalence. Therefore, entomological surveys assigned to the duringe survey . The 100Clinical cases were defined as individuals with fever  or a history of fever in the past 2 days, and malaria parasite infection was confirmed by a rapid diagnostic test or microscopy. The incidence of clinical malaria was calculated for each catching site by aggregating data from all individuals living in households located within a buffer zone  around the catching site. For each entomological survey, the incidence was aggregated over the month before the entomological survey . For eacIn this area, the rainy season usually starts in May and ends in November.Statistical analyses were performed using R software, version 3.3.2. Exact Poisson confidence intervals (CIs) were estimated for count data , and exact binomial CIs were estimated for proportions . Estimated EIRs were modeled using the generalized estimating equations (GEE) framework with a negative-binomial link to estimate incidence rate ratios (IRRs), an exchangeable correlation structure, and robust standard errors to account for overdispersion and repeated individual-level measurements. HBR, prevalence, and incidence data were divided into quartiles and introduced as categorical variables, to avoid bias from nonlinear relationships between EIR and these covariates. The results of univariable analyses are presented in This study protocol was reviewed and approved by OxTREC (reference 1017–13 and 1015–13); by the Ethics Review Committee for Research Involving Human Research Subjects, Health Science Group, Chulalongkorn University (COA 154/2014); by the Tak Community Advisory Board; and by village committees. All participants provided their written consent to participate in this study.P. vivax SI was 2.9 events/1000 mosquitoes , yielding an average P. vivax EIR of 0.61 infective bites/person/month  and low in villages B1-TPN and B2-HKT . In contrast, the baseline prevalence of P. vivax infection ranged from 12% in village B1-TPN to 31% in village A2-TOT. Among these infections, individuals in only 15% had an elevated aural temperature . The geometric mean parasitemia levels observed in afebrile and febrile individuals infected with P. falciparum were 5950 Plasmodium genome equivalents/mL  and 34 360 Plasmodium genome equivalents/mL , respectively. The corresponding figures for P. vivax infections were 7110 Plasmodium genome equivalents/mL  and 10 170 Plasmodium genome equivalents/mL , respectively , and the mean  and EIR . The basectively .P. vivax EIR was associated positively with the prevalence of asymptomatic malaria and the HBR of mosquito vectors. There was no association between the P. vivax EIR and the incidence of symptomatic vivax malaria . In this village, the HBR was also lower after MDA than during baseline surveys (124 vs 228 bites/person/months). In all villages, the rise in symptomatic malaria cases after MDA preceded or coincided with the rise in infected vectors, suggesting a causal association  in the variates . The resociation . This dePlasmodium was high. This led to a sustained reduction in the incidence and prevalence of P. falciparum malaria but only a transient reduction in the incidence and prevalence of vivax malaria [P. vivax preceding or coinciding with an increase in the prevalence of infected vectors but without concomitant return of P. falciparum strongly suggests that relapse, rather than external reintroduction of infected vectors, was the main source of reinfection.In malaria-endemic settings, it is usually not possible to ascribe causal relationships between vector carriage and human infections. An argument against the use of MDA as an elimination accelerator has been that submicroscopic parasite densities do not transmit malaria. This study describes a unique opportunity to assess the contribution of asymptomatic malaria parasite infections to the vectorial transmission of malaria in a low-transmission setting of Southeast Asia. Three rounds of MDA with dihydroartemisinin-piperaquine and a single low dose of primaquine rapidly interrupted malaria transmission in villages where the prevalence of submicroscopic carriage of  malaria . The timP. falciparum vector infection. In the study area, the prevalence, incidence, and number of P. falciparum–infected mosquitoes were too low to use P. falciparum EIR as a primary outcome measure. The intensity of vivax malaria transmission in the study area was much higher than that of falciparum malaria [P. vivax–infected vectors to assess the contribution of asymptomatic infections to transmission and the impact of MDA. Vantaux et al have suggested that asymptomatic carriage contributes to the transmission of P. vivax but not P. falciparum [P. falciparum to the low densities of gametocytemia, but the sample was small (60 participants), and the follow-up was only 2 months. In low-transmission settings of Southeast Asia, malaria parasite densities measured by uPCR in asymptomatic individuals fluctuate over 6 orders of magnitude, and carriage may persist for many months [P. falciparum and P. vivax infections [P. vivax are transmissible, but not those with P. falciparum.The main limitation of this study is the very low level of endemicity of falciparum malaria, which prevented accurate characterization of  malaria , providilciparum . They aty months , 9. Succy months , and gamy months . This cofections . It seemAnopheles minimus sensu stricto and Anopheles maculatus sensu lato were the most abundant species collected inside the study villages [Anopheles mosquitoes is similar in farm huts located outside the villages and in residential households located inside the villages and that infected malaria vectors could be collected at both sites. In the absence of an animal reservoir of human malaria parasites [Plasmodium outside the village if the coverage of MDA intervention is sufficient.We did not conduct entomological investigations in potential sites of transmission outside the villages . In this area, a history of travel outside the village and being a young male are the major risk factors for falciparum malaria , 31. Thevillages . Highly villages  and may villages . They haarasites , MDA camPlasmodium sporozoites in the salivary glands, so mosquito infections were relatively old. By multivariable GEE analysis, we estimated that MDA was associated with a 12.5-fold decrease  in the P. vivax EIR. The exception was village A2-TOT, in which the response to MDA was much worse than elsewhere and the P. vivax EIR increased during MDA. It was not possible to estimate the P. vivax EIR accurately during the baseline survey in A2-TOT, because the sample size was small . MDA participation was poor in this village [P. vivax remained low after MDA, the HBR was significantly lower after MDA than before. This suggests that human-vector contact, as well as relapse, contributes to the submicroscopic carriage of P. vivax. By contrast, P. falciparum does not relapse and so is not expected to rebound following effective MDA campaigns, provided that community-wide access to early diagnosis and treatment prevents reestablishment of local transmission from imported cases.The carriage of malaria parasites by malaria vectors was associated positively with the prevalence of human malaria parasite infections determined by uPCR. We specifically detected  village , and theP. vivax gametocytes and young gametocytes of P. falciparum [P. falciparum, sterilizing transmissible infections within hours [The impact of MDA on the entomological indices is explained by the pharmacology of the antimalarial drugs used. The posttreatment prophylactic effects of a treatment regimen of piperaquine lasts approximately 30 days . Particilciparum , and priin hours , 41. Matin hours , 14, so in hours  found thP. falciparum is established. Without primaquine radical cure, vivax malaria rapidly returns because of relapse.In low-transmission settings of Southeast Asia, asymptomatic parasite reservoirs contribute to the transmission of malaria and its persistence in affected communities. Three monthly rounds of MDA with dihydroartemisinin-piperaquine and a single low dose of primaquine is an effective intervention that interrupts the transmission cycle of malaria rapidly in these areas, where the prevalence of infection is relatively high and where artemisinin-resistance in The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.Supplementary Table 4Click here for additional data file.Supplementary Table 5Click here for additional data file.Supplementary Table 6Click here for additional data file.Supplementary Table 7Click here for additional data file.