Malaria and schistosomiasis represent two of the most prevalent and disabling parasitic infections in developing countries. Few studies have evaluated the effect of maternal schistosomiasis and malaria in the peri-conceptional period on infant’s risk of infection.In Benin, women were followed from the preconception period until delivery. Subsequently, their children were followed from birth to 3 months of age. Pre-pregnancy malaria, malaria in pregnancy (MiP)—determined monthly using a thick blood smear—and urinary schistosomiasis—determined once before pregnancy and once at delivery using urine filtration—were the main maternal exposures. Infant’s febrile infection  was the main outcome. In a secondary analysis, we checked the relation of malaria and schistosomiasis with infant’s hemoglobin (Hb) concentration. Both effects were separately assessed using logistic/mixed linear regression models.st trimester, and the prevalence of schistosomiasis was 21.8%. From birth to 3 months, 25.3% of infants had at least one episode of febrile infection. In multivariate analysis, MiP, particularly malaria in the 1st trimester, was significantly associated with a higher risk of infant’s febrile infection . In secondary results, pre-pregnancy malaria and schistosomiasis were significantly associated with a lower infant’s Hb concentration during the first 3 months.The prevalence of MiP was 35.7% with 10.8% occurring during the 1We evidenced the deleterious effect of maternal parasitic infections on infant’s health. Our results argue in favor of the implementation of preventive strategies as early as in the peri-conception. According to the Developmental Origins of Health and Diseases (DOHaD) concept, a deleterious environment during peri-conception, gestation and the early postnatal period could lead to a predisposition to childhood and adult-onset diseases . MaternaMalaria remains a major infectious disease for population in low and middle-income countries (LMICs). In 2017, 210 million cases of malaria were estimated, mainly occurring in sub-Saharan Africa (SSA) (92%) . MalariaOn another note, schistosomiasis represents one of the most prevalent and disabling parasitic infections in LMICs. According to the World Health Organization (WHO), schistosomiasis affects more than 200 million people in LMICs in 2016 . The preWe aimed to assess the effect of maternal urinary schistosomiasis and malaria in the peri-conceptional period on the risk of febrile infection in infant during the first 3 months of life. In a secondary analysis, we also checked the effect of these two conditions on infant’s hemoglobin (Hb) concentration.Between June 2014 and August 2018, a preconceptional mother-child cohort was conducted in Southern Benin in the districts of Sô-Ava and Akassato, 25 km from Cotonou. Sô-Ava is a lakeside area where fishing is the main activity of the population, while Akassato is a semi-rural area. Both areas are hyperendemic for malaria and schistosomiasis . This stAt enrolment, demographic, socioeconomic, anthropometric  data and household characteristics were collected. Women were screened for urinary schistosomiasis and malaria. All women who were positive for schistosomiasis and malaria were immediately treated. Hemoglobin (Hb), c-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP) levels were determined. Each month, women were visited at home, the first day of the last menstrual period was recorded and a urinary pregnancy test was performed.Once pregnant, women were followed up monthly at the maternity clinic. At each antenatal care visit, anthropometric data, clinical data  and obstetrical data were collected. Women were screened monthly for malaria as well as for proteinuria and glycosuria. Maternal Hb, CRP and AGP levels were determined in the first and the third trimester of pregnancy. Urinary schistosomiasis was determined at delivery, and all women who were positive were treated after delivery. Malaria was also screened (using thick blood smear) at delivery in placental blood. In addition to the scheduled visits, women were invited to attend the maternity clinic anytime in case of symptoms.A subset of infants (born from April 2016) was followed monthly from birth to 3 months of age. At birth and at each scheduled visit, anthropometric data , breastfeeding and dietary practices, vaccine coverage and clinical data  were collected. Infants’ Hb concentration and malaria status (using a thick blood smear) were determined each month from birth until 3 months of age . During follow-up, mothers were encouraged to attend the health facility in case of any symptoms in the child. Clinical symptoms were recorded by the study’s nurses and then confirmed by the study’s referent physician. No biological investigation was carried out for etiological purposes of clinical symptoms.S. haematobium eggs in urine, using the filtration method [®. CRP and AGP levels were determined by enzyme-linked immunosorbent assay (ELISA) technique [Microscopic malaria was diagnosed by thick blood smear (TBS), and the parasitaemia was quantified using the Lambaréné method . Diagnosn method . Hb concechnique st trimester and not later on), late-MiP  and combined-MiP . Schistosomiasis was defined as at least one S. haematobium eggs-positive urine sample before or during pregnancy (yes/no). Malaria-schistosomiasis coinfection was defined as the detection of both malaria and schistosomiasis at least once before or during pregnancy. Anemia during pregnancy was defined as an Hb level less than 11g/dL at least once during pregnancy (yes/no). Socioeconomic level was approximated using a score combining occupation and ownership of assets, which was then categorized according to the tertiles into low, medium and high categories. Gravidity was categorized as primi / secondigravidae (1–2) or multigravidae (≥3). Ethnicity was categorized as Toffin (main ethnic group) or others . Educational level was categorized as literate if ≥ primary school or illiterate if not. Body mass index (BMI) was categorized as underweight (< 18.5 kg/m2), normal (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2) and obesity (≥ 30 kg/m2) according to WHO standards. Gestational age estimation was based either on last menstrual period (LMP) if the difference between LMP and ultrasound scan (US) performed between 9 and 13 weeks of gestation was less than 7 days or on US if the difference was >7 days. At delivery, placental malaria was defined as a positive TBS of placental blood.Pre-pregnancy malaria was defined as a positive TBS before pregnancy (yes/no). MiP was defined as at least one positive TBS during pregnancy (yes/no). In addition, we categorized malaria according to its timing during pregnancy as early-MiP  with respiratory , gastrointestinal , or cutaneous (skin fungi or skin rashes) clinical signs, or as clinical malaria (fever + positive thick blood smear). Infants were classified as whether they had at least one febrile infection during follow-up (yes/no). Hb concentration was considered as a quantitative and time-dependent variable from birth to 3 months of age. Feeding mode was categorized according to WHO standards into maternal breastfeeding , mixed feeding and exclusive formula feeding . NutritiFirst, we described the general characteristics of the mother-child pairs according to maternal malaria and schistosomiasis.Second, we used a logistic regression model to assess the effect of maternal schistosomiasis and malaria before and during pregnancy on the risk of infant’s febrile infection during the first 3 months of life.The main exposure variables were maternal schistosomiasis, pre-pregnancy malaria and MiP. The adjustment variables were maternal age, gravidity, ethnicity, socioeconomic and educational level, anemia (before and during pregnancy) as well as infant’s sex, term at birth, breastfeeding, nutritional status and study center. In addition, sensitivity analyses were conducted to assess the effect of malaria-schistosomiasis coinfection (as the main exposure) on infant’s Hb concentration and risk of febrile infection.In a secondary analysis, we assessed the effect of maternal schistosomiasis and malaria before and during pregnancy on the infant’s Hb concentration during the first 3 months of life using a mixed linear regression model with a random intercept at the individual level (considering that successive Hb concentrations in the same infant were correlated).All variables with a p-value below 0.2 in univariate analysis were included in the multivariate analysis. Then, the variables with a p-value less than 0.05 after a step-by-step backward selection procedure were kept in the multivariate model. Malaria and schistosomiasis were forced in all final models. Statistical analyses were done with Stata version 13.1 for Windows .The Ministry of Health in Benin and the Ethics Committee of the Institut des Sciences Biomédicales Appliquées in Benin approved RECIPAL (decision no. 39 of 05/16/ 2014) and SEPSIS (decision no. 85 of 04/05/2016) projects. Women were included in RECIPAL after providing a signed written informed consent, and the newborns were included in SEPSIS after both parents have provided a signed written informed consent. All infections detected  were immediately treated and all medications were paid by the projects.Out of a total of 1214 women of reproductive age enrolled in the RECIPAL study, 411 became pregnant. Among them, 273 were followed up until delivery and gave birth to 287 newborns . A subset of 161 newborns born from April 2016 was followed up from birth to 3 months of age as part of the SEPSIS study. Among them, one died on the first day of life, and for two more, their parents withdrew the informed consent just after delivery .Mean (SD) gestational age and weight at birth was 39.5 (1.6) weeks and 3006 (376) grams, respectively. During the first 3 months of life, most infants were exclusively or predominantly breastfed (92%). Mean infant’s Hb concentration at birth and at 3 months of age was 14.7 (2.8) and 10.7 (1.5) g/dL, respectively. During the 3 month-follow-up, 25.3% (40/158) of infants had at least one febrile infection. Respiratory, gastrointestinal and cutaneous infections as well as clinical malaria accounted for 53% (n = 21), 48% (n = 19), 5% (n = 2) and 5% (n = 2) of all cases, respectively.During the first 3 months of life, the proportion of infants with at least one febrile infection varied according to the infected or uninfected status of mother . Indeed,Overall, Hb concentration tended to decrease over time, with a plateau from 2 months . The decTo our knowledge, this is the first study conducted in SSA, which assesses the effect of maternal schistosomiasis and early-MiP on infant’s risk of infection. We evidenced that early-MiP–infection occurring only in the first trimester and not after–was significantly associated with a higher risk of infection in infant during the first 3 months of life.Thanks to the women’s follow-up from the preconceptional period, we were able to assess the effect of malaria before and in early pregnancy on infant’s infection. The outcomes were collected prospectively and repeatedly from birth to 3 months of age. In addition, important determinants of infant’s infection, such as his nutritional status and feeding practices have been taken into account in the analysis.st trimester of pregnancy was associated with a higher risk of febrile infection. This result is concordant with the literature. Indeed, the association between MiP, particularly placental malaria, and a higher risk of infant’s malaria and non-malaria fever during the first 18 months of life has already been reported [in utero exposure to soluble Plasmodium antigens. Fetal exposure to malaria may induce an alteration of immune responses leading to a higher susceptibility of infants to subsequent infections, not only to malaria but to infections in general [st trimester of pregnancy and subsequent infections in the child, with a 4 times higher risk of infection in infants born from mothers infected in the 1st trimester only. It has been suggested that early malaria in pregnancy may impair placentation and then contribute to the pathogenesis of low birthweight (LBW) and fetal growth restriction, which are high risk factors for morbidity in the child [In our study, MiP, especially in the 1reported . These o general –22. In the child . In our Schistosoma haematobium infection in schoolchildren in Benin, which were 17.6% at national level and 59.6% in Sô-Ava district [The prevalence of maternal urinary schistosomiasis was high (21.8%), particularly in Sô-Ava district (41%). This result is in line with prevalences that were reported during a recent mapping of district . The prodistrict . In our district .Looking at the effects on Hb concentrations, we showed that maternal schistosomiasis and pre-pregnancy malaria were significantly associated with a lower infant’s Hb concentration during the first 3 months of life. Maternal schistosomiasis has been related to maternal anemia and LBW, but in a very limited number of studies –29. AnemOur study has some limitations that should be considered. First, urinary schistosomiasis was detected before pregnancy and all infected women were treated immediately. It is likely that schistosomiasis detected at the end of pregnancy may have occurred later in women, resulting in a limited effect on the fetus. Second, because of our small sample size, we could not assess the effect of schistosomiasis, malaria and malaria-schistosomiasis coinfection according to the intensity of these infections.In conclusion, our results highlight the critical effects of maternal infections during the first 1,000 days of life in line with the DOHaD concept. We showed that maternal schistosomiasis and malaria in the peri-conceptional period were independently associated with a higher risk of infant’s febrile infection and Hb concentration during the first 3 months of life. These results underline the need for measures to prevent maternal infections as early as in the peri-conceptional period.S1 FigSchisto: schistosomiasis.(TIF)Click here for additional data file.S1 TableMalaria and schistosomiasis have been forced in all final models; Breast: breastfeeding included exclusive and predominant feeding . Infant’(DOCX)Click here for additional data file.S2 TableMalaria and schistosomiasis have been forced in all final models; Breast.: breastfeeding included exclusive and predominant feeding . Infant’(DOCX)Click here for additional data file.


Since 2004, the Kingdom of Saudi Arabia has pursued a policy ofmalaria elimination. The distribution of malaria at this time wasconstrained to regions located in the South Western part of the country. Thepresent study aimed to understand the risk of malaria infection and factorsassociated with these events between March 2006 and August 2007 in one partof Aseer region.The study was carried out in Tihama Qahtan area in the farsoutheastern part of Aseer, historically the most malaria endemic area ofthis region. The area covers 54 villages served by three primary health carecentres . Malaria cases were detectedusing passive case detection (PCD) at the three health centres for 18 monthsfrom March 2006, each positive case was investigated using patient andhousehold level enquiries. In addition, four cross-sectional surveys in 12villages were undertaken using rapid diagnostic tests within the catchmentsof each health centre coinciding with malaria transmission seasons.Plasmodium falciparum casesand one was a P. vivax case. The majority of theseinfections were likely to have been acquired outside of the area andrepresent imported cases, including those from the neighboring region ofJazan. Among the 18 locally acquired cases, the majority were adult maleswho slept outdoors. 3623 individuals were screened during thecross-sectional surveys, 16 (0.44%) were positive and infections onlydetected during peak, potential transmission periods.Among 1840 individuals examined in the PCD survey, 49 (2.7%) werepositive for malaria, most were There was evidence of local malaria transmission in the Tihama Qahtanarea in 2006-2007, however prevalence and incidence of new infections wasvery low, making the future ambitions of elimination biologically feasible.The constant source of imported infections must be considered in thearea’s elimination ambitions, alongside strong behavioural communitymessages about sleeping outdoors unprotected and travel to malaria endemicareas outside the region. Plasmodium falciparum transmission ispredominantly maintained by Anopheles arabiensis [Since the epidemics of malaria in the South Western regions of the Kingdom ofSaudi Arabia in the late 1990s early 2000s , the Minabiensis . These aabiensis , continuabiensis ,7.An. arabiensis populations [P. falciparum cases detected in the region wasonly 18, compared to over 700 cases five years earlier.The worst affected area of Aseer region is the Tihama Qahtan lowlands area,described through community-based malaria surveys as hypo-endemic during the 1970s. Malariaulations ,11. The ulations . Howeverulations . By 2005Identification of the risk factors associated with developing malariainfections and clinical disease is an important step in the process of evaluatingthe needs of elimination ambitions. The present study describes the characteristicsof malaria cases detected at three Primary Health Care Centres (PHCC) andcross-sectional surveys in the Tihama Qahtan area of Aseer region.The study was carried out in the Tihama Qahtan area with an estimatedpopulation of about 18,000 living in 54 villages , sharingAll patients attending the three outpatient clinics of Alfarsha, Albugaaand Wadi Alhayah PHCCs during the period March 2006 to August 2007, complainingof a febrile illness and suspected of being malaria were included in the study.Thick and thin blood films were prepared at the laboratory directly from afinger-prick blood sample. The history of illness, present symptoms andduration, body temperature, residence, age, gender, nationality, and blood testresults including parasite species were recorded on a case record form developedspecially for the study. All microscopists in the health centres were employedand trained by professional staff from Ministry of Health, all clinicalexaminations were undertaken by physicians.The home of each positive malaria case detected during the PCD surveywas visited within 48 hours of the notification. At each household, details wererecorded on the travel history, sleeping patterns (indoors versus outdoors),presence of migrant laborers in close proximity to the household, type ofhousing, number of household inhabitants, sleeping habits, history of fever inthe family, history of anti-malaria treatment used, history of bloodtransfusions and use of malaria vector control . Every household member was asked to provide afinger prick blood sample for microscopic examination of malaria parasites.Each village location was extracted using combinations of Google Earthand maps provided by the Provincial General Directorate of Health Affairs. Thelocation of the household was used to compute risk factors such as distance fromroad networks, distance to the PHCC, distance from dams and rivers.Population based, cross-sectional, active malaria case detection surveyswere conducted in the three PHCCs areas. Two surveys were performed before theonset of malaria transmission seasons in May-July 2006 and May-July 2007, andtwo during the transmission seasons August–December 2006 andSeptember-November 2007. The sample selection was based on the distance from therelevant PHCC. Four villages were randomly selected by balloting from the listof each Centre’s catchment villages, selecting two villages at a distance>5 km and two villages within a distance <5 km from each PHCC. The12 villages selected for the four cross-sectional surveys are shown in . Based oData were entered and analyzed using simple descriptive frequenciesusing EPI-INFO version 6.0.Ethical approval was provided by Aseer General Directorate of HealthAffairs (50/38/721) for the community cross-sectional surveys, and verbalconsent was obtained from each participant, all data remained anonymous andlinked only to subject numbers.A total of 1840 febrile patients suspected of malaria were screenedbetween March 2006 and August 2007 at the three PHCs. The majority of thescreened patients came from Alfarsha PHCC (60%), 30% were screened at WadiAlhayah PHCC, and only 10% were screened from Albuqaa PHCC (10%). Among thosescreened older children (37%) and adults (37%) were more likely to present withfever and symptoms suggestive of malaria than younger children (26%).P. falciparum infections and one case ofP. vivax detected at Wadi Alhaya PHCC. The majority ofcases (34) were detected at Alfarsha PHCC resulting in an overall slidepositivity rate of 3.1% at this PHCC. Across all PHCCs, infections were detectedin all age groups, however they were more common in males and those aged above15 years  cases of malaria infection were detected among thosescreened, 48 15 years . Cases w15 years .31 . 92% ofthose sampled were Saudi nationals. No positive RDT result was recorded duringthe examination of 1756 individuals during the low transmission seasons. Duringthe high transmission season 16 (0.9%) RDT positive individuals were found tohave a positive RDT for P. falciparum was occurringduring 2006-2007, albeit at very low levels. Malaria infection rates among febrilepatients presenting to clinics was only 2.7%, however only 18 of these 49 infectionswere likely to have been the direct result of local transmission. Defining thelocation of infections versus the residence of detected cases is complex [The present study is the first to describe in detail the characteristics ofmalaria cases detected through the PCD system and the first cross-sectional surveyof infection prevalence for thirty years in the Tihama Qahtan area. From thecombined findings of the cross-sectional surveys and the PCD investigations, isevident that local transmission of  complex . It was An. arabiensis, isexophilic, biting outdoors in the early to late evening. During the maintransmission season night time temperatures can rise to as high as 30°C[Among the 18 infections likely to have been acquired within the area, themajority were males and sleeping outdoors was mentioned a normal behavior. This isimportant because the vector in this area,  as 30°C forcing More effective might be to target larval stages of vectors, which is currentpractice by the Aseer Vector Borne Disease Administration.P. falciparum. Despitebeing a traditionally stable endemic area for P. falciparumtransmission, and a major public health threat, by 2006/2007, local transmission hadreached very low levels. Importantly, none of the 16 infections detected oncross-section were symptomatic at the time of the survey. While residual infectionscan be detected with RDTs, these asymptomatic carriers might continue to contributeto malaria transmission unless radically treated. It is not clear whether theseindividuals would have become symptomatic in the days after the survey had they notbeen treated or would have remained asymptomatic. If the latter this would pose acontinued problem when aiming to identify and remove the last active residual fociof transmission [Infection prevalence, as one might expect, was lower among those seen duringactive case detection in 12 villages that surround the three PHCCs. Across foursurveys, and 3623 person observations, only 16 positive RDT results were recorded,all positives were from 10 of the 12 villages and only detected during the maintransmission seasons. All infections were smission .The epidemiology of malaria in Tihama Qahtan, has changed considerably overthe last thirty years. Reasons for the broader decline in malaria incidence acrossAseer will be examined elsewhere , and likely tobe a result of increased investment in malaria control operations since 2004,improved living standards, housing structures and increased networks of PHCCfacilities and roads. By 2007, residual transmission was still present and thepurpose of the present study was to interrogate factors likely to contribute to thelocally acquired infections. Such investigations require minimal additional data,organized here as a study, but possible to integrate into routine PCD and ACDsystems. Further analysis might, in future, be possible where adequate“controls” could be simultaneously defined, for example febrile,patients from the same geographic area, week of detection but without the evidenceof malaria infection. This would provide opportunities to under-take a more formalrisk factor analysis within a case-control framework . In addiLocal malaria transmission was evident in the Tihama Qahtan area in2006-2007, however prevalence and incidence of new infections was very low, makingthe future ambitions of elimination biologically feasible. The constant source ofimported infections must be considered in the area’s elimination ambitions,alongside strong behavioural community messages about sleeping outdoors unprotectedand travel to malaria endemic areas outside the province. Being able to distinguishthe origin of new infections in the area is a critical step to defining the basis offoci targeting of intervention during the elimination phase.


Studies of the association between the level of anti-malarial antibody and protection from malaria infection can yield conflicting results if they fail to take into account differences in the malaria transmission rate. This can occur because high malaria exposure may drive high antibody responses, leading to an apparent positive association between immune response and infection rate. The neonatal period provides a unique window to study the protective effects of antibodies, because waning maternally-derived antibodies lead to different levels of protection with time.Plasmodium falciparum infection (detected by PCR). In addition, mathematical models were used to assess how the frequency of malaria infection varied between the cohorts with different exposure levels.This study uses data from two well-defined infant cohorts in Western Kenya with different burdens of malaria transmission. Survival models were used to assess how the magnitude of maternally derived malaria-specific IgG antibody  affected the time-to-first Despite differences in underlying malaria incidence in the two regions, there was no difference in time-to-first malaria infection between the cohorts. However, there was a significant period of protection observed in children with high initial MSP1 (42 kDa fragment)-specific antibody levels, but this protection was not observed in children with low antibody levels. Children from the high transmission cohort had both longer initial periods of protection from malaria , but more rapid time-to-first-infection once malaria specific maternal antibodies declined below protective levels (attributable to higher exposure rates).P. falciparum infection.This study demonstrates the complex interaction between passive  immunity and the degree of malaria exposure in infants. Children from regions of high malaria transmission had higher levels of maternally-derived antibodies in early life, which led to a significant protection for several months. However, once this immunity waned, the underlying higher frequency of infection was revealed. A better understanding of the interaction between malaria exposure, immunity, and transmission risk will assist in identifying protective immune responses in The online version of this article (10.1186/s12936-019-2657-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum antigens that are targets of naturally-acquired protective antibodies has been the goal of many studies on malaria immunoepidemiology [The identification of emiology –5. A comemiology , 7, highemiology , 9. Thisemiology .P. falciparum infection. That is, mothers may vary in their levels of exposure to P. falciparum infection and hence their levels of anti-malarial antibody. Similarly, it is expected that maternal malaria exposure is likely a predictor of infant exposure. Thus, children of highly exposed mothers may initially experience both high exposure and high protection . However, as maternal antibody protection wanes with time, early immune protection may be followed by a period of reducing antibody levels and increased susceptibility to infection. This may provide a ‘window’ in which the underlying level of exposure can be assessed in the absence of interference from different levels of maternal acquired immunity.The trans-placental transfer of maternal IgG antibodies to infants plays a critical role in protection of infants from common childhood infections including malaria . ImportaP. falciparum DNA detection (indicative of infection) in two well-defined infant cohorts in Kenya with different intensities of malaria transmission [In this study, the interactions between antibody levels, exposure, and time-to-first infection were investigated. This study was based on the previous longitudinal study of malaria-specific antibody levels and frequency of smission , 16. Givhi  and malarialo . Both serve patients from a predominantly rural area. Mothers of newborn infants who were attending post-natal clinics were approached for participation in the study. Enrollment from both sites occurred in between April and May 2006 and infants from both sites were followed simultaneously to eliminate any effects of seasonality in malaria exposures. The description of this longitudinal cohort was previously reported [Two cohorts of children were established at the Chulaimbo Sub-District Hospital in Kisumu County and at the Mosoriot Sub-District Hospital in Nandi County, Kenya. These cohorts will be referred to as malariareported . InclusiP. falciparum antigens were used in a Luminex multiplex assay : liver stage antigen 1 (LSA1); circumsporozoite protein (CSP); cell-traversal protein for ookinetes and sporozoites (PfCelTOS); serine repeat antigen 5 (SERA5); RIPR CT (C terminus) and NT (N terminus); reticulocyte binding protein-like homologue (Rh2 and Rh4.9); merozoite surface protein ; MSP1 42-kDa fragment (referred to as MSP1 for simplicity); erythrocyte binding antigen 140 (EBA 140), EBA175, and EBA181; and apical membrane antigen 1 (AMA1 3D7 and FVO). Proteins were conjugated to non-magnetic microsphere bioplex beads (Bio-Rad) according to the manufacturer’s instructions with slight modifications. Briefly, varying concentrations of each protein were coupled to beads to obtain an optimal antibody signal in a linear range against a serially diluted positive control pool of plasma from healthy malaria immune adult residents of Western Kenya. Plasma was incubated with conjugated beads at a 1:1 ratio for final dilutions of 1:100 and 1:1000 to select the appropriate dilution within the linear range of the positive control pool. The secondary antibody was R-Phycoerythrin AffiniPure F(ab’)2 fragment goat anti-human IgG F(ab’)2 (Jackson ImmunoResearch Laboratories). A more detailed description of the use of this assay to test P. falciparum antibodies has been described previously [The following 20 and stored at − 20 °C. Plasmodium falciparum DNA was detected using PCR primer and probes as described [Plasmodium falciparum DNA was quantified by including a TopoII plasmid that contained the P. falciparum PCR product to generate a standard curve. The sensitivity of this assay was 4 copies of P. falciparum DNA/ml.DNA was extracted from up to 200 µl blood using Qiagen DNAeasy kit  according to the manufacturers’ protocol. DNA was eluted off the column in an equivalent volume of Hescribed . Plasmodsegmented package in R [Thr), the significance was assessed by using a time-dependent Cox regression model in the form of:i(t) is the antibody level of child i at time t. Thus, the hazard ratio for antibody level increases by Hazard ratios (HRs) for time-to-first infection between regions and for antibody level was measured with a cox proportional hazards model. Three different Cox models were used throughout this study. First, a standard Cox model was used to investigate if the level of antibody at first detection is associated with time to infection , and infants from the malariahi setting (Kisumu) were analysed with the non-parametric Mann–Whitney U test. These analyses were carried out in R: A language and environment for statistical computing (version 3.3.3).Differences in antibody levels between US adult controls, infants from the malariaB. This can be written as:Levels of inherited maternal antibody levels (R function nlme in library nlme (v3.1-122), with fixed effects for A, R function intervals in library nlme. In this study, the initial antibody levels in children from Nandi were not significantly different from that of unexposed controls, and the level was fixed to the baseline value B.To account for within child correlations due to the longitudinal nature of the data, the model was fitted using a non-linear mixed effect model using Ton and k between high and low malaria groups were also tested (using an F-test).The time-to-first malaria infection in different populations was modelled using a parametric exponential survival with an added delay term as follow:hi cohort experienced more malaria infections than children in malarialo cohort [P. falciparum DNA in peripheral blood (as measured by qPCR). Given the higher malaria infection rate in malariahi, it is expected that time-to-first infection with P. falciparum should be shorter in this cohort. However, unexpectedly there was no significant difference in the time to detection of P. falciparum DNA between the malariahi and malarialo groups . However, the reverse was the case after 6 months, as children form the malariahi group showed higher infection rates . That is, there is evidence for a difference in time-to-first infection before/after 6 months which, when averaged over the entire study period, leads to no observable effect. This first 6 months coincides with a period of maximum protection due to maternal antibodies [Previous analyses of the same cohort have shown that children in malariao cohort , 16. In tibodies . Thus, thi and malarialo region) and uninfected US adult controls. Then, the levels at first detection of various antibodies were also tested to see if they were also different between the malariahi and malarialo group. Ultimately, the levels of various antibodies were also analysed to see if they could be used to predict the time to first malaria infection (detectable by PCR). The results are summarized in Additional files hi vs malarialo group). This was also correlated with the time to first malaria infection. Thus, the analysis will be focused on the two allelic forms measured in this study—3D7 and FVO. Antibody levels to both antigen alleles were highly correlated, thus only 3D7 results are presented .The children from the malariahi children is a possible explanation as to why this group had lower risk of infection during the first 6 months, but it does not explain the greater risk of infection after 6 months , these levels decayed at a half-life of 1.75 months (95% CI = 0.94–5.92 months), until it returned to a plateau levels  by approximately 8 months after birth. Therefore, it was likely that although children in the high malaria region experience an initial protective effect from maternal antibodies inherited from their frequently exposed mothers, this protection waned with time after which these children then experienced high exposure to P falciparum infection. In contrast, children in the low malaria region did not demonstrate significantly higher antibody levels than unexposed controls . However, a model with continuous linear protection by antibody level describes this data better . The increased complexity of these Cox-based models failed to provide further insight possibly because MSP1 3D7 was not solely responsible for all causative protection, but rather serving as an indicator of overall protection inherited from the mother. Considering this, a parametric survival function  and (2) those with similar initial antibody levels from each region   from the malarialo group. Subset 2 was picked by including the lowest 50% from the malariahi group and the highest 50% from the malarialo group. Comparing children with divergent levels of initial inherited antibodies (subset 1), the delay to first infection was significantly higher in high malaria region compared to the low malaria region . However, once the ‘protection’ waned (after the delay), the force of infection was higher for children from the high malaria region with high antibody levels  Fig. . Subset lo area vs 1.64 months in malariahi area, shared value of 1.44 months, P = 0.21, F-test) after which there was a shorter time-to-first infection in the high malaria region , and different mosquito biting preference in infants vs adults (reviewed in ). BecausThe strengths of this study are the nature of the cohorts and rigorous quantitative analyses that can be used to untangle the effects of inherited (but decaying) antibody immunity and exposure. These findings set the framework for future cohort studies of malaria immunity by demonstrating how the exposure-outcome relationship is confounded by the presence of malaria immunity and malaria exposure and, therefore, exposure needs to be taken into account for accurate estimation of antibody driven protection. However, accurate measurement of individual risk of malaria exposure is not trivial. Many studies attempt to adjust for the heterogeneity in exposure, but this was mainly estimated with a proxy variable such as distance to water, baseline of antibody concentration, or ethnicity (reviewed in ). WithouP. falciparum infection.Overall, this study demonstrates the need to disentangle the complex relationship between transmission intensity, antibody responses, and observed time-to-infection to correctly identify protective immune responses in Additional file 1. Distribution of the first time of sampling.Additional file 2. Cox proportional hazard regression using various antibodies to predict time to first malaria infection (detection by PCR).Additional file 3. Risk table for infants living in Kisumu .Additional file 4. Risk table for infants living in Nandi .Additional file 5. Comparing the levels of first detectable antibody in high malaria, low malaria, and uninfected US adult control.Additional file 6. Kinetics of MSP1-FVO IgG.Additional file 7. Time-to-first P. falciparum infection in Nandi vs Kisumu based on the observed levels of maternal antibody (MSP1-FVO).


Following successful malaria control during the last decade, Ethiopia instituted a stepwise malaria elimination strategy in selected low-transmission areas.Plasmodium falciparum and Plasmodium vivax apical membrane antigen-1 (AMA-1).Cross-sectional surveys were conducted in Babile district, Oromia, Ethiopia from July to November 2017 to evaluate malaria infection status using microscopy and nested polymerase chain reaction (nPCR) and serological markers of exposure targeting P. falciparum and 0.4% (5/1135) and 3.6% (41/1143) for P. vivax by microscopy and nPCR, respectively. Antibody prevalence was associated with current infection by nPCR for both P. falciparum (p<0.001) and P. vivax (p=0.014) and showed an age-dependent increase . Seroconversion curves indicated a decline in malaria exposure 15 y prior to sampling for P. falciparum and 11.5 y prior to sampling for P. vivax, broadly following malaria incidence data from district health offices, with higher antibody titres in adults than children for both species.Parasite prevalence was 1.2% (14/1135) and 5.1% (58/1143) for Malaria transmission declined substantially in the region with continuing heterogeneous but measurable local transmission, arguing in favour of continued and tailored control efforts to accelerate the progress towards elimination efforts. As transmission intensity declines, efforts to find and treat the sparse and heterogeneously distributed remaining infections results in operational challenges,5 in part because a large fraction of these infections is present at densities below the detection limit of standard diagnostics.6 In order to consolidate gains and progress to elimination, improved tools for infection monitoring are needed.Ethiopia achieved a remarkable reduction in malaria morbidity and mortality following the major scale-up of control efforts in 2004/2005,7 Uniquely, serology allows retrospective examination of exposure history, including the effects of interventions and the absence of recent exposure in elimination settings.9 In the present study, the current and past burden of malaria in Babile, a district nominated for elimination by the Federal Ministry of Health (FMoH) of Ethiopia, is examined by microscopy, molecular assays and serology.Serological assays that detect human antibody responses to malaria parasite antigens can be used to characterize current and previous parasite exposure.4 The 14 villages were selected using a computer-randomized list. Within these villages, convenience sampling was undertaken, with participants being invited without any formal random selection. Study approval was obtained from the Ethics Review Committees of the College of Natural Sciences at Addis Ababa University (CNSDO/205/10/2017), Armauer Hansen Research Institute (PO24/17) and Oromia Region Health Bureau (BEFO/AHBIFH/1-8/2700). Informed written consent was obtained from every participant or the parent/guardian of children <18 y of age.A cross-sectional study was conducted from July to November 2017 in 14 villages that are located at an altitude range of 1280–1450 m above sea level in Babile district within the East Hararghe zone of the Oromia region of Ethiopia. Babile is one of the 239 districts nominated by the FMoH for malaria elimination efforts.Participants were asked about demographic characteristics, intervention utilization, household characteristics and travel history. Finger prick blood samples were collected from participants to prepare thin and thick blood films and dried blood spots (DBSs) on Whatman 3MM filter paper . The DBS samples were air dried and stored individually in sealed plastic bags with self-indicating silica gel beads  and transported under ambient conditions right after collection from the field site and stored at −20°C until further processing. Giemsa-stained thick blood films were considered negative if no parasites were observed after examining 200 fields.10 Proper elution of plasma from DBSs was assessed by the colour change of the spots (to white) as well as the elution (to red/brown) after soaking them 1–2 nights in saponin solution at ambient temperature while on a horizontal shaker. If blood spots did not change colour, still retaining the brownish blood colour, spots were soaked further until a colour change was observed or excluded from analyses as reported before.11Plasmodium species were identified using nested polymerase chain reaction (nPCR) targeting the small subunit 18S ribosomal RNA gene.12 Antibody responses (immunoglobulin G) against apical membrane antigen-1 of P. falciparum (AMA-1-3D7) and P. vivax  were tested using enzyme-linked immunosorbent assay (ELISA)13 using blood from malaria-naïve Europeans as negative controls and World Health Organization (WHO) positive control sera .15 The WHO reference positive control sera were included on every plate in six, with fourfold serial dilution starting at the concentration of 1/100 in 0.05% phosphate-buffered saline Tween-20 to translate optical density (OD) values to titres as described before.16A combined approach for serum elution and DNA extraction from 6-mm diameter DBS punches was used.11 The log-likelihood of a catalytic conversion model that allowed for a change in transmission occurring at iterative years was plotted to evaluate the time at which seroconversion rates changed for each species.13 The time point at which a change in transmission most likely had occurred was determined using maximum log-likelihood. A likelihood ratio test was used to determine if a model with a change in transmission vs one without fitted the data best . Reverse cumulative distribution plots were used to evaluate differences in the geometric mean antibody titres. Wilcoxon rank-sum test with Bonferroni correction for multiple comparisons was used to test between two continuous variables.Analyses were performed using the survey command in Stata 13  to take into account the sampling approach and in GraphPad Prism 5.0 . The cut-off for seropositivity among samples was determined as the mean OD of the negative control sera plus 3 standard deviations. Seroconversion curves were modelled using a simple reversible catalytic model fitted by maximum likelihood for all study villages combined.P. falciparum AMA-1 (Pf-AMA) and P. vivax AMA-1 (Pv-AMA). The median age of participants was 12 y (interquartile range [IQR] 7–26) and 52.1% (596/1144) of participants were female. None of the participants had measured fever at the moment of sampling. There was substantial variation in long-lasting insecticidal net ownership, ranging from 25.0% to 93.5% between villages   of P. vivax infections, whereas nPCR detected 5.1% (95% CI 2.5 to 10.1) of P. falciparum and 3.6% (95% CI 2.2 to 5.7) of P. vivax infections. Antibodies against Pf-AMA were detected in 11.2% (95% CI 6.5 to 18.6) of study participants; for Pv-AMA this prevalence was 13.0% (95% CI 5.3 to 28.6)  nPCR positivity among seronegatives (p<0.001). Similarly, P. vivax parasite prevalence by nPCR was higher among Pv-AMA seropositive (8.1% [11/149]) compared with seronegative individuals . Details on concordance between tests are presented in P. falciparum (p=0.3559) or P. vivax (p=0.1443).Microscopy detected 1.3% (95% CI 0.5 to 3.3) of P. falciparum infection was detected by nPCR in three of them, thus demonstrating current malaria exposure in this population. Among 14 children <5 y of age who tested positive for Pv-AMA, infection was detected by nPCR in only 4; the single 1-y-old child who was seropositive for Pv-AMA antibodies also tested positive for P. vivax infection using nPCR.When samples from all villages were combined, the likelihood of individuals testing seropositive increased with age for both Pf-AMA  and Pv-AMA , which was also apparent from the age-seroprevalence curves  as first-line treatment,18 large-scale distribution of long-lasting insecticidal nets, improved indoor residual spraying campaigns and training of 30 000 health extension workers in malaria diagnosis and treatment.19 This effort was followed by a decline in new malaria cases by 66% in 2011 as compared with the pre-intervention period.1 In line with this, the serological investigation in the present study revealed a significant decline in malaria transmission 15 y prior to the study for P. falciparum and 11.5 y for P. vivax. Higher antibody titres were detected in adults compared with children, in line with other studies and supporting the notion of antibody acquisition following repeated exposure to malaria infections. Serological responses to parasite antigens are informative tools to reflect the history of malaria and monitor malaria control programmes and have the potential to uncover heterogeneity in the effectiveness of malaria control interventions.21Following the epidemics in the early 2000s, a substantial reduction in malaria incidence was reported in Ethiopia.P. falciparum infections in the present study is earlier than the major scale-up of malaria interventions in the country. Of note, improved malaria control started before 2002 with the replacement of chloroquine with sulphadoxine–pyrimethamine as the first-line treatment for uncomplicated P. falciparum malaria around 1998 and improved access to insecticide-treated nets around the same time.17 In line with this, a nationwide decline in malaria incidence and death rates has been recorded since 2001.17 A similar rate of decline in the years prior to the massive scale-up was reported in a recent serological study in the northwestern part of the country that even showed an earlier predicted time point than the present study.22 The estimate of time since changes in malaria burden in the current study may thus be realistic. Moreover, rainfall and other climatic patterns may affect transmission regardless of government-imposed interventions.24 Variation in the change point estimate from seroconversion curves also depends on sample size and how sudden transmission changed.25The change time predicted for 27 malaria exposure might have been missed by only measuring responses to a single antigen. Further information could be generated by measuring short-lived antibodies to indicate recent exposure to infection across all ages.28A strength of the current study is that it utilized both routine tests (microscopy) and improved tools (PCR and serology) to concurrently estimate current and historic levels of malaria transmission. Shortcomings include the limited number of examined villages and villagers and the lack of formal random selection in participant recruitment. The asymptomatic status of study participants give us confidence that no systematic bias was introduced. An additional limitation is the reliance on AMA-1 as the sole antigen. Although AMA-1 is a well-characterized marker of historical exposure at the population level,Despite these shortcomings, the current findings support evidence for a decline in malaria burden. The current data further demonstrate that malaria transmission is still ongoing in the study area. While importation of malaria is possible, and an important source of infection in several low-endemic settings, our findings of detectable serological responses in children, often accompanied by PCR-detected infection, suggest that there is still non-negligible local transmission. Continued efforts are required to further reduce malaria in the region. Novel tools that can interrupt transmission and improved strategies on enhanced community case management to find the few remaining infections that plausibly maintain the infectious parasite reservoir are of paramount importance in accelerating progress towards elimination. The striking variation in malaria indicators between villages highlights the need for a better understanding of (variation in) the uptake of interventions and potentially tailor interventions to local needs to accelerate elimination efforts in the region.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.


Over the last 15 years, the majority of malaria drug discovery and development efforts have focused on new molecules and regimens to treat patients with uncomplicated or severe disease. In addition, a number of new molecular scaffolds have been discovered which block the replication of the parasite in the liver, offering the possibility of new tools for oral prophylaxis or chemoprotection, potentially with once-weekly dosing. However, an intervention which requires less frequent administration than this would be a key tool for the control and elimination of malaria. Recent progress in HIV drug discovery has shown that small molecules can be formulated for injections as native molecules or pro-drugs which provide protection for at least 2 months. Advances in antibody engineering offer an alternative approach whereby a single injection could potentially provide protection for several months. Building on earlier profiles for uncomplicated and severe malaria, a target product profile is proposed here for an injectable medicine providing long-term protection from this disease. As with all of such profiles, factors such as efficacy, cost, safety and tolerability are key, but with the changing disease landscape in Africa, new clinical and regulatory approaches are required to develop prophylactic/chemoprotective medicines. An overall framework for these approaches is suggested here. One of the challenges of malaria control is to be able to provide protection for vulnerable populations. In recent years, there has been great progress in the use of drug regimens to prevent infections in children. However, these require frequent drug administration. One alternative that has been tested is the use of long-acting injectable formulations. Although there are currently no long-acting injectable medicines in development for malaria, new formulation technologies, similar to those developed for prophylaxis against HIV, might point the way to new approaches against this disease. In addition, recent developments in monoclonal antibody technology may be applicable to protect against malaria, especially in vulnerable populations. This paper discusses how such treatments would fit the target product profiles for malaria, and a regulatory pathway for their development. Since they pose similar challenges and possibilities from this perspective, the discussion of small molecules and antibodies was combined.Over the last decade, there has been a considerable increase in the portfolio of new molecules which are being developed for the treatment of malaria , 2. New Plasmodium mitochondrial bc1 inhibitor binding to the Q0 site of the complex), pyrimethamine and cycloguanil and its prodrug proguanil , sulfadoxine  and the 8-aminoquinolines primaquine and tafenoquine are the only causal prophylactics with proven clinical efficacy. In suppressive chemoprophylaxis, the parasite is killed once it enters the erythrocytic stages of the lifecycle, stopping the infection at a low level of parasitaemia before it becomes clinically significant. Inhibitors of beta-haematin formation, such as chloroquine and mefloquine, and (plastid) ribosomal inhibitors, such as doxycycline, are suppressive chemoprophylactics, and must be given for at least 2 weeks after leaving a malaria-endemic area to clear any parasites that emerge from the liver after a person has left the endemic area. Currently, the predominant oral prophylactics used by travelers are (rarely) mefloquine, doxycycline and atovaquone–proguanil. These were initially developed as treatments for uncomplicated malaria [Malaria chemoprophylaxis can be achieved by a variety of different mechanisms. Causal prophylactics target the asexual hepatic stages of malaria. Atovaquone . AlthouPlasmodium blood stages [Plasmodium infection, have been identified [Plasmodium falciparum, and progress in this direction is being made. This is particularly important in order to confirm activity of compounds without asexual blood stage activity, and to avoid a focus on murine-malaria specific prophylaxis.Identifying new classes of anti-malarial chemoprotective molecules targeting either blood- or liver-stage has historically been limited by the lack of known molecular targets and of high-throughput screening methods. However, in the last 10 years there has been a dramatic increase in the deployment of high density, phenotypic, cell-based high-throughput screens of d stages  and new entified . These fentified , 27, theentified ) and theentified ), now alentified  have gooentified . More reentified , increasPlasmodium vivax and P. falciparum  and for a single-dose radical cure indication , as Krintafel®, by GSK. Both uses require a test for a patient’s G6PD (glucose-6-phosphate dehydrogenase) deficiency status as a safety measure. No further clinical work on injectables has been reported. In the 1980s, Chinese scientists investigated the use of pyronaridine given by injection, with a total intramuscular dose of 300–400 mg being given in two or three injections. The drug was rapidly absorbed and provided an efficacious concentration that remained for an extended period given the elimination half-life of 60 h [Work on injectable depot anti-malarials was a key part of the previous malaria eradication campaign with the development of 4-4′-diacetylaminosulphone (DADDS), a long-acting prodrug of the DHPS inhibitor dapsone, and cycloguanil pamoate . Cl of 60 h .More recently, there has been a significant effort to develop anti-virals that provide protection against HIV infection in high risk individuals using daily oral dosing, termed pre-exposure prophylaxis, PrEP . HoweverWhen considering new molecules that could be used in chemoprotection, it is important to consider their ultimate use, since this drives the regulatory strategy. As mentioned earlier, previous generations of medicines, such as mefloquine and atovaquone–proguanil, were first approved for case management, and were only subsequently approved for use at lower doses for prophylaxis. However, if the priority is to deliver new medicines for prophylaxis rapidly, one alternative approach would be to pursue a path towards initial registration directly for prophylaxis; this would require a new clinical and regulatory approach. The key question is to define how large and diverse a population will need to be exposed to a new medicine before the risk–benefit balance is established and considered adequate, and this ratio is clearly different between a medicine used for treatment and one for prophylaxis. Another question relevant to development is how to protect the new drug; any deployment strategy must also take into consideration the need to protect the drug against the emergence of resistance . The assprevention could be replaced by a more easily delivered chemoprotection regimen . A once-per-season injectable regimen would certainly be worth considering. Moving to chemoprotection (prophylaxis), rather than simply using a full treatment course monthly, requires a new regulatory and clinical approach, and early discussions on this strategy have taken place with regulators during the past 2 years.Previously, targets have been proposed for the characteristics of molecules which could be used as oral medicines in prophylaxis, and a target candidate profile for molecules with hepatic stage activity (TCP-4); ). It seeIn a discussion of potential new medicines there are always many moving parts. A common framework for discussion and agreement on the ideal and minimally acceptable protective efficacy and other qualities of new medicines is important, given the lengthy development required from discovery to launch. In the world of injectable chemoprophylaxis, such a discussion is especially important, given the overlap between the potential uses for injectable small molecules, therapeutic antibodies, and vaccines. Based on these insights, the Target Candidate and Product Profiles (TPPs) presented here will need to be refined and updated in discussions within the malaria community, as and when new data become available.There are multiple uses for an injectable anti-malarial prophylaxis Table . The firThe second use is to protect a non-immune population within their area of residence, who are suddenly exposed to a malaria outbreak or epidemic. This is one of the major concerns late in any eradication effort, in places where ‘maintaining zero’ is the priority . In a siThe third potential use for an anti-malarial chemoprotective agent is to protect vulnerable populations in areas of high malaria incidence, and this is somewhat distinct from the other uses. Over recent years, there has been a massive deployment of sulfadoxine–pyrimethamine plus amodiaquine (SP–AQ) for SMC. Chemoprevention is defined as giving a full curative course of treatment. This is given over 3 days, to each child under 5 years old, monthly throughout the rainy season in the Sahel (to 13 million children in 2016), to reduce the incidence of symptomatic malaria in this vulnerable population. Since a large part of the cost of deployment of SMC is the delivery, a better treatment would be a once-monthly, single oral medication. A longer-acting injectable would have to offer significant advantage, which is why the ideal threshold for this use has been set at 3 months in the target product profile shown in Table Plasmodium falciparum malaria’. This would be defined as prevention of malaria infection in subjects travelling from geographical areas with no, or very low risk of malaria infection, to geographical areas with significant risk of malaria infection. The clinical development strategy supports the filing by collecting data to demonstrating acceptable efficacy, safety and tolerability in the target populations.The regulatory strategy in each case would be to file for an indication of ‘Prophylaxis against ®  has been approved by the European Commission after having been given a positive scientific opinion by the CHMP , and is about to undergo Phase IV in-country studies. The current course of vaccination involves four 0.5 mL intramuscular injections with a 25-gauge needle, at a cost of around $5 per injection in infants. Second, in HIV infections, where an effective vaccine has also been a major challenge, pre-exposure prophylaxis, or PrEP, using an injectable is being studied [The discussion of a TPP for an injectable prophylactic medicine is informed by the uses described above. The final acceptance of an injectable product is guided by experience with two other therapeutic or preventative approaches. First, site injection tolerability, in terms of needle size, and injection volume should be driven by a favourable comparison to vaccines. The most advanced malaria vaccine, Mosquirix studied , 50. Alt studied , a new m studied  with a vThe level of efficacy required for a new product can be gauged by the efficacy of current prophylaxis. In comparative, randomized clinical trials, nonimmune adults, adolescents and children (≥ 11 kg) visiting malaria-endemic regions and receiving once-daily atovaquone/proguanil (250/100 mg in adults and dosage based on body weight in children < 40 kg), had no cases of falciparum malaria for 28 days , comparaThe clinical safety requirements of a long-acting injectable for prophylaxis are much more difficult to define precisely in advance of data. The two overriding principles are that first, the drug will be given to subjects with no overt disease, and so the safety thresholds should in general be similar to those required for vaccines. This will inevitably lead to the need for considerable phase IV activities. The second aspect relates to the long plasma residence time of pharmacologically active material. Appropriate safeguards have to be in place for the care of subjects, should a severe adverse event occur, and clearly such an event would preclude further development of the medicine for populations in low-resource settings. However, it is important to underline the fact that the safety considerations related to an injectable drug driving exposure over several months are very similar to the safety considerations related to an oral drug driving exposure over several months. The ideal medicine is one that can be used to protect all ages; however the development of the medicine for children will require particular care, and the availability of substantial safety data in adults before proceeding to the more vulnerable younger populations.A target product profile (TPP) including other parameters for an injectable prophylactic medicine for malaria is proposed in Table P. falciparum parasites. The definition of a hit and a lead still follow the criteria discussed previously [50 of less than 1 µM being considered as potential starting points for medicinal chemistry, aiming for a final potency of less than 10 nM. The key data however are those that increase our understanding of how well this translates into in vivo efficacy, and allowing an early prediction of the human effective dose.Compounds with a TCP-4 profile would normally be shown to be highly potent against the hepatic schizonts of eviously , with coPlasmodium berghei sporozoites in mice. This assumes equipotent activity between P. berghei and P. falciparum. A newer model is the FRG huHep chimeric mouse model with engrafted human hepatocytes and erythrocytes. FRG stands for the lack of functional Fah, Rag-2 and Interleukin 2 receptor common gamma chain genes in these animals. This model allows an endpoint based on asexual blood stage infection from the liver [7-fold, or a 200-fold reduction in the bioluminescence signal [4-fold reduction in this model, arguably defining the lower threshold of efficacy. During lead optimization, it is important to be able to assess the human effective dose, i.e. the dose required to produce a similarly effective exposure in humans. In the absence of imaging technologies to determine the time-dependent concentration of a drug within the liver, the definition of the prophylactic concentration has to be based on the plasma concentration, assuming that the plasma to hepatocyte concentration ratio is invariant with plasma exposure. The minimum protective plasma concentration in the mouse model can then be used as the basis for pharmacokinetic modelling of the human exposures. For a pre-clinical orally active compound the ideal threshold is an adult dose of < 100 mg, or < 2 mg/kg. Similar constraints apply to injectables, but driven by a need to be able to deliver the drugs in an acceptable injection volume of < 0.5 mg per drug, as discussed below. Two other biological factors are important. First, the demonstration that clinical selection of mutations is a rare event, or that these mutations are not transmitted. It has been shown that is relatively easy to select for mutations against atovaquone both in vitro and in patients, but that these mutations are difficult to transmit [P. falciparum, activity against other species would be an advantage, since many patients have mixed infections that include P. vivax, Plasmodium malariae and Plasmodium ovale.In vivo measurements of efficacy have been performed traditionally using infection with GFP- (green fluorescent protein) or luciferase-expressing he liver . Here, te signal . DSM265,transmit . Second,Selection of a formulation, which may also involve the use of pro-drugs, is critical to enable the maintenance of long-lasting protection. The ideal use case would be a 3-month coverage from a single injection, allowing potential protection for a season in some countries. Thus, compounds having high potency and very low clearance, and with formulations delivering long-term drug release into the systemic circulation are favoured. When considering the product presentation, a number of factors need to be taken into account, including but not limited to: cost of goods, a therapeutic product that can be stored at room temperature in climate zones III and IV, a formulation with a potential for local production, and a formulation that is easy to administer. As such there is no published decision tree that maps the technology path for selecting long-acting injectable formulations. A range of different formulation technologies have been applied in commercial products, such as oil solutions, aqueous micro- and nanosuspensions, in situ forming gels, and micro-particular systems. There is, hence, a good industrial basis to define the most likely options that would fulfil both the technological elements as well as the cost of the product. In resource-limited settings, the first line of formulation will most likely be an oil solution or suspension of the micro- or nano-particulate drug, due to the simplicity of development and production of the formulation, low cost of excipients and potential for thermal sterilization, which would allow local production. The use of an oil-based solution or suspension requires clinically acceptable oils, such as fractionated coconut, castor, corn, cottonseed or olive oils. A key consideration here will be local tolerability .The second option for a long-acting injectable for prophylactic treatment of malaria would probably be an aqueous suspension of either micro- or nanoparticles. The advantage of an aqueous suspension is that there is a well-established relationship between particle size and rate of dissolution. This allows much better control of the rate of release of the drug and, therefore, better matching of the final pharmacokinetics, once the human clearance is known. In the case of oil solutions of the drug, the oil is metabolized by macrophages in the muscle over a period of days to weeks, resulting in an amorphous depot of drug in the tissue. Data from preclinical animal models and in humans have demonstrated that appropriate slow release of a drug can be obtained, although the choice of oil is largely empirical at this stage. The technology associated with aqueous suspensions is well established, i.e. production facilities and capacity are available, however, cost of goods is a bit higher than the oil solutions, and the development complexity the same.Selection of compounds for long-acting injectable formulations have historically been an afterthought, so the physico-chemical properties needed for success have not been well mapped. The selection criteria depend on the final technology; for an oil based solution, a crystalline material with high solubility  and high melting point of the free form (> 150 °C) are favoured. Ironically, these are properties usually avoided by medicinal chemists when developing an oral product. The definition of the range for logD is under discussion as recent investigations have shown that there is limited correlation between this parameter and the ability to formulate in a lipid based vehicle . The choIdeally, the drug should be administered either sub-cutaneously or intramuscularly. The former has the advantage that it is easier to administer, and requires less training, but there is a greater constraint on the volume that can be delivered. Given that the ultimate product will target the protection of children, infants and adults, the proposed final volume is 0.5 mL for infants and children, and 2.0 mL for adults.The needle size is provisionally suggested at 27-gauge, to minimize discomfort during the injection. Significantly larger volumes of administration are routinely given, such as the 4 mL of microcrystalline penicillin-G benzoate through a 21-gauge needle , which iAlternatives to injections are implant systems, as used in long-term contraception. These have the potential advantage of controllability, with the possibility of removal should an adverse event occur. But this approach requires potent molecules that allow limited dosage: contraceptive etonogestrel implants only contain 68 mg of drug . Implantmax and an increase in the duration of significant plasma concentration. As such, the initial first-in-human study can be de-risked by the selection of an appropriately low starting dose .The first tier would consist of molecules for which extensive human systemic safety data already exist. This would apply to new formulation or a prodrug of a molecule already used for prophylaxis, such as atovaquone–proguanil. Here the safety and tolerability of the combination is well understood for oral administrations of up to 60 days, enabling an adequate assessment of the risk–benefit balance, and giving a relatively straightforward, albeit cautious path to human studies. In the initial single ascending dose studies in humans, the increase in dose should produce an increase in CThe second tier would be prodrugs of new chemical entities with oral formulation for which initial human safety data can be collected using the oral route, allowing dosing to be halted in case of adverse events of concern. Testing of parenteral formulations could follow, but there would be no understanding of rare serious adverse events until after Phase III trials.The third and most difficult tier would be molecules for which no oral formulation of the active parent is possible due the physico-chemical properties of the agent. Given the required long plasma residence time, the starting dose in initial clinical trials would have to be extremely low, and the dose escalation between cohorts extremely conservative.The safety of the molecules is of paramount importance, whether considering long-acting injectables or long-acting oral molecules. In the case of any safety concern, removal of an active drug from plasma would require extensive dialysis, if feasible for the molecule, and lack of access to such a procedure could be life-threatening in resource-poor settings. It is, therefore, important to consider potential long-acting injectables within three tiers of risk:In some of the uses described above, the population at risk from malaria will include a considerable number of women of childbearing potential, whose pregnancy status may not be determined. For new molecules coming forward for prophylaxis, early analysis of the developmental and reproductive toxicology (DART) would be an essential part of the development strategy. Absence of any signals in such preclinical studies would facilitate the inclusion of women of childbearing age in clinical studies. This could facilitate early use of such an agent in the larger population. As discussed below, monoclonal antibodies offer a considerable advantage, since the materno–fetal transfer of IgG during the first trimester is minimal , 65.The preclinical candidate will be a single molecule. There is still debate within the community as to whether the final product should be a single molecule or a combination. The potential for the selection of resistance is much lower than when a molecule is used for the treatment of uncomplicated malaria, since the molecule encounters far fewer parasites . HoweverIn this review a conservative approach of taking two active molecules has been adopted. This is important in early development discussions, as early human volunteer studies require the availability of formulations for both molecules that are compatible. The additional twist in this compatibility discussion is that the drug exposure profile of each molecule in the blood needs to be compatible and since this is modulated by the kinetics of release from the initial depot it may be difficult to do this for two individual molecules with different physicochemical properties. Matching pharmacokinetics is important both for duration of efficacy and for mitigating the risk of emergence of resistance during the extended tail of exposure. In theory, separate injections of the two drugs is possible, although this would be a large disadvantage. Seriously mismatched pharmacokinetics would limit the use to applications where subjects are outside of high transmission zones during the tail exposure period; again, this is possible, but not ideal. As such, any preclinical safety and toxicology studies may have to be repeated with the final formulation.Initial proof of concept in man can be performed in volunteer infection studies with sporozoites. Recent studies with oral small molecule inhibitors of parasite liver stages have demonstrated clinically useful activity with relatively small cohorts, either using direct injections of sporozoites or insect-delivered sporozoites , 27. ForThe TCP-4, for a single molecule suitable for development as part of a new injectable, combination regimen for prophylaxis against malaria is detailed in Table An alternative approach to protecting vulnerable populations with an injectable small molecule would be the use of a monoclonal antibody . Even thGiven the high commercial price of many existing antibody therapies, it is often assumed that the cost of antibody therapy would be prohibitive in most malaria-endemic countries. Provided antibodies are expressed at high levels (4 g/L), and are stable, hence the production costs can drop from around $100–300/g  to estimAs with small molecules, an intramuscular or sub-cutaneous presentation is probably preferable to intravenous injection, and so a limiting factor is the volume of administration. The amount of antibody is, therefore, limited by the achievable concentration. Although concentrations as high as 200 mg/mL have been reported for subcutaneous injection, a more conservative target is 100 mg/mL . Based oTo achieve long-term prophylaxis, mutations in the Fc region are needed. Monoclonal antibodies tend to be IgGs, with plasma half-lives of 20–25 days , 73. Thib), a much higher number than seen with small molecules. Therefore, allowing for the difference in body weights for humans and mice, this translates approximately into a twofold difference in the mg/kg dose between humans and mice, setting an efficacy threshold in mice of an 80–200 mg total dose. This could be related back to a cellular potency target EC50 < 100 pM (< 15 ng/mL), based on modelling of various affinity and dissociation rates [An antibody could be compared directly with a small molecule in the animal model. The goal would be an 80% protection from infection, minimally for 1 month and ideally for 3 months. Given the restrictions on injection volume, this would require administration of 2 mg/kg of each active molecule in adults and 2–5 mg/kg in children. As for small molecules, the simplest preclinical model for therapeutic antibodies would be the FRG chimeric mouse model with engrafted human hepatocytes, discussed earlier. Allometric scaling for a series of antibodies  suggestson rates , 78. It on rates .As mentioned above, the exquisite selectivity of antibodies means that off-target effects are minimized. Anti-sporozoite or anti-merozoite antibodies do not target host antigens. Therefore, toxicology studies should be less complicated compared to other indications. Fetal transfer in the first trimester is reported to be minimal; the required Fc receptor for the transfer of IgGs is hardly detectable in the placental syncytiotrophoblast during the first trimester . SpecifiAntibody discovery tends to be much faster than for small molecules, with a much higher probability that an individual project will deliver a development candidate . HoweverFor new chemical entities and antibodies the Phase I single ascending dose study in healthy volunteers would aim to achieve the plasma concentration predicted from studies using the FRG-mice . At thisIn parallel, it is important to investigate the potential combinations of two molecules that can be brought forward. Simulations using data from preclinical cytochrome P450 and transporter assays can help eliminate combinations with likely problematic drug–drug interactions. Additivity and possible synergy between two molecules can be studied in the FRG SCID mouse. Exposure–response analyses of these studies also allow modelling of the doses proposed for Phase II trials. In addition, the modelling has the potential to demonstrate the contribution of the individual compounds to overall efficacy, addressing the ‘combination rule’ required by the US FDA. The safety of combinations needs to be investigated in healthy adult volunteers combination studies before moving to Phase II trials in infected but asymptomatic subjects. A flow chart for the selection and optimization of new molecules with potential chemoprotective activity is shown in Fig. directly. Such a development plan for prophylaxis was presented for initial discussion with regulators  in the context of an oral drug combination therapy. Similar considerations would apply for antibodies. Two main clinical trial designs are proposed to support registration for a prophylaxis indication .The use of a placebo in adults and children in the Phase II studies in areas of high malaria incidence was strongly recommended in initial discussions with regulatory authorities because this is the only circumstance in which the true attack rate is known, and hence the true protective efficacy can be measured. The population enrolled in this study would ordinarily be exposed to sub-clinical malaria infection which would otherwise remain untreated. All subjects would receive an initial sterilizing cure for this sub-clinical malaria, and benefit from close clinical safety monitoring during the study, as well as standard-of-care treatment upon detection of positive parasitaemia during study conduct. The acceptability of placebo controlled trials in adults and in children will need additional discussion with the Ethics Committees at individual sites, to take account of local concerns on this topic. There are sometimes ethical objections to placebo injections, especially if repeated blood sampling was undertaken. An alternative would be to combine the placebo or experimental drug with a proven vaccine against a different disease.‘attack rate’) would be expected in this population, meaning that very large numbers of subjects would be required to demonstrate non-inferiority of the new medicine. For this reason, in the development of atovaquone–proguanil, the primary endpoint in Phase III studies was the overall frequency of any adverse events assessed at 7 days after leaving the endemic country, with protective efficacy as a secondary endpoint. In these comparator studies, confirmed P. falciparum malaria occurred in 0/486 and 0/477 subjects receiving atovaquone–proguanil and mefloquine, respectively [The protective efficacy, safety and tolerability would then be confirmed in a Phase III study in the target population. These are adults and children older than 5 years travelling from geographical areas with no, or a very low risk of malaria infection, to geographical areas of significant risk of malaria infection. Ethically, these studies need to be active controlled studies; oral atovaquone–proguanil being the most likely comparator. The protective efficacy of both atovaquone–proguanil and the NCE  is expected to be high, therefore a very low rate of malaria infection (ectively , and in ectively , illustrDemonstrating non-inferiority of protective efficacy would as mentioned above require very large trials. Making a number of assumptions about the risk of developing malaria for travellers from Europe, Canada and South Africa to East Africa who do not take prophylaxis, the average duration of travel and the efficacy of a prophylactic treatment, Høgh et al.  estimateIn migrant populations a number of potential approaches have been published, including enrolment of subjects in Travel Clinics , 87, proversus children was not considered critical as long as there is a good spread of age across the study.In initial discussion with MHRA, a total safety population of approximately 3000 subjects exposed to the clinical dose was deemed a sufficient safety data package for registration of an oral prophylaxis, provided no significant safety signals are detected. The breakdown numbers of adults First, the general acceptance that countries that are undergoing elimination will have internal populations migrating from low transmission zones to those of high transmission, for example going from the south to the north of Zambia. In the past, the prophylaxis of migrant populations was considered only to be commercially relevant for protecting western tourists and soldiers. However, it is now clear that cost-effective solutions must be found to protect migratory populations in low- and middle-income countries.Second, in countries that have eliminated malaria and are ‘maintaining zero’ , there iThird, there have been tremendous successes in the last 5 years using SMC, which involves giving a full treatment course of anti-malarials every month. A regimen which was truly optimized for prophylaxis, and available as an injection would have potentially major benefit. This latter point has come into focus recently because of the stagnation in the reduction of malaria incidence globally . One of Several factors have driven a renewed evaluation of the role of prophylaxis in the malaria elimination agenda over the last few years:First is the availability of new chemotypes with activity against the malaria parasite, which opens up the possibility of focusing on new medicines specifically designed for prophylaxis. This is an appealing situation since, ideally, the same medicine should not be used for both the protection and the treatment of malaria within the same geographic locale. To date, none of the new molecules has a half-life equivalent to that seen from oral doses of 4-aminoquinolines or the 8-aminoquinoline tafenoquine, hence the need for slow-release injectable formulations, to provide the appropriate duration of cover.P. falciparum liver stage assays and animal models, including a murine model of the hepatic infection of P. falciparum allows the comparison of different molecular classes in vivo. Standardization of protocols for such assays will be an important driving factor over the coming years, allowing head-to-head comparisons of small molecules and monoclonal antibodies.Second, the availability of better in vitro Third, in experimental medicine, the arrival of robust supplies of GMP-standardized sporozoites has enabled testing of new chemical series in human volunteer infection studies. These data, or alternatively those obtained in insect-driven infection studies, allow early identification of compounds with activity in humans, and estimation of human effective doses for full-scale clinical studies.Finally, new developments in formulation technologies allow the development of some of the molecules with prophylactic activity as long-acting injectables. This can be achieved by formulation of the parent compound or development and formulation of pro-drugs. Developments in HIV, oral contraception and antipsychotic medicines have shown that slow release allowing protection for one or even several months is possible, with acceptable dosing volumes and needle sizes. These studies also underline the need for highly potent molecules, to minimize cost, and to maximize acceptability.Several factors lead to some optimism for the future:Taken together, there are many reasons to be optimistic about the probability of identifying and developing long-acting injectable formulations. What is important is that a common language and common standards are applied to assess the different candidates, and on this basis Target Candidate Profiles are proposed. In addition, the route to registration will be a new one, not necessarily proceeding via the approval of medicines for a treatment indication. As such, new target product profiles and the use cases supporting their delivery in the field are important. This document contains proposals for such profiles, in the full knowledge that over the next few years clinical data will become available providing many lessons that aid in further refinement of the next generation of profiles. More discussion is needed with experts in the field and the bigger malaria community e.g. on the clinically relevant level of efficacy to be targeted. Furthermore, MMV is preparing clinical development strategies for new prophylactic medicines and engaging stringent regulatory authorities in early discussions.