Plasmodium (P.) falciparum species, particularly in sub-Saharan Africa, and it normally has an incubation period of seven to 14 days. Dormant disease secondary to P. vivax and P. ovale is well-reported, yet only a handful of cases report dormant malaria secondary to P. falciparum. Even though malaria is significantly less common in the United States in comparison to other parts of the world, it is still a growing concern given international travel from endemic regions and a growing immunocompromised population. Here, we present a case of Plasmodium falciparum malaria in a patient with systemic lupus erythematosus (SLE) with neuromyelitis optica spectrum disorder (NMOSD) and renal transplant without travel to sub-Saharan Africa in 10 years.Malaria is a global health concern with high morbidity and mortality. It is often attributed to the  Plasmodium (P.) falciparum, P. malariae, P. vivax, P. ovale, and P. knowlesi, and their primary mode of transmission is the female Anopheles mosquito. Other, less prevalent, modes of transmission include blood product exchange such as transfusions, organ transplants, needle sharing, or congenital transmission [Malaria is a complex, multisystem infection with a high level of global morbidity and mortality. In 2020, 241 million cases were reported worldwide, with mortality of 627,000 people [P. vivax and P. ovale but it is rare in P. falciparum malaria [In vector transmission, the Anopheles mosquito must have digested infected blood via a previous blood meal. The mosquito then infects the next human host with sporozoites, which mature into schizonts in liver cells, where they may stay dormant for multiple years. This most commonly occurs with the species  malaria . Eventua malaria . This st malaria ,4.P. falciparum, the species most common in sub-Saharan Africa, often causes the most severe infections and contributes to the majority of global malaria deaths. Usually, it has an incubation period of 7-14 days prior to symptomatic disease, and latency is rare [More severe sequelae are cerebral malaria, respiratory failure, acute renal failure, liver dysfunction, and severe anemia.  is rare .Plasmodium falciparum malaria in a patient with systemic lupus erythematosus (SLE) with neuromyelitis optica spectrum disorder (NMOSD) and renal transplant without travel to sub-Saharan Africa in 10 years.While malaria is much rarer in the United States than in other parts of the globe, approximately 2,000 cases are reported in the United States each year . Most maA 46-year-old woman with SLE complicated by membranous proliferative glomerulonephritis (MPGN), leading to end-stage renal disease (ESRD) requiring renal transplant and neuromyelitis optica spectrum disorder (NMOSD) presented to a Bronx, New York, hospital after a syncopal episode in the setting of subacute fatigue. She was found to have pancytopenia with white blood cells (WBC) of 2.4 k/μL, hemoglobin (Hgb) of 4.9 g/dL, with a mean corpuscular volume (MCV) of 80.1 fL, and platelet count of 19 k/μL. Bloodwork from an outpatient clinic visit showed the patient had a WBC of 3.8 k/μL, Hgb of 7.5 g/dL, and platelet count of 76 k/μL one week prior. She denied any overt bleeding, menorrhagia, symptoms of active SLE, or acute infection. Her SLE/NMOSD was currently inactive on maintenance rituximab, and she was taking tacrolimus and prednisone for her kidney transplant. She received 2 units of packed red blood cells (pRBCs) and the tacrolimus was quickly tapered. Further studies revealed total bilirubin, direct bilirubin, and haptoglobin were within normal limits, lactate dehydrogenase was mildly elevated at 275 U/L, and the reticulocyte index was low.Within hours of admission, she developed a fever of 102.8 degrees F without associated chills or diaphoresis and remained hemodynamically stable. Broad-spectrum antibiotics and an infectious workup were initiated. Chest X-ray, urinalysis, urine culture, bacterial and fungal blood cultures, sputum cultures, cryptococcal and Histoplasma antigens, serum fungal markers, respiratory pathogen panel, as well as Epstein Barr and Parvovirus B19 polymerase chain reaction (PCR), HIV antibodies, and PCR were unrevealing. Cytomegalovirus PCR revealed a low viral load of 107 IU/mL. Procalcitonin was >50 ng/mL, C-reactive protein was 20.2 mg/mL, and erythrocyte sedimentation rate was 43 mm/h. Double-stranded DNA was negative and complements 3 and 4 were within normal limits. Chest CT revealed a focus of branching and ground glass opacity seen in the right lung apex and right upper lobe with bronchiectasis  due to recurrent transfusion demands. However, on day 10 of hospitalization, a repeat CBC was collected while the patient was febrile to 100.7 degrees F. Later that day, the CBC was flagged for a manual parasite screen by the automated system. Thin film microscopy of a blood smear confirmed the presence of Outcome and follow-upThe patient was seen in the hematology clinic one week after discharge. Laboratory data from this time revealed her leukopenia had resolved, and her platelet count was improving (now 80 k/μL). Hemoglobin stabilized at 7.7 g/dL without any transfusions and the reticulocyte index at this time was consistent with hyperproliferative bone marrow. Repeat blood smear evaluation with microscopy and malaria antigen testing three months after treatment were both negative.P. falciparum malaria without recent travel to or visitors from endemic regions. Latent malaria secondary to P. ovale and P. vivax is well-documented, with some cases re-emerging after more than a decade of dormancy [P. falciparum is much rarer and its reporting is limited to case reports and series. One Canadian case was of a West African male with a history of childhood malaria who presented with P. falciparum malaria in the setting of diabetic ketoacidosis two years after his last known travel to West Africa [P. falciparum malaria with a dormancy range of three months to 10 years. All three patients had immigrated from sub-Saharan Africa and were immunocompromised due to HIV infections or pregnancy [Here, we present the case of a 46-year-old West African female with a history of SLE and NMOSD requiring renal transplant who presented with pancytopenia and cyclical fevers and was found to have dormancy . Howevert Africa . Dauby’sregnancy . These cP. falciparum malaria eight years later since travel to an endemic region without an identifiable trigger [P. falciparum remained, even in individuals whose last exposure was 13-16 years prior [However, another case by Szmitko et. al. presented a healthy young woman without a past medical history who was born in an endemic region of Africa and had a delayed presentation of  trigger . In all rs prior . Others P. falciparum, however, reported cases presented within the expected incubation period [P. falciparum and presented soon after transplant without dormancy [In the United States, the majority of malaria cases stem from VFRs, like in other non-endemic countries . Our patn period ,16. Our dormancy ,18. Our Lastly, another unique aspect of this presentation is our patient’s autoimmune history, which added non-infectious inflammatory syndromes to the differential such as Adult Still’s disease and Familial Mediterranean Fever. Given her immunocompromised state and transfusion-dependent state, finding the correct diagnosis in a timely manner was crucial.P. falciparum malaria. Despite a lower incidence of malaria in non-endemic countries, malaria must still be considered in a patient with cyclical fevers and a travel history to an endemic malaria region, even if it was remote. Vulnerable populations, such as those on immunosuppressive medications, are at particular risk for these infectious diseases and a delay in diagnosis could have significantly poor outcomes.In summary, our case highlights individuals with partial immunity to malaria may have a delayed presentation or re-emergence of P. falciparum malaria in an immunosuppressed individual living in the United States without recent travel or visitors from an endemic region. Partial immunity to malaria from childhood exposure may have played a role in this atypical presentation. Additionally, although less common, P. falciparum may be transmitted via blood transfusion or organ transplant. Therefore, cyclical fevers should raise suspicion of malaria in patients who are immunosuppressed or from endemic malaria regions, even in the absence of recent travel to or visitors from endemic regions. The population of patients who travel between endemic and non-endemic malaria regions and those who are immunocompromised is growing. Thus, the number of patients vulnerable to malaria infections is also increasing, and a delay in diagnosis could have detrimental outcomes.Our report presents a unique case of latent


Plasmodium falciparum malaria may be protective of COVID-19 severity and mortality. The number of COVID-19 cases and deaths, however, may be vastly undercounted. Very little is known about the extent to which the Tanzanian population has been exposed to SARS-CoV-2. Here, we investigated the seroprevalence of IgG to SARS-CoV-2 spike protein in two Tanzanian rural communities 1½ years into the pandemic and the association of coinciding malaria infection and exposure.African countries stand out globally as the region seemingly least affected by the COVID-19 pandemic, caused by the virus SARS-CoV-2. Besides a younger population and potential pre-existing immunity to a SARS-CoV-2-like virus, it has been hypothesized that co-infection or recent history of P. falciparum; CIDRα1.1, CIDRα1.4 and CIDRα1.5 of PfEMP1 and GLURP and MSP3.During a malariometric survey in July 2021 in two villages in north-eastern Tanzania, blood samples were taken from 501 participants (0–19 years old). Malaria was detected by mRDT and microscopy. Levels of IgG against the spike protein of SARS-CoV-2 were measured by ELISA as well as IgG against five different antigens of p < 0.001). P. falciparum malaria prevalence by blood smear microscopy was 7.9% in Kwamasimba and 2.1% in Mkokola. 81.3% and 70.5% in Kwamasimba and Mkokola, respectively, showed recognition of minimum one malaria antigen. Residing in Kwamasimba was associated with a broader recognition . The recognition of malaria antigens increased significantly with age in both villages . Being SARS-CoV-2 seropositive did not associate with the breadth of malaria antigen recognition when adjusting for age .The seroprevalence of SARS-CoV-2 IgG was 39.7% (106/267) in Kwamasimba and 32.5% (76/234) in Mkokola. In both villages the odds of being seropositive increased significantly with age (AOR = 1.12, 95% CI 1.07–1.17, More than a third of the children and adolescents in two rural communities in Tanzania had antibodies to SARS-CoV-2. In particular, the adolescents were seropositive but being seropositive did not associate with the status of coinciding malaria infections or previous exposure. In Tanzania, natural immunity may have developed fast, potentially protecting a substantial part of the population from later variants.The online version contains supplementary material available at 10.1186/s12879-022-07820-6. The coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19, was first reported in Wuhan, China in December 2019 , 4. The In Africa, when the pandemic occurred, there were major public health concerns due to the existing high prevalence rates for both infectious and non-infectious diseases and the putative adverse consequences of COVID-19 preventive lockdown measures on the prevention and management of other infectious diseases such as malaria .The latest WHO world malaria report revealed an estimated 241 million malaria cases worldwide in 2020 ; an incrPlasmodium falciparum malaria infection may be protective of COVID-19 severity and mortality [Intriguingly, African countries including Tanzania, stand out globally as the region seemingly least affected by the COVID-19 pandemic as compared to North America and Europe. Besides a younger population and potential pre-existing immunity to a SARS-CoV-2-like virus , 13, it ortality , 15. Theortality , and a nortality .Little is known as to what extent the Tanzanian population has been exposed to SARS-CoV-2. So far around 39,000 confirmed cases and 845 deaths due to COVID-19 have been reported to the WHO since the 3rd of January 2020 with the majority  reported on the 2nd of August 2021 . Here, aP. falciparum. These antigens were three cysteine-rich interdomain region (CIDR) domains  of P. falciparum erythrocyte membrane protein 1 (PfEMP1) associated with the sequestration of infected erythrocytes and severe malaria [We further investigated the seroprevalence of spike protein-specific IgG in relation to the prevalence of malaria and the breadth of IgG reactivity to five different antigens of  malaria , 24, and malaria . Lastly,.The economic activities are mainly subsistence farming and petty trading. The study communities have been participating in annual surveys since 2003 when the prevalence of P. falciparum malaria was higher in Mkokola (78%) than in Kwamasimba (25%) [®  were obtained on site and blood smears for microscopy were prepared at the National Institute for Medical Research, Korogwe Research Laboratory. Study participants who were diagnosed with malaria (mRDT) and with or without anaemia were given appropriate drugs administered as per existing treatment guidelines. Additionally, other diseases such as diarrhoea and skin fungal infections were managed as per the clinician’s discretion.A cross-sectional survey was conducted in two rural villages, highland Kwamasimba (KWA) (700 m above sea level) and lowland Mkokola (MKL) (300 m above sea level). The villages are located in Korogwe District, about 100 km from the Indian Ocean coastline and Tanga City in north-eastern Tanzania. According to an updated census survey the population sizes of Kwamasimba and Mkokola were around 2000 each  Census survey 2018) during the cross-sectional surveyba (25%)  whereas ba (25%) . The ovePlasmodium and for species determination, respectively. The blood slides were prepared using a premade template for quantification of parasite densities and determination of Plasmodium species, respectively. The blood slides were fixed with methanol and stained with 5% Giemsa for 20 min and depending on parasite density, parasite count was reported against 200 or 500 white blood cells. Two independent microscopists blinded of the mRDT results, who participate in malaria microscopy proficiency testing, read the blood smears. Plasma samples were obtained by centrifugation of the venous blood and stored at − 80 °C until further testing. During the survey, all national guidelines for the prevention of COVID-19 were followed, including wearing face masks, handwashing, use of hand sanitizers, and distancing whenever possible.The cross-sectional survey was conducted from the 19th to the 30th of July 2021 to determine the malaria point prevalence in children and adolescents aged between 0 and 19 years old as previously done . During manuscript in prep.) diluted in PBS. Plates were blocked with 3% skimmed milk in PBST (PBS and 0.1% Tween 20) for 1 h at room temperature. Plasma samples were diluted 1:200 with 1% skimmed milk in PBST and 50 µL was added into each well followed by 2 h incubation at room temperature. Microtiterplates were washed three times with PBST, and to detect bound human IgG, rabbit anti-human IgG horseradish peroxidase (HRP) conjugated antibody  was then added (1:4000) and incubated at room temperature for 1 h. TMB was added following manufacturer’s instruction and optical densities (ODs) were read at 450 nm. One hundred samples collected from the same two villages in the years 2015 to 2019 were randomly selected and used as negative control and seropositivity was determined as values above three standard deviations of the mean of the 2015 and 2019 samples [Plasma samples were analysed for SARS-CoV-2 spike IgG titres as described elsewhere  with sli samples . To vali samples , a positIgG levels against recombinant domains of three different PfEMP1s , GLURPR2 and MSP3 , 32 werep < 0.2 in the univariate analysis. A p-value of < 0.05 was considered statistically significant.The demographic and clinical data were collected on a paper-based questionnaire and then double-entered into a Microsoft Access database, validated and transferred into Stata v13.0  for cleaning and merging with ELISA OD values. Statistical analyses were done in STATA and GraphPad Prism v8.4.2 . In proportional tests, data are presented as percentages, medians, and Chi-square. The Wilcoxon rank-sum (Mann–Whitney) was used to assess the relationship between SARS-CoV-2 spike IgG OD values and malaria prevalence. The correlation between ordered age groups and seropositivity of COVID-19 and malaria was assessed using the Trend test across ordered groups. Multivariate logistic regressions were performed to assess the relationship between SARS-CoV-2 spike seropositivity with malaria prevalence, breadth of malaria antigen recognition, demographic, and clinical data. In the multivariate analysis, a forward selection was done for variables with . In 2021, the P. falciparum malaria prevalence by mRDT was 15.4% in Kwamasimba and 6.4% in Mkokola, primarily among children above 5 years of age  and diarrhoea  than in Kwamasimba, otherwise no marked differences in the clinical characteristics in the two villages was observed; such as haemoglobin levels, coughing, body weakness, headache, body or abdominal pain, loss of appetite, vomiting, and yellowness of eyes  were from the highland village, Kwamasimba and 234 (46.7%) were from the lowland village, Mkokola , however, the seropositivity increased with age in both villages from around 20% in the 1–4-year-olds  to at least 50% in the older age group of 15–19 years; 68.6% and 50% in Kwamasimba  and Mkokola , respectively  in Kwamasimba and Mkokola, respectively, showed recognition of minimum one antigen  and MSP3  was similar between the two villages. On the contrary, the serorecognition of the three PfEMP1 domains was significantly higher in Kwamasimba than in Mkokola: CIDRα1.1: 24.7% and 15.8% ; CIDRα1.4: 38.6% and 24.4% ; CIDRα1.5: 3.8% and 0.8% , respectively. The number of malaria antigens recognized was significantly increased with age in both villages  (Table The IgG reactivity in the 501 plasma samples to five p = 0.04) but in the multivariate regression analysis the association disappeared  .The association of SARS-CoV-2 seropositivity and demographic and clinical characteristics was also explored by logistic regression Table . The SARp < 0.001) and the recognition increased significantly with age in both villages AOR = 1.12; 95% CI 1.08–1.16, p < 0.001). The breadth of malaria antigen recognition was inversely associated with a previous 2-day clinical history of having fever  and associated with not having body pain .The association between the breadth of malaria antigen recognition and demographic and clinical data was explored by ordered logistic regression Table . ResidinSerum antibody testing has the potential to detect previous COVID-19 infections. We therefore measured the seroprevalence of SARS-CoV-2 spike protein IgG in blood samples from two rural villages in Tanzania during the pandemic, July 2021. Around a third of the participants were positive for antibodies recognizing the spike protein and the seroprevalence increased with age, from ~ 20% in the 1–4-year-olds to ~ 60% in the 15–19-year-olds. Global seroprevalence has been estimated to be ~ 60% by September 2021 from either infection or vaccination, and children under 10 years and adults above 60 were less likely to be seropositive than 20–29-year-old . In AfriThe antibody titers were at similar levels as the tested convalescent samples used. COVID-19 in children and adolescents is associated with asymptomatic or mild illness and much lower mortality than in adults . WhetherIn countries in North America and Europe public health measures, such as school closures, have minimized children’s exposure to SARS-CoV-2 but seroprevalence of SARS-CoV-2 antibodies seem similar irrespective of age , 37. IntThe seroprevalence of SARS-CoV-2 varied markedly among geographic regions early in the pandemic and was higher in high-income countries with very high human development index levels , howeverPrevious exposure to SARS-CoV-2-like virus inducing cross-reactive antibodies have been shown using ELISA in both Sierra Leone (52% cross-reactivity) and the Democratic Republic of Congo (19.2% cross-reactivity) , 13, resP. falciparum. It has been hypothesized that co-infections or a recent history of malaria infection could be protective of COVID-19 severity and mortality [Medical conditions such as cancer, chronic lung diseases, heart conditions, diabetes, but as well as infectious diseases such as tuberculosis  and HIV ortality , 15. HowThis current study was limited by an overall malaria prevalence of only 5.2% by blood smear microscopy (11.2% by mRDT) and by not investigating coinciding SARS-CoV-2 infections using PCR or IgM, or severe illness from COVID-19 infection. Furthermore, the used questionnaire was not designed to capture clinical information linked to a potential SARS-CoV-2 infection since this study was done on already collected samples from a malariometric cross-sectional survey. A longitudinal cohort study would have been more appropriate to investigate an association of the history of malaria exposure and the incidence of COVID-19 infections.We did, however, find a higher serorecognition of malaria antigens in Kwamasimba, in particular among the 1–4-year-olds, which indicates a higher prior exposure to malaria parasites  as refleWe found that more than a third of children and adolescents in rural Tanzania were having antibodies to SARS-CoV-2. This was almost one and a half year into the pandemic, few days prior to the highest number of recorded cases in the country, prior COVID-19 vaccination, and prior to the rapid spreading of the omicron variant (B.1.1.529) . In a coAdditional file 1. Baseline characteristics of the study populations from before the COVID-19 pandemic.Additional file 2 The number of SARS-CoV-2 seropositive and negative infants in Kwamasimba and Mkokola villages based on age in months.


Arterialsclerosis caused by hypertension can lead to many complications, such as heart attack, stroke and so on. Early diagnosis and treatment of arterialsclerosis can prevent cardiovascular and cerebrovascular diseases and improve the prognosis. The present study aimed to explore the value of ultrasonography in evaluating the early lesion of the local arterial wall in hypertensive rats and identify useful elastography parameters.A total of 24 spontaneously hypertensive rats (SHR), 10-, 20-, 30-, and 40-weeks-old, were used in this study, with 6 rats in each group. Blood pressure was recorded using the Animal Noninvasive Blood Pressure Measurement System , and the local elasticity of the abdominal aorta of rats was measured using a ultrasound diagnostic instrument . According to the histopathological results, SHR were divided into two groups: the normal arterial elasticity and the early arterial wall lesions. Mann–Whitney U test was used to compare the differences in elastic parameters and influencing factors between the above two groups, and receiver operating characteristic curve (ROC) was used to analyze and judge the value of each elastic parameter in evaluating early arterial lesions.A total of 22 cases were divided into two groups: 14 in the normal arterial elasticity and 8 in the early arterial wall lesions. The differences in age, blood pressure, pulse wave velocity (PWV), compliance coefficient (CC), distensibility coefficient (DC), and elasticity parameter (EP) between the two groups were compared. The differences in PWV, CC, DC and EP were statistically significant. Subsequently, the ROC curve analysis was performed for the above four evaluation indexes of arterial elasticity; the results were as follows: the area under the curve of PWV, CC, DC, and EP was 0.946, 0.781, 0.946, and 0.911, respectively.Early arterial wall lesions can be evaluated by ultrasound measurement of local PWV. PWV and DC can accurately evaluate the early arterial wall lesions in SHR, and the combined application of the two can improve the sensitivity and specificity of the approach. The incidence of hypertension has increased in recent years, and high blood pressure is the primary factor leading to ischemic heart disease and premature death. High blood pressure also leads to heart failure, stroke, atrial fibrillation, chronic kidney disease, and peripheral vascular disease and is a major risk factor for cognitive decline . ArterioThe early manifestations of atherosclerosis include decreased arterial wall elasticity, hyperplasia of collagen fibers, and formation of endodermal lipid vacuoles . The indAlthough PWV has been shown to evaluate arterial elasticity in hypertensive patients, only some clinical studies have been conducted in human populations –19. The According to the international, national, and institutional rules, the study was performed considering animal experiments, clinical studies, and biodiversity. The study protocol was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University, Beijing, China.A total of 24 spontaneously hypertensive rats (SHR)  were purchased from Beijing Huafucang Biotechnology Co, Ltd. The rats were fed under a specific pathogen-free (SPF) grade environment in the Animal Experimental Center of Beijing Friendship Hospital, Capital Medical University. SHR rats were divided into four groups: 10-, 20-, 30-, and 40-weeks-old, with six animals in each group, according to the rule that blood pressure increases with increasing age.The rats were preheated at 40 ℃ for 15 min and the systolic blood pressure (SP) and diastolic blood pressure (DP) of the caudal artery was measured by the American Kent Company Animal Noninvasive Blood Pressure Measurement System (model CODA) at 10, 20, 30, and 40 weeks after resting and wakefulness . Each anThe elasticity of the abdominal aorta in rats was measured using a VINNO M80 ultrasonic diagnostic instrument with probe X6-16L (frequency 6.5–18 MHz). The main abdominal aorta was identified in the left abdomen of the rats under anesthesia. The abdominal aorta was maintained parallel to the probe to display the long axis of the abdominal aorta. After the position was determined, the “PWV” key was started. The movement track of the abdominal aortic wall on each sampling line was presented on the image in the form of an expansion wave, showing the expansion wave of the movement track of the abdominal aortic wall in at least five stable cardiac cycles. The measuring cursor was used to indicate the position of “trough to trough” in one cardiac cycle. The local PWV, CC, DC, and EP of the vessel wall were measured five times for each measurement. Then, the maximum and minimum values were removed, and the average value of the remaining three times was calculated  curve. P < 0.05 for the area under the curve (AUC) was statistically significant.Since the ultrasound imaging of the abdominal aorta was unsatisfactory and the parameters of elastography deviated greatly, 22 slices from SHR rats were subjected to Masson staining. The final lesion degree was indicated by the total score; the score of no collagen fibrous hyperplasia and lipid vacuoles was 0, the score of mild collagen fibrous hyperplasia and no lipid vacuoles was 1, the score of moderate and severe collagen fibrous hyperplasia and no lipid vacuoles was 2, and the score of collagen fibrous hyperplasia and lipid vacuoles was 3 Fig. .Fig. 4HiAccording to the pathological results, the score 0 was divided into the normal group, and the score > 1 was divided into the early arterial wall lesions group. The two groups were compared in terms of age in weeks, blood pressure, and ultrasonic elasticity indices. The results showed significant differences in PWV, CC, DC, and EP Table .Table1MaAccording to the Mann–Whitney U-test results of the above two groups of rats, the ROC curves were drawn to evaluate the accuracy of PWV, CC, DC, and EP in predicting the early arterial wall lesions of SHR. The level of diagnostic value was indicated by the AUC. In this study, the AUC of PWV for diagnosis of early arteriosclerosis was 0.946  0.857–1.000, P < 0.05), the AUC of CC was 0.781 , the AUC of DC was 0.946 , the AUC of EP was 0.911 . Also, the cutoff values of each parameter were calculated by Youden index. Table , Fig. 6.In this study, local PWV was measured using ultrasound equipment to assess arterial elasticity. Invasive and non-invasive methods are applied to assess local PWV. The non-invasive evaluation methods include ultrasound and magnetic resonance imaging (MRI) [11.12]. The two main methods for ultrasound assessment of local PWV reported in the literature are vascular echo tracking (ET)  and PWV In addition, multiple vascular elastic parameters were obtained simultaneously in this study. This is different from the above two ultrasonic devices.The parameters obtained by ultra-high speed ultrasound are the pwv at the beginning and at the end of systole (PWV-BS and PWV-ES), and the parameters obtained by ET are arterial compliance (AC), the arterial stiffness index (β), and the single-point pulsed wave velocity (PWVβ) , 15, 22.Many clinical factors, such as age, blood pressure, heart rate, and other cardiovascular risk factors, can affect the measured values of vascular elasticity parameters . Among tFurthermore, no significant difference was observed in age between the normal and the early arteriosclerosis groups. This phenomenon was different from that reported previously. Some studies have shown that PWV increases with age as arterial stiffness increases in humans , which cA new ultrasound device was used to evaluate the elastic function of the abdominal aorta in rats in this study. Four elastic parameters were obtained, including PWV, CC, DC, and EP. Significant differences were observed in the above four elastic parameters between the normal and the early arterial wall lesions groups. The ROC curve analysis showed that the sensitivity and specificity of PWV and DC were high, which could be used in combination to evaluate the arterial elasticity. Also, no statistically significant difference was detected in blood pressure and weeks of age between the normal and the early arterial wall lesions groups, which is different from previous studies, indicating that elastography parameters can accurately assess arterial elastic function without being affected by other factors.


Plasmodium vivax-induced malaria. The unique aspect of the parasite’s biology and interactions with the human host make it harder to control and eliminate the disease. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Duffy-negative blood groups are two red blood cell (RBC) variations that can confer protection against malaria.Over a third of the world’s population is at risk of Molecular genotyping of G6PD and Duffy variants was performed in 225 unrelated patients  recruited at a Reference Centre for Infectious Diseases in Manaus. G6PD and Duffy variants characterizations were performed using Real Time PCR (qPCR) and PCR–RFLP, respectively.FY*A/FY*B was predominant in both uncomplicated (45.3%) and severe malaria (39.2%). Only one Duffy phenotype Fy(a-b) was found and this involved uncomplicated vivax malaria. The G6PD c.202G > A variant was found in 11 (4.88%) females and 18 (8.0%) males, while c.376A > G was found in 20 females (8.88%) and 23 (10.22%) male patients. When combined GATA mutated and c.202G > A and c.376A > G mutated, was observed at a lower frequency in uncomplicated (3.7%) in comparison to severe malaria (37.9%). The phenotype Fy(a−b +) (p = 0.022) with FY*B/FY*B (p = 0.015) genotype correlated with higher parasitaemia.The Duffy blood group system showed a phenotypic distribution Fy(a + b−) of 70 (31.1%), Fy(a + b +) 96 (42.7%), Fy(a−b +) 56 (24.9%) and Fy(a−b−) 1 (0.44%.) The genotype P. vivax and this deserves to be further investigated and explored among this population.A high prevalence of G6PD c202G > A and c.376A > G and Duffy variants is observed in Manaus, an endemic area for vivax malaria. In addition, this study reports for the first time the Duffy null phenotype Fy(a-b-) in the population of the Amazonas state. Moreover, it is understood that the relationship between G6PD and Duffy variants can modify clinical symptoms in malaria caused by The online version contains supplementary material available at 10.1186/s12936-022-04165-y. Plasmodium vivax cases of 118,651  and Plasmodium falciparum of 22,182 (15.8%) [Malaria is one of the most serious public health problems worldwide, and in the tropical and subtropical regions, this parasitic disease is the leading cause of social and economic problems , 2. In 2 (15.8%) .Plasmodium sp., the parasite load, the host genetics factor, the immunological status of the host [P. vivax infections are endemic, clinical complications and mortality are reported and this has led to the characterization of P. vivax malaria as a serious or even fatal disease [Susceptibility or resistance to malarial infection depends on the host-parasite interactions, the the host , 5. The the host . In regi disease , 8.P. vivax malaria is mainly associated with anaemia, which is occasionally due to severe haemolysis [Although little is known of the pathophysiology, the progression and aggravation of emolysis –11. Otheemolysis , 13.P. vivax, and blood system antigens act as receptors for P. vivax merozoite ligands that contain Duffy-Binding-Like (DBL) domains through alleles. These Duffy blood group system antigens  are encoded by two co-dominant allelic forms FY*A and FY*B that differ by the single nucleotide polymorphism (SNP) in position c.125G > A of the exon 2 [FY*BN.01 that silences Fyb expression in erythroid cells, and homozygous individuals (FY*BN.01 / FY*BN.01) bear the Fy(a−b−) phenotype that is known to be a protective factor against vivax malaria infections [The Duffy Antigen Receptor for Chemokines (DARC), recently renamed Atypical Chemokine Receptor 1 (ACKR1), acts as a possible facilitator in the process of erythrocyte invasion by e exon 2 . The SNPfections .b antigen expression is determined by SNP c.265C > T and c.298G > A. These polymorphisms occur within the first intracellular loop of the Duffy protein, which result in reduced expression of the Fyb antigen. The frequency of Fyb weak, characterized as FY*BW.01 is approximately 2% in Caucasians [a weak (FY*AW.01) allele are yet to be observed in inhabitants of the Amazonas state, Brazil [Another phenotype associated with weak Fyucasians . Some po, Brazil , 18.Glucose 6-phosphate dehydrogenase (G6PD) is an enzyme involved in the pentose monophosphate pathway. Deficiency of this enzyme leads to free radical-mediated oxidative damage to erythrocytes, thus causing haemolysis. G6PD deficiency, linked to the inheritance of X chromosome(s) with disease causing variant(s), is mostly prevalent in people of African, Asian, and Mediterranean descent , 20. In P. vivax malaria, comorbidities such as inherited haemoglobinopathies and G6PD deficiency require investigation since they may also be factors that affect heterogeneity in clinical manifestations, and may be characterized by subtypes or endophenotypes [The clinical severity between malaria endemic areas can be complex with multifactorial influence and genetic factors . In endeenotypes .P. vivax to infect the vector before symptoms occur, favours the perpetuation of the parasitic life cycle and, due to subpatent infections, this causes difficulty in tracking infected individuals [P. vivax to oxidative stress in G6PD deficiency, with negative influence on the parasite, as well as the association of different phenotypes of the Duffy blood group in the invasion of RBCs [Vivax malaria is a challenge for malaria control strategies and its elimination . The uniividuals . Many stividuals , 31, 32. of RBCs –35. FurtPlasmodium infections, viral infections and pregnant women were excluded. Patients were classified as either uncomplicated malaria or severe malaria following the World Health Organization (WHO) recommendations , a reference center for infectious diseases in Manaus, capital of the Amazonas state, Brazil. Blood samples were collected from all the participants. Inclusion criteria were patients over 18 years of age, of either sex and of any skin colour with severe  or uncomplicated (outpatient) malaria, and without any associated diseases. All patients were treated at the clinical research ward at the hospital. All the patients included in the study are unrelated individuals, non-smokers and non-diabetic. Patients with comorbidities, haemoglobinopathies, mixed Approximately 2.5 mL of peripheral blood were collected in tube with EDTA  at a concentration of 1.5 mg/mL for blood counts. Aliquots of 0.3 mL blood in 1.5 mL tubes were kept for extraction of nuclear DNA. An additional 2.5 mL were collected in a tube without anticoagulants for biochemical analyses. Immediately after blood collection, haematological determinations were performed using an automated counter  and biochemistry was performed on a Beckman Coulter .Plasmodium species  was done by polymerase chain reaction (PCR) and Real Time PCR (qPCR) methods [The diagnosis of the malaria infection followed the standardized protocol established by FMT-HVD, which is a nationally recognized centre for the diagnosis of malaria. In summary, the malaria diagnosis was performed under light microscopy with multiple readings for the parasitaemia. Identification of  methods . The det methods :\documenDNA was extracted from 200 µL of whole blood according to the QIAamp DNA Mini Kit  manufacturer's protocol . After extraction, the DNA was quantified using a spectrophotometer , and then stored at − 20 ºC.FY*BN.01 allele from c.1-67 T > C mutation [Duffy genotyping was performed using conventional PCR for amplification of the sequence of interest, followed by restriction enzyme digestion; one for Duffy blood group (Duffy PCR) and one for the GATA box variant (GATA PCR). The Fy(a − b −) phenotype is known as “erythrocyte silent” and arises from homozygosity mutation . This mumutation , 40.BanI restriction enzyme digestion and incubated for at least 4 h at 37 ºC. The enzyme digestion product was electrophoresed on a 1.5% agarose gel stained with ethidium bromide for discrimination of the alleles.Genotyping for Duffy blood system groups was performed with synthetic oligonucleotides FYAB1 (5 'TCC CCC TCA ACT GAG AAC TC 3') and FAB2 (5 'AAG GCT GAG CCA TAC CAG AC 3'). Amplified products were visualized on a 1.5% agarose gel stained with ethidium bromide. After PCR amplification was confirmed, the products were processed using http://www.isbtweb.org/fileadmin/userupload/Workingparties/WPonRedCellImmunogeneticsand/008FYAllelesv4.1.pdf) [Duffy genotypes, as well as phenotype characterizations, were named according to the FY (ISBT 008) Blood Group Alleles (4.1.pdf) .FY*A/FY*B and FY*B/FY*B genotype samples were used. Synthetic oligonucleotides FYN1 (5 'CAA GGC TGA CCC TA 3') and FYN2 (5 'CAT GGC ACC GTT TGG TTC AG 3') were used for the GATA PCR.For verification of c.-67 T > C SNP in the GATA box promoter region, StyI restriction enzyme and incubated for at least 4 h at 37 °C. The enzyme digestion product was observed on a 2.5% agarose gel. GATA normal genotypes appeared as 108 and 81 bp bands, while GATA mutated showed an additional 61 bp band. Samples with genotypes FY*A/FY*A and FY*A/FY*B were used to verify the SNPs c.265C > T and c.298G > A in the FY*AW.02 coding region and for Fyx.The GATA PCR product was treated with an MspAI in order to verify the SNP c.265C > T, and was then incubated for 4 h at 60 °C. For the SNP c.298G > A, the Mwol restriction enzyme was used and then incubated for 4 h at 37 °C. Both were discriminated on an 8% polyacrylamide gel. The mutated genotypes for the SNP c.265C > T had an additional band of 161 bp, and c.298G > A had an additional band of 343 bp.The PCR product was treated with the restriction enzyme For characterization of the variants, Real Time PCR (qPCR) was performed  using TaqMan® probes that were specific for each polymorphism. The amplification reaction was performed for a final volume of 12 uL/reaction, which contained 5 μL of 2 × TaqMan Universal Master Mix, 0.3 μL of 20 × SNP genotyping assay, 4.8 μL of sterile water and 2.0 μL of DNA (~ 100 ng) of the sample. The G6PD variants analysed in this project were chosen based on globally observed frequencies and their clinical importance according to WHO classification. The inclusion of the G6PD variants c.202G > A and c.376A > G was based on several studies that showed that both account for more than 95% of all mutations demonstrated in the Brazilian population, as well as in the population of the Amazonas state. For the qPCR technique, the (A-) V68M (202G > A) (rs1050828) and (A +) N126D (376A > G) (rs1050829) probes were used. To confirm the mutations found, amplification by PCR of the relevant DNA segments was performed and was followed by DNA sequencing  –44.Data were entered into a database using Graphpad-Prism 5.0 software  and IBM SPSS Statistics, version 19 , and organized by variable type. The analysis of qualitative or categorical variables of three or more groups was performed using the non-parametric Chi-square test (χ2) test using Yates' continuity correction. For the analysis categorical or dichotomous data was used Fisher's exact test when the number of subjects per cell was less than 5. All the statistical tests were performed using a significance level of 5%. The Hardy–Weinberg equilibrium (HWE) was evaluated by comparing expected genotypic frequencies with the observed ones using a Chi-square test.P. vivax malaria were included in the study. Ninety-seven patients (43.1%) had uncomplicated malaria and 128  had severe malaria. Table P. vivax malaria stratified by gender. Most of the severe malaria male cases had pallor (82.5%), choluria (77.8), headache (76.2%), loss of appetite (74.6%) and hepatosplenomegaly (71.4%). Among the female patients, headache (92.9%), loss of appetite (85.7%), pallor (83.3%), vomiting (83.3%) and choluria (73.8%) were the common symptoms.A total of 225 patients diagnosed with ients 43.% had unc6 uL), haemoglobin  and haematocrit . High bilirubin levels were also observed  (ANOVA—p < 0.001). Hypoglycaemia (< 70 mg/dL), a clinical and laboratory features of severe malaria, was detected in 16% of patients with severe malaria  and severe malaria (39.2%)  Fig. . The pheTwenty-nine patients were carriers of the G6PD c.202G > A variant; 18 (8.0%) males were hemizygous, 10 (4.44%) and 1 (0.44%) female was heterozygous and homozygous, respectively. The c.376A > G was identified in 43 (19.11%) patients. Eighteen women (8.0%) were heterozygous and 2 (0.88%) were homozygous. Twenty-three (10.22%) males were hemizygous. Both SNPs were in HWE as calculated from the frequencies of the alleles among the females, since the G6PD gene is located on the X chromosome.FY*BN.01/FY*BN.01, responsible for the Fy(a−b−) phenotype, was observed in one patient  with uncomplicated malaria who was not a carrier of the G6PD variants. Among patients with uncomplicated malaria, the frequency of c.202G > A and c.376A > G variants was low, particularly when these polymorphisms were concomitant. Severe malaria patients showed no variations in frequency for the c.202G > A variant. However, in the presence of A376G, there was a slight decrease in frequency.The 3, is shown in Figs. FY*A/ FY*A and FY*B/ FY*B, with lower parasitaemia in the presence of the FY*A/FY*BN.01 (p = 0.015).When combined with the GATA variant, the c.202G > A and c.376A > G variants were observed at a lower frequency in uncomplicated (3.7%) malaria in comparison to severe malaria (37.9%) Table . The levPlasmodium species, is not related to gender, though children and pregnant women are more susceptible to malaria. However, socio-economic and cultural factors play a central factor in determining differences in gender vulnerability to malaria [Death due to malaria, regardless of the  malaria –47.Six (19.35%) female patients and sixteen (39.02%) male patients had both G6PD c.202G > A/c.376A > G mutations concomitantly c.202G > A and c.376A > G are responsible for the majority of the observed prevalence of G6PD deficiency in Brazil. The occurrence of these concomitant mutations, which can be in heterozygous or homozygous states in females and hemizygous in males, deserves attention in genetic association studies in order to investigate possible clinical phenotypes of this deficiency among patients receiving treatment with malaria medication –50.The higher the parasitaemia in the host, the higher the consumption of glucose that may lead to hypoglycaemia, mainly in severe malaria. Hypoglycaemia may provoke dizziness, palpitations, tremors and loss of consciousness . CarefulFY*A/FY*B was the most frequent genotype in both malaria patients, uncomplicated (45.3%) and severe (39.2%). These findings corroborate the study reported by Cavasini et al. [FY*A and FY*B alleles among P. vivax malaria patients. The FY*A/FY*B and FY*A/FY*A genotypes were associated with a high frequency of P. vivax infection, thus suggesting that these individuals have a higher risk of developing malaria disease. The FY*A/FY*A and FY*A/FY*B genotypes are associated with increased frequency of P. vivax infection, while FY*A/FY*BW.01 and FY*B/FY*BW.01 were associated with low parasitic density levels [In this study, i et al.  which coy levels .P. vivax malaria infection [FY*A and FY*B antigens.The presence of a single case with null Duffy phenotype Fy(a−b−) is first described in the population of the Amazonas state. In endemic areas, the Duffy negative blood group is reported as a protective factor against nfection . In a stnfection . These r6 epitopes was significantly lower in reticulocytes and RBCs from individual carriers of the FY*B/FY*B genotypes compared with individuals of the FY*A/FY*A or FY*A/FY*B genotypes. Another similar study showed that the presence of the FY*BN.01 allele resulted in a 50% reduction of that protein when invaded by P. vivax [For the process of parasite invasion into RBCs, Duffy protein is functionally important. In regions of high malaria transmission rates, as in the inhabitants of the Amazon, Duffy protein is naturally immunogenic. Woolley et al.  demonstrP. vivax .P. vivax malaria patients, the FY*A and FY*B alleles were found to have low, medium, and high parasitic density [FY*BN.01 and FY*BW.01 alleles were found only in patients with low parasitic density and low symptomatology.In a study with  density . HoweverFY*BES (ES: erythrocyte silent). The frequency of the FY*BES allele was 41.0% [A study performed in the state of Pará-Brazil, with a population of African descent demonstrated that the presence of the c.202G > A variant was 0.060, the Duffy blood group included 24.3% Duffy negative and 41.3% of the individuals were heterozygous for as 41.0% . These fP. vivax infections [Several studies have revealed that the complexity of phenotypic and genotypic variation of the Duffy system and the variants of G6PD (A-) is geographically variable across human populations in areas in which malaria is endemic , 58–61. fections .One of the major confounders and a limitation in this study is the inherent disadvantage of G6PD deficiency due to the associated haemolysis, which may be one of the factors that account for the absence of consensus on the G6PD-malaria protection hypothesis. In some cases, this can potentially protect against uncomplicated malaria, but not severe malaria , 64. DesP. vivax and this deserves to be further investigated and explored among this population.A high prevalence of G6PD c202G > A and c.376A > G and Duffy variants is observed in Manaus, Amazonas state, Brazil, principally in severe vivax malaria patients. This presents a new viewpoint in the protection or not in vivax malaria. In addition, this study reports for the first time the Duffy null phenotype Fy(a−b−) in the population of the Amazonas state. Moreover, it is understood that the relationship between G6PD and Duffy variants can modify clinical symptoms in malaria caused by Additional file 1: Table S1. World Health Organization diagnostic criteria for severe falciparum malaria. Adapted from: Guidelines for the Treatment of Malaria, World Health Organization, 2015.Additional file 2: Table S2. Phenotypic frequency distribution of Duffy blood group among uncomplicated and severe vivax malaria according to gender. No significant correlations were demonstrated in the frequency analysis of the Duffy blood group phenotypes between the gender and uncomplicated and severe P. vivax malaria patients.


Plasmodium vivax (molFOB) may provide a detailed assessment of malaria transmission. Understanding the epidemiology of malaria through the molecular force of the blood-stage infection of Plasmodium infection and clinical malaria episodes in three peri-urban communities of Manaus, Western Brazilian Amazon. In this study, we investigated risk factors and spatial-temporal patterns of incidence of Plasmodium species. molFOB was calculated by counting the number of genotypes observed on each visit, which had not been present in the preceding two visits and adjusting these counts by the respective times-at-risk. Monthly samples were collected in a cohort of 1,274 individuals between April 2013 and March 2014. DNA samples were subject to P. vivax and P. falciparum infection. Expected heterozygosity for P. vivax was 0.69 for MSP1_F3 and 0.86 for MS2. Multiplicity of infection in P. vivax was close to the value of 1. The season was associated with P. vivax positivity [adjusted hazard ratio (aHR) 2.6 (1.9-5.7)] and clinical disease [aHR 10.6 (2.4-47.2)]. P. falciparum infection was associated with previous malarial episodes [HR 9.7 (4.5-20.9)]. Subjects who reported possession of a bed net [incidence rate ratio (IRR) 1.6 (1.2-2.2)] or previous malaria episodes [IRR 3.0 (2.0-4.5)] were found to have significantly higher P. vivax molFOB. Respectively, 77.8% and 97.2% of the population remained free of P. vivax infection prevailed in the area and infections were mostly observed as monoclonal. Previous malaria episodes were associated with significantly higher P. vivax molFOB. Overall,  Plasmodium falciparum predominates.Plasmodium vivax is the predominant species and caused over two thirds (68%) of all cases in 2020. Despite most endemic countries in Latin America have achieved a reduction of incidence since 2010, there were still more than half a million  cases in 2020, with Venezuela and Brazil accounting for more than half of these cases (65%).,,,,,The global burden imposed by malaria remains high, with an estimated 241,000,000 cases and 627,000 related deaths in 2020.P. vivax, underscoring the challenges, namely relapses, yet to be addressed in control and management of this species.,,,,P. vivax gametocytes, along with asymptomatic cases, which enables the transmission of parasites even before the infected individual develops clinical symptoms. Thirdly, P. vivax parasite densities are generally lower than those seen in P. falciparum infections, especially in hypoendemic areas such as those in Brazil, and therefore require more sensitive diagnostic tools.P. vivax infections usually exist in such settings, and that these infections can easily be missed by routine active and passive case detection.,,,As in other regions, recent progress in malaria control in Brazil has been accompanied by an increasing proportion of cases caused by Plasmodium infections as well data on gametocyte carriage from low-transmission settings. Therefore, in this study, we investigated the risk factors and spatial-temporal patterns of the incidence of Plasmodium infection and clinical malaria episodes in a peri-urban area of Manaus, Western Brazilian Amazon. We measured the prevalence of blood-stage P. falciparum and P. vivax infections using polymerase chain reaction (PCR), the incidence of clinical cases, and estimated the molecular force of P. vivax blood-stage infection (molFOB) derived from molecular detection and genotyping of infections. molFOB is the observed number of new blood-stage infections, as identified by individual Plasmodium spp. genotypes, divided by the time-at-risk . molFOB is an important parameter for estimating exposure at the individual level and provides a detailed assessment of malaria transmission by measuring individual exposure and its burden.,In view of these challenges to the elimination of malaria, it is important to gather data regarding spatial and temporal patterns of asymptomatic Ethics statement - This study was approved by the Brazilian National Ethics Committee (CONEP) (349.211/2013) and the Ethics Committee for Clinical Investigation of the Barcelona Hospital Clinic (7306/2012). All participants were informed about the objectives of the study, as well as the potential risks of and benefits from their participation in the study. An informed consent form was signed by all study participants or by a parent or legal guardian in case of participants that were under 18 years of age. Children between 12 and 17 years of age signed an additional assent form. Patients were treated according to the protocols of the Brazilian Ministry of Health.Study design and subjects - This cohort study was conducted between April 2013 and March 2014 in the Brasileirinho, Ipiranga and Puraquequara communities, which are located in the peri-urban area of Manaus . A detailed description of the study area has been presented elsewhere.f Manaus A. The raAll of the houses in the communities received visits. In the case of participants present at the residence, these were invited to participate in the study and be part of our sample. A total of 1,274 participants of any age were enrolled into the study.Sample collection - For each study participant, a questionnaire was completed containing personal information such as age, gender, occupation, pregnancy, history of travel , as well as information on malaria preventive measures, previous malaria episodes and current health status. Upon enrolment and during monthly follow-ups, finger-prick blood samples (~300 μL) were collected using Microtainer® tubes containing EDTA and sodium fluoride . In infants, blood was obtained by either heel or toe puncture. Within one hour of collection, 50 μL of blood were transferred into a reaction tube containing 250 μL of RNAprotect  in order to preserve RNA for downstream analyses, and 200 μL of whole blood was transferred to another reaction tube and was separated into a red blood cell (RBC) pellet and plasma.Clinical symptoms - In the case of symptoms related to malaria, or in the case of increased body temperature (> 37.5ºC), a thick blood smear (TBS) was prepared according to World Health Organization’s guidelines.Plasmodium spp. infection, clones and gametocyte carriage - Pelleted RBCs, obtained from 200 μL of whole blood, were re-suspended in phosphate-buffered saline (PBS), and genomic DNA was extracted using a FavorPrep 96-well Genomic DNA kit , according to the manufacturer’s instructions. DNA was eluted with 2 x 100 μL of elution buffer and stored at -20ºC until assayed using PCR. RNA from 50 μL of whole blood, stored in RNAprotect, was extracted using an RNeasy Plus 96-well kit  and eluted in 50 μL of RNase-free dH2O, as described previously.Plasmodium species (QMAL) quantitative polymerase chain reaction (qPCR), which targeted a conserved region of the 18S rRNA gene.P. falciparum and P. vivax, as previously described.,P. falciparum, a modified reverse primer was used.2, 104 and 106 copies/μL). For the genotyping of individual P. vivax clones, the molecular markers MSP1F3 and MS2 were typed using capillary electrophoresis for highly precise fragment sizing, which allowed for longitudinal follow up of individual parasite clones. Details of the genotyping methods have been described previously.All DNA samples were subjected to a generic P. vivax and/or P. falciparum positive samples to detect gametocyte-specific transcripts of the pvs25 (P. vivax) and pfs25  genes. For quantification of pvs25 and pfs25 transcript numbers, control plasmids containing the amplified region were included as standards in each run. All qPCR and RT-qPCR assays were performed on a real-time PCR System .Reverse transcription real-time polymerase chain reaction (RT-qPCR) assays were performed on RNAs from all Statistical analysis - Data from questionnaires were imported into databases using the Cardiff TeleForm version 10.4.1 (Cardiff Software). Individual databases were combined in Microsoft Access 2010. For incidence calculations , data from the subjects were censored on the last visit before two consecutively missed scheduled follow-up visits in order to reduce bias.2 test with continuity correction. To achieve normal distribution, qPCR densities were expressed as log10-transformed 18S rRNA genomic copies/μL blood for asexual parasites, and log10-transformed pfs25 or pvs25 transcripts/μL blood for gametocytes. Geometric means of densities were calculated. Differences in densities of asexual or sexual-stage parasites were tested for statistical significance using Welch’s two-sample t-test.P. vivax was determined for both genetic markers combined. Negative binomial regression models were used to assess the influence of different risk-factors on the incidence of P. vivax and P. falciparum gametocyte positivity as previously described, using positivity counts and times-at-risk over the entire period of observation.The molFOB was calculated by counting the number of genotypes observed on each visit that had not been present in the preceding two visits (0-0-1 patterns) and then adjusting these counts by the respective times-at-risk. molFOB for Supplementary data. Versions of each model’s analysis were implemented with backwards-selection to eliminate non-significant covariates and resulted in the most parsimonious models. Statistical analyses were conducted using R v3.1.1 or STATA v14.Incidence rate ratios (IRR) and adjusted IRR (aIRR) were calculated with their respective 95% confidence intervals. Because using the collapsed data to model molFOB for everyone does not allow for the analysis of time-changing covariates, factors influencing frequency of parasite positivity and frequency of clinical episodes within the study period were explored using multiple failure time models that allowed for time-changing covariates.Study population - In all, 1,274 individuals were enrolled at the beginning of the study. A total of 51.1% (651) were males and approximately half were aged between 18 and 59 years old . Nearly 40% (504) of the subjects claimed to have had more than three malaria episodes during their lifetime. A total of 17 individuals (1.3%) had experienced a malaria infection in the preceding two weeks and 40 (3.2%) had taken antimalarial drugs in the past two months. A total of 1,201 subjects (94.9%) had resided in the study area for more than two months. The number of individuals enrolled was similar in the three communities, Brasileirinho (n = 430), Ipiranga (n = 416) and Puraquequara (n = 428). The characteristics of the study population are summarised in Prevalence by active case detection - Monthly P. vivax prevalence by qPCR ranged from 2.5% in June 2013 to 6.5% in November 2013  occurred in March 2014 ; of asymptomatic was 65.6% (42/64), and 54.5% (12/22) of symptomatic P. vivax infections were submicroscopic. A total of 75.0 % (12/16) of the P. falciparum infections were asymptomatic, and four (35.7%) asymptomatic P. falciparum infections were submicroscopic. The geographic means of P. vivax and P. falciparum parasitemia in symptomatic participants were 258.07 and 372.76 parasites per mm3, respectively. P. vivax asymptomatic infections predominated in the Puraquequara community, although clinical cases were mostly seen in Ipiranga. For P. falciparum, asymptomatic infections and clinical cases were both predominant in the Ipiranga community. In the rainy season, fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In the dry season, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive.ber 2013 A. P. falrch 2014 B. Both sy season C-D. The Risk factors associated with P. vivax positivity - In the multivariate analysis shown in P. vivax infection was associated with the rainy season (Dec-May) , and community of residence (p = 0.002), with individuals living in Puraquequara having an aHR = 2.4 (1.1-2.3), when compared to Ipiranga. Age was associated with P. vivax infection (p = 0.001), with individuals aged 20-60 years having an aHR = 2.2 (1.1-2.5), when compared to children under 10 years. Bed net usage was significantly associated with higher P. vivax positivity . Indoor residual spraying (IRS) in the house was associated with lower risk of P. vivax infection [Supplementary data (Table I)].Risk factors associated with P. vivax clinical disease - Plasmodium vivax clinical disease was associated with the rainy season  (Supplementary data (Table II)]. 0.002)] . Living Risk factors associated with P. falciparum positivity - Plasmodium falciparum infection was associated with living in a community (p < 0.001), with individuals living in Brasilerinho [aHR = 0.04 (0.01-0.26)] and Puraquequara [aHR = 0.23 (0.07-0.71)] having lower positivity than individuals in Ipiranga. Individuals with previous malarial episodes had significantly higher P. falciparum positivity  [Supplementary data (Table III)].Genetic diversity and multiplicity of infections - The heterogeneity in the incidence of malaria infections is shown in P. vivax and P. falciparum infections were restricted to a small proportion of the study population. Overall, 77.8% of the population remained free of P. vivax infection and 97.2% of the population remained free of P. falciparum infection over the course of the study period. Based on the two markers (MSP1 F3 and MS2), expected heterozygosity for P. vivax was 0.69 (MSP1_F3) and 0.86 (MS2). Overall, the multiplicity of infection was close to the value of 1 as determined by both markers (1.06 for MSP1_F3 and 1.04 for MS2), indicating that infections were mostly monoclonal. P. vivax molFOB during the entire year of follow-up. Similarly, new P1s. vivax infections were restricted to approximately 20% of the study population, whereas 80% did not contract new infections. Of the individuals who had any type of P. vivax infection, the majority had a molFOB = 1 to 2. The maximum number of genetically distinct infections/individual/year was molFOB = 5.Factors associated with P. vivax molFOB - P. vivax molFOB and it was significantly associated with living in a community (p < 0.001). P. vivax molFOB was lowest in the Brasileirinho community with an aIRR = 0.47 (0.31-0.71), when compared to Ipiranga. No significant difference in P. vivax molFOB was observed between the Ipiranga and Puraquequara communities. Increased P. vivax molFOB was significantly associated with reported bed net possession  and previous malaria episodes  [Supplementary data (Table IV)].P. vivax and P. falciparum and evaluated the incidence of Plasmodium spp. and clinical cases of malaria in peri-urban communities in the Brazilian Amazon. It also estimated the P. vivax molFOB in low transmission settings in Manaus. Understanding malaria epidemiology through P. vivax molFOB may provide a detailed assessment of malaria transmission by measuring individual exposure and its burden. Such information is of paramount importance when measuring intervention efficacy, host susceptibility and transmission patterns in low transmission and pre-elimination settings such as Brazil.The present study highlights well-known differences in the epidemiology of ,P. falciparum molFOB in these earlier studies were seasonality, village of residence and age.,,P. vivax, molFOB was strongly associated with incidence of clinical episodes and a high molFOB likely resulted in rapid acquisition of immunity against P. vivax in children.Previous studies have measured molFOB in Papua New Guinean children in observational and randomised clinical trial cohorts, and have related incidence of clinical infection and other factors for this measurement.P. vivax infections.,,P. vivax positivity were age (20-60 years), seasonality, use of mosquito nets and IRS. In the Brasileirinho, Ipiranga and Puraquequara communities, the distribution of mosquito nets and IRS is mainly focused on areas where there are many cases of malaria . However, this measure may not have been effective in preventing infection in these areas, or it may not be sufficient to decrease P. vivax positivity, since many infections may derive from hypnozoites rather than new infections, as suggested by a study in Papua New Guinea.Recent studies in the Amazon region and other malaria endemic areas in the world have shown a high proportion of submicroscopic ,P. vivax clinical disease found in our study were seasonality and, marginally, frequency of travel and use of mosquito nets. Koepfli et al.P. vivax. Protective factors against clinical disease, such as being over 60 years old and working in agriculture, may be associated with prolonged exposure to P. vivax infection during one’s lifetime. Due to age- and exposure-dependent acquired immunity, clinical presentation of malaria becomes rarer, thus increasing the number of asymptomatic carriers.,A higher prevalence of malaria infection in the rainy season was also shown in previous studies, thus corroborating what has already been described for other regions of the Amazon.P. vivax and the molFOB in the study area were higher and less affected by seasonality when compared to P. falciparum. Whereas prevalence of P. vivax and the molFOB peaked in November in the Ipiranga community, transmission indicators remained more stable throughout the entire observational period in the other two communities, with almost no seasonality observed in Brasileirinho.We also observed that prevalence of P. falciparum prevalence profile was characterised by a very sharp peak in January in the Ipiranga community during which the observed prevalence increased by almost 10-fold, alongside a sharp rise of clinical malaria cases. In fact, nearly all clinical cases observed in the present study occurred during this period and in the Ipiranga community. The outbreak caused by P. falciparum subsided by April, and the remainder of the observational period was characterised by very low P. falciparum prevalence in all three communities. These observed differences in the seasonality profiles of the two species indicate a more stable transmission of P. vivax in contrast to a more unstable transmission of P. falciparum.In contrast, the annual P. vivax parasite densities are close to the threshold of detection of even molecular diagnostic tests, thus hampering the characterisation of the true incidence of new infections detected by genotyping, as reported elsewhere.,This study has limitations, since the overall lower P. vivax infection prevailed in the area and infections were mostly observed as being monoclonal. High proportions of symptomatic and submicroscopic infections were also found. Previous malaria episodes were associated with significantly higher P. vivax molFOB, likely indicating that effective radical cure is an important strategy to be addressed in these endemic communities. Asymptomatic and submicroscopic infections pose substantial challenges for P. vivax malaria control and hamper accurate surveillance efforts that are needed to pursue elimination, especially in low transmission settings.In conclusion,


Controlled infection studies in malaria-naive adults suggest increased vaccine efficacy for fractional-dose versus full-dose regimens of RTS,S/AS01. We report first results of an ongoing trial assessing different fractional-dose regimens in children, in natural exposure settings.E at month 0 and month 1, followed by either full doses at months 2 and 20 (group R012-20 [standard regimen]), full doses at months 2, 14, 26, and 38 (R012-14), fractional doses at months 2, 14, 26, and 38 (Fx012-14), or fractional doses at months 7, 20, and 32 (Fx017-20). The fractional doses were administered as one fifth (0·1 mL) of the full RTS,S dose (0·5 mL) after reconstitution. All vaccines were administered by intramuscular injection in the left deltoid. The primary outcome was occurrence of clinical malaria cases from month 2·5 until month 14 for the Fx012-14 group versus the pooled R012-14 and R012-20 groups in the per-protocol set. We assessed incremental vaccine efficacy of the Fx012-14 group versus the pooled R012-14 and R012-20 group over 12 months after dose three. Safety was assessed in all children who received at least one vaccine dose. This trial is registered with ClinicalTrials.gov, NCT03276962.This open-label, phase 2b, randomised controlled trial is conducted at the Malaria Research Center, Agogo, Ashanti Region (Ghana), and the Kenya Medical Research Institute and the US Centers for Disease Control and Prevention site in Siaya County (Kenya). We enrolled children aged 5–17 months without serious acute or chronic illness who had previously received three doses of diphtheria, tetanus, pertussis, and hepatitis B vaccine and at least three doses of oral polio vaccine. Children were randomly assigned (1:1:1:1:1) using a web-based randomisation system with a minimisation procedure accounting for centre to receive rabies control vaccine (M012 schedule) or two full doses of RTS,S/AS01E group and 321 to the rabies vaccine control group). 1500 children received at least one study vaccine dose and the per-protocol set comprised 1332 children. Over 12 months after dose three, the incremental vaccine efficacy in the Fx012-14 group versus the pooled R012-14 and R12-20 groups was −21% . Up to month 21, serious adverse events occurred in 48 (16%) of 298 children in the R012-20 group, 45 (15%) of 294 in the R012-14 group, 47 (15%) of 304 in the Fx012-14 group, 62 (20%) of 311 in the Fx017-20 group, and 71 (24%) of 293 in the control group, with no safety signals observed.Between Sept 28, 2017, and Sept 25, 2018, 2157 children were enrolled, of whom 1609 were randomly assigned to a treatment group (322 to each RTS,S/AS01E regimens provided substantial, similar protection against clinical malaria, suggesting potential flexibility in the recommended dosing regimen and schedule. This, and the effect of annual boosters, will be further evaluated through 50 months of follow-up.The Fx012-14 regimen was not superior to the standard regimen over 12 months after dose three. All RTS,S/AS01GlaxoSmithKline Biologicals; PATH's Malaria Vaccine Initiative. The unprecedented deployment of malaria interventions since 2000 led to a considerable decrease in malaria morbidity and mortality. However, progress in malaria control has stagnated in recent years. WHO estimated that 241 million cases of malaria occurred in 2020, resulting in 627 000 deaths, of which 77% were in children younger than 5 years and approximately 95% occurred in the WHO African region.Evidence before this studyWe did a systematic literature search of PubMed on Nov 12, 2021, for any clinical trial of the RTS,S/AS01 malaria vaccine with a fractional dose, using the search string  AND ) and clinical trial (article type). We restricted the search to publications in English. Of the four manuscripts identified, three reported results of phase 2a controlled human malaria infection (CHMI) studies assessing vaccine efficacy in adults, and one evaluated the effect of a change in the vaccine regimen on the quality of antibody responses.Added value of this studyE in children aged 5–17 months, under conditions of natural exposure. We evaluated whether immunisation regimens with a delayed fractional third dose or an early fractional fourth dose are efficacious or increase the protective efficacy and effect of RTS,S/AS01E against malaria in countries with moderate-to-high malaria endemicity. We additionally evaluated whether timing of the dose administration affects vacine efficacy and provides new information on the immunogenicity and safety of two fractional-dose regimens of RTS,S/AS01E in children. Vaccine efficacy against clinical malaria of a fractional regimen (full doses at 0 and 1 month and one fifth of a full dose at month 2) was not superior to that of the full-dose schedule over 12 months after the first three doses. Improved vaccine efficacy was not observed for the delayed fractional-dose  regimen either. However, we observed a similar vaccine efficacy for all RTS,S/AS01E groups. All regimens were immunogenic and well tolerated.To our knowledge, this is the first study to evaluate proof-of-concept for a fractional-dose regimen of RTS,S/AS01Implications of all the available evidenceE confer high protection against clinical malaria. Although the primary endpoint or our study was not met, our results suggest that the use of a fractional RTS,S/AS01E regimen (with some flexibility around the time of administration of the third or fourth doses) provides protection against clinical malaria similar to the standard month 0, 1, and 2 full-dose regimen, over an initial 20-month period of follow-up from first vaccination. If these findings are confirmed up to 50 months of follow-up, they might confer large public health benefit, as a fractional dose regimen could potentially allow vaccination of more children with the same amount of vaccine as compared with a full dose.The phase 2a CHMI studies in malaria-naive adults showed that regimens containing a delayed third or early fourth fractional dose of RTS,S/AS01E  is the only vaccine currently recommended against malaria. A phase 3 clinical trial was conducted at 11 sites across seven sub-Saharan Africa countries between 2009 and 2014. Vaccine efficacy against all episodes of clinical malaria in children aged 5–17 months when administered according to a 0, 1, and 2 month primary schedule (M012) was 55·1% (95% CI 50·5 to 59·3) over 12 months of follow-up.Plasmodium falciparum transmission.RTS,S/AS01P falciparum infection.Concomitantly, efforts continue to improve vaccine efficacy, durability of protection, and availability of RTS,S. Several controlled human malaria infection (CHMI) studies in malaria-naive adults have suggested that a regimen containing a fractional dose of different RTS,S formulations confers high protection against We report results up to 20 months of follow-up of an ongoing phase 2b trial that aims to establish proof-of-concept for the use of fractional-dose regimens of RTS,S in children aged 5–17 months at first vaccination, under conditions of natural exposure. Therefore, we compared a fractional-dose regimen to the currently recommended standard full-dose schedule (M012). We also explored the effect of a delayed third dose in a fractional-dose regimen (M017), and the effect of an early full or fractional fourth dose (at month 14) on vaccine efficacy, over 1 year of follow-up after dose three and up to month 20. Immunogenicity and safety of all doses were also assessed up to month 21.P falciparum prevalence by microscopy was 17% in 2016 in the Ashanti Region and 39% in 2015 at the Kenya trial site; reported use of long-lasting insecticidal nets the night before the survey was 51% in the Ashanti Region and 91% at the Kenya site among children younger than 5 years.11This open-label, phase 2b, randomised controlled trial is conducted by the Malaria Research Center, Agogo, Ashanti Region (Ghana), and the Kenya Medical Research Institute and the US Centers for Disease Control and Prevention (KEMRI/CDC) site in Siaya County (Kenya). Both sites have perennial, moderate-to-high malaria transmission. We enrolled children without serious acute or chronic illness aged 5–17 months if they had previously received three doses of diphtheria, tetanus, pertussis, and hepatitis B vaccine and at least three doses of oral polio vaccine. Recruitment procedures and full inclusion and exclusion criteria are described in appendix pp 1–2. The families of all screened children were educated on malaria prevention and provided with a long-lasting insecticidal net. Parents or guardians provided written informed consent.The trial protocol was approved by local and national regulatory authorities, institutional review boards, and independent ethics committees . An indeChildren were randomly assigned (1:1:1:1:1) to receive one of four different RTS,S vaccination regimens or a rabies control vaccine (M012 schedule) with an adaptive procedure using a web-based randomisation system with a minimisation procedure accounting for centre. Those in the RTS,S groups received two full doses at month 0 and month 1 and either full doses at month 2 and month 20 (group R012-20), full doses at month 2, month 14, month 26, and month 38 (group R012-14), fractional doses at month 2, month 14, month 26, and month 38 , or fractional doses at month 7, month 20, and month 32 . Parents or guardians were encouraged to bring the child to the study-designated health-care facility in case of illness (passive case detection) at any point in time. Blood samples for efficacy analyses were taken to prepare a blood smear for microscopy in children with fever at the time of presentation or within the previous 24 h.We assessed P falciparum asexual parasitaemia more than 5000 parasites per μL and fever (axillary temperature ≥37·5°C)P falciparum asexual parasitaemia more than zero parasites per μL and fever or history of fever within 24 h of presentation . Prevalent infections were defined as all episodes of parasite density of more than zero parasites per μL during cross-sectional visits  were read at KEMRI/United States Army Medical Research Directorate-Africa, Malaria Diagnostics Center  according to the site's protocols and WHO standards were ensured. Blood films were read by two independent microscopists using a methodology previously described (counting against known blood volume), with discrepant readings resolved by a third reader.16For patient care, additional blood slides or rapid diagnostic tests were read at the respective sites in real time. We treated participants positive for malaria according to national guidelines in each country. Children were not presumptively treated for malaria at baseline.Blood samples for immunogenicity assessments were collected as shown in appendix p 4. Anticircumsporozoite protein and anti-HBs antibody geometric mean concentrations (GMCs) and seropositivity or seroprotection rates were assessed in a subset comprising 250 children . Assays are described in appendix p 9.We analysed solicited adverse events after doses three and four in children in the reactogenicity subset (the same as the immunogenicity subset). Trained personnel collected solicited local and general adverse events on diary cards for 4 days after vaccination. All adverse events in children not in the reactogenicity subset were reported as unsolicited adverse events. We collected unsolicited adverse events for 30 days after vaccination through passive surveillance at inpatient and outpatient facilities and during home visits, from all children in the exposed set, which comprised children who received at least one vaccine dose. All solicited adverse events were graded by intensity, with grade 3 indicating severe adverse events. All solicited local adverse events were considered causally related to vaccination, and the investigators assessed the causality of all other adverse events.Cases of severe malaria and cerebral malaria, serious adverse events, including adverse events of specific interest , and adverse events leading to withdrawal, were reported throughout the study. Additionally, we analysed seizures occurring within 30 days after vaccination. To support diagnosis of potential immune-mediated diseases, when indicated, samples were sent to Clinical Laboratory Sciences South Africa for further testing . For cliP falciparum infections from study start to month 20  compared with the M012 schedule with full RTS,S doses (pooled R012-14 and R012-20 groups) was demonstrated if the lower limit of the 95% CI for the incremental vaccine efficacy estimate was more than zero. The first secondary endpoint was occurrence of clinical malaria meeting primary and secondary case definitions in the Fx012-14 group from month 2·5 up to month 14. Other secondary outcomes related to vaccine efficacy assessment were occurrence of clinical malaria meeting the primary and secondary case definitions in RTS,S groups at different follow-up periods up to month 20 and the prevalence and incidence of month 20 . We alsoP falciparum infection was anticipated to be higher than that of clinical malaria, resulting in adequate power for endpoints evaluating vaccine efficacy against infections.The study had at least 90% power to detect significant incremental vaccine efficacy against the first or only episode of clinical malaria of the Fx012-14 regimen over the standard full-dose regimen (primary endpoint), assuming 250 evaluable children per group. Expected incidences were at least 0·5 episodes per person-year at risk in children in the control group, 0·29 episodes per person-year at risk in the pooled R012-14 plus R012-20 groups, and 0·16 episodes per person-year at risk in the Fx012-14 group . The incidence of The primary analysis of efficacy was done in the per-protocol set, including children who received all three first vaccinations as per protocol and who contributed to efficacy surveillance starting 14 days after dose three. In groups R012-14 and Fx012-14, children not receiving dose four per protocol were censored at the last documented visit if available or at 13 months after dose one if not; these children contributed to the per-protocol analysis up to censoring. Secondary analyses were carried out in the per-protocol set, unless otherwise specified.P falciparum infection were calculated and presented as 100 × (1 – hazard ratio) from the Cox proportional hazards model, stratified by country. Vaccine efficacy against all episodes of clinical malaria was calculated as 100 × (1 – incidence rate ratio), overall (adjusted for country as a fixed effect) and by country, and analysed by negative binomial regression allowing for interdependence between episodes within the same child.Vaccine efficacy estimates were calculated for an RTS,S regimen  compared with the control group, whereas incremental vaccine efficacy was estimated by comparing one RTS,S group with another. Vaccine efficacy and incremental vaccine efficacy estimates against first or only episode of incident clinical malaria or P falciparum infections at each calendar month was calculated as the proportion of participants reporting at least one infection from the total number of participants in each group for whom parasitaemia density was available for the considered month .Vaccine impact was defined as the estimated number of cases of clinical malaria averted over the relevant period per 1000 children vaccinated. The estimated number of cases was calculated as the area under the 3-month incidence curve of clinical malaria for each group . In post-hoc analyses, 95% CIs for the incidence of clinical malaria episodes by 3-month periods were computed using a generalised linear model with a Poisson distribution, the log as the link function, an offset (log time), and deviance as the scale, with the group variable as fixed covariable (factor with the four active groups and the control group being the reference group). In addition, the prevalence of All endpoints were analysed sequentially, and any conclusion on the first secondary endpoint was conditional to reaching the primary endpoint. All other secondary endpoints should be interpreted descriptively.ClinicalTrials.gov, NCT03276962.All analyses were done with SAS (version 9.4) This trial is registered with GlaxoSmithKline Biologicals was involved in study design and oversight, coordinated data collection, data analysis, data interpretation, and writing of the report. PATH's Malaria Vaccine Initiative contributed to study design and data interpretation but was not involved in data collection.Between Sept 28, 2017, and Sept 25, 2018, 2157 children were enrolled, of whom 1609 were randomly assigned to a treatment group . 1500 children received at least one study vaccine dose and the per-protocol set comprised 1332 children . BaselinDuring the 12 months after dose three, the incidence of the first or only episode of clinical malaria meeting the primary case definition was 0·36 per person-year in the pooled R012-14 plus R012-20 groups, 0·45 per person-year in the Fx012-14 group, and 0·61 per person-year in the control group. The incremental vaccine efficacy in the Fx012-14 group versus the pooled R012-14 plus R12-20 groups was −21%  in the pooled R012-14 plus R012-20 groups, 35% (13–51) in the Fx012-14 group, and 54% (38–66) in the Fx017-20 group. Vaccine efficacy estimates were similar for each group irrespective of whether they were assessed for first or only episodes or all episodes and meeting the primary or secondary case definitions .Plasmodium malarie (143 [5%]) or Plasmodium ovale (74 [3%]) by microscopy.Vaccine efficacy against all episodes of clinical malaria (secondary case definition) from day 0 to month 20 after four vaccinations in the R012-14 group and the Fx012-14 group, and after three vaccinations in the R012-20 group and the Fx017-20 group is shown in Over the 12 months after dose three, incremental vaccine efficacy of a third fractional dose using the secondary case definition  was −13% (95% CI −46 to 12) in the Fx012-14 group compared with the pooled R012-14 plus R012-20 groups . IncremeP falciparum infection up to month 20 in all RTS,S groups are shown in appendix p 16. Estimates of vaccine efficacy against all episodes of prevalent P falciparum infections are shown in appendix p 17.Parasite density was similar between study groups . EstimatUp to month 20, 1490 cases of clinical malaria (secondary case definition) per 1000 vaccinated children were averted in the Fx012-14 group and 1288 cases per 1000 vaccinated children were averted in the Fx017-20 group. The number of averted cases was different between the groups receiving the full-dose regimens . HoweverP falciparum infections tended to be higher in Ghana (where incidence was lower); the same was observed for vaccine efficacy against all episodes of prevalent infections  than for first or only episode (primary case definition) of clinical malaria. Vaccine efficacy and incremental vaccine efficacy estimates varied between Ghana and Kenya for all endpoints . Point efections .Figure 4Up to month 20, the number of clinical malaria cases averted  in RTS,SThe distribution of anti-circumsporozoite protein antibody concentrations after vaccination is shown in appendix p 5. 1 month after dose three, anti-circumsporozoite protein antibody GMCs tended to be higher in the Fx012-14 than in the Fx017-20 group but similar GMCs were observed between the groups 1 month after dose four. Overall, the fourth dose did not increase anti-circumsporozoite responses to levels observed after dose three . There wA summary of reported adverse events is shown in Unsolicited adverse events occurring within 30 days from any vaccination up to month 21 are shown in Serious adverse events up to month 21 were reported in 48 (16%) children in the R012-20 group, 45 (15%) in the R012-14 group, 47 (15%) in the Fx012-14 group, 62 (20%) in the Fx017-20 group, and 71 (24%) in the control group . Four deMalaria cases up to month 21 were reported in 89 (30%) children in the R012-20 group, 86 (29%) in the R012-14 group, 84 (28%) in the Fx012-14 group, 100 (32%) in the Fx017-20 group, and 101 (35%) in the control group . Severe Cause-confirmed viral meningitis was detected in six children: one (<1%) each in the R012-20 and Fx012-14 groups and two (1%) each in the control and Fx017-20 groups. At least one seizure episode within 30 days after any vaccination occurred in 12 children . Convulsive seizures levels 1–2 P falciparum infections varied greatly by calendar month from enrolment to study month 21, with no clear seasonal pattern observed . However, this was not the case over the initial 12 months of follow-up after dose three, when vaccine efficacy point estimates were very similar across groups. Interestingly, vaccine efficacy in the delayed fractional-dose group (Fx017-20) was similar to that in the other groups despite the 5-month delay in the administration of the third dose and the fact that the fourth dose (given at month 20) also did not contribute to the assessed efficacy. This might indicate either that the fractional RTS,S dose can be delayed by 5 months without losing protection against malaria, or that the initial lower protection in the Fx017-20 group after dose two is compensated by a higher efficacy of the delayed third fractional dose.We observed a difference in the background incidence of clinical malaria between Ghana and Kenya, similar to previous reports for malaria incidence in areas close to the study sites.Over the initial 12 months of follow-up after dose three, there was a discrepancy between groups receiving the same full-dose regimen (R012-20 and R012-14) when assessing vaccine efficacy against all episodes of clinical malaria. This discrepancy is driven by results from Kenya. However, a post-hoc analysis showed that, during this period, the incidence of clinical malaria episodes did not differ significantly between the R012-20 and R012-14 groups, although point estimates were lower in the R012-14 group than the R012-20 group. Therefore, we concluded that this was most probably a chance finding, which could nevertheless have contributed to the overall efficacy over 20 months of follow-up. Therefore, any conclusion on the potential benefit of a fourth dose given 12 months after dose three compared with 18 months after dose three should be drawn with caution this early in the study.Anti-circumsporozoite protein antibody responses to RTS,S were similar between groups. Consistent with previous observations, the fourth dose did not boost anti-circumsporozoite protein antibodies to higher concentrations than those achieved after dose three.In this study, we did not observe a difference in antibody avidity between the fractional-dose and full-dose regimens. In a previous CHMI study, the delayed fractional dose was shown to increase circumsporozoite protein-specific antibody avidity, which was hypothesised to be a potential contributor to the improved vaccine efficacy of an Fx017 regimen over an R012 regimen.All regimens were well tolerated, supporting the acceptable safety profile of RTS,S. The incidence of solicited adverse events was similar to that observed in the phase 3 trial assessing the full-dose M012 regimen in African children aged 5–17 months.Our study has several limitations. Because the primary objective was not demonstrated, any further group comparisons should be interpreted with caution. Comparison between the delayed fractional regimen and groups receiving a M012 schedule might be hindered by seasonal variations in malaria incidence and transmission; in addition, the study did not include a R017-20 full-dose group to allow a non-biased comparison with the fractional Fx017-20 regimen. The study was only powered for overall results and not by site; this, together with the heterogeneity observed for vaccine efficacy estimates, known difference in malaria transmission even within the sites, and the multiplicity of objectives, limit the interpretation of results by country. Due to the open-label design, some bias in safety assessments is possible, although the study was designed to minimise it .In conclusion, the Fx012-14 regimen did not show superior vaccine efficacy against clinical malaria after the three first doses when compared with a standard full-dose M012 schedule, and improved vaccine efficacy was also not observed for the delayed Fx017-20 regimen. However, all fractional-dose and full-dose RTS,S regimens provided substantial and similar protection against malaria.Our results suggest that the use of a delayed fractional-dose regimen does not affect protective efficacy over 20 months of follow-up compared with a standard regimen, and that the timing of the third and fourth RTS,S vaccinations might be flexible. Although not confirmatory, if borne out over the 50-month follow-up, these findings might portend substantial public health benefit, as reduced vaccine volume requirements and flexibility in dose timing would allow increased access to the vaccine and decreased malaria-specific morbidity and mortality. Continued follow-up to month 50 will provide further insight into the efficacy of different RTS,S regimens, including the effect of multiple annual booster doses.www.clinicalstudydatarequest.com (study ID 204889).Anonymised individual participant data and study documents will be available at study end, when they can be requested for further research from The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention. OO-A, LS, ML, DM, ABo, FR, and EJ are employees of the GSK group of companies. OO-A, LS, DM, FR, and EJ have restricted shares in the GSK group of companies. All other authors declare no competing interests.