In low malaria transmission areas, many people acquire multiple malaria infections within a single season. This study aimed to describe the pattern and epidemiological profile of malaria recurrence in a hypoendemic area of western Thailand and identify factors associated with having multiple malaria episodes.Plasmodium species and age groups. The ordinal logistic model was used to determine factors associated with multiple malaria episodes, and to compare with single episodes, and those with no malaria infection.An open cohort of 7000 residents in seven clusters along the Thai-Myanmar border was followed during a 6.5-year period (2011–mid 2017). Symptomatic and asymptomatic malaria infections were detected by passive case detection (PCD), weekly household visit, and mass blood surveys every 4–6 months. Malaria recurrence was defined as subsequent parasitaemic episodes occurred later than 7 days after receiving anti-malarial treatment. This study focused on analysis of recurrent episodes that occurred within 1 year after treatment. Numbers of malaria cases with single and multiple episodes were compared between clusters. Kaplan–Meier curve was performed to determine the intervals of recurrent episodes by Plasmodium species as the primary infections. The median interval and interquartile range (IQR) between the first and second episode was 88 (43–175) days for all parasites, 56 (35–133) days for two Plasmodium falciparum episodes, and 90 (59–204) days for two Plasmodium vivax episodes. The interval between the episodes was increased with age. Factors significantly associated with multiple episodes of malaria infection included male sex, young age, Karen ethnicity, forest-related occupation, and having other malaria infected persons in the same house in the same period.The cumulative incidence of malaria in the study area was 5.2% over the 6.5 years. Overall, 410 malaria patients were detected. Of these patients, 20% and 16% had multiple malaria episodes during the entire period and within 1 year after initial treatment, respectively. About 80% of repeated malaria episodes were caused by the same People who have multiple malaria episodes may play an important role in maintaining malaria transmission in the area. Understanding epidemiological profiles of this group is important for planning strategies to achieve the elimination goal. Plasmodium falciparum attacks every 4 to 6 weeks over many years [P. falciparum and Plasmodium vivax are symptomatic, found that the cumulative proportions of patients having recurrent P. falciparum and P. vivax infections within 63 days after treatment of acute P. falciparum malaria were 21.5% and 31.5%, respectively [People living in malaria endemic areas may experience more than one malaria attack even in a single season. The distribution and determinants of the multiple malaria attacks depend on the local epidemiological settings. In the hyperendemic areas of Africa, children may suffer repeated ny years –3. Even Plasmodium species when the blood-stage parasites are not completely eradicated and subsequently re-expanded in number after drug concentrations in the blood decline. Relapse occurs mostly in P. vivax and Plasmodium ovale when parasitaemia and clinical manifestations reappear due to re-activation of dormant hypnozoites in the liver. Reinfection is an infection acquired from a new mosquito bite. Although secondary infection is usually genetically different from that of the primary infection, it is difficult to distinguish precisely between relapse, recrudescence and reinfection in the case of P. vivax infection [Several terms are used to define the repeated episodes of malaria in a patient. Recurrence of parasitaemia after treatment can result from: (a) recrudescence from asexual parasitaemia, (b) relapse from hypnozoites, and (c) reinfection by a new mosquito inoculation –8. Recrunfection –9.P. falciparum, P. vivax is seemingly much less virulent, thus the term benign tertian, but there are studies showing that recurrent patients with this parasite could become difficult to treat and sometimes fatally ill [Plasmodium vivax infection may result in a decrease in peripheral blood lymphocyte subsets, and the number of recurrent episodes was found to be correlated with an unbalanced and exacerbated immune response and unbalanced ratio of the CD4+/CD8+ T-cells [Recurrence after treatment leads to a new clinical episode with a risk of complications for the patient . Compareally ill . Plasmod T-cells . Malaria T-cells , 14. Thu T-cells . The str T-cells , 17.The determinants of malaria infection can be observed at different levels: (a) individual level including biological and disease-related factors; (b) household and community levels covering social and economic factors, and (c) environmental and institutional factors –20. At tCountries in the Greater Mekong Subregion (GMS) are moving toward regional malaria elimination. In the GMS, malaria distribution is highly heterogeneous on both large and small geographical scales. On a regional scale, an upsurge in malaria incidence has been observed in Cambodia in 2017 , despitePlasmodium species, mosquito vector species, and human populations. One goal of the ICEMR programmes is to generate basic epidemiological information across a range of epidemiological settings [This study was part of The International Center of Excellence for Malaria Research (ICEMR) project that was established to evaluate the impact of malaria control interventions across endemic regions that differ in the dominant settings . This stThe study was conducted in seven clusters (subsets of 7 villages) of an open cohort for 6.5 years (2011-mid 2017) following approximately 7000 people in Tha Song Yang District, Tak Province where malaria incidence has been persistent for decades. The seven clusters varied in population sizes representing semi-rural, rural, and remote areas along the Thai-Myanmar border. The study employed both passive case detection (PCD) and active case detection (ACD). For PCD, symptomatic malaria cases were detected at hospitals, clinics and malaria posts, while ACD was performed during weekly home visits to collect data on symptomatic and asymptomatic cases as well as population movement within the cohort. In addition, MBS in the cohort also was performed three times a year during 2011–2015 with the aim to detect both hidden symptomatic and asymptomatic cases.At the beginning of the project, household visits by field workers and/or trained community health volunteers were performed. Informed consent was obtained from all study participants in each cluster of the cohort. A unique study identification number was assigned to an individual in each cluster who agreed to participate. Demographic variables included gender, age, occupation, education level, ethnicity and nationality. Households were also given house codes along with their spatial coordinates. As an open cohort, besides the fixed numbers of member in the initial cohort, there might be residents who moved out or new comers who moved in the cluster. New members at each household visit were registered into the cohort upon their consent. If the persons moved out from the clusters, they were censored from the study.All malaria cases in this study were diagnosed by microscopy following the standard malaria diagnosis procedure. During the ACD and MBS, axillary temperature and fever history were collected. Those with fever or reported a history of fever received an on-site malaria testing using a rapid diagnostic test kit (RDT). Malaria cases detected by PCD were diagnosed by microscopy, the routine malaria diagnostic method at the local malaria clinics and hospitals. Microscopic confirmation by experienced microscopists was also performed for all blood samples collected via ACD, MBS, and PCD activities. All confirmed malaria cases were treated according to the national standard guidelines, and were followed in the next weekly home visits.P. falciparum parasites are normally cleared within 5 days of artemisinin-based combination therapy (ACT) [P. vivax cases treated with the chloroquine-primaquine combination is < 150 h [Data on malaria episodes of individuals in the cohort were combined from ACD, MBS, and PCD activities. People in the cohort with no infection detected during ACD, PCD or MBS were considered “not infected”, while those with malaria infections detected only once in the study period were “single episode” cases. For people with multiple malaria episodes, subsequent episodes were defined as those that occurred after 7 days to 1 year from the first episode. This is based on the clinical efficacy of the anti-malarial drugs: py (ACT) , and the < 150 h . The recMajor risk factors examined here included gender, age, ethnic groups, education, occupational risk, and having other infected person(s) in the same household at the same time. These variables represent potential malaria risk factors among individuals in the area. Age was classified into five different age groups: young child (< 5 years), child (6–10 years), young adult (11–20 years), working age adult (21–60 years), and older person (> 60 years). There were three types of ethnic groups in the study areas: Thai, Karen and others (mostly Myanmar). Occupational risk was classified by characteristics of work environment in the study area and chance of having an infection either as indigenous or imported cases including: low-risk work, child/student, factory labourer, plantation worker, high-risk work and unspecified. Low-risk work included housekeeper/housewife, shopkeeper, monk, government officer, retired and unemployed. Plantation work included those who worked in different types of plantations including cassava, corn, rice and sugar cane in the study areas. High-risk work included hunter, lumberjack/forest worker, orchardist, rubber tree plantation worker, and soldier/police. Having other infected person in the same household at the same time was enumerated among cases residing in the same residential address and the dates of malaria positive test results of the cases were within ± 7 days. Furthermore, risk factors at the household level also included positive cases residing in the same cluster within the same month.Pattern of malaria infections may be due to variation at individual and household or cluster level . This stP. vivax infection, while 181 (34.3%) and 6 (1.1%) episodes were caused by P. falciparum and mixed infections, respectively. Of the 410 malaria cases, 83 patients (20%) had multiple malaria episodes over the study period, ranging from 2 to 5 malaria episodes. About 80% (N = 67) of patients with multiple malaria episodes had the recurrent episodes within 1 year after the primary infection.The study cohort comprised of 7812 people in 1196 houses residing in different sizes of the selected seven clusters ranging from 3315 to 64 people  had multiple malaria episodes within 1 year. The proportion of multiple episode cases varied across clusters and did not correlate with cluster’s baseline malaria incidence. In Cluster 2 with the lowest malaria incidence level (1.7%), about 10% of malaria cases had multiple malaria episodes, whereas in Cluster 4 with the baseline malaria incidence of 8.5%, the proportion of cases with multiple episodes was observed only in 8%. In Cluster 6 and 7, more than 30% of malaria cases had multiple episodes.When considering malaria occurrence at the household level, malaria cases were observed in 21.9% of the houses (N = 262) over the 6.5 years  of the subsequent infections were also due to P. falciparum. Similarly, 31/38 (81.6%) patients with P. vivax at the first episode also had P. vivax at the recurrent episodes. Mixed infection  was observed in five cases, two of which had repeated episodes with P. vivax.For the 67 individuals with multiple episodes of malaria, 73% (N = 49) had two malaria episodes within 1 year. It is noteworthy that one individual had five malaria episodes within 1 year. The majority of the repeated malaria episodes were due to infections by the same P. falciparum infections showed the shortest interval with a median of 56 (IQR: 35–133) days. For two repeated P. vivax episodes, the median time was 90 (IQR: 59–204) days. A longer interval was observed among those who were infected with different parasite species—the median time was 109 (IQR: 45–189) days. Trend in time from the first to the second episode was shown when exploring different age groups. For patients aged < 5 years, the median time was 59 (IQR: 35–182) days, whereas for those aged 6–10 years the median time was 70 (IQR: 36–183) days. For patients aged 11–20 and 21–60 years, the median time was 89 (IQR: 62–109) and 112 (IQR: 51–189) days, respectively.As shown in Fig. Different statistically significant risk factors for repeated malaria attacks were identified Table . Males hPlasmodium spp, vectors, and environment, vary considerably.Recurrent malaria infection could have an impact on both individual’s health and malaria transmission in the community. Even in malaria low-transmission areas, recurrent parasitaemia after initial treatment is still observed. In this area along the Thai-Myanmar border, the incidence of malaria occurred in 5.2% of the studied cohort over 6.5-year period. Yet, ~ 20% of malaria cases still had recurrent episodes; most of recurrent episodes occurred within 1 year after the initial infection. The maximum number of malaria episodes an individual had within 1 year was 5 times. Previous studies conducted in high malaria transmission areas in Africa reported that malaria episodes among children ranged from 0 to 40 per child over a 5-year period . Even inRecurrent malaria episodes may be due to recrudescence, relapses, or new infections. This study, however, did not differentiate the causes of these recurrent episodes. Yet, in low-malaria transmission areas, the annual incidence of new infections is typically low and estimated to be less than one per person . This suP. falciparum parasites. Per the NMCP anti-malarial drug policy, falciparum cases were treated by ACT (in this case artesunate-mefloquine: AS-MQ). However, due to the emergence and spread of artemisinin-resistant P. falciparum parasites in the GMS [P. falciparum followed by another P. falciparum episode occurred within 35 days, highlighting the possibility of recrudescence of falciparum malaria due to drug resistance. For vivax malaria, the standard treatment chloroquine-primaquine (CQ-PQ) for uncomplicated P. vivax infection remained generally effective in the GMS, although sporadic CQ-resistant P. vivax parasites have been reported in this region [P. vivax occurred within 35 days, which also suggests deteriorating CQ efficacy in this region. This demands close monitoring of anti-malarial drug resistance in this area.Inadequate anti-malarial treatment and drug resistance may result in recrudescence and relapse. Recrudescence is caused by the remaining blood-stage parasites, which failed to be completely eliminated by anti-malarial treatment. The GMS is known as an epicenter of multidrug-resistant  the GMS , treatme the GMS . In thiss region . In thisP. vivax and P. ovale infections. Duration of relapses varies by the parasite strains; in the tropical region of Southeast Asia, P. vivax strains, such as the Chesson strain, relapse within 1–5 months [P. vivax recurrent events occurred between day 51 and 110, and 65.5% of these subsequent episodes were genetically classified as relapses [P. vivax infection was 90 days, which are likely due to relapse. In addition, this study also identified that 3.7% of P. falciparum primary infection had subsequent episode of P. vivax infections. Plasmodium vivax recurrence after treatment of primary P. falciparum infection has been reported in previous studies conducted along the Thai-Myanmar border [P. vivax infections, the subsequent episodes of vivax infection after P. falciparum treatment are also likely due to relapses. Adequate PQ treatment can substantially reduce the risk of relapses [P. vivax recurrence [Relapse is a result of hypnozoite reactivation during 5 months . A study5 months . Similarrelapses . Identifrelapses , 36. In r border . Since Arelapses . In the currence . TherefoMany factors have been associated with malaria infections –44. ThisLike what has been found in earlier studies , 8, chilThe majority of patients in this study belongs to the Karen ethnic group, one of the largest ethnic minority groups along the Thai-Myanmar border. Karen people mainly work as workers in local factories or in the plantations/orchards near the border areas. Among those who work in the agricultural settings, some maintain farming plots on the Myanmar side of the border, whereas some might reside in agricultural huts only during peak harvesting times of the year . Such prAnopheles vectors are abundant and effective in malaria transmission [Anopheles minimus and Anopheles maculatus are the main malaria vectors. Previous study has shown that monthly Anopheles capture rate was associated with P. vivax monthly incidence, suggesting a potential role of these vectors in local malaria transmission [Having other infected person in the same household at the same period was a significant risk factor for having multiple malaria episodes in this cohort study. In areas where smission , 50, humsmission . In thissmission . A sociosmission . HoweverIn this study, there was no information available to differentiate between recrudescence, relapse, and re-infection. Molecular studies may allow us to identify the types of recurrence. In addition, this study did not cover the issue of drug resistance in the study area that might be related to transmission intensity, multiplicity of infections, and long-term persistence of resistant parasites . MoreoveIn a low-transmission area where the cumulative malaria incidence was only 5.2% over the 6.5-year period, 16% of all infected patients were still found to have multiple malaria episodes within 1 year. Children, male, Karen ethnicity, working in forest-related occupation, and living currently in the same house with malaria case were significant risk factors associated with multiple malaria episodes. These people with multiple malaria episodes could serve as potential human reservoirs that require special attention, particularly during the malaria pre-elimination phase. Although the causes of the recurrent episodes were not genetically identified, inadequate malaria treatment either due to anti-malarial drug resistance or poor drug compliance should be closely monitored to prevent recrudescence and relapses. Understanding of the local epidemiology in terms of distinguishing locally acquired infection versus cross-border infection is important to guide successful interventions in moving toward malaria elimination. Further intensive studies are needed on adaptive strategies for different areas to achieve the ultimate elimination goal.


The relative contribution of bacterial infections to febrile disease is poorly understood in many African countries due to diagnostic limitations. This study screened pediatric and adult patients attending 4 healthcare facilities in Ibadan, Nigeria, for bacteremia and malaria parasitemia.Salmonella subtyping was performed by polymerase chain reaction. Antimicrobial susceptibility was tested by disk diffusion.Febrile patients underwent clinical diagnosis, malaria parasite testing, and blood culture. Bacteria from positive blood cultures were isolated and speciated using biochemical and serological methods, and Staphylococcus aureus (69 [59.0%]) and Salmonella enterica (34 [29.1%]) being the most common species recovered. Twenty-seven (79.4%) of the Salmonella isolates were serovar Typhi and the other 7 belonged to nontyphoidal Salmonella serovarieties. Thirty-four individuals were found to be coinfected with Plasmodium falciparum and bacteria. Five (14.7%) of these coinfections were with Salmonella, all in children aged <5 years. Antimicrobial susceptibility testing revealed that most of the Salmonella and Staphylococcus isolates were multidrug resistant.A total of 682 patients were recruited between 16 June and 16 October 2017; 467 (68.5%) were <18 years of age. Bacterial pathogens were cultured from the blood of 117 (17.2%) patients, with The study demonstrates that bacteria were commonly recovered from febrile patients with or without malaria in this location. Focused and extended epidemiological studies are needed for the introduction of typhoid conjugate vaccines that have the potential to prevent a major cause of severe community-acquired febrile diseases in our locality. Streptococcus pneumoniae, Staphylococcus aureus, typhoidal and nontyphoidal Salmonella enterica serovars, Haemophilus influenzae, Neisseria meningitidis, Klebsiella pneumoniae, and Escherichia coli ) and S. enterica (34 [29.1%]) being the most common species identified  were enrolled in the study . Seven oentified . Twenty-Plasmodium parasite infections. Salmonella Typhi was detected more than twice as frequently in children aged 6–12 years than in other age groups  patients, which included malaria coinfection with 6 S. Typhi infections and 1 NTS infection  patients coinfected with bacteria and malaria parasites were <18 years of age. All but 1 of the 7 patients with Salmonella and Plasmodium coinfection were children.All 682 samples were blood cultured, and malaria testing was performed on all but for 64 of the 682 patients. Of the 618 patients tested for malaria, 171 (27.7%) tested positive, and nfection . MalariaSalmonella was cultured at higher frequencies from specific LGAs: S. Typhi recovery of from patients recruited from Akinyele, Ibadan northeast, and Egbeda LGAs was between 9.6% and 10.3%, whereas invasive Salmonella were detected at a prevalence of 0–1.6% in residents recruited from Ido, Ibadan north, Ibadan southwest, and Ibadan northwest  isolates were resistant to 6 additional different agents. The median number of antimicrobials to which the Salmonella, both Typhi and iNTS, were resistant was 5 . Resistance was similarly common among S. aureus isolates, with 64 (92.8%) being β-lactam resistant and 26 (37.7%) demonstrating resistance to cefoxitin . In addition to our inability to perform malaria testing for 64 patients, particularly those recruited out of hours, there were other limitations to the study. Malaria parasitemia was detected by thick- and thin-film microscopy. More sensitive methods that measure parasite density are better suited to identifying clinical malaria in hyperendemic locations like ours but were not used in this study, nor did we collect data on malaria parasitemia in healthy individuals for comparison. Blood culture volumes were recorded for most, but not all, of the enrollees and a contamination rate of 7.8%, which could have obscured some infections, was recorded.Salmonella [Febrile patients in Nigeria and other malaria-endemic countries are often presumed to have malaria and are managed accordingly. Limitations notwithstanding, this study confirms findings from other parts of Nigeria and the African continent, pointing to bacteremia as a principal cause of febrile illness, and the need for blood culture facilities and/or alternate diagnostics , 29–31. lmonella , 33. Addlmonella , 34.Salmonella Typhi, an organism for which blood culture is only 40%–60% sensitive [E. coli, S. pneumoniae, K. pneumoniae, Enterobacter aerogenes, and H. influenzae—were less commonly recovered, and Candida albicans was detected in 1 positive culture. A similar study from more than a decade ago [E. coli, S. pneumoniae, and H. influenzae to be the most predominant species recovered from children aged <5 years. In Lagos, Nigeria, a 2005 study of 74 patients with pyrexia of unknown origin identified S. Typhi (16.2%), Proteus species (14.9%), E. coli (12.2%), and Klebsiella species (9.5%), and also detected Plasmodium species in 44.6% of specimens [S. pneumoniae to be the leading cause of death in children [S. Typhi [ensitive , 33, wascade ago , which specimens . A more S. Typhi . SimilarS. Typhi .S. Typhi detection rates ranging between 0.5% and 5% in Abuja (2009–2017) and between 4% and 10% in Kano (2013–2017) by blood culture [Salmonella was lower (29.1%) in this study compared with the aforementioned Abuja study (60%), but comparable to the 24% recorded in Kano. Notably, S. Typhi predominated in all studies, but like previous investigations we also recovered a smaller number of NTS isolates, half (n = 3) of which were Salmonella Enteritidis. The approximately 4:1 ratio of S. Typhi to iNTS compared with a the ratio contained in recent report from northern Nigeria [Salmonella in this study are worrisome, they are at the lower end of recent reports from Nigeria, and differences in our data from a western Nigerian city point to possible within-country geographical differences in the etiology of community-acquired bacteremia.Few studies in Nigeria have reported automated blood culture isolates from bacteremia patients, and those that have focused on children aged <5 years. In this important age group, the bacteremia recovery rates and range of pathogens in this study were remarkably similar to those recorded in Ibadan almost a decade previously . A more  culture . These d culture , 38. The Nigeria . AltogetSalmonella Typhi was generally more commonly isolated from the blood of residents from the northern and eastern parts of Ibadan city. As shown in S. Typhi recovery showed intermediate recovery rates. Altogether, the data point to clustering of typhoidal infections, and as has been shown at other locations [Within our Ibadan study area, we recorded the residential LGA of the study participants. ocations , spatialSalmonella disease, whereas 94 (47%) had malaria and there were no coinfections [Salmonella from 6.3% of the blood culture isolates and detected Plasmodium species in 44.6% [Malaria was diagnosed in 171 (27.7%) of the tested febrile patients in this study, including 6 of the patients with enteric fever. The 2003 report from Nsutebu et al from neighboring Cameroon [in 44.6% . Altogetin 44.6% , co-deteS. Typhi, and other bacteria are common in patients with community-acquired febrile disease in Ibadan, with both pathogens most commonly recovered from young children. The comparable clinical presentation of malaria and bacterial infections and limited patient overlap point to the need for laboratory verification of fever etiology to appropriately manage such infections. For this study, routine blood culture for febrile conditions, including those typically treated empirically, was implemented in a Nigerian hospital. While the time to diagnosis from blood culture is slow, the resulting etiologic information was extremely valuable. Rapid and cheaper tests for bacterial infections that can be implemented at nontertiary points of care and used parallel with malaria rapid diagnostic tests are urgently needed.In conclusion, we find that plasmodia, S. Typhi infection detected in this study suggests that suspected typhoid needs to be rapidly and effectively treated and that even patients with laboratory-verified malaria infection should be screened for bacteremia. Interventions targeting these febrile diseases, such as vector control, water, sanitation, and hygiene, along with diagnostic test development and vaccine deployment need to be priorities in Nigeria. This study also creates a range of research questions, in particular source attribution and spatial risk for typhoidal Salmonella and the longitudinal trajectories for malaria and typhoid. These research areas can expand on this short study to measure the true burden of these diseases and the value of any future interventions.The high prevalence of


Plasmodium lactate dehydrogenase (pLDH) have been less widely used but have higher specificity, mostly due to a much shorter time to become negative.Rapid diagnostic tests (RDT) for malaria are common, but their performance varies. Tests using histidine-rich protein 2 (HRP2) antigen are most common, and many have high sensitivity. HRP2 tests can remain positive for weeks after treatment, limiting their specificity and usefulness in high-transmission settings. Tests using A prospective, health centre-based, diagnostic evaluation of two malaria RDTs was performed in rural Niger during the high malaria transmission season  and during the low transmission season . All children under 5 years of age presenting with fever (axillary temperature > 37.5 °C) or history of fever in the previous 24 h were eligible. Capillary blood was collected by finger prick. The SD Bioline HRP2  and the CareStart pLDH(pan)  were performed in parallel, and thick and thin smears were prepared. Microscopy was performed at Epicentre, Maradi, Niger, with external quality control. The target sample size was 279 children with microscopy-confirmed malaria during each transmission season.In the high season, the sensitivity of both tests was estimated at > 99%, but the specificity of both tests was lower: 58.0% (95% CI 52.1–63.8) for the pLDH test and 57.4% (95% CI 51.5–63.1) for the HRP2 test. The positive predictive value was 66.3% (95% CI 61.1–71.2) for both tests. In the low season, the sensitivity of both tests dropped: 91.0% (95% CI 85.3–95.0) for the pLDH test and 85.8% (95% CI 79.3–90.9) for the HRP2 test. The positive predictive value remained low for both tests in the low season: 60.5% (95% CI 53.9–66.8) for the pLDH test and 61.9% (55.0–68.4) for the HRP2 test. Performance was similar across different production lots, gender, age of the children, and, during the high season, time since the most recent distribution of seasonal malaria chemoprevention.The low specificity of the pLDH RDT in this setting was unexpected and is not easily explained. As the pLDH test continues to be introduced into new settings, the questions raised by this study will need to be addressed. Plasmodium falciparum, and Plasmodium lactate dehydrogenase (pLDH), which is present in all Plasmodium species; currently available tests detect one or both antigens. HRP2-based tests have been preferred in areas where P. falciparum is predominant, due to a higher reported sensitivity [Rapid diagnostic tests (RDT) for malaria have become the in-the-field diagnostic standard in most countries in Africa. Current malaria RDTs are immunochromatographic tests that detect the presence of circulating parasite antigens. The two most commonly targeted antigens are histidine-rich protein 2 (HRP2), which is specific to sitivity . There isitivity . This lisitivity . These fThe CareStart™ Malaria PAN (pLDH) Ag RDT  is among the highest-scoring RDTs in the most recent WHO-sponsored evaluation of RDTs . In the A formal clinical diagnostic evaluation of the newly introduced pLDH-based RDT, in parallel with the previously used HRP2-based RDT, was performed in field conditions in rural Niger. Off-site microscopy by at least two blinded microscopists was the gold standard comparator.Malaria transmission in Niger has a marked seasonality, with a large peak between July and October, that follows the rainy season (June–September). As of 23 December, a total of 3,484,069 cases of malaria were reported across the country in 2018, including 106,004 cases in Magaria District .A prospective, health centre-based, clinical diagnostic evaluation was performed in two phases: first during the peak malaria transmission season  and then during the low malaria transmission season . In October 2017, the timing ensured that inclusions happened over the course of an entire monthly cycle of SMC, which has been offered in the study area since 2013. In the high season, the study took place in the Magaria Integrated Health Centre (Centre de Santé Intégré (CSI)), an outpatient facility in Magaria, Niger  presenting to study sites with fever (37.5 °C axillary temperature) or a history of fever in the preceding 24 h were enrolled. Children with any sign of severity (including but not limited to decreased consciousness and seizure) were excluded and immediately referred to the District Hospital for treatment. During the high transmission season, in order to spread inclusions throughout the period between SMC distributions, only the first 20 febrile children presenting each day were enrolled. During the low transmission season, all febrile children presenting at the study CSI were offered enrolment.After obtaining written informed consent from a participant’s caregiver, a study nurse collected demographic and clinical information about the participant, including age, gender, village of origin, bed-net use, receipt of most recent SMC (high season only), and information about the RDT used . The study nurse collected capillary blood by a fingerstick, preparing thick and thin smears in duplicate. The study nurse wrote the patient ID number and time of testing on the RDT cassettes. The two tests (SD Bioline Ag P.f. (HRP2), Standard Diagnostics, Giheung-ku, Republic of Korea, catalog number 05FK50; and CareStart pLDH(pan), AccessBio, Somerset, NJ, USA, catalog number RMNM-02571) were performed following manufacturers’ instructions. A photograph of the RDT was taken at the time of reading (15 min for the HRP2 test and 20 min for the pLDH test), and the results were recorded in the study register. Invalid RDTs were repeated for clinical purposes but only the first result was considered for the diagnostic evaluation.Any child with a positive result on either test (pLDH or HRP2) was treated, free of charge, following national protocols, with antipyretics, artemether–lumefantrine, and any other treatment deemed necessary by the treating clinician. Febrile children with a negative RDT were treated according to the clinician’s best judgment, and all treatment was also free of charge.At the end of each day, the slide of each participant that appeared to be of the best quality was stained (10% Giemsa solution for 15 min) and the lesser-quality slide was discarded. Slides were stored and regularly transported to the Epicentre Laboratory in Maradi, where microscopy was carried out according to WHO standards by a team of expert microscopists with over 20 years combined experience in malaria microscopy . In brieFollowing manufacturer’s instructions, the RDTs were stored at temperatures < 30 °C between shipment and their arrival to the study site. At the study sites, RDTs were stored at ambient temperature in a ventilated, locked room for the duration of the data collection period . At least two different production lots were used during each transmission season.The target sample size was calculated based on the number of true-positive cases needed to ensure that sensitivity and specificity (lower bound of the 95% CI) are above 90%, with 90% power and alpha = 5%, under the null hypothesis that the sensitivity and specificity are 95% and 98%, respectively. Under this scenario, a target of 279 children with microscopy-positive malaria during each transmission season was set. In the high transmission season, when, based on previous observational data from health clinics in the area, the assumed prevalence of microscopy positivity was 60% among febrile children, the target sample size for enrolment was therefore 465 febrile children. In the low-transmission season, assuming the prevalence of microscopy positivity would be 30% among febrile children, the original target sample size was 930 children. Under those assumptions, this would provide enough microscopy-negative samples to meet the desired precision. During the low season, it became obvious that the true prevalence was lower than 30%, and that enrolling only 930 participants would not give the 279 true positives necessary. (The rate of RDT positivity was only 10% after the first week). Inclusions were therefore extended through 31 March, 2018, the longest period logistically possible to carry out the study, and a second study site was also added at the Dantchiao CSI.P. falciparum (mono-infection or co-infection) were considered positive for analytic purposes. Comparisons in sub-groups should be regarded as exploratory, as the target sample size was not set with the goal of making these comparisons. Data were analysed using Stata version 15 .The performance characteristics of the RDTs , negative predictive value (NPV)) were calculated independently during each transmission period, and 95% CIs were calculated using the exact binomial method. Only infections with P. falciparum parasitaemia by microscopy, and 1407 participants were enrolled in the low transmission season, including 155 (11%) with P. falciparum parasitaemia by microscopy  with In the high season, for the pLDH test, there were 361 positive results, 169 negative results, and 9 invalid results; for the HRP2 test, there were 370 positive results and 169 negative results. In the low season, for the pLDH test, there were 233 positive results, 1173 negative results and 1 invalid result; for the HRP2 test, there were 215 positive results and 1192 negative results.In the high season, the sensitivity of both tests was estimated at > 99%, but the specificity of both tests was around 58% Table . The poiDuring the high season, the sensitivity of both tests was 100% at all except the lowest levels of parasitaemia (< 200 parasites/µl); during the low season, the sensitivities at the lowest level of parasitaemia were similar to those seen during the high season, but inferior in the intermediate levels of parasitaemia Table . NonetheA series of exploratory analyses not originally planned was undertaken to explain the modest performance of the pLDH test. As Table There were no differences in performance characteristics of the test by participant gender. Performance characteristics were broadly similar by age in both transmission seasons, although the number of participants in each age group was small and the study was not designed to evaluate this Table . ImportaP. falciparum), and 1 which was interpreted as P. ovale was read as P. falciparum by the external laboratory. During the high season, the overall difference in parasite densities between the laboratories was 8%, and there were no differences in the classification of parasite density into the categories shown in Table P. falciparum, both with a parasite density of 40 parasites/µl. Among 17 positive slides, there was only one discordance: a sample with a parasite density of 24 parasites/µl in the study laboratory was read as negative in the reference laboratory. Otherwise there was no difference in the classification of parasite density into the categories shown in Table Regarding external quality control of microscopy, during the high transmission season, among 25 negative slides, there was 100% concordance with the reference laboratory. Among 30 positive slides, 29 were read to have the same species  were generally fainter and slightly less crisp than the lines (control and test) given by the HRP2 test. This difference was noticeable when looking at the tests side by side, as illustrated in Additional file Among the participants during the high season with positive RDTs but negative microscopy results, a majority had test bands of equal intensity to the control band , the test bands were generally weak on both tests, as exemplified by Additional file The specificity and PPV of the pLDH RDT were low, much lower than previously described, and very similar to the HRP2 test, which was unexpected considering the different performance and characteristics of these types of tests reported previously , 3–5, 9.The prevalence of parasitaemia during the high transmission season was slightly lower than expected , which may be due to the protective effect of SMC. Previous evaluations of the pLDH and HRP2 tests have not been performed in areas receiving SMC. Nonetheless, it is unclear how SMC would affect the performance of an RDT. One possibility would be that SMC could lead to high levels of sub-microscopic parasitaemia, some of which would be detected by the rapid tests but not by microscopy, a phenomenon described elsewhere outside the setting of SMC . This coThe second surprising result of this study is the poor sensitivity of the pLDH  and particularly HRP2  tests during the low malaria transmission season. These sensitivities are lower than would be expected , 12. EveThe photographs of RDT cassettes provide a valuable record of what tests look like when performed on ‘real-life’ samples, and are an added value for monitoring, especially given the surprising results obtained. The intensity of bands should not be correlated with severity of infection, but nonetheless, in this evaluation, interesting patterns emerged. The visual appearance of the pLDH test is different from the HRP2 test, with a fainter, slightly hazier line than the HRP2 test. While this does not call into question the accuracy of the test, the experience in the field was that this was less desirable for staff accustomed to the clearer lines of the HRP2 test.Plasmodium [Another hypothesis for the discrepant results between the two tests in the low season would be the presence of HRP2 deletions in the parasite genome, which in turn cause HRP2-based RDTs to be negative even in the presence of asmodium . This haasmodium , 15. A nasmodium . The absOne of the limitations of this study is that information about recent use of artemisinin combination therapy (ACT) was not collected. One hypothesis to explain the high false-positivity rate would be that persistent antigenaemia remained high after effective ACT treatment, but given the characteristics of the time to become negative of these tests, all of these false positives would have had to present to the clinic very quickly after finishing their ACT, which seems improbable. The RDTs were stored at ambient temperatures for relatively short periods of time, and those temperatures may have surpassed recommended storage temperatures, so degradation might have been of concern. On the other hand, this mimics usual storage condition for the vast majority of RDTs used in routine clinical care in the Sahel.PCR was not performed on these samples, which may be a limitation of this study. PCR comes with certain advantages, including that it relies on a standardized process, with less room for human error in interpretation of a result than microscopy, and also that it has a higher ability to detect low-level parasitaemia , 18. ForIn summary, this clinical diagnostic evaluation was undertaken to support the routine use of the pLDH RDTs, but these results showed that the tests had similar performance to an HRP2 test, particularly regarding specificity during the high transmission season. This likely led to a significant amount of over-treatment of malaria, though in this setting, over-treatment is preferable to under-treatment. Nonetheless, as the pLDH test continues to be introduced into new settings, many questions remain. It should be monitored in areas with and without SMC, and future evaluations should include additional PCR analyses, possibly including consideration of HRP2 deletions.Additional file 1: Table S1. Performance characteristics of two malaria rapid diagnostic tests when considering children with asexual forms as well as those with gametocytemia  as being parasitemic, Magaria, Niger, 2017–2018*.Additional file 2. Photograph of HRP2 (blue text) and pLDH (black text) malaria RDTs, high transmission seasons, Magaria, Niger. This participant had a negative blood smear.Additional file 3. Photograph of HRP2 (blue text) and pLDH (black text) malaria RDTs, high transmission season, Magaria, Niger. This participant had a negative blood smear.Additional file 4. Photograph of HRP2 (blue text) and pLDH (black text) malaria RDTs, low transmission season, Magaria, Niger. This participant had a negative blood smear.Additional file 5. Photograph of HRP2 (blue text) and pLDH (black text) malaria RDTs, low transmission season, Magaria, Niger. This participant had 24 parasites/mcl on blood smear.


CareStart™ malaria HRP2/pLDH (Pf/pan) combo test is one of the several rapid diagnostic tests (RDT) approved for diagnosis of malaria at the point of care in Tanzania. However, there are limited studies on the diagnostic performance of RDT after wide scale use in primary health care facilities in Tanzania. Therefore, this study was carried out to determine the diagnostic performance of RDT when compared with blood smear (BS) microscopy as a reference standard.A cross-sectional study was conducted between March and August 2019 at Kibiti Health Centre, Pwani region, Tanzania. Blood samples for malaria tests were collected from patients with malaria symptoms. Diagnostic performance parameters of RDT, i.e. sensitivity, specificity, positive and negative likelihood ratios (LR+/−), diagnostic accuracy and predictive values were determined using contingency table. An agreement between RDT and microscopy was statistically determined by Cohen’s kappa test.Plasmodium falciparum. The geometric mean parasite density was 2921parasites/µl, whereas majority (68.6%) of patients had parasite density greater than 10,000/µl. The sensitivity, specificity, positive and negative predictive values of CareStart™ were 99.8%, 87.6%, 89.8%, and 99.8%, respectively. The LR+ and LR− were 8.0 and 0.002, respectively. The diagnostic accuracy was 0.5. There was a strong agreement between the results obtained using CareStart™ and BS microscopy .Of 980 patients screened, 567 (57.9%) were found to be malaria positive by RDT, whereas 510 patients (52%) were positive by microscopy. Of the 510 microscopy-positive patients, 487 (95.5%) were infected with CareStart™ malaria HRP2/pLDH (Pf/pan) had high sensitivity and strong agreement with microscopy results. However, moderate specificity of RDT resulted in a substantial number of patients with false positive malaria test. Wherever available, microscopy should be used to confirm RDT test results. Global malaria control and elimination programme advocates test and treat as one of the key strategies toward its achievements , 2. In T(RDT) is mostly used in countries with limited resources for diagnosis of malaria due its advantages in these settings. The advantages of RDT include easy to use, provision of results from whole blood within 20 min, easy to carry diagnostic kits and require less skilled personnel  age was 6 3–18.5) years whereas 394 (40.1%) participants had age below 5 years. A total of 894 participants (91.3%) reported the use of insecticide-treated bed nets. The mean [(Standard deviation (± SD)] haemoglobin level was 10.5 (1.7) g/dl and 776 (79.2%) participants had mild anaemia. A majority (65.6%) of the participants had fever. Of the 510 BS positive patients, 487 (95.5%) were 8.5 yearsThe sensitivity and specificity of CareStart™ were 99.8% [(95% CI 98.9%–99.9%) and 87.6% (95% CI 84.4%–90.3%). The positive predictive value was 89.8% (95% CI 87%–92%) while the negative predictive value was 99.8% (95% CI 98.6%–99.9%). The LR+ and LR− were 8.0 and 0.002, respectively (good diagnostic tests have LR+ > 10 and LR− < 0.1). The diagnostic accuracy for malaria was 0.5 (95% CI − 0.193 to 1.193). There was a strong agreement for the accuracy malaria diagnosis between CareStart™ and BS Microscopy   compared to microscopy.CareStart™ combo performance has been evaluated using microscopy as the reference standard in different malaria transmission settings , 27. HigP. falciparum [In this study, the sensitivity and specificity for detection of malaria by RDT was 99.8% and 87.9% respectively. This is slightly different the overall sensitivity and specificity of 95% and 94.2%, respectively that was reported from Ethiopian patients  respectilciparum , 21, 27.lciparum , 15. HowFor RDT to have good specificity during diagnosis of malaria, screening procedure, which is clinical examination, should be robust. This study used clinical malaria symptoms such as fever, general body weakness and headache as recommended in the national malaria treatment guidelines to guide diagnosis . HoweverOver diagnosis of febrile infections which lead to irrational use of anti-malarial drugs has been reported in Tanzania , and thiPlasmodium species [Plasmodium vivax (98.26%) compared to P. falciparum (88.52%) when using CareStart™ kit’ [Plasmodium species using CareStart™ kit’ was also higher for P. vivax (100%) compared to P. falciparum (90.77%). In this study, the most prevalent Plasmodium species was P. falciparum (95.5%) and, therefore, a comparison of diagnostic performance of CareStart™ RDT in individuals infected with different species of Plasmodium could not be made.Another probable cause for discrepancies in diagnostic accuracy of RDT for malaria is the differences in the prevalence of  species , 15, 27.Plasmodium species are considered. In this study, the geometric mean parasite density was 2921 parasites/µl whereas majority of patients had parasite density greater than 10,000/µl. Although the current study did not further perform statistical analysis to establish an association between parasite density and RDT sensitivity to malaria parasites, however findings from the study which was conducted in Madagascar reported low sensitivity (60.0%) of P. falciparum at densities of < 100/μl and increasing sensitivity (100%) at higher parasite densities of > 500/μl [The diagnostic accuracy in terms of specificity (87%) results observed in this study (when using CareStart™ combo test are similar to the specificity of 84% which was reported in the study conducted in Rwanda when HRP2 + pLDH RDT was used (14). These results are an indication that RDT perform better when all of > 500/μl .In this study we compared the diagnostic performance of CareStart™ malaria HRP2/pLDH (Pf/pan) combo test in comparison with standard microscopy for detection of malaria infection among symptomatic patients. Although the results of this study have shown good performance of RDT for detection of falciparum malaria in line with the national malaria treatment guidelines , the useCareStart™ malaria HRP2/pLDH (Pf/pan) had high sensitivity and strong agreement with microscopy results. However, moderate specificity of RDT resulted in substantial number of patients with false positive malaria test. It is recommended that in areas with moderate to high malaria transmission, BS microscopy should be performed to confirm RDT positive results.


Plasmodium parasites that cause malaria, of which Plasmodium falciparum is the most pathogenic. The deformability of RBC is markedly modified by invasion and development of P. falciparum. Surface membrane area is potentially impacted by parasite entry and development, the cytoskeleton is modified by parasite proteins and cytosol viscosity is altered by parasite metabolism. RBC hosting mature parasites  are abnormally stiff but the main reason for their absence from the circulation is their adherence to endothelial cells, mediated by parasite proteins exposed at the infected-RBC surface. By contrast, the circulation of non-adherent rings and gametocytes, depends predominantly on deformability. Altered deformability of rings and of uninfected-RBC altered by malaria infection is an important determinant of malaria pathogenesis. It also impacts the response to antimalarial therapy. Unlike conventional antimalarials that target mature stages, currently recommended first-line artemisinin derivatives and the emerging spiroindolones act on circulating rings. Methods to investigate the deformability of RBC are therefore critical to understand the clearance of infected- and uninfected-RBC in malaria. Herein, we review the main methods to assess the deformability of P. falciparum infected-RBC, and their contribution to the understanding of how P. falciparum infection causes disease, how the parasite is transmitted and how antimalarial drugs induce parasite clearance.Despite a 30% decline in mortality since 2000, malaria still affected 219 million subjects and caused 435,000 deaths in 2017. Red blood cells (RBC) host  Red blood cells (RBC) are essential for oxygen delivery to organs and thus must circulate in narrow blood vessels without being destroyed. These cells, devoid of nucleus and organelles, have unique properties of deformability, i.e., exquisite ability to shape modification upon mechanical constraints. This enables their circulation in blood capillaries, which are narrower than the RBC main diameter. The biconcave shape of RBC increases their surface-to-volume ratio . The defPlasmodium falciparum. Plasmodia are protozoan parasites that cause malaria. During the intra-erythrocytic stage of infection, RBC undergo marked changes. Upon parasite internalization (invasion), RBC undergo a very transient shape change (echinocytosis) before recovering a normal biconcave shape , most attributable to ve shape . During ve shape . As the ve shape , and itsve shape . More th spleens  and have spleens . These r spleens . These s spleens . Retenti spleens . Drug-inance see .P. falciparum-infected RBC was first monitored by a rheoscope (counterrotating transparent cone-and-plate chamber to measure elongation of RBC under shear stress) which assesses the shape of individual RBC  use a highly focused laser beam to generate a three-dimensional gradient of electromagnetic energy resulting in the trapping and controlling of microscopic objects. The force applied depends on the displacement of the beam waist. Two silica microbeads attached to diametrically opposed ends of an RBC are trapped by two laser beams and displaced to stretch the cell . Another®) combines flow cytometry with microscopy. Ten to hundreds of thousands of single-cell pictures can be acquired in a matter of minutes including brightfield, scatter and fluorescent images. Hydrodynamically focused cells are trans-illuminated by a brightfield light source and orthogonally by laser(s). As a result, each cell image is broken-down into separate sub-images based on a range of spectral wavelengths. Semi-automatic analyses of RBC samples can identify discrete subpopulations based on morphological features. The morphology and size  of RBC are related to their ability to persist in circulation through their impact on the surface-to-volume ratio  had also been observed using IFM and correlated with retention in microsphiltration , directly pathogenic through their ability to adhere to endothelial cells, were preferentially studied and were the target of most antimalarial drugs. It is now clear that the loss of RBC deformability begins soon after parasite invasion. Alteration of the biomechanical properties of infected and uninfected RBC potentially enhances their clearance by the spleen. RBC deformability-oriented approaches may therefore play a role in current and future strategies for malaria elimination (P. falciparum, induces swelling of iRBC, impacting their deformability and inducing the fastest parasite clearance ever observed in humans (Early works demonstrated that iRBC deformability is an important determinant of malaria pathogenesis . This nomination . Enhancemination . Artemismination . In thismination , 2018. Tn humans .This review of methods to study the deformability of RBC highlights the different information and outcomes they can each provide. Importantly, their combination enables a better understanding of deformability changes induced by parasite growth and/or by drugs. Confronting outputs from methods that challenge RBC deformability in qualitatively different ways (elongation, cylindrical squeezing, dumble-shape squeezing, membrane indentation, see MD wrote the manuscript under the direction of PN and PB. BH, PN, and PB wrote and corrected the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The pathophysiology of malaria-related anaemia is not fully understood although increased destruction of parasitized and nonparasitized erythrocytes, as well as inadequate erythropoiesis, has been proposed. Circulating antierythropoietin (anti-EPO) antibodies have also been implicated in malaria and malaria-related anaemia in mice. However, studies on this association have not been investigated in humans. This study therefore determined the prevalence of anti-EPO antibody production and assessed its association with malaria and malaria-related anaemia in humans. A total of 86 children aged 1-10 years , all residents of Duayaw Nkwanta, Ghana, were recruited for this case-control study. Venous blood was collected for thick and thin films for malaria microscopy, full blood count by automated haematology analyzer, and antierythropoietin antibody and erythropoietin estimation by sandwich ELISA method. Out of the 86 participants recruited, only 3 (3.5%) were positive for anti-EPO antibody; 2.3% of the case group; and 1.2% of the control group. There was no association between the cases and the controls in the production of anti-EPO antibodies. Erythropoietin concentration was significantly higher in malaria-related anaemic subjects (p=0.032). Antierythropoietin antibodies are not associated with malaria infection and malaria-related anaemia in humans. Erythropoietin concentration is associated with malaria-related anaemia. Plasmodium berghei, where they established that malaria infection was associated with anti-EPO antibody production in some strains of mice [Recent studies have also shown an association between antierythropoietin antibodies and malaria-related anaemia in murine models. A study by Tsubata et al. found that treating infected mice with exogenous antierythropoietin (anti-EPO) antibodies provides protection against malaria infection . Another of mice . These sThis study observed the expected high temperature among participants with malaria compared to the controls , consistVarious mechanisms of malarial anaemia have been investigated including increased RBC destruction through rupture of parasitized red blood cells (PRBCs) , phagocy Plasmodia sp. do not affect only red blood cells but also platelets as these cells originate from a common stem cell [α is elevated in malaria due to inflammation [α has also been reported to inhibit EPO production and EPO-induced erythroid progenitor cell proliferation [α in this study, we could not conclude about the impact of TNF- α on EPO concentration; thus this association requires further studies.Furthermore, as an obligate intracellular parasite,tem cell . A signitem cell , Patel etem cell , and Shatem cell . Additioammation , 41. On feration . This co Plasmodium berghi Anka was the malaria parasite examined in the study by Helegbe et al. in mice; however, the predominant Plasmodium species responsible for malaria infection in humans in Ghana is Plasmodium falciparum.The prevalence of anti-EPO antibodies production among the entire study population was 3.5%; 2.3% in the case group; and 1.2% in the control group . Chi-squAdditionally, during falciparum malaria infection, there is loss of both parasitized and nonparasitized erythrocytes through parasite maturation and antibody sensitization, respectively, leading to anaemia. Erythropoietin (EPO), on the other hand, is crucial to sustain the proliferation and differentiation of erythroid cells. EPO is bound by circulating erythrocytes and studies have shown that reduction in circulating RBCs results in a relatively high concentration of unbound EPO which in turn stimulates erythrocyte production in the bone marrow , 44. In This study is however limited by the small sample size used which may have encumbered the full exploration of the association between anti-EPO and malaria. Future studies may focus on a larger sample size.Antierythropoietin antibodies are not associated with malaria infection and malaria-related anaemia in humans. Erythropoietin concentration is associated with malaria-related anaemia.


Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.Malaria, an infectious disease caused by the parasite  Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria.The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs. Plasmodium falciparum, accounting for 435,000 annual deaths worldwide in 2017  families involved in malaria pathogenesis and immune evasion   , includi1254100) . The two1254100) .Sera from Malian adults reacted more intensely to all six STEVORs than the pediatric sera did. In contrast, the levels of RIFIN seroreactivity were comparable between the two groups, with no significant differences .For each of the three RIFINs, Malian adult sera recognized significantly more total peptides than the pediatric sera did . For alln = 4; Sera collected at day 90 during the transmission season from children who had already experienced at least one clinical malaria episode showed serorecognition of significantly more total peptides for each of the three RIFIN proteins than paired preseason pediatric sera . These s10.1128/mSphere.00097-19.1FIG S1n = 10). (A) Pediatric sera at day 90  recognized significantly more RIFIN and STEVOR peptides than pediatric sera from before the malaria season . (B) Sera from day 90 of the malaria transmission season (gold) differed in seroreactivity for only a few peptides compared to sera at the start of the season (sky blue) . Download FIG S1, TIF file, 2.7 MB.Peptide microarray serorecognition of and reactivity to three RIFINs and six STEVORs using matched pediatric sera before and during the malaria transmission season . (A) Day 90 pediatric sera that did not experience a clinical malaria episode  were not significantly different from matched sera collected at the start of the malaria season . (B) For sera obtained during the malaria transmission season (magenta), seroreactivity differed for only a few peptides compared to the start of the season  . Download FIG S2, TIF file, 1.4 MB.Peptide microarray serorecognition and reactivity of matched pediatric sera of children who did not experience a clinical malaria episode during the malaria transmission season   , which, The semiconserved domain has not previously been identified as a target of the human immune response. A recent study used peptide microarrays to identify binding sites of rabbit antibodies directed against RIFINs and STEVORs and localized this reactivity to the C-terminal, intracellular conserved domain . A subseWe have previously shown that malaria-exposed sera react to the intracellular portion of PfEMP1s . Althougin vitro  (This study had some limitations. Antigens on the protein microarray were fabricated with a cell-free (ELISAs) . Finding(ELISAs) , 37, 38.(ELISAs) , and theProtein and peptide microarrays populated with a panel of RIFINs and STEVORs associated with severe malaria vulnerability revealed that the semiconserved domains of both RIFINs and STEVORs serve as markers of malaria exposure. To comprehensively identify epitopes associated with severe malaria vulnerability and whether differences in exposure are truly correlated with protection, protein and peptide microarrays populated with a comprehensive set of RIFINs and STEVORs should be probed with sera from severe malaria case-control studies. Further analyses and characterization of relevant STEVORs and RIFINs through invasion inhibition, rosetting, and phagocytosis assays will provide insight into the role each protein plays in the development of severe malaria.Both the protein and peptide microarrays included three full-length RIFIN antigens  and three full-length STEVOR antigens  associated with cerebral malaria vulnerability; PF3D7_0832000 is also associated with vulnerability to severe malarial anemia . We alsoP. falciparum open reading frame, (ii) recombination cloning, (iii) in vitro transcription/translation (IVTT), and (iv) microarray chip printing (Protein microarray construction followed a four-step process: (i) PCR amplification of each complete or partial printing . This IVprinting . The pepprinting . Serum sThe amino acid positions of the start and finish of RIFIN and STEVOR domains for each antigen were defined using the PEXEL motif and transmembrane domains as anchors for bioinformatic alignment, based on previous analyses and the PlasmoDB database , 34, 40.P. falciparum infections represent 97% of the malaria infections in this region. Malaria transmission is sharply seasonal, with minimal transmission at the height of the dry season in March; fewer than one infected mosquito bite per person per month at the start and end of the transmission season in June and December, respectively; and a peak of up to 60 infected bites per person per month in September  in the Dogon region in east-central Mali. eptember . Childreeptember . The aveeptember , and theeptember .n = 18) (n = 75)  trial (n = 18)  and sera(n = 75) , 46. TheP. falciparum parasites on a blood smear. The average age for these six children at enrollment was 3.1 ± 0.5 years (mean ± standard error), compared to 4.3 ± 0.2 years for the remaining four children .A high-density peptide microarray was probed with sera from a subset of randomly selected individuals whose sera was used for the protein microarray, including sera from 10 Malian adults and 10 Malian children and 5 malaria-naive North American control samples. Pediatric sera included time points before (day 0) and during (day 90) the malaria transmission season. Among the 10 children, 6 subjects experienced at least one clinical malaria episode before the day 90 time point, defined as having malaria symptoms and The protocols were approved by institutional review boards of the University of Bamako Faculty of Medicine; the University of Maryland, Baltimore; and the U.S. Army Surgeon General. Written informed consent was obtained before screening and enrollment in the trials. Verbal consent of illiterate parents or guardians was provided and documented using thumbprints and verified by independent witnesses.P. falciparum protein microarrays (E. coli system. Given the synthetic fabrication process for peptide microarrays, no adjustment for background fluorescence intensity was necessary; raw fluorescence intensity represented peptide seroreactivity. Group serorecognition of a protein or peptide fragment was defined as detection of a level of fluorescence intensity significantly greater than that of the malaria-naive North American control group, based on a two-sample Kolmogorov-Smirnov test. Serorecognition differences between two populations for the peptide array were calculated with a two-tailed McNemar’s test. Levels of seroreactivity of a protein fragment or peptide were compared among groups using a nonparametric Wilcoxon rank sum test for unmatched group-wise comparisons. Levels of seroreactivity for paired samples across time points were compared using a Wilcoxon signed-rank test. Finally, we identified peptides that not only were serorecognized by a single population but also had differentially increased seroreactivity in a given comparison. All P values presented represent two-sided values determined without correcting for multiple comparisons, as was previously done in other microarray analyses (We applied data analysis techniques previously used to evaluate serologic responses with roarrays , 37, 47.analyses , 24, 38.


Plasmodium falciparum malaria illness is generally estimated using one of two distinct approaches: either by transforming P. falciparum infection prevalence estimates into incidence estimates using conversion formulae; or through adjustment of counts of recorded P. falciparum-positive fever cases from clinics. Whilst both ostensibly seek to evaluate P. falciparum disease burden, there is an implicit and problematic difference in the metric being estimated. The first enumerates only symptomatic malaria cases, while the second enumerates all febrile episodes coincident with a P. falciparum infection, regardless of the fever’s underlying cause.The disease burden of P. falciparum infection, fever, and care-seeking to estimate the fraction of P. falciparum-positive fevers amongst children under 5 years of age presenting at health facilities that are attributable to P. falciparum infection versus other non-malarial causes. A Bayesian hierarchical model was used to assign probabilities of malaria-attributable fever (MAF) and non-malarial febrile illness (NMFI) to children under five from a dataset of 41 surveys from 21 countries in sub-Saharan Africa conducted between 2006 and 2016. Using subsequent treatment-seeking outcomes, the proportion of MAF and NMFI amongst P. falciparum-positive febrile children presenting at public clinics was estimated.Here, a novel approach was used to triangulate community-based data sources capturing P. falciparum infection was estimated to be the underlying cause of only 37.7%  of P. falciparum-positive fevers, with significant geographical and temporal heterogeneity between surveys.Across all surveyed malaria-positive febrile children who sought care at public clinics across 41 country-years in sub-Saharan Africa, P. falciparum burden amongst children under 5 years of age and indicate that for many children presenting at health clinics, a positive P. falciparum diagnosis and a fever does not necessarily mean P. falciparum is the underlying cause of the child’s symptoms, and thus other causes of illness should always be investigated, in addition to prescribing an effective anti-malarial medication. In addition to providing new large-scale estimates of malaria-attributable fever prevalence, the results presented here improve comparability between different methods for calculating P. falciparum disease burden, with significant implications for national and global estimation of malaria burden.These findings highlight the complex nature of the The online version of this article (10.1186/s12936-019-2830-y) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria remains a significant global health burden, causing approximately 216 million cases and 445,000 deaths globally in 2016 [Plasmodium infection [P. falciparum prevalence [Despite significant gains in reducing malaria prevalence,  in 2016 . For estP. falciparum infection, before adjusting to produce estimates of the total number of community cases. By enumerating P. falciparum-positive fevers, no distinction is made between P. falciparum-attributable fevers and non-malarial fevers coincident with a diagnostically patent, asymptomatic P. falciparum infection, and both are reported as confirmed malaria cases by the clinic to the HMIS.The first step of the adjusted case-count approach enumerates the number of fever cases presenting at clinics that are coincident with a patent P. falciparum-attributable fevers, and not fevers that are coincident with, but not caused by, a patent P. falciparum infection. Burden estimates derived using this methodology will therefore produce estimates of malaria cases that are systematically lower than those produced using the adjusted case-count approach.Conversely, the cartographic approach relies on mechanistic models of the proportion of infected individuals who go on to experience a symptomatic infection , 7–9. ByP. falciparum prevalence (required to utilize the cartographic approach) are sparse or parasite prevalence is low (e.g. in much of Asia and the Americas) [P. falciparum prevalence, there is increasing interest in adopting the adjusted case-count approach into burden estimation frameworks to benefit from the higher cadence of updates and the new digital health information systems technologies in these countries. To examine compatibility between the two approaches, in the most recent World Malaria Report, a number of sub-Saharan African countries reported increasing numbers of malaria cases when estimated via the adjusted case-count approach [P. falciparum-attributable fevers and P. falciparum-positive fevers  is required to estimate the true burden of malaria.The adjusted case-count approach is typically used where observations of community mericas) , 2, 10. mericas) , 10, 11.approach . When esP. falciparum-positive fevers that are causally attributable to the P. falciparum infection, amongst febrile children under 5 years of age presenting at public health facilities in a number of sub-Saharan African countries. Henceforth, the following terminologies are used: (i) malaria-positive fever, defined as a P. falciparum-positive fever, without confirmation of the infection being the underlying cause, (ii) malaria-attributable fever (MAF), defined as a P. falciparum infection where infection has been determined to be the fever’s underlying cause, and (iii) non-malarial febrile illness (NMFI), defined as a fever with an underlying non-malarial cause, which may or may not be co-incident with a P. falciparum infection.In this analysis, a model is presented to estimate the proportion of P. falciparum parasite prevalence (PfPR) was modelled. This relationship was used to calibrate a Bayesian hierarchical model, which was fit to household survey data to predict whether febrile children under 5 years of age in this dataset had a NMFI, a MAF, or co-symptomatic MAF and NMFI. Individuals’ subsequent treatment-seeking behaviour was then tracked to generate estimates of the proportion of febrile individuals under 5 years of age presenting at health clinics with fevers due to the following underlying causes: (i) MAF, (ii) co-symptomatic MAF and NMFI, (iii) NMFI coincident with a rapid diagnostic test (RDT)-patent asymptomatic P. falciparum infection, and (iv) NMFI without an RDT-patent P. falciparum infection. These estimates were compared to malaria case counts in children under 5 years of age obtained from Ministry of Health reports from three malaria-endemic countries, to estimate the proportion of malaria-positive fevers with a non-malarial underlying cause. These findings highlight the difference between P. falciparum-positive fevers presenting at clinics (considered “cases” under the adjusted case-count approach) and the true burden of symptomatic malaria-attributable infections (the metric estimated under the cartographic approach). From a clinical perspective, both malaria-positive and malaria-attributable fevers should still receive anti-malarial medication, but the results presented in this analysis indicate the proportion of fevers coincident with P. falciparum infections that would not be resolved by anti-malarial medication alone. Many of these fevers are likely to be caused by self-limiting infections and not require further treatment, but nevertheless these findings highlight the importance of clinicians checking for signs of other infections even when P. falciparum infection is diagnosed.Here, estimates of malaria-positive and malaria-attributable fevers were collated from a wide variety of sources  and the relationship between the proportion of malaria-positive fevers that are causally attributable to malaria in a given community and the corresponding P. falciparum prevalence in children under 5 years of age (PfPR0–5). The four datasets were chosen to encompass the variety of definitions of fever , and differing methods for defining MAF. The datasets fell broadly into two categories: defining MAF using a pyrogenic parasite density threshold for malaria-infected individuals, above which malaria-positive fevers are defined as MAF (one dataset); and datasets which record paired observations of malaria positivity and fever  allowing for empirical estimation of MAF (three datasets). Full details of the four datasets are given in Additional files Four different primary datasets were assembled and used to measure the relationship between MAF and P. falciparum infections above and below a pyrogenic parasite density threshold, was derived from active case detection studies. In this dataset, the proportion of malaria-infected individuals under and over the pyrogenic threshold was used in conjunction with the PfPR at the study site in the modelling approach described below. Details of the parasite density threshold dataset can be found in Additional file The first dataset, comprised of observations of P. falciparum-positive and P. falciparum-negative populations, and as such the “excess” fevers within the febrile P. falciparum-positive individuals not explained by the background NMFI rate are considered to be fevers that are causally attributable to the P. falciparum infection. Full details of each of these three datasets and the formula for empirical calculation of MAF are given in Additional files PfPR0–5 was measured, and paired observations of malaria positivity and fever were recorded in children under five. The first dataset consisted of household survey data from the Demographic and Health (DHS) Program  study [The remaining three datasets consisted of paired records of fever and malaria positivity. From these, the proportion of malaria-positive fever cases where the malaria infection was the underlying cause of the fever (MAF) was derived empirically by accounting for the rate of malaria-negative fever in the population. This empirical calculation assumes that the rate of fever caused by NMFI is the same in both the M) study   and PfPR0–5. Next, the remaining three datasets  were used in a second fitting step to re-scale the relationship learned in the first step. Full details of the model fitting methodology are given in Additional file Using the four datasets described above, the relationship between MAF in children under 5 years of age and PfPR0–5 were also investigated: (i) the effect of residual exposure-dependent immunity, measured using recent declines in PfPR0–5, and (ii) the effect of background treatment rate. Data sources for each of these factors can be found in Additional file Two additional factors which may impact the relationship between MAF and P. falciparum infection, and (iv) NMFI without an RDT-patent P. falciparum infection. Each child’s treatment-seeking behaviour was tracked in order to establish the proportion of malaria-attributable and non-malarial fevers presenting at health facilities. To inform the model, the DHS Program household survey dataset described in the previous sub-section was utilized  MAF, (ii) co-symptomatic MAF and NMFI, (iii) NMFI coincident with a rapid diagnostic test (RDT)-patent asymptomatic PfPR0–5, (ii) suitability for fever without a malaria infection in children under 5 years of age, and (iii) caregivers’ propensity to seek treatment for fever of any cause for children less than 5 years of age. All three covariates were available for each year of the study period at a 5 × 5 km spatial resolution across areas of stable P. falciparum transmission in sub-Saharan Africa. The value of each of the three covariates was extracted for each child based on their GPS location and the time of survey. Full details of each covariate are given in Additional file In the Bayesian hierarchical model, three model-derived spatial covariates were used as predictors of MAF or NMFI. These three predictor variables were: (i) PfPR described in the previous subsection. By using this recalibration , the moFor country-years where a contemporaneous Ministry of Health report was publicly available, the modelled proportion of malaria-positive fevers that were/were not causally attributable to malaria was applied to the reported number of malaria-positive fevers amongst children under five from each report. Ministry of Health reports were identified via Google search for all countries with a survey, and included if annual confirmed malaria cases were reported for children under five.PfPR0–5 ~ 0, until PfPR0–5 > 0.4 after which the proportion declines much more slowly. The coefficients for the fitted model are given in Table The proportion of malaria-positive fevers that are causally attributable to malaria was estimated to decline exponentially from 0.82 at PfPR0–5 , compared to lesser declines (< 5% reduction) or PfPR0–5 increases. Differences in the fitted relationship by age were observable with older children , showing non-significant differences between the two subsets.Background treatment-seeking rate for fever  was not found to have a significant impact on the relationship between malaria-attributable fever  and PfPR0–5 was implemented in the Bayesian hierarchical model to attribute fevers amongst the surveyed individuals to either malaria or non-malarial causes. For each survey, the treatment-seeking rate of MAF and NMFI cases (which can be RDT-positive or RDT-negative) was collated to produce an estimate of the proportion of each fever type presenting at public health clinics in that country-year.The final fitted relationship between malaria-attributable fever  and P. falciparum malaria if all malaria-positive fevers were counted as malaria cases.Averaging across all surveys, Fig. PfPR0–5 over the survey time period in those countries [PfPR0–5 shown in Fig. Amongst the surveyed country-years, six countries were surveyed three or more times between 2006 and 2016 . The proportion of malaria-positive fevers that were predicted to be causally due to malaria rose in Angola, Madagascar, Senegal (with the exception of 2010–2011), and Tanzania between the first and last surveys. This was consistent with a decrease in Confirmed malaria cases  amongst children under 5 years of age were available for three country-years with near-contemporaneous household surveys  are not considered.The analysis presented here indicates that a significant proportion of confirmed P. falciparum and NMFI. Many of the surveys from countries with the greatest predicted overestimates for cases using the adjusted case-count approach were completed in the first half of the study decade, coinciding with higher malaria burden  and worimmunity . These cPfPR0–5 over the study period) [PfPR0–5 decreased over the study period). This increase in MAF in the six countries could also be explained by a decrease in NMFI prevalence relative to malaria prevalence over the time period, but this was not observed in the current study [Many of the 21 countries included in this study were surveyed multiple times between 2006 and 2016, with three surveys in Angola, Liberia, Madagascar, Tanzania and Uganda, and four surveys in Senegal. In most of these countries, a larger fraction of malaria-positive fevers were causally attributable to malaria in later surveys compared to earlier surveys within the same country (with the exception of 2016 surveys in Tanzania and Uganda). These findings may be explained by a loss of exposure-related immunity amongst children under 5 years of age as malaria prevalence decreases   resultinnt study ; indeed P. falciparum and other illnesses are common, the results presented here do not directly indicate any requirement for upscaling of antimicrobial medications, especially in areas where bacterial infections are less common. Evidence from literature suggests that clinicians show good adherence to integrated management of childhood illness (IMCI) guidelines when prescribing ACT after a positive RDT [Averaging over the 41 surveyed country-years, approximately two-thirds of malaria-positive fevers amongst children under 5 years of age would not be resolved with effective anti-malarial treatment. If clinicians do not investigate other causes of fever given a positive RDT, non-malarial fevers may be systematically missed and remain untreated, potentially leading to missed opportunities to reduce the burden of other important non-malarial febrile illnesses. It is, however, also important to consider that many of the underlying causes of these non-malarial fevers may not warrant treatment, for example if the underlying cause is a self-limiting illness. As such, although this analysis suggests that clinicians should be aware that co-infections with tive RDT , 20, buttive RDT . Studiestive RDT . Diagnostive RDT . This prPfPR were investigated. No effects of recent PfPR declines or differential underlying treatment rates were observed on the relationship between MAF and PfPR . In addition to biases in recall of recent fever , 26 the Plasmodium vivax malaria is also, by definition, grouped within NMFI in this analysis, confounding the definition of NMFI. P. vivax is not prevalent in Africa , exceptgativity , 30. Duesmission ), the efPfPR model inputs.The results presented here suggest a high prevalence of co-infections between malaria and NMFI amongst febrile children presenting at clinics. Here, the effects of co-morbidity (or modulating effects of co-infection on febrile illness) are not accounted for, owing to the lack of diagnosis for NMFI. Future studies may aim to investigate the effects of co-morbidity if differential diagnosis of NMFI is available, or to include spatial distributions of known NMFI-causing pathogens, which were precluded from the current analysis given the lack of availability of cartographic NMFI estimates at the same scale as the P. falciparum burden [The Ministry of Health reports used to generate the number of paediatric malaria-positive fevers that would and would not be resolved by anti-malarial treatment alone in three sub-Saharan African countries Fig.  are subjm burden , or thosThese findings indicate a need for standardizing approaches for estimating malaria burden. Epidemiologists and public health professionals comparing burden estimates made using the adjusted case-count or cartographic approaches should be aware that these approaches estimate fundamentally different metrics. Refinement of burden estimation approaches to generate estimates of both malaria-attributable fever and malaria-positive fever would improve cross-comparability and allow assessment of the quantity of non-malarial febrile illness amongst malaria-positive fevers. The findings presented here are not intended to suggest that malaria-infected individuals should not be treated with effective anti-malarial medication; but rather the intention of this analysis is to increase understanding of the complex nature of the malaria burden, and to highlight the vast burden of non-malarial paediatric fever.Additional file 1. Studies and data used for parasite density threshold dataset. For each study, the study P. falciparum prevalence in children under 5 years of age (PfPR0–5), parasite density threshold used, upper and lower age bounds, and the number or proportion of malaria-positive febrile individuals over and under the parasite density threshold is given.Additional file 2. Household survey data utilised for (i) modelling relationship between MAF and PfPR, and (ii) modelling proportion of MAF and NMFI cases seeking treatment in clinics. P+/− refers to the number of children P. falciparum positive/negative at time of interview. F+/− refers to the number of children for whom fever was/was not recalled in the 2 weeks preceding the survey.Additional file 3. Literature review and PRISM datasets utilised for modelling relationship between MAF and PfPR. For each study, the fever and parasite positivity status for each individual was extracted. Not all datapoints listed here were used in the final model; analysis was restricted to children under 5 years of age. *Search methodology for datapoint, as documented in “https://clinepidb.org/.Additional file 4. Datasets and modelling approach for estimation of the relationship between malaria-attributable fever and Plasmodium falciparum prevalence in children under 5 years of age.Additional file 5. Details of covariates used in MAF–NMFI modelling approach.Additional file 6. Model for individual-level attribution of MAF and NMFI to febrile children within the household survey dataset.Additional file 7: Figure S1. (a) Estimates of proportion of fevers amongst malaria-positive presenting to public health clinics that are causally due to MAF (red sections), NMFI accompanied by an asymptomatic P. falciparum malaria infection (blue sections) and co-symptomatic MAF and NMFI (yellow sections); and (b) as (a), also including NMFI not accompanied by a P. falciparum malaria infection (grey sections); amongst febrile children under 5 years of age presenting to public health clinics amongst children from household surveys in 41 country-years in sub-Saharan Africa, 2006–2016.Additional file 8. Proportion of each type of fever presenting at public health clinics in each survey, with associated 95% credible intervals. MAF = malaria-attributable fever; NMFI = non-malarial febrile illness; Pf+/Pf− = P. falciparum RDT positive or negative (at time of seeking treatment). “MAF and NMFI” indicates febrile children with co-symptomatic MAF and NMFI.