Research on Gestational Malaria (GM) is scarce in America's. In the few available studies in Colombia, the analysis of immunological or parasitological aspects predominates, with few analyzes of epidemiological aspects. The objectives were to determine the frequency of GM and submicroscopic infections (positive with PCR and negative with thick blood smears), to identify obstetric and malaria history associated with GM, and to describe maternal and neonatal outcomes associated with GM, in northwestern Colombia. A retrospective study with records of 825 pregnant women was conducted. qPCR and thick blood smear were performed. Frequencies were determined with 95% confidence intervals. Comparisons were made with the Chi-square test, Mann–Whitney U test, and prevalence ratios adjusted in a log-binomial model. The frequency of GM was 35.8% (95% CI 32.4–39.1) of submicroscopic infection was 16.2% (95% CI 13.7–18.8). According to the multivariable model, the subgroups with the highest frequency of GM were pregnant women without healthcare coverage (32.3%), in the third trimester of pregnancy (30.5%), nulliparous (35.6%), and with a previous diagnosis of malaria in the current pregnancy (64.0%). GM was associated with more frequency of gestational anemia, infection in neonates, and lower birth weight. The results indicate in a precise and direct way that malaria control in this northwestern region of Colombia is far from adequate, which is even more serious considering the affectations for the mother and the neonate. Specifically, in America's, there has been an increase in cases since 2015. For example, Colombia registered an increase of 28.2% in 2019, with 66,581 infected2. In Colombia, malaria has an unstable pattern characterized by variable interannual and inter-monthly prevalence; it is very common in adults, generally symptomatic, with a wide clinical spectrum ; it causes anemia with high frequency; and produces mortality in all age groups3.During the year 2019 around the world, 228 million cases of malaria and 405,000 deaths occured, and 11 million pregnancies were exposed to infection in countries with moderate and high endemicity6. The persistence of malaria in Colombia cannot be explained by Plasmodial resistance to drugs or resistance of Anopheles to insecticides6. The official reports do not specify the morbidity or mortality cases from gestational malaria (GM); however, with data from the National Institute of Health (NIH) of Colombia, it is possible to say that: (a) in the rural population exposed to malaria in Colombia  there are 58,752 births, which, we assume, would correspond to the number of pregnant women exposed; (b) based on the data on severe malaria from the NIH 2007–2020, there is 1 case of severe GM for every 2833 total reported cases of malaria; (c) between 2007 and 2020, there were 380 cases of severe/complicated malaria in pregnant women in Colombia, 27.1 cases/year5.In Colombia, malaria cause significant outbreaks along the Pacific Coast, the Magdalena and Amazon basins, and is associated with clusters in municipalities of the Pacific Coast, the border with Panama, the north of Antioquia, municipalities of Córdoba, Vichada and Antioquia9. The few studies carried out in Colombia have reported an increased risk of anemia (55.6%); severe malaria with liver dysfunction, acidosis or thrombocytopenia (13.5%); alteration of the development of the immune response to the anti-tetanus toxoid vaccine; and lower birth weight13.Beside, GM constitutes a disease with different control and prognosis from malaria in the non-pregnant population. GM produces serious consequences on maternal , fetal , neonatal , and infant health 14. In America's, studies on the prevalence of GM are meager18, and particularly in Colombia, there is no clear and precise measurement of the event. In three municipalities of Urabá Antioqueño , a prevalence of 10.39% was found with thick blood smears (TBS)12. In 129 parturient from Turbo-Antioquia and Puerto Libertador-Córdoba, the prevalence of GM was 3.1% with TBS and 14.0% with PCR (Polymerase Chain Reaction)19. Other studies have reported frequencies of 13% or 14% with TBS21 versus 32% with PCR, demonstrating a high frequency of submicroscopic GM 22.Concerning the magnitude of GM, most of the evidence is concentrated in Sub-Saharan Africa. In this continent, a meta-analysis with 171 studies between 1990 and 2011 with 340,904 pregnant women found a prevalence of 32% (95% CI 25.9–38.0) in the East and South, and 38.2% (95% CI 32.3–44.1%) in the central and western part of the continentThe evidence presented reveals several problems that justify the need for more studies on this topic: (a) heterogeneity in the frequency of GM and difficulties in generalizing the available evidence, given that most studies are based on non-probability samples, (b) the low number of studies in Colombia, (c) malaria control programs focused on TBS which generates underreporting and does not detect submicroscopic infections, (d) there are few studies on associated factors to guide other research, and prioritize higher-risk groups, and (e) the primary outcomes of GM on maternal and neonatal health are unknown.Plamosdial species; to identify the obstetric and malaria history associated with GM; and to describe the principal maternal and neonatal outcomes of GM in the most endemic region in northwestern Colombia, 2009–2020. The human population of the region, during these years, has not registered significant changes in demographic, socioeconomic and cultural characteristics. Malaria data indicate a decreasing trend between March 2010 and March 2014, when the “Colombia Malaria Project” was executed in 43 municipalities with a high endemicity, and it was possible to reduce the cases about 60%, compared to the previous years. Once this Project was finished, the State did not continue it, and malaria increased again, with growing trend until 2020.The objectives of this investigation were to determine the general frequency of GM, the specific frequency of its submicroscopic form and of each 5.In 2019, Colombia had 241 municipalities without effective malaria control, especially in rural areas. The rural population residing in these areas with the active transmission is 3,334,116 inhabitants (6.6% of 50.4 million Colombians), located in the five natural regions of Colombia and 21 departments. Based on the adjusted data for births in Colombia in 2019 , the population of pregnant women exposed to malaria in 2019 in these 241 municipalities was 58,186 . In general, the living conditions of the population residing in these areas with active malaria transmission are very poor and demonstrate great economic poverty5.The study region has an extension of 34,848 km, and it is made up of the areas of Urabá Antioqueño, the upper basins of the Sinú and San Jorge rivers, and the Bajo Cauca Antioqueño. The region has had a high malaria incidence  since 1950 and generates at least 60% of Colombia's total annual malaria cases. This region comprises 25 municipalities, distributed in the departments of Antioquia  and Córdoba . According to official DANE data, the total population of this region was 1,114,000; all of them are a population exposed to malaria, of which approximately 5414 are exposed pregnant womenPlasmodial infection in pregnant women and neonates; the epidemiological and histopathological aspects of placental and gestational malaria; the epidemiology and consequences of submicroscopic Plasmodial infection; and the comparison of the outcomes of microscopy, polymerase chain reaction, and histopathology in the diagnosis of the infection. Given that in Colombia this type of research does not receive much money to finance it, at the beginning of these investigations, epidemiological information was collected, in order to accumulate a good amount of data that allowed the development of the current study, for which the same diagnostic tests, observational designs, and epidemiological surveys were applied.A retrospective study was carried out based on the records of pregnant women who participated in five investigations between 2009 and 2020. The objectives of these investigations were to describe the following aspects of gestational, placental and/or congenital malaria: the parasitic genetic variability, the relationship of placental damage with the expression of cytokines; the immunomodulatory effect of placental The pregnant women met the following inclusion criteria: stable residents (at least one year before the beginning of each study) of the study region, with TBS and qPCR results for malaria diagnosis, apparently healthy as recorded in their clinical history , attend or not the prenatal consultation but have the delivery in any of the public hospitals of the municipalities of the region; with voluntary participation in the study and signing of the informed consent. The research does not carry out any specific activity to promote attendance at prenatal consultation or delivery at the local hospital . Exclusion criteria were being under antimalarial treatment in the previous 2 weeks, presence of any disease or infection according to the criteria of the health personnel caring for pregnant or laboring women.In total, the data of 825 pregnant women were analyzed; it corresponds to the following parameters: expected proportion of GM 25%, confidence 95%, sampling error 3%, and estimated population of pregnant women exposed to malaria based on the number of births 15,000, correction of 8%.Plasmodial infection using qPCR24. In addition, a questionnaire was filled out to extract the epidemiological information from the clinical history, and it contained obstetric information and a history of malaria. The questionnaire was designed by malaria, GM, and epidemiology experts, guaranteeing its appearance (applicability and acceptability) and content validity.A peripheral blood sample was taken from each pregnant woman by venipuncture. Slides were made for the microscopic diagnosis of malaria by TBS, and Whatman No.3 filter paper circles were blotted for DNA extraction with the Saponin-Chelex method and subsequent molecular diagnosis of maternal Selection bias was controlled by including all subjects who met the eligibility criteria. Information biases were controlled by training the field team, standardizing the fieldwork, internal quality control in the laboratory, implementation of the test manufacturer's instructions, double data entry, and appearance and content validity of the questionnaire. Confounding variables were analyzed using generalized linear regression.The general frequency of GM (positive by TBS or PCR) and the specific frequency by type of diagnostic test, as well as the frequency of submicroscopic GM, were calculated, all with their 95% confidence interval. Due to the fact that no statistical differences were found in the frequency by year and municipality of study, the specific frequencies for these two variables were not presented.26.The obstetric variables and malaria history were described with absolute (n) and relative (%) frequencies for categorical variables, and central tendency and dispersion measures for the continuous variables. The possible associations of these variables with GM were explored with Pearson's Chi-square test for nominal variables, Chi-square test for trend for ordinal variables, and Mann–Whitney U test for continuous variables . The strength of the association was established with prevalence ratios (PR) with the Katz method and its 95% confidence intervals. For polytomous variables, dummies were constructed, taking as a reference group the one with the lowest frequency of GM. A multivariable generalized linear model was performed with the variables that showed a bivariate association with the frequency of GM to identify possible confounding factors. Furthermore, the variables with the most significant explanatory potential for GM were identified through a multivariable generalized linear model with the logarithm transformation and binomial family Among women with and without GM, the frequency of gestational anemia (hemoglobin < 11.0 g/dL), abortions, fetal death, neonatal death, low birth weight (less than 2500 g), and neonatal malaria  was compared using Pearson's Chi-square test or Fisher's Exact test (when the expected frequency was less than 5), and the number of abortions, fetal and neonatal deaths, hemoglobin and weight at birth were compared through the Mann–Whitney U test.All analyzes were performed in SPSS 27.0 with a significance of 0.05.The guidelines of the Declaration of Helsinki and Resolution 8430 of Colombia for research with pregnant women were applied. All pregnant women signed the informed consent (or assent for those under 18 years of age). The project was endorsed by the Bioethics Committee of the “Sede de Investigación Universitaria SIU”, Minutes # 21-101-961, and was classified as a minimal risk study.The frequency of GM was 35.8% (95% CI 32.4–39.1); with TBS was 19.5% (95% CI 16.7–22.3) and the submicroscopic infection was 16.2% (95% CI 13.7–18.8).The mean time of residence in the malarious area was 8.8 ± 8.2 years . The mean age of the pregnant women was 23.3 ± 6.4 years . The average of age gestational in weeks was 30.2 ± 9.8 . The highest proportion of pregnant women were adolescents or young women under 25 years of age (63.8%), primigravidae (31.6%), multiparous (31.2%), 24.2% with previous episodes of malaria during the current pregnancy, 34.0% with malaria in the last year, and 43.7% mentioned the use of mosquito net. Affiliation to the General System of Social Security showed that 93.4% were affiliated to the subsidized regime , trimester of pregnancy (higher frequency in the third trimester), number of deliveries (higher in nulliparous women) and previous diagnosis of malaria in the current pregnancy Table .Table 3MGM was related with anemia, being 87% higher in women with GM compared to pregnant women without malaria; and higher frequency of malaria in neonates, being 3.91 times higher than what was found in pregnant women without malaria. Although low birth weight did not present a statistical association with GM, a statistically lower median birth weight was found in pregnant women with malaria than in pregnant women without the infection Table .Table 4C27; (c) higher risk of infection in some population subgroups that should be prioritized ; and (d) high heterogeneity in the frequency of GM according to the presence of some associated factors, which shows the importance of conducting local studies that allow a deeper understanding of clinical, epidemiological, cultural, and socioeconomic conditions that increase the risk of developing the disease29.The frequency of GM was high (35.8%) with many cases of submicroscopic infection (16.2%), which aggravates the situation, because the diagnostic test of malaria used routinely in the area is TBS. Gestational anemia, neonatal malaria and birth weight are significantly associated with presence of GM. These data show several relevant aspects to study GM: (a) its high frequency, which implies a high risk for maternal, fetal, neonatal, and infant health; (b) very high endemic levels despite reports from national authorities indicating that malaria is a controlled infection at the beginning of the pre-elimination stage14. Other studies from America's have documented smaller proportions than the current study. In 449 pregnant women from the state of Bolívar-Venezuela, it was 27.4%17 and in a study in Brazil with data from the passive surveillance system, it was 7.5%; however, in this latter case, the low proportion was attributed to the absence of an active search for cases and the lack of registration of asymptomatic and submicroscopic cases, which constitutes a clear detection bias18. In the specific case of Colombia, previous studies have reported frequencies of GM ranging from 10.4% with TBS to 32% with qPCR21. In a meta-analysis of the prevalence of malaria associated with pregnancy in Colombia 2000–2020 the prevalence of GM was 5.8% (95% CI 3.8–8.7) by TBS, 16.7% (95% CI 9.0–28.8) with PCR, and 8.5% (95% CI 3.4–19.7) of submicroscopic infections; with a high heterogeneity in the magnitude of the infection, attributable to the level of endemicity of the different regions studied, and variations in the study designs30. The high frequency reported in different populations, shown the high gynecological-obstetric risk attributable to GM, and it indirectly demonstrates problems in the control of the infections in prenatal care programs. This situation is aggravated when considering that among the pregnant women of this research, some subgroups exceeded these frequencies and, therefore merit priority actions in healthcare, control, and prevention.The frequency of GM was 35.8% (95% CI 32.4–39.1), which is statistically the same as the one reported in a meta-analysis of 171 studies with 340,904 pregnant women treated in prenatal care programs in sub-Saharan Africa, where the frequency was 32% (95% CI 25.9–38.0) in the East and South part of the continent, and 38.2% (95% CI 32.3–44.1%) in the central and west part of the continent22, demonstrating greater risks to the health of the mother–child pair since this type of infection is not treated, which means that it can progress to cases of severe fetal, congenital or neonatal malaria, and other consequences mentioned in the introduction12. Previous studies in the same endemic region confirm the pathological ability of microscopic or submicroscopic infections to produce malaria in the mother, the placenta, and the neonate33. Some researchers refer beneficial aspects of persistent and repeated submicroscopic malaria; this has been described for cases of asymptomatic malaria (without specifying whether they are submicroscopic cases) associated with decreased incidence of malaria, but without protection against disease when new infection occurs34. Other studies show otherwise, since persistent asymptomatic P. vivax and P. falciparum infections, with low-density parasitaemias, produces higher density infections at a later time, with the subsequent clinical outcomes35. Other authors have indicated that the evidence available in this topic does not show beneficial to the individual, even, this should be named as chronic malaria infections, which is a construct with major scientific, operational, and ethical challenges36. It is crucial to detect and treat submicroscopic malaria to control endemicity, disrupt transmission, and to target all malaria infections, irrespective of their density or presentation37.Also, it is essential to emphasize the high frequency of submicroscopic infection , which has been documented in previous studies38.The standard test applied in Colombia's endemic areas is TBS, whose diagnostic ability is low compared to qPCR, which leads to a high proportion of false negatives. Added to this is the high number of pregnant women who live in remote areas and are not detected. For this reason, it is crucial to improve diagnostic efforts, especially considering that in Colombia, the high cost-effectiveness of some strategies for GM diagnosis has been demonstrated39, without association with gravidity18; while it differs from other that have reported the association with having a higher number of pregnancies; although in this last study the association was detected in a bivariate analysis, without adjusting for possible effect modifying variables in a multivariate analysis12. At this point, it is essential to clarify that the explanatory factors found in this research are difficult to contrast with the available literature for several reasons:21.Most studies focus on parasitological or diagnostic variables, without obstetric factors39.The most of observational studies in GM has been done in Africa with other independent variables  and where the obstetric factors are not comparable with the current study population40.The majority of studies investigating factors associated with GM take the frequency of malaria as independent variables for different obstetric outcomes, which means that the GM effects and not its triggers are studied41.The studies on risk factors generally compare what happens in pregnant women versus non-pregnant women, which is different from the design and population of the current studyThe multivariable analysis identified the following explanatory factors of GM: healthcare coverage, trimester of the pregnancy, number of deliveries, and previous diagnosis of malaria in the current pregnancy. These findings coincide with other studies that have identified as the main risk factors for GM the weeks of gestation and previous diagnosis of malariaDespite the limited comparison level of the explanatory factors of GM in this research with the previous literature, this type of multivariable analysis shows the importance of identifying central subgroups for targeting disease prevention, care and healing actions.9 and with the few previous reports from Colombia12. However, it is essential to clarify that no association was found with other events related to GM, such as abortion, fetal death, and stillbirth. Abortions presented a high frequency in pregnant women with GM and those without the infection, which indirectly shows the need to investigate other causes of this problem. The presence of fetal death was higher in pregnant women with GM; however, this difference was not statistically significant. Considering that the statistical power in this comparison was 80%, it can be stated that in the South of Córdoba-Colombia, GM is not related to this type of mortality, exposing to the need to investigate other causes. In pregnant women with GM, a higher frequency of stillbirth was also found, without registering a statistically significant difference, but in this case, the statistical power of the comparison was 54%, so it is not possible to rule out malaria as a cause of this problem in the study group.The frequency of GM was related to a higher frequency of anemia, infection in newborns, and lower birth weight, which is consistent with the available evidence in this field42.Added to the above, there are other problems for the control of GM, analyzed in other investigations, such as the nonspecific clinical presentation, insufficient facilities for an efficient diagnosis, lack of trained personnel, absence of clear regulations for mass implementation of diagnostic tests, and low allocation of financial resources for diagnosis and control programsAmong the limitations of this study, some are attributable to the type of study, such as the exploratory nature of the associations . Others are related to the information sources used by the Colombian health system that are not exhaustive for all factors associated with GM, and structural problems in the study area where poverty, armed conflict, and other social phenomena prevented an approach with greater external validity. A favorable aspect to highlight of this work is having gathered all the investigations carried out by this research group in 12 years, which allowed us to obtain 825 records, which means a large sample, all of them coming from prospective investigations.A high frequency of GM, especially submicroscopic, was found. GM increased the frequency of gestational anemia and infection in neonates and reduced birth weight. The results clearly, precisely, and directly indicate that malaria control in this northwestern region of Colombia is far from adequate, which is even more serious if it is considered that those affected by GM are both the mother and the neonate.


Plasmodium falciparum protein HRP-2 are being developed with increasingly lower limits of detection. However, it is currently unclear how parasite stages that are unaffected by standard drug treatments may contribute to HRP-2 detectability and potentially confound RDT results even after clearance of blood stage infection. This study assessed the detectability of HRP-2 in periods of post-treatment residual gametocytaemia.In some settings, sensitive field diagnostic tools may be needed to achieve elimination of falciparum malaria. To this end, rapid diagnostic tests (RDTs) based on the detection of the P.falciparum infected, gametocyte positive individuals were treated with or without the gametocytocidal drug primaquine (PQ), alongside standard artemisinin-based combination therapy (ACT), in the context of a randomised clinical trial in Ouelessebougou, Mali. A quantitative ELISA was used to measure levels of HRP-2, and compared time to test negativity using a standard and ultra-sensitive RDT (uRDT) between residual gametocyte positive and negative groups.A cohort of 100 Time to test negativity was longest by uRDT, followed by ELISA and then standard RDT. No significant difference in time to negativity was found between the treatment groups with and without residual gametocytes: uRDT , RDT  or ELISA . Similarly, no difference was observed when adjusting for baseline asexual parasite density. Quantified levels of HRP-2 over time were similar between groups, with differences attributable to asexual parasite densities. Furthermore, no difference in levels of HRP-2 was found between individuals who were or were not infectious to mosquitoes .Surviving sexual stage parasites after standard ACT treatment do not contribute to the persistence of HRP-2 antigenaemia, and appear to have little impact on RDT results.The online version contains supplementary material available at 10.1186/s12936-022-04387-0. Historically, diagnosis of malaria infection has been dependent on a visual detection of blood stage parasites by blood film microscopy. However, the 2011 Malaria Eradication Research Agenda  process concluded that novel and improved tools are needed to detect and target transmission at its lowest levels for use in elimination contexts. This conclusion stemmed from the growing body of evidence that submicroscopic parasite densities were capable of maintaining a viable reservoir of infection in humans , p = 0.077), nor was any significant difference observed when adjusting for baseline gametocyte density and day of study visit   and mosquito infectious (red) individuals. Table S1. Cox proportional hazards ratio by test, between infectious and non infectious individuals. Reported with and without adjustment for baseline gametocyte density.


Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue.A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist.Plasmodium falciparum samples , and in 92% of Plasmodium vivax samples . Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides.In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9–92.7), and specificity 75.6% (95% CI 73.1–78.0). With good quality slides sensitivity was similar , but specificity increased to 85.1% (95%CI 82.6–87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200–200,000 parasite/µL. Species were identified accurately in 93% of The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations.Trial registration ClinicalTrials.gov number NCT03512678.The online version contains supplementary material available at 10.1186/s12936-022-04146-1. Plasmodium falciparum from all other plasmodial species, and are challenged by parasite lineages with deletions in genes coding for antigens detected by RDTs. The information on parasite density and species is required for clinical management of patients and is particularly important in the context of research studies on malaria prevention, diagnostics and treatment . HoweveUpon completion of the image scanning and analysis, an automatically-generated report indicates the diagnosis , WBC count, and the species and parasite density if the slide diagnosis is positive. The report also displays a mosaic of suspected parasite thumbnails, and clicking on a thumbnail brings up the relevant field-of-view to allow easy examination by a clinician. A screenshot of a typical report, with a selected field-of-view, is shown in Additional file During the study, an earlier algorithm version (1.11.6) was run, but it can be viewed as a placeholder. At the conclusion of the study, the saved image sets were re-analysed in the field with an updated version . For bohttps://www.wwarn.org/obare-method-calculator) was used to identify discrepancies in the microscopy results requiring tie-breaker reads and to determine the consensus result [All sites used a standardized laboratory manual and standard operating procedures (SOPs) for malaria microscopy. Blood slide reading was performed independently by two qualified microscopists, who were blinded to individual microscopy results. The Obare Method Calculator , assuming that up to 10% of subjects would be excluded due to missing or incomplete data . This saP. falciparum, 327 (33.7%) with P. vivax, 8 (0.8%) with mixed P. falciparum and P. vivax infections and the remaining 10 (1.0%) with other mixed or non P. falciparum/P. vivax infections. The results presented here were obtained using the most recent version of the algorithm available at the time the analyses were performed (version1.15.17b).In total, 2,250 slides were evaluated, of which 969 (43.1%) tested positive by light microscopy , 96.5% , and 100% , respectively  and specificity was 75.6% (95% CI 73.1–78.0)  of P. vivax samples . For P. falciparum and P. vivax species identification, kappa coefficients were 0.76 (95% CI 0.69–0.83) and 0.73 (95% CI 0.66–0.80); Table Among those positive samples that were labelled as positive, parasite species was identified accurately in 93% (499/537) of Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% (196/845) of slides. A Bland–Altman plot for assessing agreement of parasite density estimations between microscopy and EasyScan Go, revealed a wide interval agreement ranging from − 1.098 to 1.021 in a logarithmic scale, corresponding to 0.08 to 10.50 in original scale with a mean difference of − 0.038  .P. falciparum species identification, kappa values remained similar (0.77 in first tier vs 0.74 in second tier), but for P. vivax species identification, kappa values were lower in the first tier sites (0.68 vs. 0.79). Quantitations were more accurate in first tier sites: 27% (141/521) of the parasite counts derived from the EasyScan Go were within ± 25% of the microscopic reference counts vs. 17% for second tier sites, a 10% difference . Figure There were 1478 slides from first tier sites  and 705 slides from second tier sites . Diagnostic sensitivity of the EasyScan Go was slightly lower on the first tier vs second tier sites, at 89.1% vs. 94.2% (5.1% difference (95% CI 2.6–7.4%), P = 0.0001), whereas specificity was much higher, at 85.1% vs 51.5% (33.6% difference (95% CI 29.4–37.7%), P < 0.0001). For This large field evaluation, conducted in varied geographical and epidemiological settings, represents the real-life performance of a fully automated digital microscope with thick-film-only malaria detection algorithms. Because none of the slides from the current evaluation were used in the training of the algorithms, the results given are true hold-out performance. An important aspect of this study was the inter-site slide preparation variability. Some sites (“first tier sites”) had overall higher quality slides than others (“second tier sites”) as assessed during EQC, enabling assessment of the effects of slide quality on algorithm performance.P. falciparum or P. vivax,and vice versa), and 27% of quantitations within 25% error using expert microscopy as reference. Though this does not correspond to the criteria for expert microscopy, it represents reasonable performance, potentially useful in clinical settings.On the 1478 slides from first tier sites, the device had overall parasite detection sensitivity 89%, specificity 85%, species identification accuracy 84% on positive-labelled slides  based on assumptions about the expected numbers of parasite-like artefacts. If a slide presents many more parasite-like artefacts than expected, the noise floor is exceeded and an incorrect Positive slide disposition results .P. vivax samples was that second tier sites had a higher kappa value than first tier sites (0.79 vs. 0.68) due to fewer vivax samples being misclassified as falciparum. This might have been due indirectly to higher levels of late-stage-like artefacts in second tier sites—the algorithm determines species of a slide based largely on the ratio of ring-stage to late-stage suspected parasites, since late stages are so rare in falciparum samples. A few extra late-stage suspects caused by artefacts could push a sample to the vivax label, despite possibly high numbers of ring-stage suspects, increasing the odds that a low-quality vivax sample would be labelled vivax.A somewhat strange finding pertaining to species identification of This field evaluation demonstrated a parasite detection sensitivity of 89% and species identification accuracy of 84% on slides with acceptable quality when compared to expert microscopy. This corresponds to Level 2 criteria (80–90% for both sensitivity and species identification) as defined in the WHO-TDR Research Malaria Microscopy manual . For comThe differential performance of the digital device depending on slide quality highlights the need for high quality slide preparation when performing automated microscopy, which was not achieved in roughly 30% of the slides from the sites, despite procedures and adequate training conducted by research groups. Considerable variations were observed in staining and blood film preparation across study sites. Expert microscopists were able to accurately process the “poor quality” slides in this study, consistent with findings in . Thus, tStandardization of blood film staining and preparation by means of automated staining might improve consistency in slide quality and thus aid automated systems. The automated staining technique with Giemsa stain, i.e. use of auto-spreading and auto-staining slides is challenging, expensive and not widely used. The EasyScan GO algorithms have a module that detects and flags potentially poor quality blood films. Such feedback to field clinicians might improve the device’s diagnostic accuracy by helping clinics address preparation issues affecting the device.Parasight using fluorescent stain, have advanced to commercial platforms recently [Automated malaria microscopy platforms based on computer vision and machine-learning have been in development for over the past two decades , 28. Somrecently . The EasP. vivax, P. ovale and P. malariae) since it processed only thick films. A thin film module has since been added to the EasyScan GO [This study had several limitations. The algorithm does not perform parasite staging, nor does it distinguish sexual from asexual stages. Although the software tested in this study readily distinguishes falciparum from non-falciparum species, it does not differentiate between the non-falciparum species  programmes (7). This could be achieved through supplementing the 1st microscopy reading by the EasyScan Go 2nd reading, as well as cross-checking of blood slides in the context of a QC procedure in therapeutic efficacy studies. The QC for malaria microscopy is a tedious process that should be performed independently to verify microscopy results and identify systematic errors in anti-malarial drug efficacy assessments. Despite the importance of quality assurance of slide readings in the context of malaria research and routine surveillance, there are vast differences in operating procedures, including identifying and resolving discrepant results , 31. TheThe digital malaria microscopy device EasyScan Go has the potential to facilitate cross-checking of blood slides as part of quality assurance. Further software improvement is required to improve parasite density estimations and sensitivity at low parasite densities. High quality of smears and staining is paramount to allow machine-learning-based image analysis to perform adequately. The aforementioned technological barriers need to be overcome prior to implementing the EasyScan Go as a diagnostic tool in malaria research settings.Additional file 1. STARD-2015-Checklist.Additional file 2. Parasite Density Estimation.Additional file 3. EasyScan Go.Additional file 4. Thick Film EasyScan Go Output.Additional file 5. Study Enrolment.Additional file 6. Site Specific Results.


E  is the first malaria vaccine to reach Phase IV assessments. The World Health Organization-commissioned Malaria Vaccine Implementation Programme (MVIP) is coordinating the delivery of RTS,S/AS01E through routine national immunization programmes in areas of 3 countries in sub-Saharan Africa. The first doses were given in the participating MVIP areas in Malawi on 23 April, Ghana on 30 April, and Kenya on 13 September 2019. The countries participating in the MVIP have little or no baseline incidence data on rare diseases, some of which may be associated with immunization, a deficit that could compromise the interpretation of possible adverse events reported following the introduction of a new vaccine in the paediatric population. Further, effects of vaccination on malaria transmission, existing malaria control strategies, and possible vaccine-mediated selective pressure on Plasmodium falciparum variants, could also impact long-term malaria control. To address this data gap and as part of its post-approval commitments, GSK has developed a post-approval plan comprising of 4 complementary Phase IV studies that will evaluate safety, effectiveness and impact of RTS,S/AS01E through active participant follow-up in the context of its real-life implementation.Following a 30-year development process, RTS,S/AS01P. falciparum genetic diversity in the periods before and after vaccine implementation.EPI-MAL-002 (NCT02374450) is a pre-implementation safety surveillance study that is establishing the background incidence rates of protocol-defined adverse events of special interest. EPI-MAL-003 (NCT03855995) is an identically designed post-implementation safety and vaccine impact study. EPI-MAL-005 (NCT02251704) is a cross-sectional pre- and post-implementation study to measure malaria transmission intensity and monitor the use of other malaria control interventions in the study areas, and EPI-MAL-010 (EUPAS42948) will evaluate the E vaccine safety, effectiveness and impact. The studies are currently being conducted in the MVIP areas. Their implementation has provided opportunities and posed challenges linked to conducting large studies in regions where healthcare infrastructure is limited. The results from these studies will support ongoing evaluation of RTS,S/AS01E’s benefit-risk and inform decision-making for its potential wider implementation across sub-Saharan Africa.GSK’s post-approval plan has been designed to address important knowledge gaps in RTS,S/AS01 E (GlaxoSmithKline (GSK), Belgium) is a pre-erythrocytic Plasmodium falciparum malaria vaccine developed for routine immunization of young children living in malaria-endemic countries. In the pivotal Phase III trial, 4 doses of RTS,S/AS01E administered to children aged 5 months or older reduced clinical malaria by 39% and severe malaria by 29% over 4 years of follow-up [E was associated with a reduction in overall hospitalizations, and hospitalizations due to malaria, severe anaemia and the need for blood transfusion [E was generally well tolerated and although more reactogenic than control vaccines, local and systemic symptoms were generally transient and mild-to-moderate in intensity [E recipients versus controls after vaccination in children aged 5 months or older, with the risk mainly during the first 3 days after vaccination [E-vaccinated children than in children vaccinated with control vaccines [E compared to girls vaccinated with control vaccines, without differences in risk factors, time to death or causes of death that could explain the results. No such difference was observed in boys. Detailed case evaluation indicated that these imbalances were likely to be chance findings due to unexpectedly low rates of meningitis in the control group , or a low mortality rate in girls in the control group, or the lack of biological plausibility to explain the causal relationship to RTS,S/AS01E vaccination [RTS,S/AS01E received a positive scientific opinion from the European Medicines Agency [E impact and safety remained. The WHO, therefore, adopted the recommendations of the Strategic Advisory Group of Experts on Immunization and the Malaria Policy Advisory Committee who jointly endorsed pilot implementation of the vaccine in 3–5 settings in sub-Saharan Africa [In 2015, RTS,S/AS01n Africa . Under tn Africa .E would be first introduced in selected areas in Ghana, Kenya and Malawi by the respective routine national immunization programmes in the framework of the Malaria Vaccine Implementation Programme (MVIP). Authorization for use of RTS,S/AS01E in this context was granted on 24 April 2018 by the Ghana Food and Drug Board, 11 May 2018 by the Kenya Pharmacy and Poisons Board, and 16 May 2018 by the Malawi Pharmacy and Medicines Regulatory Authority. Vaccination started on 23 April 2019 in Malawi, 30 April 2019 in Ghana and 13 September 2019 in Kenya [E is the first vaccine to be implemented as a complementary tool to existing interventions under the Global Technical Strategy for Malaria, 2016–2030 [E should be used for the prevention of P. falciparum malaria in children living in regions with moderate to high malaria transmission as part of a comprehensive malaria control strategy [In April 2017, the WHO announced that RTS,S/AS01E. Key aspects of the MVIP have been summarized by the WHO in an on-line series of question and answers, in a summary of key milestones in the journey to vaccine implementation, and in the 2021 SAGE report of the RTS,S/AS01E vaccine [E vaccine doses necessary to the MVIP (up to 10 million doses) [E introduction, the MVIP evaluates the vaccine safety, impact, and effectiveness in order to generate information necessary to inform potential future policy for the deployment of RTS,S/AS01E on a broader scale. A first step is the WHO-commissioned Malaria Vaccine Pilot Evaluation (MVPE). This consists of household surveys, and sentinel hospital and community mortality surveillance, building on routine systems. The MVPE will measure the programmatic feasibility of delivering a 4-dose vaccine schedule, vaccine safety in routine use, and the impact of the malaria vaccine on severe malaria and all-cause mortality. This evaluation is largely based on passive follow-up and comparison of the occurrence of vaccine safety and impact study endpoints between vaccine implementation areas (exposed clusters) and areas where the vaccine is not yet implemented (unexposed clusters) [E vaccine safety, effectiveness, impact, and the potential effect of vaccination on the genetic diversity of circulating parasite strains. This paper presents an overview of GSK’s post-approval plan currently being conducted in the MVIP areas [E benefit-risk profile, and to informing decisions for its potential wider implementation across malaria endemic areas of Africa.The MVIP is a collaboration between selected countries and international private and public partners established by the WHO to coordinate, support and evaluate the introduction of RTS,S/AS01 vaccine –12. GSK n doses) . In addilusters) . Second,IP areas . The chaE, GSK’s post-approval plan is designed to assess vaccine safety, impact, and effectiveness in a real-life setting. It comprises 4 GSK-sponsored Phase IV studies , and the post-vaccine introduction study, EPI-MAL-003 (NCT03855995), are compared. For operational reasons, the before-after comparison is being conducted in Ghana and Kenya only. In addition, because the Ministries of Health of the implementing countries are not introducing RTS,S/AS01E into all national areas, vaccine safety and impact will also be assessed using a contemporaneous comparison between exposed and unexposed areas of the study endpoints. Moreover, since annual and/or geographical variations in malaria incidence may occur as a result of changes in malaria transmission intensity or in malaria control intervention coverage, these potential confounders are monitored in the EPI-MAL-005 study (NCT02251704).Many low and lower-middle income countries where malaria is endemic have little or no data on background incidence rates of rare diseases such as those that may be reported as adverse events following immunization. This may prevent robust post-authorization vaccine safety and effectiveness monitoring, and can lead to delays in detecting safety signals, potentially contributing to vaccine hesitancy related to the new vaccine, or to other vaccines introduced in the future. In these settings, disease surveillance studies conducted prior to vaccine introduction can be used to determine reliable background rates of specific diseases/events that can be compared with post-introduction observations. In order to further monitor the benefit-risk profile of RTS,S/AS01P. falciparum parasite prevalence (as a proxy for transmission intensity) and malaria control intervention coverage over 10 annual surveys in the EPI-MAL-002 and EPI-MAL-003 study areas. Results from EPI-MAL-005 will be used to adjust the temporal and contemporaneous comparison analyses for potential year-to-year variation during the conduct of EPI-MAL-002 and EPI-MAL-003. Finally, because P. falciparum has evolved multiple mechanisms to vary cell surface antigens and evade the host’s immune response, an ancillary study to EPI-MAL-005, EPI-MAL-010 (EUPAS42948), has been designed to monitor parasite genetic diversity before and after vaccine implementation.EPI-MAL-005 is an observational cross-sectional study assessing E vaccine introduction. To assess vaccine safety, effectiveness and impact, these baseline incidence rates will be compared with rates documented in the post-implementation study EPI-MAL-003 which commenced when RTS,S/AS01E vaccination was introduced by the Ministries of Health  but not others (unexposed clusters). Thus, in addition to the before-after comparison, EPI-MAL-003 also includes a contemporaneous comparison of endpoints of interest between vaccinated and unvaccinated study participants.Study EPI-MAL-002 is designed to collect incidence data of pre-defined health outcomes, i.e., adverse events of special interest (AESI). These include rare events potentially associated with vaccination Table , meningiGSK’s Phase IV study package is fully embedded in the MVIP. Therefore, selection of participating clusters depended on the cluster identification process led by the Ministries of Health according to the WHO guidance . EPI-MALHaemophilus influenzae type b vaccine, through direct invitation or when hospitalized before routine immunization. Similarly, EPI-MAL-003 is enrolling children presenting for routine immunization, regardless of their future vaccination status with RTS,S/AS01E.To allow direct comparisons of endpoints, EPI-MAL-002 and EPI-MAL-003 are strictly identical in terms of design and conduct. Both are multi-country observational studies with prospective cohort event monitoring. Children < 18 months of age are enrolled during routine immunization with the pentavalent diphtheria-tetanus-pertussis-hepatitis B and E in children in the exposed clusters in EPI-MAL-003) consists of active surveillance for outpatient and inpatient visits by each enrolled participant and of 10 home visits conducted according to a specific time frame (prospective cohort). In addition, hospital-based disease surveillance is organized across the entire study area for infants and children who are not enrolled in the prospective cohort. In other words, throughout the whole study period, all hospitalized children under the age of 5 years who are not already enrolled in the prospective cohort and who live within the study areas are eligible for enrolment in the hospital-based disease surveillance.In both studies, follow-up until approximately 5 years of age  [E will compare malaria-related events in vaccinated and unvaccinated children from exposed clusters enrolled in active surveillance. Vaccine impact  will be investigated by comparing the incidence of malaria-related events in unvaccinated children (either from EPI-MAL-002 or from EPI- MAL-003 unexposed clusters) with the incidence of events in children from EPI-MAL-003 exposed clusters .The study objectives are to estimate the incidence of protocol-defined events, including AESI, meningitis, other adverse events leading to hospitalization, death, and malaria . AESI Table  are evenrtality) , 3. EstiE vaccine introduction.The studies are conducted in the setting of routine medical practice and local laboratory testing (first-line testing), and include a strong support component comprised of study-specific trainings, telemedicine support, second-line laboratory testing, and consultation with an external expert panel for final case classification. These tools are likely to enhance case detection and ascertainment rates. Both EPI-MAL-002 and EPI-MAL-003 use identical surveillance in order to allow a robust comparison of study outcomes before and after the RTS,S/AS01Freely given and written or witnessed and thumb-printed informed consent is obtained from each study participant’s parent/legal representative prior to study participation. Protocol-specified procedures include the collection of demographic data, vaccination records, medical history, medical care episodes, adverse events, use of malaria control measures, development delays or death, and a physical examination when contacts between study staff and subjects occur. The protocol requires collection of a sample of 5 ml of blood for testing at an external laboratory (second-line laboratory) in all suspected cases of AESI and meningitis. All study participants are treated according to good medical and routine practices . In caseIncidence rates of safety endpoints will be calculated and compared based on both before-after and between cluster comparisons using a univariable and multivariable Poisson regression model adjusted for specific covariates, including if applicable, those identified in EPI-MAL-005. The vaccination status of participants will be confirmed using data from individual vaccination cards, vaccination registers and health and demographic surveillance system (HDSS) or equivalent surveillance system. Incidence rates will be computed using person-time denominators for group of interest. Each child will contribute person-time until study end or 5 years of age, whichever occurs first.In addition, the potential risk of meningitis is being monitored in near real time using the maximized sequential probability ratio test (MaxSPRT) method . MaxSPRTE on the incidence of malaria will be estimated [Vaccine effectiveness (direct effect) and impact  of RTS,S/AS01stimated . Cases oE vaccination [EPI-MAL-002 started in January 2016 and an interim analysis has been conducted to provide preliminary results from 14,329 children who participated in the prospective cohort monitoring prior to the introduction of RTS,S/AS01cination . EPI-MALE will lead to a reduction in the incidence of malaria disease in vaccinated subjects in EPI-MAL-003 compared to baseline rates recorded in EPI-MAL-002. However, annual fluctuations in malaria incidence occur due to changes in transmission intensity influenced by rainfall patterns or changes in how malaria control interventions are used. By monitoring malaria transmission intensity and the coverage of malaria control interventions in the EPI-MAL-002 and EPI-MAL-003 study sites during the conduct of these studies, EPI-MAL-005 will allow a more accurate estimate of the true impact of RTS,S/AS01E vaccination. This study will contribute to the analysis of vaccine effectiveness and vaccine impact by identifying variables to be used as covariates to adjust the before-after and between cluster comparisons.It is expected that the use of RTS,S/AS01EPI-MAL-005 has a cross-sectional design with yearly surveys coinciding with the recruitment and follow-up periods of the EPI-MAL-002 and EPI-MAL-003 studies. At each survey, 600 participants aged 6 months to < 10 years are randomly selected from the sites participating in the survey in that year, stratified by age group. The selection process will be repeated every year meaning that the subjects will be different in each cross-sectional survey except if they are re-selected in a subsequent survey by chance. The surveys occur during the period of peak malaria transmission that varies from end September to mid-December in western African sites, from end April to mid-August in eastern and southern African sites , 20.P. falciparum parasite prevalence , and of the use of malaria control interventions .The co-primary study objectives are estimation of At each survey, demographic information, medical and vaccination history, information on healthcare-seeking behaviors in the previous 14 days, fever in the last 24 h, and use of malaria control measures , are recorded for all participants. During the survey visit, axillary body temperature is measured, and a capillary blood sample is collected for malaria testing by microscopy and nucleic acid amplification tests . In the event of fever at the time of the visit, or fever or other symptoms/signs of clinical malaria reported in the previous 24 h, a rapid diagnostic test is conducted, and the participant treated if the test is positive. Participants identified as being parasite-positive following microscopy are treated according to national guidelines.E vaccination status and gender. Annual fluctuations in parasite prevalence are estimated using the Cochran-Armitage trend test. The agreement between parasitaemia as measured by microscopy versus nucleic acid amplification tests is assessed using the Cohen’s Kappa coefficient and the Landis and Koch scale. A risk factor analysis for malaria infection is conducted using a multivariable logistic regression analysis.Parasite prevalence and use of malaria control measures are computed by study site, age group, RTS,S/AS01The study started in October 2014 and the first two surveys have been published . At the P. falciparum is a pathogen with high variability and a high number of different circulating haplotypes. The parasite uses numerous mechanisms to vary cell surface antigens and evade the host immune response. For this reason, there is a potential concern that widespread implementation of RTS,S/AS01E could drive the selection of specific parasite variants or alter the number of parasite haplotypes over time by exerting selective pressure. EPI-MAL-010 is an ancillary study to EPI-MAL-005 that will monitor the genetic diversity of circumsporozoite protein sequences in the P. falciparum parasite population before and after vaccine implementation.Although the central NANP amino acid repeat sequence of the circumsporozoite protein used as a major component of the RTS,S vaccine antigen is normally well conserved across parasite strains, P. falciparum parasitaemia confirmed by microscopy and/or nucleic acid amplification tests, and collected before and after RTS,S/AS01E implementation in two study sites: Kintampo (Ghana) in Western Africa and Kombewa (Kenya) in Eastern Africa.EPI-MAL-010 has a longitudinal, cross-sectional study design and uses capillary blood samples collected from participants enrolled in EPI-MAL-005 over 7 survey years. Samples are from participants aged 6 months to < 5 years with P. falciparum haplotype prevalence  and frequency  in participants aged 6 months to < 5 years vaccinated or not with RTS,S/AS01E.This study is estimating P. falciparum by microscopy and/or nucleic acid amplification tests. Trends in the prevalence of specific P. falciparum haplotypes with a frequency of at least 5% will be assessed by a logistic regression model. Multinomial logistic regression will be used to describe the annual fluctuations in haplotype frequency using the 3D7 haplotype as the reference group.Amplicon sequencing is conducted on samples tested positive for Considering the specificities of the study setting, GSK together with its local scientific partners, conducted comprehensive study feasibility assessments in which both scientific and operational aspects were carefully balanced to allow generation of robust data. During the planning phase, global experts provided advice on study design and execution. Evolving circumstances, external constraints and the involvement of multiple stakeholders required adaptability and flexibility, balancing an optimal study design with real-world constraints. Designing, setting-up and conducting a complex post-approval plan in sub-Saharan Africa brings some important setting-specific considerations: (1) data collection needs to be performed using prospective follow-up because existing data collection systems and databases may be sub-optimal or absent; (2) vaccine safety, effectiveness and impact data have to be collected in a healthcare environment where case detection and ascertainment may be challenging because of limited healthcare infrastructure and diagnostic capability; (3) there may be limited access to health care in remote settings; (4) background incidence rates of many diseases including study-specific endpoints are limited or not available; (5) standard laboratory procedures may not exist across all study sites; and (6) sample storage and transportation from remote locations can be challenging.E, and have developed capability in terms of training, experience and quality of healthcare that resulted from previous study participation. However, for some sites without such past experience, additional investment was required to set up baseline structures and procedures; (2) the existence of a HDSS, part of the International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH), or of an equivalent surveillance system. HDSS sites have a demographic database in place that updates, on a regular basis, the number of births, deaths, immigrations and emigrations, and potentially vaccinations and population health outcomes. The number of HDSS sites in Ghana, Kenya and Malawi being limited, a population census had to be fully established or partially enhanced in approximately half of the study sites.More information on key parameters that were considered in the framework of the study feasibility assessment is provided below. Study site selection was based on specific criteria: (1) the existence of pre-existing scientific research infra-structure capable of expanding beyond routine data collection. Several of the finally selected study sites have research experience in conducting clinical trials with RTS,S/AS01The EPI-MAL-002 and EPI-MAL-003 studies are mainly based on data collection in the framework of routine medical practice, which may hamper the ascertainment of diseases requiring more advanced diagnostic tools. A full understanding of the structure and capacity of the healthcare system in each country where the studies were planned was required, including the assessment of the capacities for case detection and the ascertainment of study endpoints in each study site. As an outcome of this assessment, specific tools were put in place during the study preparation and conduct to enhance case detection and diagnostic capabilities. These include regular and ongoing medical and pharmacovigilance trainings, telemedicine support , the distribution of job aids, the support of local laboratories and the set-up of a central reference laboratory  for blood and CSF testing. These enhancement tools should increase the likelihood of reaching the highest possible level of diagnostic certainty. For the key endpoint of meningitis, medical and non-medical staffs have received training on meningitis case detection and ascertainment, which includes information on the national guidelines for case management (including lumbar puncture and testing of CSF) with secondary testing to be performed at a central laboratory when sample volume permits.This multi-country initiative involves key local and global public health partners. The scientific and operational constraints and the complexity of the study set-up provide opportunities for collaborations and alignment in healthcare approaches between countries to promote best practice. However, turnover of trained personal in the study areas is likely to be an ongoing challenge.Within countries, the MVIP, and more specifically the study design, set-up and conduct, promote collaboration between different authorities, such as routine healthcare system, diagnostic services, national immunization services, epidemiological research, and National Malaria Control Programmes.E vaccination has been observed during the pandemic thus far, although it might be expected that hospitalization practices may change in order to limit admissions to children with serious or critical conditions and avoid hospital crowding. However, the potential impact of COVID-19 on the evaluation of AESI as determined in pre-vaccination pre-COVID-19 period requires continuous monitoring of the situation. Sensitivity analyses may be conducted considering COVID-19 pandemic periods for both studies.Despite best laid plans, the coronavirus disease 2019 (COVID-19) pandemic in Africa continues to unfold and its impact is evolving. In this environment, the MVIP is continuing and measures are being taken to protect the welfare and safety of participants and study staff, and to ensure data integrity . No majoE in a real-life setting is a unique and complex undertaking that requires the establishment of large-scale and strong partnerships. Assessing the benefit-risk profile of a 3-dose primary schedule vaccine with a 4th dose booster administered beyond the usual Expanded Programme on Immunization schedule in low and lower-middle income countries requires the establishment of ad-hoc tools and quality control methods to allow collection of robust and reliable data. Whilst technical and operational expertise is key to achieve this goal, human and financial resource needs should not be underestimated. The effort has been based on the WHO-led MVIP, existing research platforms and expertise in the implementing countries, and significant commitment by GSK. The experience with RTS,S/AS01E in sub-Saharan Africa highlights important aspects to be considered when planning and implementing a vaccine post-approval plan in low and lower-middle income countries. The RTS,S/AS01E experience will pave the way for the development and implementation of new generation malaria vaccines, and of other vaccines for the developing world.The implementation and the safety, effectiveness and impact evaluation of RTS,S/AS01


Malnutrition among older age people is becoming significantly higher in spite of improvements in the health care system. Life expectancy of Ethiopian elders is increasing; but reliable and valid tools for screening and diagnosis of malnutrition in this subgroup are limited. This study aimed to assess the validity of anthropometric measurements: Mid Upper Arm Circumference (MUAC), Body Mass Index (BMI), and Calf Circumference (CC) in detecting malnutrition status of older age people in Ethiopia.A community based cross-sectional study was conducted in Borena District from January to March, 2020. A total of 421 participants aged were systematically included in the study. To test reliability and validity of the measurements,Cronbach’s α coefficient and Pearson’s correlations were used, respectively. The full Mini-Nutritional Assessment (MNA) tool was used to diagnosis malnutrition. Overall accuracy, sensitivity and specificity of BMI, MUAC and CC were estimated using Receiver Operating Characteristic curves. The Youden Index was used to determine the best cut-off point.r = 0.56, p < 0.01); MNA, MUAC revealed. The area under the curve (AUC) of BMI, MUAC and CC were found: 0.98, 0.94 and 0.96 indicating the overall accuracy respectively. The sensitivity and specificity of BMI, MUAC and CC using established cut off points were found: 90%, 96%; 78%, 94% and 84%, 95% respectively. However, using the Youden index the best cut-off point, the sensitivity and specificity of MUAC and CC were 88%, 86%; 92% and 89% respectively and adjusted for age and sex.The reliability of BMI, MUAC and CC by Cronbach’s alpha was found 0.847. Significant positive correlations between MNA, BMI(The current study demonstrated that BMI was a reliable and valid method to identify the malnutrition status of older age people. A MUAC value of 19 cm and CC of 30 cm were simple and efficient cut-off points for the determination of malnutrition in the older age people. A future study is needed to validate the validity of BMI, MUAC and CC against biochemical tests as gold standard.The online version contains supplementary material available at 10.1186/s12877-022-03467-9. As medical advances have increased the availability and access to better preventive care, people have been living longer, and ageing has imposed a global public health challenge , 2. AccoIncreasing numbers of older age people in the global community require reconsideration of the suitability of health infrastructures . This poTo address the health problems among older people, provision of improved healthcare, both in hospital and in the community, from early detection to preventive care, is imperative . The MinCompared to the use of MNA, the assessment of nutritional status by a single or multiple anthropometry measurement is simple, non-invasive and time saving in health facilities and as part of community-based screenings of malnutrition. Anthropometry also remains the most practical tool for the assessment of nutritional status among members of the community in developing countries where scarced resources and high patient load exist, in spite of some limitations, such as do not provide uniform body composition information and recent nutritional disturbance . MoreoveThis study was conducted with the aim of determining the reliability and validity of anthropometric measurements in identifying malnutrition status among older age people living in Borena District, South Wollo Zone, Ethiopia. This study will help health care providers to assess the malnutrition status of elders using appropriate tools and thus provide appropriate care to improve nutritional status.th January and 5th March 2020. Borena District is 180 km away from the zonal town, Dessie and 580 km away from Addis Ababa, the capital city of Ethiopia. The District administration has 39 Kebeles, s and the population in 2019/20 was estimated to be 186,173. Of these, 3.2%, that is 5958 people, were estimated to be aged 65 years and over [A community-based cross sectional study was conducted among older age people living in Borena District, South Wollo Zone, Ethiopia, between 25edematous and/or severely ill were not included in this study.All people aged 65 and over years old living in Borena District were potential study participants. The final subjects need to have been living in the Kebele that was randomly selected for at least 6 months. Those who were The sample size was determined by using the prevalence of malnutrition among older age individuals in Gondar, Ethiopia which was previously determined by Mesele  to be 21A pre-tested structured interviewer administered questionnaire that comprised of socioeconomic, demographic, environmental, and diet-related measures was used. Anthropometric measures were also taken. Five data collectors were trained to collect the data.Malnutrition was assessed using the full MNA tool which comprised of anthropometric measurements, recent dietary intake, general condition and subjective assessments , 25. A tHeight was measured using portable height stadiometer. Participants were barefoot, legs straight, shoulders relaxed and look straight ahead in the horizontal plane. Participants were also asked to inhale deeply, hold their breath and maintain an erect position prior to taking the measurement. Height measurement was taken to the nearest 0.1 cm. For those people with a kyphosis, thus making it difficult to measure height, the arm span was used as an alternative.The arm span is the distance from the middle finger tip of the left hand to that of right hand when both arms are stretched out horizontally. This length is then converted to a height equivalent . Weight To measure participants’ MUAC, non-stretch adult MUAC tape was used. The left arm was flexed at the elbow to 90 degree, and the midpoint between the lateral acromion and distal olecranon identified and marked. Then the arm was relaxed and the MUAC tape placed around the marked midpoint of the arm. The measurement was recorded to the nearest 0.1 cm. An MUAC of less than 21 cm was considered as malnourished .The CC was measured at the widest point of the calf circumference, between the ankle and knee in a sitting position with the leg bent 90° at the knee to the nearest 0.1 cm using non-stretchable tape. The tape was manipulated to maintain close contact with the skin without compressiing the underlying tissues. A CC less than 31 cm was considered to be malnourished .A questionnaire was prepared in English and translated into Amharic for the field work and then back to English for checking the language consistency. The interviewers and supervisors were trained in data collection, interviewing and measurement techniques for two days. A pre-test (from 5% of older age people from non-seleced Kebeles) was undertaken prior to undertaking the data collection work to assess the consistence of responses, technical measurement error, precision and accuracy of anthropometric measurements. A digital scale was used to reduce frequent calibration and was standardized with a constant weight. The collected information was reviewed on a daily basis and possible errors were returned to the data collectors for correction.The data were entered into Epi-Data version 3.1, and after cleaning and coding, imported to STATA version 15  for analysis. MNA was computed from the 18- items and used as a reference to compare the reliability and validity of BMI, MUAC and CC. The data wre explored for missing values and outliers before categoring variables. Characteristics of study subjects were expressed using frequency and percentage; and differences in distribution were demonstrated for categorical variables using Pearson’s chi-square test.The reliability of the BMI, MUAC and CC was calculated using coefficient of Cronbach’s α and a Cronbach’s α value of 0.60, 0.70 & 0.80 were considered acceptable, adequate and good respectively . CriteriSensitivity and specificity were calculated using (ROC) curves. The Youden Index (Sensitivity + specificity—1) was used to conclude the best cut-off point of BMI, MUAC and CC .Ethical approval was obtained from the Research Ethics Review Committee of the College of Health Sciences and Medicine, Wollo University with reference number WU-CMHS-3412/08/2019. All methods were carried out in accordance with the Declaration of Helsinki and regulations. Informed written consent to participate was obtained from each selected participant confirmed by their signinature for those who are literate and by their finger stamp for illiterate participants. Participants were advised that they were free to withdraw or discontinue participation at any time without any form of prejudice. Privacy and confidentiality of the collected information was maintaned throughout the process. All information gained during the study was kept strictly confidential.p-value < 0.001), while more women has no formal education (88.7% vs 67.5%) and higher proportion of women were widowed than men . The proportion of partners who were able to read and write are higher among women than men .The overall response rate for the study was 96.6%. The characteristics of the sample are presented in Table 2, 69.1% were malnourished  based on the MNA score but no association was observed between sex and malnutrition status by BMI, MUAC and CC.Based on an MNA score of < 17, 71.5% (95% CI 67.2–75.3) of respondents were malnourished and 16.2% (95% CI 11.4–19.7) were at risk of malnutrition. Based on a MUAC of < 21 cm, 58.7% were malnourished, whereas 65.8% were malnourished based on CC < 31 cm. Based on BMI of < 18.5 kg/m. However, deleting one of the BMI, MUAC or CC from the measurement significantly lowers the Cronbach’s α value to 0.783, 0.777 and 0.792 respectively. Anthropometric measurements  and the total MNA were correlated to show the criterion related validity of the tool  ), 0.937  and 0.956  in their place of order.. The overall accuracy of MUAC and CC showed a slight variation in women and men. MUAC has higher accuracy in screening malnutrition in women than men  for each anthropometric measurement BMI, MUAC & CC were illustrated in Fig. men Fig. .Fig. 2ROBased on the established cut-off points using the ROC curve, the sensitivity and specificity of BMI, MUAC and CC in both sexes were found to be 90% , 96% 95% CI, 92.9%-100%); 78%  94%  and 84% 95% CI, 76.2%-91–8%), 95%  respectively. Using the Youden index the best cut of points were 19 cm for MUAC and 30 cm for CC with their sensitivity and specificity of 88%, 86% , 92%  and 89%  in both sexes respectively. Using a MUAC of 19 cm for screening malnutrition among older age s increases the sensitivity from 78 to 88% , whereas and MUAC < 21 cm and CC of < 31 cm had a lower sensitivity, compared to the proposed these new cut-off points (MUAC < 19 cm and CC < 30 cm). In areas where the use of the MNA is impossible, application of BMI, MUAC or CC can address the health demands posed by older people with malnutrition, particularly in resource-limited countries where the cases to service providers ratio is high. This is likely to have large public health and clinical practice implications, especially with respect to nutritional interventions.The current study demonstrated that BMI has good sensitivity and specificity to identify malnutrition status among older people with the established cut-off points (BMI < 18.5 kg/mAdditional file 1.


Plasmodium falciparum parasites with deletions of the histidine-rich protein 2 and/or 3 (pfhrp2/3) genes. Studies have reported pfhrp2/3 gene deletion prevalence in Ethiopia that exceeds the WHO recommended threshold to switch to non-HRP2 targeted RDTs for detection of P. falciparum. Therefore, RDTs that target alternative antigens, such as P. falciparum lactate dehydrogenase (PfLDH) are increasingly in programmatic use.Malaria rapid diagnostic tests (RDTs) have expanded diagnostic service to remote endemic communities in Ethiopia, where 70% of malaria services per annum are reliant on them. However, diagnostic strategies are threatened by  (HRP2/Pv-LDH) and SD-Bioline Pf (HRP2 for Pf/LDH for Pf) RDTs. Dried blood spot (DBS) samples were collected from selected patients for molecular and serological analysis. The clinical data and RDT results were recorded on standard forms, entered into EpiInfo, and analysed using STATA. The Pf-LDH detecting RDT results were compared with real-time PCR and bead-based immunoassay to determine their diagnostic performance.Malaria suspected patients visiting health facilities of Amhara, Tigray, Gambella, and Oromia regions of Ethiopia were screened by community health workers using Carestart Pf/PvP. falciparum RDT positive. A subset of samples (n = 820) were previously tested using P. falciparum lactate dehydrogenase (pfldh) quantitative real-time PCR, and 456 of these further characterized using bead-based immunoassay. The proportion of samples positive for P. falciparum by the PfHRP2 Carestart and SD-Bioline RDTs were 66% (539/820) and 59% (481/820), respectively; 68% (561/820) were positive for the PfLDH band on the SD-Bioline RDT. The sensitivity and specificity of the PfLDH RDT band were 69% and 38%, respectively, versus pfldh qPCR; and 72% and 36%, respectively, versus PfLDH detection by immunoassay. Among samples with results for RDT, qPCR, and immunoassay, higher proportions of P. falciparum were recorded by pfldh qPCR  and PfLDH immunoassay  compared to the PfLDH band on the SD-Bioline RDT .The 13,172  study participants were enrolled and tested with PfHRP2 and PfLDH detection RDTs; 20.6% (95% CI: 19.6 to 21.6) were P. falciparum cases than PfLDH, and fewer cases than qPCR or immunoassay. The poor sensitivity and specificity of the PfLDH RDT compared to qPCR and to immunoassay in this study raises concern. Continuous operator training and RDTs quality assurance programme to ensure quality diagnostic services are recommended.Both PfHRP2 RDTs detected fewer  Plasmodium [Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi are the aetiologic agents of human malaria, of which P. falciparum is responsible for the most severe forms of the disease [Human malaria is a mosquito-borne parasitic disease caused by protozoan parasites belonging to the genus asmodium . Plasmod disease . Over th disease .In Ethiopia, malaria is one of the most important public health problems with more than 60% of Ethiopians at risk . The cou), parasite-specific lactate dehydrogenase (LDH) (species specific and pan specific), and Plasmodium aldolase, of which HRP2 antigen is the preferred and most commonly used for the detection of P. falciparum, due to its high abundance in the blood, high heat stability and species-specificity [P. falciparum [P. falciparum, 30–40% of cases are caused by P. vivax, and < 1% by both P. ovale and P. malariae based on routine facility report [P. ovale and 7% P. malariae antibody exposure history [P. ovale curtisi cases imported from Ethiopia [Malaria case management is a crucial elimination strategy to reduce morbidity, mortality, prevent subsequent transmission and manage non-malarial febrile illnesses . Diagnoscificity . In sub-lciparum . WhereasEthiopia .P. falciparum and P. vivax, respectively [P. ovale and P. malariae parasites. Besides this limitation, the malaria diagnostic programme is threatened by the emergence and spread of P. falciparum parasites that have single or dual deletion mutations of the pfhrp2/3 genes and hence not detected by the widely used HRP2-detecting RDTs [Light microscopy is the gold standard for diagnosis of clinical malaria . Howeverectively , with deing RDTs .P. falciparum histidine-rich protein 2 (hrp2) genes [. Because this estimate exceeds the WHO-recommended 5% prevalence threshold for transitioning to non-HRP2 detecting RDTs, the national malaria programme is evaluating RDTs that detect alternative antigens for programmatic use. The only available alternatives P. falciparum-specific RDTs that meet procurement criteria detect P. falciparum-specific parasite lactate dehydrogenase (PfLDH) [pfhrp2 and/or pfhrp3 gene deletions has been described. In this paper, the analysis includes the performance of two WHO pre-qualified RDTs, to include comparison to bead-based immunoassay results for HRP2, aldolase, and parasite LDH antigens and to include additional samples collected from the Oromia region that were not included in the original study.A recent large survey conducted in three regions of north and west Ethiopia and estimated that HRP2-based RDTs would miss 9.7% of symptomatic falciparum malaria cases owing to deletion of the 2) genes . Because (PfLDH) . In the ) RDT (product code RM 103 VM-02571) and SD Bioline Malaria Ag Pf (HRP2 for Pf/LDH for Pf) (product code 05FK90). The RDTs were performed using finger-prick fresh whole blood following the manufacturer's instructions. Dried blood spot (DBS) samples were collected from selected positive patients for advanced testing run based on the published standard protocols for immunoassay [Plasmodium species, quantify the parasite and test P. falciparum  target genes. Whereas, the bead based immunoassay [This cross-sectional survey was conducted in selected health facilities of Amhara, Tigray, and Gambella regions of Ethiopia Fig.  between unoassay  at the Cunoassay  in NorthP. falciparum positivity was measured using the T-test. The sensitivity and specificity of PfHRP2 and PfLDH detecting RDTs (screening tests) were performed against its qPCR and immunoassay (reference tests) results.All enrolled subjects provided informed consent, and any subject with a positive test for malaria received treatment as directed by national guidelines. The study was carried out after the ethical approval is obtained from the Ethiopia Public Health Institute Institutional Review Board  and the WHO Ethics Review Committee Protocol number: ERC.0003174. Data cleaning and analysis was performed using Stata SE version 14 software. The association of the clinical and demographic variables with the P. falciparum positives either by one or both RDTs. The patient’s information on clinical sign and symptoms showed a 91.4% fever cases (reported), 63.5% joint pain, 58% poor appetite, 57.4% feeling cold, 45% nausea and 18.4% headache. In addition, 75.7% were rural residents and, 6% (n = 777) of them had history of anti-malarial treatment a month before enrolled in the study, of which 31% received ACT, 11% chloroquine, 7% quinine, and 51% other drugs. All the variables including sex, clinical signs and symptoms, house location and anti-malarial treatment history had strong association with P. falciparum positivity (P < 0.001) , Tigray (50%), Oromia (4%), and Gambella regions (16%) were enrolled in the study. Of all malaria suspected patients, 20.6% (2714/13172) were P. falciparum cohort samples. The proportion of P. falciparum in PfHRP2 detecting CareStart RDT and SD-Bioline RDT were 66% (539/820) and 58.7% (481/820), respectively (Table pfldh PCR result showed 68% (561/820) and 86% (701/811) P. falciparum, respectively  from 0 from P.pfldh PCR assay was 69% and 38%, respectively, as previously reported [The sensitivity and specificity of the PfLDH targeted RDT result compared with the reported  , 90% (411/456) and 88% (363/413) positives, respectively , 55% (251/456), 57% (260/456) and 65% (297/456) P. falciparum is the major cause of malaria mortality and morbidity in Africa, effective case management is needed to reach the goal of elimination. The use of malaria rapid diagnostic tests for the national diagnostic programme since the 1990th has been a backbone with significant increase of use over the last two decades which accounts for over 70% of diagnosis in Ethiopia [pfhrp2/3 gene mutated parasites in Ethiopia [pfhrp2/3 gene deletions causing false negative HRP2-RDT results, which is above the 5% WHO prevalence threshold to change the diagnostic strategy [Malaria case management through accurate diagnosis and prompt treatment is vital especially in elimination setting. In this study, relatively low malaria cases (21%) in spite of high fever prevalence (91%) in 13,172 self-presenting malaria suspected patients are reported which implies the need for accurate parasitological confirmation of cases for the rational use of treatment and management of non-malaria illnesses . Given Pstrategy , as a reP. falciparum in HRP2 and PfLDH targeted RDTs, qPCR and multiplex assay results. The results show that generally higher proportion of P. falciparum is reported by the PfLDH RDT, pfldh qPCR and PfLDH multiplex assays , with more cases are identified by PCR and bead-based immunoassay, compared to HRP2 RDTs and HRP2 bead-based immunoassay  which implies that the PfLDH target antigen is performing better than HRP2 antigen detection for P. falciparum prevalence estimation.This study evaluates the performance of PfLDH detection RDT for programme use and presents the proportion and sensitivity of https://extranet.who.int/pqweb/vitro-diagnostics/vitro-diagnostics-lists). However, the performance of malaria RDT is dependent on several factors in addition to parasite densities [Both RDTs used during the survey are WHO prequalified and, therefore, should meet requirements for safety and quality . This study design could introduce selection bias that limits the generalizability of our findings to the broader population. Nonetheless, analysis of multiple diagnostic assays applied to each sample provides a valuable opportunity to evaluate the performance of PfLDH RDTs. In addition, the field-level RDT tests were carried out by community health workers in remote health posts which could have impacted the results related to operators limited skills, inaccessibility for adequate supervision and, uncontrolled storage and transportation conditions. For example, faint test bands are not uncommon with the PfLDH-detecting RDT evaluated here and might be detected by expert readers, but missed by community health workers.Subjects were enrolled using an adaptation of the WHO protocol for P. falciparum cases detected by PfLDH detecting RDT, pfldh qPCR and a PfLDH immunoassay is generally higher than HRP2 detection by RDT or immunoassay. However, the sensitivity and specificity of PfLDH detection RDTs compared to both pfldh PCR and to PfLDH bead-based immunoassay was lower than expected. As the country is preparing to switch to non-HRP2 detection in response to pfhrp2 deletion problem, a strong RDT quality assurance system and adequate in-service training for operators is needed before introducing alternative RDT products for national programme use.The field-level performance of PfLDH detection RDTs results showed that the overall proportion of