Plasmodium vivax remains a global health problem and its ability to cause relapses and subpatent infections challenge control and elimination strategies. Even in low malaria transmission settings, such as the Amazon basin, where progress in malaria control has caused a remarkable reduction in case incidence, a recent increase in P. vivax transmission demonstrates the continued vulnerability of P.vivax-exposed populations. As part of a search for complementary approaches to P.vivax surveillance in areas in which adults are the majority of the exposed-population, here we evaluated the potential of serological markers covering a wide range of immunogenicity to estimate malaria transmission trends. For this, antibodies against leading P. vivax blood-stage vaccine candidates were assessed during a 9 year follow-up study among adults exposed to unstable malaria transmission in the Amazon rainforest. Circulating antibody levels against immunogenic P. vivax proteins, such as the Apical Membrane Antigen-1, were a sensitive measure of recent P. vivax exposure, while antibodies against less immunogenic proteins were indicative of naturally-acquired immunity, including the novel engineered Duffy binding protein II immunogen (DEKnull-2). Our results suggest that the robustness of serology to estimate trends in P.vivax malaria transmission will depend on the immunological background of the study population, and that for adult populations exposed to unstable P.vivax malaria transmission, the local heterogeneity of antibody responses should be considered when considering use of serological surveillance. Plasmodium vivax representing 64% of all malaria cases [Plasmodium vivax challenges control strategies because of its ability to cause relapses [Malaria remains a public health problem and its global distribution depends on environmental, economic, social and political factors affecting low-income tropical countries . In the ia cases . Plasmodrelapses , and sigrelapses .P. vivax, spatial clustering of transmission and subclinical reservoirs, strongly suggest that public health authorities should reformulate malaria control strategies to include a more sensitive parasite detection method for active surveillance [In the Amazon rainforest, high heterogeneity in risk factors for eillance ,9. Althoeillance . As moreeillance –13. Accoeillance –16.P. vivax merozoite-stage vaccine candidate antigens: the Apical Membrane Antigen-1 (AMA-1), that is expressed by merozoites and sporozoites, as a type I integral membrane protein [19), which is a processed fragment of the MSP-1 polypeptide that remains on the merozoite surface and is carried into the parasitized erythrocyte [P. vivax reticulocyte invasion pathway [In order to determine whether serology is useful for monitoring temporal changes in low transmission malaria setting, we evaluated, over a 9 year study period, the antibody response of adult individuals from a well-characterized rural Amazon population –19 again protein ; the 19-throcyte ; and the pathway . Since D pathway ,24, we i pathway . During P. falciparum malaria incidence has decreased drastically in recent years, and P. vivax is now responsible for all malaria cases reported , in the municipality of Presidente Figueiredo, state of Amazonas, Brazil. The study site and malaria transmission patterns have been described in detail elsewhere –19. Inhareported .The ethical and methodological aspects of this study were approved by the Ethical Committee of Research on Human Beings from the Research Institute René Rachou , according to the Resolutions of the Brazilian Council on Health (CNS-196/96 and CNS-466/2012). In November 2008, 701 residents from the settlement were invited to participate in our study, of which 541 (77.2%) accepted through giving written informed consent, obtained either directly, or from the next of kin, caregivers, or guardians on the behalf of participating minors.A population-based open cohort study was initiated in November 2008. Six (May-June 2009) and 12 months later (November-December 2009), two similar cross-sectional surveys were undertaken, using the following procedures –19: (i) In the study area, the number of malaria cases fluctuated during the 9-year follow-up period, reflecting periods of high (I and III) and low (II) malaria transmission . The num18S rRNA gene of human malaria parasites, was performed. For this, genomic DNA was extracted from either whole blood samples collected in EDTA, or from dried blood spots on filter paper using either the Puregene blood core kit B  or the QIAmp DNA mini kit (Qiagen), respectively, according to manufacturers’ instructions. The real-time PCR was performed as previously described [At the time of each cross-sectional survey, malaria infections were diagnosed by optical microscopy of Giemsa-stained thick blood smears. The Giemsa-stained smears were evaluated by experienced microscopists, according to the malaria diagnosis guidelines of the Brazilian Ministry of Health. In addition, a non-quantitative real-time PCR using the species-specific amplification of the escribed .Recombinant DBPII proteins included amino acids 243–573 of either the Sal-1 reference strain   or the BP. vivax (MSP-119), which represents amino acids 1616–1704 of the full length MSP-1 polypeptide, has been described elsewhere [The 19-kDa C-terminal region of the Merozoite Surface Protein-1 of lsewhere .P. vivax Apical Membrane Antigen-1 . For each protein, the results were expressed as ELISA reactivity index (RI), calculated as the ratio of the mean optical density (OD at 492 nm) of each sample to the mean OD plus three standard deviations of samples from 30 unexposed volunteers. Values of RI > 1.0 were considered seropositive.Conventional enzyme-linked immunoassays (ELISA) for total IgG antibodies to escribed , using shttp://www.epidata.dk). The graphics and the analysis were performed using GraphPad Prism version 7  and the R statistical software (version 3.3.2). In the statistical analyses, the antibody response was defined either as a binary categorical variable  or as a continuous variable . Both type of analyses were necessary since antibody levels do not saturate, while seroprevalence does, such that, if transmission intensity declines, antibody levels will drop while seropositivity may remain high [2) test or Fisher’s exact tests, as appropriate. The Shapiro-Wilk test was performed to evaluate the normality of the variables. Differences in means and medians were tested, respectively, using either one-way ANOVA, with turkey’s post hoc test, or the Mann-Whitney U test or Kruskal-Wallis test, with Dunn’s post hoc test, as appropriate. The linear correlation between variables, such as levels of antibodies and recent episodes of malaria, were determined using the Pearson’s correlation coefficient. For statistical purposes, a recent episode of malaria was defined as a confirmed malaria infection occurring within the 6 month period prior to each cross-sectional survey; for that, all confirmed P.vivax cases were assessed from the Brazilian Malaria Surveillance System Registry . k-means clustering was used to group the intensity of antibody responses to either the DBP-related antigens , or MSP-119 and AMA-1 combined. A logistic regression model using maximum likelihood was built to describe the independent associations between the intensity of antibody response to each group of antigens  and malaria-exposure variables, such as age, time of residence in the Amazon area, dwelling location (riverine or not), and whether recent vivax-malaria episodes had been reported in the 6 months prior to each cross-sectional survey. The dependent variables of the models were treated as binary values, where the high and moderate response groups was defined as “1”, and the low response group as 0. Two separate logistic regression models were built: one using the k-means cluster groups identified using the antibody responses to DBPII, and other using the groups based on MSP-119 and AMA-1. Only variables associated with statistical significance at the 5% level were kept in the final models. As described in the Results, we also used a second higher-level classification to categorize responders according to their combined response to both groups of antigens .A database was created using Epidata software . Overall, antibody responses to MSP-119 and AMA-1 were higher than those to DBPII-related antigens .Antibodies to the antigens . While rP. vivax blood-stage antigens varied, with greater variation observed among the more reactive proteins, MSP-119 and AMA-1  . Similarly, antibodies to MSP-119 were boosted by P. vivax episodes . Despite this, at the end of the follow-up study (Phase III), the average levels of either AMA-1 or MSP-119 antibodies did not reach the baseline levels  and low (II) malaria transmission . Accordind AMA-1 . In the ct test) . The levct test) . Confirme levels .19/AMA-1, the DBPII-related  antigens presented a similar profile of antibody responses, including a decrease in both the proportion of responders and relative antibody levels at the time of low malaria transmission (Phase II). On the other hand, the general profile of antibody response to the engineered DEKnull-2 vaccine remained relatively stable throughout the study , suggesting that the influence of recent malaria episodes was relevant only in the group of responders who present relatively significant levels of antibody response. The time of malaria exposure, evaluated here by subject’s age and time of residence in the Amazon area, were not significantly associated with the levels of antibody response to any of the panel of antigens used.In order to identify variables that could be involved in the presence, intensity and persistence of the antibody responses, we analyzed those 57 individuals who had participated in all phases of the long-term cohort study. The ELISA results were clustered  according to antigen group, which were grouped as either (i) DBPII-related proteins , or (ii) MSP-1proteins . Using lproteins , the tot19/AMA-1), we also used another higher-level classification to categorize responders according to their combined response to both groups of antigens (low responders (LR), 33 out of the 57 (58%) individuals who present an unstable response of antibodies and whose plasma samples were clustered as “low” to both group of proteins (DBPII-related and MSP-119 & AMA-1); (ii) intermediate responders (IR), 13 (23%) of subjects, exhibiting heterogenous antibody responses, which varied in duration , as well as intensity ; and (iii) high responders (HR), including here 11 (19%) individuals, with persistent long-lived antibody responses, which clustered within the “moderate/high” group for both antigens. Although the HR group was slightly older than the other groups (median 59 vs. 44–53 years), this difference was not statistically significant. However, there was a significant difference between the groups of responders in relation to dwelling location, with the majority of HR members (90%) living close to vector breeding sites (riverine population) as compared with IR or LR groups     low 0.0001) . Through= 0.004) .P. falciparum [P. falciparum asexual blood-stage antigens, most previous studies estimate seroconversion rates from age-dependent seroprevalence, and have found it a usefull tool for inferring malaria transmission intensity [P. falciparum transmission, equivalent data on P. vivax malaria are scarce [P. vivax studies have assessed IgG antibodies to a panel of P. vivax proteins, longitudinal studies are required to better understand the role of serological markers as tools to detect temporal fluctuations in P.vivax transmission [P. vivax blood-stage antibody dynamics, we assessed antibodies to leading P. vivax merozoite-stage vaccine candidates–at 6 time-points during a 9 year study period–among malaria-exposed adults living in an area of low but unstable malaria transmission[P.falciparum in Africa [P. falciparum-endemic areas young children bear the major burden of disease [P. vivax blood-stage antibodies and either the age of studied subjects or their time living in the endemic area.Serology has become an increasingly important tool for the surveillance of a wide range of infectious diseases, including the well-characterized epidemiology of lciparum ,34. Usinntensity ,36. Althe scarce ,15,37,38nsmission–19,39. Wn Africa ,13,40. I disease , while i19 were comparable in immunogenicity, the current study confirmed previous findings showing that AMA-1 seems to be a sensitive marker to detect fluctuations in areas of unstable malaria transmission [19, provided to be a much more precise estimator of malaria transmission [P.falciparum studies, recent findings also confirm that AMA-1 is a useful surrogate marker in low transmission settings [19/AMA-1 cluster (HR and IR groups) exhibited a straightforward relationship between AMA-1 antibody boosting and P. vivax blood-stage episodes. Also, while HR and IR groups could be differentiated by breadth (specificities for multiple antigens) of antibody responses, the profile of AMA-1 antibodies remained relatively similar between these groups of responders. These findings suggest that in the studied subjects, the levels of AMA-1 antibodies (but not the breadth of antibody responses per se) is useful as a serological marker of long-term changes in malaria transmission. On the other hand, it has been suggested that an increasing breadth of antibody specificities could be dependent upon the level of antigenic input [P. vivax malaria is probably much higher in the HR group than in IR group. The majority of HR subjects live close to vector-breeding sites (riverine population), being more exposed to malaria transmission, and heterogeneity in mosquito exposure contributes considerably to heterogeneity in infection risk [P. vivax neutralizing antibodies [Although we demonstrated here that AMA-1 and MSP-1smission ,42–44. Asmission . In P.fasettings . Additioic input , with hiic input . Althougion risk . In our ion risk . Consequion risk ,48. Howetibodies .Plasmodium vivax antigens spanning a range of immunogenicities were included in the current study in order to build up a detailed picture of the immunological background of the studied individuals. At both individual and population levels, DBPII-strain-specific antigens were not clearly associated with variation in malaria transmission over time. This finding was not completely unexpected since in low transmission settings, such as the current study area, serological markers based on highly immunogenic antigens seem to be more appropriate than less immunogenic antigens [P. falciparum transmission [P. vivax transmission. In agreement with these findings, it has been suggested that antibody titers to P. vivax antigens tend to reach their corresponding plateau sooner than its P. falciparum counterparts [P. vivax antigens tend to reach their corresponding plateau sooner, it has been speculated that it may be related to hypnozoite infection [P. vivax compared to P. falciparum [P. vivax blood-stage infections. At the individual level, DEKnull-2 immune responses confirmed the highest acquired immunity of HR group as compared with IR or LR group; all HR subjects were positive to DEKnull-2, and remained seroreactive until the end of the 9 year follow-up study. Despite of that, the levels of antibodies reacting to DEKnull-2 declined during the study, including in the high transmission phase III.antigens . To overantigens . Howeversmission . By usinterparts . Althougnfection , and thelciparum . Aditionlciparum . Thus, wP. vivax transmission intensity, as levels are boosted by re-exposure to parasite blood-stages, and dropped when transmission is reduced. In an area of unstable malaria transmission, such as the Amazon area, its efficiency will depend on immunological status of the studied individuals, since a certain level of prior exposure appears to be necessary for adequate boosting of the antibody response. Thus, for adult populations exposed to unstable P. vivax malaria transmission, the heterogeneity of responders needs to be considered when planning whether to use serological markers as a complementary approach to increase the effectiveness of estimating malaria transmission.As the current study involved a relatively long-term follow-up, it was only possible to obtain samples from 57 out of 102 individuals for the full 9 year study period, which resulted in a decrease in the statistical sample power from 80% to 63%. Despite this, the profile of antibody response remained as expected. Even though our limited sample size precluded a more robust statistical analysis, the results presented here confirm that in the study area the levels of AMA-1 IgG antibodies fulfill the prerequisite to be used as a marker of S1 Fig19 and AMA-1 (B)Clustering of the antibody responses to DBPII variants (A) and MSP-1. The k-means clustering method was used the identify clusters according the ELISA reactivity index (RI). For the analysis, the 57 malaria-exposed subjects who participated until the end of the follow-up period , and for who there were multiple consecutive samples, were included. For each group of proteins, three clusters were identified . C and D show the RI  for DBPII-related antigens and MSP19 & AMA-1, respectively, according to the k-cluster of reactivity.(TIF)Click here for additional data file.S2 Fig19 and AMA-1 (A) but not to DBPII-related antigens (B).Logistic regression models describing the association between vivax malaria episodes and antibody responses to MSP-1 While the predicted probability of the MSP-119 and AMA-1 antibodies levels increases significantly with new episodes of P. vivax malaria (*P < 0.01), DBPII-related antigens were not associated. The variables age, time of residence in Amazon Region, and dwelling location were also included in the logistic models but were not significantly associated (data not shown).(TIF)Click here for additional data file.S3 Fig19, and DBPII-related antigens . For each antigen, ELISA results were expressed as the median of the Reactivity index (RI). The cross-sectional surveys were carried-out as described in legend of Three groups of responders were identified as Low (LR), Intermediate (IR) and High (HR) responders, as described in the legend to (TIFF)Click here for additional data file.S1 File19, and also recent malaria episodes, for each cross-sectional survey .Code, Reactivity Indexes (ELISA assay) of DBPII Sal1, DBPII Brz1, DEKnull-2, AMA-1 and MSP1(XLSX)Click here for additional data file.


P. falciparum malaria can cause multiple disease complications that differ by patient age. Previous studies have attempted to address the roles of parasite adhesion and biomass in disease severity; however, these studies have been limited to single geographical sites, and there is limited understanding of how parasite adhesion and biomass interact to influence disease manifestations. In this meta-analysis, we compared parasite disease determinants in African children and Indian adults. This study demonstrates that parasite biomass and specific subsets of var genes are independently associated with detrimental outcomes in both childhood and adult malaria. We also explored how parasite var adhesion types and biomass play different roles in the development of specific severe malaria pathologies, including childhood cerebral malaria and multiorgan complications in adults. This work represents the largest study to date of the role of both var adhesion types and biomass in severe malaria. Plasmodium falciparum malaria differs between children and adults, but the mechanistic basis for this remains unclear. Contributing factors to disease severity include total parasite biomass and the diverse cytoadhesive properties mediated by the polymorphic var gene parasite ligand family displayed on infected erythrocytes. To explore these factors, we performed a multicohort analysis of the contribution of var expression and parasite biomass to severe malaria in two previously published pediatric cohorts in Tanzania and Malawi and an adult cohort in India. Machine learning analysis revealed independent and complementary roles for var adhesion types and parasite biomass in adult and pediatric severe malaria and showed that similar var profiles, including upregulation of group A and DC8 var, predict severe malaria in adults and children. Among adults, patients with multiorgan complications presented infections with significantly higher parasite biomass without significant differences in var adhesion types. Conversely, pediatric patients with specific complications showed distinct var signatures. Cerebral malaria patients showed broadly increased expression of var genes, in particular group A and DC8 var, while children with severe malaria anemia were classified based on high transcription of DC8 var only. This study represents the first large multisite meta-analysis of var expression, and it demonstrates the presence of common var profiles in severe malaria patients of different ages across distant geographical sites, as well as syndrome-specific disease signatures. The complex associations between parasite biomass, var adhesion type, and clinical presentation revealed here represent the most comprehensive picture so far of the relationship between cytoadhesion, parasite load, and clinical syndrome.The clinical presentation of severe  Plasmodium falciparum remains responsible for more than 400,000 deaths every year  from ery year . In aread adults . Whereasd adults . The parP. falciparum is sequestration of infected erythrocytes (IEs) within the microvasculature  family, encoded by var genes. PfEMP1 proteins are expressed in a clonally variant fashion on the surface of IEs  or erythrocytes .var gene family and malaria disease is the immense diversity of var genes in the parasite population , a parasite protein released upon merozoite egress, is used as a blood surrogate marker for the combined amount of both circulating ring-stage and sequestered mature-stage P. falciparum-IEs. PfHRP2 levels are associated with severe malaria in both children and adults  by applying an integrated multicohort analysis to better understand how both parasite burden and binding phenotype are associated with severe malaria in both adults and children and (ii) by examining how parasite burden and parasites expressing different  cohorts , 34, alo cohorts . This wovar gene transcript profiles (var profiles) for this work were obtained from two previously published studies of pediatric malaria in Tanzania (TZ) (The nia (TZ)  and Malania (TZ)  and one nia (TZ)  cohort wPrevious work has shown that malaria disease presentation is influenced by both transmission intensity and patient age , 44. The10.1128/mBio.00217-19.3TABLE S1Table S1, PDF file, 0.1 MB.Demographic and clinical characteristics of the study cohorts. Download Copyright © 2019 Duffy et al.2019Duffy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the var adhesion types associated with malaria disease severity at the three sites, var transcript profiling was performed using a set of 41 PCR primer pairs designed to quantify transcripts of defined var domain subtypes  remain closely correlated at all three sites , all but DC5 retain their close association across every site, and DC5 primers remain the most closely associated in Tanzania  and C-terminal domains . In contrast, cluster B contained var genes encoding predicted EPCR binding domains (subcluster iii) and var genes associated with unknown/rosetting function (subcluster iv). Exceptions in cluster B were subtypes relating to DBLα0.9 of DC20 and DC6 (subcluster v). Although DC6 domains can be found in all three var groups  were the highest expressed transcripts across the three sites  (see P = 1.00), other domain subtypes showed site-specific differences in expression distributions. These differences are not solely explained by differences between patient age and ethnicity. For example, while adult GMC and pediatric BLZ patients shared 26 similarly distributed domain subtypes (P = 1.00), only 14 subtypes met this threshold when comparing pediatric TZ versus BLZ patients. These results were unexpected, as pediatric and adult severe malaria patients present different organ complications.To explore 000) see . While s10.1128/mBio.00217-19.2FIG S2var domain subtypes targeted by the 41 primer sets vary according by study site and malaria status. Expression density plots for each var domain subtype were stratified by site. BLZ, Malawi; GMC, Goa; TZ, Tanzania. The var domain subtypes are shown in the same order as in FIG S2, EPS file, 8.1 MB.Transcript levels of the Copyright © 2019 Duffy et al.2019Duffy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 1.00), the main site-specific expression differences were observed for CD36-binding and C-terminal domains  in the combined severe versus uncomplicated malaria cases. Five primer pairs were identified that passed this threshold: primer pair DBLα2/α1.1/2/4/7 targets all EPCR binders (DC8 and group A), primer pair DBLα not var3 targets group A PfEMP1 , primer pairs DBLβ12 and DBLβ3/5 mostly target DC8 (EPCR), and primer pairs CIDRγ and CIDRα2.3/5/6/7/9/10 target CD36-binding var genes. Notably, the rank order of the top 5 transcribed domains was the same in both SM and UM patients across the three sites, but the var transcript levels tended to be higher in SM patients , while neither of the CD36 high expressers showed any significant difference. In all three sites, UM patients showed a bimodal distribution of the DC8 and group A var transcripts, including two peaks representing low-expression and high-expression individuals  models were trained on var profiles to classify samples as severe or uncomplicated malaria. This was performed for all sites individually and for Tanzania and Malawi together to derive a model representing all childhood cases.In order to more precisely evaluate the contribution of var repertoire . Although we previously reported that only DC8-EPCR binders were predictive of adult SM  contribDBLα0.16 ) ranked C6 (DBLγ ), which SM model , still r10.1128/mBio.00217-19.5TABLE S3var domain subtype in adult and childhood UM versus SM models. Download Table S3, PDF file, 0.1 MB.MDCA and mProbes FWER for each Copyright © 2019 Duffy et al.2019Duffy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the var profile models significantly discriminate UM from SM. Notably, the combined childhood SM model  performs very similarly to the individual Tanzanian and Malawian models  and the adult Goan model . Taken together, predictive performance was highly consistent across all sites.Receiver operating characteristic (ROC) curves and the accompanying 95% confidence intervals (CIs) representing the unbiased out-of-bag (OOB) predictions made during model training are shown in P = 0.002) and adults on children . In other words, adult SM could be predicted by var profiling of pediatric samples and vice versa. This demonstrates that, despite presenting highly distinct severe disease manifestations, similar var subsets are predictive of SM in both adults and children.To further explore pathogenic mechanisms in children and adults, we made blind predictions, using the adult model to classify the child samples and the childhood model to classify the adult samples. Importantly, both models showed significant ability to cross-classify  for bothP = 4 × 10−9) and Goa   curves for these models are shown in var profiles alone for both Malawi  and Goa . Statistical analyses indicate substantial improvement in all prediction metrics in both sites when var profile models are combined with PfHRP2 measurements   . To eval10.1128/mBio.00217-19.6TABLE S4var profile models with PfHRP2 measurements for Malawi and Goa samples. Download Table S4, PDF file, 0.1 MB.Summary statistics for combining Copyright © 2019 Duffy et al.2019Duffy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.00038 , where a Tu value of 1 represents a 0 level of expression.All participants had quantitative reverse transcription-PCR (qRT-PCR)-based escribed . BrieflyPfHRP2 quantification was performed as previously described , 37. BriAll statistical and machine learning analysis was carried out using the R language for statistical computing . All pri(i) Clustering and distribution analysis. Primer-primer correlation coefficients were calculated as Spearman’s rho (ρ), and hierarchical clustering was performed using the R hclust function. Significant differences in primer distribution between sites were evaluated using the Kolmogorov-Smirnov test, using the R ks.test function, and P values were adjusted for multiple testing using the Holm approach.(ii) Machine learning modeling and significance testing. Machine learning models were used to assess the ability of var transcript profiles to classify subjects as SM or UM or to distinguish specific SM severity criteria. RF models were trained to distinguish SM from UM in all three sites using the R randomForest package . To avoid bias due to overfitting, unbiased model predictive performance was evaluated based on the consensus RF out-of-bag (OOB) predictions generated during model training. Receiver operating characteristic (ROC) curves were constructed based on the OOB predictions. Strength of predictive performance was measured as area under the ROC curve (AUC). This takes the form of a number between 0 and 1, where 0.5 represents random performance and 1 represents perfect classification. The R pROC  function. Sensitivities, specificities, and odds ratios were calculated from confusion matrices of binary predictions made at the optimal point on the ROC curve, defined as the point that minimized the Euclidean distance to a sensitivity and specificity of 1. package . Missinge R pROC  package var subtypes to model performance, the mean decrease in classifier accuracy (MDCA) was used. This statistic is calculated during the resampling training procedure of the RF model and is the percentage of decrease in accuracy in random data subsamples that contain the var subtype versus those that do not. Significance of model var domain subtypes was calculated using the mProbes algorithm  that combine related information, such as a common binding phenotype , gene group , domain organization , or part of a tandemly arrayed domain combination  were developed (var transcript levels. The summed var transcript abundance (var set) was calculated by summing the reported Tu values for each var domain subtype per sample and subtracting 1 for each additional primer beyond the first. This was done to ensure that the set’s summarized Tu value was equal to 1 if no expression was observed for any var domain subtypes in a particular sample. Machine learning and importance estimates were created for the summarized features, as described above.Sets of eveloped  and appl


Anopheles vector. In addition, updates on current anti-gametocyte, anti-gamete, and anti-mosquito transmission blocking vaccines are given. We conclude with our views on some important future directions of research into P. falciparum sexual stage immunity relevant to the search for the most appropriate transmission-blocking vaccine.Malaria infections remain a serious global health problem in the world, particularly among children and pregnant women in Sub-Saharan Africa. Moreover, malaria control and elimination is hampered by rapid development of resistance by the parasite and the vector to commonly used antimalarial drugs and insecticides, respectively. Therefore, vaccine-based strategies are sorely needed, including those designed to interrupt disease transmission. However, a prerequisite for such a vaccine strategy is the understanding of both the human and vector immune responses to parasite developmental stages involved in parasite transmission in both man and mosquito. Here, we review the naturally acquired humoral and cellular responses to sexual stages of the parasite while in the human host and the  Malaria is one of the most important parasitic infections with the highest burden of mortality and morbidity in sub-Saharan Africa. Despite progress and advances in the strategies to control the disease, malaria claimed the lives of approximately 445,000 people from among 216 million clinical cases globally in 2016; mostly in children under 5 years and pregnant women as reported by WHO  suggest Plasmodium parasites that are essential for transmission from humans to mosquitoes. Initially, a certain proportion of the erythrocytic stage parasites undergoes a permanent differentiation also referred to as sexual commitment into both male (microgametocyte) and female (macrogametocyte) gametocytes . TRI is based on observations of naturally acquired antibodies against gametocytes that are produced in the human host in response to proteins of gametocytes that were not taken up by mosquitoes . When thExtensively studied antigens to date include gametocyte/gamete proteins such as Pfs230 and Pfs45/48 and the zygote/ookinete proteins Pfs25 and Pfs28  also knoNaturally occurring antibodies targeting Pfs230 and Pfs45/48 have been observed in field studies in The Gambia, Kenya, and Cameroon and were associated with reduced malaria transmission  or with A recent study by Stone et al. using field-based mosquito-feeding assays found mosquito infection rate to be significantly reduced for people harboring naturally acquired anti-Pfs48/45 and anti-Pfs230 antibodies. In addition, these antibodies were shown to be host gametocyte density-dependent and mechanistically associated with transmission reducing activity (TRA) , 48. In in vitro experiments and are currently being evaluated in clinical trials , distinct from those recognizing internally expressed gametocyte and gamete antigens, in natural human infections. This is largely due to the indirect effect of asexual stage immunity on the prevalence and density of gametocytes.P. falciparum gametocyte-infected erythrocyte surface antigens in human peripheral circulation (anti-gametocyte immunity)  , 53. Themmunity) , 52–54.In the first investigation of its kind, plasma antibodies from gametocytemic Gambian children donated after antimalarial treatment were used to detect antigens on the surface of 3D7 cultured mature stage V gametocytes. Surprisingly, no antibody recognition of the surface of erythrocytes infected with developing gametocytes, stages I-IV, representing the stages known to be sequestered in deep tissues, were found , 54. In P. falciparum gametocyte rates were reduced among semi-immune native Papuans, independent of immune control of asexual parasitemia, when compared to a transmigrant Javanese population with a history of lower malaria exposure. These findings suggest specific immune control of gametocytemia as the observations could not be explained by differences in the frequency or grade of parasitemia, illness or by known patterns of antimalarial treatment. Further, immunofluorescence tests with acetone-fixed whole gametocytes showed a correlation between antibody levels and reduced gametocytemia among the native Irianese  of parasite-negative children and adults recognized the surface of mature gametocyte-infected erythrocytes /50 of pa. Since sPlasmodium species are limited. However, there is evidence that such immunity exists. The transfer of T-cells from gamete-immunized mice was shown in the 1980s to markedly reduce gametocytemia in the recipient mice using the rodent species P. yoelii nigeriensis (A. stephensi as demonstrated by direct blood feeding (up to 95% transmission reduction) suggesting the direct impact of T-cells on transmission. However, the T-cell transfer had no effects on asexual stages  have been shown to be able to stimulate the proliferation and activation of lymphocytes from dividual . In this malaria . HoweverPlasmodium species and other pathogens  (A. gambiae (AgTEP1) has been shown to be valuable for the initiation of immune defense against P. berghei. TEP1 plays the role of opsonins and facilitates the interaction between the parasite and the hemocytes with subsequent encapsulation, and killing of the parasite  . TEP of parasite . Double  gambiae . This veP47-like . This prertility  but in Pnt lysis .A. gambiae mosquito by ookinetes of P. berghei has been demonstrated to modulate the mosquito's immune system by up-regulating the expression of the antibacterial peptide defensin and a putative gram-negative bacteria-binding protein , which equally confers resistance to invasion by Plasmodium  and an immunolectin, FBN39 for P. falciparum and the antimicrobial peptide gambicin and a novel putative short secreted peptide, IRSP5 for P. berghei —modified Lickenase carrier (LiKM)—Virus-like particle (VLP)—Alhydrogel. Only two of these vaccine candidates, namely Pfs 230 and Pfs25, have entered clinical trial stage  in the sense that they do not protect against disease in the vaccinees. However, they reduce the risk of transmission to other people by the mosquito vector and by so doing favor herd immunity; as such they have sometimes been referred to as altruistic vaccines . Two groal stage  and are al stage . Althougal stage . Hence tal stage , 121P. falciparum with a truncated chitinase 1 (PfCHT1) gene which could be due to the inability of affected ookinetes to invade the mosquito midgut .Pfs230 is a 363 kDa protein and a potent antigen of malaria TBV. It is a main component in the fertilization process as male gametes with impaired Pfs230 gene are incapable of interacting with red blood cells (RBCs) and forming exflagellation centers . This reo midgut . Adminiso midgut . It shouo midgut . Signifio midgut , 46, 124o midgut . This vao midgut  has entehttps://clinicaltrials.gov/ct2/show/NCT02013687). It appeared that the combination is safe with no serious adverse reactions observed in healthy volunteers even when higher doses are administered. However, the antibodies generated showed low TRA hence the necessity to prioritize vaccine adjuvant formulations for further investigations , and the P. falciparum ortholog Pfs25 in a mouse model. Anti-Pvs25H antibody levels peaked after the third vaccination and vaccine-induced antibodies were functional, giving significant TRA (Another combination of Pfs25 and EPA (Pseudomonas aeruginosa ExoProtein A) plus Alhydrogel® was also demonstrated to be well tolerated by naïve individuals after several doses in a phase 1a dose-response clinical trial in the US which correlated with antibody titers . A Phasecant TRA . This cocant TRA .The main limitations of these sexual stage vaccine candidates have been the systemic reactogenicity observed in some clinical trials, short-lasting antibody responses owing to the fact that the host has no or limited exposure to the antigens requiring multiple boosting doses and strong adjuvants. In addition the recombinant antigens are difficult to express in their native form . To circSome mosquito components are invaluable for the sporogonic development of malaria parasites as they are involved in parasite invasion through interaction with parasite receptors. Antibodies raised against these components could be very useful in blocking parasite development in the mosquito vector . Thus, tA. gambiae as previously shown by Dinglasan et al.  is a Glycosylphosphatidyl inositol (GPI)-anchored antigen which plays a valuable role in ookinete invasion in n et al. . Adminisn et al. . The TRA strains . More iml trials . This anl trials , 115. Hol trials . Antibodn. Dirus . HoweverA. gambiae (cpbAg) has been shown to be up-regulated following P. falciparum gametocyte ingestion by the vector  and the mosquito Saglin proteins is a prerequisite for vector salivary gland invasion  within these vectors (An. gambiae serpin-2 (AgSRPN2) have been shown to be P. berghei-specific in An. gambiae and An. stephensi as they failed to interfere with P. falciparum oocyst formation   , CX3CL1- (PfSRA) , Gametoc (PfSRA)  have beeJK-O and BD designed and drafted the manuscript. CS, FB, GA, and BU reviewed and edited the manuscript. All authors approved the final version of manuscript for publication.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.


Malaria remains a leading cause of childhood mortality in sub-Saharan Africa. Identifying patients who are at risk for severe manifestations at presentation still remains challenging. This study examines whether a semi-quantitative test on C-Reactive Protein (CRP) could be useful for rapidly predicting the presence or absence of malarial parasitemia in febrile children.Data were collected from children with fever or a history of fever at the Agogo Presbyterian Hospital in the Ashanti Region of Ghana. Haematological measurements, microscopic detection of plasmodium species and semi-quantitative CRP measurements with a membrane–based immunoassay for whole blood were performed. CRP was classified as positive when the measured level was ≥ 10 mg/l.Plasmodium spp. Whereas malaria parasites were detected in only a few patients (7.1%) with normal CRP levels (< 10mg/l), more than a half of patients with an increased CRP concentration (≥ 10 mg/l) were parasite positive . Patients with increased CRP levels had more than an eight-fold likelihood for parasitemia after correction for other parameters . Sensitivity, specificity as well as positive predictive and negative predictive values of CRP for malaria were 99.3% (CI 96.2%-100%), 9.2% (CI 6.4%-12.8%), 31.7% (CI 27.4%-36.1%) and 97.0% (CI 84.2%-99.9%), respectively.During 548 visits, thick blood film results could be obtained from 541 patients, 270 (49.3%) yielded parasitemia with The semi-quantitative method of measuring CRP is cheap, rapid and easy to perform but not useful in predicting parasitemia and malaria. However, due to its high negative predictive value, it could have a role in identifying those patients unlikely to be presenting with clinical malaria. Plasmodium falciparum malaria is among the leading causes of childhood mortality in most sub-Saharan African countries [ountries . In 2017The case definition of malaria requires at least an increased body temperature and the presence of asexual stages of malaria parasites. However, as a result of the intermittent nature of malaria fever , some maIn highly endemic regions, parasitemia can be a common finding and clinical manifestations can be due to causes other than malarial infection. The WHO therefore recommends that malaria microscopy or rapid diagnostic test (RDT) be carried out in suspected malaria cases . In a coBlood glucose, lactate concentration and base excess have long been used in assessing severe malaria and its complications. Biomarkers related to the immune activation pathways such as procalcitonin, angiopoeitin and von Willebrand Factor (vWF) have recently been studied ,9. ThougC-Reactive Protein (CRP) has also been studied lately in patients with malaria in Africa and other parts of the world ,10,11. CCRP can be measured qualitatively, semi-quantitatively and quantitatively. The qualitative method measures the absence or presence of a certain level of CRP in a serum sample . The semThe aim of this study was to evaluate the value of a semi-quantitative CRP test to predict clinical malaria among febrile children in a highly endemic area of Ghana.Plasmodium detection were performed and analysed. All samples were collected at the Agogo Presbyterian Hospital in the Asante Akyem North district in Ghana. The predominant malaria parasite in this region is P. falciparum [Patients were recruited between January and October 2010. Data were collected from children aged up to 15 years old with fever or a history of fever during the 24 hours preceding the consultation. Haematological measurements, semi-quantitative CRP measurements and microscopic lciparum . All staR KX-21N Haematological Analyzer .Body temperatures were recorded for each child, and fever was defined as an axillary temperature > 37.5°C. Venous blood was drawn using Vacutainer tubes  containing EDTA as an anti-coagulant. A blood smear was immediately made and later stained with Giemsa. The full blood count (FBC) analysis for each participant`s sample was analysed using a SysmexThe study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Ethical approval was obtained from the Ethics committees of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi.Written informed consents of all study participants were obtained from mothers or guardians after been informed about the aim of the study in presence of a witness.Plasmodium species and counts (number of parasites per 200 leukocytes) were analysed by microscopy and recorded. A slide was considered negative when no malaria parasite was seen in 100 highpower fields (HPF). A slide was considered positive when at least one parasite was seen in the thick film. All slides were double-read by different readers to ensure accuracy. Slides which were discrepant were excluded from the analysis.CRP was measured using the CRP Test Kit CRP-K10 , a semi-quantitative membrane–based immunoassay for the determination of CRP in whole blood, serum or plasma. The test was standardized to detect CRP levels at or above 10 mg/l, with a reported sensitivity of 94.8% and a specificity of 98.3% . Between 10 and 12 drops of the buffer solution included in the test kit were put in a test tube, this was held vertically to prevent air trapping. A capillary tube was filled with approximately 3.5 μL of the patient`s serum and transferred into the tube filled with buffer solution and mixed thoroughly. Three drops of the specimen mixture were then put into the specimen well and the results were interpreted after 5 minutes. The appearance of a line in the “C” (control) marked zone confirmed that the test had worked properly. In the middle part of the test is the reference line “R”, with the test line “T” closest to the test field “S”. The results were then interpreted based on the coloured lines which appeared in the field lines. A test was said to be positive when three distinct colours appeared in the lines “T”, “C” and “R”. The CRP concentration of a sample is interpreted as 10–30 mg/l when the test line (T) is fainter than the reference line (R), around 30 mg/l when the test (T) is close to the reference line (R) in intensity and over 30 mg/l when the test line (T) is brighter than the reference line (R). The CRP concentration in the sample was less than 10 mg/l and the test was considered negative when two coloured lines appeared in the control (C) and reference (R) areas, with no distinct colour line appearing in the test area (T). A test was considered invalid when there was no control line (C) and/or reference line (R). This was then discarded and a new test was performed. .The association between the outcome variable parasitemia and possible impact variables were analysed performing a univariate analysis and then a multivariable logistic regression with backward stepwise selection. Parameters with potential relevance such as age, sex, tachycardia, tachypnea, blood culture result and white blood cell count were included in the final logistic regression model to assess their independent effect on parasitemia. For the stepwise estimation, a p-value of <0.1 was set as statistical cut-off meaning that variables with a p-value <0.1 were further analysed. Odds ratio (OR), 95% confidence interval (CI) and p-values were then calculated.Overall, 541 patients aged up to fifteen years were included in the study . One patPlasmodium spp. and 278 (50.7%) were negative. Among patients above 12 months of age who were screened, 88.5% had a parasitemia, whereas among those aged up to 12 months, the prevalence was 11.5%. 76.6% of positive blood smears were found in children below 5 years of age.Out of the 548 visits, thick blood smear results were available for all the patients. 270 (49.3%) blood smears yielded parasitemia with CRP levels were low (< 10 mg/l) in 42 (7.7%) visits and high (> 30 mg/l) in 368 (67.2%) visits. Few patients with negative CRP were malaria positive (7.1%), whereas more than half of patients with an increased CRP concentration (≥ 10 mg/l) were malaria positive  . SimilarBlood culture results were available for 505 patients. Of them, 31 (6.1%) patients were positive for bacteria . CRP levPatients with an increased CRP level had more than an eight-fold likelihood for positive parasitemia  in the adjusted multivariable model . Sex, taIn assessing the quality of the test, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of increased CRP for parasitemia, malaria and septicemia were calculated . For parPlasmodium parasitemia was assessed in an area where malaria infection is very prevalent. The intention was to find out if CRP could help in distinguishing clinical malaria manifestations from other causes. As recommended by the WHO, all febrile patients should be tested for malaria and those who test positive for parasitemia should be treated for malaria. CRP must therefore not be considered as a replacement of proper parasitological diagnosis.The usefulness of CRP as a biomarker for the prediction of In the univariate analysis, elevated CRP levels were seen in patients with parasitemia and those with malaria. CRP was also associated to parasitemia in the multivariate analysis. Almost all of the patients with a detectable parasitemia  and those with clinical malaria  had elevated CRP levels. This finding is consistent with results from studies conducted in The Gambia, Tanzania, Mozambique and Malawi , 23 wherHowever, the positive predictive values of elevated CRP for parasitemia and malaria were only 52.8% and 31.7%, respectively. Accordingly, in patients with elevated CRP levels, about a half had no parasitemia and two-thirds had no malaria. This is similar to the background frequency of parasitemia and malaria found in the patients recruited for the study, indicating that there was no additional information provided by an increase of CRP levels in such an area.CRP is known to rise during bacterial infections , 17. In Negative predictive values of elevated CRP were high for parasitemia (92.9%) and malaria (97.0%), implying that the likelihood that patients with normal CRP levels have no parasitemia or malaria is high. Based on this finding, a normal CRP may be used in ruling out malaria in febrile children in a context where malaria microscopy and RDT may not be readily available. However, CRP levels were normal only in a minority of patients.In a recent study from Malawi the usefulness of different biomarkers was examined, namely C3a, C5a, angiopoientin-1, angiopoientin-2, soluble tie 2 (sTie-2), soluble Endoglin (sEndoglin), vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), tissue factor and leptin to identify plasmodium parasites in pregnant women. Individually, all these markers showed only a moderate ability (areas under ROC between 0.62 and 0.72) to identify malaria . SimilarPlasmodium spp. infection from septicemia due to its poor specificity. However, in a setting like sub-saharan Africa where other febrile illnesses play an important role, a negative CRP can nearly rule out parasitemia or malaria due to its high negative predictive value. The high negative predictive value results from the relative low frequency and cannot be generalised for situations with very high prevalence. Nevertheless, this finding could be helpful for clinicians in making decisions, especially in highly malaria-endemic areas, where fever is frequently equivalent to malaria and treated accordingly [From our results, though CRP is elevated in parasitemia and in malaria, it lacks the ability to differentiate a ordingly , leavingordingly .A limitation of this study was the use of only microscopy at 100 HPF to detect parasitemia without the use of PCR-diagnostics. As PCR is known to be more sensitive than microscopy examination , the useThe semi-quantitative method of measuring CRP is cheap, rapid and easy to perform, but not useful in predicting malaria or parasitemia in febrile children. Furthermore, it is unable to differentiate malaria from other febrile illnesses like septicemia due to its poor specificity. Nevertheless, a negative CRP has a high negative predictive value in few patients with normal CRP, meaning a negative CRP might be indicative of those patients unlikely to be presenting with clinical malaria. However, CRP cannot replace parasitological testing in febrile patients.S1 Data File CRP(XLS)Click here for additional data file.


Malaria is known to cause acute and deadly complications. However, malaria can cause unforeseen pathologies due to its chronicity. It increases the risk of endemic Burkitt Lymphoma development by inducing DNA damage in germinal centre (GC) B cells, and leading higher frequency of Epstein–Barr virus (EBV)-infected cells in GCs. EBV is well known for its tropism for B cells. However, less is known about EBV’s interaction with T cells and its association with T cell lymphoma.+ T cell lymphoproliferative disorder with skin involvement. Patient was treated and discharged. However, a pathologist noticed unusual structures in skin tissue samples. Careful evaluation of skin biopsy samples by polarized microscopy revealed birefringent crystalloid structures resembling malarial haemozoin mainly loaded in macrophages and giant histiocytes. After purification of DNA from the skin biopsy samples, nested PCR was performed for the detection of Plasmodium parasites and Plasmodium falciparum DNA was amplified. Because, the co-presence of EBV infection with malaria is a well-known aetiology of lymphoma, EBV-early RNA (EBER) transcripts were investigated in paraffin-embedded tissue samples and found to be positive in macrophage-like histiocytes.A 43-year-old Sudanese male admitted to hospital in Istanbul, Turkey, a non-endemic country, with hyperpigmented painful skin rashes on his whole body. A complete blood count and a peripheral blood smear during admission revealed large granular lymphocytes (LGLs) with abnormally higher CD8 T cell numbers. Additional skin biopsy and pathology results were compatible with CD8Plasmodium infection should be examined in patients from malaria endemic regions by pathological and molecular investigations.This is a unique case of malaria and EBV infection in a T-LGL lymphoma patient who presented in a non-endemic country. This case emphasizes the clinical importance of EBV monitoring in T-LGL patients with skin involvement. Notably,  The role of EBV infection as an etiological agent in T cell lymphomas, especially together with CD8+ T cell lymphoproliferative disorder, has recently gained attention [+ T cell lymphoma with skin involvement, Plasmodium falciparum malaria, and EBV infection, which has not been reported previously.Malaria affects mostly tropical and sub-tropical regions of the world and is known to cause acute and deadly complications such as cerebral malaria, respiratory distress and severe anaemia. However, malaria can cause unforeseen pathologies because of its chronicity , 2. For ttention . Here, a6/µL) and Hb levels (13.9 g/dL) with a high white blood cell levels  and low neutrophil (10.500/µL) and platelet (128.000/µL) levels. Investigation of a peripheral blood smear revealed 29% large granular lymphocytes (LGLs). Flow cytometric analysis of peripheral blood confirmed that 95% of lymphocytes (CD3+/TCRαβ+ population) were positive for pan-T antigens  and CD8, but negative for CD4 and CD56. Ultrasonography and FDG-PET-CT evaluation of the abdominal area found hepatomegaly, splenomegaly, and hypermetabolic supra-infradiaphragmatic lymph nodes as well as a hypermetabolic spleen. He had a history of malaria, but HCV and HIV tests were negative. These results were compatible with CD8+ T cell lymphoproliferative disorder with skin involvement. Therefore, a 0.5-cm-deep skin punch biopsy was performed in an inner part of the leg showing lesions.A 43-year-old Sudanese male was admitted to Acibadem University Hospital in Istanbul, Turkey with hyperpigmented painful skin rashes on his whole body. He was experiencing these symptoms intermittently for a year and self-medicated himself with non-steroid anti-inflammatory drugs with no fever or other health problems. He had recently experienced joint pains. A complete blood count during admission showed normal erythrocyte counts  for 6 weeks, and further immunopathological parameters were evaluated in skin lesions.LGL leukaemia is a rare lymphoproliferative disease and presents with anaemia, neutropenia, and an increase in the number of LGLs . About 8features . Althoug+, CD2+, and CD5+, among which 90–95% were positive for CD8  staining showed that the epidermis was minimally spongy and the upper dermis was oedematous with mild perivascular lymphocyte infiltration. However, the deep dermis was infiltrated by intra- and peri-vascular small lymphocytes Fig. a. StandaAfter this final diagnosis, methotrexate and Prednol were administered for 6 weeks. Six weeks later, blood values had normalized, skin and arthritis symptoms subsided, and the patient was discharged. No other follow-up could be performed because the patient returned to his country. However, because the patient was from Sudan, a malaria endemic region, and the unusual presence of small intra-cytoplasmic microorganism-like structures in histiocytic cells, the pathologist, who had no experience with malaria cases, consulted a malaria specialist at Osaka University, Japan.Plasmodium parasites and readily captured by macrophages and the reticuloendothelial system of the host, which can be easily recognized as birefringent crystals under polarized light [Plasmodium parasites [P. falciparum DNA was amplified  transcripts were investigated in paraffin-embedded tissue samples and found to be positive in macrophage-like histiocytes Fig. .Fig. 3EBP. falciparum and EBV is a major risk factor for endemic Burkitt’s lymphoma (eBL) development [Here, an unexpected and surprising diagnosis of malaria and EBV infection in a T-LGL lymphoma patient who presented in a non-endemic country was reported. Malaria infection has been restricted to the tropical and sub-tropical areas of the world , hence, elopment . In thiselopment .+ T cells in blood, and the presence of skin lesions with similar immunopathological features suggested that this case was not primary cutaneous CD8+ T cell lymphoma, but a more general and rare version of T-LGL. Therefore, EBV was not suspected in the aetiology during the diagnosis, although EVB has been investigated in the aetiology of cutaneous T cell lymphomas [+ T cell lymphoma.This case has several unique and noteworthy features that complicate the diagnosis and etiology of the multimorbidity. First, the presence of increased LGLs, a higher number and percentage of CD8ymphomas . FurtherP. falciparum infection in the patient by specific nested PCR.Second, previously unseen microorganism-like structures inside the macrophages and giant histiocytes, which could not be stained by any dye, were beyond the expertise of the pathologist for the differential diagnosis of the case. Malaria has been eradicated for more than 50 years ago from many parts of the world including Turkey, resulting in clinicians/pathologists lacking knowledge for its diagnosis and related complications. In the current case, the diagnosis of birefringent haemozoin crystals in the skin tissue led to the suspicion and confirmation of P. falciparum and EBV is a major risk factor for eBL development, although the aetiological relationship is somehow enigmatic [+ T cell populations that contain less central memory cells, which might allow EBV reactivations. Hence, repeated/continuous malaria co-infection with EBV reactivation may influence T cell maturation and differentiation for an increased risk of cancer. Although it is a speculation, one of these scenarios might be present in the current case.The co-infection of nigmatic . One of nigmatic  has showThe third and last unique feature of this case is the presence of EBER in macrophage/macrophage-like giant histiocytes. Although B cells, T cells, and epithelial cells are major targets for EBV, monocytes and macrophages may also play a role in the primary infection or reactivation of EBV . NotablyIn addition, methotrexate as an immunosuppressive therapy agent is known to activate EBV infection and lymphoproliferative disorders including oral mucosa lesions , while rPlasmodium infection should be examined in patients from malaria endemic regions by pathological and molecular investigations. EBV co-infection and its possible involvement in T cell lymphoma risk could be another unforeseen complication of chronic malaria infection. Furthermore, the molecular mechanisms underlying the co-presence of Plasmodium, EBV infections, and CD8+ T cell lymphoma require further investigation in animal models [In conclusion, this case report emphasizes the clinical importance of EBV monitoring in T-LGL patients with skin involvement. Furthermore, l models .


Plasmodium falciparum infection in the Mount Cameroon area and predictors associated with RDT positivity.Malaria remains a diagnostic challenge in many endemic communities. Although rapid diagnostic tests (RDTs) are presently widely used for malaria diagnosis, there is a dearth of information on post-marketing surveillance on its efficacy in Cameroon. The present study evaluated the performance characteristics of CareStart™ Malaria HRP2 (histidine-rich protein 2) antigen (Ag) RDT in diagnosing Plasmodium falciparum (G0141) Ag RDT was evaluated in a cross-sectional community-based survey involving 491 children of both sexes aged 6 months to 14 years between April and May 2018. Malaria parasitaemia was confirmed by light microscopy. Sensitivity (Se), specificity (Sp), positive (PPV) and negative (NPV) predictive values of the RDT, and the corresponding accuracy and Kappa value (κ) were determined using microscopy as the gold standard. Haemoglobin (Hb) concentration was obtained using an auto-haematology analyser. Results were compared using the chi-square test and associations between predictor variables, and RDT results were assessed using logistic regression analysis.The CareStart™ Malaria HRP2 κ = 0.52). RDT positivity was significantly associated with fever (P < 0.001), children less than 5 years (P = 0.02), history of fever within a month (P < 0.001) and anaemia (P = 0.002).Microscopically confirmed malaria parasite prevalence was 27.7%, and geometric mean density was 187 parasites/μL of blood (range 70–1162). Se, Sp, PPV, NPV and accuracy were 82.4, 76.6, 57.4, 91.9 and 78.2%, respectively. Sensitivity depended on parasitaemia and reached 96.1% at densities ≥ 200 parasites/μL of blood. The accuracy of malaria parasitaemia (as assessed by the area under the receiver operating characteristic curve) to predict malaria by RDT was 75.4% (95% CI 70.6–80.1). The agreement between microscopy and RDT was moderate (P. falciparum infection is moderate and is most useful at parasitaemia ≥ 200 parasites/μL of blood and presentation with fever. While RDT is effective as a diagnostic test for confirmation of clinical cases of malaria, its applications in population screening with a higher proportion of asymptomatic cases are limited.The overall concurrence of CareStart™ Malaria HRP2 pf Ag RDT with microscopy in the detection of  Plasmodium falciparum infections are asymptomatic. Falciparum malaria continues to negatively impact human life and fragile economies. Globally, malaria is still a public health concern as 445,000 deaths were caused by malaria in 2016 and Cameroon alone accounted for 3% of this number [Plasmodium falciparum histidine-rich protein 2 (PfHRP2), that can be captured by monoclonal antibodies raised against these antigens in the form of a RDT [In malaria-endemic countries, many s number . Howevers number  cites Cas number  cites Cas number , 11. Malof a RDT .Some communities in malaria-endemic areas lack healthcare facilities, and diagnosis of malaria relies predominantly on its clinical presentation which is nonspecific. Although presumptive diagnosis of malaria is less expensive , the accThe examination of Giemsa-stained blood smears for the detection of malaria parasites using light microscopy therefore remains the gold standard for malaria diagnosis, as it provides information on both parasite species and density . HoweverIn 2008, WHO and the Foundation for Innovative Diagnostics (FIND) jointly implemented an evaluation programme, which coupled product testing with a post-purchase lot verification service, to assess, review and compare the performance of malaria RDTs in a standardised manner . Until rIn many malaria-endemic countries, post-marketing surveillance is non-existent and there are no practical tools that can be used by central reference laboratories or at point-of-care to find out if RDTs are still performing optimally after delivery. Despite persistent high malaria prevalence, and the continuous influx of RDTs into our markets, the surveillance of RDTs in the Mount Cameroon area seems virtually ignored, hence making this study necessary.P. falciparum. The antibodies are mixed with colloid gold which conjugate and react with the HRP2 antigen in the patients’ samples. The HRP2 is highly abundant and heat stable; however, the HRP2 antigen remains in circulation for up to 4 weeks after the malaria parasites have been cleared [The CareStart™ Malaria HRP2 (G0141) Ag RDT is inexpensive, stable at high temperatures (40 °C) and mostly used in health units in the Mount Cameroon area . It cont cleared . AlthougSo far, several studies conducted in various parts of the country have determined the sensitivity and specificity of assorted brands of malaria RDT kits, including the CareStart™ , 23, 24,P. falciparum is the main malaria parasite species accounting for over 90% of reported malaria, and Anopheles gambiae is the main vector species. Malaria transmission is perennial with two peaks—the beginning and end of the rainy season [The study was carried out in Dibanda, a rural community located in the Mt. Cameroon area, Fako Division of the South West Region of Cameroon between April 2018 and May 2018. The coordinates of Dibanda ranged from altitude 358 m, latitude 04°06.447′ N, longitude 009°18.725′ E to 400 m, 04°07.179′ N and 009°18.464′ E. The study community comprised of seven residential quarters spanning the entire Dibanda community. The community has no Health Centre and the closest centre offering health services, found in the next town Mutengene, is the only government-owned institution that offers affordable health services to both the Mutengene community and other neighbouring communities. This study area is subjected to a Cameroonian-type equatorial climate characterised by a temperature range of 18–27 °C [y season .Although the indigenes of this area are of the Bakweri tribe and part of the Bantu ethnic group , its ferThe participants included pre- and school age children of both sexes aged 6 months to 14 years old. They weighed > 5 kg and were free from other clinical conditions not related to severe malaria and the sickle cell disease. Exclusion criteria in this study included children with severe malaria  [n = Z2pq/d2 [n = the sample size required, z = 1.96: which is the standard normal deviate , p = 66.2%: proportion of malaria prevalence, q = 1–p: proportion of malaria negative children and d = acceptable error willing to be committed. The minimum sample size was estimated as n = 255. The sample size was then increased by 25% to a minimum of 319 participants to account for anticipated non-respondents, incomplete data entry and loss of samples due to blood clotting. A convenience sampling method was used in all the blocks within the study area until the required sample was attained.The study was a cross-sectional community-based design carried out during the peak malaria transmission period in the Mount Cameroon area of April and May 2018 . Followi Z2pq/d2  where n Information on malaria knowledge and preventive methods was obtained from the participants using a pre-tested, structured questionnaire. The questions included demographics , malaria knowledge  and prevention methods as well as the history of fever.The axillary temperature was measured using an electronic thermometer and fever was defined as temperature ≥ 37.5 °C .Plasmodium were observed on the blood film. Parasite density was determined on thick blood film by systematically counting the number of parasites per 200 white blood cells and multiplying the parasite count with the participants’ white blood cell count obtained from the full blood count analysis.Three to four millilitres (ml) of venous blood was collected from each child using sterile disposable syringes. Drops of whole blood were dispensed immediately on RDT kits using a sample applicator and on slides for the preparation of thick and thin blood films. The remaining blood was dispensed into labelled ethylenediamine tetra-acetate tubes and placed in a cool box for transportation to the Malaria Research Laboratory, University of Buea, for a full blood count analysis. The air-dried thin blood film was fixed in absolute methanol, and both thick and thin blood films were stained using 10% Giemsa solution for 20 min. Each blood film was independently examined microscopically by two well-trained and well-experienced parasitologist, following standard procedure for the detection and identification of malaria parasites . Slides A complete haematological assessment of each blood sample was performed using the MINRAY 2800 BC auto-haematology analyser, following the manufacturer’s instructions, and anaemia was defined as Hb < 11.0 g/dL .P. falciparum.The CareStart™ Malaria HRP2 pf  Ag RDT is a chromatographic test for in vitro diagnosis. It has a long shelf-life of more than 1 year, with storage conditions ranging from 1 to 40 °C. Each kit is composed of 25 chromatographic test strips fixed in a cassette, lysis buffer, a pack of 25 lancets and disposable alcohol swabs saturated with 70% isopropyl for disinfection. The CareStart™ Malaria Pf (HRP2) Ag RDT contains a membrane strip pre-coated with a monoclonal antibody as a single line across the test strip conjugated to a signal, typically colloidal gold. The monoclonal antibody is specific to HRP2 of P. falciparum.Each CareStart™ Malaria Pf (HRP2) Ag RDT device was labelled with an identification code like that used on the slide for each study participant. Approximately 5 μL of venous blood was added onto the test device window using the sample applicator provided with the kit. This was followed by adding 2 drops (60 μL) of reagent buffer. The device was allowed untouched for 20 min at room temperature, and the result was recorded as per the instruction of the manufacturer. Negative results were recorded when only a band appeared on the control area whereas the presence of two bands, one band in the control area and the other band in the test area were recorded as a positive result for Plasmodium with an axillary temperature of < 37.5 °C.Asymptomatic malaria parasitaemia was defined as the presence of Plasmodium, with an axillary temperature of ≥ 37.5 °C, joint pains, vomiting, headache, diarrhoea, chills.Clinical malaria parasitaemia was defined as the presence of True positive (TP): Have the disease and test positive.False positive (FP): Do not have the disease but test positive.True negative (TN): Do not have the disease and test negative.False negative (FN): Have the disease but test negative.Sensitivity (Se) of the test is the ability of the test to identify correctly those who have the disease (true positive rate). Se = TP/(TP + FN) .Specificity (Sp) of the test is the ability of the test to identify correctly those who do not have the disease (true negative rate). Sp = TN/(TN + FP) .Positive predictive value (PPV) is the probability that a disease is present when the test is positive. PPV = TP/(TP + FP) .Negative predictive value (NPV) is the probability that the disease is not present when the test is negative. NPV = TN/(TN + FN) .Accuracy (Acc) is the overall probability that a patient will be correctly classified. Acc = (TP + TN)/(TP + TN + FP + FN).χ2). Group means were compared using analysis of variance (ANOVA), and parasite density was log-transformed before analysis. Using light microscopy of Giemsa-stained thick blood film as the gold standard, the RDT Se, Sp, false positive rate, false negative rate, PPV, NPV and Acc were calculated. Inter-test agreement for both results of positive and negative readings was expressed by the percentage of overall agreement and kappa value (κ) for the agreement between malaria RDTs and the reference method (microscopy). The detection limit was calculated from the sample with lowest parasitaemia but highest sensitivity and specificity. Associations between predictor variables and primary outcomes were assessed using both bivariate and multivariate logistic regression analysis. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed. Any covariate with a P value < 0.2 in the bivariate analysis was subsequently included in the final multivariable logistic model. Significant levels were measured at 95% CI with the level of significance set at P < 0.05.Data collected was cleaned up and analysed using the IBM-Statistical Package for Social Sciences (IBM-SPSS) version 20 and Epi-info version 7. Continuous variables were summarised into means and standard deviations (SD), and categorical variables reported as frequencies and percentages were used to evaluate the descriptive statistics. The differences in proportions were evaluated using Pearson’s chi-square  and females (42.9%) was not statistically significant (P = 0.111). Conversely, the geometric mean parasite density (GMPD) was significantly higher (P = 0.025) in males (218 parasites/μL of blood) than females (172 parasites/μL of blood). A significant difference in P. falciparum infection by microscopy (P = 0.002) and RDT (P = 0.001) was observed with age, with the ≤ 4 years age group having the highest malaria prevalence (31.9% by microscopy and 47.4% by RDT), followed by the 5–8 years age group (29.9% by microscopy and 38.1% by RDT) and least by the ≥ 9 years age group (11.9% by microscopy and 23.8% by RDT). In addition, children ≤ 4 years of age had the highest GMPD (224/μL of blood), followed by the 5–8 years age group (159/μL of blood) and lastly the 9 years age group (141/μL of blood).The prevalence of falciparum malaria among the 491 children varied with sex as shown in Table P. falciparum infection was highest in children whose parents had no formal level of education (63.2% by microscopy and 57.4% by RDT), while children whose parents had tertiary level of education had the least prevalence (9.0% by microscopy and 14.1% by RDT), and the difference was significant at P < 0.001 and P < 0.001 respectively  when compared with children who did not use a bed net (48.4% by microscopy and 46.7% by RDT).Children who used mosquito bed net had a significantly lower P. falciparum infection prevalence was significantly higher by microscopy (P = 0.039) and RDT (P < 0.001) in children with anaemia (30.3% by microscopy and 44.5% by RDT) than those non-anaemic (20.9% by microscopy and 26.9% by RDT) as shown in Table P. falciparum, while the false negative rate was 8.1% (24) among the children as shown in Table κ) between microscopy and CareStart™ Malaria HRP2 pf Ag RDT (G0141) was 0.52.The CareStart™ Malaria HRP2 pf Ag RDT had a sensitivity of 82.4% (95% CI:74.9–88.4%) and specificity of 76.6% (95% CI 71.9–80.9%). The PPV, NPV and Acc were 57.4% (95% CI 52.4–62.3%), 91.9% (95% CI 88.7–94.2%) and 78.2% (95% CI 74.3–81.8), respectively. False positive results were observed in 42.6% (83) of children with microscopy negative results for P < 0.001), fever (P < 0.004) and parasite density (P < 0.001). The positivity of the RDT was 100% for clinical malaria parasitaemia  and 72.2% among febrile children . The RDT demonstrated a sensitivity of 96.1% for parasite densities above 200 parasites/μL of blood , less than 5 years old (P < 0.02), those who had fever within a month (P < 0.001), and those anaemic were more likely to have a positive RDT result. Febrile children were 6.8 times more likely to have a positive RDT for P. falciparum than non-febrile children while those under 5 years of age were 2 times at odds of having a positive RDT than their contemporaries. In addition, children with a history of fever within a month at the time of the survey were 4.57 times more likely to be positive for P. falciparum infection by RDT when compared with children without fever. Moreover, those with anaemia were 2 times at higher odds of having a positive outcome with RDT than non-anaemic children as shown in Table A multivariate analysis demonstrated that children who were febrile . The optimal cutoff for the diagnosis of malaria by RDT was 77 parasites/μL of blood.The determination of the diagnostic performance characteristics of the rapid immune-chromatographic methods is essential given the need for accurate and prompt diagnosis before treatment. With the availability of various RDTs, a limitation of comparative field trials and the heterogeneity of the transmission and epidemiology of malaria in Cameroon, the findings of the study provide essential information for an informed decision to be made by the NMCP.Anopheles mosquitoes and consequently malaria transmission [A prevalence of 27.7% for malaria was observed by microscopy in this study population. This prevalence was similar to the 29.6% reported by Apinjo et al.  in childsmission , 36. Alssmission  to cemensmission  and provsmission . In addismission .Malaria prevalence was higher in females than males by microscopy and the findings are in line with Kimbi et al.  who repoThe high prevalence of anaemia 72.7%) in children less than 15 years is not an uncommon finding in this part of the country, and this highlights anaemia as a major public health problem among the population in this area. The relationship between malaria parasitaemia and anaemia is well established in previous studies .7% in ch, 39. HowP. falciparum to be 84.8–92.0%. Findings from this study demonstrated a higher CareStart RDT (G0141) performance characteristics when compared with a previous study carried out by Ndamukong-Nyanga [In the present study, CareStart RDT sensitivity (82.4) and specificity (76.6) did not reach the values indicated by the manufacturer . The PPV was 57.4%, and the NPV was 91.9%. This could be explained by the difference between populations used to estimate these parameters. In the present study, we evaluated a population with all levels of parasitaemia, whereas the manufacturer’s tests were done on samples with a density of > 200 parasites/μL of blood. The results from this study are consistent with similar studies reported in Africa. A study by Wanja et al.  in Westeg-Nyanga  in the Mg-Nyanga .On the other hand, a study by Maltha et al.  in BurkiThe sensitivity of the RDT in this study increased with an increase in parasite density achieving a sensitivity of 96.1% at parasite density greater than or equal to 200 parasites/μL of blood, as recommended by WHO . This isFindings from the study showed a low positive predictive value (57.4%) indicating a high false positive rate. This could probably be due to prior infection and subsequent effective treatment as reported in the structured questionnaires. This study indicated that children who had a history of fever within a month prior to the survey were 4.6 times more likely to be positive by RDT. The HRP2 antigen detected by this RDT has been demonstrated in several studies to persist in the blood stream between 14 and 30 days before being cleared completely , 47. MorThe relatively high negative predictive value (91.9%) in this study indicates the likelihood of the RDT to correctly identify a child without malaria as true negative, hence showing a substantive ability to differentiate between children who had malaria and those without. Hence, this RDT could be confidently used to confirm negative test patients as non-malaria patients. The false negative results produced by CareStart (G0141) could be accounted for by deletions or mutations of the HRP2 gene such that the parasite no longer produces the antigen or produces the mutant antigen that is not recognised by antibodies on the test strip .This study revealed that children under 5 years were 2 times more likely to be positive for RDT when compared with older children. Similar results with a decrease in sensitivity of the Paracheck-Pf RDT in older age groups have been reported in Tanzania . This isPlasmodium infection and a higher parasite load. Malaria has been reported by several authors to contribute substantially to anaemia in malaria-endemic regions [Findings in this study also revealed higher odds of having a positive RDT among anaemic than non-anaemic children. This is probably because of the likelihood of anaemic children having  regions , 51 and P. falciparum infection in the community is moderate and is most useful at parasitaemia ≥ 200 parasites/μL of blood and presentation with feverin in this malaria-endemic region. The RDT has proven to be very effective as a diagnostic test for confirmation of clinical cases of malaria. However, its applications in population-based screening in the community with a higher proportion of asymptomatic cases are limited. Control measures involving population screening should be complemented with microscopy for non-clinical cases for proper case management. Considering that RDT positivity is significantly associated with fever, age less than 5 years, history of fever within a month and anaemia, its wide use can significantly improve the quality of malaria case management by avoiding indiscriminate use of antimalarial drugs for parasite negative patients in malaria-endemic regions. As a recommendation, the NMCP should ensure more assessment for the performance evaluation of the different malaria RDT brands available in the Cameroon market for good management of the disease.The overall concurrence of CareStart™ Malaria HRP2 pf (G0141) with microscopy in population screening for


Plasmodium falciparum and Plasmodium vivax samples diluted to two densities, along with malaria-negative samples from healthy individuals, and from people with immunological abnormalities or non-malarial infections. Three measures were established as indicators of performance, (i) panel detection score (PDS) determined against low density panels prepared from P. falciparum and P. vivax wild-type samples, (ii) false positive rate, and (iii) invalid rate, and minimum criteria defined. Over eight rounds of the programme, 332 products were tested. Between Rounds 1 and 8, substantial improvements were seen in all performance measures. The number of products meeting all criteria increased from 26.8% (11/41) in Round 1, to 79.4% (27/34) in Round 8. While products submitted to further evaluation rounds under compulsory re-testing did not show improvement, those voluntarily resubmitted showed significant increases in P. falciparum (p = 0.002) and P. vivax PDS (p < 0.001), with more products meeting the criteria upon re-testing. Through this programme, the differentiation of products based on comparative performance, combined with policy changes has been influential in the acceptance of malaria RDTs as a case-management tool, enabling a policy of parasite-based diagnosis prior to treatment. Publication of product testing results has produced a transparent market allowing users and procurers to clearly identify appropriate products for their situation, and could form a model for introduction of other, broad-scale diagnostics.Malaria rapid diagnostic tests (RDTs) emerged in the early 1990s into largely unregulated markets, and uncertain field performance was a major concern for the acceptance of tests for malaria case management. This, combined with the need to guide procurement decisions of UN agencies and WHO Member States, led to the creation of an independent, internationally coordinated RDT evaluation programme aiming to provide comparative performance data of commercially available RDTs. Products were assessed against  Plasmodium parasites causes clinical presentation indistinguishable from other fever-causing pathogens, rapid, accurate diagnosis is a crucial component of effective case management [Plasmodium falciparum and/or Plasmodium lactate dehydrogenase (pLDH) expressed by all human malaria species [Malaria continues to be a serious threat, responsible for approximately 435,000 deaths in 2017 [The first malaria RDTs emerged in the early 1990s , and theP. falciparum and Plasmodium vivax samples was undertaken in 12 countries in Africa, Asia, and South America [P. falciparum and P. vivax and had antigen above a minimum threshold consistent with clinical infection were included [Development of a coordinated effort to quality control malaria RDTs pre-purchase (product testing) and post-purchase (lot testing) began in 2002 at the WHO Regional Office for the Western Pacific (WPRO) as a collaboration with the Special Programme for Research and Training in Tropical Diseases (TDR) and the WHO Roll Back Malaria Programme. In 2003 WPRO convened a multi-partner consultation including the Philippines Research Institute for Tropical Medicine (RITM), the Institut Pasteur du Cambodge (IPC)/Cambodian National Malaria Centre (CNM), TDR, WHO-RBM, US Centers for Disease Control and Prevention (CDC), and the Hospital for Tropical Diseases (HTD) . Subsequ America . Samplesincluded , 11.P. falciparum, P. vivax and negative samples, to enable WHO to develop evidence-based recommendations on product selection  to a PDS of ≥ 75% against the 200 parasites/μL density for both species in all transmission settings [After 4 years of development, specimen collection and piloting, in 2007, the WHO and the Foundation for Innovative New Diagnostics (FIND) implemented lot testing services (testing a sample of a production lot) on a limited basis at RITM and IPC/CNM. Soon after, WPRO issued recommendations that procurers only purchase products manufactured under the ISO 13485 standard, and submit a sample from each production lot, for lot-testing. However, comparative performance assessment was still needed to guide initial procurement decisions. Therefore, in 2008, the WHO invited ISO 13485-certified manufacturers to participate in the first round of ‘product testing’ to be conducted at the CDC, which assessed detection accuracy, reliability, and heat stability of commercially available RDTs, against a large panel of P. falciparum parasites diluted in whole blood to 200 parasites/µL, with each sample being tested on two RDTs from each lot. A higher density of 2000 parasites/µL was also tested on one RDT from each lot. Products needed to meet a PDS of ≥ 80% against the 2000 parasites/µL density samples to proceed to Phase 2.Prior to each round of product testing, WHO issued a call for expression of interest to invite manufacturers to submit products for assessment. Manufacturers must have had a valid ISO 13485:2003 certificate to participate, and those accepted needed to submit more than 1000 RDTs from 2 lots, for each product. Evaluation was performed using cryo-preserved blood samples, with testing divided into two phases. During Phase 1, products were screened against 20 cultured P. falciparum samples consisting of paired dilutions at 200, and 2000 parasites/µL, , 35 wild type P. vivax pairs, and 100 microscopy and PCR malaria negative samples from transmission-free populations with no recent history of exposure to malaria and half containing no known pathogens or immunological factors (clean negatives), and the other half containing pathogen and immunological factor-containing blood (dirty negatives). When wild type samples were depleted following a testing round they were replaced with new samples ensuring no statistical difference in the distribution of panel antigen concentration between rounds [The Phase 2 panel comprised approximately 100 wild-type During evaluation, RDT results were read by two trained personnel; the first reader determined results at the minimum manufacturer stated time and the second reader as soon as possible thereafter (< 30 min). The second reader was blinded to results from the first read. Test line intensity was recorded on a scale of 0 (no band) to 4 (strong band) using standard colour charts, with intensities 1–4 classified as positive. The PDS was used as the performance measure to score products in each phase. Since Phase 1 acted as a screening step, only PDS measured in Phase 2 was used for product assessment. Results from the first read were used to determine PDS.The PDS measure was developed to reflect both product sensitivity and reproducibility. It required all four tests, two from each of two manufacturing lots, against the same sample (at 200 parasites/µL) to be positive to register as “detecting” the sample, and quantifies the percentage of samples the product detected  overall, (ii) against each type of negative specimen, and (iii) as incorrect species detection. An invalid rate was reported for all products, with an invalid test defined as an absence of control line at the time of reading. Invalid tests were not repeated during product testing.The number of requests from manufacturers to submit products for testing generally increased over the eight rounds Fig. . In fiveIn total 332 products were evaluated over the eight rounds of testing; 227 were unique , with thP. falciparum detecting RDTs with just under half  the products having PDS ≥ 75% in Round 1 compared to 88.2% (30/34) in Round 8  of Round 1 products had a PDS ≥ 75%, which increased to 91.7% (22/24) in Round 8 , 4 pan only, one product detecting P. falciparum on one line with a separate line detecting P. falciparum and P. vivax together and one product detecting P. falciparum on one line with a separate line detecting P. vivax and pan.. Between Rounds 1–8, the proportion of products eligible for procurement based on performance indicators more than tripled from approximately 25% to > 80% . Only eight of these 19 products detected P. vivax, and all except one were above the recommended PDS threshold of ≥ 75%. There was no significant change in the P. vivax PDS . Overall there was a significant decrease in median false positive rate of 1.6% . Seventeen out of 19 products met the procurement criteria on either initial or repeat evaluation, with 12 meeting the criteria at both evaluation points.Twenty-two, 19, 30 and 27 products were due for compulsory resubmission in Rounds 5 through 8. However, only 19 of these were actually resubmitted; 10 in Round 5, two in Round 6, five in Round 7 and two in Round 8. Results from the first and last evaluations are summarized in Table P. falciparum PDS of 9.7% , and a non-significant decrease in the mean false positive rate of 0.1% . Among the 37 P. vivax detecting products, significant P. vivax PDS improvements were observed with a mean change of 35.5% . Fifteen products met the procurement criteria on initial evaluation, compared with 31 on repeat evaluation; 13 products met procurement criteria at both evaluation points.Of the 53 products voluntarily resubmitted, there was a significant improvement in mean Spawned by challenges of field studies, weak IVD regulation, and the need to expand access to high quality malaria diagnosis, the WHO Malaria RDT Product Testing Programme has over the past decade generated performance data on 332 products. Through direct feedback to manufacturers and global stakeholder dissemination and communication efforts, the Round 1 report catalysed an evolution of malaria diagnostic testing by revealing a subset of high-performing products . This prThe product testing results also provided detailed information for manufacturers which sometimes resulted in changes in the instructions for use (IFU). For instance, observations from Round 1 showed the results from the second RDT read were often better than the first read at the manufacturers’ recommended reading time. This information was fed back to manufacturers, with many subsequently changing their IFU to increase the recommended reading times from 15 to 20 min.The comprehensive testing protocol and transparent reporting of results not only facilitated product selection, but generated performance-based competition between manufacturers so as to capture a larger market share. A substantial improvement in test performance was associated with this, while prices have fallen , 19. AftBeyond positive changes in RDT performance, uptake and use in practice, there is evidence that the program has influenced the RDT marketplace. Specifically, FIND conducted a manufacturer survey which showed the proportion of RDTs sold with a PDS ≥ 75% more than doubled from 23% in 2007, to 57% in 2009 and tripled by 2010 to 78%, coinciding with the release of the first and second product testing reports . SimilarP. falciparum-only HRP2 RDTs in 2018 and also for RDT combination tests in 2019. An exception exists in which non-WHO prequalified RDTs, that meet performance criteria and specifically target non-HRP2 antigens, can be used in areas where pfhrp2 deletions are prevalent as an interim measure [Between 2009 and 2019, all major public sector procurers have continuously had in place policies stating diagnostic test budgets can only be spent on RDTs that are recommended by the WHO. WHO recommendations on procurement of RDTs have evolved over the past decade being initially based on the results of product testing between 2009 and 2017, followed by a requirement for WHO prequalification for  measure , 22–24.  measure . The resLot performance variation is an issue for all diagnostics. The product testing program tested RDTs from two different lots selected and supplied by manufacturers. There is no guarantee that results for the two lots submitted for evaluation are representative of every subsequent lot. Therefore, the WHO recommends both proactive and reactive post market surveillance to identify sub-standard lots prior to and/or post field deployment and continues to support needs of the global community through centralized testing at the Research Institute of Tropical Medicine, Philippines and the WHO has supported local capacity development for lot verification for malaria RDTs in Nigeria  and India  , 27.The objective of the WHO malaria RDT product testing programme was to provide independent comparative performance data to guide procurement decisions of UN agencies and WHO Member States. Through the close collaboration with FIND, CDC and several other partners, this objective has not only been repeatedly fulfilled, but the programme has influenced policy, clinical and manufacturer practice and helped shape the global market. Ultimately, it has driven improved product performance by establishing broadly accepted minimum performance criteria , 28, 29,Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi, and P. falciparum lacking HRP2.The RDT evaluation programme also served as a model for establishing and ensuring performance standards for RDTs detecting other diseases. To date, a leishmaniasis  and Ebol