Plasmodium spp. was determined using a rapid diagnostic test (RDT) and a polymerase chain reaction (PCR). Demographic, house locations, medical history, and biological data were analyzed. Factors associated with Plasmodium spp. carriage were analyzed using logistic regression, and the carriage localization was investigated through spatial cluster analysis. Of the 1,501 samples analyzed with PCR, positive results totaled 90 and 10 for Plasmodium vivax and Plasmodium falciparum, respectively. The general PCR prevalence was 6.6% [5.3–7.9], among which 74% were asymptomatic. Only 13/1,549 were positive by RDT. In multivariate analysis, participants older than 15 years, living in a remote neighborhood, with a prior history of malaria, anemia, and thrombocytopenia were associated with an increased odds of Plasmodium spp. carriage. High-risk clusters of P. vivax carriage were detected in the most remote neighborhoods on the village outskirts and two small foci in the village center. We also detected a hot spot for both P. vivax and P. falciparum symptomatic carriers in the northwestern part of the village. The present study confirms a wide-scale presence of asymptomatic P. falciparum and P. vivax carriers in this area. Although they were more often located in remote areas, their geographic distribution was spatially heterogeneous and complex.To implement future malaria elimination strategies in French Guiana, a characterization of the infectious reservoir is recommended. A cross-sectional survey was conducted between October and December 2017 in the French Guianese municipality of St Georges de l’Oyapock, located along the Brazilian border. The prevalence of  French Guiana is a French overseas territory and a malaria-endemic country on the South American continent. Historically, malaria was mainly due to Plasmodium falciparum, but in the past 15 years, Plasmodium vivax infections have become predominant.3 This area is committed to a regional program for malaria control.4 In remote areas, diagnosis and treatment are free of charge and administered in health centers using rapid diagnostic tests (RDTs) and artemisinin combination therapies. Vector control is also conducted in all active transmission areas. In-house residual spraying is conducted in households where malaria cases are detected. In addition, permethrin insecticide–treated nets are distributed for free to pregnant women, symptomatic malaria cases, and their family members.In 2016, the incidence of malaria worldwide increased for the first time since 2000.6 Although French Guiana’s coastal and urban areas have very low malaria transmission, the country’s inland sites experience higher levels.7 Illegal gold miners working deep inside the rainforest are massively infected, mostly by P. falciparum.8 Amerindian villages along the main rivers at the borders between Suriname and Brazil represent the second largest population affected by symptomatic malaria, mostly due to P. vivax.9Although the number of malaria cases has declined over the past 10 years, the region still faces major challenges in controlling malaria transmission among socially marginalized and/or isolated populations, such as gold miners and autochthonous populations.Plasmodium spp. provide a reservoir of infection in areas of transmission. Considering a parasitemia generally above 1 parasite/µL of blood, they may contribute to continuous transmission of the disease and are potentially responsible for the genesis of outbreaks.11 Therefore, to interrupt transmission, it is important to focus actions on identifying residual malaria transmission foci. Those efforts should then be intensified to eliminate any remaining foci. For P. vivax malaria infections, it has been clearly shown that both symptomatic and asymptomatic carriers are rapidly infectious.12 However, the scarcity of studies makes it difficult to identify the Amazonian region’s residual transmission areas, referred to as “malaria hotspots.” French Guiana’s population, notably gold miners, is expected to have strong malaria immunity, and many of them present asymptomatic parasitemia.13 Symptomatic malaria cases in this context occur throughout the year.7 In addition, prevalence of Plasmodium spp. is highly heterogeneous among the different mining sites.8 Apart from these, most of the remaining cases in French Guiana are detected at the border with Brazil (Northeastern French Guiana) and at a lower rate along the Surinamese border (Southwestern French Guiana), more specifically in Amerindian villages.7 Malaria transmission among these autochthonous populations is mostly seasonal, demonstrating an unstable pattern.7 Within these populations, it is likely that asymptomatic carriage exists.6Asymptomatic carriers of 7 In this municipality, Anopheles Darlingi was previously described as the predominant species.14 The vector density and number of breeding sites are high beginning a few weeks before the onset of symptomatic cases.15 However, the density of An. darlingi is heterogeneous, depending notably on the distance to the dense forest.16This study was conducted in the village of Saint Georges de l’Oyapock (STG), a persistent malaria-endemic area along the Brazilian border . Two peaPlasmodium spp. carriage in this Amazonian area, and the risk factors associated with malaria parasite carriage remain unclear. The aim of this study was to provide a better understanding of the distribution and contribution of Plasmodium spp. carriage to the overall parasite reservoir.To date, no study has investigated the distribution of Plasmodium spp. carriers to decrease malaria prevalence in the study area. This period of the year was chosen to cover the epidemic peak observed annually in this municipality.This transversal study was carried out between October and December 2017 in the municipality of STG , and wasPlasmodium spp. infection in this transmission area, 13 STG neighborhoods were selected based on their history of an elevated incidence of symptomatic cases over the last 2 years. These neighborhoods also included three remote villages located in the south of the municipality, 10 minutes by canoe for Blondin, or in the north, 1 hour by canoe for Trois-Palétuviers  in STG, mainly in the municipality center. Three neighborhoods  are particularly isolated and only accessible by canoe. The entire population size of the 11 studied neighborhoods was ∼2,727 inhabitants, all ages and nationalities included.6 Because of the good participation and mobility of the population in this area, we targeted a sample size of 1,500 participants for the first step of the study, expecting to finish a year later with a minimum of 1,200.6Designed for the “Palustop” before–after study, the sample size was calculated at 1,200 .® Malaria Ag Pf/Pan test . Malaria PCR detection was performed using a real-time method derived from Shokoples et al.17 Detection and identification of four Plasmodium species  were performed using a Taqman®  probe strategy, with a sensitivity of 1 parasite/µL for all the species except P. vivax at 0.5p/µL. Positive and negative controls were systematically added to the experiments. This method is certified according to the International Organization for Standardization 15189 requirements for medical examinations. Complete blood count, reticulocyte, and the glucose-6-phosphate dehydrogenase (G6PD) activity tests were also performed.Malaria RDTs were performed to immediately treat participants who tested positive. We applied the most commonly used RDT in French Guiana, the SD BIOLINE18Participants were interviewed using a questionnaire designed to gather information on knowledge, attitudes, and practices regarding malaria. Data on demographic factors such as gender, age, and occupation were collected. Participants were also questioned about their travel habits such as frequency and location of hunting, fishing, and farming in the border area. For children, parental consent was obtained and parents were interviewed on behalf of the child. The questionnaire was administered by trained cultural mediators in the language of the participants. Their houses were georeferenced. Participants’ body temperatures were measured by an ear thermometer, and fever was defined as any temperature ≥ 38°C. All participants received a medical examination. If RDT or PCR results were positive, participants were treated according to national treatment guidelines.Plasmodium spp. carriage was defined as a Plasmodium PCR-positive result, regardless of body temperature.Symptomatic cases were defined as RDT- or PCR-positive results associated with a history of fever in the last 48 hours or during the inclusion process. 19 were applied via SaTScan software . Clusters of high or low rates with maximum likelihood ratios were selected, and any overlapping clusters were excluded. A multi-scale analysis was performed by considering the study area as a whole . Then, because of their relative distances, the neighborhoods of Trois-Palétuviers, Blondin (1 and 2), and the STG downtown area were considered separately . Different analyses were also performed according to the Plasmodium species  and the presence or absence of symptoms . Map analyses were conducted in QGIS 2.12 software.Bernoulli methods proposed by Kulldorff et al.Plasmodium spp. carriage were investigated using logistic regression models. Bivariate analyses were used to select potential covariates (P < 0.1) for the multivariate model. A backward selection procedure was then applied to keep only significant associated factors (P < 0.05) in the final multivariate model. Odds ratios and associated 95% CIs were estimated. Statistical analyses were conducted with SAS version 9.4 software .Associated factors of Comité de Protection des Personnes du Sud-Ouest et Outre-Mer 4 N° AM-36/1/CPP15-024 (French ethics committee). The database was anonymized and declared to the French Regulatory Commission . Samples collected by the National Reference Center were registered by the French Ministry for Research . Duly signed, informed consent was obtained from all participants involved in the study, following explanations about the study in their local languages. Treatments and medical follow-up of malaria infection were offered.The study was approved by the A total of 1,566 inhabitants were included in the study (57.4% of the study area inhabitants). Of these, 1,501 (95.8%) individuals had a blood sample taken. Venipuncture failure or fear of blood sampling was the primary reason why certain participants did not contribute to the blood samples. The median age was 22.8 years (min–max: 0.2–92.9) and the sex ratio was 0.88 . A major9/L) were associated with Plasmodium spp. carriage  were screened for G6PD deficiency (N > 10 U/g Hg). The median value of G6PD was 11.9 [11.7–12.2] in males and 12.2 [12.0–12.5] in females. According to the WHO classification for G6PD activity ranges, the majority of them  had a normal G6PD activity (> 80%), 67 females  had an intermediate activity (30–80%), and only three females  and one male  had an activity less than 30% and 10%, respectively.20Severe anemia (< 8g/dL) was found in only two participants (0.14%). Anemia (< 10g/dL) and thrombocytopenia . In comparison, malaria prevalence using RDTs was 0.84% . The median age of the PCR-positive population was 23.5 years (min–max: 0.8–74.3), significantly higher than that of the PCR-negative population  and 38.5% (n = 5/13) of the PCR- and RDT-positive population, respectively. Asymptomatic carriage also varied according to the Plasmodium species: 100% (n = 10/10) for P. falciparum and 70.0% (n = 63/90) for P. vivax (P < 0.005). A strong heterogeneity of Plasmodium spp. carriage between neighborhoods ranged from 0.0% to 29.5%  or erroneous (n = 8) global positioning system localizations.Spatial data from thirteen individuals, all associated with negative PCR results, were removed from the analysis because of either missing , and Plasmodium spp. asymptomatic cases, respectively.At the whole study area scale, the Trois-Palétuviers neighborhood alone formed a high-risk cluster for asymptomatic P-value = 0.038) for symptomatic cases (considering Plasmodium spp.).The study of the Trois-Palétuviers neighborhood alone  indicated that the “center” of the neighborhood formed a low-risk cluster , 6.67 (P < 0.005), and 10.71  for the positive Plasmodium spp. cases, P. vivax cases only (asymptomatic and symptomatic), and Plasmodium spp. asymptomatic cases, respectively. No other significant results were derived from the local scale analysis for this neighborhood.At the whole study area scale, the southern part of the Blondin neighborhood (Blondin 2) formed a high-risk cluster for P. vivax carriers (two asymptomatics and two symptomatics) of the five household members. The second significant high-risk cluster, located in the Philogène quarter, included two neighboring houses, which had four asymptomatic P. vivax carriers among the eight household members. Except for these two very small hot spots, the surrounding neighborhoods presented significantly low relative risks. One of the most significant (P < 0.005) low-risk clusters (RR = 0.14), denoted as CL2, is represented in Two high-risk clusters in the Saint Georges downtown area corresponded, in fact, to specific homes . All the symptomatic cases in this area were P. vivax cases. What is more, among the total number of cases in this cluster (12), only one was associated with P. falciparum. Thus, these results reveal that this cluster predominantly comprised symptomatic P. vivax cases.By considering symptomatic cases for Plasmodium falciparum cases were pooled in the eastern part of the Saint Georges locality (CH4 in P = 0.417).y CH4 in . HoweverPlasmodium spp. carriage are shown in Plasmodium spp. carriage in the final multivariate model.Independent associated factors of Plasmodium spp. prevalence, ranging from 0.0% to 29.5% depending mainly on the neighborhood. The general prevalence was 6.6% IC95% (5.3–7.9). The majority carried P. vivax (90.0%) and was asymptomatic (74.0%).Active case detection of malaria in this border area revealed a heterogeneous Anopheles-vector biting time (4). The present study surprisingly revealed that the use of bednets is associated with Plasmodium spp. carriage in bivariate analysis. Also, when bednets were used, the presence of holes was associated with Plasmodium spp. carriage. In the same way, participants who reported the use of plants for medicinal purposes had a higher Plasmodium spp. carriage prevalence. This may be due to a confounding bias: Amerindian and Creole communities were the hardest hit by malaria and had the greatest numbers of users of traditional medicine and bednets.Numerous behavioral factors can increase the risk of infection. The present study showed that people aged from 14 to 25 years formed the most significant at-risk group. People older than the age of 14 may have more at-risk behaviors such as night hunting or fishing. Other contributing factors include activities in high transmission sites such as gold mines or even staying outside with friends during sunset, a known 22 The situation in this part of French Guiana reflects a similar trend. Blondin 2 and Trois-Palétuviers, the most remote neighborhoods, appeared to be the largest hot spots. Indeed, these results indicated that living near the forest and vector-breeding sites is associated with elevated Plasmodium spp. carriage, as previously identified along the Oyapock River.23 Concordantly, in the Trois-Palétuviers neighborhood, symptomatic cases were reported more significantly in the outlying areas, near high vector density sites. The role of vector density and human behaviors in sustaining symptomatic cases in this micro-area deserves to be explored. It is worth noting that complex malaria transmission foci were also found in the STG village center within Amerindian families. In this context, travel or certain practices (frequent fishing and hunting expeditions in this community) would explain those cases. Similarly, most P. falciparum carriage appeared in Onozo, a fishing neighborhood that may have hosted gold miners. Determining additional risk factors may help guide effective control programs among these border populations. Such risk factors include travel to specific regions or villages linked to imported malaria cases and potentially resulting in the start of transmission chains.24 In addition, the present study describes a fine-scale spatial distribution of Plasmodium spp. carriage, but further studies are needed to determine whether or not these hot spots are stable and potentially responsible for the seasonal transmission observed each year.As the current global context moves toward malaria elimination, shifts toward heterogeneous transmission of the disease are generally observed.Plasmodium spp. carriage, notably asymptomatic malaria, has significant health consequences. For example, anemia and thrombopenia are observed in both symptomatic and asymptomatic infections. Asymptomatic carriage can result in a low-grade hemolysis.25 In areas where asymptomatic carriage is highly prevalent, malaria-attributable anemia can be challenging. Helminth infections are common and can exacerbate anemia while simultaneously providing a degree of protection from symptomatic malaria.26 However, in our study, hypereosinophilia, which is a marker of helminth infection, was not associated with a lower rate of Plasmodium spp. carriage. Thrombocytopenia in asymptomatic carriage was previously described in Africa.27 The mechanism behind thrombocytopenia may be attributed to peripheral destruction and consumption of platelets.28 Thus, some authors propose replacing the term “asymptomatic malaria” with “chronic malaria infection” and suggest treating all cases of Plasmodium spp. carriage because of their health and social consequences.29Plasmodium species, and degree of immunity. In our study, the PCR deployed to detect parasite carriers was not an ultrasensitive method. With its detection limit around parasite/µL of blood, we could assume that patients detected positive have a high probability to transmit the disease. Moreover, P. vivax parasites generally generate gametocytes earlier than P. falciparum.A challenge is to identify asymptomatic individuals with a level of gametocytemia sufficient to transmit infection to mosquitoes. Asymptomatic carriers could transmit malaria even at low submicroscopic parasitemias. The limits for transmission are difficult to determine because of a combination of variables, including gametocyte maturity, Plasmodium spp. carriage is challenging. Control programs based on passive case detection do not detect asymptomatic infections, and, therefore, these carriers remain untreated. Because of the absence of symptoms, most infected people do not go to health facilities for testing or are under the detection limit of RDTs. Therefore, one option is to identify and treat asymptomatic patients. In our study, two remote neighborhoods represented 66.0% of the asymptomatic cases of this municipality. Such a high concentration of cases can positively influence efforts to implement control measures. Despite their distance from the STG village center, health authorities should consider these transmission hot spots if they expect better malaria control in this region. Remote-area residents were previously described as hard-to-reach populations often missed by control measures.30 Nonetheless, Trois-Palétuviers and Blondin 2 achieved the highest participation rates . These rates suggest that these communities have a high level of concerns regarding this disease and expect action from health authorities. This highlights the importance of providing easier access to diagnosis and care in these areas. Improvements to consider include training community residents in malaria testing, as is already carried out in Brazil, and facilitating travel between Trois-Palétuviers and health centers. Finally, there are additional elimination strategies worth examining. For example, intermittent preventive treatment should be considered, in particular for certain high-risk parasite carriage populations such as Trois-Palétuviers and Blondin 2 inhabitants older than 14 years. Other strategies to consider could also include seasonal malaria chemoprevention and mass drug administration.33 However, these approaches require rigorous risk-benefit analysis before any implementation. In fact, the associated risks include an increased drug pressure that may impose selection in favor of resistance and eventual side effects in asymptomatic patients. Benefits could lead to a decreased morbidity for participants and to malaria elimination in the community. However, at this time, in France and Brazil, WHO recommendations are followed and diagnostic testing for malaria is a prerequisite to receiving treatment in symptomatic cases.4 Detection of asymptomatic cases is not part of the control programs. To be addressed, policymakers must commit to elimination. In addition to these asymptomatic carriage hot spots, small foci around households are observed in other neighborhoods. Generally, in low-endemic settings, reactive case detection is a viable option for febrile and RDT-positive malaria patients.34 In these neighborhoods, reactive case detection by PCR should be accompanied by cultural mediator-assisted discussions with the entire household. The objective is to explain the purpose of this screening and to facilitate adherence to treatment and health recommendations.Whatever the country, Plasmodium spp. carriage in this area, where data were scare. These identified risk factors can help policymakers develop targeted intervention strategies along this border area in relation to the heterogeneous distribution. These findings may support a scientific-based choice for the future malaria program in France and Brazil, such as outreach initiatives in isolated neighborhoods that offer new points of community information, better access to malaria diagnosis, and treatment adapted to the level of transmission.This study allowed us to identify the main risk factors for


In non-endemic settings, expertise in malaria microscopy is limited and rapid diagnostic tests (RDTs) are an adjunct to malaria diagnosis.We performed an External Quality Assessment (EQA) on reading and interpretation of malaria RDTs in a non-endemic setting.Participants were medical laboratories in Belgium and the Grand Duchy of Luxembourg using malaria RDTs; they received (i) 10 high-resolution photographs presenting test line combinations of RDTs with interpretations listed in a multiple choice format and (ii) a questionnaire about their practices of malaria diagnosis.P. falciparum–pan-Plasmodium RDTs (113 (82.5%) products, 6 brands), most frequent errors were (1) disregarding faint test lines (18.6%), (2) reporting invalid instead of P. falciparum (16.8%) and (3) reporting “Plasmodium spp., no further differentiation possible” without mentioning the presence or absence of P. falciparum (11.5%). For four-band RDTs (21 (15.3%) products, 1 brand), errors were (4) disregarding faint P. vivax test lines (47.6%) and (5) reporting “Plasmodium spp., no further differentiation possible” without mentioning the presence of P. falciparum and P. vivax (28.6%). Instructions for use (IFU) of only 4 out of 10 RDT products mentioned to interpret faint-intensity test lines as positive (conducive to errors 1 and 4) and IFU of 2 products displayed incorrect information (conducive to errors 2 and 5). Outside of office hours, 36.1% of participants relied on RDTs as the initial diagnostic test; 13.9% did not perform microscopic confirmation.Among 135 subscribing laboratories, 134 (99.3%) used 139 RDT products (11 different products from 8 brands). After exclusion of the results of one laboratory, analysis was done for 133 laboratories using 137 RDT products. Scores of 10/10, 9/10 and 8/10 were achieved for 58.4%, 13.1% and 8.0% of 137 RDT products respectively. For three-band Reading and interpretation of malaria RDTs was satisfactory, but errors were embedded in the instructions for use of the products. Relying on RDTs alone for malaria diagnosis (about one third of participants) is not a recommended practice. Plasmodium, which is transmitted by the bites of the female infected Anopheles mosquito. There are five Plasmodium species affecting humans: Plasmodium falciparum , Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi [P. falciparum (99%) in children aged under 5 years (70%) [Malaria is a disease caused by the parasite knowlesi , 2, 3. Irs (70%) . The preP. falciparum malaria can quickly evolve to life-threatening complications. Outcome depends on timely diagnosis and prompt treatment [Plasmodium species and parasite density. This information is relevant for reasons of selection of antimalarial drugs and hospital admission  and eradication of liver forms  [Imported malaria is a rare but potentially fatal event: in the UK, 1300 to 1800 malaria cases are yearly reported with 2–11 deaths [reatment ,6,7,8,9.Plasmodium antigens in the blood. The present antigens migrate along a nitrocellulose strip and are captured by Plasmodium-specific antibodies coupled to a signal  which generates a visible cherry-red test line that appears within 30 minutes. RDTs also provide a so-called control line which indicates correct migration. Depending on the number of lines (“bands”) that appear on the strip in the cassette , RDTs are categorized as “two-, three-, or four-band RDTs, see Malaria microscopy requires considerable expertise , 11. In External Quality Assessment (EQA) of malaria RDTs remains difficult to perform on clinical blood samples as high volumes of blood are needed to be distributed to a large number of participants . The preth June 2013.The rapid diagnostic tests used to create the photographs for this study were obtained from patient’s tests. The ITM travel clinic adopted the presumed consent or opt-out procedure by which a written objection is given if they do not wish their biospecimens to be retained for research (or data used for research). The study protocol had been approved by the ITM institutional review board on 4Concerning the information of the patients, this work was conducted following the ISO 17043 standard indicating that: “the identity of participants was known only to people involved in coordinating the EQA programme, and was maintained confidential”. The Institute of Public Health is accredited ISO17043.Participants were diagnostic laboratories in Belgium and the Grand Duchy of Luxembourg which participated to the EQA (Proficiency Testing) program of microscopic malaria examination previously organised by the Institute of Public Health (IPH), Brussels, Belgium. Prior to the survey, diagnostic laboratories (further referred to as “participants”) were asked which malaria RDTs product(s) they used; a total of 11 different RDT products were used. These are all commercial tests.CareStart Malaria HRP2/pLDH (pf/pan) Combo test Carestart pLDH 3 line malaria Binax Now malaria Palutop 4+ optima OptiMAL-IT Malaria Total  Nadal Malaria Test SD Bioline Malaria Ag P.f./Pan (FK60) SD Bioline Malaria Ag P.v. (FK70) SD Bioline Malaria Ag P.f./Pan (FK40) Malaria -test cassette The EQA consisted of high-resolution photographs  in real-life dimensions presenting combinations of control and test lines at different line intensities. The reference (correct) interpretation was embedded in a multiple choice list of six options. Separate sets of photographs were made for each of the 11 RDT products and each laboratory received the set of photographs corresponding to the RDT product(s) it used. Two sets of photographs were used for multiple RDT products (one for three RDT products and one for two RDT products). In addition to the set of photographs, participants received a questionnaire about their practice of malaria diagnosis in particular with regard to the place of RDTs in the diagnostic algorithm for malaria.rd 2013 and participants were asked to return their (paper-based) results by post mail before September 17th 2013. Results were subsequently entered in an Excel spread sheet .Prior to distribution, photographs, questions and questionnaire were validated by an expert panel of participants, in line with IPH EQA procedures. The IPH sent out the EQA on September 3P. falciparum, or (iii) missing an invalid result. Minor errors included (i) not reporting the presence of Plasmodium non-falciparum  when reporting malaria, (ii) not reporting the possibility of a mixed Plasmodium species infection, (iii) reporting an incorrect species identification for non-falciparum species or (iv) reporting a negative result as positive.Answers submitted by the participants were compared to the reference results. Errors were categorised as major or minor depending on their potential impact on patient care. Major errors included (i) missing the diagnosis of malaria, (ii) not reporting the presence of P. falciparum line and a pan-Plasmodium line . The four-band RDT (one product) represented 21 (15.3%) of RDTs used; this RDT detects antigens specific to P. falciparum and P. vivax as well as a pan-Plasmodium antigen. Other RDT products were rare and included a two-band P. vivax RDT  and a single two-band P. falciparum RDT.Out of the 172 diagnostic laboratories which performed malaria diagnosis in 2013, 135 (78.5%) had declared to use a total of 11 different RDTs and were subscribed to the RDT EQA; participation rate was 99.3% (134/135). Most participants  used one RDT brand; three laboratories used two RDT products of different brands and one laboratory used three RDT brands. One laboratory reported combined answers for the two RDT products used–this answer was subtracted from analysis. As a result, the total number (denominator) of interpretable results for the photographs was 137 (numbers of RDT products used) from 133 participants and for the survey 132 ; Overall (n = 137), results were good to very good. Scores of 10/10, 9/10 and 8/10 were achieved for 58.4% (n = 80), 13.1% (n = 18) and 8.0% (n = 11) of 137 RDT products respectively; the lowest score was 5 (1 participant). There were no errors (major nor minor) for the SD BIOLINE Malaria Ag P.v (05FK70) or the SD BIOLINE Malaria Ag (05FK40).i.e. the three-band P. falciparum–pan-Plasmodium detecting RDT, two-thirds of RDT products  obtained the maximum score. The most frequently reported major error was disregarding a faint P. falciparum test line and reporting a negative result . This error was made by 2/16 CareStartTM HRP2/pLDH Combo Test users, 1/3 NADAL® Malaria Test users and 10/14 OptiMAL users but by none of the participants using Binax Now®. Although the P. falciparum test lines of the concerned products appeared slightly weaker compared to the Binax Now® product, they were still visible. Reviewing the instructions for use (IFU) showed that IFUs of only 4 out of 11 RDT products mentioned to consider test lines of weak or faint intensity as positive .For the main type of RDT used, P. falciparum malaria . This error occurred among all but one (13/14) OptiMAL users. Of note, the OptiMAL IFU mentioned that in case of co-presence of the control line and P. falciparum- (Pf-pLDH) test line without a visible pan-Plasmodium (pan-pLDH) test line, the result should be interpreted as “invalid” , however this answer did not disclose the presence  nor absence  of P. falciparum  related to the interpretation of the presence of both P. falciparum and non-falciparum test lines, for which the correct answer is “P. falciparum, possibility of a mixed infection”: errors occurred in both senses (i.e. not mentioning the possibility of mixed infection when both lines were present (n = 6) as well as mentioning the possibility of mixed infection in the case of only the P. falciparum line visible (n = 10)). Additional errors occurred sporadically among products and participants and were random, suggesting clerical errors.In addition, “P. vivax–which represented a major error. Another major error (committed by more than a quarter (28.6%) of participants) consisted of reporting “Plasmodium spp., no further differentiation possible” instead of mentioning the presence of both P. falciparum and P. vivax. The latter error was embedded in the IFU, which mentioned to report “Presence of Plasmodium” for this combination of test lines  missed a faint test line and reported negative instead of st lines . There w the IFU .P. falciparum . The two users of the SD BIOLINE Malaria Ag P.v (05FK70) test did not make any errors.For the two-band ulti med product, the single user reported two negative results instead of In total there were 28 minor and 59 major errors for the 3-band tests; 32 minor and 21 major errors for the 4-band tests.The number of requests for malaria diagnosis as well as the exposure per laboratory technician was low, with 112 (84.8%) of 132 participants processing ≤ 100 and 46 (34.8%) even ≤ 20 requests per year. Most participants  declared to perform malaria diagnosis during as well as outside of office hours, but the strategy differed: during office hours, most  performed microscopy in combination with a RDT  and 12.1% (16/132) of participants respectively). A minority of participants left the decision about performing RDT versus microscopy to the clinician or used microscopy only for confirmation (1.5% (2/132) of participants each). Outside of office hours, 85.2% (104/122) of participants consistently performed microscopy  and 19.7% (24/122) of participants respectively). More than one third  of participants relied on the RDT as an initial test; 13.9% (17/122) used the RDT as the only diagnostic tool and 22.1% (27/122) provided microscopy for confirmation .P. falciparum/non-falciparum three-band products were the most widely used products (more than three-quarters of participants).The present EQA session assessed the competence of reading and interpretation of malaria RDTs in a non-endemic setting and surveyed their use in the diagnostic strategy. More than three-quarters 78.5%) of laboratories performing diagnosis of malaria declared to use malaria RDTs, which is slightly more compared to a previous survey in the same area (72.7%) . These p% of laboP. falciparum HRP2 antigen, as part of a prozone effect [Overall results of reading and reporting were good to very good. A major error–occurring among 18.6% and 47.6% of three- and four-band product users respectively—was disregarding the presence of faint test lines and reporting the result as negative. Although this incorrect answer was noted mostly for RDT products for which photos displayed the faintest lines, the present EQA design did not allow to trace whether this error was related to observation versus interpretation. Faint test lines of malaria RDTs should be interpreted as positive , 20. Weae effect , 23, 24.e effect , 25,26. i.e. reporting an invalid result in case of a P. falciparum (OptiMAL) and reporting “Presence of Plasmodium” without mentioning the presence of P. falciparum and P. vivax in the case of PALUTOP. This is striking, as for both RDT products, errors in the IFU had been noted previously [i.e. risks that cannot be mitigated by adaptations and improvement of product design) of product failure and test limitations [Two other major errors were associated with incorrect IFU information about the interpretation of combinations of test lines eviously . These oitations , 28.P. falciparum was not fully exploited, for instance by reporting “Plasmodium spp., no further differentiation possible” instead of either P. falciparum or Plasmodium non-falciparum (11.6% among the three-band users). Indeed, an asset of RDTs is their ability to rule in or out the presence of P. falciparum and this conveys important information to the clinician. Part of these errors may have been caused by non-familiarity with the options for answering the EQA questions; alternatively operator misunderstanding or misinterpretation of test line patterns is a known limitation of RDTs also in non-endemic settings [Another observation of this EQA was that the information about the presence (or absence) of settings .P. falciparum densities), most authors recommend to use RDTs as a complement and not as substitute for microscopy in non-endemic settings [The survey further revealed that, outside of office hours, more than one third 36.1%) of participants relied on the RDT either as the single diagnostic tool (13.9%) or with subsequent microscopy (22.1%). This observation is in line with what was previously recorded in the same setting . Given t% of partsettings ,29, 30. settings ,31.e.g. reporting P. falciparum in the case of a clearly negative test result). Among the strengths, there was the high response rate (99.3%) in the present EQA and questionnaire versus survey response rates of 60.3% and 72.3% in the United Kingdom and the US respectively [Our study had some limitations. First, the photograph-based design did not allow to assess the pre-analytical and large parts of the analytical phase (including the operator’s and RDT’s performance). Second, as stated above, the EQA design did not allow to trace errors to either misreading of test lines versus misinterpretation. Further, the idea of offering each participant with “their own” RDT product conveyed the challenge of harmonizing panels and test line intensities among the different RDT products. Next, non-familiarity with pre-formatted answers may explain some anecdotal errors which in retrospect might have been clerical  ,32. AlthP. falciparum (one of the assets of RDTs) was not always exploited. Relying on RDTs alone for malaria diagnosis was practiced by nearly one third of participants but is not a recommended practice.In conclusion, the present EQA results showed that diagnostic laboratories performed well in reading and interpretation of malaria RDTs. Systematic errors comprised disregarding faint test lines and reporting them as negative as well as interpretation errors of some test line patterns, part of these errors were embedded in the instructions for use of the products. Further, the information about presence or absence of S1 Fig(PDF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


P. vivax and P. knowlesi infections. As a result, these patients are not able to receive anti-malarial therapy in a timely manner. The objective of the present study was to investigate if patients presenting with AKI harbored any of the five human Plasmodium species  within their renal tissues.Acute kidney injury (AKI) is a frequent presentation in malaria infections. Several cases of AKI that are accompanied by clinical symptoms of malaria infection, such as fever, nausea, respiratory distress, and anemia remain undiagnosed due to challenges in accurate diagnosis using peripheral blood microscopy and rapid diagnostic tests that are currently used in clinical settings. This is particularly true for P. falciparum, P. vivax and P. knowlesi as mono- and mixed species infections. Presence of microvascular injury in a majority of the malaria associated AKI cases suggested vascular involvement of P. vivax and P. knowlesi. This research note also highlights P. knowlesi as an emerging pathogen in the Indian subcontinent.We found that renal biopsies from malaria associated AKI patients harbor the human malaria parasites  Plasmodium–P. falciparum, P. vivax, P. knowlesi, P. malariae and P. ovale. Acute kidney injury (AKI), is a frequent presentation in severe malaria which is associated with mortality [P. falciparum is found all over the tropical world, P. vivax is highly prevalent in South America, India and South East (S. E.) Asia, and P. knowlesi has been shown to cause malaria predominantly in S.E. Asia [P. vivax infection in patients with AKI [Plasmodium species within their renal tissue and, (b) To investigate the presence of all five species of Plasmodium known to infect humans  in malaria AKI cases since most diagnostic methods are suitable for the detection only of P. falciparum and P. vivax. We report our observations here.Human malaria is caused by five different species of ortality –5. Overaortality , 7. Whil.E. Asia –11 with .E. Asia . In many.E. Asia –16. An ewith AKI . HoweverThis study was a retrospective case–control analysis carried out as collaboration of the Department of Biochemistry with the Departments of Pathology, Pediatrics and Nephrology at All India Institute of Medical Sciences (AIIMS), New Delhi, India. The ethics committee of the All India Institute of Medical Sciences, New Delhi has approved this study. The overall study design is presented as a flowchart in Additional file Cases: AKI patients presented with abdominal pain, fever and low urine output with suspected or confirmed malaria diagnosis. Both peripheral smear malaria positive cases with confirmed malaria diagnosis, as well as peripheral smear malaria negative cases with malaria-like symptoms have been included as cases.Controls: Renal biopsies from AKI cases showing acute tubular injury or necrosis (ATN) and/or acute cortical necrosis (ACN) without clinical, laboratory, or histopathological evidence of malaria. Details of malaria AKI cases are presented in Table Archival formalin-fixed paraffin-embedded (FFPE) renal tissue blocks from 2011 to 2018 were retrieved from the Department of Pathology, AIIMS, New Delhi, India. Geographical distributions of patients are presented in Additional file The individuals who performed the experiments were blinded to the identity of the controls and cases until the PCR outcomes had been obtained and documented.Initial evaluation of patients included urine analysis, ultrasound, complete blood counts and measurements of creatinine, urea, electrolytes, pH and bicarbonate. Renal biopsies were processed for light microscopy by standard techniques. Diagnosis of malaria was based on peripheral blood smears.Histological sections of all FFPE tissues were stained with Giemsa, hematoxylin, periodic acid schiff (PAS) and Jones methenamine silver stains . The gloPlasmodium species in the peripheral blood smear (thick and thin smears stained by Giemsa). Species identification was done on thin film microscopy (Nikon Ni-E). In the cases where peripheral blood smear did not yield any parasites, diagnosis was carried out by physicians based on clinical presentation.Clinical diagnosis of malaria was based on microscopic detection of Plasmodium species  was carried out. The primers used for PCR amplification are listed in Additional file P. knowlesi primers were designed to target Pkr140 gene, which is present in 7 copies distributed across 6 different chromosomes in the P. knowlesi genome [P. knowlesi specific DNA segments which are not present in the other human Plasmodium species and therefore, prevent cross reactivity with either P. vivax or P. falciparum. At least 150 ng of DNA was used in a 20 µl PCR reaction.For detection of malaria parasite from renal tissue, DNA was extracted using the Qiagen QiAmp DNA FFPE tissue kit as per the manufacturer’s protocol. PCR amplification of extracted DNA for all the five i genome . These phttp://www.indianremotesensing.com/2017/01/Download-India-shapefile-with-kashmir.html. Then, the map was visualized and saved using map shaper tool https://mapshaper.org/. The zoomed in image of Delhi was by using R package “rgdal” only. The final composite map was generated using Microsoft Powerpoint.Indian map shapefile (.shp) was downloaded from Plasmodium species . Among the 12 cases, 5 cases were found to have P. vivax, 1 had P. knowlesi, 2 had mixed infections of P. vivax and P. knowlesi, and 4 had mixed infections consisting of three parasites P. vivax, P. falciparum, and P. knowlesi . Table S1. Oligonucleotide sequences for Plasmodium species used for PCR amplification using the standardized conditions. Table S2. BLAST results of Sanger sequenced PCR products of Plasmodium knowlesi.


Plasmodium falciparum cases for at least 1 year, despite the presence of multidrug-resistant parasites.In Cambodian villages, 3-month mass drug administration with high coverage using dihydroartemisinin-piperaquine was safe and was followed by the absence of clinical  Plasmodium falciparum in Southeast Asia suggests a need for acceleration of malaria elimination. We evaluated the effectiveness and safety of mass drug administration (MDA) to interrupt malaria transmission.The increase in multidrug-resistant Four malaria-endemic villages in western Cambodia were randomized to 3 rounds of MDA (a 3-day course of dihydroartemisinin with piperaquine-phosphate), administered either early in or at the end of the study period. Comprehensive malaria treatment records were collected during 2014–2017. Subclinical parasite prevalence was estimated by ultrasensitive quantitative polymerase chain reaction quarterly over 12 months.Plasmodium falciparum incidence in intervention and control villages was similar over the 12 months prior to the study: 39 per 1000 person-years (PY) vs 45 per 1000 PY (P = .50). The primary outcome, P. falciparum incidence in the 12 months after MDA, was lower in intervention villages . Following MDA in 2016, there were no clinical falciparum malaria cases over 12 months (0/2044 PY) in all 4 villages. Plasmodium vivax prevalence decreased markedly in intervention villages following MDA but returned to approximately half the baseline prevalence by 12 months. No severe adverse events were attributed to treatment.MDA coverage with at least 1 complete round was 88% (1999/2268), ≥2 rounds 73% (1645/2268), and all 3 rounds 58% (1310/2268). P. falciparum cases for at least 1 year.Mass drug administrations achieved high coverage, were safe, and associated with the absence of clinical NCT01872702. Plasmodium falciparum in Southeast Asia has decreased substantially over the last decade, probably related to changes in land use, wide deployment of insecticide-treated bed nets, and, particularly, early diagnosis and treatment of symptomatic disease with artemisinin combination therapies (ACTs). However, partial resistance to artemisinins emerged in western Cambodia in the first decade of the millennium and was subsequently also detected in other regions of the Greater Mekong subregion. The reduced susceptibility of P. falciparum to artemisinins is increasingly compounded by ACT partner drug resistance causing high treatment failure rates ) and control villages (60/1322 [45/1000 per year]). The control villages received MDA at the end of the study period, and clinical cases were monitored during the poststudy period from July 2016 to June 2017. No falciparum malaria cases were reported in any of the 4 study villages during the poststudy period (Over the 12 months following the start of MDA (July 2015 to June 2016), falciparum malaria incidence was fell to 1 in 659 (1.5/1000 per year) in the intervention villages compared with 50 of 1348 (37.1/1000 per year) in control villages that had not yet received MDA  prior to MDA vs 1 of 659 (1.5/1000 per year) after MDA . In villages that received MDA in 2016, falciparum malaria incidence was 50 of 1348 (37/1000 per year) before MDA vs 0 of 1373 after MDA  and from 39 of 1348 (29/1000 per year) to 9 of 1373 (6.5/1000) per year in the control villages  and control villages (39/1348 [29/1000 per year]) during the study period from July 2015 to the end of June 2016  with 4 months after the start of MDA, the prevalence of subclinical P. falciparum infections assessed by uPCR was 5 of 543 (0.9%) vs 2 of 470 (0.4%) in intervention villages, and 17 of 701 (2.4%) vs 7 of 696 (1.0%) in control villages. The odds ratio (OR) was 0.15 , with no significantly different change against baseline. By 12 months, the prevalence of subclinical P. falciparum infections was 1 of 512 (0.2%) in intervention villages, compared with 10 of 1090 (0.9%) in control villages  became negative throughout the follow-up period. One study participant with a subclinical P. falciparum infection before MDA remained infected after receiving 3 rounds of MDA.The prevalence of subclinical P. vivax infections were more prevalent than subclinical P. falciparum infections in all surveys. At 4 months, there was a large decline in the prevalence of subclinical P. vivax infections in intervention villages, 57 of 543 (10.5%) to 3 of 470 (0.6%), and only a small decline in control villages (60 of 701 [8.6%] to 70 of 696 [6.4%]) . By 12 months, subclinical P. vivax infections in intervention villages had returned to approximately half of the baseline prevalence, and subclinical P. vivax infection was no longer significantly lower than in control villages  remained free of infections throughout the 12-month follow-up period whereas 23 (40%) had subclinical P. vivax infections. The status of 10 (18%) participants was unknown as they did not attend subsequent surveys.Of the 57 individuals with uPCR-detected P. falciparum parasite strains detected by uPCR, 27 (48%) were sequenced for kelch 13 mutations, and all 27 were positive for the C580Y haplotype. None was positive for other K13 markers. Among 35 falciparum malaria cases detected up to April 2016, all had a mutation associated with artemisinin resistance in the Kelch 13 gene: 34 had the K13 C580Y mutation and 1 had the K13 F446I mutation.Molecular markers for artemisinin resistance were detected in both subclinical and clinical falciparum malaria episodes. Of 56 In total, 14845 DHA-PQ tablets were administered during MDA, and adverse events were reported by 45.5% (909/1999) of participants who received treatment . The mosP. falciparum infections decreased in the study villages but did not reach zero. The subclinical falciparum infections did not correlate with clinical malaria incidence, which might be related to the remaining asymptomatic carriers.Deployment of 3 rounds of MDA reduced falciparum malaria incidence dramatically and rapidly. A single case of falciparum malaria was detected in the year after MDA, compared to 24 cases in the same villages in the previous year, and 50 cases in untreated villages. No clinical falciparum malaria cases were detected in intervention or control villages in the year after MDA, with the exception of a forest worker who did not participate in the MDA. Forest work is a well-documented risk factor for malaria in the study region . FollowiPfKelch13 marker for artemisinin resistance. All low-density subclinical P. falciparum infections but one were cleared by the DHA-PQ regimen. Increasing drug resistance is, however, an important concern for the future use of DHA-PQ for MDA in this area.Our study employed 3 rounds of DHA-PQ, which facilitates the capture of short-term travelers and the elimination of infections circulating in vectors . A full Plasmodium vivax incidence and prevalence decreased substantially following MDA, but rebounded by 6 months and had returned to approximately half the pre-MDA level 1 year after the MDA. This has been observed also in other MDA trials [P. vivax causing relapses of the primary infection. Hypnozoites are not affected by the MDA regimen and require a prolonged course of primaquine.A trials , and is The MDA was remarkably safe and well accepted. Few severe adverse events and no drug-related severe adverse events were reported. As the study did not include a control group receiving placebo, it is not possible to assess reliably the true adverse event frequency attributable to the study drugs. Factors affecting acceptability have been reported earlier, and confirmed that the perceived side effects of the drug administration were common but mild .Plasmodium infections into villages after the interruption of malaria transmission. MDA conducted in geographically distinct villages well connected by roads, as in this study, cannot prevent the reintroduction of falciparum malaria unless a wider region is targeted.MDA was conducted in individual villages surrounded by untreated villages. Malaria can be reintroduced by visitors from neighboring villages or through residents who are exposed when staying outside their village. Forest workers are at high risk to reintroduce A major limitation of this study is the number of randomized clusters. With only 2 clusters per arm, statistical significance testing is unstable, yet our results still suggest a real difference between study arms and before vs after the intervention. The study is part of a larger multicenter trial in Myanmar, Vietnam, and Lao People’s Democratic Republic. The combined analysis of the studies is expected to have sufficient power to detect statistically significant differences. Another limitation is the absence of an entomological research component that could have helped demonstrate interruption of transmission. Detailed entomological observations were collected in Myanmar, one of the other studies in the project . FinallyP. falciparum elimination in low-endemicity regions including Southeast Asia [Plasmodium infections by screening and treating visitors and immigrants, immunization of residents with a malaria vaccine candidate, and reducing vector density by adding ivermectin to MDAs are under discussion.A similar impact of the same MDA regimen as an addition to establishment of a network of malaria posts has recently been demonstrated in Myanmar . MDA effast Asia , 11, 36.P. falciparum cases for at least 1 year. The impact on the prevalence of subclinical P. vivax infections was much more limited. Additional interventions including radical cure with primaquine will be needed to eliminate vivax malaria. The careful selection of quality drugs in the most appropriate drug regimens, the targeting of suitable villages, and allocation of adequate resources needed to achieve high coverage will be critical to address during the expansion of MDA programs in Southeast Asia.In our preelimination setting, MDA  resulted in absence of clinical Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.


Plasmodium ovale wallikeri seems to have a longer latency period and produces deeper thrombocytopaenia than Plasmodium ovale curtisi. Prospective studies were warranted to better assess interspecies differences.Few previous retrospective studies suggest that P. ovale spp. infection diagnosed by thick or thin film, rapid diagnostic test (RDT) or polymerase chain reaction (PCR) were recruited between March 2014 and May 2017. All were confirmed by DNA isolation and classified as P. o. curtisi or P. o. wallikeri using partial sequencing of the ssrRNA gene. Epidemiological, analytical and clinical differences were analysed by statistical methods.Patients with imported P. o. curtisi and 44 P. o. wallikeri) were correctly genotyped. Males predominate in wallikeri group (72.7%), whereas were 48.6% in curtisi group. Conversely, 74.3% of curtisi group were from patients of African ethnicity, whilst 52.3% of Caucasians were infected by P. o. wallikeri. After performing a multivariate analysis, more thrombocytopaenic patients (p = 0.022), a lower number of platelets (p = 0.015), a higher INR value (p = 0.041), and shorter latency in Caucasians (p = 0.034) were significantly seen in P. o. wallikeri. RDT sensitivity was 26.1% in P. o. curtisi and 42.4% in P. o. wallikeri. Nearly 20% of both species were diagnosed only by PCR. Total bilirubin over 3 mg/dL was found in three wallikeri cases. Two patients with curtisi infection had haemoglobin under 7 g/dL, one of them also with icterus. A wallikeri patient suffered from haemophagocytosis. Chemoprophylaxis failed in 14.8% and 35% of curtisi and wallikeri patients, respectively. All treated patients with various anti-malarials which included artesunate recovered. Diabetes mellitus was described in 5 patients (6.32%), 4 patients of wallikeri group and 1 curtisi.A total of 79 samples (35 P. o. wallikeri infection may be more frequent in males and Caucasians. Malaria caused by P. o. wallikeri produces more thrombocytopaenia, a higher INR and shorter latency in Caucasians and suggests a more pathogenic species. Severe cases can be seen in both species. Chemoprophylaxis seems less effective in P. ovale spp. infection than in P. falciparum, but any anti-malarial drug is effective as initial treatment. Diabetes mellitus could be a risk factor for P. ovale spp. infection.Imported  Plasmodium falciparum and Plasmodium vivax, to which most of the research resources are devoted [Malaria remains a global health problem with more than 212 million new cases in 2016 and 429,000 annual deaths worldwide, mostly driven by Plasmodium ovale spp. infection can be considered a neglected disease and little is known on its real incidence, geographic distribution, global disease burden or interactions with other Plasmodium species. The main problems for its study have been that it is frequently misdiagnosed because of usual submicroscopic or low parasitaemia levels, low performance of malaria rapid diagnostic tests (RDT) [Plasmodium spp. [Conversely, ts (RDT)  and a maium spp. .P. ovale spp. epidemiology reporting infections in most of sub-Saharan Africa, Southeast Asia, and the Indian subcontinent [P. ovale spp. malaria [P. ovale spp. infection seems to be increasing among travellers returning from West Africa [P. falciparum or P. vivax [Nowadays, polymerase chain reaction (PCR) techniques have expanded the knowledge on ontinent –6, but nontinent . In addiontinent , spleen  malaria . Lastly,t Africa –11, in wt Africa  comparedP. vivax .P. ovale is considered to comprise two genetically different subspecies, named P. ovale curtisi and P. ovale wallikeri that could have diverged 1–2 million years ago in their evolution [Since the study by Sutherland et al. was published in 2010, volution . They bovolution –17.Plasmodium o. curtisi and P. o. wallikeri do not seem to differ in parasitaemia levels [P. o. wallikeri but only in 30% of blood samples [P. o. wallikeri may have a shorter period of latency [Once established the genetic differences, the next question was to find out if there were any other differences in epidemiology, microbiology or clinical features between both subspecies. a levels , 9, 13 aP. o. curtisi and 14 P. o. wallikeri infections confirmed by PCR and gene sequencing [P. o. wallikeri infection than among those with P. o. curtisi (p = 0.031). However, non-significant trends showing shorter latency, lower albumin level, higher temperature and markers of more severe haemolysis were found in P. o. wallikeri group. Because of the retrospective design and small number of patients, larger, prospective studies were warranted to confirm these findings. Therefore, the results of a multicentre, prospective, comparative study of imported P. o. wallikeri and P. o. curtisi infections conducted in several European countries during the period 2014–2017 are presented below.Recently, a multicentre, retrospective study in Spain was reported, comparing 21 imported quencing . The onlA prospective, observational, un-randomized, open-label, multicentre study was performed at European hospitals of TropNet Europe, European Network for Tropical Medicine and Travel Health. Participants were recruited between March 2014 and May 2017. The study was approved by the University Hospital Príncipe de Asturias Ethical Board and at each site as needed. Written informed consent was obtained from the participant or a legal representative before enrolment in the study. The trial was conducted in accord with the Declaration of Helsinki and Good Clinical Practice.P. ovale spp. infection was made up by thick and/or thin film and/or second generation RDT and/or PCR available in each hospital. Blood smears were stained by a standard technique with Giemsa solution for 30 min and were reviewed by an expert microbiologist. Parasite count was measured by determining the proportion of parasitized erythrocytes or the number of trophozoites per microlitre. Mixed infections with other Plasmodium species were excluded.The initial diagnosis of imported P. ovale spp. had already been isolated, this was sent into a 1.5 mL screw cap tube with complete closure to the same Reference Laboratory.For cases tested positive, three drops (≈ 50 μL) of full blood left after routine laboratory tests, were dotted on filter paper (Whatman™) and sent by regular mail to the Reference Malaria & Emerging Diseases Laboratory of the National Centre of Microbiology in Madrid. If DNA of ®) from whole blood following the manufacturer’s protocol. Molecular diagnosis confirmation of P. ovale spp. was carried out by semi-nested multiplex malaria PCR [DNA isolation was performed using the QIAamp DNA blood mini kit  and PLF (5′-AGTGTGTATCCAATCGAGTTTC-3′) primers [Characterization of  primers . The sec2, 50 mM KCl, 20 mM (NH4)2SO4, 200 μM of dNTP, 0.075 µM of each primers, 1.25 units of Taq DNA polymerase , and 5 μL of DNA, extracted by QIAgen kit, was used as template in a reaction volume of 50 μL. For the second reaction mixture, 2 µL of the PCR product of the first reaction was used as template. For both reactions, a GeneAmp® PCR System 2700 thermal cycler (Applied Biosystems Laboratory) was used, beginning with 7 min at 94 °C, followed by (first-round) 40 cycles of 20 s at 94 °C, 20 s at 62 °C, and 30 s at 72 °C, or (second-round) 35 cycles of 20 s at 94 °C, 20 s at 53 °C, and 20 s at 72 °C. The final cycle was followed by an extension time of 10 min at 72 °C.The PCR mixture in both reactions consisted of 75 mM TrisHCl (pH 9.0), 2 mM MgClGeneral Electric Healthcare) and sequenced with the Big Dye Terminator v3.1 Cycle Sequencing in an ABI PRISM® 3700 DNA Analyzer. All amplified products were sequenced in both directions twice.The amplified products were purified using Illustra DNA and Gel Band Purification Kit  if travelled to a country endemic of malaria after a year of stay in a non-malarial area, and VFR traveller if visited his/her first-degree relative’s country of birth having been born in a non-malaria endemic country. Recent Plasmodium infection was defined as probable or definite malaria infection in the previous 12 months before P. ovale spp. diagnosis.An anonymized database was designed to input all the medical information and laboratory registries in a prospective way. Data collected included gender, age, ethnicity, underlying diseases, type of patient, dates and purpose of travel, countries visited, malaria chemoprophylaxis, date of admission and diagnosis, presenting clinical signs or symptoms and complications of severe malaria according to criteria of the World Health Organization (WHO)  where noLaboratory results included microbiological data with parasitaemia count, full blood count with white blood cells, haemoglobin and platelets, values of glucose, creatinine, albumin, transaminases, lactate dehydrogenase (LDH), total bilirubin (tBR) in plasma, the coagulation parameters activated partial thromboplastin time (APTT) and international normalized ratio (INR), glucose-6-phosphate dehydrogenase (G6PDH) activity in red blood cells, as well as serological studies of infection with human immunodeficiency virus (HIV), hepatitis B (HBV) and hepatitis C (HCV). Thrombocytopaenia was defined as a platelet count under 150,000/µL. Treatments, clinical and microbiological evolution and duration of hospital stay of those admitted were recorded. Treatments were performed following the guidelines of each country and health centre without a common recommendation.t-test for independent samples if the normal distribution could be assumed. In the Student’s t test for independent samples, the Levene’s test for homogeneity of variances was used. If normal distribution was not valid, the non-parametric Mann–Whitney U-test was performed. To test for normality, either the Shapiro–Wilks for small samples or the Kolmogorov–Smirnov with Lilliefors’ correction for large samples were used. Values are reported as number of patients and percentage or, for non-parametric distributions, as median and interquartile range (IQR). A multivariate linear regression analysis was performed for continuous variables and multivariate logistic regression for categorical variables to confirm if real differences were found when non-homogenous population between both groups were identified. A two-sided p value of 0.05 or less was considered to indicate statistical significance. Statistical analysis was performed using the SPSS version 21 .Differences of proportions were evaluated by the Chi-squared-test if less than 20% of cells had five or less expected values. If more than 20% of cells had five or less expected values we used the Fisher’s exact test for categorical variables. Differences of means between groups were calculated by the Student’s P. falciparum, 1 with P. vivax and 1 with Plasmodium malariae), 2 samples did not get amplified and data from another 2 were not obtained. At the end, 79 correctly genotyped and with complete patient information were included for statistic analysis. Of them, 35 were identified as P. o. curtisi and 44 as P. o. wallikeri.During the period of the study, a total of 96 blood samples from 29 hospitals were sent to the reference laboratory. Of them, 13 were excluded because of mixed infections , which would generally result in less clinical and analytical involvement [P. o. wallikeri who travelled by international cooperation were male and Caucasians, which would bias the characteristics of this group.On the results obtained, significant differences were found in the epidemiological characteristics of sex, ethnicity and type of patients between both groups. Ethnic differences may be the main confounding factor since there is a greater probability that African patients retain some semi-immunity against olvement . On the This distribution could be a result of chance alone or due to the fact that actually Caucasians patients are more susceptible to infection by wallikeri parasites. However, a previous retrospective study did not show these differences in sex, ethnicity or type of patient  and the P. o. wallikeri and INR values were higher in the wallikeri group. The comparation of the rest of analysis variables were not finally significant, over all those that indicate a greater degree of haemolysis, such as LDH, tBR or haemoglobin level.When performing the multivariate study adjusted by ethnicity to reduce this confounding factor, the number of patients with thrombocytopaenia and two of the analytical alterations remained significant. Thrombocytopaenia was more pronounced in P. o. wallikeri infection [Plasmodium species ranging from 24 to 94% of indicence, although spontaneous haemorrhages are infrequent and limited to very severe cases [P. ovale spp. infection. Of the 79 patients analysed, 53 (67%) had under 150,000 platelets/µL, which corresponds to our previous series findings of 66.6% in 16 imported ovale infection patients [In the case of thrombocytopaenia, these results confirm the main finding of a previous retrospective study where the only difference was more severe thrombocytopaenia in nfection . Thrombore cases . Also itpatients  and a reThe pathogenesis of thrombocytopaenia in malaria is not fully understood but is accepted that encompasses several different mechanisms that lead to increased platelet destruction or consumption, such as increased attachment to endothelium and adherence to Von Willebrand factor, clumping and agglutination of infected and uninfected erythrocytes, consumption into the coagulation process and haemolysis, increased diffuse platelet sequestration, decreased nitric oxide bioavailability, and immune complexes-mediated destruction .P. falciparum or P. vivax [P. o. wallikeri it could indicate a greater intrinsic pathogenicity of this species compared to P. o. curtisi, although without evidence of any major significantly worse clinical criteria of severity.Although thrombocytopaenia is not among the severity criteria of the WHO, evidence is accumulating that it can predict an adverse outcome, which seems to be driven by a greater severity of illness in P. vivax , 24 and P. vivax . TherefoP. o. wallikeri compared to P. o. curtisi, this increase was always mild and in no case exceeded the value of 1.54 nor was related to any bleeding. On the other hand, the APTT values were similar in both groups. No other coagulation factors, fibrinogen levels, fibrin degradation products or d-dimer were measured that could argue in favour of a significant coagulation disorder.Regarding the other significant finding of this study, the higher INR elevation in P. falciparum malaria is associated with significant coagulation activation. The increase in INR or its equivalent, prolongation of prothrombin time, is produced by the activation of the extrinsic pathway of the coagulation cascade and can lead to disseminated intravascular coagulation in the most severe cases of malaria. Plasmodium falciparum seems to trigger coagulation activation through multiple different pathways although the mechanisms involved are not well understood [P. ovale spp. infection. In the case of INR, the finding of this study might support the hypothesis of a more pathogenic wallikeri parasite, but does not seem to pose a significant clinical difference or severity.It is well known that derstood . Much leP. o. wallikeri compared to P. o. curtisi infection, but only in Caucasians patients. This difference is more significant since only one of the seven Caucasians patients who suffered infection by P. o. curtisi had taken adequate prophylaxis versus 11 out of 22 (50%) of the wallikeri group, which should have lengthened their latency. The retrospective study by Nolder et al. in the UK also found significant shorter latency period in wallikeri cases but did not report the ethnicity or patient characteristics [P. o. curtisi and P. o. wallikeri [In the initial analysis, no differences were found in the latency period, but when the multivariate analysis was performed a significant shorter time appeared in eristics . On the allikeri  importedut of 22 0% of theP. ovale spp. infection. Finally, note that in both species latencies greater than 1 year have been found as described previously [These results of different latency times in only Caucasians patients suggest a strong influence of the previous partial immunity to malaria in the time of symptoms of eviously .P. o. wallikeri, a high percentage if compared with data from large series of imported malaria as in USA 2014, where only 7.8% of travellers correctly took prophylaxis [Plasmodium species, these results are similar to the study by Nolder et al. which stated that the proportion of ovale malaria which occurred in patients reporting chemoprophylaxis use (33%) was significantly higher than for P. falciparum (6.4%) and P. vivax (23.7%) [P. ovale spp. used an effective prophylaxis [P. ovale/P. vivax malaria, these patients had also taken chemoprophylaxis significantly more frequently than those with non-P. ovale/P. vivax malaria (35.5% vs. 2.7%) [P. ovale spp. infection, since it is ineffective against its hypnozoites and, therefore, does not prevent a delayed primary attack or true relapses from occurring when there is no prophylactic medication in blood.A complete chemoprophylaxis was reported in 22.8% of ovale spp. infections and up to 35% in phylaxis . If anal (23.7%) . Also, iphylaxis . Moreoves. 2.7%) . These rP. ovale, another ovale relapse, a mixed infection with other Plasmodium or simply a non-ovale malaria unrelated to the current episode. Since it is not feasible to obtain a previous diagnostic sample, it is impossible to know if the current episode represented a primary infection or a relapse by P. ovale. Primary infection could only be diagnosed in the cases of first malarial episode and very short latency period.Although the information cannot be completely reliable, over 40% of patients reported an episode of malaria in the previous 12 months. This could have been a primary infection by P. ovale infection 3 out of 16 patients (18.7% with mean age 30.6 years) were also diabetics [P. falciparum and 2.5% in 398 imported malaria in a Spanish Tropical Medicine Centre [P. falciparum infections from 2006 to 2015 in Hospital Universitario Príncipe de Asturias (HUPA), 2.6% were diabetics (unpublished data). As diabetes is a known risk factor for malaria due to P. falciparum in endemic areas [P. ovale studies might indicate that would also be a risk factor, and deserves further investigation to confirm this finding.Regarding the underlaying conditions, it is worth focusing on diabetes mellitus and drepanocytosis. The prevalence of diabetes mellitus is increasing worldwide, especially in low income countries, as are found in sub-Saharan Africa . The numiabetics . A recenic areas , the higP. ovale spp. [P. falciparum infection. In this study, 2 out of 47 African patients (4.2%) had drepanocytosis. Moreover, in the previous comparative retrospective study, 8.3% (2 out of 24) showed drepanocytosis and up to 23% (3/13) in the series of 16 imported P. ovale spp. [P. falciparum in HUPA during the period 2006–2015 (unpublished data) significant difference can be found (p < 0.005) that would support the results of the study conducted in endemic area.In endemic areas, carrying the sickle cell trait represents a risk factor for infection by ale spp.  and a paale spp. . GatheriP. o. wallikeri, was not studied.As in previous studies, no differences were detected in parasitaemias between both groups , 9, 32. P. ovale spp. diagnosis.There was also no difference in the sensitivity of RDTs, which was low as described in the literature  without P. falciparum the rate of severe cases was around 2–17% [P. ovale spp. infection [P. falciparum and P. vivax infection [P. ovale spp. Analysing the six patients with severe malaria or complications, two had drepanocytosis  and one was diabetic, which might indicate the possible importance of these risk factors in the severity of P. ovale spp. infection.This study confirms that this infection can cause malaria with severity criteria or complications, in this case 7.6% of all patients with no differences between both species. In series of imported malaria by nd 2–17% , 34, butnfection . A rare nfection , but notP. falciparum and > 20,000/µL in P. vivax and Plasmodium knowlesi, but is not known in the case of P. ovale spp. In this study, all four cases with tBR > 3 mg/dL had parasitaemias over 4000/µL. In addition, four patients received parenteral artesunate, two of them with tBR > 3 mg/dL and one with unusually high parasitaemias of 2%. It is described that host response may reach full strength at lower parasitaemia in P. vivax and P. ovale spp. infection than in P. falciparum [P. ovale spp. in the face of treatment decisions and outcome.In cases of malaria with tBR > 3 mg/dL, the WHO severity criteria add parasitaemia levels above 100,000/µL in lciparum  so, perhP. ovale spp. described for multiple antimalarials [The treatments were very varied, including artemisinins alone or in combination, atovaquone/proguanil, chloroquine or quinine-doxycycline, all with good initial clinical evolution, which is compatible with the sensitivity of alarials . Over 70P. ovale species. Second, only strains of P. ovale from Africa, and patients from Africa and Europe were analysed; a study of infections and patients from Asia, Oceania or other places might show different results. Third, the behaviour of these species in endemic countries may be different but it would be difficult to gather a significant number of patients with P. ovale spp. monoinfection due to the high prevalence of mixed Plasmodium infections and malaria reinfections. Fourth, the date of infection is a minimal approximate and it is virtually impossible to distinguish a primary infection from a relapse. Last, a mix of ethnicities, non-immune and semi-immune patients led to heterogeneous study groups, although the multivariate statistical study has reduced this bias.The prospective study design has minimized some of the limitations of the previous retrospective study but some of them still remain. First, although it is one of the largest studies carried out worldwide, the number of patients may still lack sufficient statistical power to show other differences between infections with both P. o. wallikeri infection was more frequent in males and Caucasian patients than in P. o. curtisi group. Malaria caused by P. o. wallikeri produced more thrombocytopenic patients, more marked thrombocytopaenia and a higher INR than P. ovale curtisi, although without other clinical or severity differences. Plasmodium o. wallikeri malaria had shorter latency, but only in Caucasian patients. These findings suggest that wallikeri species might be somewhat more pathogenic than P. ovale curtisi. Some cases with severity criteria were seen in both species and required treatment with intravenous artesunate. The clinical evolution with any anti-malarial drug was usually good, however, chemoprophylaxis seemed less effective in P. ovale spp. infection than in the case of P. falciparum. Diabetes mellitus might also be a risk factor for P. ovale spp. infection such as drepanocytosis. Current RDT was shown to have low diagnostic sensitivity in both species and PCR was essential for diagnosis in a significant percentage of cases.In this study, imported