Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon.Population-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P < 0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic.Two cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.This study confirms a substantial proportion of asymptomatic and submicroscopic The online version of this article (10.1186/s13071-018-2787-7) contains supplementary material, which is available to authorized users. Plasmodium falciparum, with Plasmodium vivax predominance progressively increasing to peak at 88.4% of total malaria cases in 2015 [Anopheles darlingi is the main malaria vector [The past two decades have witnessed impressive success in the reduction of malaria incidence in Latin America, with the number of confirmed cases decreasing by 31.0% from 2000 to 2015. Nevertheless, malaria remains a major public health concern in endemic countries, with approximately 90 million people being at risk of infection and an estimated 216 million cases worldwide in 2016 [ in 2015 . In BrazLatin America is considered a low to moderate malaria transmission region. Recent population based surveys reported prevalence rates below 6.0% by LM –14. PCR Plasmodium spp. in the Amazon region were carried out in small rural or riverine settlements. Additionally, very few studies assessed the prevalence and density of gametocytes, the parasite stage responsible for transmission from human to mosquito vectors. This study aimed to determine the prevalence of Plasmodium spp. and the gametocyte burden in symptomatic and asymptomatic infections in a peri-urban area in the Brazilian Amazon.Most studies examining prevalence of Plasmodium spp. infected individuals contributing to malaria transmission, we conducted two large-scale cross-sectional surveys in a peri-urban area of Manaus, located in the western part of the Amazonas State, Brazil. The presence of sexual parasite stages in both symptomatic and asymptomatic infected individuals was also investigated.In order to estimate the reservoir of The municipality of Manaus is located on the river Rio Negro banks, in the northeast of the Amazonas State. The municipality has an estimated 2,094,301 inhabitants of whom the great majority resides in urban and peri-urban areas . IntensiThe three communities in which this study was conducted, Brasileirinho, Ipiranga and Puraquequara, are located in the peri-urban area of Manaus Fig. . The maiTwo cross-sectional surveys were conducted on a household-basis, including all inhabitants of all ages of the study area that were willing to participate in the study. In case of absence of any family members, the field team returned to the respective house for a second time. For each study participant a questionnaire was completed, containing personal information , information on malaria preventive measures, previous malaria episodes and current health status. The first cross-sectional survey (CS1) was conducted from mid-November 2012 to beginning of January 2013, at the beginning of the rainy season, and the second one (CS2) from the beginning of August 2013 to mid-September 2013, at the end of the dry season.via finger puncture using Microtainer® tubes containing EDTA and sodium fluoride . In infants, blood was obtained by puncture of the heel or toe. Within 1 h of collection, 50 μl of blood was transferred into a reaction tube containing 250 μl of RNAprotect®  in order to preserve RNA for downstream analyses [Upon participant enrolment, a 300 μl blood sample was collected from each patient analyses  and 200 In order to assess the submicroscopic proportion of infections, during CS1 two thick blood smears were prepared for all participants. One slide was analysed at the local malaria outpost (for diagnostics purposes only) and the second was analysed by a microscopist at FMT-HVD for confirmation of species and parasitemia. In cases of a positive result, participants were treated by health post staff within 24 h according to the national guidelines of the Ministry of Health . During P. vivax and P. falciparum by qPCR  to determine positivity of infection. Individuals with body temperature above 37.5 °C at blood collection or in the past 48 h were considered as febrile. An asymptomatic infection was defined as the presence of a malarial infection in the absence of fever and any other malaria related symptoms  at the moment of sample collection, or anytime during the preceding 48 h. Individuals developing symptoms within 30 days after blood collection  were also considered symptomatic and therefore excluded from the asymptomatic group.All participants were tested for Thick blood smears were Giemsa-stained according to World Health Organization guidelines . NumbersTM 96-well Genomic DNA Kit  according to the manufacturer’s instructions. DNA was eluted with 2 × 100 μl of elution buffer and stored at -20 °C until analysed by PCR. If the amount of whole blood available for DNA extraction was ≤ 100 μl, the DNA volume was vacuum concentrated  according to the volume originally collected. RNA from whole blood stored in RNAprotect was extracted by means of the RNeasy® Plus 96 kit  as described elsewhere [Pelleted RBCs obtained from 200 μl blood were resuspended in PBS and genomic DNA was extracted using FavorPreplsewhere .Plasmodium spp. by targeting a conserved region of the 18S rRNA gene [18S rRNA gene of P. falciparum and P. vivax, as previously described [P. falciparum a modified reverse primer was used . All samples positive for P. vivax and/or P. falciparum were included in RT-qPCR assays detecting the respective transcripts of p25 gene , which is specifically expressed in gametocytes. For quantification of pvs25 and pfs25 transcripts, standard plasmids containing the region targeted by the RT-qPCR were included in each run. All qPCR and RT-qPCR assays were ran in the 7500 Fast Real-Time PCR System . Primer and probe sequences, composition of reaction mixes, PCR profiles and the detection limit for each assay are listed in Additional file All DNA samples were subjected to QMAL Taqman qPCR to detect escribed . For detχ2) test (replaced by Fisher’s exact test if necessary). Univariable logistic regression was used to determine risk factors for P. vivax infection and gametocyte positivity. The crude odds ratios (OR) with their respective 95% confidence intervals (95% CI) were determined considering P. vivax infection and gametocyte carriage as the dependent variables. Logistic regression was used for the multivariable analyses and the adjusted ORs (AOR) with 95% CI were also calculated. All variables associated with the outcomes at a significance level of P < 0.20 in the univariable analysis were included in the multivariable analysis. Median comparisons for copy number between surveys and between symptomatic and asymptomatic individuals were made using Mann-Whitney test. Differences were considered statistically significant for P < 0.05. Association between P. vivax parasite density and P. vivax gametocyte density was analysed by linear regression.Questionnaire data were imported into databases using Cardiff TeleForm version 10.4.1 (Cardiff Software). Individual databases were combined in Microsoft Access 2010. Statistical analyses were performed with STATA® 13 . Differences in participant characteristics, parasite prevalence and carriage of gametocyte between the two surveys were analysed by Chi-square  or gender  between surveys. Besides pre-school (15.0%) and school children (24.4%), 15.4% of participants were adults working in agriculture/fishing, 14.3% worked in an office or pursued high education studies, while 15.6% were house wives and 5.0% retired  and few had travelled outside the village in the month prior to the survey  , 1110 only during the 2013 dry season (CS2) and 963 at both time points . In CS1, 50.1% (1008/2010) used LLINs, 56.7% (1140/2010) lived in an IRS treated house, and 33.1% (666/2010) of participants had benefited from both interventions. As LLIN distributions and IRS spraying campaigns were conducted in the months prior to CS1, the proportion of people that reported benefitting from either intervention was significantly higher in CS1 than for CS2 (Table P < 0.0001). In addition, considering data from both CS1 and CS2, 19.2% (784/4083) of participants lived in households with screened windows.The majority of participants lived in households that had benefited from vector control activities. In CS1 73.7% (1482/2010) of participants were using LLINs (long-lasting insecticide-treated nets) and/or lived in a house that was treated with indoor spraying of residual insecticides (IRS) in the preceding six months, in CS2 it was 54.5% (1131/2073) of participants   carried P. vivax gametocytes when detected by RT-qPCR, whereas 56.3% (18/32) of P. vivax infections detected by LM were positive for gametocytes. A modestly lower prevalence of P. vivax infections was observed in CS2 with 3.4%  of participants having qPCR-detectable infections, although this difference between surveys did not reach statistical significance, irrespective of considering all participants  or only those that were surveyed twice . A comparable number of P. vivax infections were gametocyte positive in both surveys . Total P. vivax parasitemia was comparable in both surveys  but pvs25 transcript numbers were significantly higher in gametocyte positive samples in CS2 compared to CS1 . Overall, P. falciparum infections were rare ; in CS2 a single mixed P. falciparum/P. vivax infection was detected . Among P. falciparum positive samples in CS1, 81.8% (9/11) showed presence of gametocytes by light microscopy and 93.3% (14/15) by qPCR. In CS2, the only P. falciparum positive sample was also positive for gametocytes by qRT-PCR.In CS1, 86 of 2010 (4.3%) participants were infected with P. vivax was about twice as high in Brasileirinho and Puraquequara (4.3%) than in Ipiranga  . Adults  had a higher risk for infection than children (2.4%) . The risk for infection was significantly higher for workers from the agriculture sector than for office workers and individuals pursuing higher education .Combining the results of both CS1 and CS2, prevalence of quequara .3% than χ2 = 0.035, df = 1, P = 0.852). Individuals with recent travel history showed a lower prevalence (2.4%) than individuals that did not leave the study area  and for participants living in IRS-treated houses  as compared to persons not using either intervention measure (3.1%). The proportion of P. vivax infected individuals was lower, but not significant, if the individual´s home had screened windows (3.2%), compared to those individuals living in houses with unscreened windows .Prevalence amongst new residents (4.1%) was only slightly higher than that for individuals that lived in the study area for more than two months . Furthermore, P. vivax prevalence was higher in individuals that experienced a recent malaria episode (7.0%) than in individuals with no recent episode . Individuals presenting fever the day of the visit or during the past 48 h had a significantly increased risk for infection by P. vivax . Individuals with symptoms other than fever (up to 48 h before visit) had a moderately increased, albeit not-significant, prevalence (5.4%) as compared to asymptomatic individuals   than participants with one or more previous malaria infections . Individuals that reported to have had one or more previous malaria episodes in their lives were at much higher risk for P. vivax infection than individuals that had no previous infection (Table P < 0.001). Another factor strongly associated with P. vivax infection was fever (during visit or during the past 48 h) .After adjusting for potential confounders  gender, occupation, history of malaria and fever remained as independent predictors for P. vivax infected individuals  were positive for gametocytes, detected by RT-qPCR. Both in the univariate  and in the multivariate  analyses the only risk factor associated with detectable carriage of gametocytes was P. vivax parasite density. We modelled the probability for carriage of gametocytes (defined as positivity by RT-qPCR in a 50 μl blood sample) depending on P. vivax density using a generalised additive model  the probability was below 30.0% and increased only slightly. At 100 copies/μl the probability to carry gametocytes was 50.0% and increased dramatically reaching a probability of 90.0% at 10,000 copies/μl. Gametocyte density (measured as pvs25 transcripts/μl) also increased with P. vivax density   Fig. .Fig. 2ReP. vivax blood-stages and prevalence of P. vivax gametocytes increased with age. The density of P. vivax blood-stages and pvs25 transcripts was highest in children younger than 12 years  of infected individuals did not report fever or any other symptoms (the day of visit or up to 48 h before) nor did they report with symptoms to a malaria clinic during the 30 days before visit. Of these asymptomatic individuals, 11.1% (13/117) developed symptoms within 30 days after the visit. Thus, 67.3% (104/156) of all P. vivax infected individuals were and remained asymptomatic. There was no significant difference between CS1 (56/86) and CS2 (48/70) . Similarly, 76.7% (56/73) of P. vivax gametocyte positive individuals were afebrile and 64.4% (47/73) were asymptomatic. 53.4% (39/73) of individuals carrying P. vivax gametocytes were asymptomatic (at visit and past 30 days) and did not develop any symptoms within 30 days after the visit  than afebrile infections . Individuals showing any malaria-related symptoms up to the past 48 h also exhibited a significantly higher parasite density  as compared to asymptomatic infections   of all people infected with 6 of all P. falciparum infections (including 1 mixed infection), 12 (75.0 %) were asymptomatic (at visit or up to 48 h before). Of the 15 individuals carrying P. falciparum gametocytes, 11 (73.3%) were asymptomatic. None of the P. falciparum infected individuals developed symptoms within 30 days after the visit  and 1.4 % (CS2), respectively, were caused by P. falciparum, in accordance with a decreasing trend in P. falciparum cases in Brazil since 1989 [P. falciparum infections dropped from 40.8% in 1986 to 6.5% in 2003 [P. falciparum than P. vivax [P. vivax often jeopardizes the adequate management of this species because of compliance issues. As a consequence, in many co-endemic settings successful control has resulted in a shift from P. falciparum to P. vivax being the most prevalent parasite [Most infections were caused by nce 1989 . In the  in 2003 . In addiP. vivax , as the parasite , 28.P. vivax, with male individuals being at higher risk as compared to female. This difference can possibly be explained by different occupation and leisure related behaviour. For instance, one third of adult men were working in the agricultural sector, which was associated with an elevated risk for infection . One fifth of adult men had stated to be unemployed or pursue other not classified occupations. That group was significantly associated with infection. Among the different occupation groups housewives were at highest risk for P. vivax infection, probably because many houses in the study area include an outside area where activities such as laundry or gardening are performed. Our observations are in agreement with a previously published study conducted in a farming settlement in the South of the Amazon State where an increased risk of infection for individuals working in agriculture or housekeeping occurred [P. vivax infections, showing an elevated risk for adolescents and adults, although this is likely associated with behaviour rather than age. The strongest predictor for P. vivax infection was fever. However, one has to keep in mind that in areas with such high proportion of asymptomatically infected individuals, in some cases the fever potentially might be caused by other concurrent (often unrelated) infectious diseases.Multivariate analysis revealed gender and occupation as key factors associated with infection by occurred . In the P. vivax. This can probably be explained by increased attention and control measures by both health post agents and inhabitants in known transmission hotspots. Whether LLINs and IRS can reduce malaria transmission in South America is not fully clear yet, as the main vector in the region, An. darlingi, is to a large extent exophilic, with its main peak of activity occurring at nightfall [P. vivax infections might result from reactivated hypnozoites rather than from recent mosquito bites. According to a study carried out in Papua New Guinea, relapses may be the cause of about 80% of P. vivax episodes [We were not able to find a protective effect for LLINs or IRS. Univariate analyses showed instead that both intervention measures were associated with a higher risk for infection by ightfall . In addiepisodes . A recenepisodes .P. vivax, we identified P. vivax blood-stage parasite density as the only risk factor for carrying gametocytes. Other studies also identified  blood stage density as a major predictor for carriage of gametocytes [P. vivax, gametocytes appear early in infection and their production continues throughout the infection, thus explaining increased risk for gametocyte carriage and increasing gametocyte densities with increasing P. vivax parasitaemia [Amongst individuals positive for etocytes , 32. Fursitaemia .P. vivax positive individuals were positive for gametocytes. Other reports show higher rates of gametocyte carriers; however, these symptomatic individuals had high parasite densities that correlated with better detection of gametocytes [About half of all etocytes , 34, 35.etocytes , while ietocytes . GametocP. vivax infected individuals appeared to be free of symptoms on the day of blood sampling. The majority of them also remained free of symptoms for the following 30 days, as previously reported [Up to three quarters of all reported , 15. We reported .P. vivax gametocytes, the proportion of asymptomatic individuals was smaller. This can be explained by the lower chance to detect gametocytes in low blood-stage parasite densities, such as observed in asymptomatic individuals. Despite having lower parasitemia, and therefore lower gametocytemia, asymptomatic infections had been shown to be able to infect mosquitoes [P. vivax infections (62.8%) were submicroscopic, as opposed to only 26.7 % of P. falciparum infections. Two recent reviews reported similar proportions of subpatent  P. vivaThis work is the first large-scale survey assessing the infectious reservoir, and therefore the malaria transmission potential, with molecular methods in the peri-urban area of Manaus. We identified a large proportion of asymptomatic and submicroscopic malaria infections implicating that these might play an important role in malaria transmission in the study area. These infections are not only a burden to the local population, but also responsible for infections in  visitors . ClassicP. vivax infections in the studied area. The majority of gametocyte carriers did not present symptoms, thus indicating that asymptomatic infections might significantly contribute to malaria transmission in this region. Therefore, efficient control and malaria elimination tools need to include approaches also targeting the asymptomatic reservoir. Longitudinal studies are needed in these locations to analyze the incidence of malaria infection and the risk factors for P. vivax gametocyte carriage.This study shows a substantial proportion of asymptomatic and submicroscopic Additional file 1:Table S1. Primers and probes used for amplification of 18S rRNA, pvs25 and pfs25, using qPCR or RT-qPCR assays including reaction efficiency and detection limits. (DOCX 16 kb)Additional file 2:Table S2. Reaction mixes for amplification of 18S rRNA, pvs25 and pfs25, using qPCR or RT-qPCR. The table describes the reagents, concentrations and template volume used in the qPCR and RT-qPCR assays. (DOCX 13 kb)Additional file 3:Table S3. Conditions for amplification of 18S rRNA, pvs25 and pfs25, using qPCR or RT-qPCR assays. (DOCX 14 kb)Additional file 4:Table S4. Univariate analysis for predictors of P. vivax infection. (DOCX 22 kb)


Plasmodium vivax, while Plasmodium falciparum malaria incidence has decreased in recent years. This review discusses current malaria data, policies and challenges in four South American Amazon countries: Brazil, Colombia, Peru and the Bolivarian Republic of Venezuela. Challenges to continuing efforts to further decrease malaria incidence in this region include: a significant increase in malaria cases in recent years in Venezuela, evidence of submicroscopic and asymptomatic infections, peri-urban malaria, gold mining-related malaria, malaria in pregnancy, glucose-6-phosphate dehydrogenase (G6PD) deficiency and primaquine use, and possible under-detection of Plasmodium malariae. Some of these challenges underscore the need to implement appropriate tools and procedures in specific regions, such as a field-compatible molecular malaria test, a P. malariae-specific test, malaria diagnosis and appropriate treatment as part of regular antenatal care visits, G6PD test before primaquine administration for P. vivax cases , single low dose of primaquine for P. falciparum malaria in Colombia, and national and regional efforts to contain malaria spread in Venezuela urgently needed especially in mining areas. Joint efforts and commitment towards malaria control and elimination should be strategized based on examples of successful regional malaria fighting initiatives, such as PAMAFRO and RAVREDA/AMI.In spite of significant progress towards malaria control and elimination achieved in South America in the 2000s, this mosquito-transmitted tropical disease remains an important public health concern in the region. Most malaria cases in South America come from Amazon rain forest areas in northern countries, where more than half of malaria is caused by  Although significant advances have been made towards malaria elimination in several endemic countries in the Americas, malaria is still an important public health concern. In Latin American tropical and sub-tropical areas there are still several malaria endemic regions that impose a considerable burden on local populations. The majority of malaria cases in South America occur in the Amazon region. In 2015, the four countries reviewed here accounted for 83% of malaria cases in the Americas: Brazil (24%), Bolivarian Republic of Venezuela (30%), Colombia (10%), and Peru (19%) [Within the Americas, populations in the Amazon are the ones at highest risk of malaria infection . Here foPlasmodium falciparum elimination plan was launched a couple of years ago. This review covers the current malaria landscape of these four countries, focusing on specific challenges that need to be addressed in order to advance towards malaria elimination in this region with Amazon countries. The WHO 2016 World Malaria Report [In South America, Argentina and Paraguay are in elimination phase with malaria elimination certified by the World Health Organization (WHO) after three consecutive years of no indigenous cases 2013–2015) [3–2015 [1P. falciparum 39.7%  by Plasmodium vivax, and 3.3% (2804) were mixed infections [Most countries in the Americas have shown an impressive decreasing trend in malaria incidence over the first decade of this Century/Millennium (reviewed in ), includnd 3.3% 204 were mPlasmodium: P. falciparum, P. vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. In most endemic regions, malaria is due mainly to infection by either P. falciparum or P. vivax. The proportion of these two parasite species varies geographically, depending in part on susceptibility to infection of specific populations. In Africa, Duffy-negative (Fy−) blood group is widespread [P. falciparum over P. vivax infections. This has been attributed to the model by which P. vivax parasites require attachment to the Fy antigen (receptor) on erythrocytes, therefore the absence of Fy antigen results in protection from P. vivax malaria infection, although there are reports of vivax malaria in Duffy-negative individuals in Africa. In the Americas, including the four South American countries reviewed here, there has been an increasing relative predominance of P. vivax infections [Human malaria is caused worldwide by at least five different species of the genus despread  and therfections  althoughP. vivax accounts for the majority of malaria cases. In the countries reviewed here there is also a majority of P. vivax over P. falciparum cases, except for some regions in Colombia where the proportion of these parasite species changes and a greater proportion of P. falciparum was observed overall in 2016 .Table 1P. vivax and P. falciparum make P. vivax particularly challenging to control and eliminate in malaria endemic regions [P. falciparum infections) which go undetected and, therefore, sometimes untreated resulting in higher levels of transmission to mosquitoes , a faster growth capacity in the mosquito vector and at lower temperatures, the presence of hypnozoites  that allow relapses to occur at later times, and natural immunity acquired earlier in life compared to than against P. falciparum. Moreover, although malaria caused by P. vivax was for a long time considered benign in terms of severity of disease compared to falciparum malaria, recently this perception has changed, and vivax malaria has emerged as a neglected potentially severe disease, in particular in Southeast Asia and South America [Recent review highlights why biological differences between  regions –15. Thes America  with an In Latin America, previously neglected vivax malaria is now emerging as an important focus of research in regions where the majority of malaria infections are caused by this parasite (reviewed in ). In PerP. falciparum/P. vivax mixed infections [Plasmodium spp. infections at four malaria endemic areas of the Pacific coast only 7.5% of the cases were classified as clinically severe malaria, caused by both P. vivax and P. falciparum [P. vivax or P. falciparum infections at tertiary hospitals in malaria endemic areas of Colombia with special emphasis on the Pacific coast, severe thrombocytopaenia (43%), hepatic dysfunction (40%), and severe anaemia (34%) where the most common complications .In Colombia, studies point to higher morbidity and mortality for fections , 24. Amolciparum . In an oPlasmodium malariae is however another malaria-causing parasite. It differs from other Plasmodium species by exhibiting a lower growth rate and very low levels of parasitaemia. Human infection by P. malariae is often asymptomatic and long-lasting (up to last 40 years in the absence of re-infection) with blood stages probably persisting for a lifetime [Plasmodium-infected blood, as observed in Latin America [Although not usually acknowledged because of its low incidence in Latin America (<1%), lifetime –29. Patilifetime  or when  America .P. malariae is considered a milder pathogen compared to P. falciparum and P. vivax, except it can result in cases of chronic nephropathy. In Africa, the most commonly observed malaria corresponds to P. malariae and P. falciparum. When P. malariae is diagnosed as a mono-infection in Brazil, it is treated with chloroquine (25 mg/kg over 3 days); for coinfections the treatment is chloroquine with the addition of primaquine for P. vivax coinfection  fever cycles, as opposed to all other human malaria parasites whose infection results in 2-day (tertian fever) cycles. P. malariae infections in Latin America was only 0.1%  [P. malariae in the region may be underestimated due to difficult microscopic differentiation from P. vivax as well as to low parasitaemia in coinfections; microscopists are not well trained in identifying this parasite due to its very low occurrence. In the early 1970s, P. malariae was identified in over 80% of all Plasmodium positive samples in an isolated Amerindian population in southeast Peru, with some cases of P. malariae coinfection with P. vivax [P. malariae cases while microscopic examination of the same samples showed only 1.2% (6 out of 497) [P. malariae infections in the population [P. malariae infections were almost half (43.8%) of all symptomatic malaria cases tested by nested PCR [P. vivax/P. malariae combination occurred in 79.2% of coinfections. Because no P. malariae cases were reported in Colombia in 2015, the authors of this study suggest under-detection by the gold standard method of thick smear microscopy and poor detection regarding mixed infection and low parasitaemias. A very recent report from Colombia showed that P. malariae infections were not detected by microscopy, only by PCR; P. malariae infections were both single as well as mixed with P. vivax or P. falciparum [P. malariae is present in small foci in communities of the Colombian Pacific coast [According to the Amazon malaria initiative (AMI) annual report for 2015, in 2014 the percentage of P. vivax . Using m of 497) . A 2005 pulation . A very sted PCR . The stulciparum . Althougic coast  extensivPlasmodium species was identified were caused by P. malariae (58.4%), with 20.7 and 17.9% due to P. vivax and P. falciparum infection respectively [Plasmodium species found was only identified in 3 cases, all caused by P. malariae.A very recent review of reports published between 1971 and 2016 of blood transfusion-transmitted malaria in the Americas showed that more than half of the malaria cases in which the ectively . Most caPlasmodium parasites, and thus they might constitute a natural reservoir for human malaria. The simian Plasmodium brasilianum, closely related to P. malariae, causes quartan fever in New World monkeys [P. brasilianum in the Venezuelan Amazon, affecting remote Yanomami indigenous communities [P. malariae and P. brasilianum are the same quartan malaria parasite species—an anthropozoonosis—that moves freely between NHPs and humans. Natural infections of monkeys in Panama, Venezuela, Colombia, Peru, Brazil, and French Guiana have been reported [Plasmodium simium, genetically related to P. vivax [P. simiun [Non-human primates (NHPs) are also infected with  monkeys . A very munities . The autreported , 40. PlaP. vivax , is anotP. vivax . Whether. simiun , requireIn each of the four countries except for Brazil, the Ministry of Health (MoH) issues weekly epidemiological bulletins reporting on incidence of different diseases including malaria, for which data are shown as number of malaria cases, type of infection (parasite species), distribution per state and per district/municipality, distribution per age groups, risk maps, and often reporting number of detected imported cases Table . These bP. vivax patients [In Latin America, chloroquine (CQ) is still widely used for vivax malaria treatment of asexual stages . A total adult dose of 1500 mg/base is given over 3 days, in the countries reviewed here as a first day dose of 10 mg/kg followed by 2 days of 7.5 mg/kg each for a total dose of 25 mg/kg , 46. Pripatients . Identifpatients , whereasP. falciparum in the Brazilian Amazon reported recently [For falciparum malaria, ACT is used in the four countries, given over 3 days Table . Artesunrecently  along wiP. falciparum transmission in Brazil and Venezuela, with the recent addition in 2015 of Peru [P. falciparum infections. The P. falciparum PQ dose has been recently reduced by a new WHO recommendation from 0.75 mg/kg to a dose considered safe for individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency  [P. malariae, when diagnosed, consists of CQ without the need for PQ.A single dose of PQ as a gametocytocidal is added to ACT to prevent  of Peru . Colombie below) , howeverP. falciparum elimination launching a National Elimination Plan aiming at reducing cases by 90% by the year 2030. In November 2015, the country’s NMCP was recognized as the 2015 Malaria Champion of the Americas [During the 2000–2014 period, several countries in Latin America have achieved remarkable reductions in malaria incidence  in 1990, most of them in Bolivar state (86.2% of the country cases), and three municipalities in “epidemic” and two in “alarm” status . In AmazIn a document prepared for and presented in September 2016 at the PAHO 68th session of the Americas regional committee for the malaria elimination project (action plan) 2016–2020, Venezuela was shown to have had a 205% increase in number of malaria cases in 2014 as compared to 2000 Fig. . Except Although no official publications were available since the government stopped publishing malaria epidemiological reports in late 2014, some have been later published online including all weekly reports for the years 2015 and 2016 which appeared in 2017. The re-emergence of malaria has resulted in a crisis that has been profiled in important international newspapers. A recent article in The New York Times indicated that currently cases show high morbidity and mortality due in part to the lack of available anti-malarial drugs such as CQ and PQ in affected regions, including gold mining areas in which mining is mostly illegal (see below) . Men comP. vivax and 20% P. falciparum infections .Thirteen academic and public health Venezuelan institutions have come together to call attention to the current malaria epidemic in the country; with 146,670 cases registered until by August 27th for the year 2016, they have issued an alert that this is higher than the number of cases for the same time period in 2015  and PAHOThe situation in Venezuela underscores the need for concerted malaria control efforts by Amazon countries in the region. About 78 and 81% of Brazil and Colombia malaria imported cases, respectively, are estimated to come from Venezuela .P. falciparum resistance to CQ . Active case detection strategies were implemented, and identified crucial strategies were strong community involvement, an extensive microscopy network and ongoing epidemiologic investigations at the local level. Regional partnerships in the region supported these efforts, including the Amazon network for the surveillance of anti-malarial drug resistance (RAVREDA), which along with the Amazon malaria initiative (AMI) have contributed substantially to malaria control efforts in South America, including the crucial policy change to ACT treatment of uncomplicated falciparum malaria of all these countries by 2006 [Inter-country collaboration efforts at border areas may be needed, as demonstrated by the example of Ecuador and Peru eliminating malaria in recent years from adjacent coastal border regions . Local p by 2006 .P. falciparum and 63% for P. vivax  between 2005 and 2010 implemented interventions at the border areas of four countries  that led to important reductions in malaria incidence. This project, called PAMAFRO , was part of an effort to work towards social integration of Andean countries particularly focusing on health and education. Malaria was chosen because of its international disease status of high priority in the region, affecting poor populations usually located in border areas. Among the malaria control strategies implemented there was critical community participation with local health workers involved and health education campaigns. During the period PAMAFRO activities were implemented, malaria declined in Peru by 95% for iewed in ).Urban and peri-urban malaria, as opposed to the exclusively rural malaria in poor and isolated settings, is now a recognized phenomenon due in part to traveling but mostly to bigger population moves, especially from rural to urban and peri-urban areas. The origin of malaria cases found (diagnosed) in urban areas is not however clearly defined. In order to implement appropriate malaria control measures, it is important to determine whether these cases are being transmitted within the urban settings where they are detected or have been imported from rural settings. Urban malaria cases have been reported in Brazil, Colombia, and Venezuela .Anopheles mosquitoes in the house, similar Plasmodium clones among humans and vectors in the urban area, as well as detection of gametocytes or sporozoites in infected mosquitoes [A recent report on urban malaria across several Centres of Excellence for Malaria Research (ICEMR) including one each in Brazil and Colombia, and several others in Asia and Africa emphasizes that this determination requires epidemiological as well as entomological information about transmission . They susquitoes .In Brazil, the “free economic zone” economic model in the Amazon encouraged in the 1970s brought multinational companies to the region because of tax exemptions, resulting in uncontrolled waves of migration to the periphery of the city of Manaus from the interior of the Amazonas and other northern states . There hIn Peru, peri-urban malaria has also been reported in different cities in the Amazon (Iquitos ), three Mobile populations such as miners, migrants, labourers are at high risk of malaria infection in the Americas. Malaria infection among miners in Brazil contribute about 6% of the country’s total cases, 3% in Colombia and 47% in Venezuela . In the Anopheles darlingi. Migration of previously unexposed to malaria populations into malaria endemic areas due to mining jobs has led to spikes in malaria cases in mining locations in all countries in this review. Furthermore, these areas usually lack good health care and disease prevention/control measures. The workers, upon return to their home villages, may introduce malaria parasites to new regions that could then be at risk of malaria depending on climate, activities and vector species present.Gold mining, often illegal in developing countries, affects ecosystems greatly as it starts with vast deforestation. Development of mining activities results in turn in highly mobile populations that migrate in search of jobs, usually young males, that work outdoors and are exposed to mosquito bites for long periods of time daily and sometimes live in camps with incomplete walls, allowing for an even wider exposure to mosquitoes. Deforestation and still water favour mosquito proliferation, especially In Venezuela, gold mining has been associated with high malaria incidence in specific municipalities such as Sifontes where this activity is important, situated in the Bolivar state in the eastern region of the country . The samgarimpeiros work long hours and live in conditions of very poor hygiene and nutritional deficiencies, leading to poor health. Medical care is free in the French health centres, but the remoteness of the mines (sometimes 4 days by boat) and the fear of law enforcement hampers effective access to care by the miners. In Suriname, also with a considerable proportion of Brazilian miners (66%) [In French Guiana, recent data show that malaria affects many Brazilian gold miners working illegally in numerous mining sites . Most ofrs (66%)  malaria rs (66%) , 80.P. vivax infections in pregnancy.Reports of malaria in pregnancy in South America are limited (reviewed in ). MalariP. vivax) and 3.3/16.5% for placental malaria  [There are a few recent reports on malaria in pregnancy from malaria endemic areas in Colombia that underscore the need for malaria diagnosis (preferably with a molecular method) and infection control as part of antenatal care in these areas. Two studies showed that malaria in pregnancy in these regions may be under diagnosed when evaluated by microscopy instead of PCR (some cases are asymptomatic). In a northwest Colombia area bordering Panama, the frequency of malaria in pregnancy for 2004–2007 detected as maternal blood infection by microscopy was 13% of women; however when assessed by PCR this rose to 32% . Anotherciparum) .P. falciparum and P. vivax infections, and although most cases that progress into severe malaria are due to P. falciparum, P. vivax can also result in severe malaria. Although Colombia uses PQ for vivax malaria, it is contraindicated in pregnancy and pregnant women may, therefore, have recurrent infection due to relapses of the dormant liver stages (hypnozoites) which can only be eliminated with PQ. A report on a retrospective study from pregnancies in 2005–2006 in the Colombian northwest found severe malaria in pregnancy with hepatic dysfunction and spontaneous bleedings as the most frequent complications present in >70% of severe cases [P. falciparum and P. vivax infections in pregnancy, with an almost 20% of P. falciparum infections being afebrile (asymptomatic), at risk of being undetected. Another report from three malaria-endemic areas of Colombia of a descriptive study of 34 malaria in pregnancy cases from 2011 to 2013 included three (8.8%) severe P. falciparum malaria cases with moderate anaemia (mean Hb 8.7 ± 0.1 g/dL) and an additional two severe malaria cases in women who developed a second malaria episode during pregnancy  [P. falciparum infection presented hepatic dysfunction with hyperbilirubinaemia and clinical jaundice, while the P. vivax case showed severe anaemia (Hb 4.4 g/dL) with general pallor. The second malaria episode during pregnancy in six women was due to the same parasite species as the first infection  but with lower parasitaemia in the P. vivax infections  and lower Hb levels. The patients had received treatment without PQ for P. vivax as it is contraindicated in pregnancy.In Colombia, malaria in pregnancy can be caused both by re cases . This st. vivax) . Of the P. vivax. Out of the 13,308 malaria cases reported in Manaus from 2003 to 2006 among women aged 10–49 years, 6.1% were in pregnant women and 80% of these were due to P. vivax [P. vivax infection [P. vivax and P. malariae  with most cases from a rural area near the forest [In Brazil, where PQ is also not prescribed to pregnant women (who receive anti-relapse prophylaxis consisting of CQ 7.5 mg/kg weekly throughout pregnancy) studies have shown that malaria in pregnancy has a higher prevalence than malaria in non-pregnant women of similar age in endemic regions, and most of it (about 80%) is due to P. vivax . In 2007nfection . A recene forest . Brazil e forest .P. falciparum compared with non-pregnant male and female counterparts in the same age groups [P. vivax infection in severe malaria in pregnancy in Peru is highlighted in a case report that occurred in 2007: a 19 year-old pregnant woman with a history of travel was admitted to the hospital and later diagnosed with severe P. vivax infection when transferred to the intensive care unit (ICU) with multiple organ failure and stillbirth, leading to death [In contrast, a retrospective analysis of pregnant women living in the Amazon region of Iquitos in 2004–2005, Peru, showed a lower prevalence of clinical malaria in pregnant women, although the authors suggest this may be due to passive case detection and surveillance bias, and community-based data including active case detection suggested that this population may have a higher frequency of asymptomatic infections and higher incidence of e groups . The usee groups . The impto death .P. vivax with almost three quarters presenting symptoms [P. vivax infected mothers (3/5); and one case of P. vivax placental malaria (0.8%) was registered.In the southwestern Bolivar state of Venezuela , a descriptive malaria epidemiological study including pregnant women conducted in 2005–2006 revealed an incidence of malaria in pregnancy of 27.4%: 87% symptoms . In thisP. falciparum or P. vivax. In the early 1960s, CQ resistance in P. falciparum appeared in migrants working in endemic malaria areas along the Thailand–Cambodia and Colombia and Venezuela border areas and later spread to Africa in the late 1970s probably imported by labourers from Southeast Asia [P. falciparum has arisen in Southeast Asia [kelch13 (k13) gene [Anti-malarial drugs, especially when given as monotherapy in the past for a period of time, have resulted in drug resistance due to mutations arising in target parasites such as ast Asia –95. Curr13) gene , however13) gene , althoug13) gene .P. falciparum infection treated with ACT (N = 1554) was reported by day 4 in 1528 (98.3%) patients, whereas there was delayed clearance (between days 4 and 7) in the remaining 26 patients (1.7%) (unpublished observations). Although a recent global survey of ACT resistant parasites which included South American samples from the Peruvian and Brazilian Amazon as well as from the Colombian coast indicated that all parasite isolates analysed from this region lacked k13 mutations associated with resistance [P. falciparum isolates without k13 mutations, indicating that there may be additional genes that when mutated contribute to artemisinin resistance in these parasites [A retrospective survey using medical records from 1992 to 2008 in Manaus, Brazil, showed that parasite clearance detected by microscopy in patients with sistance , these rarasites .P. vivax.In the Americas, CQ is still used widely for vivax malaria. Although there are reports of CQ resistance in the Brazilian Amazon , 103 wheP. falciparum and P. vivax infections. Rapid diagnostic tests (RDTs) are being encouraged to some extent in Brazil and Colombia especially in remote areas with no easy access to microscopy facilities . However, reports from Peru starting in 2010 revealed the existence of P. falciparum parasites containing deletions of the pfhrp2 gene in the Peruvian Amazon region [P. falciparum isolates with pfhrp3 deletions as well, and some parasites containing both hrp2 and hrp3 gene deletions. In areas of low malaria transmission such as the four countries reviewed here, P. falciparum parasite densities in infected individuals are usually low, further increasing the probability of a false negative result. An alternative P. falciparum test in regions known to have pfhrp2 deletions is one based on detection of P. falciparum lactate dehydrogenase (LDH). This test performs well in the areas reported to be affected by pfhrp2 deletions in Peru [pfhrp2 deletions, WHO has recently recommended to further analyse P. falciparum isolates from infections showing a discordance between HRP2-RDT and microscopy results (≥10–15% higher positivity microscopy rates) by PCR first to confirm the presence of P. falciparum and then proceed to confirm a possible pfhrp2 gene deletion by PCR and antigen analysis [Most n region , 105 resn region . These r in Peru , althouganalysis .Plasmodium—specific nucleic acids based on their amplification by ultrasensitive techniques, including using a high-volume sample quantitative polymerase chain reaction (qPCR) method for the detection of low-density parasitaemias (>20 parasites/mL) [An increasing number of studies are being published that performed molecular diagnosis of malaria by detection of ites/mL) . In Asiaites/mL) –113 (Tabites/mL) , 115.P. vivax infections [P. vivax carriers from the Brazilian Amazon were able to infect mosquitoes at a rate of about 30% . Ongoing studies in Amerindian communities indicate that asymptomatic infection prevalence may be even higher, also with a vast majority of P. vivax infections; upcoming studies will similarly address asymptomatic infections in gold mining populations .The PCR-positive cases that are microscopy- or RDT-negative are usually asymptomatic infections that go undetected also due to low parasitaemias in subjects that may be screened by non-molecular methods as part of malaria surveys in endemic regions. The parasites in these infections constitute a reservoir that contains infective gametocytes that can indeed be transmitted to mosquitoes, as shown by a recent study from Colombia with natural or experimentally-induced fections . Asymptoout 30%  and mixed infections were revealed by PCR analysis that had been regarded as single infections, including those of P. vivax or P. falciparum with P. malariae (see above); the later parasite was only detected by PCR [A molecular method that has been evaluated as an alternative to the more expensive PCR technique is the loop mediated isothermal DNA amplification (mLAMP) , a fasteominates . The stud by PCR .P. vivax as determined by qPCR, 2.4-fold higher than prevalence detected by microscopy (1.8%). Furthermore, from all P. vivax infected individuals, about half (46.8%) were positive for gametocytes as detected by qRT-PCR and 82.7% did not report a concurrent febrile illness (asymptomatic). In the same study, P. falciparum infections were rare (0.8% by qPCR) of which 73.3% were asymptomatic.In a cross-sectional study conducted in 2012 in the Manaus area , 4.3% ofAnopheles mosquito species In the Americas, with several acting as malaria vectors. Their ecology and biting behaviour are not well studied, and show regional variation. The major malaria mosquito vector in the countries reviewed here is Anopheles darlingi [P. falciparum and P. vivax. It is the main malaria vector species in the western Brazilian Amazon [Anopheles species have been reported to be involved in malaria transmission including An. darlingi along with An. nuneztovari and An. albimanus [There is a great diversity of darlingi , a very n Amazon . In Cololbimanus .In Latin America, including the four countries reviewed here, vector control methods have been implemented in endemic areas, mostly the use of long-lasting insecticide treated nets and indoor residual spraying (IRS). However, there is a knowledge gap on the impact that these measures have on suppressing mosquito vector populations and parasite transmission.All four countries recommend the use of insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs), which are distributed free of charge to all age groups Table . IRS is Glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDd) is a widespread enzyme deficiency that affects approximately 400 million people worldwide (reviewed in –129). G6Two recent reviews of G6PDd provide some prevalence estimates worldwide and for Latin America (LA)  which sometimes result in extreme degrees of deficiency. G6PD A− is considered to be less severe in comparison. The G6PD A− variant is believed to have came to the Americas with West African populations during the transatlantic slave trade. The G6PD A-202A mutation is the variant most broadly distributed, identified in 81.1% of the G6PDd individuals surveyed in the Americas . ReportsA recent study from four malaria endemic areas in Colombia found a 6.56% of G6PDd overall (N = 426) including both intermediate and severe deficiency . PreviouP. falciparum transmission and P. vivax relapses, therefore knowledge of local G6PDd prevalence should be taken into account. Although none of the malaria endemic countries in the Americas has currently as policy a requirement for G6PD testing prior to PQ administration, Brazil is currently considering to introduce a recommendation to test, whenever possible, for a P. vivax diagnosis before PQ treatment. An alternative safer treatment for positive G6PDd cases identified would be PQ at 0.75 mg/kg/week for 8 weeks. The lack of a good qualitative rapid G6PD activity test to be used in the field is a challenge. The chromatographic CareStart™ test has been recently tested in the Brazilian Amazon in 674 individuals (320 of whom where infected with P. vivax) and shown to have low sensitivity in detecting mild and intermediate G6PDd, however its high sensitivity in detecting severe deficiency makes it a good candidate as a RDT for G6PDd [Implementation of malaria pre-elimination and elimination programmes will rely on the use of PQ to prevent or G6PDd . A recenor G6PDd .P. falciparum infections   Vivax Working Group, a collaboration of 18 countries that has worked successfully in a joint effort to build knowledge, tools, expertise, evidence and regional consensus based on identified knowledge gaps to effectively change practice . In SoutHard to reach isolated populations  for whom traditional health system practices are largely unavailable remain a considerable challenge for case detection. On the other hand, urban malaria requires further investigation into possible transmission occurring within cities where cases are reported.Implementation of a field-compatible molecular test for malaria diagnosis in rural areas;P. malariae infections in addition to P. falciparum and/or P. vivax in areas where this parasite has been reported;Testing that can detect Malaria in pregnancy, an important population for malaria control and elimination programmes, calls for malaria diagnosis and appropriate treatment for positive cases as part of regular antenatal care visits, especially in remote malaria endemic areas.P. vivax infection which causes the majority of malaria cases in the endemic areas reviewed here with.Implementation of G6PDd field-compatible diagnostic tests to guide PQ administration, especially for An alternative (8 weeks) weekly PQ regimen for vivax malaria for G6PDd individuals, preferably as DOT and/or with specific instructions to patients regarding alarming haemolysis signs such as dark urine.P. falciparum infections in Colombia, especially on the Pacific coast where there is a higher incidence of falciparum malaria.Addition of SLD PQ for The data presented here supports the following recommendations:


Plasmodium knowlesi occurs throughout Southeast Asia, and is the most common cause of human malaria in Malaysia. Severe disease in humans is characterised by high parasite biomass, reduced red blood cell deformability, endothelial activation and microvascular dysfunction. However, the roles of intravascular haemolysis and nitric oxide (NO)-dependent endothelial dysfunction, important features of severe falciparum malaria, have not been evaluated, nor their role in acute kidney injury (AKI). In hospitalised Malaysian adults with severe (n = 48) and non-severe (n = 154) knowlesi malaria, and in healthy controls (n = 50), we measured cell-free haemoglobin (CFHb) and assessed associations with the endothelial Weibel–Palade body (WPB) constituents, angiopoietin-2 and osteoprotegerin, endothelial and microvascular function, and other markers of disease severity. CFHb was increased in knowlesi malaria in proportion to disease severity, and to a greater extent than previously reported in severe falciparum malaria patients from the same study cohort. In knowlesi malaria, CFHb was associated with parasitaemia, and independently associated with angiopoietin-2 and osteoprotegerin. As with angiopoietin-2, osteoprotegerin was increased in proportion to disease severity, and independently associated with severity markers including creatinine, lactate, interleukin-6, endothelial cell adhesion molecules ICAM-1 and E-selectin, and impaired microvascular reactivity. Osteoprotegerin was also independently associated with NO-dependent endothelial dysfunction. AKI was found in 88% of those with severe knowlesi malaria. Angiopoietin-2 and osteoprotegerin were both independent risk factors for acute kidney injury. Our findings suggest that haemolysis-mediated endothelial activation and release of WPB constituents is likely a key contributor to end-organ dysfunction, including AKI, in severe knowlesi malaria. Plasmodium knowlesi is an important emerging zoonotic infection in Southeast Asia, and is now the most common cause of human malaria in Malaysia2 and regions of western Indonesia4. The risk of severe disease in adults is at least as high as in falciparum malaria6, and fatal cases occur7. Features of severe knowlesi malaria are similar to those of severe falciparum malaria in adults, and include hyperparasitaemia, jaundice, acute kidney injury (AKI), respiratory distress, shock and metabolic acidosis6. However, in contrast to P. falciparum, P. knowlesi-attributed coma has not been reported to-date, and endothelial cytoadherence, a key pathogenic feature of severe falciparum malaria, does not appear to occur9. Thus, alternative pathogenic mechanisms may play a greater role in severe knowlesi malaria. We have recently reported that, as with falciparum malaria, disease severity in knowlesi malaria is associated with parasite biomass, endothelial activation, and microvascular dysfunction10, as well as reduced red blood cell (RBC) deformability11. However, the roles of intravascular haemolysis and nitric oxide (NO)-dependent endothelial dysfunction, important features of severe falciparum malaria16, have not yet been reported.The monkey parasite 2+) to ferric (Fe3+) haemoglobin. Ferric haemoglobin (methaemoglobin) then releases haem17, which due to its hydrophobic nature readily intercalates into cell membranes and increases susceptibility to oxidant-mediate damage18. Free haem mediates a range of other pathogenic effects, including increased production of reactive oxygen species and proinflammatory cytokines20, and upregulation of endothelial cell adhesion molecules21. CFHb is also able to quench nitric oxide (NO), and in adults with falciparum malaria is associated with reduced NO-dependent endothelial function, and hyperlactatemia, suggesting a role in impaired tissue perfusion12.In conditions associated with intravascular haemolysis, such as severe falciparum malaria, the cell-free haemoglobin (CFHb) released during erythrocyte rupture is readily oxidised from ferrous , angiopoietin-2, P-selectin, and osteoprotegerin (OPG), are considered key markers and mediators of endothelial activation. Plasma concentrations of vWF have been shown to increase soon after inoculation of P. falciparum into human volunteers24, and both vWF and OPG increase early in P. berghei-infected mice26, suggesting that endothelial activation is an early host response in malaria. In both knowlesi and falciparum malaria, endothelial activation is associated with disease severity and measures of impaired organ perfusion27.CFHb has also been shown to stimulate degranulation of endothelial Weibel–Palade bodies (WPBs) via TLR4 signalling28. However, the contribution of this process to disease severity, and the association with endothelial activation and dysfunction, has not been evaluated. We therefore measured CFHb in Malaysian adults with severe and non-severe knowlesi malaria, and assessed associations with the endothelial WPB constituents angiopoietin-2 and osteoprotegerin, endothelial and microvascular function, and other markers of disease severity.In severe knowlesi malaria, high parasitaemias can develop rapidly, and intravascular haemolysis has been reported29, in addition to 50 healthy controls. Clinical and pathophysiological data from a subset of these patients have been previously reported10. Baseline demographic, clinical and laboratory features of patients and controls are shown in Table A total of 202 patients with knowlesi malaria were enroled, including 154 with non-severe and 48 with severe malaria by WHO criterian = 24, 50%), jaundice , respiratory distress , severe AKI by WHO criteria , shock , metabolic acidosis , severe anaemia  and abnormal bleeding . Nineteen patients (40%) had one severity criterion, 17 (35%) had two and 12 (25%) had three or more. Using KDIGO criteria to define AKI , AKI was present on admission in 40 (83%) patients with severe malaria and 44 (29%) patients with non-severe malaria. AKI developed during admission in another two (4%) patients with severe and two (1%) patients with non-severe malaria.Among the 48 patients with severe knowlesi malaria, WHO severity criteria included hyperparasitaemia (p < 0.0001 for both comparisons), and higher in those with severe compared to non-severe disease (p < 0.0001) . Haptoglobin was lower in patients with severe and non-severe knowlesi malaria compared to controls , and lower in patients with severe compared to non-severe knowlesi malaria (p = 0.004). In patients with severe and non-severe knowlesi malaria, there was no significant difference in CFHb or haptoglobin in those enrolled prior to, compared to post, commencement of antimalarial treatment . Endothelial function was associated with P. knowlesi parasitaemia , and with lactate , with the latter remaining significant after controlling for parasitaemia (p = 0.041). There was no association between endothelial function and CFHb.Endothelial function, as measured by the reactive-hyperaemia index (RHI), was lower in patients with severe knowlesi malaria compared to those with non-severe knowlesi malaria . There was a positive correlation between endothelial and microvascular function as measured by RHI and NIRS, respectively , remaining significant after controlling for parasitaemia and age .Microvascular function, as measured by NIRS, was reduced in patients with knowlesi malaria in proportion to disease severity  , and with microvascular dysfunction , with both correlations remaining significant after controlling for parasitaemia . CFHb correlated with creatinine , remaining significant after controlling for parasitaemia (p = 0.001). Using logistic regression and controlling for parasitaemia, log CFHb was associated with risk of AKI by KDIGO criteria on or during admission . There was no association between CFHb and patient age.CFHb was correlated with parasitaemia , likely reflecting release of this enzyme from RBCs31. There was no association between CFHb and the other liver aminotransaminase, alanine transaminase.CFHb was correlated with aspartate transaminase . Similarly, OPG was increased in severe compared to non-severe knowlesi malaria , and increased in both groups compared to controls (p < 0.0001 for both comparisons). No increase in P-selectin was seen in knowlesi malaria patients overall compared to controls, and VWF, although increased in knowlesi malaria patients compared to controls, was not increased in severe compared to non-severe disease .As CFHb has been shown to stimulate degranulation of WPBsse Table 0. Similar = 0.33, p < 0.0001) and OPG , with both correlations remaining significant after controlling for parasitaemia , independent of parasitaemia. OPG also correlated with age in healthy controls . Age is a known risk factor for severe knowlesi malaria10. However, in a backward stepwise logistic regression model controlling for age and parasitaemia, both OPG and angiopoietin-2 (but not CFHb) remained as independent risk factors for severe malaria (with hyperparasitaemia removed as a severity criterion), and for AKI (Table p = 0.040); however, this did not remain significant after controlling for parasitaemia and age.Both angiopoietin-2 and OPG correlated with age in knowlesi malaria patients (10), OPG was also associated with ICAM-1  and E-selectin , with both correlations remaining significant after controlling for parasitaemia and age (p ≤ 0.0001 for both correlations). OPG was also correlated with IL-6 , remaining significant after controlling for parasitaemia and age (p < 0.0001). The correlations between OPG and E-selectin, ICAM-1 and IL-6 were also independent of angiopoietin-2 , lactate , microvascular dysfunction  and endothelial dysfunction   is increased in knowlesi malaria in proportion to disease severity, and to a greater extent than that seen in falciparum malaria. Furthermore, intravascular haemolysis is independently associated with markers of disease severity, including lactate, microvascular dysfunction, and creatinine, suggesting that haemolysis likely contributes to impaired tissue perfusion and organ dysfunction in knowlesi malaria. With the apparent paucity of P. falciparum may reflect the 24-h erythrocytic life-cycle of P. knowlesi, or may reflect poor adaption of P. knowlesi to the human host. Massive destruction of RBCs has been previously reported in P. knowlesi-infected rhesus macaques (Macaca mulatta), another unnatural host for this parasite. As well as the red cell agglutination and sludging reported in these early studies32, haemolytic phenomena such as haemoglobinuria, renal tubular acidosis and haemoglobin casts within tubular lumens were frequently noted as pre-terminal events34. The marked haemolysis associated with P. knowlesi makes this parasite an ideal model to study the pathophysiological consequences of haemolysis in severe human malaria.As with falciparum malaria, the cause of intravascular haemolysis in severe knowlesi malaria is likely multifactorial, with lysis of infected and uninfected red blood cells both contributing. The greater severity of intravascular haemolysis in severe knowlesi compared to 12, and with oxidative damage which contributes to AKI15. In addition, we now show that in knowlesi malaria, intravascular haemolysis is independently associated with the endothelial cell WPB constituents angiopoietin-2 and OPG, suggesting that endothelial activation is likely a key mediator of haemolysis-induced end-organ damage. An association between CFHb and angiopoietin-2 has been previously demonstrated in adults12 and children13 with falciparum malaria; however, we now extend these findings by demonstrating an association in P. knowlesi between CFHb and both angiopoietin-2 and OPG that is independent of parasite biomass. Our findings are consistent with previous in vitro and murine reports demonstrating that CFHb stimulates degranulation of WPBs23. In the current study, the lack of independent associations with CFHb and markers of end-organ damage, after controlling for angiopoietin-2 and/or OPG, further supports the role of endothelial activation in mediating pathophysiological consequences of intravascular haemolysis.The mechanisms by which haemolysis mediates end-organ damage in severe malaria are incompletely understood. In severe falciparum malaria, haemolysis is associated with impaired NO-dependent endothelial dysfunction27, a notable finding of our study was the marked elevation of the other key WPB constituent, OPG, in severe knowlesi malaria. OPG is a member of the tumour necrosis factor (TNF) receptor superfamily, and is a soluble decoy receptor for the receptor activator of NF-кB ligand (RANKL), thus modulating the interaction between RANKL and its receptor RANK35. OPG has a widespread tissue distribution, including in vascular and immune tissues. In vascular tissues, release of OPG from endothelial cells is upregulated by cytokines including TNF, IL-1a and IL-1b37. OPG has been shown to stimulate endothelial cell migration38, to increase leukocyte adhesion to endothelial cells both in vitro and in vivo39, and to upregulate endothelial cell adhesion molecules in the presence of TNF40. This latter effect of OPG is consistent with the results of our current study, with OPG independently associated with endothelial adhesion molecules ICAM-1 and E-selectin. Upregulation of adhesion molecules by OPG may also explain in part the association between OPG and mortality in a recent study of African children with cerebral malaria25.While increased angiopoietin-2 is well documented in severe malaria, and known to be a key marker of disease severity41, Hashimoto’s thyroiditis42 and type 1 diabetes mellitus43. In addition, OPG is elevated in other conditions associated with endothelial dysfunction, such as cardiovascular disease, and in patients with diabetes mellitus is associated with adverse cardiovascular outcomes and mortality44. Endothelial dysfunction is a key feature of severe malaria, resulting from reduced NO bioavailability16. OPG is known to block RANKL-induced activation of the intracellular eNOS pathway in vitro, and to reduce endothelial NO production45. The association between OPG and endothelial dysfunction in severe knowlesi malaria suggests OPG likely exacerbates endothelial NO deficiency, contributing to severe disease. OPG inhibition of eNOS and endothelial NO production is reversed in vitro by RANKL45. Taken together, these findings raise the possibility that RANKL may be a candidate adjunctive treatment to improve NO bioavailability in conditions associated with elevated OPG and endothelial dysfunction such as severe malaria.We also found an independent association of OPG with endothelial and microvascular dysfunction, as measured by RHPAT and NIRS, respectively. This is consistent with other studies which have demonstrated an association between OPG and endothelial dysfunction in other conditions, including hyperuricemia46. As RANKL has been shown to reduce macrophage production of proinflammatory cytokines47, inhibition of RANKL by OPG may be expected to increase inflammatory responses. In keeping with this, in our study, OPG was independently associated with IL-6. This is also consistent with a murine study, in which inflammatory cytokines, including IL-6, TNF, IL-1B and MCP-1, were reduced in OPG knockout mice and in WT mice infused with RANKL46.In addition to its release from endothelial cells, OPG is also expressed in immune cells, including dendritic cells and macrophages, and may modulate inflammatory responses through inhibition of RANKL/RANK signalling48), and new treatment strategies to prevent malaria-associated AKI are needed. In falciparum malaria, haemolysis has been linked to AKI from oxidative stress and lipid peroxidation49, and further studies are warranted to determine if haemolysis-induced oxidative stress also contributes to AKI in knowlesi malaria. The pathogenic pathways of CFHb may present targets for adjunctive treatments to protect against AKI in both falciparum and knowlesi malaria51.In this study we found that OPG and angiopoietin-2 were both independently associated with AKI, suggesting that haemolysis-induced endothelial activation is an important mechanism of malaria-associated AKI. AKI is common in knowlesi malaria, occurring in 44% of all patients in this study, and in 88% of those with severe disease. AKI is now recognised to have significant long-term consequences, including increased risk of chronic kidney disease, cardiovascular disease and death . Thus, we cannot confirm that endothelial cells are the source of the increased plasma OPG. However, the consistent finding of increased endothelial activation in severe malariaIn conclusion, we have demonstrated that intravascular haemolysis is increased in severe knowlesi malaria, and to a greater extent than falciparum malaria. Furthermore, we demonstrate that CFHb is independently associated with angiopoietin-2 and OPG, and that OPG is associated with endothelial cell adhesion molecules and microvascular and endothelial dysfunction, as well as with clinical biomarkers of severity, including lactate and AKI. These findings suggest that haemolysis-mediated endothelial activation and release of WPB constituents, including OPG, is likely a key contributor to end-organ dysfunction in severe knowlesi malaria.The study was approved by the Ethics Committees of the Malaysian Ministry of Health and Menzies School of Health Research. Informed written consent was provided by all participating adults, and by the parent or guardian of any participant aged <18 years.10. For the current study, patients enroled between September 2010 and December 2012 were included if they had PCR-confirmed P. knowlesi monoinfection, were non-pregnant, ≥12 years old, had no major comorbidities or concurrent illness and were within 18 h of commencing antimalarial treatment. Severe malaria was defined according to modified WHO criteria, as previously described10. Renal function was further assessed using the kidney disease: Improving Global Outcomes (KDIGO) criteria for AKI. Using this definition, AKI is defined as an increase in serum creatinine of ≥26.5 µmol/L within 48 h, or to ≥1.5× baseline56. Baseline creatinine was estimated using modification of diet in renal disease (MDRD) equation56, with an assumed eGFR of 100 mL/min per 1.73 m2. Healthy controls were visitors or relatives of malaria patients, with no history of fever in the past 48 h and with blood film negative for malaria parasites.Patients were enrolled as part of a prospective observational study of all malaria patients admitted to Queen Elizabeth Hospital, an adult tertiary-referral hospital in Sabah, Malaysia58. Patients with severe disease were treated with intravenous artesunate, while those with non-severe disease received oral artemisinin combination treatment, as previously described5.Standardised history and physical examination were documented. Haematology, biochemistry, acid–base parameters and lactate  were obtained on enrolment. Parasite counts were determined by microscopy, and parasite species identified by PCRVenous blood collected in lithium heparin and citrate tubes was centrifuged (including a second high-spin speed for the citrate tube) within 30 min of collection and plasma stored at −70 °C. Plasma CFHb and vWF were measured on the citrated platelet-free plasma by ELISA . Haptoglobin was measured on lithium heparin plasma by ELISA (ICL Laboratories). Plasma concentrations of angiopoietin-2, P-selectin and adhesion molecules ICAM-1 and E-selectin were measured on lithium heparin plasma using quantikine ELISA kits from RnD. OPG was measured on lithium heparin plasma using a duoset ELISA from RnD. IL-6 was measured by flow cytometry .16. The RHPAT index is at least 50% dependent on endothelial NO production59 and has been shown to be L-arginine responsive in falciparum malaria16. Measurement of endothelial function was discontinued on patients with non-severe malaria in July 2011. Microvascular function was assessed on enrolment as previously described60, using near infra-red spectroscopy  as previously reported10.Endothelial function was measured non-invasively on enrolment using peripheral arterial tonometry (EndoPAT) by the change in digital pulse wave amplitude in response to reactive hyperaemia, giving a reactive hyperaemia peripheral arterial tonometry (RHPAT) index, as previously describedt-test or Wilcoxon–Mann–Whitney tests were used for two-group comparisons. Categorical variables were compared using χ2 or Fisher’s exact tests. Associations between continuous variables were assessed using Spearman’s correlation coefficient. Partial correlation was used to evaluate associations between variables after adjusting for parasitaemia, with non-normally distributed variables log-transformed to normality. Backward stepwise regression was used to evaluate predictors of severe malaria and AKI, with variables removed at a significance level of >0.05. For this analysis, patients with hyperparasitaemia as a sole severity criterion were reclassified as having non-severe malaria. For comparison of intravascular haemolysis in P. knowlesi vs. P. falciparum malaria, median plasma CFHb in patients with severe knowlesi malaria was compared to previously published CFHb measurements from patients with severe falciparum malaria enroled contemporaneously in the same study cohort30.Statistical analysis was performed with STATA software (version 14). For continuous variables, intergroup differences were compared using analysis of variance or Kruskal–Wallis tests depending on distribution. Student’s Data will be available on request from the corresponding author.Supplementary Tables


Plasmodium infection, endothelial cell activation, and loss of blood-brain barrier integrity. However, the sequence of events, which initiates these pathophysiological processes as well as the contribution of their complex interplay to the development of cerebral malaria remain incompletely understood. Several cytokines and chemokines have repeatedly been associated with cerebral malaria severity. Increased levels of these inflammatory mediators could account for the sequestration of leukocytes in the cerebral microvasculature present during cerebral malaria, thereby contributing to an amplification of local inflammation and promoting cerebral malaria pathogenesis. Herein, we highlight the current knowledge on the contribution of cytokines and chemokines to the pathogenesis of cerebral malaria with particular emphasis on their roles in endothelial activation and leukocyte recruitment, as well as their implication in the progression to blood-brain barrier permeability and neuroinflammation, in both human cerebral malaria and in the murine experimental cerebral malaria model. A better molecular understanding of these processes could provide the basis for evidence-based development of adjunct therapies and the definition of diagnostic markers of disease progression.Cerebral malaria is among the major causes of malaria-associated mortality and effective adjunctive therapeutic strategies are currently lacking. Central pathophysiological processes involved in the development of cerebral malaria include an imbalance of pro- and anti-inflammatory responses to  Plasmodium species P. falciparum and P. vivax account for the majority of malaria cases and are transmitted by the bite of an infective Anopheles mosquito. Despite considerable progress in malaria eradication over the past 15 years  is one of the major manifestations of severe malaria  present on the erythrocyte surface to endothelial cell surface proteins, such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 36 (CD36), or endothelial protein C receptor (EPCR)  infection reliably causes signature symptoms of CM in susceptible C57BL/6 mice  exposed to the immune system before they rapidly enter new erythrocytes -infected mice during ECM than in those isolated from P. yoelii-infected (non-ECM) mice  immune response, and cytokines, such as IFN-γ, TNF, and LTα, and immune cells, e.g., CD4+ and CD8+ T cells together with NK cells are involved in ECM  activate human endothelial cells in vitro /CCL2, RANTES/CCL5, and IL-8/CXCL8, upon stimulation by pro-inflammatory cytokines or lipopolysaccharide (LPS) post mortem MIP-1β/CCL4, IL-8/CXCL8, and IP-10/CXCL10 levels were significantly elevated in cerebrospinal fluid (CSF) of fatal P. falciparum-induced CM cases when compared to fatal severe malarial anemia cases and non-malaria deaths  of CXCL10 in the brain during P. berghei infection remain to be conclusively determined, and could include neurons, astrocytes, or endothelial cells , a protein which correlates with parasite biomass, as well as regulators of endothelial activation angiopoietin-1 and -2 (summarized in Sahu et al., Together, reliable biomarkers which predict disease outcome and allow for potential prophylactic measures are yet to be identified. Several clinical studies have reported potential diagnostic and prognostic biomarkers for CM, which apart from chemokines such as IP-10/CXCL10 and PF4/CXCL4 also include All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it 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.