Epidemiologic data on malaria are scant in many high-burden countries including the Democratic Republic of the Congo (DRC), which suffers the second-highest global burden of malaria. Malaria control efforts in regions with challenging infrastructure require reproducible and efficient surveillance. We employed new high-throughput molecular testing to characterize the state of malaria control in the DRC and estimate childhood mortality attributable to excess malaria transmission.P. falciparum was the most prevalent species, either as monoinfection  or combined with P. malariae  or P. ovale . Only 7.7% (95% CI 6.8–8.6) of households with children under 5 owned an insecticide-treated bednet (ITN), and only 6.8% (95% CI 6.1–7.5) of under-fives slept under an ITN the preceding night. The overall under-5 mortality rate was 147 deaths per 1,000 live births (95% C.I. 141–153) and between clusters was associated with increased P. falciparum prevalence; based on the population attributable fraction, 26,488 yearly under-5 deaths were attributable to excess malaria transmission.The Demographic and Health Survey was a cross-sectional, population-based cluster household survey of adults aged 15–59 years in 2007 employing structured questionnaires and dried blood spot collection. Parasitemia was detected by real-time PCR, and survey responses measured adoption of malaria control measures and under-5 health indices. The response rate was 99% at the household level, and 8,886 households were surveyed in 300 clusters; from 8,838 respondents molecular results were available. The overall prevalence of parasitemia was 33.5% ; P. falciparum prevalence is substantial in the DRC and is associated with under-5 mortality. Molecular testing offers a new, generalizable, and efficient approach to characterizing malaria endemicity in underserved countries.Adult  Plasmodium falciparum malaria has galvanized efforts to define its spatial epidemiology, which can guide the allocation of resources to areas in greatest need P. falciparum transmission intensity Renewed interest in the eradication of P. falciparum malaria in sub-Saharan Africa but there is a paucity of clinical and epidemiologic data th century, when Marlowe, the narrator of Joseph Conrad's Heart of Darkness, could remark of what was then the Congo Free State: “At that time there were many blank spaces on the earth … zbut there was one yet – the biggest, the most blank, so to speak – that I had a hankering after The Democratic Republic of the Congo (DRC) accounts for an estimated 11% of cases of Herein, we endeavor to populate a blank area of the malaria map by incorporating molecular diagnostics into a nationally-representative household survey in the DRC. By leveraging the infrastructure of the Demographic and Health Survey (DHS), we generate estimates of malaria prevalence, control, and burden in the DRC. Because this approach represents a new, low-cost, and generalizable method of robust malaria surveillance, it offers an opportunity to generate quality malaria epidemiologic data for countries with challenging infrastructure and thus reduce disparities in the quality of malaria surveillance between countries.The 8,886 households from 300 clusters surveyed  yielded Malaria control measures targeting children were poorly deployed . Of 5,52Strategies to prevent pregnancy-associated malaria were also rarely implemented . Only 7%P. falciparum, either as monoinfection  or as coinfection with P. malariae , P. ovale , or all three species . P. malariae was present in 2.9% (95% C.I. 2.4–3.5) of respondents and 8.7% (95% C.I. 7.1–10.3) of parasitemias. P. ovale parasitemia was rare overall, as were moninfections with either P. malariae  or P. ovale .Of 8,838 adult respondents, 2,682  were parasitemic . Among pP. falciparum prevalences were low in the major cities of Kinshasa, Mbuji-Mayi, Lubumbashi, and Mbadanka, and in the mountainous eastern regions bordering Rwanda and Uganda and the southern region bordering Zambia. Clusters with higher prevalence were marked in rural areas in the interior and in areas bordering Angola in the south and the Central African Republic and the Sudan in the north. P. falciparum was present in 284 of 300 clusters, among which the prevalence of P. falciparum parasitemia varied from 2.3% to 81.8% (median 31.9%). P. malariae was present in 143 clusters distributed across the DRC, and prevalence in these clusters varied from 2% to 16.9% (median 4.3%). P. ovale was present in only 28 clusters, largely in the interior and southeast.There was substantial heterogeneity in parasite rates between clusters . P. falcP. falciparum endemicity , the mean cluster altitude decreased and the proportion of rural communities increased substantially .From the lowest to the highest level of antially . AdditioThe penetration of malaria control measures showed mixed associations with endemicity. However, increasing malaria endemicity was associated with increased proportions of fever in children under 5 in the preceding two weeks (p = 0.01). Malaria endemicity was not associated with the frequency of other childhood symptoms, the proportion of children for whom treatment was sought, or the frequency of the receipt of any type of childhood vaccination.P. falciparum prevalence was significantly associated with increased U5MR   was 147 deaths per 1,000 live births (95% C.I. 141–153); between clusters, the U5MR ranged from 0 to 378 (standard deviation 77) . In a li4–0.127) . Between4–0.127) . In otheAmong children born in the preceding 5 years, after adjustment for covariates and compared with children born in clusters with parasite prevalences of 0–9.9%, children under 5 died more frequently in clusters with prevalence rates of 10–24.9% , 25–49.9% , and ≥50%  . Based oHigh throughput PCR analyses of leftover dried blood spots enabled us to map and quantify malaria endemicity in the DRC without the need for a dedicated and complex malaria indicator survey. Overall, the point prevalence of malaria parasitemia was over 33% in adults, and parasite prevalence within survey clusters was associated with traditional determinants of malaria endemicity and with increased under-5 mortality. These associations confirm the utility of parasite prevalence as a measure of malaria endemicity.To our knowledge, this is the first nationally-representative malaria survey in sub-Saharan Africa to employ molecular diagnostics, and the first attempt to describe the epidemiology of malaria in this geographic area since 1951 falciparum species Our method of parasite detection and mapping offers substantial advantages compared with existing methods. Molecular assays for malaria are substantially more sensitive than detection using either blood smears or rapid diagnostic tests Successful malaria control has been highly dependent on preventive measures and effective therapy P. falciparum prevalence and under-5 mortality (U5MR). Reducing U5MR is a global public health priority but progress in the DRC is lacking The adult parasite rates of 34% overall and 82% in some clusters concur with previous studies of PCR-detectable parasitemia in other highly-endemic sub-Saharan African settings Though unmeasured factors may account for these differences in U5MR between levels of malaria endemicity, we incorporated covariates that address other major contributors to childhood mortality – lower respiratory tract infection and diarrhea; treatment-seeking behavior for fever, cough, and diarrhea; and the receipt of any vaccination – in order to capture a reasonable degree of variation in clinical syndromes, health system access, and public health infrastructure that influence childhood mortality. Additionally, the association between malaria endemicity and U5MR may be complicated by the complexity of the relationship between endemicity and the clinical consequences of malaria infection This report is subject to several additional limitations. Comparing our results with other prevalence studies is complex because we measured parasite rates using PCR testing in adults, while other studies typically measure microscopic parasitemias in children The integration of molecular malaria testing Demographic and Health Surveys offers a cost-effective opportunity to generate robust data in order to guide the allocation of resources and measure control initiative effectiveness. Such initiatives are succeeding in multiple sub-Saharan African settings, including Kenya All survey respondents provided verbal informed consent for the collection of blood spots in one of the five main languages spoken in the DRC per DHS protocol. Consent was verbally acquired owing to both limited literacy levels in rural provinces as well as the need for immediate de-identification of all data at the conclusion of the interview to ensure the confidentiality of the publicly-accessible survey datasets. Consent procedures, survey administration, and blood sample collection were approved by the Ethics Committees of Macro International and the School of Public Health of the University of Kinshasa, and testing for malaria parasites was approved by the Institutional Review Board of the University of North Carolina.The 2007 DHS was designed to provide nationally-representative data on population, health, and social indices for program planning and impact evaluation. It was the first such survey conducted in the DRC and represented a collaboration between the DRC Ministries of Health and Planning and Macro International, with funding provided by the United States' Agency for International Development United Nations, the World Bank, and the United Kingdom's Department for International Development.The DHS employed a stratified two-stage cluster design based on DRC census data obtained before the national election in 2006. The survey's sampling units were 300 randomly selected geographic clusters , from whBlood spots were collected from adult women and men in half of the surveyed households, primarily for estimating the seroprevalence of HIV. After consent was obtained, blood was collected in multiple spots on a single filter paper from each participant and placed in a drying box with desiccant overnight. After transfer to sealed plastic bags with desiccant and humidity indicators, they were placed into coolers prior to transport to Kinshasa. From each card, three 0.3 inch discs were punched and deposited in a single well of a plastic 96-well sample plate, which, when full, was sealed with a plastic cover, placed in a sealed plastic bag with desiccant, and stored at room temperature.Plates of punched blood spots were transferred from Kinshasa to the University of North Carolina, where genomic DNA (gDNA) was extracted using the invitrogen PureLink 96 Kit  using a vacuum manifold. In the final step, gDNA was eluted using 200 uL of elution buffer into a fresh 96-well sample plate, and sample plates were sealed securely and stored at -20C prior to further processing.P. falciparum, P. ovale, and P. malariae, as well as that of a human control gene Molecular testing employed two real-time PCR assays that target the 18S ribosomal DNA sequence of Plasmodia. Samples were initially amplified in a real-time PCR assay that detects all species of Plasmodia P. falciparum gDNA for the pan-species assay and mixtures of P. falciparum, P. ovale, P. malariae, or human DNA for the speciation assay, as appropriate. The first 867 samples were tested in duplicate; because of the lack of discordant results, the balance of samples was tested singly in each assay. For quality control, a random set of 5% of all gDNA samples was retested in the pan-species assay, and discordant repeat pan-species results were resolved by repeating the speciation assay. Ultimately, 8 specimens (of 520 re-tested) that were initially pan-species negative were determined to harbour P. falciparum after quality control testing. All reactions were performed on an ABI 7300 System  and analyzed using the ABI 7300 Sequence Detection Software (v1.3). Reaction plates were prepared using an epMotion 5070 system . To minimize the risk of sample contamination, filtered pipet tips were exclusively employed in all steps, and separate work areas were maintained for punching discs from blood spots, extracting gDNA, preparing reaction mixtures, and assembling reaction plates.All reaction plates included negative controls (with molecular-grade water in place of template) and positive controls: Plasmodium by both the pan-species and the speciation real-time PCR assays.In subsequent analyses, malaria parasitemia positivity was defined as the presence of any species of Population-level proportions of the penetration of malaria control measures , the prevalence of childhood illnesses, and the prevalence of parasitemias were calculated using sampling weights to account for survey design. For children born in the preceding 5 years, women were queried whether the child slept under a bednet the previous night; ever received any vaccination; experienced fever, cough or diarrhea in the preceding 2 weeks; was administered treatment for fever, cough, or diarrhea; and was administered an antimalarial for fever. Additionally, heads of households were queried about the residence of children under 5 and the possession and type of bednet, and women were queried about pregnancy status and their personal bednet use.To analyze geographic variation of the adoption of malaria control measures in children and pregnant women, cluster-level indicators were generated. Within each cluster, we calculated the prevalence of parasitemia with each species and, using survey data, the cluster-level frequencies of adherence to malaria control measures among children and pregnant women. Using ArcGIS , individual maps of parasite rates and rates of uptake of control measures were generated using inverse-distance weighting to smooth surfaces across unmeasured locations between survey clusters. Additionally, we tested the cluster parasite prevalences by species for spatial autocorrelation by examining the significance of global Moran's I values based upon row-standardized inverse distance weights matrices. Prior to analysis, cluster coordinate locations were randomly offset by 2 kilometers (in urban clusters) or 5 kilometers  to maintain privacy.P. falciparum served as an index of malaria endemicity. Clusters were grouped into categories of P. falciparum prevalence that approximated quartiles: 0–9.9%, 10–24.9%, 25–49.9%, and ≥50%. Bivariate associations between malaria endemicity (the main independent variable) and cluster-level demographics, adoption of malaria control measures, and childhood illnesses were assessed with one-way analysis of variance or the chi-squared test for continuous or categorical outcome variables, respectively.To examine associations between malaria endemicity and cluster-level indicators, the proportion of adults in each cluster that were parasitemic with Surveyed women were queried about their total number of births and about the vital status of all births. From complete birth histories, the under-5 mortality rate (U5MR) was calculated on national and cluster levels as the fraction of children ever born to surveyed women that died prior to 60 months of age. Cluster-level U5MR was mapped as above. This approach is biased towards underestimating U5MR because it does not account for the foreshortened time of exposure among children less than 5 years of age at the time of survey. Nevertheless, due to the small sample size at the cluster level, it was deemed a more appropriate method than the life table technique, and it has been validated as an accurate estimate compared with more traditional methods To assess potential confounders of the main effect of malaria endemicity and U5MR, cluster-level proportions were generated for prevalence of the receipt of any vaccination ; the prevalences of fever, cough, and diarrhea ; and the prevalence of medical treatment sought for fever, cough, or diarrhea (among children with respective symptoms).P. falciparum prevalence) on cluster U5MR, linear regression was employed; the independent variable was the cluster parasite prevalence as a continuous measure and the dependent variable was the cluster U5MR. Differences in median U5MR by cluster malaria endemicity (categorized as above) were compared by the Kruskal-Wallis analysis of variance due to the non-parametric distribution of cluster U5MRs.To evaluate the overall influence of malaria endemicity (as reflected by the cluster p) of parasite prevalence on under-5 mortality, adjusted odds ratios (aORs) for under-5 death were calculated using logistic regression. The model incoporated child health covariates that were significantly associated with U5MR in bivariate testing. Because the data were clustered and the model included cluster-level independent variables and an individual-level dependent variable, a multilevel random-intercept logistic regression model was employed to account for the potential interdependence of observations within cluster groups  p for under-5 deaths by each parasite prevalence category was then estimated using the aORs and the fraction of births within each parasite prevalence category To estimate the population attributable fraction  and later merged with survey data in Stata/IC; all statistical analyses were subsequently performed with Stata/IC.


Plasmodium falciparum pre-erythrocytic antigen ME-TRAP (multiple epitope string with thrombospondin-related adhesion protein) is a leading malaria vaccine candidate, capable of inducing sterile protection in malaria naïve adults following controlled human malaria infection (CHMI).Heterologous prime boost immunization with chimpanzee adenovirus 63 (ChAd63) and Modified vaccinia Virus Ankara (MVA) vectored vaccines is a strategy recently shown to be capable of inducing strong cell mediated responses against several antigens from the malaria parasite. ChAd63-MVA expressing the We conducted two Phase Ib dose escalation clinical trials assessing the safety and immunogenicity of ChAd63-MVA ME-TRAP in 46 healthy malaria exposed adults in two African countries with similar malaria transmission patterns.ChAd63-MVA ME-TRAP was shown to be safe and immunogenic, inducing high-level T cell responses (median >1300 SFU/million PBMC).ChAd63-MVA ME-TRAP is a safe and highly immunogenic vaccine regimen in adults with prior exposure to malaria. Further clinical trials to assess safety and immunogenicity in children and infants and protective efficacy in the field are now warranted.Pactr.org PACTR2010020001771828 Pactr.org PACTR201008000221638 NCT01373879ClinicalTrials.gov NCT01373879 NCT01379430ClinicalTrials.gov NCT01379430  Plasmodium falciparum remains a leading cause of childhood morbidity and mortality, predominantly in Africa, in spite of the implementation of extensive control measures Malaria caused by Analysis of the immunological correlates of immunity induced by the RTS,S vaccine in both phase IIa sporozoite challenge studies Increasing data from animal models, fieldwork and inoculation of volunteers with irradiated sporozoites support an important role for CD8+ T cells in mediating pre-erythrocytic immunity, even in the absence of antibodies P. falciparum malaria vaccine using the sporozoite and liver stage antigen ME-TRAP. This antigen contains a fusion protein of multiple epitopes  and the P. falciparum pre-erythrocytic antigen, thrombospondin-related adhesion protein (TRAP) The Jenner Institute has been working to develop a T cell inducing pre-erythrocytic Multiple vectors for this antigen have been clinically tested including DNA, fowl pox (FP) and the orthopox virus modified vaccinia virus Ankara (MVA) Ewer et al. submitted).Most recently, heterologous prime boost immunization with chimpanzee adenovirus 63 (ChAd63) followed by MVA, both expressing ME-TRAP, has been shown to be the most immunogenic vaccine regimen to date, inducing more than 2000 IFNγ producing T cells post MVA boost in malaria naïve volunteers The safety, immunogenicity and efficacy of malaria vaccines may be affected by the intensity and pattern of local malaria transmission which determine pre-existing natural immunity to malaria and the potential natural ‘boosting’ of the vaccine induced immune responses The protocols for these trials and supporting CONSORT checklist are available as supporting information; see The objective of the studies was to assess the safety and immunogenicity of ChAd63 ME-TRAP and MVA ME-TRAP administered in a heterologous prime boost regimen to healthy malaria-exposed adults.P. falciparum malaria transmission occurs The first trial  was conducted at the Medical Research Council (MRC) Unit field site located within Sukuta Health Centre in Kombo North district of The Gambia, West Africa. Sukuta village has an estimated population of 17,000 (2003 census). The climate is typical of sub-Saharan Africa with a long dry season lasting from December–June followed by a relatively short rainy season from July–November when the majority of P. falciparum malaria coinciding with the long monsoon rains (April to June) and the short rains (October to December) The second trial  was conducted in Vipingo, Kilifi County, Kenya, East Africa. Participants were recruited from the Rea Vipingo Sisal Plantation Estates in Kilifi which has over 1000 employees and a land area of 3,950 hectares. In Kilifi, there are two seasons of high transmission of M. Afolabi personal communication).Recent studies have reported a decline in malaria transmission in both sites P. falciparum at screening was not an exclusion criterion.Healthy males aged 18–50 years were invited to participate in the studies. There was no selection of participants on the basis of pre-existing neutralizing antibodies (NAb) to the ChAd63 vector prior to enrolment. Volunteers were considered eligible if they were consenting adult males aged 18–50 years in good health who were likely to remain resident in the study area for the study duration. Exclusion criteria included any evidence of any acute or chronic illness or hematological, renal or hepatic pathology. Specific exclusion criteria included; prior receipt of an investigational malaria vaccine, recent or planned use of any investigational drug, vaccine, immunoglobulin or any blood product, confirmed or suspected immunodeficiency, history of surgical splenectomy, concurrent participation in another clinical trial or within 3 months of this study (see 10 vp) and high dose (5×1010 vp) ChAd63 ME-TRAP. Trial B also compared intramuscular and intradermal routes of administration of 2×108 pfu MVA ME-TRAP. The same lot of each vaccine was used in both trials. The trials were conducted independently however the same Data and Safety Monitoring Board (DSMB) was used for each trial. The clinical trial protocols and supporting CONSORT checklist are available as Supplementary Information; see We conducted two Phase Ib open-label, dose-escalation malaria vaccine trials . Both cl10 viral particles (vp)  or ChAd63 ME-TRAP 5×1010 vp  administered intramuscularly in the deltoid. All participants were subsequently vaccinated in the opposite arm 56 days later with 2×108 plaque forming units (pfu) MVA ME-TRAP administered intramuscularly. The first participant in group 1 to receive ChAd63 ME-TRAP 1×1010 vp was vaccinated in isolation. 48 hours later, two further participants were enrolled in group 1. Prior to dose escalation of ChAd63 ME-TRAP from 1×1010 vp to 5×1010 vp, safety data from group 1 up to 14 days post ChAd63 ME-TRAP was reviewed by the DSMB. There was a protocol-required interval of at least 14 days between immunization of groups 1 and 2. Details of clinical follow-up and safety monitoring are given in In Trial A, eligible participants were allocated to receive either ChAd63 ME-TRAP 1×1010 viral particles (vp)  or ChAd63 ME-TRAP 5×1010 vp  administered intramuscularly in the deltoid. All participants were subsequently vaccinated in the opposite arm 56 days later with 2×108 plaque forming units (pfu) MVA ME-TRAP. Participants in each group were randomised 1∶1 to receive MVA ME-TRAP administered intramuscularly (IM) or intradermally (ID). The first 3 participants in group 1 were vaccinated with ChAd63 ME-TRAP 1×1010 vp 7 days ahead of the remaining 7 participants in this group. There was an 8 day interval between enrolment of group 1 and group 2. Details of clinical follow-up and safety monitoring are given in In Trial B, eligible participants were allocated to receive either ChAd63 ME-TRAP 1×10For both trials, a time window ranging between 1 and 28 days depending on the visit was allowed for vaccination and follow-up visits. Throughout the paper, study day refers to the nominal time point for a group and not the actual day of sampling.10 vp ChAd63 ME - TRAP or 5×1010 vp dose ChAd63 ME in a ratio of 1∶2. 8 weeks later participants were randomised 1∶1 to receive 2×108 pfu MVA ME-TRAP administered intramuscularly (IM) or intradermally (ID). The randomization sequence was generated by an independent statistician using STATA programme. Group allocations were kept in sealed opaque envelopes stored in a locked cabinet by the study coordinator who gave them to the research nurses on day of vaccination. Participants and clinical study staff were un-blinded to group allocation, however, field workers were blinded to group allocation.30 participants were systematically allocated to receive either 1×10These were observational and descriptive studies to assess the safety and immunogenicity of ChAd63 ME-TRAP and MVA ME-TRAP in malaria exposed adults. The sample sizes were chosen to allow estimation of the magnitude of the primary outcome measures, especially of serious adverse events (AEs) rather than assessment of statistically significant differences between groups.The clinical trial protocols and associated documents were approved by Gambia Government/MRC Joint Ethics Committee for Trial A and The Kenya Medical Research Institute National Ethics Review Committee for Trial B. Documents for both clinical trials were reviewed and approved by the Oxford Tropical Research Ethics Committee (OXTREC). Regulatory approval was given by the Medicines Board of The Gambia for Trial A and The Pharmacy and Poisons Board of Kenya for Trial B. All participants gave documented informed consent prior to any study procedure being undertaken. The study was conducted according to the principles of the Declaration of Helsinki (2008) and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. An independent DSMB and local safety monitors provided safety oversight and GCP compliance was independently monitored by an external organization at both trial sites .Generation of the recombinant vectors has been previously described P. falciparum pre-erythrocytic thrombospondin-related adhesion protein (TRAP). The ‘ME’ is a string of 20 epitopes, mainly CD8 T cell epitopes from P. falciparum pre-erythrocytic antigens, fused to the thrombospondin-related adhesion protein. The individual cytotoxic T lymphocyte (CTL) epitopes which constitute the ‘multiple epitope’ part of ME-TRAP represent six potentially protective target antigens and are included to try to broaden the immune response rate in the vaccinated population. The ME string is fused to the entire sequence of the T9/96 strain of P. falciparum TRAP and the ME-TRAP hybrid is a 2398 base-pair insert which encodes for a single polypeptide of 789 amino acids The antigen ME-TRAP contains a fusion protein of multiple epitopes (ME) and the In each trial participants were observed for 1 hour post each immunization. Following each immunization participants in both trials were reviewed at home by a trained field worker and findings recorded on standardised case report forms. Local and systemic vaccine reactogenicity was evaluated and graded for severity, outcome and association to vaccination as per the criteria outlined in xg). Serum was stored at −80°C.Blood samples were collected into lithium or sodium heparin-treated vacutainer blood collection tubes . PBMC were isolated and used within 6 hours in fresh assays as previously described Peptides were purchased from NEO Peptide . The peptides, 20 amino acids (aa) in length and overlapping by 10 aa covered the entire ME-TRAP insert present in the viral vectored vaccines. Peptides were also synthesised for the sequence of TRAP from the 3D7 strain. Peptides were reconstituted in 100% DMSO at 50–200 mg/mL and combined into various pools for ELISPOT and flow cytometry assays. Peptides are listed in ex-vivo IFN-γ ELISPOT assays performed on blood samples taken at each clinic review . Ex-vivo IFN-γ ELISPOT assays were performed with an 18–20 hour stimulation of PBMC with peptides pools containing up to 10 peptides per pool, including peptides representing the T9/96 and 3D7 strains. Fresh PBMC were used in all ELISPOT assays using a previously described protocol, except that 50 µL/well ME-TRAP peptide pools  were added to duplicate wells, 50 µL/well R10 and DMSO control were added to negative un-stimulated wells, and 50 µL/well Staphylococcal enterotoxin B (SEB)  plus phytohemagglutinin (PHA)  was added to positive control wells. Spots were counted using an ELISPOT counter (Autoimmun Diagnostika (AID), Germany). Results are expressed as the mean of the duplicate IFN-γ spot-forming units (SFU) per million PBMC. Background responses in un-stimulated control wells were almost always less than 20 spots, and were subtracted from those measured in peptide-stimulated wells. Responses are shown as the summed response to all the ME-TRAP (T9/96) peptide pools (unless otherwise stated).The kinetics and magnitude of the T cell response to ME-TRAP were assessed over time by s) for non-parametric data. Collated immunology data was analysed by multivariate linear regression using log-transformed ELISPOT results. A value of P<0.05 was considered significant.Data were analyzed using GraphPad Prism version 5.03 for Windows  and Stata 10.0 . Geometric mean or median responses for each group are described. Significance testing of differences between two groups used the two-tailed Mann-Whitney U test or Wilcoxon signed rank test as appropriate. Correlations were analyzed using Spearman's rank correlation co-efficient .Heterologous prime boost with ChAd63-MVA ME-TRAP induced high frequencies of antigen-specific IFNγ-secreting T cells in both trials as measured by ex-vivo IFNγ ELISPOT. Peak IFNγ ELISPOT responses were detected 7 days post MVA ME-TRAP when a positive response (defined as responses above the lower limit of detection and at least double the response measured at Day 0) was detected in 90% and 100% of recipients in Trial A and Trial B respectively . ResponsIn Trial A there was no statistically significant difference in peak IFNγ ELISPOT response between individuals receiving 1×1010 vp ChAd63 ME-TRAP  and individuals receiving 5×1010 vp ChAd63 ME-TRAP  . In contrast in Trial B, the median peak IFNγ ELISPOT response elicited in individuals receiving 5×1010 vp ChAd63  was significantly greater than the peak response in individuals receiving 1×1010 vp ChAd63 ME-TRAP .In Trial B there was no significant difference in peak immune response between volunteers receiving MVA administered intramuscularly or intradermally for either dose of ChAd63 ME-TRAP  .ELISPOT data was combined from both trials and analysed using a multivariate linear regression model. Data was stratified by dose of ChAd63 ME-TRAP and trial site. Only dose of ChAd63 ME-TRAP had a significant effect with a 2.3 (95% CI 1.4–3.8) fold increase in mean ELISPOT response in the individuals receiving 5×1010 vp. Of note, route of administration and trial site did not have significant effects on outcome .In these two Phase Ib trials we have shown in healthy, malaria-exposed adult volunteers that a recombinant ChAd63-MVA heterologous prime-boost immunization regimen encoding ME-TRAP is safe as well as very immunogenic for T-cell induction. ChAd63 ME-TRAP demonstrated an excellent safety profile, inducing only a small number of AEs, all of which were mild in intensity. ChAd63 ME-TRAP had a similar reactogenicity profile in our malaria exposed population to that seen in UK volunteers who received comparable doses of ChAd63 ME-TRAP 8 pfu MVA ME-TRAP have previously been assessed in malaria exposed adults 8 pfu is associated with an increase in frequency but not severity of local AEs, causing relatively short-lived injection site pain, discoloration, warmth and swelling (with the exception of one case of moderate injection site swelling lasting 30 days). The increased dose of MVA ME-TRAP did not however translate into an increase in systemic reactogenicity. MVA ME-TRAP administered intramuscularly in our subjects was associated with considerably fewer local AEs than intradermal administration; however, short-lived injection site pain was still reported by 97% of volunteers. This finding was also seen in UK volunteers The safety and immunogenicity of intradermally administered MVA ME-TRAP at doses of up to 1.5×1010 vp). Of note, there was no statistically significant difference in peak immune responses between individuals in Trial B who received MVA ME-TRAP administered intradermally or intramuscularly. Given this finding and the increased frequency of local AEs associated with intradermal administration, future studies in adults will use 2×108 pfu MVA ME-TRAP administered intramuscularly.In agreement with data from previous clinical studies of ChAd63 vectored vaccines,10 vp ChAd63 ME-TRAP were comparable with Phase Ia data . However, importantly the vaccine-induced T cell responses reported here appear to be the most potent reported to date in Africa for any vaccine type. Responses exceeding 1000 SFU /million PBMCs are very difficult to induce with any vaccination strategy and the levels attained here in this first study of ChAd63-MVA in Africa are therefore encouraging for more widespread use of these vaccine vectors.Whilst a previous study of vectored malaria vaccines observed a reduction in T-cell immunogenicity in malaria exposed populations compared to UK volunteers  Ia data  [35]. FuConcerns have been raised that pre-existing anti-vector immunity could limit the immunogenicity or compromise safety of adenoviral vectored vaccines in exposed populationsHanke et al unpublished), Hepatitis C Gilbert et al. unpublished) as well as for liver-stage and blood-stage malaria Chimpanzee adenoviruses were first used as a vaccine in humans in 2007 P. falciparum infection and disease in the field.Future Phase Ib studies will now assess the safety and immunogenicity of ChAd63-MVA ME-TRAP in children and infants. If these data are favourable, field efficacy studies will be undertaken in infants to assess whether strong cellular immunity against ME-TRAP can translate into significant efficacy against naturally acquired Table S1TRAP T9/96 and 3D7 peptide sequences and peptide pools. The sequences in bold represent the 3D7 strain sequences that differ from the T9/96 strain. When no sequence is present for the 3D7 strain, it means that both the 3D7 and T9/96 sequences are identical and the T9/96 peptide has been used in the 3D7 pool.(PDF)Click here for additional data file.Table S2Local and systemic AEs deemed definitely, probably or possibly related to ChAd63 ME-TRAP or MVA ME-TRAP. Only the highest intensity of each AE per subject is listed. Data are combined for all AEs for all volunteers receiving the same vaccine at the stated dose. Number  =  number of volunteers experiencing named AE. %  =  percentage of immunised volunteers experiencing named AE. There were no immunization related serious AEs. IM  =  intramuscular administration. ID  =  intradermal administration.(PDF)Click here for additional data file.Table S3Laboratory abnormalities post immunization deemed definitely, probably or possibly related to ChAd63 ME-TRAP or MVA ME-TRAP. All were mild, deemed possibly related to vaccination and resolved fully with no long term sequelae. None of the laboratory abnormalities were deemed clinically significant. All laboratory abnormalities resolved by time of next venepuncture* . ALT  =  alanine aminotransferase.(PDF)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Clinical trial protocol for Trial A.(PDF)Click here for additional data file.Protocol S2Clinical trial protocol for Trial B.(PDF)Click here for additional data file.


To the Editor: In January 2005, the chief surgeon in a squadron of French policemen reported a cluster of Plasmodium vivax malaria attacks in troops returning from a 108-day operation in French Guiana. We conducted a retrospective cohort study to describe the malaria attacks and determine factors related to them.A self-administered questionnaire was drawn up, with questions concerning operations in French Guiana  and preventive measures implemented against malaria. A malaria case was defined by the association of clinical signs and Plasmodium parasites in blood smears or quantitative buffy coat tests (per definition of military epidemiologic surveillance).The 40-person mission in French Guiana (Operation Anaconda) took place from July 26, 2004, to November 6, 2004 (108 days of exposure). This mission against clandestine gold panning was conducted in a deep-forest environment where the troops were temporarily housed in villages of Brazilian gold panners. Occasionally, they washed themselves late in the evening in stagnant water near the river and patrolled outside during maximum biting periods. All troops received a chemoprophylaxis (doxycycline 100 mg daily) during the mission and for 4 weeks afterward.>1 malaria attacks (attack rate 25%) for a total of 18 malaria attacks (incidence 13/100 person-months of exposure). P. vivax was isolated for 17 attacks and P. falciparum for 1 attack .>1 malaria attacks (attack rate 61%). Of these, 30 had >1 attacks caused by P. vivax; occasionally an attack was associated with P. falciparum (The incidence rate for this study was 10 times higher than the maximum incidence rate observed for French troops deployed in Côte d'Ivoire (1.3/100 troop-months in 2004). During an earlier Operation Anaconda, 37 of 62 persons deployed near the Sikini area had Our results suggest that the Sikini area was the high-risk area for malaria transmission . The operation dates (15–28 September) are compatible with the duration of the first cases of malaria occurrence.French Guiana is the only French territory, except for Mayotte, where malaria is endemic, with nearly 5,000 cases per year, occurring mainly along the rivers bordering Suriname and Brazil (The Sikini area is located near the Oyapock River (Brazilian border). The mean annual incidence in Amerindians there is 48.6%, mainly due to P. falciparum (incidence 24.8%) and P. vivax (incidence 25.9%) . The relapses were treated with 50-mg daily doses of primaquine for 4 patients and by chloroquine for the fifth patient. Two patients had relapses after receiving primaquine. Primaquine resistance information was not available. However, resistance to primaquine has emerged in P. vivax strains (Initial malaria attacks were treated with chloroquine or quinine. Five patients experienced We recommended that pre-impregnated battlefield uniforms be available for French policemen and chemoprophylaxis adherence be reinforced by directly observed intake by supervisory staff. Relapses of P. vivax malaria are a major therapeutic problem, particularly after primaquine therapy.


Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18–40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5–333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/µL (range 2–44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8–116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/mosquito . Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 NCT00744133ClinicalTrials.gov NCT00744133  Controlled human malaria infection (CHMI) using mosquitoes infected on cultures containing Pf gametocyte-infected erythrocytes has been shown to be safe and effective for testing the efficacy of anti-malarial drugs and malaria vaccine candidates for more than 25 years, including early studies of subunit malaria vaccines and atovaquone efficacy in vitroThe exposure of malaria-naïve persons to the bites of infected mosquitoes has been performed for almost 100 years The traditional CHMI model requires the bites of 5 infected, insectary-raised mosquitoes to reliably induce malaria. Disadvantages of this model include the requirement for an insectary and entomology expertise, secure transportation from the insectary to the clinical trial site, precise timing of mosquito rearing to coordinate with malaria candidate vaccine dosing, and the theoretical risk of participant exposure to microorganisms other than malaria that may be carried by standard laboratory-raised mosquitoes Anopheles stephensi mosquitoes We previously reported on an improved CHMI system using the bites of 1, 3 or 5 aseptically-raised mosquitoes. This study demonstrated that the aseptic model is safe, associated with a precise pre-patent period, and transmitted malaria to all 6 participants (100%) bitten by 3 The study was approved by the University of Maryland School of Medicine Institutional Review Board and was conducted in compliance with the Declaration of Helsinki. Independent site monitoring was conducted to ensure that human subject protection, study procedures, laboratory procedures, study intervention administration, and data collection processes met GCP/ICH and regulatory guidelines. An independent safety monitoring committee was convened to review study participant safety data and clinical results. The clinical trial was registered on clinicaltrials.gov, registry number NCT00744133.A. stephensi required to achieve 100% Pf parasitemia in malaria-naïve adults The main objective of this study was to validate in a larger study our preliminary results on the minimum number of aseptic, PfSPZ-infected A. stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf at the Center for Vaccine Development at the University of Maryland School of Medicine in Baltimore, Maryland. All participants provided written informed consent prior to study screening procedures, including documentation of medical history, assessment of cardiovascular risk Healthy, malaria-naïve U.S. volunteers aged 18–40 years were recruited to be infected by the bites of 3 aseptically raised -infected A. stephensi mosquitoes Details of the administration of the study product including dosage, preparation, and quantification of salivary gland assessment; post-exposure assessment; evaluation of safety and tolerability; and malaria diagnostics were identical to those reported previously All adverse events were graded for severity and relatedness and solicited symptoms and signs were assessed as previously described Daily malaria diagnostic screening began 5 days after CHMI by direct examination of thick blood smears for presence of Pf parasites. All therapeutic decisions were based on thick blood smear results. If a participant developed symptoms or signs consistent with malaria, additional thick blood smears were evaluated at 8–12 hour intervals.Blood smears were prepared as previously described qPCR was performed on DNA extracted from 0.5 mL of venous blood collected contemporaneously with blood smears, using published methods with minor adaptation Sample size calculations factored in the 6 individuals from the previous study Thirty-six potential participants underwent screening, and 20 who met study inclusion criteria were scheduled for CHMI . One indA total of 142 mosquitoes were required on the day of CHMI (mean 7.5 per participant). Of these, 88 mosquitoes (62%) took a blood meal and were dissected to determine the presence and quantity of PfSPZ. Fifty-seven mosquitoes (65%) were found to have detectable PfSPZs with a geometric mean PfSPZ load of 37,894 per mosquito . No mosquito had fewer than 1,000 PfSPZs per paired salivary glands. Participants were exposed to 3 mosquitoes with a combined total geometric mean PfSPZ load of 132,269 . This was a greater number of PfSPZ per 3 mosquitoes than the 6 participants exposed to 3 mosquitoes in the previous study Local symptoms and signs were solicited from days 2–7 after CHMI and are presented in All 19 participants (100%) developed patent parasitemia by microscopy, with 12/17 (71%) detected on Day 11 after mosquito exposure. Two participants missed study visits including Day 11 just before patency detection by microscopy and were not included in analyses of patency by microscopy. The geometric mean time to patency by microscopy was 258 hours (range 211–334) or 10.8 days (range 8.8–13.9). Geometric mean parasite density by microscopy at patency was 9.5 parasites/µL (range 2–44). Compiled microscopy data from 23 participants bitten by 3 mosquitoes, incorporating results from the previous study, shows similar results, with 17 (74%) developing patent parasitemia 11 days after exposure, a geometric mean time to patency of 257 hours (range 210.5–333.8) or 10.7 days (range 8.8–13.9), and geometric mean parasite density of 10.8 parasites/µL at diagnosis (range 2–44).2 = 0.05).Parasitemia by qPCR was detected in all 19 participants (100%); 14 (74%) became positive on day 7 after exposure. The geometric mean time from exposure to detection of parasitemia by qPCR was 177 hours (range 162–286) or 7.4 days (range 6.7–11.9). Geometric mean parasite density by qPCR analysis on the first day of qPCR positivity was 1.66 parasites/µL (range 0.11–18.21). Combined qPCR data from all 25 participants bitten by 3 mosquitoes shows similar results with 17/25 (68%) turning positive on Day 7 post-exposure. For these same 25 participants, the geometric mean parasite density on the first day of qPCR positivity was 0.88 parasites/µL (range 0.42–18.21). Combined data from 23 participants of both studies exposed to 3 aseptic mosquitoes demonstrated that qPCR detected patency an average of 79.0 hours before microscopy (95% CI 70.0–88.0). Before smear detection, no false positive or false negative qPCR assays were detected. Geometric mean parasite density on the first day of qPCR positivity in the current study was greater than the 6 participants exposed to 3 mosquitoes in the initial study We compared our malaria diagnostic results in the 25 study participants exposed to 3 mosquitoes in the current study and our previous study While we performed the qPCR analysis primarily for diagnostic purposes, we calculated the parasite multiplication rate for all 25 participants exposed to 3 mosquitoes reared aseptically. Using the sine wave method described by Bejon et al. A total of 25 participants combined from the previous and current study were evaluated. We removed two individuals from diagnostic analysis due to a missed study visit, which would alter conclusions. Eight of the remaining 23 participants (35%) bitten by 3 mosquitoes developed clinical symptoms consistent with malaria before diagnosis by thick blood smear, while 11/23 (48%) developed symptoms on the day of diagnosis and 6/23 (17%) of the participants were symptom-free until the days after blood smear diagnosis. Among the 8 volunteers who were symptomatic before diagnosis, symptoms developed a mean of 2.8 days (range 1–5) before diagnosis by microscopy. All symptoms were reported as mild and did not interfere with daily activities. No individuals experienced temperature above 37.5°C (lower threshold for Grade 1 fever) on the days before malaria diagnosis. All of the remaining 6 volunteers developed symptoms or signs consistent with malaria within 24 hours after diagnosis and initiation of treatment. Fever ≥37.5°C was recorded in 19 participants (76%) on the day of diagnosis. The mean duration of fever was 2.3 days (range 1–4 days). Severe fever (>39.0°C) was the only solicited severe symptom recorded during the time of malaria illness and was present in 11/25 participants (44%). The most common solicited symptoms and signs during the malaria illness period were headache (88%), measured fever (76%), malaise (76%), chills (76%), and myalgia (72%) . SolicitFour of 25 participants (16%) bitten by 3 mosquitoes developed clinical symptoms at least possibly related to malaria exposure before or at the time of qPCR positivity. A total of 7 solicited symptoms were recorded with the most common being headache. In the time between positive malaria diagnosis by qPCR and microscopy (mean 79 hours), an additional 64 solicited symptoms at least possibly related to malaria exposure were recorded.3/µL. No hypoglycemic events were recorded. All laboratory abnormalities resolved after malaria treatment. All results of ECG testing and serum troponin levels were normal.Laboratory abnormalities were noted in 20 of 25 (80%) participants challenged by the bite of 3 aseptically-reared mosquitoes, with mild aspartate aminotransferase (AST) and alanine aminotransferase (ALT) elevations being the most common  . Twelve A. stephensi mosquitoes bites safely and effectively transmitted malaria to all 25 participants (100%) tested in this and a previous study. Preliminary results from a previous smaller study had suggested that the 3 mosquito regimen was reliable and safe in 6 participants The use of 3 Pf-infected, aseptically-reared The use of 3 aseptically-raised mosquitoes bites for CHMI studies appears to be safe and well-tolerated. Symptoms and signs experienced by participants were generally mild or moderate, and the only severe symptom or sign experienced in the post-CHMI was fever >39.0°C (maximum 39.9°C) in 44% of participants. This is comparable to severe fever and other signs and symptoms seen in traditional 5 mosquito CHMI There has been controversy in the field as to whether the intensity of salivary gland infections with PfSPZ affects the infectivity and/or pre-patent period after CHMI. In our earlier study, 6 participants were exposed to a geometric mean total of 57,187 PfSPZ  corresponding to 16,645 PfSPZ/mosquito . In the current study, 19 volunteers were exposed to more than 2 times as many total PfSPZ  and PfSPZ/mosquito . Yet both studies achieved 100% infectivity and the pre-patent periods, incubation periods, and parasite density in blood by microscopy at time of diagnosis were similar. Thus, despite the presence of 2–3 times as many PfSPZ in the mosquito salivary glands, we did not see measureable differences in patency parameters, suggesting that similar numbers of PfSPZ were inoculated effectively into the volunteers. This may be because volunteers were exposed for a defined period of time (5 minutes) and based on the anatomy and diameter of the salivary duct, the PfSPZ generally move in single file down the duct and into the skin A. stephensi mosquitoes reared in standard insectaries are contaminated with bacteria and fungi, which may reduce mosquito fitness and infection by the malaria parasite P. yoelii has proven to be highly variable, ranging from 0–1,297 per bite (mean 123) and is only weakly correlated to sporozoite gland quantity Compared to infectivity controls in a recent study of CHMI utilizing the traditional 5 mosquito methodology in malaria-naïve adults, the 3 mosquito aseptic CHMI resulted in detectable malaria patency by qPCR an average of 1.1 days earlier  with qPCIf these earlier results are borne out in further studies, the predictable pre-patent periods, earlier diagnosis of malaria and earlier antimalarial treatment administration of aseptic 3 mosquito CHMI will allow termination of the infection sooner than in the traditional model. As more experience is gained in conducting CHMI trials the use of qPCR to achieve earlier diagnoses is attractive, but it also carries risks. A false negative or false positive result in a small challenge trial of a malaria vaccine could profoundly alter study results. Moreover, the presence of Pf DNA does not necessarily prove the presence of live, blood-stage asexual parasites. Although DNA fragments are quickly eliminated from the bloodstream, making it less likely that PCR would detect dead parasite forms The accuracy of microscopy-confirmed diagnosed malaria in parallel with qPCR diagnosis has been studied in the context of CHMI by mosquito bite We report, herein, successful application of an aseptic CHMI technique and 100% malaria infectivity using 3, rather than 5, mosquitoes in a CHMI study. Although tested in only 25 participants to date, the 5 mosquito CHMI was introduced as the standard after assessment in only 6 volunteers The novel use of 3 aseptically reared mosquitoes is a safe and effective means to transmit malaria infection to malaria-naïve adults in a controlled setting. Confirmatory studies would potentially set a new standard for evaluation of malaria vaccines and antimalarial drugs in non-endemic areas using this model. Microscopy continues to be the gold standard used for the diagnosis of malaria in centers conducting CHMI, but qPCR diagnoses parasitemia several days earlier than thick blood smear. Further work to establish the specificity, practicality, rapidity, and standardization of qPCR should be considered with the intent of eventually transitioning from thick blood smear to qPCR for diagnosis in CHMI.Figure S1Participant Flow Diagram.(TIFF)Click here for additional data file.Table S1Quantitative Polymerase Chain Reaction Results.(DOCX)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.Protocol S1Study Protocol.(DOC)Click here for additional data file.


Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite.Two cases of  In Cambodia, malaria ranks among the leading causes of illness and death. Mostly affecting the ≈3 million persons (23% of Cambodia’s population) who live near forested areas, malaria remains an occupational disease in specific high-risk groups, such as forestry workers and migrant populations who have come into forested areas. However, for the past decade, the number of reported malaria cases has generally decreased but in a sawtooth pattern of periodic increases   cases in 2000 to 6,250 (25%) in 2009. In several areas of low transmission, the proportion of P. vivax infections has increased up to 50%  by using PCR (cytb and cox1 genes) . Because these animals were confiscated by Wildlife Alliance rescue patrols from illegal traders, the locations where they were trapped in Cambodia are unknown.No studies in humans  febrile illness in selected outpatient clinics, where they had been tested for malaria by using rapid diagnostic test . A subset of nonfebrile patients were used as controls; patients in a critical clinical condition were excluded. After we obtained informed consent, each patient’s history was taken, a physical examination was conducted, and blood and throat swab samples were collected. The defined test battery, including the following pathogens, was performed by using a PCR/sequencing approach: Plasmodium spp., Leptospira spp., Rickettsia spp. (including Orientia tsutsugamushi), dengue viruses, Japanese encephalitis virus, and influenza viruses. HIV infection and tuberculosis were not evaluated. DNA and RNA from the erythrocyte pellet or throat swab were extracted by using the QIAamp kit , according to the manufacturer’s instructions. Malaria parasites were detected by using a Plasmodium spp.–specific nested PCR based on the cytb gene followed by sequencing , comprising 1,193 febrile and 282 nonfebrile persons. For 564 (38.2%) patients, no pathogens were detected. A total of 754 patients  were infected with malaria parasites, and the distribution of P. knowlesi infections in 2 patients from Ou Chra health center in Pailin Province  and was cured in 3 days. He further added that he used to hunt and spent most of his time in forests around Pailin, where long-tailed macaques, the natural hosts of P. knowlesi  was detected by the medical staff, but the patient reported a history of fever and had self-treated with acetaminophen. Results of rapid diagnostic test were negative, but microscopy results were positive (33 parasites/µL) , panel CP. knowlesi infections were confirmed by additional PCR amplification and sequencing of the ldh, tufA, and cox genes (P. knowlesi single infections by real-time PCR (Both patients’ P. knowlesi infections occur in humans in Cambodia, thereby increasing the number of countries in Southeast Asia with cases in humans (P. knowlesi malaria cases in humans and monkeys. Such studies would enable an assessment of the public health relevance of this zoonotic malaria parasite and characterization of P. knowlesi malaria epidemiology in this region. They would be particularly useful in determining whether this simian species has been imported from neighboring countries by humans or whether P. knowlesi parasites are already circulating and are transmitted from monkey reservoir hosts to humans. Moreover, to address this issue, development of new tools, such as specific serologic markers, is urgently needed, in addition to molecular biology methods.Our findings confirm that


A more detailed understanding of the profiles of cytokines, chemokines, and endothelial activation may be useful in developing biomarkers and novel therapeutic approaches for malaria.Clinical observations suggest that Canadian-born (CB) travellers are prone to more severe malaria, characterized by higher parasite density in the blood, and severe symptoms, such as cerebral malaria and renal failure, than foreign-born travellers (FB) from areas of malaria endemicity. It was hypothesized that host cytokine and chemokine responses differ significantly in CB The patient population for the study (n = 186) was comprised of travellers returning to Toronto, Canada between 2007 and 2011. The patient blood samples’ cytokine, chemokine and angiopoietin concentrations were determined using cytokine multiplex assays, and ELISA assays.Plasmodium vivax infections had significantly higher mean cytokine levels for IL-6 and macrophage colony-stimulating factor (M-CSF) than other adults with P. vivax (ages 18–55). Patients with P. vivax infections had significantly higher mean cytokine levels for monocyte chemotactic protein-1 (MCP-1), and M-CSF than patients with Plasmodium falciparum. Angiopoietin 2 (Ang-2) was higher for patients infected with P. falciparum than P. vivax, especially when comparing just the FB groups. IL-12 (p40) was higher in FB patients with P. vivax compared to P. falciparum. Il-12 (p40) was also higher in patients infected with P. vivax than those infected with Plasmodium ovale. For patients travelling to West Africa, IFN-γ and IL-6 was lower than for patients who were in other regions of Africa.Significantly higher plasma cytokine levels of IL-12 (p40) were observed in CB compared to FB travellers, while epidermal growth factor (EGF) was observed to be higher in FB than CB travellers. Older travellers (55 years old or greater) with P. vivax infected patients may also prove useful in understanding age-associated and species-specific host immune responses, as could the species-specific differences in Ang-2. Regional differences in host immune response to malaria infection within the same species may speak to unique strains circulating in parts of West Africa.Significantly higher levels of IL-12 (p40) and lower levels of EGF in CB travellers may serve as useful prognostic markers of disease severity and help guide clinical management upon return. IL-6 and M-CSF in older adults and MCP-1, IL-12 (p40) and M-CSF for  Almost half the global population is at risk of contracting malaria, with 300–500 million new clinical cases arising each year that result in between one and three million deaths ,2. In coPlasmodium falciparum-infected patients, but has been seen, defined under the same criteria, in Plasmodium vivax-infected patients [Plasmodium  infection. The resulting data was analysed to identify immune factors associated with disease severity, and other patient sample characteristics.Severe malaria is defined by the WHO as a situation in which a patient suffering from asexual parasitaemia, with no other obvious causes of symptoms, has one or more clinical or laboratory features that include but are not limited to hyperparasitaemia, impaired consciousness or unrousable coma, renal impairment, and prostration ,9. Severpatients . It was patients -13. To aThe study acquired ethics approval from the Toronto Academic Health Sciences Network Research Ethics Board (REB#10-0094-E).in vitro immunochromatographic assays , and quantitative PCR as required, samples positive for malaria were transferred to molecular research for storage and further analysis.The malaria specimen database at Public Health Ontario Laboratories (PHOL) comprises patient blood samples received for analysis between 2006 and 2011 coupled to basic patient information. Samples from patients suspected to have malaria were obtained via venipuncture by physicians from hospitals and clinics throughout the Greater Toronto Area and transported to PHOL in EDTA vacutainer (BD) tubes for processing in the clinical parasitology department. Following sample analysis using Giemsa-stained thin and thick smear microscopy, Only samples with complete demographic information, defined as patient age, gender, parasitaemia, country of birth and species of infection, were selected for the study. The sample set was stratified into patients born in Canada, and those who recently immigrated to Canada from malaria endemic regions. The samples from CB patients were considered to be more likely to have severe malaria, as defined by WHO guidelines and the RIFLE criteria for acute renal failure ,9. An apP. vivax, P. ovale, and P. malariae were immediately transferred into 110μL whole-blood aliquots. The aliquots were stored in 0.5 mL screw top tubes at −80°C. Specimens positive for P. falciparum were first separated into two approximately equal aliquots after homogenizing. One aliquot was further separated into cellular and plasma fractions via centrifugation at 2,000 rpm for 5 min. The plasma fraction and the remaining whole blood aliquot were separated into 110 μL volumes and stored at −80°C in the same manner as the other Plasmodium spp.After homogenization through repeated inversions, specimens delivered to Public Health Ontario, which were positive for Patient samples arrived at PHOL along with specimen data sheets filled out by their attending doctor specifying age, gender, travel history, and other personal data. Clinical parasitology test results confirmed species, immunochromatography test results, parasitaemia , and microscopy as well as PCR observations. The data was de-linked from the patients’ names and other personal identifiers, and the remaining details were added to a computer database, with an original copy of the specimen data sheet being placed under lock and key for reference. Follow-ups with the patients’ physicians by the medical microbiologist occasionally provided more specific patient information.P. falciparum samples designated for use in the experiment were from plasma aliquots. The aliquots were removed from −80°C storage, and thawed on ice until they were ready for aspiration. The samples were then separated into two 55μL aliquots and placed back in −80°C storage until required for assays.Samples selected for use in the study required additional processing prior to being used in the assays. All P. vivax, P. ovale, and P.malariae were removed from −80°C storage and after thawing on ice, treated with lysis buffer in order to isolate the plasma fraction. This step was required because repeated freeze/thawing of the blood at −80°C leads to some cell lysis, releasing haemoglobin into the plasma fraction and inhibiting proper separation by pipetting and visible plasma fraction. To ensure the samples contained no intact erythrocytes, they were incubated in ACK lysing buffer (Invitrogen) at a ratio of 10:1 sample to buffer volume for 3 min at room temperature. This step was followed by centrifugation of the samples at 4°C and 2,000 rpm for 5 min. Following this, the supernatant was removed and stored in 55μL aliquots back at −80°C until required for assays.Whole-blood samples of The prepared samples were transported to collaborators on dry ice and immediately placed at −80°C upon arrival.in vitro or in vivo with malaria infection, 20 cytokines were selected to evaluate in this study [Based on prior studies that established a link either is study -19. ThesBased on prior studies establishing a link between severe malaria and angiopoietins, Ang-1 and Ang-2 were also evaluated ,19.To do this, 96-well plates were coated with 50 μl of capture antibody  per well and left to incubate overnight at room temperature. Plates were then washed seven times with 0.05% Tween-20 in PBS pH 7.2–7.4, and blotted on paper towel. Two hundred μl of reagent diluent (PBS pH 7.2–7.4/0.1% BSA mixture) was then applied to each well and left to incubate for 2 h. Afterwards, the diluent was drained and 100 μl of standard solution  diluted in reagent diluent was applied to standard wells at the top of the standard lanes. The remaining wells in the standards lane were filled with standards serially diluted, so that each well contained half the concentration of standards compared to the well above it, with the last lane containing only reagent diluent. The remaining wells were filled with 50 μl of sample diluted at a ratio of 1:5. The plates were then left to incubate at 4°C overnight.Ninety-six-well plates were then washed seven times with 0.05% Tween-20 in PBS pH 7.2–7.4, and blotted on paper towel. Fifty μl of detection antibody (R&D: part 840684) reconstituted in reagent diluent was then applied to each well and left to incubate for 2 h at room temperature. Plates were then washed seven times with 0.05% Tween-20 in PBS pH 7.2–7.4, and then blotted on paper towel. Fifty μl of extravidin alkaline phosphatase  that was diluted 1:1,000 in reagent diluent was applied to each well and left to incubate for 45 min at room temperature. Plates were washed another seven times with 0.05% Tween-20 in PBS pH 7.2–7.4, then rinsed with distilled water and blotted on paper towel. One hundred μl of PNPP Sigma fast  was then applied to each well and the plate was left to develop in the dark until ready. The top standards well should be between two and three at an absorbance of 405 nm. The optical density was then determined at a wavelength between 405–570 nm, and a final cytokine concentration was determined in pg/mL.t-test. Additional comparisons were made for the data set based on patient travel history, age, gender, and species of infection. Samples for which cytokine or Ang-1/2 testing did not yield data due to technical difficulties were excluded. Each comparison was completed using the student’s unpaired t-test to assess significant differences between the related data sets defined by a p value <0.05   compared to FB travellers (p = <0.01). A significantly higher level of EGF (p = 0.0315) was found to exist in FB compared to CB travellers , and M-CSF (p = 0.0433), than those aged 19–55  and M-CSF (p = <0.0001) than patients infected with P. falciparum. Patients infected with P. vivax had higher levels of IL-12 (p40) (p = 0.0478) than patients infected with P. ovale. CB patients infected with P. vivax also had higher levels of MCP-1 (p = <0.0001), and M-CSF (p = 0.0367), when compared to CB patients infected with P. falciparum. FB patients infected with P. vivax had higher levels for MCP-1 (p = 0.0020), M-CSF (p = <0.0001), and also IL-12(p40) (p = 0.0467), compared to FB patients infected with P. falciparum  and IL-6 (p = 0.0426) were significantly lower in patients who travelled to West Africa compared to those who had travelled to other regions in Africa  was observed in patients infected with P. falciparum, compared to those infected with P. vivax. The Ang-2 levels were also significantly different (p = 0.0218) between the FB patients infected with each species, with a higher mean present for FB P. falciparum than for FB P. vivax  and EGF are potential markers of disease severity. It was noted that IL-12 (p40) was significantly increased and EGF significantly decreased in CB. If CB are more vulnerable to severe diseases as has been suspect clinically, then high IL-12 (p40) and low EGF may predict worse outcomes. Consistent with this observation, IL-12 (p40) is a pro-inflammatory cytokine produced by mononuclear phagocytes that activates T-helper 1 cells (Th1) and is important for assisting in innate immune responses against intracellular microbes . HoweverP. falciparum surface proteins have been studied in great detail for vaccine research [EGF binds to the epidermal growth factor receptor (EGFR) where it plays a key role in regulating cellular differentiation, proliferation, and survival . EGF-likresearch ,24,25, bP. vivax infections, adults over age 55 had more pronounced IL-6 and M-CSF production. IL-6 is a pro-inflammatory cytokine. It is produced by numerous cell types including monocytes, endothelial cells and fibroblasts [P. falciparum malaria [P. falciparum infections, where the role of IL-6 was expected to be more pronounced.Studies focusing on older adults , as wellroblasts  and has roblasts . IL-6 ha malaria , in a st malaria , and has malaria . The finP. falciparum malaria, where platelet destruction and the release of P-selectin plays a role in thrombocytopaenia associated with severe disease [P. vivax cases [P. vivax infection appear reasonable. It was noted that when all patient data was divided by species of infection, P. vivax had a higher mean cytokine response for M-CSF than P. falciparum. This corresponds for the tendency of P. vivax to elicit a stronger immune response in patients [P. vivax compared to P. falciparum, as well as just CB patients.Macrophage colony-stimulating factor (M-CSF) enhances macrophage activity, and it has been suggested that when elevated in malaria, it may lead to an increase in macrophage-mediated platelet destruction . Elevate disease . M-CSF hax cases , so the patients , though P. falciparum-infected patients from Thailand and India [P. vivax compared to P. falciparum within the full data set, and in comparisons of just FB individuals, and just CB individuals. This finding again corresponds to the tendency of P. vivax to have a greater inflammatory response than P. falciparum, as does the results with just FB blood samples where the P. vivax patients again had a higher level of IL-12 (p40) than P. falciparum patients. The increased levels of circulating IL-12 (p40) in P. vivax compared to P. ovale again attests to the substantial inflammatory response associated with P. vivax.Monocyte chemo-attractant protein 1 (MCP-1) is required for migration of monocytes and macrophages to sites of inflammation , and hasnd India ,36. The P. falciparum blood samples were from travellers who had been to Western Africa, with most having visited Ghana and Nigeria. All those individuals who had contracted malaria in Western African countries (which included Nigeria and Ghana), were grouped and compared against travellers who were known to have been in other regions of Africa. Significantly lower levels of IL-6 and IFN-γ were found in the Western Africa subset. One study has indicated that there can be reductions in total cases of severe malaria with increasing transmission rates [Infections obtained through travel to different areas of Africa also showed a significant difference in patient immune response. A substantial amount of the on rates , possiblP. falciparum malaria to have elevated levels of IFN- γ [P. falciparum malaria in India among patients aged five to 75 [P. vivax-infected patients from the Brazilian Amazon indicate plasma levels of IFN- γ increase linearly with a gradual augmentation of disease severity [Of the cytokines that were found to be associated with travel, Il-6 has been shown to often be elevated in severe cases of malaria ,30,31 asf IFN- γ ; this isve to 75 . Studiesseverity .P. falciparum infections, a higher level of Ang-2 is present than in P. vivax infections. These findings appeared in a comparison of all the patient data, as well as in a comparison of just FB patients with P. falciparum compared to FB with P. vivax. P. falciparum is generally considered to be more virulent than other causes of malaria, and has been observed to more commonly lead to severe cases of malaria [P. falciparum infections are consistent with this endothelium activation factor as Ang-2 and P. falciparum are both associated with more severe disease instances. A previous study reported that Ang-2 was more strongly elevated in the peripheral blood of individuals with P. vivax malaria compared to individuals with P. falciparum infection [Ang-2 levels have been reported to be elevated in cases of complicated severe malaria compared to uncomplicated malaria ,19. Ang- malaria . Higher nfection , which cP. vivax-infected patients, and Ang-2 in P. falciparum-infected patients may also prove useful in understanding age-associated and species-specific host immune responses. Regional differences in host immune response to malaria infection within the same species  may be related to unique strains circulating in West Africa.The significantly higher mean levels of IL-12 (p40) and significantly lower levels of EGF found in CB travellers may serve to be useful prognostic markers of disease severity and help guide clinical management upon their return. IL-6 and M-CSF in older adults, MCP-1, IL-12(p40) and M-CSF in CB = Canadian-born; FB = Foreign-born; Pf = Plasmodium falciparum; Pv = Plasmodium vivax; Po = Plasmodium ovale; Pm = Plasmodium malariae.The authors declare that they have no competing interests.GM and RM performed the experiments, carried out the data analysis, and GM wrote the initial draft of the manuscript. RL assisted with sample preparation and helped to coordinate the study. JK preformed the cytokine multiplex assays. NR carried out the Angiopoietin ELISA assays. DRP, HZ, and WCL conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.All comparisons made between various subsets of the data look for significant differences. Those italicized in red were significant, all others were not significant. Blanks represent instances where insufficient data was present (~1–5 data points available) to make relevant comparisons.Click here for file


Plasmodium vivax infection has been considered a benign and self-limiting disease, however, recent studies highlight the association between vivax malaria and life-threatening manifestations. Increase in reactive oxygen species has already been described in vivax malaria, as a result of the increased metabolic rate triggered by the multiplying parasite, and large quantities of toxic redox-active byproducts generated. The present study aimed to study the oxidative stress responses in patients infected with P. vivax, who developed jaundice (hyperbilirubinaemia) in the course of the disease, a common clinical complication related to this species.P. vivax infected patients with jaundice, i.e., bilirubin < 51.3 μmol/L (8 patients) or without jaundice (34 patients), on day 1 (D1) and day 14 (D14) after anti-malarial therapy.An evaluation of the lipid peroxidation and antioxidant enzymes profile was performed in 28 healthy individuals and compared with P. vivax with jaundice compared to the control group on D1. However, the activity of thioredoxin reductase was decreased. The enzymes glutathione reductase, thioredoxin reductase, thiols and malondialdehyde also differed between jaundiced versus non-jaundiced patients. On D14 jaundice and parasitaemia had resolved and oxidative stress biomarkers were very similar to the control group.Hyperbilirubinaemia was more frequent among women and patients experiencing their first malarial infection, and lower haemoglobin and higher lactate dehydrogenase levels were observed in this group. Malondialdehyde levels and activity of celuroplasmin and glutathione reductase were increased in the plasma from patients with P. vivax-related jaundice was observed. These results suggest oxidative processes contributing to malaria pathogenesis, what may be useful information for future anti-oxidant therapeutical interventions in these patients.Cholestatic hyperbilirubinaemia in vivax malaria cannot be totally disassociated from malaria-related haemolysis. However, significant increase of lipid peroxidation markers and changes in antioxidant enzymes in patients with  Plasmodium falciparum is the most lethal species responsible for the major burden of malaria disease in Africa. However, Plasmodium vivax is the most abundantly distributed species worldwide. Recent reports suggest increasing clinical complications in P. vivax infected individuals in many endemic regions [P. vivax infection in the Brazilian Amazon region over the past years [P. falciparum severe disease, such as jaundice, anaemia, acute renal failure, shock and coma, being the first the most common [P. vivax mono-infection is confirmed by PCR.Malaria affects millions of people every year around the world . Plasmod regions ,3. Brazi regions . Some dast years . Part ofst years . Nevertht common -10, and P. falciparum are relatively well-established in the literature [P. vivax disease. Particularly, jaundice (impregnation of soft tissues by elevated bilirubins) in malaria may be explained by severe haemolysis (indirect bilirubin predominance) or liver cholestasis (direct bilirubin predominance). World Health Organization (WHO) most recent recommendations state that jaundice by itself  should not be used as a single marker of severity senso strictu, because it is not associated with higher fatality rates, unless there are other simultaneous organs dysfunctions [P. vivax is endemic, contributing to increase the social and economic burden of this disease [Severity criteria for terature , and recunctions ,13. Receunctions . The pro•OH) generated via mitochondrial OS, have been shown to play an important role in the liver apoptosis in a murine model of malarial infection [Despite the frequent occurrence of hyperbilirubinaemia, very little progress has been made in understanding the pathogenesis of cholestasis jaundice in patients with malaria, particularly in vivax disease. Increase in reactive oxygen species (ROS) has already been described in vivax malaria. As a result of the increased metabolic rate of the rapidly growing and multiplying parasite, large quantities of toxic redox-active byproducts are generated. Furthermore, a reduction in antioxidant enzymes such as glutathione peroxidase, catalase and superoxide dismutase has been observed in plasma of malaria-infected individuals -17. Thesnfection ,22.P. vivax infection, it was therefore hypothesized that the transitory predominantly cholestatic jaundice seen in vivax malaria could also be associated to OS.Based on previous studies demonstrating the role of OS upon other clinical complications of Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), a reference tertiary care center for infectious diseases located in Manaus (Western Brazilian Amazon). In this ward, the staff completed a standard questionnaire regarding epidemiological and clinical characteristics of the patients. Blood samples were collected before the beginning of the routine anti-malarial treatment with chloroquine (25 mg/kg over 3 days) and primaquine (0.5 mg/kg/day for 7 days), according to the National Anti-malarial Guidelines. Healthy volunteers without past history of malaria served as controls.Patients with any clinical complications attributed to malaria are systematically hospitalized in the Clinical Investigation Ward of the P. vivax mono-infection was confirmed by PCR [P. falciparum. Other common infectious diseases leading to cholestasis were also ruled out through specific antibody detection , blood culture , and RT-PCR . Abdominal ultrasound was also performed in all patients to exclude lithiasic cholecystitis or any other biliary tract abnormality. On day 14 (D14) after the beginning of therapy (D1), patients were informed to return to the Outpatient Clinics for clinical and laboratorial re-evaluation. Thick blood smear with parasitaemia count in 100 leukocytes, automatized full blood count and serum biochemical analysis  were systematically performed on D1 and D14.Patients included in this study had no diabetes or arterial hypertension history , and were systematically phenotyped for G6PD deficiency, according to the technique described elsewhere . G6PD ded by PCR , ruling 2-EDTA. Aliquots of plasma were stored at −70°C before analysis.About 15 mL of venous blood were collected on BD Vacutainer® tubes with and without KMalondialdehyde (MDA)  was measured using a spectrophotometer 70 UV/VIS Spectrometer PG Instruments Ltda  by reaction with thiobarbituric acid (TBA) in plasma . GlutathThe study was approved by the FMT-HVD Ethics Review Board (CAAE-0075.0.115.114-11), and all the patients signed a written consent after being informed about the objectives of the study.post-hoc Tukey test was performed. Kruskal-Wallis test was used for non-parametric analysis. Student and Mann–Whitney tests were used when only two groups were compared. Frequency differences were detected using chi-square. Correlations between variables were performed using the Spearman test. All tests were performed in BioStat 5.0®  and OriginPro 8.0® , and significance was considered when p < 0.05.Normal distribution was assessed through Shapiro-Wilk test. Parametric data were analysed by ANOVA-one way to estimate mean differences. When significant, P. vivax mono-infection, presenting with serum total bilirubin higher than 51.3 μmol/L (3.0 mg/dL) . During the same period, 42 patients with non-complicated vivax malaria in the same age group, with bilirubin within the normal range, were also randomly enrolled. After ruling out other comorbidities , a total of eight patients with vivax-related jaundice, 34 vivax patients without jaundice and 28 healthy volunteers were included in the final analysis. No complication other than hyperbilirubinaemia was observed after detailed clinical and laboratorial screening. On D14 a clinical and laboratorial screening was performed on seven out of eight with jaundice, and 18 out of 34 patients without jaundice. None of them presented with persistent parasitaemia, clinical jaundice or laboratory hyperbilirubinaemia on D14. None of the controls on D1 referred any clinical complication in between D1 and D14. Epidemiological, haematological and biochemical data are detailed in Table During the year of 2011, 25 hospitalized patients were enrolled with confirmed microscopic diagnosis of P. vivax malaria (with and without jaundice) group was observed compared to the control group. In addition, a significant increase of MDA was observed on D1 in the jaundiced group compared to the non-jaundiced group .P. vivax malaria [P. vivax infection increases lipid peroxidation and alters profile of antioxidant enzymes is provided. That may eventually lead to hyperbilirubinaemia, which is not independently associated to severity [A case series reported in 2010 showed that over 50% of patients admitted to a reference hospital in the city of Manaus had jaundice as complication for  malaria . In the severity , but wasseverity . In the Despite of the very small sample size, the finding of this complication more often in females contradicts previous findings showing that male children present with more life-threatening complications . No obviPlasmodium sp. does not have a triglyceride synthesis pathway; therefore, it obtains all the lipids from the host serum. The parasite also destabilize the cell membrane, in particular the erythrocyte membrane in order to obtain lipid [The significant increased levels of MDA in plasma of malarial patients with and without jaundice may indicate the damage triggered by free radicals produced against cell membranes, such as erythrocytes and hepatocytes . Recent in lipid ,32.•NO are associated with the host-parasite interaction. TrxR was shown to be inactivated with the production of nitric oxide (•NO), a reactive species of oxygen found in high levels during malaria infection [The antioxidant enzyme TrxR activity was significantly decreased when patients with malaria were compared to the control group. TrxR catalyzes the reduction of the active site disulfide using NADPH. The high levels of nfection . This maP. vivax infected patients (with or without jaundice) compared with the control group. Other studies have also demonstrated increased levels of the enzyme GR in malaria caused by Plasmodium berghei and P. falciparum[On the other hand, the antioxidant enzymes GR and CP activities were significantly increased in alciparum. GR is ialciparum. Recent alciparum,36. CP halciparum.P. vivax malaria with jaundice compared with P. vivax malaria without jaundice. Although the thiols levels in malarial patients are not significantly higher compared to the control group, results suggest that malarial patients who developed jaundice have greater oxidative stress, and thiol compounds may be trying to restore the plasmatic balance.Thiols contain the sulfhydryl group attached to a carbon atom. They are efficient antioxidants protecting cells against consequences of damage induced by free radicals ,39. In tSeveral reports in the literature suggest that drugs used to treat malaria, such as chloroquine and primaquine) lead to oxidative stress, particularly in erythrocytes -42. HoweP. vivax malaria who developed jaundice, bilirubin has deleterious effects on tissues. It develops oxidative stress by generating intracellular ROS in hepatic cells and cause lipid peroxidation [Bilirubin has antioxidant properties as well as pro-oxidant. At low concentrations, it acts as a scavenger of reactive oxygen species, reducing the damage caused to the cells. However, at high concentrations, as is the case of the patients with xidation . Furtherxidation , complemxidation ,22. Howexidation . Moreovexidation ,46.P. vivax patients presenting jaundice is increased. Levels of oxygen reactive species may be closely linked to the damage caused by the parasite and the subsequent release of high concentrations of bilirubin in the serum. Further studies are needed to understand the mechanisms involved in liver damage in jaundiced patients, and also to validate if similar findings are seen in other less frequent complications of P. vivax infection, e.g., severe anaemia, coma, acute renal failure and respiratory distress. These studies may provide further evidence for better management of P. vivax infections and possible future anti-oxidant supportive therapy.In summary, the oxidative stress in The authors declared that they have no competing interests.CF and RCMN carried out all the biochemical analysis and drafted the manuscript, together with PL. GCM coordinated and performed all the microbiological tests. BMLM and MAAA performed the full clinical characterization of the enrolled patients. CF, MVGL and ESL participated in the design of the study. MVGL and ESL conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.