Plasmodium species. We aimed to delineate the pattern of malaria-related hospitalization from birth in Timika, Papua–an area co-endemic for P. falciparum and P. vivaxIn endemic regions, the age distribution of malaria varies according to the infecting Plasmodium parasitemia and included re-presentation to hospital, requirement for hospital admission and death.Between April 2004 and December 2013, infants born at Mitra Masyarakat Hospital, or presenting within the first 7 days of life, were enrolled retrospectively into a cohort study and followed passively using routinely-collected hospital surveillance data. Outcomes were stratified by the presence or absence of Plasmodium parasitemia. The incidence of re-presentations with malaria during the first year of life was 213 per 1,000 person-years (py) for P. vivax and 79 per 1,000py for P. falciparum (Incidence Rate Ratio (IRR) = 2.69, 95% Confidence Interval (95%CI): 2.48–2.92). After the age of 5 years, the incidence of P. vivax had fallen to 77/1,000py and the incidence of P. falciparum had risen to 95/1,000py . Overall, 79.7%  of malaria re-presentations were recurrences rather than initial infections. Malaria accounted for 31.7%  of all hospital admissions. The infant mortality rate in this study was 52 deaths per 1,000 live births. Beyond the early neonatal period, 13.4% of deaths were associated with Plasmodium parasitemia.Overall, 11,408 infants were enrolled into the cohort. Median follow-up was 4.3 (maximum 9.7) years. In total, 7,847 (68.9%) infants made 90,766 re-presentations to hospital, 18,105 (19.9%) of which were associated with P. vivax over P. falciparum inverts after five years of age. Malaria is directly associated with nearly one in seven deaths after the early neonatal period.In Papua, Indonesia, malaria is a major cause of hospital presentation and admission in early life. The initial predominance of  Plasmodium. The most common species globally are P. falciparum and P. vivax [Plasmodium infections is determined primarily by the force of infection and the speed with which immunity develops. For both epidemiological and biological reasons (most notably the ability of Plasmodium vivax to relapse multiple times after a single inoculation), the age distribution frequently differs for P. falciparum and P. vivax malaria [Malaria is caused by mosquito-borne, protozoan parasites of the genus P. vivax . In mala malaria –7.Plasmodium infections in non-study settings requires an ability to differentiate malaria from other causes of febrile illness coupled with an ability to link multiple clinical episodes to a single individual.Acute malaria in children causes a wide spectrum of clinical manifestations ranging from an uncomplicated febrile illness to severe disease and death. Recurrent episodes of malaria during early life have deleterious effects on health as well as causing substantial social and economic hardship for children and their families –11. MalaThe aim of this study was to use routinely collected hospital data to delineate the pattern of malaria-related morbidity and mortality from birth in a cohort of children in Timika, Papua. We also aimed to define the proportionate contribution of malaria care to the total hospital workload for these children.This was a retrospective cohort study of children who were either born at Mitra Masyarakat Hospital (RSMM) in Timika, Papua between April 2004 and December 2013 or whose first presentation to hospital during this accrual period occurred within the first 7 days of life. Follow-up information for these children was obtained passively from routinely-collected hospital data via unique hospital identification number linkage, as described previously ,13.Anopheles punctulatus, An. koliensis and An. farauti complex. The point prevalence of microscopic parasitemia in 2005 was 7.5% for P. falciparum and 6.4% for P. vivax with an estimated incidence of malaria overall of 876 episodes per 1,000 people per year [P. falciparum and 5.7% for P. vivax [Timika is situated in the lowlands of southcentral Papua, Indonesia. The population in 2004 was 120,457 growing to 196,401 in 2013 . The maiper year . In 2013P. vivax . Malaria® database by hospital administrators. Information recorded includes patient identifiers , demographic details such as age, sex and ethnicity, clinical information including hospital department, diagnoses assigned by the attending physician  and vital status at the end of the clinical episode.Data for every patient presentation to RSMM are collected routinely and entered into an electronic Q-proHospital protocol dictates that a blood sample is taken for Giemsa staining and thick film examination for any patient presenting to outpatients with fever or history of fever and all patients who are severely ill, regardless of presumed diagnosis. Microscopy in early neonates was not routine during the study period and only requested if there was clinical concern. If parasitemia is too high to count on a thick film, the hospital microscopists will also read a thin film.P. falciparum infections and oral quinine plus low-dose primaquine  for P. vivax and P. ovale infections. The treatment of severe malaria during this era was IV quinine. In April 2006, treatment policy was changed to dihydroartemisinin-piperaquine for uncomplicated malaria due to any Plasmodium species with the addition of high dose primaquine  for P. vivax and P. ovale infections. At the same time, treatment of severe malaria was also changed to intravenous artesunate [Prior to April 2006, treatment of uncomplicated malaria at RSMM generally consisted of oral quinine for tesunate .st of December 2013 by means of unique hospital identification number linkage. Outcomes of interest were: subsequent re-presentation to hospital , admission for inpatient care, total bed occupancy (assuming 0.5 days of occupancy for outpatient and emergency department visits) and death during the hospital encounter with delineation of the absolute and proportionate contributions of P. falciparum, P. vivax and mixed Plasmodium species infections to these outcomes. Patients with no further documented re-presentation to hospital were assumed to have remained in the hospital catchment area and therefore to have been at risk of re-presentation until the 31st of December 2013. For the purposes of graphical presentation, actuarial life tables were used to determine the number of patients at risk of the outcomes of interest in each 6-month period from birth. Kaplan-Meier survival analysis was used to estimate the cumulative risk of the outcomes of interest and incidence rates were given per 1,000 person-years for the two age brackets <1 year and ≥5 years. The effect of previous hospital presentation on the risk of a further representation was explored using Kaplan-Meier survival analysis and univariable and multivariable Cox regression models for each Plasmodium species. Covariables in the multivariable Cox models included: age, sex, ethnicity  and the year of birth. The proportional hazards assumption was assessed for each variable by comparing visually observed versus predicted survival curves. When the proportional hazards assumption was violated, Cox models were either stratified or omitted altogether. Allowance was made in all Cox models for within-patient correlation by using the Huber-White sandwich estimator to calculate robust standard errors. All analyses and graphing were done in STATA® version 15.1 .Patients aged less than 7 days old at their first appearance in the hospital dataset were selected and followed passively until the 31Ethical approval for this study was obtained from the Health Research Ethics Committees of the University of Gadjah Mada, Indonesia (KE/FK/544/EC) and Menzies School of Health Research, Darwin, Australia (HREC 10.1397). Data were anonymized prior to access for this study. The ethics committees waived the requirement for informed consent and therefore individual patient consent for participation was not sought.Plasmodium parasitemia at the point of entry to the study and 101 (0.90%) had congenital malaria. The median duration of follow-up from the time of entry to the study until the 31st of December 2013 was 4.3 years and the maximum follow-up was 9.7 years.Between April 2004 and December 2013, 11,408 neonates were enrolled into the cohort, of whom 52.4%  were male. In total 48.9%  were Highland Papuan, 20.1%  were lowland Papuan and 31.0%  were non-Papuan. Of these patients, 11,307 (99.1%) either did not have a malaria diagnostic test or were negative for Fig 1). In total 93.6%  of first re-presentations occurred within the first 12 months of life .Overall 7,847 (68.8%) patients re-presented 90,766 times to hospital during follow-up. By 5 years of age the cumulative risk of at least one re-presentation was 70.7%  69.8–71.6%) with a median number of 9 presentations (maximum = 134)  were due to P. falciparum, 9,556 (52.8%) to P. vivax, 6 (0.0%) to P. ovale, 200 (1.1%) to P. malariae and 2,881 (15.9%) to mixed species infection.Overall 19.9%  of representations were attributable to malaria with a cumulative risk of at least one malaria re-presentation by 5 years of 38.1% (95%CI 37.1–39.1%); P. vivax infections  was significantly higher than that of P. falciparum infections  = 2.69, 95%CI: 2.48–2.92). After the age of 5 years the overall incidence of malaria declined to 225 events per 1,000py ) with a reduction in the incidence of P. vivax to 77 per 1,000py , but an increase in P. falciparum to 95/1,000py . Within the first year of life, 49.9%  of malaria-related presentations were first episodes whereas after the age of 5 years, only 6.0%  of presentations were first episodes. The corresponding Plasmodium species specific proportions due to first episodes were 75.3% and 15.9% for P. falciparum and 55.9% and 16.4% for P. vivax. Overall, 79.7%  of the presentations with malaria were recurrences rather than initial infections.In the first year of life, the incidence of malaria was 335 events per 1,000 person-years (py). The incidence of P. vivax monoinfections  and P. falciparum monoinfections  = 4.8, 95%CI 4.5–5.0, p<0.001) and this was still apparent after controlling for confounding factors (Adjusted Hazard Ratio (AHR) = 3.0, 95%CI 2.8–3.2, p<0.001; Plasmodium species was present in 2,126 (31.7%) of the children admitted . Within the first year of life, malaria was present in 22.6%  of inpatients whereas after the age of 5 years, the proportion with malaria had risen to 43.9% (133/303) of admissions. The incidence of malaria admissions was 67/1,000py in children under one year of age and 14/1,000py after the age of 5 years . The incidence of P. vivax-related admissions decreased from 34/1,000py in infants under one year to 3/1,000py  whereas there was a smaller reduction in the incidence of P. falciparum admissions from 23/1,000py to 8/1,000py .Overall, 3,120 children had 6,710 admissions to hospital during follow-up. The 5-year cumulative risk of at least one admission was 31.1% (95%CI: 30.2–32.1%), with 48.6%  of these patients being admitted more than once . Malaria due to any P. vivax monoinfection required admission to hospital whereas 20.9%  of initial P. falciparum presentations resulted in admission. Inpatient care of the children in this cohort resulted in 26,604 days of bed occupancy, of which 6,586 days (24.8%) were in patients with malaria: 4,149 days (15.6%) in patients admitted for the first time with malaria and 2,437 days (9.2%) in patients admitted for the second or subsequent time with malaria. The proportion of patients being admitted for the second or subsequent time with the same species was higher with P. vivax in the first 18 months, but higher with P. falciparum thereafter .Overall, 11.9%  of children presenting for the first time with a Fig 5). The cumulative risk of death by one year was 5.3% (95%CI 4.9–5.7%) and by 5 years this had risen to 6.1% (95%CI 5.7–6.6%). Overall 4.3% (29/667) of the patients had a diagnosis of malaria at the time of death: 14 (48.3%) due to P. falciparum, 10 (34.5%) due to P. vivax and 4 (13.8%) due to mixed species infection. Beyond the early neonatal period, 13.4% (25/187) of deaths were associated with Plasmodium parasitemia. The cumulative risk of a malaria related death was 0.1% (95%CI 0.07–0.21) by one year and 0.3% (95%CI 0.20–0.44) by 5 years.In total 667 children in the cohort died at the hospital of whom 480 (72.0%) were less than 7 days old , 481 (72.1%) were less than 28 days old  and 589 (88.3%) were less than one year old  . While patent parasitemia was only present in 4.3% of all children who died in hospital, this rose to 13.4% among those who died after day seven.We present a large, retrospective, hospital-based birth cohort study in southern Papua, Indonesia. Over 70% of the children in the study re-presented to the hospital during a median of 4.3 years of follow-up and almost 40% re-presented with malaria. Malaria accounted for about 20% of all hospital re-presentations, 32% of hospital admissions and 25% of total inpatient bed occupancy. The incidence of P. vivax relapses, preventable, in those who are eligible, by using radically curative courses of primaquine [P. falciparum infection also increases the risk of malaria recurrence suggests that at least part of the increased risk may be due to epidemiological risk factors such as socio-economic status, proximity to mosquito breeding areas and local vector control activity. Geolocating patients presenting for the first time with malaria may be an effective strategy to target transmission hotspots through vector control, distribution of insecticide treated bednets and/or mass drug administration. Current vector control strategies in the region are targeted at village, rather than individual, level. A third explanation for the increased risk of re-presentation in those with prior malaria is simply that an initial malaria presentation identified a subset of patients who were more likely to seek treatment for any condition at RSMM than the remaining children–potentially due to proximity or specific health care preferences. Given that previous nonmalaria-related presentations had relatively little bearing on the risk of a future re-presentation, this is unlikely to have been the sole explanation.Given that a single episode of malaria, regardless of species, identifies a child as being at significant risk for future re-presentation with malaria, the first episode of malaria represents a particularly important opportunity to intervene to prevent morbidity and mortality from multiple recurrences and substantially reduce hospital workload. Some of the increased risk of recurrence will be attributable to imaquine –21. Unsuimaquine . SuperviP. vivax infection to minimise the risk of drug-induced hemolysis. One of the barriers to primaquine use is the cost of such testing [P. vivax presentations might suggest. Nearly 85% of presentations with vivax malaria by the age of 5 years are recurrences as opposed to first episodes and G6PD status need only be assessed once.Ideally, testing for glucose-6-phosphate dehydrogenase (G6PD) deficiency should be undertaken prior to prescription of primaquine for radical cure of  testing . Our resP. vivax infection was heavily skewed towards children under the age of one year whereas P. falciparum infections predominated after the age of 5 years [P. vivax due to greater parasite exposure resulting from multiple relapses [Although presentation to hospital with malaria was dominated by recurrent infections rather than primary infections, requirement for admission to hospital  occurred mainly in patients presenting for the first time with malaria. The risk was almost twice as high in children presenting with falciparum malaria, a likely reflection of poor immunity and susceptibility to pathogenic processes. We have previously hypothesized that morbidity (particularly severe anemia) caused by vivax malaria tends to result from the cumulative effects of repeated infections rather than a single infection ,24. Apprrelapses .Perhaps the most striking finding in this study was the very high infant mortality rate of 52 deaths per 1,000 births, largely attributable to early neonatal deaths. In 2017 the mean infant mortality rate globally was 29 deaths per 1,000 live births, rising to 51 deaths per 1,000 live births in Africa . AnecdotOur study has some notable strengths. Due to the lengthy accrual period, relatively large local population and the hospital’s busy obstetric unit, our study included a large number of children and therefore our risk estimates are relatively precise. The hospital’s strict policy regarding blood film assessment ensured that the vast majority of patients with patent parasitemia presenting to the hospital will have been captured. RSMM was the only public tertiary referral centre in the district until late 2009 and therefore a very high proportion of all pediatric inpatient admissions and deaths in hospitalized patients will have been ascertained. Beyond 2009, some admissions and deaths may have occurred at the other public hospital even though birth occurred at RSMM.st of December 2013. Population growth, variations in treatment seeking behaviour and changes in the epidemiology of malaria in the region will have altered the pattern of malaria presentations at RSMM in the last 5 years.Our study has some important limitations. Patients presenting with malaria to community health care facilities  and those who migrated out of the catchment area prior to the study conclusion will not have been captured. Any ascertainment bias will have underestimated the risk of re-presentation and the true burden of symptomatic malaria in our cohort. This bias was likely to have been particularly important for non-Papuan patients who were required to pay a small amount for health care at RSMM. Our survival analyses of the risk of further re-presentation to hospital only included a relatively limited number of covariables. Data on household income and bednet usage were not available and may have influenced the risk of malaria recurrence. Attribution of events to malaria was based on the presence or absence of patent parasitemia. Some individuals may have had symptomatic subpatent parasitemia while others may have had patent parasitemia that was incidental to other comorbidities. Moreover, previous episodes of malaria may have caused morbidity that contributed to nonmalarial re-presentations. Congenital malaria was frequent in the early years of the study , prior tMalaria is a very common cause of representation to hospital during childhood in southcentral Papua, resulting in significant morbidity, mortality and hospital workload. Malaria tends to be recurrent after the initial presentation making the first occurrence a particularly important opportunity to implement preventive measures through optimal antimalarial treatment and geospatially targeted vector control activities. Despite malaria being directly associated with nearly one in seven deaths after the neonatal period, early neonatal causes of mortality predominate in this setting and warrant prospective investigation and intervention.S1 Data(XLSX)Click here for additional data file.


Plasmodium falciparum DNA from RDTs, then amplified and sequenced a region of the apical membrane antigen 1 (pfama1) using targeted amplicon deep sequencing. We determined the multiplicity of infection (MOI) for each sample and examined associations with demographic, clinical, and spatial factors. We successfully genotyped 223 of 287 (77.7%) of the samples. We demonstrated an inverse relationship between the MOI and elevation with individuals presenting from the highest elevation villages harboring infections approximately half as complex as those from the lowest . This study demonstrates the feasibility and validity of using routinely-collected RDTs for molecular surveillance of malaria and has real-world utility, especially as the cost of high-throughpout sequencing continues to decline.Molecular techniques are not routinely employed for malaria surveillance, while cross-sectional, community-based parasite surveys require significant resources. Here, we describe a novel use of malaria rapid diagnostic tests (RDTs) collected at a single facility as source material for sequencing to esimtate malaria transmission intensity across a relatively large catchment area. We extracted  The multiplicity of infection (MOI), defined as the number of concurrent parasite clones per Plasmodium falciparum-infected host, has shown promise as a surrogate measure of malaria transmission intensity4. In the more than twenty years since its first descriptions7, the MOI has been employed in a variety of roles to include estimating malaria transmission intensity between differing areas9, assessing changes in transmission intensity over time11, and evaluating the impact of interventions ranging from chemoprophylaxis13 to vaccines15.To acheieve malaria elimination, there is a need for tools that accurately measure transmission intensity to identify target areas for public health interventions16. The MOI is generally considered to be positively associated with the intensity of transmission. As shown in Table 31. Host factors such as premunition, however, also impact the MOI32. Efforts have been made to correlate the MOI with age, parasite density, and disease severity, all of which reflect host immunity, with mixed results  or the glutamate-rich protein (glurp)33. However, these techniques are labor intensive and often fail to detect all sequence polymorphisms and low-abundance variants35. Newer methods utilizing targeted amplicon deep sequencing of malaria infections provide a high-throughput, highly sensitive approach for detecting clones in polyclonal infections as well as more accurate quantitative estimates of clonal frequency36.To estimate MOI, investigators have historically collected dried blood spots (DBS), which allow long-term storage of malarial DNA. However, DBS are not collected in routine clinical practice, and require additional supplies for sampling and storage, which generally precludes estimation of MOI outside of research studies. Moroever, the MOI is traditionally determined using nested PCR (nPCR) with gel electrophoresis to detect polymorphisms in the highly variable surface antigens of the merozoite surface proteins  across a geographically diverse area of western Uganda. To achieve this, we extracted DNA from malaria rapid diagnostic tests (RDTs) collected during routine care at a single rural health facility and stored at room temperature under tropical conditions. Our primary objective was to identify spatial differences in the MOI as a surrogate marker of malaria transmission intensity over a highland area using only a facility-based sample of routinely-collected rapid tests. While RDTs have previously been used as a source of malarial DNA for PCR and the sequencing of drug resistance and mitochondrial polymorphisms, they have not been employed to estimate transmission intensity41. Our relatively efficient sampling strategy precludes the need for more resource-intensive, population-based approaches to mapping transmission intensity and parasite diversity. Secondary objectives included an investigation of associations between the MOI, age, and the clinical spectrum of malaria, as well as an exploration of the spatial micro-epidemiology of parasites based on the pfama1 haplotypes.In this study, we investigated the use of a targeted amplicon deep sequencing approach to describe the clonal diversity of the 42. The two most recent malaria indicator surveys undertaken in the region found declining parasitemia prevalence from 48.4% to 17.6% in 2009 and 2014, respectively44.Samples were collected from patients presenting with febrile illness to the Bugoye Level III Health Center in the Kasese District of western Uganda. The geography of the study area is highly varied. The westernmost villages of the sub-county are characterized by deep river valleys and steep hillsides with elevations up to 2,000 meters. In contrast, villages located to the east are defined by low-lying, level terrain Fig. . Three l45.Rapid diagnostic tests were performed as part of the Rapid Diagnostic Tests for Severe Malaria (RDTSM) study, a prospective, observational cohort study of patients with a parasitological diagnosis of malaria conducted from May to November 2015. We have reported full details of the study methodology elsewhereP.f/Pan RDT assay . Study RDT were obtained directly from the manufacturer, stored in the original packaging at room temperature, and utilized in accordance with the manufacturer’s instructions prior to the expiry date. Each day, study staff packaged completed RDTs in a polyethylene bag with desiccant. RDTs were stored at room temperature for approximately two years prior to extraction. Study staff with training in laboratory medicine prepared thin and thick blood smears for all patients with a positive RDT result. Hemoglobin levels were measured using the Hemocue Hb 201+ analyzer , and venous lactate values were obtained using the Abbott iStat analyzer .In brief, initial testing for malaria was performed using the Standard Diagnostics 05FK60 Malaria Ag Post hoc, we found that this number was more than sufficient to power the primary analysis at a level of 0.80 (α = 0.05) to detect the observed mean difference in MOI between the highest and lowest quartiles of elevation, even accounting for failed genotyping in nearly a quarter of samples.We first stratified the RDT-positive results by village. From each village-based strata, we sorted by level of parasitemia as determined by microscopy and systematically sampled starting from the highest density sample to achieve a balance of high and low parasitemias within each village-based strata. A convenience sample of approximately sixteen RDTs from each village was selected. 40. DNA was extracted using a previously described Chelex extraction method in a final volume of 100 μL46. The concentration of extracted P. falciparum DNA in individual samples was determined using a quantitative real-time PCR (qPCR) for P. falciparum lactate dehydrogenase (pfldh)47. Pfama1 was amplified from individual samples in duplicate in a hemi-nested PCR modified from Miller et al., which was shown to reproducibly and accurately determine the frequency and haplotype of variants in known mixtures in our laboratory using Ion Torrent sequencing48. In this approach, the inner forward primers are labeled with a unique barcode , and 167 nM for forward and reverse primers for each step of the hemi-nested PCR. Thus, all samples were amplified and uniquely barcoded in duplicate. Successful amplification was confirmed by visualizing the qPCR products on a 1% agarose gel and the product was quantified using Quantifluor dsDNA System  on a Synergy HT Multi-Mode Microplate Reader . Barcoded samples were then pooled in equal amounts based on concentration. Pools of MID labeled PCR products were cleaned using KAPA Pure Beads  and multiple indexed libraries were prepared for sequencing using KAPA Hyper Library Prep kits  using NEXTflex DNA barcodes . The indexed pools were then combined in equimolar concentration into a final pool that was sequenced on a part of a MiSeq (2 × 300 bp chemistry) at the University of North Carolina High Throughput Sequencing Facility. Unlike Ion Torrent, Illumina sequencers have problems handling samples with low heterogeneity. We typically use two approaches to increase thje heterogeneity on the sequencing runs. First, we include 10% PhiX spike in all of our amplicon deep sequencing runs. Second, we often run libraries for other projects on the same run as the amount of sequencing necessary for a small project is not sufficient to warrant a complete run. This library comprised an estimated 80% of the run which also contained amplicons from other studies.Modifications to the PCR protocol included using 2.5 units of Roche FastStart high fidelity pfama1 amplicons were generated for 5 controls, representing 10 MID labeled PCR reactions, and sequenced alongside the clinical samples. To ensure that we correctly estimated the frequency of the three strains in the mixture, we amplified the mixture using the same conditions using only the second round reaction for 30 cycles. We cloned the PCR product using a TOPO TA cloning kit (Invitrogen) and performed Sanger sequencing of 22 colonies using the M13F primer at GENWIZ . Sequences were analyzed using Geneious R10 . We estimated allele frequencies and confidence intervals by the bootsrap method with 1,000 replicate bootstraps. This was compared to the allele frequency determined by the four replicate control deep sequencing reactions.For these experiements, we included a positive control comprised of a known mixture of three malaria strains: 3D7 , FCR3 , and Dd2 . Using this control mixture, http://baileylab.umassmed.edu/SeekDeep), as previously described49. This approach uses the PCR replicates for each sample to reduce PCR and sequencing errors as shown in the SeekDeep Process Cluster step of Fig. et al., and has been shown to provide accurate frequency and genotype determinations with as few as 200 reads per sample49. As haplotypes in each PCR are called independently and then only haplotypes conserved between replicate PCRs are used, this provides a conservative approach for haplotype detection to determine MOI. Here, samples were included in the final analysis if they had ≥250 total reads combined between the replicates. The low read coverage in some samples did not influence the MOI estimates as seen in Supplemental Fig. 48. Haplotypes that were marked as likely chimeric were excluded. Haplotypes in each sample were compared to all identified haplotypes in order to provide population-level statistics. All data generated or analyzed during this study are included in this published article and the Sequence Read Archive available at www.ncbi.nlm.nih.gov/sra (SRA Accession Number Pending).The deep sequencing reads were de-multiplexed and clustered using the software package, SeekDeep  lactic acid levels, (b) hemoglobin levels, and (c) severe malaria. For the continuous outcomes of lactic acid and hemoglobin levels, we first performed linear regression with MOI serving as the primary explanatory variable. We then utilized negative binomial general linear regression model with a log-link function and robust standard errors to explore associations between the categorical outcomes of (a) lactic acidosis (venous lactate ≥5 mmol/L), (b) anemia (hemoglobin <7 g/dL), and (c) severe malaria. Data were analyzed with Stata 12.1 .To estimate associations between geographic factors and the MOI, we completed village-level geographic information system (GIS) mapping of the sub-county and surrounding environs, comprising an area of approximately 55 square kilometers. These data were entered into ArcGIS, Version 10.4.1  to create a reference map, from which we calculated the mean elevation and area of each village and subsequently divided the data into quartiles of elevation.Ethical approval of the study was provided by the institutional review boards of the University of North Carolina at Chapel Hill, the Mbarara University of Science and Technology, and the Uganda National Council for Science and Technology. Written informed consent was obtained from all adult study participants and the caregivers of participating children. All research was performed in accordance with relevant guidelines and regulations.do), the replicate PCRs showed a high level of agreement of allele frequencies in each of the five controls with a mean do of 0.0252. Using data from the cloned Sanger sequences as the reference standard, we evaluated the accuracy of the deep sequencing frequency estimates. We bootstrapped the frequency estimates to generate confidence intervals for the Sanger sequence data and compared it to the variation in replicate deep sequencing reactions  of RDT positive samples. Of the twelve RDTs from which we were unable to detect pfldh or pfama1, four were negative on microscopy while another four had parasite densities of <250 parasites/μl. We successfully sequenced 223 of the 287 (77.7%) samples with detectable malarial DNA. Demographic and clinical characteristics of the cohort are summarized in Table p = 0.28), sex (p = 0.85), parasitemia (p = 0.29), or disease severity (p = 0.70) between those samples successfully genotyped and those that were excluded from the analysis.We detected p = 0.003 compared to May), which marks the beginning of the second rainy season.After filtering in SeekDeep, we used 5.56 million of 8.15 million sequencing reads to call AMA haplotypes and determine multiplicity of infection in each sample. The mean number of reads used per sample was 22,763 . Read depth for each sample stratified by MOI is shown in Supplementary Fig. p = 0.001)  and Nambiaji  river valleys compared to the basin area (MOI 3.25) where the rivers converge. These results were generally consistent with the RDT positivity rates observed in each valley with both the Sebwe  and Nambiaji  river valleys being significantly lower than the basin area.The MOI varied significantly with geographic factors, including village elevation and river valley of residence. The MOI demonstrated an inverse relationship with elevation Table . On aver01) Fig. . This fiThe highest MOI was found in children age 3 to 4 years of age  compared to those aged 8–11 years of age, who had the lowest MOI . Differences by age, however, were not significant as shown in Table p = 0.41). Increasingly complex infections (≥3 identified haplotypes) demonstrated a trend towards a reduced risk of severe malaria although this finding was not significant in the multivariate model , which was adjusted for age. Of note, when the analysis was stratified by age, we found that risk of severe malaria was non-significantly higher with more complex infections  in children <5 years of age, while the risk of severe malaria trended lower with more complex infections in children and adults ≥5 years of age .When we defined severe malaria as the outcome measure of interest, we found strong correlations with male sex, age less than five years, and parasite density greater than 100,000 parasites/μl in the 21 cases of severe disease Table . There wp = 0.40) or hemoglobin levels  and the MOI in the linear regression models.Similarly, there was no association between the MOI and disease severity when we defined lactic acidosis (>5 mmol/L) or anemia (Hb < 7 g/dL) as the outcome measure of interest. While lactic acidosis and anemia were relatively rare outcomes in the cohort, we did not observe any trend between lactic acid  was found in 122 of 223 (54.7%) of the included samples and accounted for 24.6% of the population fraction. The distribution of haplotypes in the population are shown in Supplementary Fig. In the clustering analysis, we did not identify any evidence of population structure based on either haplotype prevalence (the proportion of infections containing the respective haplotype), or haplotype relative abundance , when plotting by elevation, river valley or the presence/absence of severe malaria. Fig. .Figure 327. We also found a lower MOI in two of the river valleys, which correlated with the RDT positivity rate, a crude measure of transmission intensity. Our findings are proof-of-concept that it is possible to estimate transmission intensity across a wide catchment area using only a facility-based sample of routinely-collected RDTs, thus negating the need for additional DBS collection or labor-intensive cross-sectional sampling. Such an approach has relatively broad programmatic application and may even be cost-effective when considering the labor costs associated with large cross-sectional surveys.We carried out targeted amplicon deep sequencing from DNA extracted from routinely-collected RDTs among a facility-based cohort of symptomatic individuals and were able to demonstrate a significant, inverse correlation between village elevation and the MOI, consistent with previous observationspfama1 haplotypes. This outcome is not entirely unexpected, as it likely reflects the high intensity of transmission and resulting mixing of parasite sub-populations across the study site. It is also supported by our recent work showing no population structure based on this gene between distant geographic sites in Africa48. We had hypothesized that the high ridgelines separating the three major rivers could serve as a physical barrier to vector migration, isolating each valley from the next. Our results, however, could suggest that this barrier is inadequate to maintain population structure, most likely at the lower elevation areas where the rivers merge into the basin area. In addition, human movement into lower elevation areas, where most commercial activity occurs, could also facilitate the mixing of parasite populations between river valleys. Alternatively, the lack of structure could also result from the choice of marker. Antigens like pfama1 may be under convergent evolution and thus similar patterns of diversity emerge in distant populations, as supported by our previous work54.In contrast, we found no evidence of population structure by elevation or river valley based on the p = 0.07) towards a reduced risk of severe disease with more complex infections. This finding supports the hypothesis of age-dependent immune response mechanisms and is consistent with a report from Tanzania in which children <3 years of age experienced a greater risk of a subsequent malarial episode with increasing infection complexity, whereas in older children, more complex infections were associated with a decreased risk of clinical malaria,55.We did not identify any associations between the MOI and age or the MOI and markers of disease severity . However, given the few cases of severe malaria (n = 21), our analysis was only powered to a level of 0.54 to detect a mean difference of one parasite clone between cases of uncomplicated and severe malaria. Interestingly, we did observe a divergent effect of infection complexity on the risk of severe malaria among children <5 years of age, who had a relatively negligible risk of severe disease with a more complex infection (≥3 identified haplotypes), compared to adults and children ≥5 years of age, who had a clear trend  of our samples. This is likely a result of our conservative duplicate reading thresholds. We are reassured that there were no significant differences in demographic, clinical, or laboratory parameters between those samples that were successfully genotyped and those that were not.Our study has several limitations. First, we did not utilize traditional measures of transmission intensity such as the entomological inoculation rate (EIR) or parasite prevalence against which we could compare our results. However, the relationship between elevation and transmission intensity is well establishedUsing routinely-collected malaria RDTs from a single health facility as source material, we were able to deep sequence and estimate malaria transmission intensity across a large and geographically diverse catchment area. To our knowledge, this is the first study to demonstrate the feasibility and validity of such an approach, which we believe has practical implications for malaria surveillance programs, especially as the cost of high-throughput sequencing continues to decline. Similar techniques may also be applicable to other disease conditions where lateral flow assays are commonly utilized for point-of-care diagnosis.Supplementary Information


We found that malaria parasites cross the blood-retinal-barrier and infiltrate the neuroretina, concomitant with an extensive, irreversible, and long-lasting retinal neurodegeneration. Furthermore, anti-malarial treatment with dihydroartemisinin strongly diminished the load of circulating parasites but resolved the symptoms of the retinopathy only in part. In summary, we introduce here a novel preclinical model for human cerebral malaria that is much more directly accessible for studies into disease pathophysiology and development of novel treatment approaches. In vivo retinal imaging may furthermore serve as a valuable tool for the early diagnosis of the human disease.Malaria is a causative factor in about 500.000 deaths each year world-wide. Cerebral malaria is a particularly severe complication of this disease and thus associated with an exceedingly high mortality. Malaria retinopathy is an ocular manifestation often associated with cerebral malaria, and presumably shares a substantial part of its pathophysiology. Here, we describe that indeed murine malaria retinopathy reproduced the main hallmarks of the corresponding human disease. In the living animal, we were able to follow the circulation and cellular localization of malaria parasites transgenically labelled with GFP via non-invasive  Cerebral malaria is a complication that is associated with a poor prognosis2 and can lead to irreversible sequelae, which can have a profound effect, particularly, on child development, although such sequelae often remain unnoticed4. The precise pathological processes and mechanisms leading to cerebral malaria are still unclear, hampering the development of efficacious treatments. Typically, cerebral malaria is accompanied by malaria retinopathy, which is considered as an early symptom for most forms of cerebral malaria6.Malaria is considered one of the most common and deadliest diseases that are afflicting mankindin vivo, in a non-invasive fashion7. The retina is perfused by two vascular systems, the outer retinal vasculature or the choroid and the inner retinal vasculature. The neuroretina is shielded from blood-borne toxins or infectious agents by the blood-retinal-barrier (BRB). As for the vasculature, the BRB consists of two morphologically distinct parts, the outer retinal, choroidal BRB, which is formed by fenestrated endothelial cells in conjunction with retinal pigment epithelial cells (RPE), and the inner retinal BRB, which is formed by non-fenestrated endothelial cells in conjunction with Müller glial cells8. While the outer BRB is a retina specific tissue adaptation, the inner BRB is virtually identical to the blood-brain-barrier in other parts of the central nervous system10.The retina is an integral part of the central nervous system and combines easy access with the possibility to directly visualize neurodegenerative processes Plasmodium berghei. The C57BL/6J mouse strain is particularly vulnerable to developing the symptoms of cerebral malaria, something that typically occurs within 8–10 days post-infection (DPI) and is usually fatal after 1–2 more days11. Even though the mouse is an often used model system for studies into the underlying pathophysiology as well as for drug candidate testing and treatment development12, malaria-induced murine retinopathy has not been studied in any detail13.Mice can develop neurological signs following infection with certain mouse-pathogenic species of malaria parasites including in vivo and ex vivo techniques for a comprehensive longitudinal study of disease pathogenesis. Our results demonstrate that the ocular and retinal symptoms in the mouse model are reproducing the main hallmarks of human malaria retinopathy. Moreover, we provide strong in vivo and ex vivo evidence that malaria parasites cross the BRB and infiltrate the neuroretina. We also show that malaria parasites trigger extensive, irreversible and long-lasting retinal neurodegeneration, the symptoms of which may persist even after timely anti-malarial treatment. Finally, we suggest that in vivo imaging techniques routinely used in the clinic, namely confocal scanning laser ophthalmoscopy (SLO) and spectral domain (SD) optical coherence tomography (OCT), offer a significant detection advantage for malaria retinopathy and could thus constitute a valuable new tool for the early diagnosis of cerebral malaria in humans as well as for evaluation of effective therapeutic approaches in vivo.Here, we present the first systematic investigation on malaria induced retinopathy in the mouse using both P. berghei parasites (ANKA strain) used for the experiments, were kindly provided by A. Walliker, Institute of Cell, Animal, and Population Biology, University of Edinburgh, United Kingdom.C57BL/6J mice were obtained from Charles River (Germany), were housed under standard (12 h) white cyclic lighting and had free access to food and water. Only male animals were used in this study. All procedures involving animals were performed in accordance with the ARVO Statement for the use of animals in Ophthalmic and Vision Research. The study and the protocol (Registration No. T1/10) were approved by the competent local authority, Regierungspräsidium Tübingen, based on the assessment by the appointed regional ethics board, and according to the applicable German law on animal protection (Tierschutzgesetz). The P. berghei were generated as previously described15. The transfection vector (pl0016) was kindly provided by The Leiden Malaria Research Group, LUMC, Leiden, Netherlands. Host animals were infected with frozen stocks, monitored until parasitaemia reached 1–5% and bled around midday, giving rise to mixed blood stage parasites with more than 80% rings and young trophozoites. Research for cerebral malaria typically uses sub-adult (e.g. three weeks old) C57BL/6J mice, producing a very severe disease phenotype that does not allow to follow the animals for prolonged periods of time. For our study, we used adult, male C57BL/6J mice (1–4 months old), which were infected intravenously (i.v.) with 1 Mio. parasites using standard protocols16. Previous experience indicated an onset of cerebral malaria in this age group after about 8 days post-infection (8DPI) with this infectious dose (data not shown). All infected mice were treated orally with pyrimethamine throughout the experiment to avoid parasite reversion to wild-type.GFP expressing P. berghei, retinal changes were determined by confocal scanning laser ophthalmoscopy (SLO), spectral domain (SD) optical coherence tomography (OCT) and electroretinography (ERG) at 3DPI18, i.e. before mice showed any signs of neurological involvement, followed by a second measurement at the expected onset of cerebral malaria (6DPI). In vivo imaging observations were performed and confirmed by three independent examiners . On those days, parasitaemia was determined on Giemsa stained blood smears. The in vivo analyses were performed consecutively beginning with ERG recordings, followed by OCT and SLO imaging. The mice were anaesthetized using a combination of Ketamine (66.7 mg/kg body weight) and Xylazine (11.7 mg/kg body weight) and their pupils dilated20. ERGs were recorded binocularly according to previously described procedures18. Briefly, single flash ERG responses were obtained under scotopic (dark adapted overnight) and photopic (light adapted with a background illumination of 30 cd/m2 starting 10 min before recording) conditions. Single white-flash stimuli ranged from −4 to 1.5 log cd*s/m2 under scotopic and from −2 to 1.5 log cd*s/m2 under photopic conditions. Ten responses were averaged with inter-stimulus intervals of 5 s (for −4 to −0.5 log cd*s/m2) or 17 s (for 0 to 1.5 log cd*s/m2).In a first group of five animals , infected with 18. For native fundus imaging laser wave lengths of 830 nm and 514 nm were used, fundus autofluorescence analysis (AF) was performed with 488 nm. For angiography, both fluorescein (FL) and the argon blue laser at 488 nm (barrier 500 nm), and indocyanine green (ICG) and the infrared laser with 795 nm (barrier 800 nm), were used.SLO imaging and angiography was performed with a Heidelberg retina Angiograph;  according to a previously published method21. This device features a superluminescent diode at 870 nm as low coherence light source. Scans are acquired at a speed of 40.000 scans per second, with each two-dimensional B-scan containing up to 1536 A-scans. The images were taken with the equipment set of 30° field of view, using Heidelberg Eye Explorer software . Resulting images were exported as 8-bit colour bitmap files and processed with CorelDraw X5 .Retinal layer morphology was visualized via OCT imaging with a SpectralisTM HRA + OCT , as reported previouslySince the mouse eye is very small it is only accessible to human-rated equipment (which we use) via the use of adaptor lenses that limit the observation angle to approx. 30°.The exact angle of observation is variable to about 10–15° and can only be estimated within this range. This means that also sizes and distances cannot be quantified precisely.Imaging of the retinal periphery in the mouse–where many of the malaria-induced aberrations occur – requires tilting the observation leading to further image distortion.Fluorescence intensities also vary with the illumination angle, thus only relative differences in fluorescence intensities can be evaluated.Concerning the quantification of mouse SLO and OCT datasets, we note that there are marked differences to human retinal imaging where the much larger size of the human eye, the corresponding calibration of imaging equipment, and the availability of large training sets of images of human pathology largely facilitates quantification. For mouse imaging specifically, the following limitations apply:i.v. as described above and treated with the anti-malarial drug dihydroartemisinin (DHA). Pyrimethamine was administered orally via drinking water to avoid parasite reversion to wild-type (see above). In vivo measurements  took place on 3DPI and 6DPI. Following the second measurement, all animals were treated with daily intraperitoneal injections of 30 mg/kg DHA from 6DPI to 18DPI, and were followed up with further measurements until 18DPI.To investigate reversibility of the observed retinal changes, a second group of six animals  were infected 2 asphyxiation, the eyes were enucleated and then either fixed in 4% paraformaldehyde (PFA) or not fixed and flash frozen on liquid N2 and then stored at −20 °C (i.e. unfixed). The eyes were cryoprotected in increasing concentrations of sucrose . After embedding in cryomatrix , the eyes were sectioned in a Microm HM cryostat (12 µm sections). A haematoxylin-eosin staining was used for routine histology.Following the last measurement mice were sacrificed using CO22, using the terminal deoxynucleotidyltransferase (TdT) dUTP nick end labelling (TUNEL) assay . Microscopy was performed on a Zeiss Imager Z1 Apotome Microscope; images were captured with Zeiss Axiovision 4.8 software .Detection of dying cells was performed as reported previouslyRetinal tissue sections were rehydrated in PBS and incubated at 4 °C over night in primary antibodies Table . Primary2, 1.5 mM CaCl2, 2 mM DTT, pH 7.2)23. The fluorescent calpain substrate CMAC, t-BOC-Leu-Met  was then added to the reaction buffer at a final concentration of 2 μM and sections plus reaction mixture were incubated in the dark for 2 h at 37 °C. The sections were washed twice for 10 min each in reaction buffer and then mounted with Vectashield . The activity assay generally labelled the cell membranes, while calpain-activity-positive cells additionally showed a bright labelling of the nucleus and perinuclear cytoplasm24.The whole unfixed frozen eyes were embedded in Tissue Tek and cryosectioned at 12 μm. Unfixed retinal sections were incubated for 15 min in calpain reaction buffer , pictures were taken from three to five sagittal sections, within 5–10° eccentricity from the optic nerve, for at least four different animals for each experimental condition using Axiovision 4.8 and 20 x magnification. The average ONL areas used for quantification were equivalent ranging from approx. 20.000–35.000 µm2. Uninfected, healthy adult C57BL/6J mice were used as controls. The average surface area occupied by a photoreceptor cell (i.e. cell size) for each genotype was determined by counting DAPI-stained nuclei in 9 different areas (50 × 50 µm) of the retina25. The total number of photoreceptor cells in a given ONL area was estimated by dividing area by the average cell size. The number of positively labelled cells in the ONL was counted manually.For quantification of cells positive for a given marker  for each microscope image (3–8 images per specimen) to measure the pixels showing GFP or GFAP staining above threshold intensity. Individual pixel intensities ranged from 0–16384 (14 bit), an intensity above 2048 was considered as positive labelling. The number of GFAP positive pixels in infected retina at 6DPI was arbitrarily set to 100%, the numbers of GFAP positive pixels in uninfected retina and at 18DPI were expressed as a percentage of this. In the same way, GFP positive pixels were expressed as percentage of the 6DPI GFAP values.Data were evaluated using Graph Pad Prism 7.03 software  and one-way ANOVA analysis with a Kruskal-Wallis test and Dunn’s multiple comparison post-test. Levels of significance were: ***p < 0.001; **p < 0.01; *p < 0.05. Data are presented in figures as a mean ± standard error of the mean (SEM).P. berghei ANKA strain, transgenically labelled with GFP14. At various time-points after infection  mice were examined using non-invasive morphological and functional ocular testing. Among the tests used were scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and electroretinography (ERG), as well as determination of parasitaemia. After 6DPI animals were treated with dihydroartemisinin (DHA), a standard anti-malarial drug and closely monitored for further 12 days until the end of the experiment (18DPI) and full histological work-up and analysis  with 1 Mio parasites of the mouse pathogenic in vivo detection of single cells expressing GFP26. In uninfected animals, SLO AF imaging reveals the normal grey fundus appearance with the dark overlying retinal vessels  mode in SLO imaging allows for the els Fig. , inset. els Fig. . SLO imaels Fig. .Figure 2in vivo SLO and OCT imaging  and perfusion abnormalities were correlated to areas of retinal whitening29. Using GFP expressing plasmodia and SLO in vivo imaging, we were able to directly colocalize the areas of retinal whitening  and retinal perfusion abnormalities were observed Fig.  arrows aTo quantify the degree of colocalization between retinal whitening, GFP-positive parasites, and areas of low perfusion as assessed in angiography, we counted the spots showing fundus changes in SLO images. We took these spots as reference (=100%) and found that 97.4% (±2.6 STD) of these were also GFP positive. When comparing fundus changes to dark, non-perfused areas seen in angiography, we found an overlap in 85.9% (±1.6) of cases.in vivo observations were corroborated by histological sections that revealed sequestration of parasitized red blood cells in the choroidal vasculature . Thus, the local retinal aberrations induced by plasmodia infection had not yet produced detectable changes in ERG responses.Already at 3DPI ies Fig. . To asseies Fig. , left anies Fig. , right cIn vivo imaging as well as in ex vivo histology, detected parasites in large retinal vessels  next to the outer segments of the photoreceptors  , which, in the healthy retina, is expressed at a low level in Müller glial cells. However, when Müller cells meet dying neurons, GFAP is strongly up-regulated, making it a very sensitive, general marker for retinal neurodegenerationCL) Fig. . In infeCL) Fig. . In uninCL) Fig. .Figure 636. DHA treatment halted further weight loss until the end of the experiment  to 18DPI , suggesting Müller cells may have shielded parasites from the DHA treatment Fig. .37 to detect dying cells. At 6DPI a significant elevation of cell death was found in the outer nuclear layer , when compared to uninfected control retina. While the DHA treatment, starting at 6DPI, brought the number of dying ONL cells down to almost control levels by 18DPI .Notably, the symptoms of malaria retinopathy were not fully reverted by the DHA treatment and in part progressed even further. While at 18DPI the area of retinal whitening Fig.  and confi.e. hypoxia), direct parasite interactions, or inflammatory processes. The data from the various in vivo and ex vivo retinal investigations is summarized in Table in vivo, non-invasive, and early detection of malaria induced structural changes to the retina.Taken together, this data suggested that the loss of outer retinal thickness was localized to relatively small areas of the peripheral retina and may have been caused, for instance, by peripheral blood vessel occlusion  for structural information, scanning laser ophthalmoscopy (SLO) for fundus analyses, angiography and (auto)fluorescent markers, and electroretinography (ERG) for functional analysis40. OCT and SLO techniques typically allow resolving features in the 7–8 µm range41 and allow for longitudinal follow-up studies. Previously, luciferase labelled P. berghei was used for detection of malaria infection in live animals and in explanted tissues and organs42. The resolution of this methodology was, however, relatively low (≈100 µM) and did not allow for the detection of individual cells. Higher resolution live imaging was afforded by an invasive confocal technique using an intracranial window in mice infected with GFP-labelled P. berghei43. Still, a major problem in malaria retinopathy research was to demonstrate a direct in vivo correlation between affected areas in retinal fundus imaging and the localization of parasites. Using GFP expressing P. berghei ANKA in the mouse14, we could effectively associate plasmodia to the pathological tissue alterations produced by the infection. To our knowledge, this is the first time that interaction of live plasmodia with a pathophysiologically relevant host tissue was monitored during the acute phase of malaria infection, using non-invasive, high-resolution in vivo imaging techniques.Among the techniques available for non-invasive 27, peripheral vascular alterations29, blood vessel occlusion44, and GFAP immunoreactivity30 were all observed in both our mouse model and in human malaria retinopathy. Thus, our mouse model appears to be a close approximation of the human pathology, even though there are certain limitations when it comes to in vivo mouse eye imaging and human imaging . However, an important difference to the human situation is the apparent absence of haemorrhages, which in our mouse model may be due to the fact that we could not investigate the very final stages of malaria retinopathy because of animal welfare concerns.The symptoms of murine malaria retinopathy observed in our study matched to large extent with corresponding human observations. For instance, retinal whitening45). Using in vivo imaging, we detected fundus alterations and retinal vascular abnormalities already at 3DPI. In the mouse, this corresponds to a time-point approximately five days before symptoms of cerebral malaria manifest themselves43, translating into a significant extension of the treatment window. Similarly, SLO angiography found very clear indications of localized low or no retinal perfusion at 3DPI. With progression of the disease further retinal alterations developed that could clearly be identified by OCT and SLO and were even more evident at 6DPI .Previous studies suggested the use of funduscopy and retinal imaging to investigate malaria pathogenesis and in particular to allow for an early diagnosis of cerebral malaria before severe symptoms such as coma and convulsions set in  and possibly also in other parts of the CNS, would be an interesting subject for future clinical studies and non-invasive OCT and SLO investigations may be particularly well suited for these.We found malaria parasites to cause significant neurodegeneration in the retina, especially in parts of the periphery that were associated with blood vessel occlusion. Remarkably, this effect was in part still visible at 18DPI, after 12 days of DHA treatment, corresponding to observation in malaria patients that may suffer from neurological deficits long after overt disease symptoms have subsidedin vivo imaging and ex vivo histology still showed GFP positive structures in the retina. This raises the question whether these GFP positive parasites have indeed been killed by the treatment and were inert, but not yet removed from the neuronal tissue, or whether they were still viable and capable to induce pathology. The latter case could be explained by parasites being partly shielded from anti-malarial drug treatment inside the neuroretina. If this was the case, novel drug delivery systems developed for the systemic treatment of brain cancer, such as GSH-conjugated, PEGylated liposomes47, could be adapted to allow anti-malarial drugs to penetrate into the CNS so as to increase on-target drug activity.Parasitaemia was strongly reduced by the DHA treatment, with no parasites detectable in the blood stream after four days. However, even after 12 days of DHA treatment The sequestration of parasites in the nervous tissue also induced local inflammation, as evidenced by the GFAP staining. This inflammatory response may in part be responsible for an aggravation of the phenotype even after parasite elimination by DHA treatment. In particular, the aneurysm-like dilations in the retinal vasculature found at 18DPI, but not at 10DPI, may have been due in part to localized inflammatory processes, which in turn also corresponded to local thinning of the retina. Remarkably, we found parasites inside some of the GFAP positive Müller cells suggesting a direct link between parasite neuroretina infiltration and inflammation.i.e. urea, creatinine, etc.)48. The BRB also protects the neuronal tissue from entering of large molecules and pathogens thus ensuring homeostasis of the brain. Although not much is known about the early pivotal pathophysiological changes that lead to cerebral malaria, there is evidence that the pathological mechanisms causing malaria retinopathy and cerebral malaria are the same or very similar27.The blood brain barrier (BBB) consists of a highly-specialized network of blood vessels, which provides nutrients and oxygen, and which removes carbon dioxide and metabolic waste . These similarities of the retina to the brain and the accessibility of the eye together with the e.g. inhibition of extravasation and retained activity in tissues. In this context, the observation that parasites apparently can use Müller glial cells to penetrate into the neuroretina is especially interesting and conforms to earlier findings on transcellular migration in the brain50. Our results furthermore indicate that in the retina, malaria parasites can infect Müller glial cells over time, even in the presence of DHA treatment. While, conversely, the parasites not associated with Müller cells  decreased upon treatment, one may speculate that the intracellular localization has a protective effect against systemic antimalarial drugs. This concept may have to be considered in future drug development, especially in the context of cerebral malaria.Particularly with respect to an improved management of complications, the verification of the passage of the parasites through the BRB may be of clinical relevance, because, anti-malarial drugs and treatment regimens may have to meet new requirements like in vivo retinal imaging methods allow for an early detection and follow-up of malaria retinopathy in the mouse. Confirmation of a similar detection advantage in future human patient studies could allow to develop retinal imaging into a clinical tool for the early diagnosis and treatment of cerebral malaria. Our work also opens a new perspective on how malaria parasites may enter the nervous tissue itself. In the retina, Müller glia cells may constitute an important port of entry. Whether the analogous cell type in the brain, the radial glial cell (Bergmann glia in the cerebellum), has a similar role in the pathogenesis of cerebral malaria may be an exciting subject for future studies. The symptoms of malaria retinopathy, as assessed in vivo and ex vivo, persisted for at least 12 days after the beginning of the DHA anti-malarial treatment. Furthermore, neurodegenerative processes and neuronal cell loss appeared concomitantly with malaria retinopathy. In the mouse model, the combination of in vivo imaging techniques with the possibility to extract diseased tissues may prove particularly useful for future investigations into disease mechanisms as well as testing of novel therapeutic approaches.Our work shows that state-of-the-art 51 may extend well beyond the acute phase of the disease. In future studies on severe human malaria, more emphasis on the identification of sequelae and their prevention shall be laid. This includes sight and hearing4, as well as other neurological deficits.Taken together, our study suggests that malaria parasites can infiltrate the neuroretina and produce long-lasting neurodegenerative effects. Since, the composition of the BRB and BBB is essentially the same; similar processes are likely to also occur in the brain and lead to long-lasting neurological deficits. This in turn implies that the overall long-term socioeconomic effects of cerebral malariaSupplemental Video 1Supplemental Video 2Supplementary information


Anopheles salivary glands proteins in the vertebrate host can reflect the intensity of exposure to Anopheles bites and the risk of Plasmodium infection. In Colombia, the identification of exposure biomarkers is necessary due to the several Anopheles species circulating. The purpose of this study was to evaluate risk of malaria infection by measuring antibody responses against salivary glands extracts from Anopheles (Nyssorhynchus) albimanus and Anopheles (Nys.) darlingi and also against the gSG6-P1 peptide of Anopheles gambiae in people residing in a malaria endemic area in the Colombian Pacific coast.The humoral immune response against An. albimanus strains STECLA (STE) and Cartagena (CTG) and An. darlingi and the gSG6-P1 peptide by ELISA in uninfected people and microscopic and submicroscopic Plasmodium carriers from the Colombia Pacific Coast. A multiple linear mixed regression model, Spearman correlation, and Mann–Whitney U-test were used to analyse IgG data.Dried blood spots samples were eluted to measure the IgG antibodies against salivary gland extracts of An. albimanus and for gSG6-P1, also IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to Plasmodium falciparum in the mixed model.Significant differences in specific IgG levels were detected between infected and uninfected groups for salivary glands extracts from An. albimanus salivary glands extracts are a potential source of new Anopheles salivary biomarkers to identify exposure to the main malaria vector and to calculate risk of disease in the Colombian Pacific coast. Also, the gSG6-P1 peptide has the potential to quantify human exposure to the subgenus Anopheles vectors in the same area.The CTG and STE  Plasmodium and is transmitted by female Anopheles mosquitoes. Although significant advances have been made towards its elimination in several previously endemic countries, malaria remains a significant public health concern [Plasmodium falciparum is the most common species (96.3%) [Malaria is caused by the protozoan parasite  concern . The Wor concern . Specifi concern . Despiteecies 96.% [3].Anopheles species in five subgenera have been reported in Colombia [Nyssorhynchus, with Anopheles (Nys.) nuneztovari, Anopheles (Nys.) albimanus and Anopheles (Nys.) darlingi as the most important malaria vectors in areas of high malaria transmission [An. albimanus is the main vector [An. albimanus lineage circulating the Southern region may be different from the one found the in the Northern part of the country suggesting that two different lineages are circulating in the country [Plasmodium vivax and P. falciparum [More than 47 smission . On the n vector , 7. Prev country . Intereslciparum  are neceAnopheles, Anopheles (An.) calderoni and Anopheles (An.) punctimacula are also important malaria vectors in the area. However, these two species are often misclassified due to their high morphological similarities [An. calderoni was found infected with both P. vivax and P. falciparum with an annual entomological inoculation rate (EIR) of 2.84 bites/human/year in Nariño between 2012 and 2013 [An. calderoni between 1.7 and 14.7 from 2009 to 2010, while EIR reported for An. albimanus during the same period was found between 0.1 and 2.6 [An. calderoni is a primary vector of malaria in Nariño. Furthermore, in the Tumaco city, located in the Narino Department), Ahumada et al., reported different malaria incidence in places where An. albimanus and An. calderoni were found in the 2011–2012 study. Specifically, they reported a high Annual Parasite Index (API) (73 cases/1000 inhabitant) in places where An. calderoi is the predominant species compared to lower (27 cases/1000) where An. albimanus was predominant [Extensive entomological research has been done in the Nariño Department , 11, 12.Plasmodium [Plasmodium parasites even in their high abundance months [To design a proper vector control method, it is necessary to accurately determine human-vector interaction and the proportion of those vectors that are infected. Vectorial capacity (VC) and EIR are quantitative entomological indicators used to determine epidemiology of vector-borne diseases such as malaria. The VC is used as the measure of a mosquito population’s proficiency to transmit an infectious agent to a susceptible population , while Easmodium . Howeverasmodium , 18. Humasmodium . Furtherasmodium . As an aasmodium . Still, e months , 12. So,Plasmodium spp. sporozoites are injected into human skin through the bite of a female Anopheles along with the mosquito salivary proteins [Plasmodium spp. [An. albimanus and An. darlingi salivary proteins have been observed in people with active malaria infection in Central and South America when compared to uninfected people living in the same region [Anopheles (Cel.) gambiae and Anopheles (Cel.) stephensi are among the most important vectors. A significant number of these studies were performed evaluating IgG responses against the An. gambiae salivary protein gSG6, a highly conserved protein among Anopheles species from the Subgenus Cellia and Anopheles [An. gambiae gSG6 sequence. IgG responses specific to this salivary peptide has been validated as a biomarker of human exposure not only in Africa but also in Asia and South America [Cellia in South America, the responses observed against the gSG6-P1 peptide could be hypothesized to result from the presence of mosquitoes belonging to the subgenus Anopheles such as Anopheles pseudopunctipennis and An. punctimacula and An. calderoni [Malaria is acquired when proteins . Previouium spp. –27. Thuse region , 28. A snopheles . The pep America , 27, 30.alderoni .Anopheles mosquitoes. Future studies are planned to identify exposure markers that include not only the primary malaria vectors but also markers for the majority of the circulating species playing an important role in malaria transmission in Latin America, even when these vectors species are in a smaller proportion. Since the use of salivary gland extract as antigen to indirectly measure exposure to mosquito species circulating in a region has been validated by several groups the main objective of this work was to measure IgG antibodies in humans living in an area where low-density P. falciparum infections are frequent. Thus, human IgG responses to Anopheles salivary gland extracts (SGE) were used to measure potential associations with low-density infections by P. falciparum and malaria risk. Additionally, it was evaluated whether gSG6-P1 peptide continues as a useful marker to detect exposure in areas where mosquitoes from the sub-genus Anopheles are important vectors of malaria in Colombia.Consequently, it is necessary to characterize a broader panel of biomarkers able to identify the risk of disease more closely in areas with a great diversity of Plasmodium infections in Colombia.The samples used in this study were collected as part of a longitudinal study in which the purpose was to evaluate the dynamic of submicroscopic ® 903 protein saver card  were collected by passive case detection in the transversal phase of the study, conducted between August 2017 to March 2018 in four villages  in Tumaco city located in the south of Colombia   in Tumaco with an API of 13.5 cases/1000 inhabitants in 2017 and 10.4 cases/1000 inhabitants in 2018. No entomological data was collected during the time of this study [Dried blood spots (DBS) in Whatmanis study .Fig. 1StP. falciparum samples were selected (n = 63) from the 958 people that were enrolled in the main study. All of these infections were afebrile (axillary temperature < 37.5 °C), and 48 (76.2%) were submicroscopic . Furthermore, 50 uninfected samples were randomly selected by age (± 5 years) and gender from the total of non-infected individuals by using an Excel random list.To compare the vector exposure between infected and uninfected individuals, all positive Anopheles albimanus and An. darlingi were maintained under insectary conditions until salivary gland dissection. Based on recent studies suggesting that time of colonization has an influence on arthropod salivary gland content [An. albimanus lineages are circulating in two geographically distant regions of Colombia, the potential differences in antibody responses against salivary content of two different strains of An. albimanus were evaluated, one from a long-stablished colony strain STECLA (STE) versus a recently colonized strain Cartagena (CTG). Briefly, An. albimanus strains originated from El Salvador (STE) and Colombia (CTG), respectively, and were maintained in the insectary at the CDC . The An. darlingi laboratory strain originated from Iquitos, Peru [An. gambiae gSG6-P1 peptide was synthesized by Genscript  and the P. falciparum Pf-MSP  peptide  was used to evaluate exposure to malaria parasites. content , and thaos, Peru , and wasos, Peru . MosquitAn. darlingi and An. albimanus SGE (1 μg/mL) or Pf-MSP (1 μg/mL) diluted 1× PBS. Plates were incubated overnight at 4 °C and blocked with 200 µL of 5% skim milk solution in PBS-tween 20 (0.05%) (Blocking buffer) for 1.5 h at 37 °C. The DBS elution was used to prepare a 1:50 sample dilution in blocking buffer, this optimal dilution had been determined by preliminary experiments and 50 µL of diluted samples were added to each well . Plates were incubated at 37 °C for 1.5 h, washed three times, then incubated 1 h at 37 °C with 50 µL/well of a 1/1000 dilution of goat monoclonal anti-human IgG conjugated with horseradish peroxidase . After three final washes, colorimetric development was carried out using tetra-methyl-benzidine (Abcam) as a substrate. In parallel, each assessed microplate contained in duplicate: a positive control, a negative control, and a blank; wells containing no sample. The positive control was a pool of DBS of people with positive malaria diagnosis. The negative control was a sample of people from US (n = 36) with no exposure to malaria parasites. The blank was composed by wells containing no sample. The reaction was stopped with 0.25 N sulfuric acid, and the optical density (OD) was measured at 450 nm.ELISA conditions were standardized as described elsewhere , 24. AlsAll data from questionnaires and forms were entered into a Microsoft Access database, and statistical analyses were conducted in STATA 14  and GraphPad Software V5. OD normalization and plate to plate variation was performed as described elsewhere . BrieflyAnopheles antigen with Pf-MSP IgG levels. Finally, a Mann–Whitney U-test was used to estimate differences between medians of each Anopheles antigen by the status of infection in the whole sample and by sites and a Kruskal–Wallis test to estimate differences between groups of infection. A multiple linear mixed regression model was constructed to determine the correlation between anti-Anopheles IgG levels  with anti Pf-MSP IgG levels. A random intercept at the village level was introduced in the model to correct the inter-village variations. The model was adjusted by Plasmodium infection, age and time of residence in a malarial endemic area; these factors showed significant p values in simple models.Odd ratios (OR) were calculated to evaluate risk of malaria. For this, the median was used to classify IgG antibody levels as high (ΔOD higher than the median) and low  and the samples were classified as cases (Asymptomatic and submicroscopic infections) and controls (uninfected). In addition, Spearman correlation coefficients were calculated to measure the strength of association between each An. albimanus (STE and CTG), An. (Nys.) darlingi or gSG6-P1 by gender, education level and occupation did not show significant differences (Mann–Whitney test p > 0.05).The exposure to mosquito bites in the area of Tumaco in Nariño (Colombia) Fig.  was studAn. albimanus salivary proteins from both strains (STE and CTG) and against the gSG6-P1 peptide was significantly higher in volunteers with Plasmodium infection  antibody levels . This is consistent with information provided by previous studies showing An. albimanus as one of the important vectors in the region.The level of antibodies against els Fig. . Howeverp = 0.0023), STE  and gSG6 = P1  but not for An. darlingi SGE .Figure Plasmodium infected patients were afebrile and considered as asymptomatic carriers. However, they were grouped according to the diagnostic test results into microscopic (if parasites were detected by LM and PCR) or submicroscopic if parasites were only detected by PCR  and gSGS-P1  between the three groups of infections. Although the tendency was also observed when using STE and An. darlingi as antigen, the differences were not significant  and gSG6-P1 peptide   = 0.105; 95% CI 0.0223–0.189 and RE = 0.070; 95% CI 0.013–0.126, respectively). In contrast with the IgG Pf-MSP, a negative association for all IgG responses to Anopheles was found with age showing there is a decreasing of IgG immune response with increased age . A similar situation occurred with the time of residence in an endemic area for malaria; IgG responses to gSG6-P1 peptide was 3.4% lower in samples from people who had lived in a malarial area for more than 5 years . Finally, no significant variation of specific anti-Anopheles IgG was observed according to the status of infection .Independent of location (random intercept at village levels), IgG response to CTG and gSG6-P1 peptide were positively associated with the IgG response to Anopheles bites have been proposed, including the detection of IgG responses to Anopheles SGE and salivary peptides. The purpose of the present study was to explore the possibility of using whole SGE from different Anopheles species as tool to detect IgG antibodies in humans that could be used as indirect estimation of exposure to Anopheles bites in a malaria-endemic area in Colombia where there is an important proportion of asymptomatic infections. Based in previous reports suggesting at least two An. albimanus lineages in Colombia [An. albimanus strains were used to try to capture potential differences in immunogenicity of salivary proteins from colony mosquitoes isolated from different geographical regions and with differences in the colonization time. Specifically, this study includes the comparison of salivary gland content immunogenicity between the CTG strain, a recently colonized strain, that could potentially resemble more closely responses to “wild mosquito antigens” in the area, to the immunogenicity displayed by the STE strain, isolated in Central America in 1974.The intensity of malaria transmission has been traditionally evaluated using the EIR, which is defined by the number of infected bites received per human per unit of time; nevertheless, this strategy has shown limitations in low endemic settings for malaria , 36. As Colombia , 9, 10, Anopheles albimanus has been reported as one of the main malaria vectors in Nariño displaying EIR up to 2.6 in recent studies. Consistent with previous studies, An. albimanus SGEs (STE and CTG) were associated with the infectious status, where people with active Plasmodium infection presented significantly higher IgG antibody levels against the salivary proteins. This study also showed that people with higher antibody levels against STE, CTG and gSG6-p1 have between 2 and 4 times more probability of suffering a malaria infection. These results agree with previous findings in Haiti were the IgG antibody levels against An. albimanus SGE were higher in patients with clinical malaria than those in uninfected people living in the same region [An. albimanus SGE is associated with Plasmodium exposure and highlights the relevance of using whole salivary content in the form of SGE as potentially useful antigen to measure risk of infection in areas of low and seasonal transmission. Interestingly, the relationship between parasitaemia and IgG antibodies against Anopheles antigens was significant when using the antigen from the CTG strain and not the STE, suggesting that the antigens contained on the SGE from the CTG may be more closely related to the one the study subjects are exposed in the field. However, no association was found between antibodies levels against An. darlingi SGE and malaria infection. This could be explained due to the low abundance (or probable absence) of An. darlingi mosquito previously reported in areas where samples were collected [An. darlingi SGE may be explained by a potential cross reactivity between salivary proteins present in mosquitoes from the subgenus Nyssorhynchus, which An. darlingi belongs to.e region . These sollected . Still, An. calderoni is a primary malaria vector in Narino [Anopheles species from the subgenus Nyssorhynchus are the main vectors of malaria in Colombia, at least six species from the sub-genus Anopheles have been described as potential malaria vectors in the region [An. calderoni, An. pseudopunctipennis and An. punctimacula) are present along the Pacific coast, the main area where P. falciparum is transmitted in Colombia [An. freeborni and An. quadrimaculatus (from the subgenus Anopheles) had between 67 and 71% of degree of identity with the gSG6 from Old World Anopheles species [An. punctimacula, which is a member of the Anopheles subgenus or could hypothetically represent exposure to minor vectors in the country [Nyssorhynchus are present.Previous studies suggest that n Narino . This man Narino . Althouge region , 38. ThrColombia  should bColombia , previou species . In the  country . Thus, tAn. albimanus among villages, it was observed that SGE from both STE and CTG, were higher in infected than uninfected people in all villages except California. This is interesting because California is an area with urban characteristics, unlike Tangareal which is a sub-urban area and Robles and Candelillas which are rural areas. To evaluate further, the multilevel analysis demonstrated that independent of site, both age and, anti-Pf-MSP IgG levels were associated not only with IgG antibody levels against the CTG strains of An. albimanus but also against the gSG6-P1. Suggesting the importance of using a panel of exposure biomarkers (mosquito antigens) and concurrent entomological data to accurately evaluate risk especially in areas where several Anopheles species are implicated in malaria transmission. Also, the current model described in this study revealed a negative association between age and IgG antibodies against all Anopheles antigens. Similar trend has been observed in other studies measuring antibody responses against mosquito salivary antigens and has been associated with the development of tolerance against certain mosquito allergens [When comparing IgG levels against llergens , 40, 41.An. albimanus lineages circulating two geographically distant regions of Colombia. Thus, the aim of this study was to determine if the risk of infection can be affected by the salivary content of mosquitoes from the same species but from different origins. So, a recently colonized strain (CTG) and a long-term established laboratory colony (STE) each isolated from a distinct geographical region  to account for potential changes in IgG responses based on salivary content were used. As the results indicate, the SGE from the CTG strain showed significant association with the Pf-MSP1 and not with the SGE from STE suggesting potential differences. Determination and confirmation of these differences are subject of further studies aimed to characterize salivary gland content of the two An. albimanus lineages circulating in Colombia and comparing those to An. albimanus isolates from other countries. This is important since the use of salivary antigens as vaccines for malaria are undergoing [Recent studies revealed important differences in salivary content in arthropods collected in the field when compared to the same species maintained in a colony . Also, adergoing  and charAnopheles IgG levels should be interpreted with caution as they do not imply causality. Second, due to the lack of a symptomatic group, it was not possible to determine the risk factors for this kind of infection and to explore the differences in the anti-Anopheles IgG levels between uninfected, asymptomatic  and symptomatic groups. Also, the lack of concurrent entomological data is a significant limitation. Since this study did not included mosquito collection or other concurrent entomological surveillance, the current results should be interpreted as an indirect measurement of disease risk  until further determination of the specific mosquitoes circulating in an area where these antibodies are measured. A future study phase will include to complete the serological data with entomological data to further validate the findings of this study. Despite these limitations, these results are useful to identify new potential biomarkers for malaria risk in Colombia.This study has several limitations. First, because this study was cross-sectional, association with the anti-An. albimanus strains CTG and STE could be a potential source of new Anopheles salivary biomarkers to determine risk of malaria in Colombia, supports previous findings that gSG6-P1 peptide has the potential to quantify human exposure to some malaria secondary vectors. All of them could be useful to estimate the risk of malaria transmission and could provide relevant tools to better understand malaria transmission dynamics and orient control strategies according to the specific characteristics in low-endemic settings.This study demonstrates that SGE from