Plasmodium knowlesi is the most common cause of human malaria in Malaysia. Acute kidney injury (AKI) is a frequent complication. AKI of any cause can have long-term consequences, including increased risk of chronic kidney disease, adverse cardiovascular events and increased mortality. Additional management strategies are therefore needed to reduce the frequency and severity of AKI in malaria. In falciparum malaria, cell-free haemoglobin (CFHb)-mediated oxidative damage contributes to AKI. The inexpensive and widely available drug paracetamol inhibits CFHb-induced lipid peroxidation via reduction of ferryl haem to the less toxic Fe3+ state, and has been shown to reduce oxidative damage and improve renal function in patients with sepsis complicated by haemolysis as well as in falciparum malaria. This study aims to assess the ability of regularly dosed paracetamol to reduce the incidence and severity of AKI in knowlesi malaria by attenuating haemolysis-induced oxidative damage.2-isoprostanes and isofurans) and markers of endothelial activation/Weibel–Palade body release  over 72 h, as well as blood and urine biomarkers of AKI. This study will be powered to detect a difference between the two treatment arms in a clinically relevant population including adults and children with knowlesi malaria of any severity.PACKNOW is a two-arm, open-label randomised controlled trial of adjunctive paracetamol versus no paracetamol in patients aged ≥ 5 years with knowlesi malaria, conducted over a 2-year period at four hospital sites in Sabah, Malaysia. The primary endpoint of change in creatinine from enrolment to 72 h will be evaluated by analysis of covariance (ANCOVA) using enrolment creatinine as a covariate. Secondary endpoints include longitudinal changes in markers of oxidative stress  contains supplementary material, which is available to authorized users. Plasmodium knowlesi, first reported as a major cause of human malaria in Malaysia in 2004 [P. knowlesi is the most common cause of human malaria, and incidence is increasing [P. knowlesi is also the most common cause of human malaria in areas of western Indonesia [The simian parasite  in 2004 , is now creasing –5. In Sacreasing . Recent ndonesia , 8.P. knowlesi malaria [As with falciparum malaria, acute kidney injury (AKI) is a common complication of knowlesi malaria. Severe AKI (serum creatinine > 265 μmol/L) occurred in 24% of patients in a prospective tertiary-hospital study of severe  malaria  and in 5 malaria . Even in malaria  occurred malaria . Importa malaria , progres malaria , long-te malaria , major a malaria  and mort malaria , 17. In  malaria . Interve3+) to ferryl (Fe4+) haemoglobin [2-isoprostanes (F2-IsoPs) and isofurans (IsoFs), which appear in free form in plasma and subsequently, after glomerular filtration, in urine. F2-IsoPs and IsoFs are considered robust measures of oxidative stress, and are associated with AKI in haemolytic conditions such as rhabdomyolysis, sepsis and post-cardiopulmonary bypass [2-IsoPs/IsoFs were associated with AKI and disease severity, suggesting that haemolysis-induced oxidative damage contributes to the pathogenesis of malaria-associated AKI [Mechanisms of renal disease in knowlesi malaria are poorly defined. In falciparum malaria, AKI has been attributed in part to haemolysis-mediated oxidative damage . Haemolymoglobin . Free-ray bypass –24. Simiated AKI .3+ state and quenching of globin radicals [2-IsoPs and improved renal function in adults with sepsis and detectable CFHb [Paracetamol has been shown to inhibit CFHb-induced lipid peroxidation via reduction of ferryl haem to the less toxic Feradicals , 25, 26.radicals . In a reradicals . In a rable CFHb . Paracetble CFHb  and chilble CFHb  undergoible CFHb .Paracetamol may also have a potential role in reducing endothelial activation and microvascular dysfunction in malaria. In previous in vitro studies, CFHb has been shown to stimulate degranulation of endothelial cell Weibel–Palade bodies (WPBs), leading to release of major WPB constituents von-Willebrand factor (vWF) and P-selectin . In addiIn adults with severe knowlesi malaria, CFHb is higher than in severe falciparum malaria, and AKI at least as common . As paraP. knowlesi malaria.We aim to test whether regularly dosed paracetamol compared with no paracetamol will reduce kidney dysfunction in patients with This is a two-arm, open-label, randomised controlled trial conducted at four centres in Sabah, Malaysia, namely Queen Elizabeth Hospital  and Keningau, Ranau, and Kota Marudu District Hospitals. Queen Elizabeth Hospital and Keningau both have well-equipped intensive care units with facilities for invasive ventilation, haemodynamic support and renal replacement therapy. Patients will be followed up at 1, 3 and 5 years post-enrolment. A summary of the trial design is shown in Fig. Patients are being identified via referral from hospital physicians and/or notification from hospital microscopists. Patients are being recruited in Emergency Departments at time of presentation, or in medical wards or outpatient departments. Patient recruitment started in October 2016 and is expected to be complete by September 2018.Age ≥ 5 yearsP. knowlesi infection1Admitted to hospital with microscopy-diagnosed Temperature > 38 ºC on admission or fever during the preceding 48 hWithin 18 h of commencement of antimalarial treatmentWritten informed consent from patient or attending relative/guardianInclusion criteria:Contraindication or allergy to paracetamol or artesunate therapyKnown cirrhosis, or more than six standard alcoholic drinks per dayPregnancy2Exclusion criteria:Participants are randomised to either the paracetamol or the control arm using computer-generated site-specific block randomisation by an independent statistician in a 1:1 ratio. Allocation tables were uploaded to the Research Electronic Data Capture (REDCap) web application hosted at Menzies School of Health Research. REDCap is a secure, web-based application designed to support data capture for research studies . StandarParacetamol arm: Paracetamol is administered orally 6-hourly for 72 h by research nurses. Patients unable to swallow receive paracetamol by nasogastric tube. Dosing of paracetamol is weight-based, with patients ≥ 50 kg receiving 1 g and patients < 50 kg receiving 12.5–15 mg/kg. If the patient vomits within 30 min of paracetamol administration, a further dose is given.No paracetamol arm (control): Patients in the control arm do not receive paracetamol; however, paracetamol may be given if temperature remains > 39.5 °C for > 30 min despite tepid sponging, or if deemed necessary by the treating clinician. There is no specific rescue medication for this trial.The date, time and dose of paracetamol administered to patients is recorded in the CRF. All patients receive standard artemisinin-based treatment for malaria. The use of intravenous artesunate and oral artemisinin-combination therapy is at the discretion of the treating clinician according to Malaysian National guidelines .Day 0, Hour 0 (enrolment) is the time of administration of the first dose of paracetamol administered by the research team. Any concomitant medications, or paracetamol doses prior to enrolment, are recorded on the CRF. There are no medications that are not permitted during the study; however, it is recommended to avoid non-steroidal anti-inflammatory drugs to avoid exacerbating any renal dysfunction.Paracil™, SM Pharmaceuticals, Malaysia).Paracetamol 500 mg tablets administered in this study are produced locally in a Good Manufacturing Practice-qualified facility  = 0.9; log-transformed estimated mean 72 h creatinine in control group = 4.38 μmol/L (estimated SD 0.36); log-transformed estimated mean 72 h creatinine in treatment group = 4.275 μmol/L (estimated SD 0.36); correlation between baseline and 72 h creatinine (data log-transformed) = 0.59. The mean 72 h creatinine in the control group, the correlation between baseline and 72 h creatinine, and the standard deviation of the creatinine was estimated using existing data from prospectively enrolled patients with non-severe knowlesi malaria at a tertiary hospital in Sabah [Our sample size for ANCOVA was calculated using the Stata command sampsi, with in Sabah . We estiin Sabah .Using the above calculation, a minimum sample size of 324 will be required. Allowing for a loss to follow-up of 10% , we will require a minimum of 360 patients enrolled in the study, 180 in each arm.Venous blood and urine will be collected on enrolment. On venous blood, standard haematology and biochemistry  will be performed through the hospital laboratories. Plasma will be separated within 30 min of collection. Both plasma and urine will be stored at –70 °C until transport for analysis.Serum creatinine will be measured on enrolment (day 0) then 12-hourly until 72 h, then at 1, 2 and 4 weeks. Follow-up creatinine is measured at 1, 3 and 5 years . Plasma CFHb concentrations will be measured at enrolment, at 12 h and then daily for 72 h. CFHb will be measured by enzyme linked immunosorbent assay (ELISA) using commercially available kits .2-IsoPs and IsoFs) and endothelial activation/WPB release ) will be assessed at admission and then daily for 72 h, with markers of endothelial activation also measured at day 28 and at 1 year. Plasma F2-IsoPs and IsoFs will be quantified using gas chromatography-mass spectrometry [Plasmodium spp. will be performed at the Sabah State Reference Laboratory [Biomarkers of oxidative stress /200 white blood cells counted.G6PD genotyping will be performed on all enrolled patients, as G6PD status may influence oxidative stress and may thus affect the beneficial effects of paracetamol.The change in log-transformed creatinine from enrolment (hour 0) to 72 h.The change in log-transformed creatinine from enrolment (hour 0) to 72 h stratified by the presence of haemolysis, as measured by CFHb .Longitudinal change in creatinine over 72 h, as measured by the area under the creatinine-time curve, and the effect of enrolment CFHb on the longitudinal change in creatinine.P. knowlesi malaria).Longitudinal change in creatinine over 72 h, as measured by the area under the creatinine-time curve, and the effect of enrolment CFHb on the longitudinal change in creatinine in patients with severe knowlesi malaria and in those with AKI (see Table Development of AKI (as defined by KDIGO criteria) at 72 h.Duration of AKI: Length of time elapsed until serum creatinine returns to measured baseline or if unavailable, presumed basline  in the absence of renal replacement therapy.Longitudinal changes in CFHb and markers of oxidative stress (plasma F2-IsoP and IsoFs) over 72 h.Longitudinal changes in markers of endothelial activation/WPB release  over 72 h.Longitudinal changes in markers of endothelial dysfunction as measured by degradation products of the endothelial glycocalyx over 72 h.Longitudinal changes in blood and urine biomarkers of AKI over 72 h, including neutrophil gelatinase-associated lipocalin and urine albumin:creatinine ratio.Fever clearance time (FCT): time taken for aural temperature to fall below 37.5 °C (FCT-A) and time taken for temperature to fall below 37.5 °C and remain there for at least 24 h (FCT-B).Parasite clearance time: Time from commencement of antimalarial treatment to the first of two consecutive negative blood films, with blood films assessed by microscopy every 6 h, and parasite half-life .Safety of paracetamol in severe and uncomplicated knowlesi malaria, as assessed by the number of patients with adverse events or severe adverse events (SAEs)See Fig. The primary endpoint will be analysed using ANCOVA with enrolment creatinine (hour 0) as a covariate. The same analysis will be performed in patients stratified by the presence or absence of significant haemolysis. Clinically significant haemolysis will be defined by using a receiver-operating curve analysis to determine the cut-off CFHb that provides maximal sensitivity/specificity for predicting AKI (KDIGO criteria). Analysis of the primary end-point will be by modified intention-to-treat, with patients’ data excluded from the primary analysis if enrolment (hour 0) or 72 h creatinine is unavailable. Patients receiving haemodialysis will be categorised as having had a creatinine rise of 132.6 μmol/L . Based oPlasmodium infection other than P. knowlesi. In addition, patient data will be excluded from the primary analysis if either 0 or 72 h creatinine measures are not available. For patients with no 72 h creatinine result excluded in the primary analysis, any data collected may still be used in secondary analyses. Patients may voluntarily withdraw from the study at any point for any reason (see below); however, data collected to the point of withdrawal may be used in the relevant analyses.Patients will be excluded if PCR confirms any t test or Wilcoxon rank-sum test, depending on distribution. Longitudinal data will be analysed using mixed-effects modelling. The parameters of primary interest will be the fixed effect interaction terms between treatment group and times, and treatment group and CFHb at enrolment, to describe whether change in secondary outcomes differs between the two groups across the observation period, and whether the treatment effect depends on enrolment CFHb, respectively. Time-to-event analyses will be used to calculate duration of AKI, fever clearance time and parasite clearance time.The secondary area under the curve endpoints will be compared by Student’s No interim analysis will be performed.The effect of paracetamol on the change in creatinine at 72 h and longitudinal change in creatinine over 72 h in patients with CKD, compared to patients without CKD.Change in creatinine at 72 h and longitudinal change in creatinine over 72 h in patients with therapeutic concentrations of paracetamol, compared to patients with absent or low concentrations of paracetamol.Population pharmacokinetics properties of paracetamol, including (1) peak plasma concentration (Cmax), (2) time to peak plasma concentration (Tmax) and (3) area under the plasma drug concentration-time curve (area under the curve).Pharmacodynamic effects of paracetamol on creatinine, fever and parasitemia.To assess the effect of intravascular haemolysis (CFHb) on AKI via specific pathophysiological pathways , a mediation analysis will be performed. Using regression modelling, the effect of CFHb on AKI mediated by each specific pathway will be assessed using the product of direct effect of CFHb on pathophysiological outcome , and direct effect of these pathophysiological outcomes on AKI.6.A description of all patients screened and reasons for non-inclusion.7.Clinical, epidemiological and laboratory features of all patients included in the study.8.The proportion of all patients lost to follow-up or withdrawn, with reasons for withdrawal.9.The number of patients with de novo CKD, hypertension and proteinuria at 1, 3 and 5 years after enrolment.Previous studies of malaria patients treated with standard dosing of paracetamol have shown no evidence of clinically significant paracetamol-related hepatotoxicity , 47, 51.All adverse and SAEs will be recorded as per standard reporting guidelines. SAEs will be reported within 1 day of awareness by the site investigator to the chair of the Safety Monitoring Committee. The Principal Investigator will report the SAE to Malaysian and Menzies ethics committee in accordance with local requirements. Any serious safety concerns identified by the Safety Monitoring Committee may result in modification or termination of the study as necessary.Each participant has the right to withdraw from the study at any time. Additionally, any patient found to have an ALT/AST of 10 times the upper limit of normal will be withdrawn from the study.The site investigator may also withdraw a participant if the treating clinician determines that further administration of paracetamol is contraindicated for any medical reason. Withdrawn patients will continue to receive standard care from the hospital clinician. The reason for withdrawal will be recorded in the CRF, and any data obtained from the participant up to the time of withdrawal from the study will be included in the analysis. Withdrawn subjects will be replaced.Clinical, biochemical and parasitological information will be collected and managed using REDCap electronic data capture tools hosted at Menzies School of Health Research, Darwin . AnalysiParticipants in the study will be reimbursed for their transport to attend all follow-up visits to the study sites. Patients will be paid MYR45 for each day of travelling. No other gifts or payments will be made.All patient information will remain confidential and be shared only by the study team. Unique identifiers are used for computer-based data entry and blood samples. In all cases, the Principal Investigator will ensure that screening forms, CRFs and the completed identification code list are kept in locked files. Subject confidentiality will be protected in any dissemination of results.This study will be the largest clinical trial to date in knowlesi malaria, and is powered to detect a difference between the two treatment arms in a clinically relevant population including adults and children with knowlesi malaria of any severity. Paracetamol is widely available and has an excellent safety profile; if a renoprotective effect is demonstrated, this trial will support the administration of regularly dosed paracetamol to all patients with knowlesi malaria. The secondary outcomes in this study will provide further insights into the pathophysiology of haemolysis-induced oxidative damage and acute kidney injury in knowlesi malaria, and other haemolytic diseases.The lack of blinding of study arm randomisation to patients and the treating team is not expected to influence the primary endpoint of change in creatinine, as this is entirely objective. The laboratory staff measuring and reporting creatinine are blinded to treatment allocation. All primary and secondary endpoints listed above are laboratory-based parameters, and therefore not able to be influenced by subjective factors.All patients will receive standard artemisinin-based therapy as per Malaysian Ministry of Health guidelines and treating clinicians are independent of the study


Accurate malaria diagnosis is critical to prevent malaria fatalities, curb overuse of antimalarial drugs, and promote appropriate management of other causes of fever. While several diagnostic tests exist, the need for a rapid and highly accurate malaria assay remains. Microscopy and rapid diagnostic tests are the main diagnostic modalities available, yet they can demonstrate poor performance and accuracy. Automated microscopy platforms have the potential to significantly improve and standardize malaria diagnosis. Based on image recognition and machine learning algorithms, these systems maintain the benefits of light microscopy and provide improvements such as quicker scanning time, greater scanning area, and increased consistency brought by automation. While these applications have been in development for over a decade, recently several commercial platforms have emerged. In this review, we discuss the most advanced computer vision malaria diagnostic technologies and investigate several of their features which are central to field use. Additionally, we discuss the technological and policy barriers to implementing these technologies in low-resource settings world-wide. Despite the availability of low-cost treatments, malaria caused 429,000 deaths in 2015 , reducing the turnaround time, and significantly improving diagnostic performance. Furthermore, automated microscopy would maintain the benefits of microscopy over RDT by providing full species determination and parasite quantitation.Over the last 15 years, numerous efforts have been made by both academic and industry groups to produce a fully integrated and highly accurate malaria diagnostic automated microscopy platform –14. A coThe standard protocol for malaria microscopy uses Giemsa stain to identify parasites in peripheral blood films. While Giemsa staining produces a very clear malaria stain, the protocol is arduous, requiring smearing the blood correctly, a short immersion in methanol, followed by a longer immersion  in Giemsa solution. The slide is then washed and dried, often requiring an hour or more to complete. Factors such as the pH of the water used and quality of Giemsa stain are critical .Many automated microscopy systems have built their platforms around Giemsa staining –18. WhilPlasmodium falciparum by assessing 22 fluorescent nucleic acid specific dyes, as well as their capability for co-stain with Giemsa. They determined Syber Green 1 to be the superior overall fluorescent stain in terms of both co-staining and intensity. The SYTO family of dyes also excelled in its capacity to stain cultured malaria positive red blood cells (RBCs). Additional fluorescent-based systems have used a dye combination to achieve accurate staining such as the Parasight system which makes use of multiple dyes in the 370 and 475 nm excitation imaging ranges . The Cellscope, developed by Partek , uses a g ranges –25.via pipette, and then automatically dispersed. While microfluidic cartridges provide an advantage by potentially creating a one-step staining process, they significantly increase design and manufacturing cost.Notably when using fluorescent staining, custom design slide carriers were also required for each of these units to accommodate the live intra-vital staining process. Carrier types include both one-step microfluidic cartridges that internally stain whole blood samples, as well multi-step units where the blood mixed with a diluent, transferred When assessing the viability of the automated microscopy malaria diagnostic systems, portability remains a significant factor. Diagnosis needs to be accessible near the patient, ideally at the point of care, as rapid identification, and treatment is critical to successful disease management , 27. SucPotential power sources for an automated microscopy system include battery packs or solar energy. The Cyscope, which is an upright microscope, uses a rechargeable battery pack. Currently, there are no automated malaria microscopes with a portable power source. However, precedence for such devices exists; several CD4+ T cell devices used in the treatment of HIV such as the BD FACSPresto and Alere Pima can be powered by battery packs that last for approximately 6 h.To reduce the power demands and add mobility to automated microscopes, several studies have focused on portable malaria diagnostic microscopes using mobile phone systems. An early system used a 20× wide field microscope with a white LED light source to illuminate brightfield images on Giemsa stain slides . Recent Studies have shown that the LOD for microscopy and RDTs range between 100 and 500 parasites/μL of blood depending on the technician and brand of RDT , 32. On P. falciparum while scanning 0.2 μL of blood.An important value proposition of computer vision systems is the potential to significantly lower the LOD. The LOD is a function of the total volume of blood scanned, since in malaria cases with low parasitemia, infected RBCs are rare. While a standard microscopist aims to scan approximately 10,000 erythrocytes at high resolution on a thin smear, automated microscopes have the potential to scan an entire blood film  with speed determined by image magnification, acquisition, and processing speeds. One of the earliest computer vision automated microscopists, the World Health Technology autoanalyzer  reportedParasight malaria detection device, showed an LOD of under 100 parasites/μL  can add further challenges which must be dealt with in distinguishing parasite and hematological features. This requires large libraries of films to be developed incorporating the full cross-section of such variation. This is dealt with in some systems using alternative staining techniques (fluorescence) or by the use of self-spreading and self-staining slides, but this has not been achieved with Romanowski-type stains (of which Giemsa is the most common). The problem of slide quality is a handicap of systems aimed at reading routine malaria blood films using classical smears, and the advantages of low-sample preparation cost and reduced workflow change are weighed against the laboratory skills and capacity required.Computer vision malaria diagnostic systems have moved from early stage research applications to mature commercial platforms that are now available in diagnostic laboratories. Initial novel microscopy applications using fluorescent dyes that did not provide a final diagnosis (such as QBC and Cyscope) succeeded in reaching the market, yet did not achieve wide acceptance, potentially since the human microscopist was still needed for verification. Moreover, they are not stand-alone systems and still require several pieces of ancillary equipment for operation. The automated microscopy computer vision systems discussed in this review (see Table The systems mentioned here are designed around either existing (giemsa smear) or novel sample preparation setups (cartridge and fluorescent stain) which produce differing target performance parameters. The stated consensus among all the systems is to achieve an LOD under 150 parasites/μL. This LOD would parallel the performance of the above average field microscopist and would offer the advantage of having a consistent automated technology that is available without issues of fatigue or retraining. Achieving detection at <5 p/μL remains challenging due to the small amount of blood analyzed by all of these platforms (<1 μL) and the presence of parasite like objects in the blood including Howell–Jolly bodies, platelets on RBCs, and staining debris. Critical to confirming the performance of these systems will be large scale multi-site studies to validate the technologies in a range of epidemiological settings.A commercial computer vision malaria diagnostic device would also benefit from additional assays to create a multiplexed platform. Computer vision systems for Tuberculosis have previously been developed –50. The Parasight and X-Rapid have already found a commercial market. Automated microscopy has the additional advantage of often providing images to clinicians, disambiguating what might otherwise feel like a black box diagnostic technique. With the case of malaria, the use of manual microscopy has retained a place despite frequent poor performance partly due to habit and tradition, but largely because it offers transparency and clinically relevant information that other techniques lack. If emerging digital systems can overcome the failings of manual systems while retaining their benefits, they should be able to grow the microscopy market.Widespread adoption of digital microscopy and computer-aided will require adequate demonstration of excellent performance, regulatory acceptability, and significant ease of use. Some devices, such as JP, AH-Y, and SS all composed the manuscript.JP, AH-Y, and SS are employees of Sight Diagnostics and declare a conflict of interest.


Placental malaria is a major cause of low birthweight, principally due to impaired fetal growth. Intervillositis, a local inflammatory response to placental malaria, is central to the pathogenesis of poor fetal growth as it impairs transplacental amino acid transport. Given the link between inflammation and autophagy, we investigated whether placental malaria-associated intervillositis increased placental autophagy as a potential mechanism in impaired fetal growth.Plasmodium falciparum infection with (n = 14) and without (n = 7) intervillositis. Western blotting and immunofluorescence staining coupled with advanced image analysis were used to quantify the expression of autophagic markers  and the density of autophagosomes (LC3-positive puncta) and lysosomes (LAMP1-positive puncta).We examined placental biopsies collected after delivery from uninfected women (n = 17) and from women with Placental malaria with intervillositis was associated with higher LC3-II:LC3-I ratio, suggesting increased autophagosome formation. We found higher density of autophagosomes and lysosomes in the syncytiotrophoblast of malaria-infected placentas with intervillositis. However, there appear to be no biologically relevant increase in LC3B/LAMP1 colocalization and expression of Rab7, a molecule involved in autophagosome/lysosome fusion, was lower in placental malaria with intervillositis, indicating a block in the later stage of autophagy. ATG4B and p62 expression showed no significant difference across histological groups suggesting normal autophagosome maturation and loading of cargo proteins into autophagosomes. The density of autophagosomes and lysosomes in the syncytiotrophoblast was negatively correlated with placental amino acid uptake.Placental malaria-associated intervillositis is associated with dysregulated autophagy that may impair transplacental amino acid transport, possibly contributing to poor fetal growth. Low birthweight (LBW), defined as a birthweight of a live infant weighing less than 2,500 grams, is a major global health issue affecting 16% of deliveries globally [Plasmodium falciparum-infected erythrocytes in the intervillous space of the placenta. This can trigger a local innate inflammatory response called intervillositis. Placental malaria with intervillositis is more strongly associated with LBW than placental malaria without intervillositis [Malaria in pregnancy is one of the leading causes of LBW responsible for up to 900,000 LBW deliveries  and overllositis .In malaria-endemic regions, up to 70% of LBW cases are due to fetal growth restriction  through The placenta is an ideal tissue to study autophagy , 10 wherP. falciparum infection on placental autophagy has not been reported.Dysregulation of the physiological autophagic process has been linked to various diseases (reviewed in ). PlacenHere, we provide unique evidence for dysregulated autophagy in placental malaria with intervillositis that is associated with reduced amino acid uptake by system A and that may contribute to LBW.P = .0001) and the placental blood parasitaemia (P = .0001) showed differences among groups.The College of Medicine Research Ethics Committee, University of Malawi, approved this study. Written informed consent was obtained from first-time mothers who delivered at the Queen Elizabeth Central Hospital, Blantyre, Malawi. We focused on first-time mothers to avoid any confounding effects from parity on susceptibility to placental malaria, placental malaria-associated intervillositis and the risk of low birthweight. Inclusion and exclusion criteria have been described elsewhere . Placentg for 15 min at 4°C. Protein concentration was determined using the Lowry assay.Placental homogenates were prepared from snap frozen placental villous tissue biopsies. Proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific) and then homogenized using Zirconia beads (Daintree Scientific) on a spiromixer for 30 sec at 4°C, followed by centrifugation at 13,000 x Placental tissue lysates were prepared in 2X Laemmli buffer. Samples containing 50 μg protein were separated on 4–12% Bis-Tris gels (Invitrogen) and transferred onto 0.45 μm polyvinylidene fluoride membrane (VWR). After blocking with 5% skim milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBS-T), membranes were incubated with primary rabbit-anti-human antibodies against autophagy markers: LC3B (Sigma), p62/SQSTM1 (Sigma), Rab7  and ATG4B . Primary antibodies were diluted to 1 μg/ml in TBS-T buffer containing 2% bovine serum albumin. To control for protein loading, the membranes were incubated with mouse anti-β-actin antibody (Sigma) at 0.2 μg/ml. Following washes with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG . Membranes were visualized by enhanced chemiluminescent substrate (Millipore). Densitometry image analysis was performed using Image J .Tissue sections (3 μm thick) of formalin-fixed, paraffin-embedded placental tissue were mounted onto poly-L-lysine-coated coverslips. Sections were deparaffinised in xylene and rehydrated. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 7.0) at 95°C for 10 minutes. The sections were then blocked with 5% newborn calf serum (Gibco) in PBS. Three primary antibodies were used: rabbit anti-human LC3B (Sigma), mouse anti-human LAMP1  and mouse anti-human CK7 (SantaCruz Biotech); a marker of the syncytiotrophoblast. Sections were first incubated with anti-LC3B and anti-LAMP1 antibodies at 10 μg/ml, and then with a secondary antibody mixture of donkey Alexa Fluor 555-conjugated anti-mouse (Molecular Probes) and donkey Alexa Fluor 647-conjugated anti rabbit (Molecular Probes) at 20 μg/ml. After washing, the sections were incubated with anti-CK7 antibodies, followed by incubation with a chicken Alexa Fluor 488-conjugated anti-mouse antibody (Molecular Probes). Sections incubated without primary antibodies were used as negative controls. Sections were mounted in Prolong Gold antifade (Invitrogen) and sealed with nail polish. Confocal microscopy was performed using a LSM 700 microscope (Zeiss) at 100x magnification. Images were captured in 3D . Alexa Fluor 488, 555 and 647 signals were acquired in separate channels sequentially.3/section; P = .085).Imaris software version 7.0 (Bitplane) was used to analyse immunofluorescence images. CK7 stained the syncytiotrophoblast, LAMP1 lysosome puncta and LC3B autophagosome puncta. One microscope field containing terminal villi was chosen as the centre of a matrix of 3x3 immediately adjacent fields automatically generated by the acquisition software. Each field was 200μm x 200μm. All terminal villi within these 9 microscope fields were analysed. The number of terminal villi enumerated per microscope field varied from 3 to 5. The CK7 signal was used to identify the syncytiotrophoblast layer and isolate LC3B and LAMP1 puncta from the syncytiotrophoblast. The number of LC3B and LAMP1 puncta in the syncytiotrophoblast was quantified in tissue sections from five randomly chosen samples from each histological group. Colocalization of LC3B and LAMP1 puncta was expressed as colocalized LC3B/LAMP1 volume, which is the volume of pixels in the syncytiotrophoblast positive for both LC3B and LAMP1. All results were normalized to the volume of the syncytiotrophoblast measured as the volume of CK7 signal. The volume of syncytiotrophoblast analysed was similar across all histological groups  was used as a well-characterized amino acid analogue substrate to measure the activity of system A amino acid transporter [14C-MeAIB uptake by MVM vesicles was measured as described previously [+-dependent uptake of 14C-MeAIB into MVM vesicles at 30 s was taken to provide an estimate of initial rate [nsporter . Microvinsporter  from theeviously . Na+-depial rate , 24.Data are presented as medians and interquartile range. Statistical analyses were performed using Stata 12 software . Given the non-normal distribution of the data (established using the Shapiro-Wilk test), two-group comparisons were made using the Mann-Whitney test and three-group comparisons using the Kruskal-Wallis test. Correlations were assessed using Spearman’s correlation test. Multivariate linear regression was conducted to test if parasitaemia and the severity of intervillositis (measured as the percentage of monocytes in the intervillous blood space) both treated as continuous variables predicted the levels of the various markers measured.Predictive models were developed using log-transformed data  and linear regression models. Model fit was assessed using R-squared statistic, checking for linearity and visualising residuals. Repeated measures from the same individual were accounted for by relaxing the assumption of independence amongst measurements within the same individual by using the stata vce (cluster clustvar) command.P = .05) (P = .82). The importance of intervillositis in driving the higher LC3-II:LC3-I ratio was further evidenced by the higher (P = .04) LC3-II:LC3-I ratio in placentas with intervillositis compared to placentas without intervillositis, regardless of their infection status (P = .04) between LC3-II:LC3-I ratio and the severity of intervillositis measured as the percentage of monocytes in the intervillous blood space. Using multivariate linear regression analysis, we found that the percentage of monocytes in the intervillous spaces and parasitaemia explain a significant amount of the variance in LC3-II:LC3-I ratio  = 25.62; P < .0001; R2 = .63). The percentage of monocytes in the intervillous spaces significantly predicted LC3-II:LC3-I ratio  = 6.56; P < .0001) but parasitaemia did not  = 0.66; P = .52). These results suggest that the inflammatory response induced by placental malaria is associated with a higher number of autophagosomes.The ratio of LC3-II to LC3-I protein levels in placental homogenates was used as the readout for the number of autophagosomes present . The LC3P = .05) . There wn status  as well P = .003) between the density of LC3B puncta measured by immunofluorescence and the LC3-II:LC3-I ratio measured by western blot.We investigated if the higher LC3-II:LC3-I ratio observed in placental malaria with intervillositis was associated with a higher density of autophagosomes (as well as lysosomes/late endosomes). Using immunofluorescence and advanced 3D image analysis, we quantified the density of LC3B puncta (identifying autophagosomes) and LAMP1 puncta (identifying lysosomes) in the syncytiotrophoblast . Our imaP = .05), or placental malaria without intervillositis (P = .02). The density of LAMP1 puncta (P = .01), or than in uninfected controls (P = .09). There was no significant difference in the density of LC3 puncta, or LAMP1 puncta, between uninfected controls and placental malaria without intervillositis (P ≥ .10). The higher parasitaemia in the group with placental malaria with intervillositis compared to the group with placental malaria without intervillositis did not confound these differences as parasitaemia correlated neither with the density of LC3B puncta , nor with that of LAMP1 puncta . When the samples were categorised based on the presence of intervillositis regardless of infection status, placentas with intervillositis showed higher densities of both LC3B (P = .01) and LAMP1 (P = .01) puncta compared to placentas without intervillositis  and LAMP1 puncta  = 2.7; P = .043; R2 = .15). The percentage of monocytes in the intervillous spaces significantly predicted the density of LC3B puncta  = 2.35; P = .026) and LAMP1 puncta  = 2.24; P = .033) but parasitaemia did not  = 0.07; P = .95; LAMP1 puncta: ß = -0.0006; t(32) = -0.13; P = .9). These findings suggest higher density of autophagosomes and lysosomes in the syncytiotrophoblast of women with placental malaria with intervillositis.The density of LC3B puncta  was high1 puncta  was alsollositis . Using mP = .0006), or placental malaria without intervillositis (P = .0004) (P = .82). The higher parasitaemia in placental malaria with intervillositis compared to placental malaria without intervillositis did not confound this difference as parasitaemia did not correlate with the LC3B/LAMP1 colocalized volume . When the samples were categorised based on the presence of intervillositis regardless of infection status, placentas with intervillositis showed higher LC3B/LAMP1 colocalized volume compared to placentas without intervillositis (P = .0001) (P = .009), but not with intervillous blood parasitaemia . Using multivariate linear regression analysis, we found that the percentage of monocytes in the intervillous spaces and parasitaemia explain a significant amount of the variance in the LC3B/LAMP1 colocalized volume  = 3.12; P = .046; R2 = .15). The percentage of monocytes in the intervillous spaces significantly predicted the LC3B/LAMP1 colocalized volume  = 2.45; P = .019) but parasitaemia did not  = -0.37; P = .71). These findings could suggest that the inflammatory response to placental malaria promotes autophagolysosome formation.We next investigated whether the higher density of autophagosomes and lysosomes observed in the syncytiotrophoblast of women with placental malaria with intervillositis translated into increased autophagosome/lysosome fusion. We measured LC3B/LAMP1 colocalized volume as a quantitative indicator of autophagosomes/lysosome fusion . LC3B/LA= .0004) . There w= .0001) . LC3B/LA2: .84 for LC3B and .89 for LAMP1). These models suggested that only ~15% of the higher LC3B/LAMP1 colocalized volume in placental malaria with intervillositis is attributable to increased autophagic process. These data suggest that a very large proportion of the higher LC3B/LAMP1 colocalized volume in placental malaria with intervillositis may not be the result of an increase in autophagic process and may therefore not be biologically relevant and alternatively might indicate a block in autophagosome/lysosome fusion.However, the higher LC3B/LAMP1 colocalized volume in placental malaria with intervillositis could also be due, at least in part, to the higher density of LC3B and LAMP1 puncta in these placentas . PredictThe increased density of autophagosomes and lysosomes and the apparent absence of biologically relevant increase in autophagolysosome formation were indicative of a block in autophagosome/lysosome fusion in placental malaria with intervillositis. In order to test this hypothesis, we investigated the expression of Rab7 , a smallP = .03) (P = .45). Placentas with intervillositis had lower Rab7 levels compared to placentas without intervillositis (P = .05) regardless of their infection status (P = .04). Using multivariate linear regression analysis, we found that the percentage of monocytes in the intervillous spaces and parasitaemia explain a significant amount of the variance in Rab7 levels  = 7.1; P = .003; R2 = .32). The percentage of monocytes in the intervillous spaces significantly predicted Rab7 levels  = -2.63; P = .013) but parasitaemia did not  = -0.65; P = .52).Rab7 expression was lower in placental malaria with intervillositis than in uninfected placentas (P = .03) . The hign status . FurtherP = .0001). This association was totally reversed to a strong negative correlation in infected placentas , which was found in placentas with intervillositis , but not in placentas without intervillositis . These findings further support our model that the inflammatory response to placental malaria is associated with a block in autophagosome/lysosome fusion.In uninfected placentas, Rab7 expression and the density of LAMP1 puncta were strongly positively correlated , suggesting normal autophagosome maturation. SQSTM1/p62 mediates the incorporation of ubiquitinated protein aggregates into autophagosomes that are then cleared during maturation into autophagolysosomes [P = .15) suggesting that the loading of cargo into autophagosomes occurs normally in all histological groups   and largP = .001) and the densities of LC3 puncta  and LAMP1 puncta . In uninfected placentas (n = 5), fetal birthweight correlated negatively with the densities of LC3 puncta  and LAMP1 puncta . These correlations were not found in infected placentas .To determine whether the block in autophagosome/lysosome fusion we identified in the syncytiotrophoblast of malaria-infected placentas with intervillositis was associated with decreased amino acid uptake, we assessed the correlation between autophagy and System A activity. System A activity correlated negatively with the LC3-II:LC3-I ratio (n = 38; R = -.51; Collectively, these findings provide strong evidence that the inflammatory response to placental malaria is associated with dysregulation in the late stages of the autophagy pathway and that this could impair placental transplacental amino acid transport, contributing to poor fetal growth.We provide the first evidence for dysfunctional autophagy in placental malaria, which appears to be largely driven by the inflammatory response to placental malaria. The locus of autophagic dysfunction was evidenced as a block in autophagosome/lysosome fusion, which could impair amino acid uptake and contribute to poor fetal growth.Our study uniquely reports the impact of a defined maternal infection on the autophagic process in the placenta. Placental autophagy has been studied in non-infectious pregnancy complications such as inflammation-induced preterm labour , idiopatAs reported for other non-infectious pregnancy complications , 17–19, Our results indicate that autophagosomes and lysosomes accumulate in the syncytiotrophoblast of infected placentas with intervillositis due to a block in autophagosome/lysosome fusion. This fusion process is highly regulated  and requOur findings that dysregulated autophagy appears to be specifically associated with the inflammatory response to malaria rather than infection itself is in keeping with the well-established bi-directional link between autophagy and inflammation . Pro-infOur findings open up new avenues of research into therapeutics or strategies to optimise fetal growth in malaria-exposed women by improving the placental autophagic process, which could restore fetal amino acid supply. For maximised efficacy, such approaches would be used in combination with established anti-malarial therapies and preventative interventions. Interventions to stimulate autophagy have been trialled in various diseases  and nanoIn summary, about 85 million women are at risk of malaria in pregnancy, resulting in ~900,000 LBW deliveries every year . Despite


Plasmodium vivax, P. malariae and P. falciparum single and mixed infections, as well as the significant socio-spatial groupings relating to the appearance of such infections. An active search in 57 localities resulted in 2,106 symptomatic patients being enrolled. Parasitaemia levels were assessed by optical microscopy, and parasites were detected by PCR. The association between mixed infections (in 43.2% of the population) and socio-spatial factors was modelled using logistic regression and multiple correspondence analyses. P. vivax occurred most frequently (71.0%), followed by P. malariae (43.2%), in all localities. The results suggest that a parasite density-dependent regulation model  was appropriate for modelling the frequency of mixed species infections in this population. This study highlights the under-reporting of Plasmodium spp. mixed infections in the malaria-endemic area of the Colombian Amazon region and the association between causative and environmental factors in such areas.Malaria outbreaks have been reported in recent years in the Colombian Amazon region, malaria has been re-emerging in areas where it was previously controlled. Information from malaria transmission networks and knowledge about the population characteristics influencing the dispersal of parasite species is limited. This study aimed to determine the distribution patterns of  Plasmodium spp. infection becomes perpetuated in a cycle of disease and poverty, contributing towards affected individuals’ worsening quality of life and limiting the possibility of eradicating such infections2.Malaria is considered as the parasitic disease that has the greatest impact on public healthAnopheles, with mammals being the definitive host3. Six species from the genus Plasmodium have been described as causing malaria in human beings: P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri and P. knowlesi5, with Plasmodium spp. being endemic in 91 countries and causing 212 million cases of infection per year 6.Malaria is transmitted by female mosquitoes from the genus 8. Colombia thus accounts for 10% of cases of malaria in the Americas11, with Colombia’s Amazon region being the focus of an outbreak of malaria during the last few years12.Current mitigation measures in disease-endemic countries have not had the desired impact since an increase in malaria cases has been reported for countries such as Colombia, where 55,866 cases were confirmed in 2015 2) is a major transmission and disease load foci14, which operates relatively independently from other Colombian regions. The Amazon region’s habitat diversity and its own climatic characteristics  determine vector presence and abundance . Such vectors are anthropophilic and highly efficient regarding parasite transmission and have facilitated the increase in cases of malaria amongst the region’s inhabitants, together with the demographics of human settlements, and clinical and housing conditions in the region and their related dynamics15.The Amazon basin covering a large part of southern Colombia ; however, these mixed-species are currently being under-diagnosed given the use of conventional techniques10. Little is currently known regarding the biology and establishment of Plasmodium mixed infections, but insight into the frequency of mixed-species infections in the population and the factors affecting their transmission is essential for developing effective disease elimination measures21. The factors involved in malaria transmission and those influencing mixed Plasmodium spp. species infection in highly endemic regions need to be determined, particularly at a time when rapid climatic changes can modify host-vector-pathogen relationship dynamics.Risk factors for acquiring malaria have been described on different levels  and influence exposure to parasitic infection, its course and outcomesPlasmodium spp. within the population, determine the distribution of mixed infections and identify infected patient profiles in the Colombian Amazon region.This study aimed to establish the frequency of three β-globin gene amplification; 1,995 subjects thus became the object of statistical analysis. The sampling region was divided into areas in accordance with the population characteristics .Of the 2,106 patients invited to participate in the study, 5.3% (n = 111) were excluded due to negative results with human ics Fig. . The avePlasmodium spp., 31.3%  for P. vivax, 6.4%  for P. falciparum and less than 1%  had mixed-species infections  than in P. falciparum .By analysing thick blood smears (TBS), 37%  of the population were identified as positive for Plasmodium spp., with P. vivax being the most prevalent species , followed by P. malariae  and P. falciparum . Mixed infection events (simultaneous infection by ≥2 species) were found in 43.2%  of the target population   of the target population were infected with P. vivax and P. malariae   Table .Plasmodium spp. parasitaemia and the distribution with respect to the area sampled. Sampling area 1 had the highest single infection frequency (48.3%) ; mixed infections appeared most frequently in area 2  , the mean rate of st) Fig. . Mean past) Fig. . Howeverst) Fig. .Figure 3Plasmodium spp. distribution with respect to area showed that P. vivax had the greatest frequency (greater than 65%) in almost all localities, except for P. vivax in Punta Brava and Yaguas    Fig. .P. falciparum infection was mostly restricted to rural settlements  was higher for rural areas (index: 57.8). Plasmodium spp. infections, whereas severe headache was most frequently reported in the population with mixed-species infections   and a rash being the least frequently reported symptom in the sample population (2.1% to 3.2%)  and diarrhoea  occurred most frequently in the study population with single Logistic regression was used to identify the association between the variables evaluated , nearby water stagnations, use of mosquito nets and use of insecticides) and the presence of mixed-species infection. Patients having 2,000 to 4,999 parasites/µL blood parasitaemia  or 5,000 to 9,999 parasites/µL blood parasitaemia  had a lower probability of acquiring a mixed infection. No significant associations were observed for the other variables included in the model , access to a public water supply and mixed P. malariae and P. falciparum infections  and triple infections  . Variables showing less significant associations were parasitaemia  in P. malariae and P. falciparum infections  and triple infection events with parasitaemia higher than 2,000 parasites/µL blood and area 1 , and the combination of parasite species revealed positive associations between area 1 and mixed Plasmodium spp. infection profiles by compiling clinical and sociodemographic variables  and those related to double infection by P. vivax and P. malariae)  was used for identifying s Tables  and 4. TP. falciparum infection  and certain symptoms . Residing in Puerto Nariño, area 4, having no stagnant water nearby, having a history of malaria and displaying mild symptoms (i.e. mild headache without shivering) correlated with on Table .P. falciparum, P. vivax and P. malariae infection, and the variables covered age, living conditions, medical history and symptoms  (Table The second profile related to triple r) Table .P. vivax and P. malariae infection) highlighted more severe symptomatology, together with parasitaemia. The variables included living in Puerto Nariño, area 4, fever, shivering, vomiting, diarrhoea, moderate headache, normal-coloured urine and >9,999 or between 2,000–4,999 parasitaemia . Their type of housing is palafitic , often but not always having palm-leaf roofs and wooden walls, thereby exposing their inhabitants to the environment and the vector’s ecosystem, thus increasing their probability of acquiring parasitic infectionsP. malariae and mixed-species malaria infections  were associated with localities in areas 1 and 4 and Puerto Nariño   is downregulated on coinfection with the second species . However, when the most prevalent species exceeds a threshold, the hosts’ immune response is triggered to limit the infection; such a mechanism is turned off once the parasite density is under control, thereby favouring population growth of the second species in mixed infections and persistence of the parasites in the host49.The coexistence of more than one parasite species in the same individual may be mediated by host and pathogen factors, such as the host immune response initially directed against the species or genotype at the highest density, thereby favouring the persistence of infection at lower density in a particular hostPlasmodium species. Fever was the symptom detected at the greatest frequency with all parasite species  and mixed ons Fig. . This pr48. Identifying whether a specific area has high disease transmission enables appropriate management strategies to be designed to effectively limit the parasite’s transmission cycle48.In-depth analysis is required for defining infection hotspots. Time series analysis should be used for parasite detection to establish whether infection events are due to transient infection or transmission foci, and risk maps and the population distribution (for host and vector) should be analysed to determine the localities of disease cases52. Although endemic countries have introduced disease mitigation measures, they have not had the desired impact as the number of malaria cases has increased, particularly in rural areas7.Control measures implemented in Colombia have focused on reducing the disease burden by the large-scale provision of insecticide-treated mosquito nets, periodic intra-household spraying and the presence of government agencies responsible for control, diagnosis and treatment22.The present study actively searched for symptomatic patients in geographically isolated localities lacking nearby healthcare posts. The average family income is less than $250 per month in these areas, so a trip to a health centre represents a considerable family expense (around $50 per trip), so many parasitic infections are not seen or treated by healthcare control programmesGreater malaria control efforts are required for progression towards the elimination of this disease; thus, understanding the distribution patterns of particular parasite species and the factors that influence malaria transmission in the Colombian Amazon region is crucial. The results of this study provide additional insight into malarial infections in the Colombian Amazon region, helping define the areas to be prioritised in terms of malaria prevention and control measures, with the aim of decreasing malarial incidence and approaching the long-term goal of eradication.54. The Amazon department has 77,088 inhabitants (population density: 1.5 inhabitants per km2)12. The town of Leticia and its surrounding communities had a projected population of 41,639 inhabitants according to the Departamento Administrativo Nacional de Estadística  2016 figures; Puerto Nariño and its neighbouring communities accounted for 8,279 inhabitants12.This transversal study was carried out from July 2015 to April 2016; it included the population of the Colombian Amazon trapezium, inhabitants from the towns of Leticia and Puerto Nariño and rural settlements located along the banks of the Amazon and Loretuyacu rivers. The Colombian Amazon region represents 42% of Colombia’s territory and is formed by the Caquetá, Putumayo, Vaupés, Guainía, Guaviare and Amazon departments, the latter comprising the greatest geographical areaFifty-seven localities were sampled and grouped into four areas, taking into account their location and mobilisation towards basins converging on major tributaries (the Amazon and Loretuyacu rivers) Fig. . Area 1 Inclusion criteria consisted of recognising symptoms related to malarial infection when taking samples, such as headache, fever during the previous 8 days and sweating. People without malaria symptoms were not included in the study (exclusion criterion). The aim of the study was explained to patients; those who accepted an invitation to participate signed an informed consent form. A survey was then conducted that compiled information regarding participants’ socio-demographic characteristics and risk factors for malaria infection. This study was approved and supervised by the Universidad del Rosario’s (Colombia) School of Medicine and Health Sciences (EMCS) Research Ethics Committee (Comité de Ética en Investigacion - CEI) (CEI-ABN026-000161). Patients under 18 years of age who accepted the invitation to participate signed an informed consent, along with their tutors’ written approval. All methods and experiments were performed in accordance with the approved guidelines.Two blood samples were collected simultaneously by capillary puncture. The first (TBS) was subjected to parasitological diagnosis by optical microscopy following Giemsa staining; the samples were processed and read on site at the time of sample collection. The second sample was stored on Flinders Technology Associates (FTA) cards and transported to the molecular biology laboratory of the FIDIC for molecular identification of the infecting parasite.β-globin constitutive gene segment10.A Pure Link Genomic DNA mini kit (Invitrogen) was used for extracting the DNA from the FTA cards, following the manufacturer’s instructions. This was followed by PCR amplification of the extracted DNA to confirm the presence of the human Plasmodium species  were identified in the β-globin-positive samples by nested PCR. Specific primers against the 18 S rRNA fragment were used in the first round of PCR for genus detection and a second amplification (using the first PCR product as template) was performed to distinguish the P. falciparum, P. vivax and P. malariae species. The amplification conditions for these PCRs have been described previously by our group10.The infecting 55. The parasite density index (PDI) was taken as the amount of confirmed cases of malaria/population at risk56. Mixed infections were defined as the simultaneous detection of two or more Plasmodium spp. Fisher’s exact or Chi2 tests were used for establishing statistically significant differences amongst the data. ANOVA was used for comparing means and Bonferroni test was used for adjusting for multiple comparisons. A t-test was used for comparing the mean values for parasitaemia with the parasite infection status (single and/or mixed infection).Descriptive statistics were used to summarise the sociodemographic variables, such as the sample-taking area, access to basic services  and risk factors ; these were presented as percentages with their respective 95% confidence intervals (95% CI). Age and parasitaemia  were reported, along with their respective means and standard deviations (SD)Logistic regression analysis was used for modelling the risk of a mixed infection, taking mixed infections as a dependent variable. Independent variables included in the model were age, residing in an urban or rural area, parasitaemia reported by TBS and housing conditions such as sewerage, gas and electricity supply, nearby stagnant water and mosquito net and insecticide use. STATA 12 software was used for analysing the data.59. In this way, groups were identified that had clinical significance from different groupings of categories of variables (i.e. this method was used to identify how sociodemographic characteristics and risk factors were grouped with single or multiple infections).Multiple correspondence analysis (MCA) was used for establishing patient profiles, taking into account the nature of the clinical, epidemiological and laboratory variables  estimated in this study. MCA was used for evaluating the degree to which each clinical and epidemiological variable participated in the compiling of profiles or groups of clinical significance in terms of similarity with or proximity to the different categories of variables, thus facilitating the incorporation of laboratory variables  into these profiles’ for observing patterns58.Two groups of variables were chosen for this analysis: active variables used for constructing factorial axes and supplementary or illustrative variables, which enriched factorial axes interpretation once they had been constructed57.Sociodemographic, epidemiological and clinical variables were considered active variables, i.e. age, gender, origin, area, mosquito net and insecticide use, nearby stagnant water, fever, headache, vomiting, shivering, diarrhoea, urine colour, abdominal pain, outbreaks on the skin and previous episodes of malaria. The contribution values for each category were analysed for interpreting the axes compiled by the active variables, and the categories with a contribution value of more than 2.5 [mean contribution of 40 categories (100/40 = 2.5%)] were selectedP. vivax, P. falciparum and P. malariae infection and parasitaemia. Cosine values were evaluated for estimating the quality of each active variable’s representation on each axis. The test values were used to determine whether the representation of each category on each axis significantly differed from 0 (≤−2 or ≥ 2 cut-off points), thus giving an evaluation of each category’s significance57.Illustrative variables were the presence/absence of The structure and formation of each profile were analysed using a bi-dimensional graphical representation. The active variables  were represented on each axis by filled boxes and the nominal illustrative variables (infection by each of the three species and parasitaemia) were represented by empty rhombuses. The test values sign indicated each modality’s position on the positive or negative pole of each axis. Square size was proportional to each modality’s contribution on the most representative axis. Possible dependence and similarity relationships were identified, taking into account the distance between the variables represented on the graph, regarding the categories thus represented. SPAD-5 software was used for MCA.Supplementary information


Community health workers (CHWs) are recommended to screen for acute malnutrition in the community by assessing mid-upper arm circumference (MUAC) on children between 6 and 59 months of age. MUAC is a simple screening tool that has been shown to be a better predictor of mortality in acutely malnourished children than other practicable anthropometric indicators. This study compared, under program conditions, mothers and CHWs in screening for severe acute malnutrition (SAM) by color-banded MUAC tapes.This pragmatic interventional, non-randomized efficacy study took place in two health zones of Niger’s Mirriah District from May 2013 to April 2014. Mothers in Dogo (Mothers Zone) and CHWs in Takieta (CHWs Zone) were trained to screen for malnutrition by MUAC color-coded class and check for edema. Exhaustive coverage surveys were conducted quarterly, and relevant data collected routinely in the health and nutrition program. An efficacy and cost analysis of each screening strategy was performed.p = 0.9484). In the Mothers Zone, there was a higher rate of MUAC agreement  and earlier detection of cases, with median MUAC at admission for those enrolled by MUAC <115 mm estimated to be 1.6 mm higher using a smoothed bootstrap procedure. Children in the Mothers Zone were much less likely to require inpatient care, both at admission and during treatment, with the most pronounced difference at admission for those enrolled by MUAC < 115 mm . Training mothers required higher up-front costs, but overall costs for the year were much lower A total of 12,893 mothers and caretakers were trained in the Mothers Zone and 36 CHWs in the CHWs Zone, and point coverage was similar in both zones at the end of the study .The trial is registered with clinicaltrials.gov (Trial number  For the past 20 years, the diagnosis and treatment of severe acute malnutrition (SAM) has become increasingly decentralized, from a strictly hospital-based approach for all cases to the current model of outpatient care for children with uncomplicated SAM and inpatient care for children presenting with complications or failing to respond to treatment.  Even thoSince the 1980s, it has been shown that mid-upper arm circumference (MUAC) can be performed by minimally trained personnel  leading Mothers are in the best position to detect signs of nutritional deterioration in their own children, and training mothers to regularly screen by MUAC and check for edema is a logical next step in this process of decentralization. This could lead to improved coverage, as well as earlier detection and treatment seeking that improves program outcomes and reduces cost per case treated. A recent study comparing CHWs and mothers performing MUAC by color-coded class rather than measurement showed the promise of such an approach . MothersFood insecurity and infections leading to childhood malnutrition is a seasonal and recurrent crisis for families in Niger, particularly in the rural areas around Maradi and Zinder where rates of global acute malnutrition , and stunting are consistently near or above emergency thresholds . In 2014The objective of this study was to evaluate the efficacy  and costs of two community-based strategies to screen children aged between 6 and 59 months for SAM by MUAC class. Mothers and caretakers (henceforth referred to as mothers) were compared to CHWs in separate health zones in Niger’s Mirriah District. It was hypothesized that training mothers to perform MUAC would lead to earlier SAM detection and attendance and lower rates of hospitalization, thus relieving pressure on a resource-poor health system by reducing the need for inpatient care.This pragmatic interventional, non-randomized efficacy study was conducted according to the guidelines laid down in the Declaration of Helsinki. The study protocol was given ethical approval by the National Consultative Ethics Committee of Niger’s Ministry of Public Health on April 10, 2013 (Deliberation Number 006/2013/CCNE.) Written informed consent was obtained from all mothers or heads of households. The trial is registered with clinicaltrials.gov .The trial was performed from May 2013 to April 2014 in Mirriah, a rural district in Niger’s southeast Zinder region bordering Nigeria. Dogo (Mothers Zone) served as the intervention zone and Takieta (CHWs Zone) served as the comparison zone where screening by color-coded MUAC class  and Bien Etre de la Femme et de l’Enfant au Niger (BEFEN) ran health and malnutrition treatment programs were examined in order to identify two areas with similar demographics, geography, SAM burden, health care provision, access barriers, and program performance. Takieta and Dogo showed the most similarities, especially for SAM prevalence at the beginning of the study and the percentage of children under 5 years of age treated for SAM over a period of 9 months prior to the study.  over the course of ten days, with the final days reserved for return visits to train mothers who were not covered during the initial pass. In order to generate awareness and mobilize communities, teams visited each village the week before trainings to explain the study to village leaders. Villages received a reminder the day before to maximize chances that mothers would be available. Women with children aged between 3 and 59 months as well as caretakers  were eligible, and on average, there were 120 mothers/caretakers per village. Training consisted of group sessions (up to 30 mothers/caretakers per session) followed by short home-based individual trainings that included obtaining written consent and distribution of MUAC tapes. An evaluation grid was used, and mothers were retrained if necessary.In the CHWs Zone, a one-day training session was conducted at the health center in Takieta. Thirty six CHWs were recruited or retained to cover 82 villages , making sure there was a proper geographic distribution of approximately one CHW per 250 children aged under five years. CHWs raised awareness about the signs of malnutrition during screenings, and gave referral slips to the mothers of children indicating why the child was referred (including for having a MUAC <115 mm). Mothers did not need a CHW referral but could go directly to the health center if they detected signs of malnutrition or were worried about their child’s health. A monthly incentive of $44 USD was provided to each CHW.Figure Exhaustive, door-to-door MUAC screenings were organized at baseline (May 2013) and three times thereafter  in all villages of both zones. This was used to determine program coverage and as a safeguard in the event that screening by mothers was not effective. Twelve teams of two investigators each were managed by four supervisors (i.e. 3 teams per supervisor) and performed MUAC screenings on all children identified as being between 6 and 59 months of age and ≥ 67 cm in length. Like many countries in West and Central Africa, Niger’s protocol includes this length restriction for MUAC eligibility. Children not already receiving treatment but identified as severely malnourished (MUAC <115 mm and/or edema) or moderately malnourished (115 mm ≤ MUAC <125 mm) were referred for SAM or moderate acute malnutrition (MAM) treatment, respectively. Mothers of children with MUAC < 115 mm and not in treatment were interviewed to identify their reasons for non-attendance.Coverage was assessed using the Semi-Quantitative Evaluation of Access and Coverage (SQUEAC) method , 16. Onlp < 0.05. Cost data was entered in the accounting software SAGE between June 1 and December 31, 2013 and used retrospectively in order to estimate the cost of implementing and monitoring each screening strategy. Supervision fees and premiums were projected for 5 additional months  in order to cover the period of the study.Data from the nutrition rehabilitation program were collected weekly from sites in both health zones. MUAC in mm at admission was collected and used to determine the distribution of MUAC at admission for children admitted by MUAC < 115 mm. Hospital referrals for medical complications at admissions and during the treatment episode were used to determine the proportion of admissions requiring hospitalization. Common descriptive statistics techniques were used for data analysis . Proportions, as well as risk ratios (RR) and/or risk difference (RD) with their associated 95 % confidence intervals (CI), were presented for each group. To measure the association between each strategy and the variable of interest, two-tailed p-value for Yates corrected chi-square test or two-tailed p-value for Fisher exact test (when indicated) were used. Differences were considered statistically significant at a A total of 12,893 mothers and caretakers were trained to screen children by MUAC color-coded class and check for edema in the Mothers Zone during the initial training sessions and subsequent coverage surveys. (Table p = 0.4471). Table p = 0.007). Consistent with earlier detection and treatment seeking, children admitted in the Mothers Zone were less likely to require inpatient care than children in the CHWs Zone, both at admission and at any point in their treatment episode, with the most pronounced difference at admission for those enrolled by MUAC < 115 mm .  .  Table  While inLate presentation of SAM (e.g. children well below the admission threshold) carries a greater risk of death, and is commonly associated with medical complications that require more costly hospital-based care.  This stuThere is growing understanding that MUAC can be used safely and effectively as the sole anthropometric criterion for admission, management, and discharge from malnutrition treatment –21. A reCoverage surveys in multiple contexts have identified major reasons why mothers do not utilize available SAM treatment, including a lack of awareness about malnutrition  and existing CMAM programs, as well as previous rejection , 24. BarThere were several limitations and possible biases to this study. The study only lasted eleven months and it takes time for members of a community to develop new health-seeking behaviors, so community mobilization is likely to improve over time through periodic trainings and MUAC tape renewals. Although it is likely that screening by mothers contributed to the observed difference in proportion of hospitalized cases in the two zones, this is not certain as hospital referrals depend on many factors (e.g. clinicians’ level of training and/or experience). Even though the two zones had similar demographics and prevalence of malnutrition at baseline, a smaller proportion of children receiving SAM treatment in the Mothers Zone required inpatient care in a nine month period prior to the study. The hospital in Mirriah is also further away from the CHWs Zone than the Mothers Zone, but the potential for bias was reduced by the fact that transport for referrals from health centers was provided by ALIMA/BEFEN ambulances in both zones. One would expect program lengths of stay to be shorter in the Mothers Zone, but comparison was not possible because the therapeutic supplementary feeding programs (i.e. programs treating MAM) in the zones were operating at different levels of capacity, causing children to be retained in the therapeutic feeding program longer in the Mothers Zone than in the CHWs Zone. The difference in availability and quality of MAM services in the two zones is another limitation to the overall analysis because it is difficult to determine the impact or influence of MAM programming on the profile of SAM cases while the presence of a well-functioning MAM program may have influenced discharge decisions.Screening by MUAC class itself also introduces error in favor of sensitivity since it does not take much to misclassify children near the 115 mm cut-off due to pulling too tightly. This can be reduced by using increased-width tapes. There were, however, few gross errors when mothers or CHWs measured MUAC, and most misclassifications in the both zones occurred either at the boundary between SAM and MAM or children screened as SAM determined to be MAM by health center agents, in line with a previous study [The length threshold for MUAC eligibility that is part of many protocols in West and Central Africa (and which changed in Niger from 65 cm to 67 cm during the study) introduced an additional bias. It systematically excludes young, stunted children who are at considerable risk of death if left untreated, which is especially problematic in an area of high stunting and in light of previous work showing such children respond well to treatment , 27. TheSome areas were identified for improvement or further exploration. Because resource-poor mothers in a rural community were shown to better identify malnutrition in their infants through pictorial rather than verbal descriptions, integrating more pictorial descriptions into the trainings would be beneficial . Future While mothers and CHWs were trained to screen for edematous malnutrition, prevalence is low in Niger and the small number of referrals for edema in either zone means that the analysis here lacks statistical power. Nevertheless, more children were referred for edema and agreement was higher in the Mothers Zone, suggesting that mothers are in a better position to detect potential cases of a deadly condition with rapid onset and resolution in either death or spontaneous recovery. In the Mothers Zone, however, fewer children were admitted to the treatment program for edema than those who were determined to be in agreement with an edema referral. Because most of the children admitted for edematous malnutrition in both zones were classified as having mild edema , this dCHWs provide valuable contributions to improved health outcomes in a community, and are being relied upon to do more and more, from diagnosis to delivery in areas as varied as malaria, malnutrition, vaccination, or childbirth assistance . These aEven with the study’s limitations, preliminary analysis of the benefits and few risks associated with the household-level screening strategy led ALIMA to train tens of thousands of additional mothers elsewhere in Niger as well as in Burkina Faso, Mali and Chad. Thus from an operational perspective integrating household-level screening into pre-existing programs was relatively straightforward.Involving mothers in screening their own children by MUAC and checking for edema is a key step in increasing access to care for children in any area where malnutrition poses a high risk of death or illness and can reduce cost per child treated. Making mothers the focal point of MUAC screening strategies should be included in regular CMAM programming. As mothers are taught how to screen their own children in countries where malnutrition is highly prevalent, important information will be gained by practitioners about how to most effectively extend the strategy.MUAC, mid-upper arm circumference; CHW, community health worker; CMAM, Community Management of Acute Malnutrition; GAM, global acute malnutrition; SAM severe acute malnutrition; MAM, moderate acute malnutrition; WHZ, weight for height Z-score; RUTF ready-to-use therapeutic food; WHO World Health Organization


The aim of the study was to analyse the trend of imported malaria cases in Parma, Italy, during January 2013–June 2017, reporting also the treatment and the outcome of cases, exploring the comparison of the three diagnostic tests used for malaria diagnosis: microscopy, immunochromatographic assay (ICT) (BinaxNOWP. falciparum, 2 P. vivax, 8 P. ovale, 1 P. vivax/P. ovale, 1 P. malariae and 2 Plasmodium sp. All samples were positive by ICT except 6. Plasmodial DNA was revealed in the 87 cases and in 2 additional cases showing P. falciparum-specific bands by ICT, as follows: 75 P. falciparum, 2 P. vivax, 6 P. ovale curtisi, 3 P. ovale wallikeri, 1 P. malariae, and 2 mixed infections. 72 patients were foreigners and 17 Italians travelling for tourism or business. The majority of these patients presented with fever at blood collection and did not have chemoprophylaxis. No fatal cases were observed and the drug mostly used was quinine observing a negative blood smear or a parasitaemia < 0.001% after 48–72 h’ therapy.Of the 288 patients with suspected malaria, 87 were positive by microscopy: 73 P. falciparum malaria missed by microscopy and correctly identifying the Plasmodium species of medical interest. The data reported in this study may stimulate the clinicians in non-endemic areas to suspect malaria also in cases, where the most typical symptoms are absent, and the parasitologists to confirm the results of microscopy, remaining the reference method, with molecular methods to avoid misdiagnosis.The study shows an update and a thorough analysis of imported malaria cases in the area of Parma during 4.5 years from the point of view of the total case management, clinical and diagnostic. The prevalence of malaria in such area in the considered period was especially due to immigrants mostly from Africa. Molecular methods were more sensitive and specific than microscopy and ICT, both detecting additional cases of  Plasmodium falciparum malaria, 1 induced Plasmodium malariae infection in a patient with a kidney transplant and 1 introduced Plasmodium vivax infection). Malaria is a mandatory notifiable disease in Italy; according to the data of the Ministry of Health [P. falciparum was the mainly detected species and the deaths were 4.Imported malaria is described as “an infection acquired in an endemic area, but diagnosed in a non-endemic country after development of the clinical disease” . The incf Health , during Plasmodium parasite from the patient’s blood in order to prevent progression of uncomplicated malaria to severe disease or death, and to prevent chronic infection that leads to malaria-related anaemia [Plasmodium sp. by microscopy, the treatment could be delayed or inappropriate [Early and accurate diagnosis and prompt treatment of clinical malaria are essential for reducing malaria morbidity and mortality, drug resistance and malaria control. The primary objective of the treatment is to ensure the rapid and complete elimination of the  anaemia . In caseropriate .Three common methods are used to diagnose malaria: microscopic examination of Giemsa-stained thick and thin blood smears, rapid diagnostic tests (RDTs), and Polymerase Chain Reaction (PCR) or other nucleic-acid based assays , 7.As reported by the World Health Organization (WHO) , microsc®, the only United States Food and Drug Administration-approved RDT for malaria, that qualitatively detects both the histidine-rich protein 2 (HRP-2), specific to P. falciparum, and aldolase, a pan-malarial antigen found in all Plasmodium species [P. falciparum infection, as well as non-falciparum species [P. vivax infections [RDTs have been developed to detect antigens derived from malaria parasites; such tests often use a dipstick or cassette format, and provide results in 2–15 min offering a useful alternative to microscopy in situations where reliable microscopic diagnosis is not immediately available . These a species . However species . Current species ; moreove species . The spe species , althougPlasmodium species, and has about 5 to 7 copies per Plasmodium genome, improving detection sensitivity [The molecular detection of malaria parasites is highly accurate and sensitive, and remains the best method for detecting multiple species and sub clinical infections , 16. Thesitivity , 20. Rea® Malaria) and Real-time PCR assays able to detect P. falciparum, P. vivax, P. malariae, Plasmodium ovale curtisi, P. ovale wallikeri and also the species Plasmodium knowlesi in subjects native or coming from Southeast Asia, endemic area for this species [The aim of this study is to analyse the trend in malaria cases at the University Hospital of Parma, Italy, a non-endemic area, during January 2013-June 2017 reporting also the treatment practices and the outcome of malaria cases, exploring the performances of the three diagnostic tests routinely used in our laboratory to diagnose malaria: microscopy, immunochromatographic assay  and by light microscope (1000× magnification), respectively. Smears were examined and were defined as negative if no parasites were observed in the whole film. Identification and count of Plasmodium sp. was done on Giemsa-stained smears: parasites concentration (parasitaemia) was calculated by counting the number of parasitized red blood cells seen in 10,000 erythrocytes on a thin-film and expressing the number as a percentage, as described for non-endemic areas [Each blood sample, containing at least 5 ml of blood drawn into sterile tubes with EDTA, arrived in the laboratory within 1 hour from the collection and immediately used to prepare thin blood films for microscopic observation; the blood smears were stained with acridine orange and with 1% Giemsa in phosphate-buffered saline (pH 7.0), according to standard procedures , 20, 24,ic areas , 20, 24.®Malaria) for the qualitative detection of the Histidine-Rich Protein II (HRPII) antigen specific to P. falciparum and a pan-malarial antigen common to P. vivax/P. ovale/P. malariae, following manufacturers’ instructions.Blood samples were also submitted to the immunochromatographic assay (ICT) , respectively. A P. falciparum positive blood sample confirmed by sequencing [Plasmodium sp. was submitted to a Taq-Man based Real-Time PCR assay specific for the human β-actin gene as previously described [An aliquot of DNA of each sample was stored at − 20 °C for further examination. Each PCR run included a negative control (a reaction mixture without DNA), and 6 positive controls consisting of plasmids containing DNA oligonucleotides with the target sequence of Plasmodium sp. were detected by microscopy: 73 P. falciparum, 2 P. vivax, 8 P. ovale, 1 P. vivax/P. ovale, 1 P. malariae and 2 Plasmodium sp. (Table For 87 (30.2%) of the 288 subjects with suspected malaria included in the study, P. falciparum positive samples by microscopy showed malaria specific antigens by ICT  , 1 P. vivax/P. ovale showed a T2 low intensity band, 1 Plasmodium sp. positive sample showed the presence of P. falciparum specific band (T1). The other 5 positive samples  by microscopy showed no bands by ICT. Two out of the 201 negative samples by microscopy showed P. falciparum specific bands and the remaining 199 negative blood samples showed no plasmodial specific bands  (Table For 89 (30.9%) of the 288 subjects with suspected malaria, the presence of plasmodial DNA was revealed by species-specific Real-time PCR  were foreigners, including 1 Romanian and 1 Greek visiting Ivory Coast and Sudan, respectively, 17 (19.1%) were Italians travelling for tourism or business in malaria endemic areas and for the remaining 8 (8.9%) the birth place was unknown but they were not European citizens; for all the patients the visiting area is reported in Table The majority of these patients presented with fever, in particular 79 (88.7%) had fever at blood collection and 10 (11.2%) were without fever Table . SeventyP. ovale malaria after 72 h of quinine therapy. The distribution of symptoms and signs among patients with malaria and subjects without malaria is detailed in Table For 85 out of the 89 patients with malaria the adopted therapy was known and it is detailed in Table Plasmodium sp. have been found: 12 systemic infections , 10 respiratory infections , 7 gastroenteritis  5 urinary infections and 4 chronic viral infections by Human Immunodeficiency Virus (HIV) or Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) viruses. In the remaining 129 subjects no infections were found.For 4 (2%) and for 28 (14.1%) out of the overall 199 negative subjects 3 and 2 samples, respectively, were examined before excluding malaria diagnosis, while for the remaining 167/199 (83.9%) subjects only 1 sample was examined. In 38 out of these 167 subjects (22.7%) infections other than Loa loa microfilariae in 1 blood sample negative for Plasmodium sp. and belonging to a patient for whom the clinical suspicion of malaria had been initially formulated.Microscopic examination revealed the presence of Malaria is no longer endemic in Italy since 1970 when the WHO officially declared Italy malaria-free where nowadays is the most commonly imported disease , 24, 25.®Malaria) and Real-time PCR assays able to detect P. falciparum/P. vivax/P. malariae/P. ovale curtisi/P. ovale wallikeri and also P. knowlesi, even if in this study such DNA was not searched for because no patients were natives or came from Southeast Asia. Most of the analysed samples arrived at the laboratory from the Emergency Unit and the diagnosis was made by the methods above described following the flow chart adopted in this laboratory previously reported [Plasmodium sp. was performed by microscopic examination (after acridine orange and Giemsa staining) within 2 h from the arrival of the sample, in order to establish the presence or absence of Plasmodium sp. and to allow the administration of a prompt therapy in case of positivity (genus identification only). The following working day the result was confirmed by a parasitologist, including species identification, stages and parasitaemia.In this study, the trend in malaria cases at the University Hospital of Parma, Italy, during January 2013–June 2017 is reported also with the treatment practices and the outcome of malaria cases; the study describe the comparison of the three diagnostic tests routinely used in our hospital to diagnose malaria cases: microscopy, ICT  both involving P. falciparum.The data reported in this study show that the malaria cases in the considered non-endemic setting have been increasing, from 20% observed in 2000 , 20 to aPlasmodium sp., while in non-African patients they were not observed. It is important to note that even if in literature splenomegaly is observed in 24% to 40% of cases of uncomplicated malaria [The symptoms of malaria are non-specific: fever, observed in this study in the 95.5% of malaria patients when they presented to the Emergency Unit was in fact present also in the 92.1% of subjects without malaria as well as headache, present in the 31.4% of malaria patients and in the 20.2% of subjects without malaria. These symptoms with malaise are commonly the first manifestations. An important consideration is that up to half of patients may not febrile when they present to the Emergency Unit and that the classic symptoms of cyclic fevers, rigors, and cold sweats are often not present , 31 as a malaria , in this malaria , 34, alsP. falciparum asexual parasitaemia and without identified alternative causes: acidosis, hypoglycaemia, severe anaemia, jaundice, and hyperparasitemia [Plasmodium species, the levels of innate and acquired immunity in the host, and the timing and efficiency of treatment, if any. WHO recommends artemisinin-based combination therapy (ACT) for the treatment of uncomplicated malaria caused by P. falciparum by combining 2 active drugs with different mechanisms of action [P. falciparum malaria. In order to prevent relapses of the hepatic hypnozoite form of P. vivax and P. ovale, primaquine is added and dose and frequency of the administration is guided by the patient’s glucose-6-phosphate dehydrogenase (G6PD) enzyme activity [P. falciparum malaria except that in a P. ovale positive patient due to lack of glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. All drugs used in the study had the same efficacy and no chloroquine resistance was observed so that all the 89 malaria patients demonstrated a favourable outcome. No cases of severe malaria were observed although, in one patient, one of the WHO severe malaria criteria  have been observed at the time of admission. All patients were discharged when a reduction of parasitaemia up to < 0.001% was observed, and this mostly occurred within the 3 days of therapy. The complete negativization after 72 h of therapy has been achieved only in 1 case and only in 14 out of the 73 P. falciparum cases gametocytes have been observed in the blood, a sign of a favorable evolution of the infection.Untreated, approximately 10% of uncomplicated imported malaria will progress to complicated or severe malaria defined as one or more of the following symptoms, occurring with asitemia . Many faasitemia , such asP. falciparum malaria and of mixed infections missed by microscopy and correctly identifying the 5 species of Plasmodium sp. of medical interest , respectively (P. knowlesi DNA was not searched for because no patients were natives or came from Southeast Asia). In fact, microscopy gave an incorrect diagnosis in the 1.12% of cases , likely due to the fact that these infections do not reach as high parasite levels as those by P. falciparum, as similarly observed in Parma from 2000 to 2012 [P. falciparum, 2 P. ovale) in which plasmodial species had not been identified by microscopy, whose results were limited to genus identification (2 cases) and to P. ovale/P. vivax in the remaining case. In such cases, the incorrect identification of species by microscopy can be explained by the low parasitaemia (< 0.001–0.26%). As regards to RDTs, in this study BinaxNOW®, contrary to what has been described in the literature [P. ovale curtisi, in 1 by P. ovale wallikeri and in 1 by P. falciparum + P. malariae. These results could be likely due to the low parasitaemia present in the samples, despite in 3 P. ovale curtisi cases the parasitaemia level was higher than that of other non-P. falciparum cases having a positive RDT result.As concerns the comparison of the diagnostic assays used for the laboratory diagnosis of malaria, as expected, and already reported in the previous years , 20, 24, to 2012 . Real-titerature , 35, is P. falciparum, followed by P. ovale, and then by P. vivax and P. malariae, showing the same picture of that observed in the period 2000–2012 [P. ovale, it is notable that 3 out of the 9 P. ovale cases were caused by the P. ovale wallikeri, species detected in the blood of 3 patients coming from Mozambique, Ivory Coast and Niger, finding that confirms the presence and circulation of this P. ovale species also in Africa, and mostly in West Africa [The plasmodial species most prevalently detected in the period included in the study was 000–2012 . Regardit Africa .Plasmodium falciparum was the most frequently observed species and 2 mixed infections, both including P. falciparum, were detected. No fatal case was observed and the drug mostly used was the quinine. As expected, molecular methods demonstrated to be more sensitive and specific than microscopy, both detecting additional cases of P. falciparum malaria missed by microscopy and correctly identifying the 5 species of Plasmodium of medical interest, respectively. Regard to RDTs, in this study BinaxNOW®, contrary to what has been described in the literature was less sensitive than microscopy and Real-time PCR.This study shows an update and a thorough analysis of imported malaria cases in the area of Parma in the past 4 and a half years from the point of view of the total case management, clinical and diagnostic. The malaria cases in Parma are increasing  are absent, and, on the other hand, the parasitologists to confirm the results of microscopy, still remaining the reference method, with molecular methods, even if they are more labour-intensive, in order to avoid misdiagnosis.