Plasmodium infections (RDTs). This study investigated whether mid-infrared (MIR) spectroscopy coupled with supervised machine learning could constitute an alternative method for rapid malaria screening, directly from dried human blood spots.Epidemiological surveys of malaria currently rely on microscopy, polymerase chain reaction assays (PCR) or rapid diagnostic test kits for −1 to 500 cm−1. The spectra were cleaned to compensate for atmospheric water vapour and CO2 interference bands and used to train different classification algorithms to distinguish between malaria-positive and malaria-negative DBS papers based on PCR test results as reference. The analysis considered 296 individuals, including 123 PCR-confirmed malaria positives and 173 negatives. Model training was done using 80% of the dataset, after which the best-fitting model was optimized by bootstrapping of 80/20 train/test-stratified splits. The trained models were evaluated by predicting Plasmodium falciparum positivity in the 20% validation set of DBS.Filter papers containing dried blood spots (DBS) were obtained from a cross-sectional malaria survey in 12 wards in southeastern Tanzania in 2018/19. The DBS were scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) spectrometer to obtain high-resolution MIR spectra in the range 4000 cmP. falciparum infections  and 85% for predicting mixed infections of P. falciparum and Plasmodium ovale  in the field-collected specimen.Logistic regression was the best-performing model. Considering PCR as reference, the models attained overall accuracies of 92% for predicting Plasmodium in both non-clinical settings  and clinical settings (diagnosis to aid case management). However, before the approach can be used, we need additional field validation in other study sites with different parasite populations, and in-depth evaluation of the biological basis of the MIR signals. Improving the classification algorithms, and model training on larger datasets could also improve specificity and sensitivity. The MIR-ML spectroscopy system is physically robust, low-cost, and requires minimum maintenance.These results demonstrate that mid-infrared spectroscopy coupled with supervised machine learning (MIR-ML) could be used to screen for malaria parasites in human DBS. The approach could have potential for rapid and high-throughput screening of  Accurate diagnosis of malaria infection in both humans and mosquitoes is essential for understanding transmission patterns, estimating epidemiological burden and informing appropriate management of cases. WHO’s Global Technical Strategy for Malaria 2016–2030 (GTS) recommends that surveillance strategies should be integrated as core interventions to provide better estimates of disease burden, improve resource allocation and accelerate progress towards elimination [Given health system challenges facing many low-income, malaria-endemic countries, there is particular interest in non-immunological point-of-care (POC) techniques that could be readily scaled up with minimum effort . EquallyPlasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale, being the most prevalent). However, microscopy-based diagnosis is labour-intensive and often requires more than one highly trained person for confirmation of conflicting results [Malaria diagnosis from human samples currently relies on light microscopy with thin and thick blood smears , polymer results . Additio results .PCR, on the other hand, is highly specific and sensitive for malaria parasite detection, but is not widely used for primary diagnosis in most malaria-endemic places because it is expensive, requires highly skilled personnel, and is therefore impractical to implement in rural and remote facilities . FurtherPlasmodium stages, i.e., gametocytes, in saliva is showing encouraging results in detecting sub-microscopic parasites in children and adults [At present, malaria rapid diagnostic tests (RDTs) are the best option for addressing the technical limitations of both light microscopy and DNA-based diagnosis. These tests target persistent specific antigens from malaria parasites , 23, 24 d adults .−1 to 400 cm−1 frequencies) and mid-infrared spectroscopy (4000 cm−1 to 400 cm−1 frequencies), combined with advanced data analysis, could provide cheaper, quicker, reagent-free, and potentially simpler options for surveys of mosquitoes and mosquito-borne infections. Examples include detection of endosymbionts such as Wolbachia bacteria, and pathogens such as Plasmodium and Zika virus in mosquitoes [Recent studies have shown that non-molecular techniques such as near-infrared spectroscopy . Sensitivity was however only 70%, possibly because the model included only a small number of negative samples. Although limited to laboratory cultures and small number of samples with low genetic variability, these studies were the first to demonstrate direct potential of MIR for malaria parasite detection.Khoshmanesh et al.  used midy et al.  used PlaThis current study has extended the approach used by Khoshmanesh et al.  and Roy Plasmodium species, P. falciparum accounts for most malaria infections Tanzania [Samples for this analysis were obtained from a cross-sectional malaria parasite prevalence survey conducted in 12 administrative wards in Ulanga and Kilombero districts in southeastern Tanzania  of Plasmodium species [The RDTs  were used to screen all individuals and results interpreted following manufacturer’s instructions . This RD samples  by diffe species , 23, 24. species .In addition to microscopy and RDTs, DBS specimens were collected from all RDT-tested patients regardless of test results. Three to four drops of blood were placed directly from finger prick onto Whatman 903™ card  . The DBSThe DBS collected from individuals for which there had been positive malaria test results (n = 147), and a separate random selection of specimens from individuals with negative results n = 149) were scanned using a Bruker ALPHA Fourier-Transform Infrared (FTIR) spectrometer equipped with Platinum ATR and a Platinum diamond sampling module [ were sca−1 wave numbers, with spectral resolution set at 5 cm−1. Each individual DBS sample was scanned 32 times in 30 s, and averaged to obtain a single representative spectrum. The anvil and crystal were cleaned after each specimen scan, using velvex tissue soaked in 70% ethanol and dried using a clean dry tissue. The spectrometer has an internal validation unit (IVU) with reference standards, and was programmed to conduct automated instrument tests for operational and performance qualification. However, additional performance and quality checks were done by conducting frequent background scans before first sample scan and after every five specimen scans. Additional details of the MIR spectrometer and the scanning process have been described elsewhere [The MIR spectra were captured from 4000 to 500 cmlsewhere , 40. AllP. falciparum, P. ovale, P. vivax and P. malariae) examined using nested PCR targeting 18S ssRNA sequences, according to the Snounou and Singh protocol [Plasmodium knowlesi [DNA was extracted from the DBS papers and the presence of any of the malaria parasites  interference bands as described in Mwanga et al. [Plasmodium-positive and 173 Plasmodium-negative individuals was considered for further analysis in the Python programming language version 3.7 with the scikit-learn library [The OPUS software  was useda et al.  and Gonza et al. . A final library . All wavPrimary assessment was done against PCR as reference because of its high sensitivity and specificity compared to RDTs and microscopy , 54. At P. falciparum occurring singly, though there were also a few cases of mixed infections consisting of P. falciparum, together with P. ovale. The MIR-based predictions were therefore done first by excluding the mixed infections, and second, without excluding them.The PCR analysis had identified mostly infections of Plasmodium species infections (N = 9) were excluded, so that the remaining dataset included only P. falciparum positives and a reduced number of negative samples . As previously described [All samples with mixed escribed , the dathttps://github.com/SimonAB/Gonzalez-Jimenez_MIRS.The training dataset was subjected to multiple rounds of randomly stratified splits into training sets (80%) and test sets (20%), to achieve rigorous classification of the different malaria-positive and malaria-negative samples. To provide a mix of data representation methods and allow for both parametric and non-parametric assessment of the data, baseline performances of seven different classification algorithms were tested. These included: K-nearest neighbours classifier (KNN), logistic regression (LR), support vector machine classifier (SVM), naïve Bayes (NB), random forest classifier (RF), XGBoost classifier (XGB), and multilayer perceptron (MLP) . All codPlasmodium positive or Plasmodium negative) using the naïve validation set separated at the beginning.The algorithms were trained on spectral features (absorbance of MIR spectra in at different wavenumbers) representing malaria-positive and malaria-negative DBS samples Fig. . The besPlasmodium species  occurring singly or together in a DBS.The full analysis was repeated, this time including the mixed infections, which had been removed from the initial model training. This way, the MIR-ML approach was evaluated for predicting infection with any of the two Plasmodium-infected and Plasmodium-free specimen were examined. Biochemical compositions such as lipids, proteins, carbohydrate and nucleic acids were explored from the sensitivity of the candidate MIR bands. Prediction coefficients of the best model were extracted and plotted its most dominant wave numbers.Lastly, based on the previous spectroscopic analysis of body fluids , and thePlasmodium positive (Npositives = 114) or Plasmodium negative (Nnegatives = 146), and the features (or wave numbers) relied upon to predict the infection status. The specific biochemical properties associated with major peaks in the spectra are shown in Table Figure P. falciparum-infected individuals were considered in the category of malaria-positive DBS cards, and any mixed infections were excluded. Logistic regression (LR) outperformed the other six candidate classifiers in accuracy and precision  where lipids, amino acids and proteins is mostly detected . Figure Plasmodium species present. Once again, LR was the best model, achieving up to 93% accuracy and 95% after bootstrapping. The final model achieved 87% overall accuracy for the validation set, correctly identifying 91% of PCR-negative individuals and 82% of PCR-positive individuals.In the second analysis, all infected individuals were considered regardless of This study is an initial demonstration, using field-collected blood specimens, that MIR spectroscopy coupled with logistic regression analysis could potentially be harnessed for detection of infectious parasitic diseases, in this case malaria. Malaria cases were identified by both MIR spectroscopy and PCR, then the results were compared. It considered 296 individual samples, to demonstrate potential application of MIR spectroscopy for malaria detection. The main finding was that spectral signatures collected from human DBS can be relied upon to identify malaria-infected and non-infected specimens.Plasmodium. Other related approaches include the use of surface-enhanced Raman spectroscopy (SERS), requiring silver nanoparticles to be added to lysed blood, and magneto-optic scanning to detect haemozoin, a waste product of Plasmodium infection of red cells pigment, haemoglobin [Validity of this approach was verified by PCR tests, and corroborates the earlier evidence by Khoshmanesh et al.  and Roy moglobin , 58. Botmoglobin .An important advancement in the work presented here is that it has demonstrated the first direct application of MIR spectroscopy on field-collected DBS specimens on filter papers. The technique therefore requires no additional reagents or pre-processing of samples. The samples analysed came from multiple age groups of both male and females in villages with varying malaria prevalence rates, thereby providing considerable variability between individual infections. When the final optimized LR model was applied to new blood samples that had not been exposed to the classifier, they correctly identified 92% of the malaria-free individuals and 93% of malaria-infected individuals compared to PCR.P. falciparum sporozoites in laboratory-reared and laboratory-infected Anopheles mosquitoes with up to 90% accuracy [Wolbachia, Plasmodium and Zika virus in different mosquito species [−1 to 400 cm−1) where the absorption intensity is due to overtone and combination bands, which are relatively weak. Spectroscopy at MIR frequencies (4000 cm−1 to 400 cm−1) captures the fundamental vibrational modes of biological samples, which are stronger and more information rich [This study adds to the growing evidence showing the potential role of infrared spectroscopy and chemometrics in surveillance of mosquito-borne diseases. In a recent study conducted using near-infrared rather than MIR spectra, it was possible to detect infectious accuracy . Earlier species –31. Manyion rich , therebyPlasmodium-specific proteins present in infected human blood [Plasmodium-infected and Plasmodium-free specimens were examined  but lower specificity (84.4 vs 91.7%) than the MIR spectroscopy approach ; (c) detailed examination and characterization of cases incorrectly identified by this new approach; and, (d) assessment of parasite detection thresholds, and factors that may influence it if MIR spectroscopy and machine learning are used.Significant improvements and field validation studies are still necessary before this technique can be deployed for actual screening and diagnosis. One avenue of improvement is the availability of more field data to validate this approach as a complementary tool for screening malaria, and potentially other blood-borne infections. Also, this current study did not include any quantification of infection intensities, which will be necessary to examine validity in areas of different transmission intensities, as well as potential role of this technology in malaria elimination settings. Such future studies should also include: (a) greater analysis on biological basis of the observed signals and how this may be influenced by the natural history of malaria infections in humans, or its manifestations, such as anaemia; (b) determination of whether the method can distinguish between the different parasite stages, such as the asexual stages It takes 30 s to scan a single blood spot under MIR spectrometer, and the approach is considerably lower cost than PCR platforms. The MIR equipment presently costs ~ US$29,000 as an initial outlay, but there are no extended costs for reagents except for occasional replacement of the desiccants. It takes less than a minute to clean the crystal and anvil, position DBS on the crystal and collect the MIR spectra, thus any experienced staff member can scan more than 250 specimens per day. Comparatively, average PCR systems cost between US$3000 and 10,000, and require reagents repeatedly. The cost of processing a DBS sample can be US$2–4 per unit and regularly takes up to 2 days to get back full results in batches typically not exceeding 100 specimens. Assuming a modest analysis of just 5000 samples per year, it would take ~ 2 years to recover full costs of switching from PCR to MIR-ML-based systems and break even. The onward costs of servicing are also low as the MIR equipment is robust. Compared to PCR, use of MIR-ML could therefore potentially be developed into a cost-effective, quick and scalable approach. Besides, the same spectra, once collected, can be analysed for multiple characteristics, potentially making this approach a one-stop system for assessing multiple disease indicators with different specimen types, such as DBS papers, blood slides, mosquitoes, and fluids. It is however not expected that the technology, in its current form, can replace current best practices for malaria surveillance or diagnostics, without further field validation.Plasmodium positive and 173 Plasmodium negative). It is expected that the quality of the predictions will improve as more data are available to train the models, especially if there is variation in localities from where the specimen originates, parasite densities, geographical locations, and demographics of infected individuals. A related limitation was that it was not possible to balance the Plasmodium-positive and Plasmodium-negative specimens by other variables such as anaemia, gender, age, period of storage, or parasitaemia prevalence in the different villages, all of which may influence malaria risk and the predictive values of test methods. Future studies should therefore test whether these factors can significantly influence outcomes of the prediction models.One limitation of this current study is that the number of samples used was low, totalling only 296  by nested PCR method.


The objective of this study was to evaluate a new commercial molecular diagnostic technique.Malaria is currently the most important human parasitic disease in the world responsible for high morbidity and mortality. Appropriate diagnostic methods are essential for early detection. Microscopy examination remains the ® Malaria + 4 RDT), microscopy examination, and two commercial molecular malaria tests , then compared against an in-house reference PCR technique.A prospective, observational, multicentre study was performed between January 2015 and April 2017. All participants were immigrants from malaria-endemic areas, who were divided into two groups: asymptomatic group and symptomatic. Samples from both groups were evaluated by a rapid diagnostic test . The commercial molecular technique had higher sensitivity than the other methods (S = 96%) and a high rate of concordance with the in-house reference PCR technique (Kappa score = 0.93).In all, 250 patients were included: 164 (65.6%) in the asymptomatic group, and 86 (34.4%) in the symptomatic group. There were seven cases of asymptomatic parasitaemia  that were detected only by molecular methods. In the symptomatic group, there were seven cases of submicroscopic malaria. The main species detected was P. falciparum in cases with low parasitaemia.The molecular techniques, although slower than microscopy, have adequate diagnostic accuracy and are very useful for the detection of  Malaria is an overwhelming problem in tropical developing countries. It is currently the most important human parasitic disease in the world and continues to be a serious public health problem . More thEffective diagnostic techniques permit early and appropriate diagnosis of malaria since this prevents the infection from progressing to a severe stage or even death. The World Health Organization (WHO) recommends confirming all clinically suspected malaria cases before treatment using a malaria-specific rapid diagnostic test (RDT) or by direct visualization of the parasites using standard microscopy , 4.gold standard method for detecting Plasmodium in blood. With this technique, under optimal conditions, an experienced microscopist can achieve a sensitivity of about 50 parasites/µL of blood, although the sensitivity of detection in most routine diagnostic laboratories is normally much lower (in the range of approximately 50–100 parasites per µL of blood) [Light microscopy requires a high level of expertise, involves examining Giemsa-stained thick and thin blood smears on glass slides. Thick blood smears remains the clinical Plasmodium antigens, such as histidine-rich protein 2 (HRP2), produced by Plasmodium falciparum, parasite-specific lactate dehydrogenase (pLDH) or aldolase enzyme, which is common to all species. Currently available RDTs have lower sensitivity than well-trained microscopists (≈ 100 parasites/µL) [P. falciparum, Plasmodium vivax or both of them [RDTs are lateral flow immunochromatographic devices that detect ites/µL)  in cases of them .Molecular techniques have been designed to minimize the problems associated with the other methods described above . They arThe main objective of this study was to evaluate a new commercial molecular technique by comparing it with other diagnostic methods: thick and thin blood film examination, RDT and an in-house reference PCR technique, in two different population groups.A prospective, observational, multicentre study was performed. Data were collected at five different hospitals in Madrid, Spain  between January 2015 and April 2017.® Malaria + 4, Biosynex), Giemsa-stained thin and thick blood films, the commercial molecular technique (C-PCR) under evaluation, and the reference molecular technique (CNM-PCR).A 5 mL blood sample was collected from all participants. All the samples were tested using all the diagnostic techniques: RDT  and pan malaria-specific pLDH (pan lactate dehydrogenase) in human blood. The tests were performed by a microbiologist at the different hospitals, in accordance with the manufacturer’s instructions.The ImmunoQuickThin and thick blood films were stained with Giemsa solution (1:10 dilution) for 10 min. All slides were examined under a light microscope with oil-immersion objectives (100×) at the Hospital 12 de Octubre. The same person, a well-trained microscopist blinded to the results of the other tests, carried out the examination in at least 200 visual fields. If the sample was positive, parasitaemia was calculated, and the result expressed in percentage form as the number of parasitized red blood cells.Plasmodium ssrDNA to detect Plasmodium in blood samples and includes an internal amplification reaction control. The second reaction, an identification reaction, is performed to enable Plasmodium species identification [CNM PCR was performed at the National Centre of Microbiology  by the same person, an expert microbiologist blinded to the results of the other tests. This technique allows for identification of the four major malaria species in human blood. The method is a semi-nested multiplex PCR involving two multiplex PCR reactions for amplification of target sequences. The first reaction amplifies fication , 9. DNA ® Compact Automated Nucleic Acid Extractor, and MagCore® Genomic DNA Whole Blood Kit 102 extraction cartridges. All samples were extracted with an internal control to ensure the validity of the extraction and amplification processes. Purified DNA was eluted in a final volume of 60 µL, which was maintained stored at − 20 °C until amplification. This PCR method  consists of two different reagent kits: a generic PCR test for detection of Plasmodium spp, and a differential PCR reagent kit, able to detect four species of Plasmodium . Plasmodium knowlesi is not identified by the differential PCR but it would be detected by the first PCR as Plasmodium spp. In both PCRs 10 µL of extracted DNA are used, as well as for negative and positive controls. The test consists of two processes in a single tube assay: PCR amplification of target DNA and Internal Control, and simultaneous detection of PCR amplicons by fluorescent dye labelled probes. Both reactions were performed using the CFX96™ Real-Time System, BioRad®thermal cycler. All samples were analysed by generic PCR, and positive samples were further analysed by differential PCR. The cost of the Plasmodium spp. PCR is 22.5 euros per reaction (DNA isolation included), if a positive result is obtained 19 euros have to be added to know the species.The same blinded microbiologist who examined blood films also performed C-PCR at the Hospital 12 de Octubre. DNA extraction from 400 µL of venous blood was carried out with RBC Bioscience’s MagCoreC-PCR is a real time PCR which only needs one amplification reaction and it can be performed at the same hospital, so it can be done faster and with less contamination risk than the CNM PCR. A formula can directly be used to quantify Ct values of differential PCR, so results can also be reported as gene copy numbers/mL.Participants were divided into two groups: asymptomatic and symptomatic group. The asymptomatic group included immigrants over 18 years of age originating from malaria-endemic areas , who presented at the hospital without fever or initial suspicion of malaria. In these cases, molecular techniques were expected to detect some submicroscopic carriers. Patients who fell into this category were located in order to offer them malaria treatment.The symptomatic group included all patients who attended one of the participant hospitals with symptoms suggestive of malaria and parasites in at least one test.The study was carried out following the ethical principles of the Helsinki Declaration. Before the samples were collected, participants were provided with an explanation of the nature of the project and given an informed consent form, which they signed. Ethical review and clearance were obtained from the Medical and Health Research Ethics Committee at each of the hospitals participating in the project.2 test. For relationships between quantitative and categorical variables, the Fisher Student’s t test or the Mann–Whitney U test were used as appropriate. Statistical significance was set at p < 0.05. Data were analysed using SPSS version 18.0  and EpiDat 3.1 software version .All variables were included in an online database “REDCap” (Research Electronic Data Capture) previously created for the study. The Kappa statistic and efficiency index were used to compare and rate RDT, microscopy and C-PCR versus the reference method, CNM-PCR. Diagnostic parameters such as sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and efficiency were calculated for each diagnostic method. All estimates were reported with 95% confidence intervals (CI). Quantitative variables were expressed as means and standard deviation (SD) or as median (25th–75th percentiles). Categorical variables were expressed as frequencies. Relationships between categorical variables were compared using the χDuring the study period, 250 patients in all were included: 164 (65.6%) in the asymptomatic group and 86 (34.4%) in the symptomatic group. All 250 blood samples were analysed by microscopy, 184 by the RDT , 235 by CNM-PCR , and 242 by C-PCR . Fifteen patients did not have enough blood to perform CNM-PCR. C-PCR could not be performed on eight patients for the same reason  and the other two were detected only by C-PCR. Six cases were due to P. falciparum and the other one was a mixed infection due to P. falciparum, Plasmodium ovale and Plasmodium malariae. All but one had come from Equatorial Guinea.In the symptomatic group, the microscopic examination was positive in 79 patients (91.8%). There were seven cases of symptomatic patients with a negative microscopy, but where molecular methods detected P. falciparum infections and were detected in patients coming from sub-Saharan Africa.Microscopy was negative in all patients in the asymptomatic group, with the exception of one patient from Nigeria, who had arrived in Spain 42 days before. Seven cases were detected only by molecular techniques: one case was diagnosed by both CNM-PCR and C-PCR, three only by CNM-PCR, and three only by C-PCR. The prevalence of asymptomatic parasitaemia was 2.8%. All were P. falciparum (87/90: 96.6%): 8 in the asymptomatic group, 79 in the symptomatic group. Other species were detected only in the symptomatic group. There were four cases of P. malariae and all were mixed infections. Plasmodium vivax was detected in three samples by PCR: in two cases it was the only species detected, and the other one was a mixed infection with P. malariae. There were eight cases of P. ovale and all but one were mixed infections, as specified in Table P. vivax infection originated from India and the mixed infection with P. malariae were from Equatorial Guinea.Overall, there were 90 cases of malaria. The main species detected by PCR was P. falciparum together with P. ovale, two to P. falciparum and P. malariae, and one to P. vivax and P. malariae. One infection was caused by three species . Eight cases showed discrepancies between the results obtained by CNM-PCR and C-PCR.There were 10 cases of mixed infections (11.1%), all in the symptomatic group. Six cases were due to The diagnostic rates obtained with each of the different techniques in this study were then compared with those of the reference, CNM-PCR Table . A totalOverall, when compared with microscopy, the molecular techniques resulted in an increase in the detection of infection, with high concordance (Kappa score 0.91) between microscopic examination of thick blood slides and PCR. There was a high concordance between the two molecular methods, showing a Kappa score of 0.93. C-PCR was able to detect more cases of malaria infection, as well as mixed infections.gold standard for malaria diagnosis and is faster than molecular techniques, it has major limitations such as low sensitivity, difficulties associated with quality control and standardization and the need for ongoing training and evaluation [It is very important to make a definitive diagnosis and confirm malaria disease. Although microscopy remains the aluation –13. For aluation , 14, 15.aluation , 15, 16.Anopheles atroparvus).As a result of globalization, the number of imported malaria cases has increased, mainly due to immigrants from malaria-endemic areas who have developed a semi-immune status –19, whic® Malaria + 4 RDT, but has lower diagnostic capacity (sensitivity 88.5%) than PCR. More specifically, 14 out of 90 patients had a negative microscopy and were diagnosed only by PCR. The thick blood slide examination was positive in only one patient with very low parasitaemia (< 0.01%) in the asymptomatic group. In malaria infections where parasitaemia is very low, diagnosis can easily be missed if it is not possible to use a molecular technique and expert microscopists are not available.For all these reasons, a new commercial technique was evaluated and its usefulness in routine malaria diagnosis by comparing it with a RDT, microscopy, and a reference PCR technique. These data show that microscopy gives a more accurate diagnosis than the dipstick RDT ImmunoQuick® Malaria + 4 RDT were both less than 90%, with a Kappa score of 0.65 against CNM-PCR. Its usefulness in malaria diagnosis lies mainly in the fact that it is quick and easy to perform and does not require trained staff. A product evaluation by the manufacturer, reporting findings from 230 samples, showed that the sensitivity and specificity of Immunoquick® Malaria + 4 versus microscopic examination of thick and thin blood smears were 70.5% and 100% for pLDH, and 98.8% and 100% for Pf HRP-2, respectively. In the present study, sensitivity and specificity were 88.8% and 84.2%, respectively, lower than those specified by the manufacturer. In the asymptomatic group, the RDT yielded two false positive results, confirmed by negative microscopy and negative PCR. This test also gives false negative results in patients with low parasite densities. Of the seven cases of asymptomatic parasitaemia, the RDT was positive in only one sample; on the other hand, the RDT detected all cases of symptomatic submicroscopic malaria except one. In P. falciparum malaria infection, HRP-2 is not secreted in the gametogony stage, so that the HRP-2 band may be absent in healthy carriers and may be overlooked.The sensitivity and specificity of RDT ImmunoQuickC-PCR is easy to perform and it can be done fast at the same hospital. Also, it is standardized and has good diagnostic parameters , which were comparable with those of CNM-PCR. Among submicroscopic infections, eight discrepant results were found between the two PCR techniques: two in the symptomatic group and six in the asymptomatic group. The discrepancies in the asymptomatic group could be due to low parasitaemia levels. The two discrepant results in the symptomatic group were detected only by RDT and C-PCR, whereas microscopy and CNM-PCR were negative. It is normal to find some variability in sensitivity scores between different PCR methods , attribuAfter considering the results obtained at the study centre, the authors conclude that molecular methods show improved sensitivity over microscopy, mainly in asymptomatic parasitaemias. The combination of microscopy and PCR upgraded the diagnosis of malaria, as the sensitivity, specificity, VPP and VPN data demonstrate. Nevertheless, PCR also has its disadvantages, since it detects non-viable parasites after malaria episodes that have been correctly treated and this may lead to false positive results and unnecessary malaria treatments. In addition, it requires a sophisticated laboratory setting and trained technicians, requires a longer time to diagnosis and incurs higher costs . For thePlasmodium falciparum was the most prevalent species diagnosed (96.6%). A low prevalence of malaria infections due to non-falciparum species was found, so that it was not possible to assess the diagnostic capacity of the methods in these cases. Mixed infections were not very frequent. Co-infections were detected in only 10 cases, all of them in the symptomatic group. Among co-infections, there were more discrepancies detected between the results obtained by CNM-PCR and C-PCR. Only two cases of mixed infections were detected by the two methods.P. falciparum. The two molecular methods were positive in only one case. It is important to bear in mind that some cases may go undetected due to variability between methods. Higher sensitivity would be desirable to ensure a proper diagnosis and prompt treatment of malaria infection.The prevalence of submicroscopic parasitaemia in the asymptomatic group (2.8%) was in line with data published by Monge-Maillo et al.  and MatiP. falciparum, so that the usefulness of the methods for non-falciparum species was not possible to assess. Third, there were too few cases of mixed infections to evaluate the usefulness of these methods for diagnosis. In this type of infection, one of the species involved is usually present in a smaller proportion and cannot be detected. Further studies are required to assess this problem.The study has some underlying limitations. It was not possible to perform all the diagnostic tests on all samples for various reasons. First, factors associated with this group of immigrant patients  made it difficult to include them in the study. Second, in most cases of malaria infection, the species responsible was The FTD-Malaria and Malaria Differentiation PCRs showed good test values for the diagnostic test parameters , as well as good inter-procedure agreement for detecting malaria infections.Plasmodium species and mixed infections that, due to the subjectivity of microscopy, can be misdiagnosed.The population of immigrants originating from malaria-endemic areas can have low parasite blood densities that non-molecular methods fail to detect. PCR is a useful tool for early detection of this type of infection. In this study, the combination of two molecular techniques enabled 14 cases (1.6%) of submicroscopic infection to be detected. Molecular techniques are also the most reliable tests to make a correct diagnosis of The data obtained in this study are in agreement with other published reports and suggest that, while microscopy is very useful and faster, PCR is more sensitive and improves the diagnosis of malaria, above all in patients with undetectable parasitaemia. These data show that PCR is a diagnostic tool with efficiency, sensitivity and specificity greater than 96% for the diagnosis of malaria infections. Further research should be carried out to evaluate the usefulness of PCR and assess the real prevalence of submicroscopic infections.


Plasmodium falciparum infections. Genetic mutations and gene deletions are important factors influencing or affecting the performance of rapid diagnostic tests.Malaria remains a public health problem in some countries of Central America. Rapid diagnostic tests (RDTs) are one of the most useful tools to assist in the diagnosis of malaria in remote areas. Since its introduction, a wide variety of RDTs have been developed for the detection of different parasite antigens. PfHRP2 is the most targeted antigen for the detection of pfhrp2 and pfhrp3 genes and their flanking regions, a total of 128 blood samples from patients with P. falciparum infection from three Central American countries were analysed through nested or semi-nested PCR approaches.In order to demonstrate the presence or absence of the pfhrp2 and pfhrp3 genes, respectively. Parasites from the three countries showed deletions of one or both genes. The highest proportion of pfhrp2 deletions was found in Nicaragua while the isolates from Guatemala revealed the lowest number of pfhrp2 deletions. Parasites collected from Honduras showed the highest proportion of phfrp3 absence (96.2%). Twenty-one percent of isolates were double negative mutants for the exon 1–2 segment of both genes, and 6.3% of isolates lacked the full-length coding region of both genes.In total, 25.8 and 91.4% of the isolates lacked the region located between exon 1 and exon 2 of P. falciparum isolates lacking the pfhrp2 and pfhrp3 genes, and their flanking regions, in Honduras, Guatemala and Nicaragua. This finding could hinder progress in the control and elimination of malaria in Central America. Continuous evaluation of RDTs and molecular surveillance would be recommended.This study provides molecular evidence of the existence of  Plasmodium vivax, and the remaining 10% to Plasmodium falciparum. Guatemala, on the other hand, has been reporting fewer than 10 cases of falciparum malaria in recent years [Malaria remains a public health problem for most tropical countries. However, there are notable differences in the incidence of malaria between geographic regions. Within the Central American sub region, two countries  reported fewer than 15 indigenous malaria cases in 2016, and, according to the World Health Organization, are projected to eliminate malaria by 2020. In Nicaragua, Guatemala and Honduras however, a total of 15,476 cases were reported in 2016, representing the largest burden in Central America. Although the prevalence of malaria is decreasing in this region, Panama and Nicaragua showed an increase in case incidence between 2010 and 2016. Approximately, 90% of malaria cases in Nicaragua and Honduras are due to Plasmodium species (pan-specific tests), but the PfHRP2 antigen (Histidine-Rich Protein-2) is produced only by P. falciparum. PfHRP2 is an abundant and heat stable antigen, which makes it a highly sensitive target for the diagnosis of falciparum malaria [As this sub-region of the Americas approaches the goal of eliminating malaria by the year 2030 [ malaria .pfhrp2 gene, located on chromosome 8 of P. falciparum, and pfhrp3 is an structural homologue of pfhrp2 [pfhrp2 or pfhrp3 genes, respectively, and 21.6% of parasites lacked both genes [PfHRP2 is a non-essential protein encoded by f pfhrp2 , which if pfhrp2 . A retroth genes . Such fith genes . All thith genes  that allth genes , 17, andth genes .pfhrp3-negative parasites and suggested that PfHRP2-based RDTs could continue to be used in Honduras, but at the same time they propose further studies including samples from a wider geographical area. Thus, this study aims to be an update of the topic for the Central American sub-region, analysing populations of the parasite from Guatemala, Nicaragua and Honduras.The only study published on this topic in Central America dates from 2015, in which samples from Honduras collected between 2008 and 2009 were analysed through a molecular approach . These apfhrp2 and pfhrp3 genes and their flanking regions in 128 falciparum malaria samples from Central America, previously diagnosed by microscopy. Twenty-one samples were collected from Guatemala and 55 samples from Nicaragua, during 2015, and 52 samples from Honduras were collected during 2011 (n = 10), 2012 (n = 11) and 2017 (n = 31). Samples from Guatemala were collected from the department of Escuintla. In Nicaragua the samples came from the North Atlantic Autonomous Region (RAAN), while in Honduras the majority of samples came from the department of Gracias a Dios and less than 10% were obtained from five other departments   were amplified a third time.In order to detect the presence or absence of a partial coding region between exons 1 and 2 of the genes pfhrp2 and pfhrp3 genes, all samples that did not amplify the exon 1–2 segment of both genes were amplified by a semi-nested PCR targeting the exon 2. Both reactions were carried out in a 50 μl volume and included 25 μl of 2× Master mix (Promega Corp.), 2.0 μl of each 10 μM primer (F1/R1 in the first round and F2/R1 in the second round), and 1.0 μl of DNA. Reactions were carried out by an initial denaturation temperature at 94 °C for 10 min, followed by 35 and 37 cycles respectively of 94 °C for 50 s, an annealing step for 50 s, 72 °C for 1 min, and a final extension step at 72 °C for 10 min  of pfhrp2 and 91.4% of the isolates lacked the homologous region of pfhrp3 (Table pfhrp2 deletions was found in Nicaragua (30.9%) while the isolates from Guatemala revealed the lowest number of deletions (14.3%). Parasites collected from Honduras showed the highest proportion of exon 1–2 phfrp3 deletion (96.2%).A total of 128 blood samples from patients with pfhrp2 negative and pfhrp3 negative). Most of those double negative parasites were detected in Honduras 13/52 (25%), followed by Nicaragua 11/55 (20%) and Guatemala 3/21 (14.3%). No significant differences were found in the frequency of the deletions between the three different years of collection in Honduras. The pfmsp-1 gene was successfully amplified in the 27 double negative isolates, showing a distribution of 52% for the K1 genotype and 48% for MAD20.A relevant result was the finding of 27 (21%) double negative isolates (pfhrp2 (MAL7P1.230 and MAL7P1.228), and pfhrp3 (MAL13P1.485 and MAL13P1.475). Most of the isolates (75.8%) revealed the presence of the MAL7P1.230 region, and almost all the isolates (99.2%) successfully amplified the MAL7P1.228 region. On the other hand, more than 62% of isolates seem to have deleted the MAL13P1.485 and MAL13P1.475 regions (Table pfhrp3 (91.4%), followed by MAL13P1.475 (68.8%). All samples amplified properly at least one of the 6 analysed loci, which indicates that the quality and quantity of DNA was sufficient to guarantee reliability in the negative results.Nested PCRs were also carried out to amplify the flanking regions of pfhrp2 sequence plus the flanking regions, while the loci associated to pfhrp3 revealed 6 different deletion patterns  seem to have deleted the entire pfhrp3 region.Five deletion patterns were recorded when analysing the exon 1–2 pfhrp2 and pfhrp3 genes, the full length of exon 2 was amplified [In order to confirm the deletion of the mplified  in thosepfhrp2, pfhrp3 and their neighbouring regions in P. falciparum parasites collected from Guatemala and Nicaragua, and this is also the second report that includes samples collected from Honduras [pfhrp2 and pfhrp3.This is the first report of deletions of the genes Honduras . MotivatHonduras , 28–30, Honduras , 12, 31 Honduras , 32–36, Honduras  with thepfhrp2 and pfhrp3 genes is the impossibility of establishing an adequate diagnosis of malaria, especially where it is solely based on RDTs that exclusively detect the PfHRP2 protein [Although RDTs are a valuable tool to establish the diagnosis of falciparum malaria, especially in areas where routine microscopic diagnosis is not available, these tests could yield false negative results due to undetectable concentrations of the PfHRP2 antigen in low parasitaemias , to the  protein , 38, 39.pfhrp2 and pfhrp3 [pfmsp-1 was successfully amplified for the samples that yielded a negative result for both gene regions. The results obtained suggest that the absence of amplification does not seem to be due to a low DNA concentration of the parasite, since it was not possible to establish any association between samples with low, moderate or high parasitaemia and absence of amplification for any loci. Also, given that all samples amplified at least one of the 6 analysed loci, these results seem to be reliable and allow to confirm the existence of deletions of the sequences associated with the genes pfhrp2, pfhrp3 and their flanking regions in the natural populations of P. falciparum circulating in Central America.Amplification of coding sequences and their flanking regions through a nested-PCR approach is a commonly used method to investigate deletions of d pfhrp3 , 38, 40.pfhrp2 and pfhrp3 genes in parasites from Central America [pfhrp2 and its flanking sequences, and therefore PfHRP2-based RDTs could be considered useful in this geographic region at the time. Interestingly, in this study 10 archival samples collected 2 years later (2011) in Honduras showed 40% of isolates with deletion in the pfhrp2 gene. This could be attributed both to the selective adaptation of the parasite in a short period of time, and to the fact that the samples were collected in some regions of the country that were not represented in the previous study . Abdallah et al. [pfhrp3 region and suggested the need to expand the research with samples collected from other geographic regions and for a longer time span. Following that recommendation, and according to the results obtained in this study, the proportion of parasites lacking pfhrp3 in this study area was higher than that previously reported in Puerto Lempira. This high percentage of pfhrp3-negative parasites is in agreement with the large genomic deletions in the chromosome 13 described in the reference line HB3, a parasite isolate collected from Honduras [Before this study, there was not enough information of deletions in the  America . The onl America . Those ah et al.  also repHonduras .pfhrp2 gene, while only 9% of the isolates were pfhrp3-positive. A WHO web mapping tool that tracks 3 of the major biological threats to malaria control and elimination compiled 53 surveys from 7 South American countries regarding pfhrp2/pfhrp3 deletions [pfhrp2 gene are less frequent than those in the pfhrp3 gene; which is consistent with the current results. These coincidences could be attributed to similar characteristics in the evolution of the parasites from South and Central America. The high percentage of deletions in the pfhrp3 gene that are observed in some localities of Colombia [When analysing all the samples collected from the three Central American countries, 75% of the isolates amplified successfully the eletions . DespiteColombia  and Perupfhrp2/pfhrp3 genes, all isolates retained the downstream locus MAL7P1.228, in a similar way to that reported in Eritrea [pfhrp2 and pfhrp3 now reveal a high number of deletions, which would support the hypothesis of the rapid appearance of deletions in these genetic regions. Nevertheless, one of the most interesting results of this study is the finding of 26 (20.3%) isolates with a double deletion (pfhrp2/pfhrp3 negative for exon 1–2). Some authors have stated that the deletion of those particular genetic sequences is the result of multiple independent events instead of the dispersion of strains originated from a single event [Regarding the flanking sequences of the  Eritrea , 40, whe Eritrea , it is nle event , 44. Howle event , as demole event . Despitele event .P. falciparum infections is considerably less than P. vivax, the impact of a few false negative results derived from RDTs detecting PfHRP2 would be low for the public health. However, although the risk of diagnostic failures due to non-detection of the parasite by RDT could be low, the countries of the region have proposed eliminating malaria by the year 2030, and this goal requires rapid and timely detection of all cases of malaria, especially in the most remote areas. Consequently, it will be necessary to evaluate the performance of those particular RDTs used in Central America and establish a routine molecular surveillance programme [In the three countries analysed in this study, the Ministries of Health use RDTs based on detection of the PfHRP2 antigen: CareStart™ Malaria HRP2/pLDH(Pf/Pv) Combo (Honduras), SD Bioline MALARIA Ag P.f/P.v (Nicaragua) and CareStart™Malaria RDT Single Kit . According to the World Malaria Report 2017 [rogramme .pfhrp2 gene seem to have intact the exon 2  that did not amplify the intron 1 segment of the  2 Table . A simil that didg et al.  In any cpfhrp2 gene searching for polymorphisms that could affect the sensitivity of the RDTs are recommended [The use of archived samples was a main limitation of the study. Samples were not analysed by serology or RDTs, as recommended by Cheng et al. and supported by the WHO , 27, 31.ommended , 48, 49.P. falciparum isolates lacking the pfhrp2 and pfhrp3 genes, and flanking regions, in Honduras, Guatemala and Nicaragua. This finding could hinder the progress made in the control and elimination of malaria in Central America. From now on, a routine evaluation of the performance of RDTs and molecular surveillance will be required.In summary, this study provides molecular evidence of the existence of


Monocytes are plastic heterogeneous immune cells involved in host-parasite interactions critical for malaria pathogenesis. Human monocytes have been subdivided into three populations based on surface expression of CD14 and CD16. We hypothesised that proportions and phenotypes of circulating monocyte subsets can be markers of severity or fatality in children with malaria. To address this question, we compared monocytes sampled in children with uncomplicated malaria, severe malarial anaemia, or cerebral malaria. Flow cytometry was used to distinguish and phenotype monocyte subsets through CD14, CD16, CD36 and TLR2 expression. Data were first analysed by univariate analysis to evaluate their link to severity and death. Second, multinomial logistic regression was used to measure the specific effect of monocyte proportions and phenotypes on severity and death, after adjustments for other variables unrelated to monocytes. Multivariate analysis demonstrated that decreased percentages of non-classical monocytes were associated with death, suggesting that this monocyte subset has a role in resolving malaria. Using univariate analysis, we also showed that the role of non-classical monocytes involves a mostly anti-inflammatory profile and the expression of CD16. Further studies are needed to decipher the functions of this sub-population during severe malaria episodes, and understand the underlying mechanisms. Pathophysiology of CM is based on the accumulation of Plasmodium falciparum-infected erythrocytes (iE) in the brain and clinically develops into coma6. Infected red blood cells adhere to vascular endothelium and other erythrocytes, cause ischemia and hypoxia, elicit endothelial activation and signalling, blood brain barrier impairment and a range of immune cell host responses to resolve the neuroinflammation process8. Here, monocytes play a central role in secreting immune cytokines, chemokines and reactive oxygen species, participating in immune cell chemoattraction and polarization and in the redox balance10. SMA is defined by a haemoglobin level of < 5 g/dL. Its aetiology is attributed to the lysis of infected and uninfected red blood cells with anaemia persisting even after successful clearance of parasitemia13. Dyserythropoiesis and bone marrow suppression explain the low replacement of red blood cells16. Early Ei elimination by monocyte/macrophages-dependent phagocytosis and reactive oxygen species production drives both the inflammatory response and inhibition of erythropoiesis, while accumulation of hemozoin-containing monocytes has been linked to suppression of erythropoiesis17.The two primary severe disease outcomes of malarial infection are cerebral malaria (CM) and severe malarial anaemia (SMA). Case fatality rates among African children with CM remain in the 15–25% range20, which has led to studies demonstrating the heterogeneity of monocytes21. Now, human monocytes are divided in three subsets based on both CD14 and CD16 expression, named classical (CD14+ CD16-), intermediate (CD14+ CD16+) and non-classical (CD14low CD16+). According to their response to LPS stimulation, classical monocytes are known for their major function of phagocytosis with capacity to produce IL-10 and low TNF; intermediate monocytes are known as inflammatory cells with high capacity to produce IL-1β and TNF; and non-classical monocytes are cells patrolling in vessels able to differentiate into anti-inflammatory macrophages during inflammation22. However, during pathological processes, the effect of count change remains unclear. We earlier established that proportions and phenotypes of monocyte subsets are related to clinical outcomes in pregnancy-associated malaria23. In that study, CD14low CD16+ non-classical monocytes increased in comparison to reported proportions of healthy pregnant women, and increased expression of the CD36 scavenger receptor on monocytes was related to higher infant birth weight.Human monocytes are identified by their lipopolysaccharide receptor CD14, a co-receptor for lipopolysaccharide (LPS) which is abundantly expressed. Inflammatory activity of monocytes expressing CD16, a Fcγ receptor, was recognized many years ago28. The relationship between monocyte subsets and levels of cytokines, plasma chemokines, and chemokine receptor expression was also investigated. Proportions and phenotypes of monocytes were first analysed by univariate analysis to evaluate their link to severity and death outcome. Second, multinomial logistic regression was used to measure the specific effect of monocyte proportions and phenotypes on severity and death, after adjustment for other variables unrelated to monocytes. Our results highlight the importance of non-classical monocytes in outcomes of severe malaria in children (CM and SMA). Multivariate analysis shows that a decreased percentage of non-classical monocytes is associated with death. Using univariate analysis, we also clarify the relationship of CD14, CD16, CD36 and TLR2 expression by monocyte subsets with severity and death outcomes.In the current study, we postulated that proportions and phenotypes of circulating monocyte subsets can be a marker of severity or death outcome in children with malaria. To verify this hypothesis, we compared monocytes sampled in children with uncomplicated malaria (UM), SMA or CM. To define monocyte phenotypes, we studied the scavenger receptor CD36 and the Toll-like receptor 2 (TLR2), two pattern recognition receptors known for their functional implication in iE eliminationP = 0.0002 for SMA compared to CM, P < 0.0001 for SMA compared to UM by Mann-Whitney U-test). Children presenting with CM were characterized by much higher parasite densities and death occurrence compared to SMA and UM children . Plasma IL-1β, IL-6, IL-10, MCP-1 and CXCL10 were also associated with severity. IL-1β and MCP-1 were specifically higher in the CM patients  . The occurrence of death was strongly related to a lower proportion of CD14low CD16+ monocytes with an odds ratio of 1.9 (P = 0.02).To determine the risk factors of severity and death occurrence, we conducted two separate backward stepwise logistic regressions Table . Variabl) Tables  and 5. S) Tables  and S2. low CD16+ monocytes was associated with more severe malaria (considering that CM is more severe than SA) and a higher risk of death; this last association was confirmed by multivariate analysis. A key role of non-classical monocytes in the evolution of diseases has rarely been reported, unlike CD14+ CD16+ intermediate monocytes. Their count consistently increases in infectious and non-infectious diseases whereas CD14low CD16+ non-classical monocyte count is more variable, increasing in infectious diseases such as tuberculosis30, HIV31, and sepsis in neonates and young children32, but decreasing in dengue fever33 and sepsis in adult patients34. During non-severe falciparum malaria episodes, classical monocytes were predominantly reported with increased frequencies of intermediate and non-classical subpopulations35. In a study with SMA and CM groups, the percentage of intermediate monocytes increased in both groups when compared with the convalescent phase, but no difference was found for non-classical monocytes36. This study, which took place in Kenya, and ours differ mainly in the age of the patients. Kenyan children with SMA were younger (mean 18 months) than children presenting with CM (mean 31.9 months), whereas our study participants had similar ages of around 30 months. Previous studies demonstrated the impact of age on the distribution of monocyte subsets with an increase of both intermediate and non-classical monocytes in adult patients versus newborns37 and in young versus older adults38. Thus, the divergence of these results obtained in children of different ages should be interpreted with caution.Here, we define and quantify the monocyte subsets during acute malaria and study the main traits of monocytes associated with increased malaria severity and mortality. We show first that a lower percentage of CD14et al. obtained results in the same direction as ours since the percentage of non-classical monocytes was lower in children with uncomplicated malaria than in healthy children of the same age39. In addition, a similar association was shown in patients presenting with stroke, the majority of whom had ischemic stroke, a neuro-inflammatory pathology, as is cerebral malaria40. The increased proportion of CD14low CD16+ monocytes at inclusion was associated with better outcomes. In patients presenting with systemic inflammatory response syndrome, lower non-classical monocytes was also associated to death whereas intermediate and classical monocyte counts were not related to patient survival or death41. Urra et al. suggest that the non-classical monocytes are involved in tissue repair as demonstrated for LY6Clow monocytes in mice, a cell population similar to human CD14low CD16+ monocytes40. However, a study aiming to describe functional phenotypes of the different monocyte subsets showed that non-classical monocytes display high cytotoxic and antigen presenting abilities42. Furthermore, they showed that non-classical monocytes were the main producers of inflammatory cytokines (TNF and IL-1β) with a strong and stable CD16 expression. Therefore, the authors propose that non-classical monocytes are pro-inflammatory cells involved in antigen presentation42.Interestingly, Dobbs 43. CX3CR1 (CX3CL1 receptor) and CCR2 (MCP-1 receptor) are two chemokine receptors involved in circulating monocyte recruitment to the inflammation site; CX3CL1 is also involved in monocyte attachment to the endothelium with subsequent transmigration43. This inverse correlation of the proportion of CD14low CD16+ monocytes and CX3CR1 expression is surprising since this cell population has been reported as CX3CR1high22. Such a profile appears rather anti-inflammatory despite the inverse correlation with plasma IL-10, a key anti-inflammatory cytokine. In addition, higher plasma levels of IL-1β, IL-6, IL-10, CXCL10 and mRNA levels of CCR2 and CX3CR1 were associated with severity and death outcome. This suggests that pro-inflammatory response, high plasma IL-10 and increased cell trafficking are markers of poor prognosis. Since non-classic monocytes were mostly inversely correlated to these parameters, these results reinforce the idea that this monocyte subset may play an important role in resolving cerebral malaria.In our study, a higher prevalence of non-classical monocytes was associated with lower plasma IL-6, IL-10, MCP-1, CXCL10, and lower mRNA expression of CX3CR1 and CCR2. MCP-1 and CXCL-10 are two chemokines involved in inflammatory monocytes trafficking and in Th1 responselow CD16+ monocytes were also defined by higher CD16 expression in comparison to CD14+ CD16+ monocytes, which is consistent with the conclusions of Mukherjee et al.42. High CD16 expression was associated with lower severity and better survival in our patients, suggesting that the role of non-classical monocytes during cerebral malaria involves the expression of CD16. The CD16 receptor is known for its critical role in immune regulation by activating the humoral response44, binding immune complexes leading to opsonic phagocytosis, and the release of proinflammatory cytokines that are toxic for Plasmodium45. CD16 expression may be implicated in parasite elimination, which could explain the relationship between the higher frequency of non-classical monocytes and lower parasitaemia. This monocyte subpopulation was also associated with increased vascular superoxide production in a context of vascular dysfunction46. A transcriptomic and proteomic study of this sub-population also showed improved aerobic respiration and ROS production capacities47. Such properties can also contribute to parasite elimination. However, to clarify the role of CD14low CD16+ monocytes during malaria, additional experiments on the functionality of isolated monocyte subpopulations from patients are needed.In our study, CD1449, and parasite clearance through non-opsonic phagocytosis of iE26, which propels CD36 as a therapeutic target50. Berry et al. showed decreased expression of CD36 in UM patients compared with healthy subjects, and with patients who had recovered from malaria51. These findings were confirmed in a later study since CD36 was less expressed by classical monocytes in acute malaria patients compared to recovery and healthy subjects39. Our data refine these results by specifying that lower CD36 expression by both classical and intermediate monocytes is related to severity and death.In our study, other parameters were also associated with severity and survival or death. Expression of CD14, CD16, CD36 and TLR2 by classical and intermediate monocytes was also related to severity and death in our study. We found that classical monocytes expressed more CD14, CD36 and less TLR2 in UM subjects in comparison to the severe groups , and that higher expression of CD14 and CD36 by these monocytes was related to survival. The receptor CD36 is known for multiple functions such as the recognition and the elimination of apoptotic cells, a process necessary for inflammation resolution52. In our study, HLA-DR expression was lower in CM patients compared to SMA and UM and this downregulation was related to death, while CD86 expression was not clearly associated with severity or death. This result, which can be compared with that of CD16, underlines the importance of the monocyte-stimulated T response in the resolution of cerebral malaria.Beyond their own functions, monocytes are also key cells for the interaction and activation of T lymphocytes and humoral response via the expression of HLA and costimulatory receptors such as HLA-DR and CD86. HLA-DR is a MHC class II receptor critical for peptide presentation to CD4 T cells, while CD86 provides costimulation to T cells for activation and proliferation. They were both shown to be downregulated in SMA and CM patients compared to uninfected children and attributed to an immunosuppressive effectlow CD16+ monocytes are necessary to limit death occurrence in SMA and CM forms, a result confirmed by multivariate analysis. Thus, a small proportion of non-classical monocytes may constitute a marker of poor prognosis in cerebral malaria. Further studies are needed to decipher the functions of this sub-population and understand the mechanisms involved.In conclusion, our data provide, to the best of our knowledge, the first evidence that non-classical CD1453. Severe malaria mainly occurs during the rainy season (April to mid-July).Patients were enrolled in Cotonou  and Sô Tchanhoué . Sô-Tchanhoué is a village located in Nokoué Lake, a permanent natural lake in the southeast of Benin, 20 km north of Cotonou. In southern Benin, malaria is mesoendemic and transmission is perennialEthical clearance was obtained from the Institutional Ethics Committee of the Institut des Sciences Biomédicales Appliquées in Cotonou, Benin. All study procedures were performed in accordance with the institutional policies, guidelines and regulations pertaining to research involving human subjects. Children under 5 years old were enrolled during the rainy season (May to July) in 2014 and 2016 after signed informed consent was obtained from the parents.P. falciparum  and for HIV  was performed, and a venous blood sample was collected for parasitaemia measurement by thick blood smear and for isolation of peripheral blood mononuclear cells (PBMC). Children who tested negative for P. falciparum and positive for HIV infection were excluded from the study. After blood sampling, an adequate antimalarial treatment was administered according to the national policy .Severe cases (SMA and CM) were recruited from the Mother and Child University and Hospital Center (CHUMEL), Cotonou, a referral hospital for severe malaria. Patients were managed by the medical team of each health facility. Uncomplicated cases (UM) were recruited in the medical centre of Saint-Joseph in Sô Tchanhoué. Each child was clinically examined at admission. A rapid diagnostic test for 54, SMA cases were defined as children with asexual P. falciparum parasitaemia, a haemoglobin (Hb) level ≤ 5 g/dl and absence of neurological signs. Cerebral malaria was defined as asexual P. falciparum parasitaemia and a Blantyre coma score ≤ 2 combined with a coma duration of six hours at least, with exclusion of other causes for coma54, particularly trauma and meningitis controlled by lumbar puncture when indicated. Children presenting UM had a positive diagnosis for P. falciparum infection combined with fever >37 °C and no sign of severity. A questionnaire was completed with the parents in order to collect social data and clinical signs and treatment received before hospital admission.As proposed by the World Health Organization (WHO)A biological assessment was carried out by the medical team at admission including haemoglobin, glycemia and blood group. For research purposes, a minimum of 4 mL peripheral venous blood was collected at admission from all patients in a vacutainer tube containing EDTA, and processed within 4 hrs. Giemsa-stained thick blood smears were prepared from venous blood collected in EDTA tubes to quantify the parasite load. Plasma was sampled after centrifugation and preserved at −80 °C. Human PBMC were isolated by a density gradient centrifugation method on Lymphoprep . Cells were frozen in RPMI supplemented with 30% foetal bovine serum and 30% DMSO in liquid nitrogen until cytometry experiments.The levels of IL-1β, IL-10, and MCP-1 in plasma samples were determined using commercially available OptiEIA kits . IL-6 and CXCL10 plasma levels were tested using R & D Systems DuoSet . As suggested by the manufacturer’s instructions, we established the lower limits of detection (LLDs) for the five parameters tested, as follows:  +2 * . Our LLDs were 14.7, 4.8, 22.2, 30.4 and 31.3 pg/ml for IL-1β, IL-6, IL-10, MCP-1 and CXCL10 respectively.th percentile 113,351, 75th percentile 233,360) resulting in a minimum of 1000 live cells of interest analysed for each sample. Acquisitions were performed on a cytometer Fortessa . Forward and side scattering parameters (FSC and SSC) were first used to gate on monocytes among other leucocytes. Three CD14 CD16 monocytes subsets were defined according to anti-CD14 and anti-CD16 labelling , anti-CD16 phycoerythrin (PE), anti-CD36 fluorescein isothiocyanate (FITC), anti-TLR2 allophycocyanin (APC) , and live-dead-violet  to exclude dead cells. For each sample, all cells were analysed in order to maximize the number of cells of interest. So, a minimum of 30,000 total events were acquired for each sample , and variables found to be independent of each other, were tested by backward stepwise logistic regression  and Prism software (version 8.1.0). Non parametric tests were used for all analyses. Mann-Whitney U-test was used to compare continuous values between the different clinical groups , after validation of significant differences between the three groups by the Kruskall-Wallis test. To study quantitative variables, the Spearman’s rank correlation test was used to assess correlations and the Wilcoxon signed-rank test to compare values. To determine the most significant parameters regarding severity and death occurrence, two multivariate analyses were then performed by logistic regression. Only variables that were found to be significant by univariate analysis with Supplementary Tables 1 and 2


Recent reductions in malaria burden have been attributed largely to long-lasting insecticidal nets (LLINs). In March–June 2017, approximately 3 years after a national LLIN distribution campaign, a cross-sectional community survey was conducted to investigate factors associated with malaria parasitaemia and anaemia, in advance of Uganda’s 2017–2018 LLIN campaign.Households from 104 clusters in 48 districts were randomly selected using two-staged cluster sampling; 50 households were enrolled per cluster. Eligible children aged 2–10 years had blood obtained for a thick blood smear and those aged 2–4 years had haemoglobin measured. Associations between outcomes and variables of interest were assessed using log-binomial regression with generalized estimating equations to adjust for household clustering.In total, 5196 households, 8834 children with blood smear results, and 3753 with haemoglobin results were included. Only 16% of children lived in households with adequate LLIN coverage. Overall, parasite prevalence was 26.0%, ranging from 8.0% in the South West to 53.1% in East Central. Limiting data to children 2–4 years of age, parasite prevalence was 21.4%, up from 16.9% in 2014–2015 following the national LLIN campaign. In a multivariate analysis, factors associated with parasitaemia included region , older age , living in a poorer household , one constructed of traditional materials , or without adequate LLIN coverage . Overall, the prevalence of anaemia (haemoglobin < 10 g/dL) was 15.1% and varied geographically. In a multivariate analysis, factors associated with anaemia included region, younger age, living in a traditional house, and parasitaemia, which was the strongest predictor .Three years after a national LLIN campaign, LLIN coverage was low and parasite prevalence had increased. Parasite prevalence varied widely across Uganda; older children, those living in poorer households, and those with inadequate LLIN coverage, were at highest risk of parasitaemia. LLINs may need to be distributed more frequently through mass campaigns or continuously through sustainable mechanisms. Targeting interventions to geographic areas and populations at highest risk should also be considered. Over the past 20 years, substantial progress on malaria has been achieved worldwide, following heavy investment in control measures . In AfriMeasuring the burden of malaria and evaluating the impact of control interventions remains a major challenge . AlthougDespite attempts to intensify malaria control, malaria remains a major problem in much of Uganda , 14, andThis cross-sectional community survey was conducted in 104 health sub-districts from 48 districts, approximately 40% of Uganda , 16. TheEnumeration areas identified in the 2014 national census served as the primary sampling unit and a two-stage cluster sampling procedure was applied , 16. TenA household survey questionnaire, adapted from prior surveys conducted in Uganda including the national Malaria Indicator Survey , 11, wasThick blood smears were made by placing a drop of blood in the middle of a barcoded slide. Slides were dried and kept in the field for no longer than 7 days to avoid auto-fixation and were periodically transported to the IDRC Molecular Research Laboratory (MOLAB) in Kampala for processing and reading. Slides were stained with 2% Giemsa for 30 min and read by experienced laboratory technologists. Parasite and densities were calculated from thick blood smears by counting the number of asexual parasites, per 200 leukocytes , assuming a leukocyte count of 8000/μL. A thick blood smear was considered negative when the examination of 100 high power fields did not reveal asexual parasites. For quality control, all slides were read by a second microscopist and a third reviewer settled discrepant readings, defined as (1) positive vs a negative thick blood smear, (2) parasite density differing by ≥ 25%. Haemoglobin measurements were made using a battery-operated portable HemoCue analyzer .Data were collected using hand-held tablet computers, as previously described , 16. AllA household wealth index was generated using principal component analysis of data based on ownership of assets, household characteristics, and type of household construction materials (excluding floor type); a single wealth index was calculated, categorized into terciles . House tFrom March to June 2017, 5200 households were enrolled in the survey, and 5196 were included in the analysis  in children aged 2–4 years was 2.5%. Given the very low prevalence of severe anaemia in this survey, risk factors were assessed for moderate anaemia (haemoglobin < 10 g/dL), which was 15.1% overall. Anaemia varied geographically and followed a similar pattern to parasitaemia; ranging from 9.6% in the South-Western region to 20.2% in the East-Central region Table . AnaemiaOver the past 10 years, Uganda’s Ministry of Health has intensified malaria control efforts, scaling-up proven interventions including LLINs, which are a key component of Uganda’s malaria control strategy. These efforts successfully reduced Uganda’s malaria burden between 2009 and 2015 . HoweverParasite prevalence is commonly used as a measure of malaria burden and transmission intensity in endemic areas , 32. TheIn this survey, increasing age among children aged 2–10 years was strongly associated with malaria parasitaemia, which has been well-described . Anti-maIn this study, children living in poorer households, and those made of traditional materials, were more likely to be parasitaemic. The complex link between malaria and poverty is well-described –61. In UPlasmodium falciparum malaria is a well-recognized risk factor for anaemia in malaria-endemic settings [Malaria parasitaemia was the strongest predictor of moderate anaemia in this study. The aetiology of childhood anaemia in low- and middle-income countries is multifactorial and complex . Howeversettings –69. Othesettings . In Ugansettings , to 61.5settings , and fursettings . These rsettings , includisettings , as wellsettings , 73.This study had several limitations. First, estimates of parasite prevalence were based on microscopy and may have underestimated the true prevalence of infection . Indeed,In 2017, approximately 3 years after LLINs were distributed in Uganda through a national campaign, adequate coverage with LLINs was low, and parasite prevalence was rising, raising concerns about the sustainability of malaria control gains. Parasitaemia and anaemia were found to vary widely across the country, highlighting the heterogeneity of malaria in Uganda. Strategies to target malaria control interventions to specific geographic areas, and to high-risk populations including older children and poorer households, should be considered.


Plasmodium vivax was the predominant species (76%), with P. falciparum responsible for the remaining 24%. Nyssorhynchus darlingi (previously Anopheles darlingi) is the main vector in Amazonian Peru, where hyperendemic Plasmodium transmission pockets have been found. Mazán district has pronounced spatial heterogeneity of P. vivax malaria. However, little is known about behavior, ecology or seasonal dynamics of Ny. darlingi in Mazán. This study aimed to gather baseline information about bionomics of malaria vectors and transmission risk factors in a hyperendemic malaria area of Amazonian Peru.Malaria remains an important public health problem in Peru where incidence has been increasing since 2011. Of over 55,000 cases reported in 2017, Ny. darlingi between watersheds. A generalized linear mixed effect model (GLMM) was constructed to model the relationship between household risk factors and the EIR.To assess vector biology metrics, five surveys (two in the dry and three in the rainy season), including collection of sociodemographic information, were conducted in four communities in 2016–2017 on the Napo  and Mazán Rivers . Human-biting rate (HBR), entomological inoculation rate (EIR) and human blood index (HBI) were measured to test the hypothesis of differences in entomological indices of Nyssorhynchus darlingi comprised 95% of 7117 Anophelinae collected and its abundance was significantly higher along the Mazán River. The highest EIRs (3.03–4.54) were detected in March and June in URC, LIB and VIB, and significantly more Ny. darlingi were infected outdoors than indoors. Multivariate analysis indicated that the EIR was >12 times higher in URC compared with SAL. The HBI ranged from 0.42–0.75; humans were the most common blood source, followed by Galliformes and cows. There were dramatic differences in peak biting time and malaria incidence with similar bednet coverage in the villages.Nyssorhynchus darlingi is the predominant contributor to malaria transmission in the Mazán District, Peru. Malaria risk in these villages is higher in the peridomestic area, with pronounced heterogeneities between and within villages on the Mazán and the Napo Rivers. Spatiotemporal identification and quantification of the prevailing malaria transmission would provide new evidence to orient specific control measures for vulnerable or at high risk populations.The online version of this article (10.1186/s13071-019-3619-0) contains supplementary material, which is available to authorized users. Nyssorhynchus darlingi  over three years even though the human blood index (HBI) ranged from 0.57 to 0.87 [Plasmodium transmission. For instance, if access to a human blood meal is blocked by a screen or a net, an avian blood meal can maintain a female Ny. darlingi by increasing daily survival, even if a meal on chicken blood is not optimal  is the arlingi, , 5–7. Ho gambiae ). If thile hosts , but itsNyssorhynchus darlingi usually rests outdoors (exophilic) and displays heterogeneous feeding behavior throughout its distribution [Ny. darlingi, the highest EIR recorded is 360.62, in eastern Brazil [ribution –13. In tribution , 14, 15.ribution  but see ribution  for noven Brazil . In Perun Brazil . An annun Brazil ; more efNy. darlingi is seasonal and its abundance is linked to rainfall and fluctuating river levels [Ny. darlingi, such as flooded forest and rainfall pools situated near or alongside lowland rivers [Plasmodium transmission of Amazonian Anophelinae. Rivers in the Amazon Basin are classified as blackwater, clearwater or whitewater [versus blackwater rivers are likely to differ. We hypothesized that one potential contributor of seasonal anopheline diversity and abundance is river type. In the present study, we chose to quantify and compare Ny. darlingi entomological indices from two villages on the whitewater Napo River and two villages on the blackwater Mazán River. We expected that the Ny. darlingi from villages along the whitewater Napo River would demonstrate higher abundance, greater diversity, and higher entomological indices compared with similar villages along the blackwater Mazán River.In many localities across the Amazon, r levels , 6, 21. d rivers , 21, 22.itewater . Generalitewater . FloodedPlasmodium vivax contributed 76.1%  of the malaria cases followed by P. falciparum, which contributed 23.8%  [Plasmodium infectivity and local risk in relation to Ny. darlingi.Regionally, malaria cases have increased from approximately 11,000 in 2012 to over 55,000 in 2017  with Lor 12,563) . The maiMosquito collections were performed in the Mazán District  located 50 km northwest of the city of Iquitos, about 40 minutes by speedboat. Salvador (SAL) and Urco Miraño (URC) are located on the Napo River and Libertad (LIB) and Visto Bueno (VIB) on the Mazán River Fig. . CriteriA population census for each community was conducted during December 2016–January 2017, with an overall 90% coverage. Each house was identified with a unique number and georeferenced using a handheld global positioning system device . In addition, a questionnaire was administered to collect baseline demographic information, health, behavioral and socioeconomic data  and information was registered on Android tablets. These data were subsequently uploaded into a database that is maintained at the Universidad Peruana Cayetano Heredia in Lima, Peru, and at the School of Public Health, Harvard, USA.Plasmodium vivax and P. falciparum) were documented from malaria episodes by means of passive case detection  at local health facilities at the village or district level from the Direccion de Salud de Loreto (DISA-LORETO) in Mazán during the time of the survey  and rainy seasons . Each survey consisted of two nights of human landing catch (HLC) and one night of barrier screens, with a total collection effort of 15 collection nights per village. Indoor and outdoor human landing catch collections were performed  for 12 h (18:00–06:00 h), one collector indoors and one collector outdoors working simultaneously, with rotation of personnel every 3 h. Each night two houses, approximately 500 m from each other, were sampled.To intercept and capture blood-seeking mosquitoes, two barrier screens approximately 15 m long and 2 m high were placed such that the distance from the house/breeding site/resting site was 2–7 m (as described in ). This mNy. darlingi. All households were surveyed by consulting residents about presence and number of their domestic animals  and observation of any feral animals  an animal census was conducted in each community in November 2016 and used to calculate the forage ratio for Nyssorhynchus species, damaged specimens, etc.) was identified using ITS2-PCR-RFLP [cox1 [http://www.boldsystems.org) or GenBank (https://www.ncbi.nlm.nih.gov/genbank). To detect the source of a blood meal, total genomic DNA of each mosquito specimen was extracted from the abdomen using the DNeasy Blood & Tissue Kit . Cytochrome b PCR-RFLP assays were performed to identify mammalian blood meal hosts  [A subsample of specimens from HLC and barrier screen collections that could not be identified morphologically  . In addiand dog) , rat andand dog) , 34. ResP. vivax (PV247-PV210) and P. falciparum using an enzyme-linked immunosorbent assay (ELISA) [Plasmodium vivax Sporozoite ELISA Reagent Kit, MRA-1028K, contributed by Robert A. Wirtz. Female Ny. darlingi (colony specimens) were used as negative controls and female Ny. darlingi infected with P. vivax as positive controls. Optical density was measured at 410 nm in a Bio-Rad ELISA plate reader  60 min after addition of the substrate. The cut-off for positive samples was determined by multiplying the average OD (negative controls) by twice (2×) the negative control for each plate. Fifty-five mosquito samples, that were at or near the limit of detectability, were retested individually by real-time PCR following the protocol in Bickersmith et al. [Heads and thoraces from specimens were analyzed in pools of 3–6 individuals (same collection site/time) to detect  (ELISA) . ELISA kh et al. .Clinical malaria records from the passive case detection of the Ministry of Health (MoH) were linked with the study dataset to identify malaria episodes in our study participants. The patients’ names were the only variable shared between the study database and the malaria notification system. Since names may have been misspelled and incorrectly entered, a normalized extension of the Generalized Levenshtein Distance was used , 38. ThiAn entry from the malaria notification system was associated with an individual from the census database if both have names with normalized GLD less or equal than 0.1. Moreover, to include multiple misspellings on the malaria notification system database, all entries were associated with the same individual if they have the same normalized GLD.Ny. darlingi bites per collector per hour. Infection rate (IR) is the proportion of Ny. darlingi that was determined to be Plasmodium positive. Parity was measured in only 40 specimens and the sample size was too small for results to be meaningfully included. These 40 individuals were used in the calculation of HBR but not tested for Plasmodium and not used to calculate IR/EIR. For calculation of entomological indices, specimens of mixed infections with P. vivax and P. falciparum were added to totals of each of these parasites [Ny. darlingi among seasons and localities we employed Chi-square analysis. To quantify blood meal source, host data recorded in the census were used to calculate the forage ratio (wi) [Bi) [Human-biting rate (HBR) was calculated as the average number of arasites . The entarasites . To testtio (wi)  and selewi) [Bi) . The weiwi) [Bi) . We emplwi) [Bi)  to generwi) [Bi) .Plasmodium spp. To assess significant differences in EIR, a generalized linear mixed effect model (GLMM) was used to handle the nested structure of sampled data: two locations (intra- and peridomestic) sampled per survey, nested within 16 households. A GLMM assuming a negative binomial distribution for the error term, and a log link function was constructed to model the relationship between household risk factors and the EIR. We estimated an incidence rate ratio (IRR) for the association between EIR and intra- and peridomestic collection, study area, number of inhabitants per household, inhabitants/room ratio, whether family was recently settled in the community, number of bednets per household, years since last bednet impregnation, and electricity supply. Statistical significance was defined as a P-value < 0.05 and 95% confidence intervals (CI) were estimated as appropriate. Factors with P-values < 0.2 for the Wald test in the univariate analysis were included in the multivariate model. Using a backward stepwise process, the final model retained all factors that were significantly associated with EIR. Interactions were systematically checked for up to order two. Likelihood ratio tests (LRTs) were used to assess statistical differences between nested models. All data analyses were conducted in STATA v.15.1 .Fisher’s exact test was used for significance testing of categorical factors for each P. vivax and P. falciparum, with the highest case numbers in SAL and LIB. Occupation as a housewife or student and having electricity were associated only with P. vivax malaria  whilst pona  vs wood wall material was associated with P. falciparum (P = 0.034). Neither P. vivax nor P. falciparum infection was associated with gender or age, education level, dwelling roof material, or ownership of bednets (LLINs and non-treated)  (Table n = 3530); Libertad (n = 2083); Urco Miraño (n = 679); and Salvador (n = 73). Nyssorhynchus darlingi Root, 1926, was the most abundant species in each of the four communities . Other Anophelinae species identified were Ny. nuneztovari Gabaldon, 1940 (s.l.), Ny. oswaldoi Peryassú, 1922 (s.l.), Ny. benarrochi Gabaldon, Cova-Garcia & Lopez, 1941 B, Ny. rangeli Gabaldon Cova-Garcia & Lopez, 1940, Ny. dunhami Causey, 1945, Ny. triannulatus Neiva & Pinto, 1922, Ny. sp. nr. konderi, An. mattogrossensis Lutz & Neiva, 1911, An. forattinii Wilkerson & Sallum, 1999, An. costai Fonseca & Silva Ramos, 1939, An. forattinii/An. costai, An. sp. nr. forattinii, and several specimens identified only to genus level (Table n = 8) vs those along the Napo River (n = 10).Overall, for both HLC and barrier screen methods, 7117 female Anophelinae specimens were collected in the five surveys in the four sites Table . By HLC,n = 5024; 78.9%) compared with inside houses  in all communities, and more biting occurred prior to midnight  was 1.6% (91/5870 specimens of Ny. darlingi tested) and this varied among communities: Libertad, 27/1895 (1.4% IR); Visto Bueno, 50/3394 (1.5% IR), Urco Miraño, 13/519 (2.5% IR); and Salvador, 1/62 (1.6% IR) were positive , followed by P. falciparum in 19 pools  and P. vivax V247 in 10 pools . Seven pools included specimens with mixed infections.All Ny. darlingi, presented a similar trend in all communities and peaked in June 2016 , > 26 in LIB  and > 56 times higher in VIB  compared with SAL  and multivariate models . Recent settlement in the community, electricity and time of the last bednet insecticide impregnation were associated with a higher risk of EIR but these factors were not retained in the multivariate model  (Table Nyssorhynchus darlingi (n = 679) was the most abundant, followed by Ny. dunhami (n = 2), Ny. benarrochi B (n = 2), Ny. rangeli (n = 1), An. sp. nr. forattinii (n = 1), Ny. triannulatus (s.l.) (n = 1), Ny. konderi (n = 1), Anopheles (Anopheles) spp. (n = 18) and several damaged specimens that could not be identified (n = 47) (Table n = 415) were captured on the village-forest barrier screen compared with the village-breeding barrier screen (n = 337). Because mosquito abundance in June accounted for > 95% of all the mosquitoes collected, data analyses comparing abundance among months or seasons were not performed. Regarding screen side, 41.2% of the mosquitoes were captured on the village side, 29.7% on the forest side and 28.9% on the breeding/river side , and then evenly distributed among the remaining three time periods: 18:00–21:00 h (n = 184); 24:00–3:00 h (n = 128) and 3:00–6:00 h (n = 96). Only 6.8% of the Anophelinae were determined by visual inspection to be blood-fed.Eight different species of Anophelinae were collected on the barrier screens (2) Table . Nyssorhfied n = 7 ; URC (n = 143); LIB (n = 107); and SAL (n = 24) (Table n = 160) were distributed among three communities: . Triple feeds were found in LIB (n = 10), URC (n = 5) and SAL (n = 3), and quadruple feeds (n = 3) only in LIB and SAL , with a higher proportion of human blood meals in VIB than in the other three sites . Forage ratio A  was highest for humans only in VIB. In URC and LIB, it was highest for cows and in SAL for pigs. In contrast, forage ratio B  was highest for Galliformes in URC, LIB and VIB, and for cows in SAL  compared with the blackwater Mazán . In fact, the trends were nearly opposite: abundance and entomological indices were higher in the Mazán River communities, and species richness was nearly identical for Anophelinae along the two river systems. There are many environmental variables that influence these aspects of the vector biology of Ny. darlingi for biting pattern, location and feeding choices at a microgeographical scale. Remarkably, our findings show that EIR is significantly higher in the peridomestic area (exophagic mosquitoes) in all study sites, but particularly elevated in the Mazán River micro-basin. Contrary to our hypothesis, our study shows that the present study sites (villages) located on the bank of blackwater rivers have higher mosquito abundance and EIR than those on whitewater rivers. Characterization of the biotic and abiotic factors that determine the distribution of Anophelinae larval habitats in both watersheds will provide a more comprehensive malaria transmission scenario. For instance, Prussing et al. [Ny. darlingi in the peri-Iquitos area, such as low forest coverage, low sunlight exposure and emergent vegetation.Entomological surveillance is imperative to characterize malaria transmission patterns that will lead to planning adequate vector control measures in a specific context. Overall, this study reflects the behavioral heterogeneity of g et al.  have outNy. darlingi to be the predominant Anophelinae species in the Mazán and Napo river communities [Ny. darlingi peak in other communities in peri-Iquitos, for example in Lupuna, Cahuide and Villa del Buen Pastor, where the highest HBR was reported in March-April for the same mosquito species [Our findings support those of a prior study that revealed munities . The abu species . Because species –7, diffeNy. darlingi prefers to bite outdoors independently of the site, although the outdoor:indoor ratio varies slightly and it decreases in March, probably when mosquitoes are seeking shelter during the rainy season [Ny. darlingi in the region [In general, y season , 13 or dy season –47. Outde region , 6, 48, e region . For inse region , nets wePlasmodium infection rate was higher in mosquito populations from the Mazán River  compared to the Napo River in specimens collected inside and outside. The EIR was also higher in VIB and LIB, with higher values outdoors than indoors and within a range comparable to previous studies in the area [The mosquito biting pattern was variable among river basins, communities and biting location. Most of the outdoor bites occurred before 23:00 h, with a peak around 9:00 h, except SAL that had a bimodal pattern (19:00 and 22:00 h). Indoor biting also differed among sites with unimodal (SAL and LIB) and bimodal (VIB and URC) patterns. The the area , 48. ThiP. vivax prevalence related to occupation  from URC and Gamitanacocha (Mazán River), reinforcing the idea that some malaria transmission might occur at some distance from the village sites [The combination of a low HBR and almost zero EIR in Salvador with a considerable number of malaria cases, raises the question of where and when the actual transmission is occurring. This study did not explicitly record all the population activities or collect mosquitoes other than in the peridomestic area or inside the houses. We believe that infective mosquito bites may take place in areas where people work or perform other activities such as bathing in creeks or social activities such as playing soccer or watching TV. Our statistical multivariate model supports this premise, estimating up to 56 times higher EIR in the peridomestic area than indoors; therefore, individuals who expend more time outdoors during malaria vector peak activity constitute a high-risk group and specific mosquito feeding deterrent measures might be considered. In line with this, a multivariate analysis detected higher ge sites . Spatiotge sites , 52.Ny. darlingi have been identified within the Iquitos area associated with habitat ecological characteristics (riverine vs highway) [Ny. darlingi appears to constitute a genetically homogeneous population [Some heterogeneities in biting behavior might be explained by intrinsic characteristics of the mosquito populations, such as genetic differentiation as a result of changed biting behavior. Two genetically distinct subpopulations of highway) . These spulation . GeneticNy. darlingi are present at the time of the survey that were not collected using human baits. In summary, HBI was higher along the Mazán River than the Napo River, with VIB demonstrating the highest and SAL the lowest, with values similar to those described previously in the Peruvian Amazon and for Ny. darlingi [P. vivax parasitemia identified a higher P. vivax prevalence associated with livestock in dwellings in URC [Our findings support the efficacy of the barrier screen methodology to intercept mosquito specimens suitable for blood meal identification analysis in this region . The higdarlingi . In agredarlingi , Gallifos in URC . In our P. falciparum cases but not for P. vivax. In contrast, higher P. vivax parasite prevalence was recorded in houses with walls made of palm leaf or straw in Gamitanacocha (Mazán River) [An association between housing structures  was detected for n River) . Even thn River) , 54.Plasmodium transmission in the Peruvian Amazon. Even with elevated coverage and use of LLINs there is a gap in malaria control that needs to be addressed in the current elimination era. Specific strategies based on behavioral aspects of Neotropical malaria vectors and the human population should be developed and tested for their efficacy in these settings.Our research revealed heterogeneity in malaria transmission and vector bionomics at a microgeographical scale and that the peridomestic area in these communities contributes substantially to maintain Additional file 1: Table S1. Animal host census in SAL, URC, LIB and VIB, Mazan district, Peru, performed in November 2016. Table S2. Summary of Anophelinae specimens collected indoors/outdoors (peridomestic). Table S3. Forage ratio (wi) and host selection index (Bi) of Ny. darlingi in Mazán District sites between 2016 and 2017. Figure S1. Number of Anophelinae mosquitoes collected with barrier screen by position in Salvador (SAL), Urcomiraño (URC), Libertad (LIB) and Visto Bueno (VIB). Abbreviations: V-F, village-forest; V-BS, village-breeding site.