Iron deficiency is the most common and widespread nutritional disorder worldwide. The automated haematology analyser XN-30  was developed to detect malaria-infected red blood cells (RBCs) in human blood samples using flow cytometry. The optical system of the analyser detects autofluorescence (AF)-emitting RBCs containing iron-deficient haem groups and would aid in the diagnosis of anaemia resulting from iron deficiency. Here, an RBC-optical (RBO) channel was devised and implemented on the analyser. In vitro analyses showed that the analyser detected AF-emitting RBCs treated with 5-aminolevulinic acid. Furthermore, the analyser detected AF-emitting RBCs in mice fed a low iron diet and infected with a rodent malaria parasite; it could also be effectively used in humans. This study demonstrates that the analyser can quantitatively and reproducibly detect AF-emitting RBCs and measure other haematological parameters, suggesting its usefulness for the initial evaluation of latent iron deficiency anaemia in conjunction with the diagnosis of malaria. In nutritionally poor regions, the diagnosis of IDA is often complicated with infectious diseases such as malaria, parasitic worms, and HIV/AIDS1. Because iron deficiency is easily treated with iron supplementation3, the World Health Organization recommends iron supplementation in conjunction with effective malaria prevention and treatment strategies5. However, iron administered to women and children without prophylaxis or access to adequate health care is often accompanied by an increase in malaria incidence, thus suggesting that IDA protects against Plasmodium falciparum malaria infection11. This contradiction generates a dilemma for policy makers in dealing with public health management in malaria-endemic areas13 and makes further clinical studies necessary. Therefore, a rapid and simple assay to detect latent IDA in malaria patients who require additional treatment would be valuable in routine laboratory testing, especially in areas where malaria is endemic and iron deficiency is common.Iron deficiency anaemia (IDA) is the most common and widespread nutritional disorder and is estimated to affect approximately two billion people worldwide14. In this system, the nucleic acids of malaria-infected RBCs (iRBCs) are stained with a Fluorocell M solution and excited by a semiconductor 405 nm laser beam. Therefore, we expected the optical system of the XN-30 analyser to detect RBCs containing iron-deficient haem (IDH), which is excited with a 402 nm laser and emits at approximately 631 nm15.The automated haematology analyser XN-30  was developed to quickly detect malaria-causing parasites and to calculate parasitaemia in human blood samples through flow cytometric analysisThis study aimed to demonstrate the applicability of the XN-30 analyser for the initial evaluation of IDA in routine laboratory testing. We devised and implemented an RBC-optical (RBO) channel on the analyser and validated detection of autofluorescence (AF)-emitting RBCs in vitro and in vivo. Finally, we assessed the ability of the analyser to detect AF-emitting RBCs in healthy human blood samples.14; in the RBO chamber, the samples were treated with CELLPACK DFL, which enables the precise determination of RBC size. The treated samples were sequentially measured in the same flow cell in the order of the M and RBO channels. The flow cell was completely washed using CELLPACK DCL after the detection of iRBCs from the M chamber and was used for the detection of AF from RBCs carried from the RBO chamber. Similarly, the flow cell was completely washed using CELLPACK DCL after the detection of AF. In addition, the M and RBO chambers were also completely washed using CELLPACK DCL every measurement. Therefore, the acquired fluorescence was the background level after every measurement (data not shown). The detected signals were automatically displayed on the M and RBO scattergrams as parasitaemias (MI-RBC%) and AF rate (AF%)  Fig. a.Figure 15. In contrast, the generation of AF was disturbed by the addition of succinylacetone (SA), which is an inhibitor of aminolevulinic acid dehydratase in the PPIX synthesis pathway , shown as the vertical axis, refers to the cell size. Therefore, the upper and lower populations represent RBCs and platelets, respectively Fig. a. In addway Fig. d. These way Fig. e.To characterise the AF obtained from the RBO channel, RBCs were treated with ALA. The AF% of iRBCs was higher than that of RBCs and increased according to ALA concentration and incubation time Fig. a. In addTo determine the factors affecting the enhancement of AF, we examined culture conditions. The analyser showed that stored RBCs increased AF% Fig. c, whereap < 0.001), suggesting the recovery of RBC size  in the liver, erythropoietin (Epo) in the kidney, and erythroferrone (Erfe) in the spleen. Two weeks after ND- and LID-feeding, the AF% of the LID-fed group was higher than that of the ND-fed group . In comparison with other haematological parameters, the AF-emitting RBC counts of the positive group was significantly higher than that of the negative group . In addition, the MCV and mean corpuscular haemoglobin (MCH) values of the positive group were significantly lower than those of the negative group , whereas the RDW-CV value of the positive group was significantly higher than that of the negative group . Although other parameters did not differ significantly , suggesting that reticulocytes or immature RBCs contain low haemoglobin content in the positive group. Similarly, the RBC-He value of the positive group was significantly lower than that of the negative group , whereas the percentage of hypochromic RBCs (%Hypo-He) and the percentage of microcytic RBCs (%Micro-R) in the positive group were higher than those in the negative group  of ALA alone20. The current study also demonstrated that the addition of 500 µM ALA partially inhibited parasite growth , which is excited by 405 nm beams and emits at approximately 630 nm, similarly to PPIX33. ZnPP persists throughout the life span of RBCs as a biochemical indicator of functional iron deficiency35. Because our methodology does not distinguish ZnPP from PPIX, the XN-30 analyser implemented with the RBO channel possibly detected RBCs containing ZnPP but not PPIX in the in vivo studies using mouse and human samples.The insertion of iron into PPIX is a pivotal step in the production of haem during erythropoiesisIn the current study, AF% was defined as the ratio of RBCs having an intensity higher than the LT Fig. a. Althou37. This accumulation leads to the development of PDD and PDT42. The principle of AF generation in cancer cells is the same as that in RBCs. Therefore, RBO technology could also have an application in cancer diagnosis and therapy.In cancer cells, treatment with high concentrations of ALA results in IDH accumulationIn conclusion, the RBO channel-implemented XN-30 analyser quantitatively and reproducibly detected AF-emitting RBCs in vitro. In addition, the analyser detected AF-emitting RBCs in mice fed an LID and infected with rodent malaria parasites; it was also effective for detection in healthy human subjects. Collectively, these results suggest that the XN-30 analyser implemented with the RBO channel would be useful for the initial evaluation of latent IDA in conjunction with the diagnosis of malaria in malaria-endemic and/or non-endemic areas.The use of blood samples from healthy Japanese volunteers was approved by the institutional review committees of the Research Institute for Microbial Diseases (RIMD), Osaka University  and Sysmex corporation . Informed consent was obtained from all participants. All experimental protocol and methods in this experiment were performed in accordance with the relevant guidelines and regulations from the Declaration of Helsinki. All animal experiments were conducted in accordance with the guidelines of “Animal experiment rules” established by the Research Institute for Microbial Diseases, Osaka University, and were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University .ALA was purchased from Cosmo Bio  and prepared as a 100 mM stock solution in saline (0.9 g/L NaCl). SA was obtained from Cayman Chemicals  and prepared as a 10 mM stock solution in saline. Ko143 was purchased from Sigma-Aldrich  and prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO).P. falciparum parasites ) was used for the detection of malaria parasites. In brief, the XN-30 analyser aspirated and diluted blood samples in a CELLPACK DCL solution at a specific dilution ratio (1:50). Subsequently, nucleic acids were stained with a staining solution (Fluorocell M) along with a lysis solution (Lysercell M), and iRBCs and WBCs were excited by a 405 nm laser beam. Approximately 100 µL of the culture suspension diluted with 100 µL of phosphate-buffered saline was added to an EDTA-K2 microtube  and loaded in the XN-30 analyser with an auto-sampler, as described in the instrument manual (Sysmex). The XN-30 analyser generated an M scattergram, which showed the developmental stages of the parasites according to DNA content and iRBC size14. The parasitaemias  were reported automatically.An XN-30 analyser (Sysmex) equipped with a prototype algorithm for cultured The RBO channel was implemented on the XN-30 analyser. The test samples were injected into the RBO chamber and then treated with CELLPACK DFL. The treated samples were measured in the flow cell in order of the M and RBO channels. For the RBO channel, approximately 60 thousand RBCs were evaluated in this study of the human peripheral blood sample. The analyser simultaneously provided an RBO scattergram, in which the dots were presented according to AF intensity and cell size see Fig. . HorizonFor in vitro analyses, RBCs and ring-form-synchronized iRBCs were prepared in parasite culture medium or in PBS with the indicated concentration of ALA, SA, and/or Ko143 and incubated under defined culture conditions (see the section: Parasite strain and culture in vitro). In in vivo analyses, the addition of ALA is not required because IDH is generated in response to diet and malaria infection.P. falciparum laboratory strain 3D7 was obtained from Prof. Masatsugu Kimura . For the assessment of antimalarial activity of the compounds in vitro, the parasites were cultured in RPMI 1,640 medium (containing 2.0 g/L glucose) supplemented with 0.5 g/L L-glutamine, 5.96 g/L HEPES, 2 g/L sodium bicarbonate (NaHCO3), 50 mg/L hypoxanthine, 10 mg/L gentamicin, 10% heat-inactivated human serum, and RBCs at a 3% haematocrit level in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 37 °C, as previously described43. RBCs infected with ring-form parasites were collected using the sorbitol synchronisation technique44. Briefly, the RBCs were collected by centrifugation at 840 g for 5 min at room temperature, suspended in a fivefold volume of 5% D-sorbitol  for 10 min at room temperature, and then washed twice with RPMI 1,640 medium to remove the D-sorbitol.16. Briefly, female ICR mice (21 days-old) were purchased from Japan SLC . For induction of IDA, 7 mice were fed a LID containing 3.6 ppm Fe. As a control, CLEA Rodent Diet CE-2, which contains 310.2 ppm Fe, was used as the ND. Both diets were obtained from CLEA Japan . The animals were fed a LID for 14 days, after which a ND was given for 7 days.Experimental IDA was induced by a LIDHamp, Epo, and Erfe mRNA were normalized to the reference 60S ribosomal protein L4 (Rpl4), hypoxanthine phosphoribosyl transferase (Hprt), and Rpl4 mRNA, respectively45. Data were expressed as relative quantification, i.e. comparison between ND and LID mice. Primer sequences are indicated in Table Total RNA was harvested from mouse liver, kidney, and spleen with the RNeasy Mini Kit . Complementary DNA was synthesized using the High Capacity cDNA Reverse Transcription Kit . Quantitative polymerase chain reaction (qPCR) was performed with Luna Universal qPCR Master Mix  and run in triplicate on the Illumina qPCR Eco system . Expression levels of 5 iRBCs with the non-lethal strain P. yoelii 17XNL. Three mice were analysed for analysing the parasite infection  were exported from the XN-30 analyser and analysed by the Flowing Software 2.5.1  using three-dimensional analysis40. RBC count, HGB value, PLT count, and WBC count were calculated according to the indicated dilution ratio (1:50), while HCT, MCV, and MPV values were used directly40.Female C57BL/6 mice (6 weeks-old) purchased from Japan SLC were injected intraperitoneally with 3 × 10Nucleic acids were stained with 20 nM coriphosphine O (CPO)  for 5 min at room temperature and then washed with saline once by centrifugation at 840 g for 5 min at room temperature. Images were captured using a BZ-X710 fluorescence microscope . A specific filter  and a BZ-X GFP filter  were used for detection of AF and CPO, respectively.RBCs were treated with 500 µM ALA (dissolved in DMSO) or with DMSO in RPMI 1,640 medium under the parasite culture condition for three days. Treated RBCs were collected by centrifugation at 200 g for 5 min at 4 °C. The collected RBCs were disrupted in water and centrifuged at 200 g for 5 min at 4 °C. The supernatants were measured with an F-4500 fluorescence spectrophotometer . The samples were excited at 405 nm  and their emission spectra were recorded between 200 and 800 nm.2 tube  and were analysed in the XN-30 and XN-1000 analysers within 4 h after blood collection without any dilution. The XN-1000 analyser provided data of RET%, RET-He, RBC-He, %Hypo-He, and %Micro-R values for evaluation of anaemia48.Twenty-one healthy Japanese female volunteers participated in the RBO analysis. Peripheral blood samples were collected by EDTA-Kt test using GraphPad Prism version 5.0.The statistical significance of differences between groups of the LID-fed mice was evaluated through a one-way analysis of variance followed by Tukey’s multiple comparison tests using GraphPad Prism version 5.0 . Statistical significance of differences between two groups in the other experiments was calculated by an unpaired two-tailed Student's Supplementary Information 1


Plasmodium spp. infection.In efforts to control malaria infection, the Democratic Republic of Congo has implemented several strategies. Studies assessing their efficiency mainly involved at-risk groups, especially children under five years of age. This study aimed to determine the prevalence and identify the risk factors associated with Plasmodium ssp.From October 2014 to March 2015, individuals aged at least 15 years were selected randomly and enrolled in a cross-sectional study conducted throughout the country. Microscopy and polymerase chain reaction (PCR) analysis were used for the detection of Plasmodium spp. infection assessed by microscopy (355/ 1870 (19%) was lower than that estimated by PCR (580/1870 (31%). In addition, the difference between the two results was statistically significant (P < 0.0001). The most prevalent Plasmodium species was P. falciparum, either as mono-infection  or combined with P. malariae . The mean parasite density was 3272739 trophozoites/μL of blood. Women had higher risks of being infected than men ].From 2286 individuals recruited, 1870 with valid laboratory results were included in the study for further analysis. The prevalence of Plasmodium spp. showed that, despite all efforts for malaria control, malaria remains a public health problem in the Democratic Republic of Congo. The high prevalence and parasite density of Plasmodium spp. in adults make this age group a potential parasitic infectious reservoir for the at-risk groups and supports the need to include this age group in further programs for malaria control.In this study, the molecular detection and species identification of  Plasmodium spp. are pathogens transmitted through the bite of infected female anophelines, causing malaria. They are a genus of protozoan (single-celled) parasites that belong to the Plasmodiidae family. Four different species of Plasmodium have been identified in humans: P. falciparum, P. malariae, P. ovale, and P. vivax. Occasionally, humans become infected with Plasmodium species that generally infect animals, such as P. knowlesi  (Table 4).Univariate analysis showed that women had a 2-fold increased risk of having Plasmodium spp. infection and sex, adjusted for the other sociodemographic variables using logistic regression analysis (adjusted ORs) is presented in The relationship between Table 5), the risk of Plasmodium spp. infection was 2-fold higher in females than in males , P = 0.041].In the adjusted analysis . Plasmodium falciparum was identified in all the samples (100%) tested positive by thick smear (microscopy). The prevalence rate found in our study was similar to that reported in Kenyan adults (22%), also using microscopy [P. falciparum and constitute a risk group for severe malaria [The overall prevalence of croscopy . Howevercroscopy . This di malaria .Plasmodium detection in DRC [Plasmodium spp. detected by PCR (31%) was significantly higher than that detected by microscopical assays (19%), with a large proportion of submicroscopic infection (42.2%). In addition, we identified P. falciparum as the most prevalent Plasmodium species infecting humans. The use of PCR for parasite detection in this study offers substantial advantages compared with the microscopical method. The sensitivity of molecular assays for malaria is substantially higher than that of microscopy. In addition, it allows for better identification of parasite species.Consistent with previous studies using molecular analysis for n in DRC , 25, 26,Table 6 summarizes the results of prior studies assessing Plasmodium spp. by PCR in DRC.et al. focused their study on pregnant women infected with P. falciparum in Kinshasa, the capital city of the DRC [et al. involved asymptomatic individuals recruited within only 6 provinces of the DRC [Although comparable results were obtained, these studies were implemented in different settings. Indeed, the study of Taylor et al.  occurred the DRC   in the DRC. The use of molecular analysis strengthens the results of this study, as PCR assays are currently recognized as the most sensitive methods for the diagnosis and identification of Plasmodium [Plasmodium spp. infection and high parasite density in this age group. This high prevalence among adults may be related to the lack of access to prevention and treatment, such as insecticide-treated nets. These groups have not traditionally been the focus of intensive detection and control strategies. A previous study showed that while mothers slept with their young children under nets, other adults used bed nets less frequently [Plasmodium spp. in adults [However, adult household surveys of malaria, at the national level, are not frequent in the DRC. Therefore, this study represents a contribution to the epidemiology of asmodium , 26. Furequently . In addin adults . Indeed,Plasmodium spp.-infected adults can play a critical role as transmission reservoirs. Adults are often asymptomatic or minimally symptomatic as many of them have acquired malarial immunity. Thus, the majority of Plasmodium-infected people are asymptomatic [ptomatic , and genptomatic .Plasmodium spp. infection and potentiality of transmission, targeting malaria interventions towards adults could maximize their impact.Given their contribution to the pool of asymptomatic reservoirs of Plasmodium infection remain very high in adults. In addition, PCR analysis revealed that the use of microscopic assays underestimates the prevalence rate. Therefore, adults infected (symptomatic or asymptomatic) represent a reservoir for parasite transmission. Efforts toward malaria control and eradication should also target adults as the elimination of the parasites in only the at-risk groups (children and pregnant women) or only symptomatic patients, will not be effective as adult carriers will continue to act as parasite reservoirs.The DRC, the second-most endemic country worldwide, has established a national policy for the fight against malaria. Strategic plans have been developed at the national level, and most of them are targeting at-risk groups (children under 5-years-old and pregnant women). Our study has shown that, despite all the efforts made in malaria control, the prevalence and parasite density of S1 FigA probabilistic sampling with four levels was carried out as sampling technique.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Questionnaire(PDF)Click here for additional data file.S2 Questionnaire(PDF)Click here for additional data file.


Severe acute malnutrition (SAM) remains a main cause of mortality among children under five years old. Vietnam needs further study to establish the optimal mid-upper-arm circumference (MUAC) cutoff for improving the accuracy of the MUAC indicator in screening SAM children aged 6–59 months. A survey was conducted at all 16 subdistricts across four provinces in Northern Midlands and mountainous areas. The data of 4,764 children showed that an optimal MUAC cutoff of 13.5 cm would allow the inclusion of 65% of children with weight-for-height z-scores (WHZs) below −3SD. A combination of MUAC and WHZ may achieve a higher impact on therapeutic feeding programs for SAM children. The MUAC cutoff of 13.5 cm (65% sensitivity and 72% specificity) should be used as the cutoff for improving and/or preventing SAM status among children under 5 in the Midlands and mountainous areas in Vietnam. SAM (weight-for-height/weight-for-length z-score [WHZ] less than −2SD) also affected nearly 52 million children under five Since 2013, the WHO guidelines have stipulated the use of MUAC at the community level to screen for SAM, while healthcare workers in primary healthcare facilities and hospitals should assess either MUAC or WHZ status and also examine the bilateral edema in infants and children aged 6–59 months. Children with medical complications and SAM are treated as inpatients in facilities and hospitals, whereas, children without medical complications and SAM are treated at home, following a community-based program for the management of acute malnutrition (CMAM) A MUAC cutoff less than 11.5 cm and/or a WHZ less than −3.0 is currently recommended to detect SAM in all children aged 6–59 months 2.2.1.A community-based cross-sectional survey was conducted in 16 subdistricts of four provinces in the Northern Midlands and mountainous area in Vietnam. Children aged 6–59 months were screened for acute malnutrition using weight, MUAC, height (or length for children aged less than 24 months), and the presence of bilateral edema. The survey included all children aged 6–59 months with no medical complications. The recumbent lengths of the children aged less than 24 months and the height of the children aged 24–59 months were measured using a microtoise (UNICEF), and the measurements were recorded to the nearest 0.1 cm. The children's weight was calculated with a SECA electronic scale following the international recommendations and also recorded to the nearest 100 g. The MUAC measurements were made using a nonstretch tape measure  provided by the National Institute of Nutrition in Vietnam. To determine the children's ages (in months), their birth dates were either extracted from official documents , or mothers/caregivers were asked. Training was conducted for all the measurers . Calibration of the measurement tool was done before the data collection. All data were recorded for each child.2.2.The WHZs (WHO 2006 growth standards) and the other anthropometric indices were calculated using the WHO Anthro for PC software Age less than 6 months or more than 59 monthsWHZ or HAZ or WAZ or MUAC was not availableAll analyses were performed in SPSS 20.0 for Windows . The sensitivity, specificity, and positive predictive values of the MUAC were determined using WHZs as the gold standard. Receiver-operator characteristic (ROC) curves were constructed to present the relationship between the MUAC and WHZ for different cutoffs. The Kappa statistic (k) was calculated, and the association test was considered excellent at k > 75%, good at 75% ≥ k ≥ 40%, or marginal at k < 40%.The data analysis was carried out at the SEAMEO Regional Center for Community Nutrition in Jakarta, Indonesia.2.3.Before agreeing to participate, the children's mothers/caregivers were informed about the purpose of the study and the name of the research institution. As research participants have the right to skip any steps of the survey or to refuse to participate without penalty if they wish, no negative consequences resulted to those who decided not to participate. The cooperation of all participants was voluntary. The collected data will be used for study purposes only. The study protocol was approved by the Ethical Committee of the National Institute of Nutrition in Hanoi, Vietnam .3.A sample of 5,098 children was included. After the removal of 182 outliers, 78 subjects aged more than 59 or less than 6 months and 74 subjects missing MUAC values, 4,764 individuals (more than 93%) contributed to the further analysis.3.1.Of the total 4,764 children aged 6–59 months, 50.3% were male. The median age was 32.6 months . The prevalence of 24–59 month-age children was two times higher than that of children aged 6–23 months, which was 34.1%. The mean MUAC was 14.0 ± 1.1 cm and ranged from 10.0 cm to 21.0 cm. The prevalence of SAM as indicated by a MUAC less than 11.5 cm was only 1.3%. In comparison, the prevalence of SAM as indicated by WHZ was two times higher, at 2.3%. However, the prevalence of MAM as indicated by a WHZ between -3SD and -2SD, was 6.1%, which was smaller than that of MAM as determined by a MUAC between 11.5 cm and 12.5 cm (8.0%). The current MUAC cutoffs for diagnosing global acute malnutrition , MAM (a MUAC between 11.5 cm and 12.5 cm), and SAM (a MUAC less than 11.5 cm) compared poorly to the same categories as defined by WHZs, with Kappas of 16.4%, 11.8%, and 5.5%, respectively.The sensitivity, specificity, and positive predictive values of each MUAC cutoff point compared to the WHZ were shown in 3.2.To determine whether the power of the MUAC to predict GAM and SAM could be improved, ROC curves were drawn against a WHZ < −3S. The sensitivity, specificity, and positive predictive values of each MUAC cutoff were calculated and showed an optimal MUAC cutoff of 13.5 cm for detecting SAM. In 4.In our data set, the MUAC cutoff of 13.5 cm resulted in an absolute increase of 60% in sensitivity, a 27% decrease in specificity, and the highest sensitivity in screening SAM compared to the 11.5 cm cutoff. Our findings are consistent with those reported. A previous study of 39 surveys conducted in a sample of children aged 6–59 months in 10 mostly African countries showed that a MUAC cutoff of 13.5 cm, which is the same as our optimal cutoff, was optimal for identifying SAM Conversely, the higher MUAC cutoff point for detecting acute malnutrition in admission to CMAM also had a lower specificity. Moreover, the data from several countries showed that the higher MUAC cutoff point should be used as a criterion for hospital discharge 2 = 1.42, p < 0.001) (the data not shown). Overall data set between the WHZ and MUACz distribution was be shown in However, identifying different subgroups of children at risk of death using the MUAC and WHZ was understandable because of the MUAC concepts. A study measuring MUAC and triceps skinfold thickness showed that MUAC was strongly related to fat mass in children but poorly related to fat-free mass or overall weight Click here for additional data file.2 = 0.19, p < 0.01) Countries having a higher proportion of shorter children have fewer children diagnosed with acute malnutrition by WHZ and thus more by MUAC alone. The positive association between longitudinal growth (stunting) and ponderal growth (wasting) would increase the proportion of children with low WHZs, while MUAC is not influenced. However, a large data set from 47 countries found a weak relationship between the prevalence of stunting and fewer incidences of GAM detected by WHZ and more by MUAC alone (rThe sitting-to-standing height ratio (SSR) can affect the WHZ, whereas the MUAC is independent. Additionally, long-leggedness may be disadvantageous when we calculate the WHZ because the legs weigh less per unit length than the torso. Thus, in some children, a low WHZ is not as serious as a low MUAC in a nutritional assessment. Some studies found a relationship between different SSRs and different livelihood zones. The SSRs of a pastoralist population were lower than those of the settled population Although the assessors, who had public health or medical doctor backgrounds, received training, and the outliers were few, the lack of testing the measurers' precision and accuracy was a limitation 5.MUAC is considered a quicker, lighter, cheaper, and more robust screening tool at the community level than WHZ. Using a broader cutoff of 13.5 cm raises the sensitivity of the MUAC indicator from 5% (at a cutoff of 11.5 cm) to 65%. Hence, the combination of MUAC and WHZ may achieve a higher impact on the therapeutic feeding program for SAM children. The MUAC cutoff of 13.5 cm (65% sensitivity and 72% specificity) should be used as the cutoff for improving and/or to preventing SAM status among children under 5 in the midland and mountainous areas in Vietnam.


Introduction: Malaria is still an important vector-borne disease in the New World tropics. Despite the recent decline in malaria due to Plasmodium falciparum infection in Africa, a rise in Plasmodium infections has been detected in several low malaria transmission areas in Latin America. One of the main obstacles in the battle against malaria is the lack of innovative tools to assess malaria transmission risk, and the behavioral plasticity of one of the main malaria vectors in Latin America, Anopheles darlingi.Methods: We used human IgG antibodies against mosquito salivary gland proteins as a measure of disease risk. Whole salivary gland antigen (SGA) from Anopheles darlingi mosquitoes was used as antigen in Western blot experiments, in which a ~65 kDa protein was visualized as the main immunogenic band and sent for sequencing by mass spectrometry. Apyrase and peroxidase peptides were designed and used as antigens in an ELISA-based test to measure human IgG antibody responses in people with different clinical presentations of malaria.Results: Liquid chromatography–mass spectrometry revealed 17 proteins contained in the ~65 kDa band, with an apyrase and a peroxidase as the two most abundant proteins. Detection of IgG antibodies against salivary antigens by ELISA revealed a significant higher antibody levels in people with malaria infection when compared to uninfected volunteers using the AnDar_Apy1 and AnDar_Apy2 peptides. We also detected a significant positive correlation between the anti-peptides IgG levels and antibodies against the Plasmodium vivax and P. falciparum antigens PvMSP1 and PfMSP1. Odd ratios suggest that people with higher IgG antibodies against the apyrase peptides were up to five times more likely to have a malaria infection.Conclusion: Antibodies against salivary peptides from An. darlingi salivary gland proteins may be used as biomarkers for malaria risk. Plasmodium spp. , which represents the number of infectious mosquito bites a person receives per night. This provides an indirect estimation of the risk of acquiring the disease. One of the challenges of this technique is the need to detect parasites in mosquitoes that are collected in the field. Mosquito trapping systems can be biased and overrepresent the most abundant species in a specific area. In addition, current methods are unable to pinpoint the exact individuals who have been bitten or whether there are differences in the number of bites different people receive. Thus, current surveillance efforts lack individualized, deployable, and cost-effective tools to measure human-vector contact and improve measurement of malaria transmission risk. Such tools would be particularly valuable in assessing the efficacy of vector control interventions in protecting people from malaria vector bites.Anopheles spp.  and designed four peptides to measure IgG responses in people living in malaria endemic areas. The objective was to evaluate whether IgG antibodies against these peptides could be reliable markers to detect the risk of disease. An. darlingi is one of the most prolific mosquito vectors of malaria in Central and South America : the API in Turbo was 1.22 in 2016 and 0.77 in 2017, while malaria incidence in El Bagre was higher, with an API of 24.7 in 2016 and 21.3 in 2017  at the Universidad de Antioquia (Colombia) following methods described previously  in Iquitos, Peru as described previously  for salivary gland dissection. Salivary glands were pooled  and placed in 1X PBS. To prepare the antigen extract, the pool of salivary glands was allowed to freeze at −80°C and thaw at 4°C four times to induce cell rupture and release of proteins; the resulting SGA was kept in PBS at −80°C until use. Protein concentration was determined using a Thermo Scientific NanoDrop™   were used in the Immunoblotting experiments performed according to Londono-Renteria et al.  for identification by mass spectrometry (MS). Briefly, the samples were reduced in solution containing 50 mM HEPES (pH 8.0) 10% acetonitrile and 5 mM DTT for 40 min at 37°C. After cooling to room temperature, the samples were mixed at 15 mM with iodoacetamide. After 15 min of alkylation, 200 ng of trypsin was added and the samples were incubated at 37°C for 15 h in a final volume of 40μl. The solution was evaporated to near dryness under a vacuum at 50°C. Twenty-five microliters of 0.1% trifluoroacetic acid was added and the pH was adjusted to 2.5 with the addition of 10% trifluoroacetic acid. Samples with an estimated protein content of <2 μg were desalted and concentrated with C18 μ Zip Tips. Samples containing up to 10μg were desalted with C18 OMIX 10 solid phase extraction tips. The digests were eluted with 0.1% TFA, 50% acetonitrile and dried under vacuum. The peptides were dissolved in 12 μl 0.1% formic acid, 3% acetonitrile which was used as the injection solvent. Digested peptides were subjected to the LC/MS analysis using an Orbitrap Fusion mass spectrometer  connected with EASY nLC 1,000 liquid chromatography (LC) system. Nano-LC was carried out with a 5 μL injection onto a PepMap 100 C18 3-μm trap column  and a 2 μm PepMap RSLC C18 column , both from ThermoFisher Scientific. The LC was operated at a 300 μL/min flow rate with a 100-min linear gradient from 100% solvent A  to 40% solvent B  followed by a column wash. A standard data-dependent acquisition performed with a full MS spectrum was obtained by the Orbitrap for m/z 400–2,000 at the resolution of 120,000 with EASY-IC calibration. The precursor ions, with charges from two to eight, were selected, isolated (1.6 m/z window), fragmented by CID, then scanned by the Ion Trap. Survey scans were performed every 2 s and the dynamic exclusion was enabled for 30 s.theGPM.org) using PEAKS v10  using a semi-tryptic search strategy with tolerances of 6 ppm for MS and 0.5 Da for MS/MS and carbamidomethylation of cysteine as a fixed modification and oxidation of methionine as a dynamic modification allowing for two missed cleavages. Peptides were filtered with a 0.5% FDR using a decoy database approach and a two spectral matches/peptide requirement.Acquisitions were searched against the NCBI-nr (05/2019) proteome and the cRAP.fasta database (ETN63669.1) and peroxidase (Protein Accession # ETN66035.1)  to Anopheles and other major culicid disease vector species using online BLAST program. Protein sequence and structure analyses were then performed for the top Anopheles-specific proteins using the Protean 3D package of the DNASTAR software   were selected using the software for 18–22 amino acid peptides (Immunogenic peptides were designed from the most abundant identified proteins: apyrase (Protein Accession # 66035.1) . Proteinpeptides . The AnDPlasmodium Merozoite Surface Protein from P. falciparum (Pf-MSP)   were used to evaluate antibody responses against the parasite.Two recombinant proteins representing the An. darlingi SGA or 2 μg/ml of each peptide in 1X PBS and incubated overnight at 4°C. Plates were blocked for 1 h at room temperature with 2% milk in 1X PBST (blocking buffer) and incubated with 100 μL/well of a 1/100 serum dilution in blocking buffer at 37°C for 2 h. Plates were washed three times with wash solution (1 × PBS and 0.1% Tween) and incubated with 100 μL/well of horseradish peroxidase (HRP)-conjugated goat anti-human IgG , antibodies  at 37°C for 1.5 h. Colorimetric development was carried out using tetra-methyl-benzidine (Abcam) as a substrate and incubated for 2 min before the reaction was stopped using 2 N sulfuric acid. Absorbance was measured at 450 nm. Each sample was tested in duplicate. Three controls were included in each plate: (1) control blank: two wells without SGA to control for non-specific induction of color for any of the reagents used in the test; (2) negative control: two wells with SGA but without human serum to control for any non-specific color induction of the coating antigen; and (3) positive control to control for plate to plate variation and normalize OD values. To determine a cut off value of exposure to Plasmodium antigens and An. darlingi, we tested serum from 12 volunteers living in the US with no history of travel to South America. The OD cut off values to establish exposure were set at 0.167 for Pf-MSP1, 0.153 for Pv-MSP1 and 0.396 for An. darlingi SGA.ELISA conditions were standardized as published elsewhere  and the Kruskal-Wallis test to compare more than two independent groups . Spearman Correlation Coefficients were calculated to measure the strength of association between IgG antibodies against salivary proteins and IgG antibodies against Plasmodium antigens as well as age. Odd ratios and Fisher's exact test were used to assess significance of risk calculation. OD values were categorized as low (less than the median OD) or high  for each peptide: AnDar_PeroX1 (median OD = 0.579), AnDar_PeroX2 (median OD = 0.6612), AnDar_Apy1 (median OD = 0.4410) and the AnDar_Apy2 (median OD = 0.4286). All tests were considered significant with a p-value < 0.05.After testing for normality, we concluded that the data did not meet the normality requirement. Consequently, we performed non-parametrical statistical tests. Specifically, we used the Mann-Whitney ETN63669.1) and a 65 kDa oxidase/peroxidase (Accession# ETN66035.1). Two peptides were designed from each of these proteins  to evaluate immune responses to these specific salivary proteins in people with active malaria infections. The specific sequence of each peptide is described in An. darlingi apyrase described in the current study has a 63% identity with an apyrase of Anopheles gambiae (AGAP011971), and 49% with an apyrase from Aedes aegypti (AAEL006347) and an apyrase of Cx. quinquefasciatus (CPIJ011010). No significant similarity was found with An. albimanus apyrases. The An. darlingi salivary oxidase/peroxidase has a 87.8% identity with the An. albimanus peroxidase (AAD22196.1). The An. darlingi oxidase/peroxidase also presents a 53% similarity with An. gambiae (AGAP010735) while similarity with Ae. aegypti (AAEL000507) and Cx. quinquefasciatus (CPIJ017579) was 48%. However, the selected immunogenic peptides align with identities between 56 and 85% with these proteins  . The mosproteins .p = 0.0097) (p > 0.05) (p = 0.0001) and El Bagre , while AnDar-Apy1 was higher in malaria positive samples only in El Bagre . Interestingly, the peroxidase-derived peptides, only showed significant differences between infected and uninfected samples from El Bagre, where AnDar-PeroX1 was significantly higher in malaria infected people . When comparing the antibody levels regardless of the field site, both of the apyrase peptides and AnDarPeroX1 were significantly higher in malaria infected samples.Serum from a total of 107 females and 72 males with ages between 2 and 83 years old (Age average = 25.1 years old) were included in this study. No significant differences were detected when comparing IgG antibody levels against any of the antigens from either Turbo or El Bagre samples with the exception of AnDarApy2 which was significantly higher in El Bagre (Mann-Whitney test  0.0097) . When co > 0.05) . Howeverp < 0.05). When divided by malaria infection status, a significant positive association between IgG antibodies against AnDarApy2 and AnDarPeroX2 was maintained with both PvMSP1 and PfMSP1 in both malaria infected and uninfected individuals  and P. falciparum  when compared against IgG antibodies in uninfected participants. In the case of IgG antibodies against the peroxidase peptides, we found only significant higher antibodies against AnDar_Perox1 in P. vivax infection , p = 0.1285 , while antibodies against AnDar_Perox2 were significantly higher only when comparing P. falciparum infected against uninfected , p = 0.0131 . No differences were found with SGE antibodies   and either PvMSP1 or PfMSP1  .p < 0.0001). Significant odd ratios were not observed when comparing IgG levels of SGA, AnDar_Perox1 or AnDarPerox2  and 5.1 (AnDar_Apy2) times higher probability of being positive for malaria than samples with lower antibody levels (Fisher's exact test, arPerox2 .An. darlingi is one of the most important malaria vectors in the Americas, and is associated with transmission of both P. falciparum and P. vivax (de Arruda et al., An. darlingi tend to bite more outdoors and in the early evening, which reduces the effectiveness of bed nets (Moreno et al., Plasmodium infection in field-captured mosquitoes. In addition, it is impossible to track individual human exposure to infective bites and estimation of risk that can be later used to determine local transmission dynamics. Tools capable of monitoring human-vector contact are needed to more accurately estimate disease risk. Here, we evaluated the IgG responses against An. darlingi salivary proteins and showed through ELISA that peptides against a salivary apyrase and peroxidase are suitable antigens to estimate the risk of suffering a malaria infection.An. darlingi salivary proteins, an immunogenic ~65 kDa protein band was identified by antibodies in serum collected from study participants. LC-MS revealed 17 salivary proteins, with apyrase and a peroxidase as the most predominant proteins. Most of the identified proteins have previously been associated with blood feeding. Salivary apyrases hydrolyze ATP and ADP inhibiting platelet aggregation. They are associated with host-seeking behavior and the ability of mosquitoes to find blood vessels (Calvo et al., P. vivax or P. falciparum changes the expression profile of apyrase or peroxidase proteins in the salivary gland of An. darlingi mosquitoes. This information would be helpful to determine a potential function of these proteins in malaria transmission by An. darlingi mosquitoes.In an effort to identify candidate salivary peptides from Plasmodium infection since immunization against these proteins induced partial protection against Plasmodium berghei infection. Protection was further improved when animals were co-immunized with saliva and parasite proteins (Dragovic et al., Ae. aegypti saliva (Londono-Renteria et al., Anopheles mosquitoes is still unknown, but it is thought to function either in the suppression of the host immune system, or as an anticoagulant in a wide range of hematophagous arthropods (Ribeiro and Francischetti, Besides the apyrase and the salivary peroxidase described above, other important salivary proteins that had been previously reported were also detected in this study. First, two members of the SG1 family were detected: the putative salivary protein SG-1B along with a SG1-like salivary protein. The SG-1B appears to be uniquely expressed in the female salivary glands, while the SG1-like can also be expressed in males (Arca et al., An. albimanus salivary peptides (Londono-Renteria et al., It is important to note that whole salivary gland proteins contained in the SGA have been successfully used as antigen to detect IgG antibodies in vertebrate serum (Waitayakul et al., Previous studies associated this increase in antibodies with a potentially higher exposure to mosquito bites (Fontaine et al., Anopheles SGA from two different mosquito colony strains, with those differences potentially associated with differences in geographical origins as well as the time of colonization (Londono-Renteria et al., An. darlingi in Iquitos, Peru, while the previous study used SGA from field-caught mosquitoes in Brazil (Andrade et al., In contrast to a previous study in Brazil (Andrade et al., An. darlingi relative to Turbo (Gutierrez et al., An. darlingi population density at the time of this study. Thus, the results presented here are more sensitive at correlating antibodies against salivary proteins with presence of malaria infection rather than An. darlingi bite intensity.Regardless, analysis of the immunogenic proteins among mosquito strains may reveal shared immunogenic proteins that represent suitable candidates to measure risk of disease irrespective of the geographical area or the strains. Thus, it was important to identify the evolutionarily conserved regions within immunogenic proteins as we seek to develop widely applicable biomarkers of malaria risk. Only AnDar_Apy2 was significantly higher in El Bagre, the area with historically higher abundance of Plasmodium antigens and the development of human immunity against malaria symptoms (Doolan et al., Plasmodium antigens (Rodriguez-Barraquer et al., An. gambiae salivary peptide, gSG6-P1 (Badu et al., An. darlingi mosquitoes are endemic. Also interesting is that the correlation between Plasmodium antigens and the apyrase peptides salivary was also significant in the uninfected population. It is important to remember that infected individuals present significantly higher IgG antibodies. People in endemic areas may encounter a significant number of bites containing infective sporozoites throughout their lives. Furthermore, in endemic areas for P. vivax, as the one where this study was carried out, is very difficult to differentiate a recent infection from a recrudescence, and people may suffer several malaria infections in a year. Since this study did not included follow up of patients, we cannot pinpoint the time of the most recent infection. However, our study showed a significant higher antibody levels against both apyrases peptides and the AnDar_Perox1 in P. vivax infected populations suggesting the relevance of these peptides to measure risk of infection with the most prevalent species circulating in this area. Further studies are urgently needed to determine the persistence of antibodies against the salivary proteins and correlate with previous/recent malaria episodes.Previous studies suggested that there is a significant association between the level of IgG antibodies against the most prominent Although further studies including a greater number of serum samples from different geographical areas are needed to validate these peptides as biomarkers of both malaria risk and human-vector contact, the current study represents an important advance in evaluating immune responses against mosquito salivary peptides in Latin America.An. darlingi salivary apyrase and peroxidases are suitable candidate markers for measuring human IgG antibody responses that may be associated with the risk of malaria infections.Peptides derived from the blondono@ksu.edu.All datasets generated for this study in the sequencing data are included in the article and the The studies involving human participants were reviewed and approved by Kansas State University and University of Antioquia. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.BL-R, AL, HV, GV, EC, MF, and MC: study design and data analysis, writing, and reviewing. JM and AT-C: sample collection. KE-V, LR, and MB: mosquito rearing and salivary gland dissection. BL-R and JM: ELISA testing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microscopy remains the gold standard for identification of malaria parasites. However, the sensitivity of malaria microscopy is low. This study evaluated the impact of repeated sampling up to 12 h in 177 children < 6 years with suspected malaria.The median age was 3 years . Eighty-nine percent (158/177) presented with hyperthermia together with one or more of the following symptoms: chills, headache, sweating, fatigue, nausea, abdominal pain, vomiting, diarrhea and cough. Baseline microscopy confirmed malaria in 29.9% (53/177) of the suspects. Repeated testing at 6 and 12 h increased the positive detection rates to 35.0% (62/177) and 41.8% (74/177), respectively. Microscopy underestimated malaria diagnosis by 11.9% on single testing. Children showing classical signs of malaria but with initial negative parasitological reports should be retested between 6 to 12 h to confirm or rule out a diagnosis of malaria. Plasmodium species, namely, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi [Globally, human malaria is caused by five knowlesi . These pknowlesi . In sub-knowlesi . In malaknowlesi . Key to knowlesi .Plasmodium parasites, and is useful in determining the degree of parasitaemia [P. falciparum infections start sub-microscopically before the detection limit is exceeded. In individuals with incompetent anti-malaria immunity such as children under 5 years, malaria-specific symptoms are usually provoked by low levels of parasitaemia. In this study, a strategy of repeated malaria testing of children up to 5 years presenting with suspected malaria was adopted, to evaluate whether smears repeated at 6 and 12 h after the initial sample improved the diagnostic yield.Microscopy remains the gold standard in the laboratory identification of malaria . Howeversitaemia . In casesitaemia . All P. An observational study was conducted at Ga West Municipal Hospital, Amasaman, in the Greater Accra Region, Ghana from March through December 2018. The study participants were children up to 5 years that were suspected of malaria. Parental written informed consent was obtained for serial finger prick samples at baseline, 6 h and 12 h. During admission, children with negative malaria smears did not receive parenteral fluids nor anti-malaria drugs. Once malaria was confirmed, anti-malarial chemotherapy was commenced immediately. Children that remained febrile received tepid sponging and antipyretic therapy. Guardians completed a questionnaire to elicit responses on malaria risk assessment.Any child with axillary temperature above 37.5 °C or rectal temperature above 38.5 °C of any duration with or without any other clinical complaints was identified as a suspected malaria case. All suspected cases of malaria were identified by pediatricians. Data on all other clinical history and/or presenting complains were collected from the patients’ folders.P. falciparum specific histidine-rich protein 2 (Pfhrp2) was done with CareStart™, following the manufacturer's instructions. Finger prick blood samples (approximately 6 µL of whole blood) were used to prepare thick blood films which were air dried, stained with 10% Giemsa for 10 min, and examined using light microscopy to quantify parasitaemia according to the WHO protocol [Detection of protocol .The frequencies of demographic parameters, malaria risk assessment, and presenting complaints were presented as percentages. Detection rate for both microscopy and RDT were calculated based on the total of tested for that category. In subsequent testing, all cases previously confirmed as malaria were excluded. The differences in baseline, 6-h and 12-h parasitological assessment were determined by Chi-square. Significant differences were defined as p < 0.05. Statistical analysis was done by SPSS Version 24 .Blood samples were taken from 177 children with suspected malaria. The median age was 3 years (interquartile range (IQR), 2.0–4.0 years), and 61.0% (108/177) were male. Most of the guardians (54.8%) were self-employed. Most (61.6%) of the patients lived close to open drains. Although 83.6% households owned long-lasting insecticidal nets (LLIN), these were not regularly used in 61.5% of households. Insecticide repellents were used in 51.4% of households. A large proportion of children (63.8%) were nocturnally active, and 21.4% slept in rooms with eaves . At 6 h repeat sampling in children with initially negative results, 9 (5.1%) and 3 (1.7%) additional positive malaria cases were detected using smear and RDT, respectively. At 12 h repeat sampling in children with negative results at the baseline and 6 h testing time points, and additional 12 (6.8%) and 5 (2.8%) positive malaria cases were detected using smear and RDT, respectively. Serial microscopy testing increased the malaria detection rate by 11.9% compared to a single smear at baseline (from 29.9% to 41.8%) which was equivalent to the detection rates from a single RDT (42.9%)  using microscopy and 42.9% (76/177) using RDT  kits is significantly higher. This is because RDT kits are manufactured to detect of blood . Even thin Ghana  and elsein Ghana , malariain Ghana –9. Howevx2 = 0.046, p = 0.83).In this study, malaria microscopy at baseline detected malaria parasites in 30% of children up to 5 years that presented with a clinical suspicion of malaria. Repeat smears, conducted at 6 and 12 h after admission increased the diagnostic yield considerably, with an additional 21 (11.9%) children diagnosed smear positive. Even though tepid sponging and the use of antipyretics were expected to significantly reduce body temperature within an hour , axillarP. falciparum to complete one cycle. Within 3–4 cycles, total falciparum parasitaemia increase exponentially from one parasite to 108 (thus 100 million) parasites, an increase of over about 520,000 parasites each hour which corresponds to an increase of 50 parasites/μL hourly [In a previous study, it was established that liver stage of merozoites double every 8 h. Hence, each hepatocyte contains thousands of merozoites . MoreoveL hourly . This lePlasmodium spp. The mean temperature in these patients was 37.6 °C, and it is possible that subsequent parasitological evaluation may have yielded further positive result. Furthermore, RDT yields improved with serial testing. These findings reveal that a single microscopic or RDT test for malaria reporting underestimates detection by 11.9% and 4.6% respectively.After the 12 h microscopy testing procedure, 103 patients with suspected malaria remained smear negative for Not every pyrexia is due to malaria. Viral, bacterial, mycobacterial, fungal, other parasitic infections, systemic inflammatory conditions and malignancies are important alternative diagnoses in children that present with fever. It is recommended that clinical and laboratory evaluation of pyrexia must be done exhaustively to unearth the actual origin of pyrexia.This study reports that one-time testing for malaria parasites underestimates malaria diagnosis by 12%. We recommend that children showing classical symptoms of malaria, but with initial negative parasitological reports be retested at 6 and 12 h to confirm or exclude the diagnosis of malaria.In order not to unduly keep the pyretic children in the health facility, parasitological assessment was done up to 12 h.


We also explored if P. falciparum genotypes assessed with these molecular markers were associated with relevant malaria epidemiological parameters using a multiple correspondence analysis. A strong association of certain local haplotypes with their geographic distribution in endemic areas, but also with parasite load and presence of gametocytes, was evidenced. Few multiclonal infections and low genetic diversity among locally transmitted P. falciparum samples were detected, consequent with the low transmission intensity of this parasite in Panamá, a pattern likely to be extended across Mesoamerica. In addition, several imported cases were genetically dissimilar to local infections and representative of more diverse extra-continental lineages.Panamá, together with all the nations in Mesoamerica, has committed to eliminate malaria from the region by 2020. As these countries approach malaria elimination and local transmission decreases, an active molecular surveillance to identify genotypes circulating along the border areas is particularly needed to accurately infer infection origin, drug resistance and disease propagation patterns in the region. This study evaluated the genetic diversity and allele frequencies of  Malaria continues to be a major, though neglected, public health challenge for health authorities in Panamá. Despite regional efforts and a national commitment to eliminate locally transmitted malaria cases by 2020 and achieve full WHO certification by 2025 , recent Plasmodium vivax has been responsible for most malaria cases in Panamá (~94% during the last 15 years) [P. falciparum resurgence has been a constant threat for malaria control and elimination. For instance, during a major malaria epidemic observed between 2002 and 2003, autochthonous P. falciparum transmission was reestablished east of the Panamá Canal, with several fatal cases—a situation not observed in the county since 1970 [P. falciparum infections [P. falciparum outbreak [P. falciparum infections in Panamá [P. falciparum transmission has again re-emerged in eastern regions of the country where malaria transmission had been previously interrupted—An event that has been related to migratory events across the Panamá–Colombia border [P. falciparum cases have been imported from endemic countries of different continents, most imported cases coming from South America [Although 5 years) , P. falcnce 1970 ,9,10. Thfections . Intensioutbreak . Nowadayn Panamá ,12. Morea border . Due to P. falciparum parasites circulating in the country can provide to the NMEP new biological insights on the dynamics of parasite transmission, resistance to malaria drugs, infection origin and connectivity between infections [Plasmodium spp. is key to optimize and effectively implement malaria elimination strategies in Panamá—a country with permeable boundaries in Mesoamerica and with a growing number of migrants entering from different malaria extra-continental endemic regions [The National Malaria Elimination Programme (NMEP) in Panamá is now implementing its strategic objectives as part of ongoing malaria elimination efforts . In thisfections . Therefo regions .P. falciparum parasites circulating in Panamá [P. falciparum circulating in the studied areas of the country [P. falciparum parasites are currently susceptible to CQ [P. falciparum epidemic. In this line, and to provide additional information to optimize the NMEP strategies, the present study evaluated the genetic diversity and allele frequencies of merozoite surface proteins 1 and 2 (msp-1 and msp-2) and the glutamate-rich protein (glurp) genes from autochthonous and imported P. falciparum human isolates collected in different endemic areas from Panamá between 2003 and 2019. We also explored if P. falciparum genotypes assessed with these molecular markers were associated with relevant malaria epidemiological parameters.In this regard, few studies have explored the genetic diversity of n Panamá ,9,10. Ov country ,10. Thisle to CQ . ResultsPanamá is in Mesoamerica, bordering the Caribbean Sea and the North Pacific Ocean, between Colombia and Costa Rica . The couP. vivax infections have been widespread throughout the country, whereas during the past decades, P. falciparum autochthonous transmission has only been observed in communities located on the eastern side of the country near the Colombian border  and Instituto Conmemorativo Gorgas de Estudios de la Salud (ICGES), the national reference diagnostic laboratory . As statby ICGES .Plasmodium spp. and parasite density following the national guidelines for malaria control of the Ministry of Health of Panamá [Plasmodium spp gametocytes in blood smears was also recorded. Parasitemia was classified as low, moderate or high according to the number of parasites observed per microscopic field [P. falciparum positivity, we used a nested PCR that amplifies the small sub-unit ribosomal ribonucleic acid (ssrRNA) genes, following a slightly modified methodology from the protocol of Snounou et al. [P. falciparum and P. vivax were included, as other Plasmodium spp.  causing malaria in humans have not been reported in Panamá in more than four decades [Capillary blood samples analyzed in this study were obtained by fingerprick from autochthonous and imported suspected malaria cases between 2002 and 2019. Blood samples were used to prepare thick blood smears and spotted onto filter paper for molecular analysis. Giemsa-stained smears were examined for f Panamá . The preic field . To confu et al. . Specifi decades . All mix decades .P. falciparum samples was assessed by genotyping the conventional molecular markers glurp, msp-1 and msp-2 [glurp and msp-1, a nested PCR approach was performed with specific primers and methods as previously described [msp-1 and the RII block of glurp [glurp and msp-1 genes were assigned according to the molecular weights of the amplified products. The detection of a single PCR fragment for each locus indicated an infection with one parasite genotype and was classified as a monoclonal infection. The presence of two or more different size bands in the same sample was considered as a polyclonal infection. To minimize bias, gels were analyzed independently by at least two researchers and gels were run with an appropriate molecular size marker for comparison.Genomic DNA was isolated from dried blood spots using a commercial kit  following the protocol suggested by the manufacturer for dried spot blood. Genetic diversity of confirmed nd msp-2 . For gluescribed . All ampof glurp . Amplifimsp-2, a previously described Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) approach was used to evaluate its allelic diversity (FC-27 and 3D7) [glurp, msp-1 and msp-2) analyzed in this study.For and 3D7) . Brieflyand 3D7) . Specifiand 3D7) ,21,22. Hmsp-2 partial sequence was further performed by the maximum likelihood method. For this purpose, msp-2 nested PCR products from the block 3 region amplified as previously described [A phylogenetic analysis based on the escribed  were eleglurp, msp-1 and msp-2 alleles was calculated as the proportion of the allele detected for each allelic family out of the total alleles detected. The frequency of polyclonal infection was calculated based on the number of samples with more than one amplified fragment out of the total samples. Single infections were those with only one allele per locus at all the genotyped loci. Multiplicity of infection (MOI) was defined as the largest number of alleles at any locus.The frequency of To analyze the pattern of association between haplotypes, built by combining genetic variants of the loci under study with malaria epidemiological parameters, a multiple correspondence analysis (MCA) was fitted with the command mca from the library MASS  using thSince the resurgence of malaria in Panamá between 2002 and 2004, the DCV-MoH and ICGES began a coordinated molecular surveillance of malaria cases with the goal of detecting and characterizing parasites that carry genetic markers associated with drug resistance . RetrospP. falciparum-confirmed blood samples collected in Panama between 2003 and 2019 as part of routine malaria surveillance. From this total number of samples, 81 (76.4%) were successfully amplified for glurp, 82 (77.4%) for msp-1 and 101 (95.22%) for msp-2. It was possible to genotype 78 (73.6%) samples with the three combined genes. Among these 78 samples, 23 (~29.9%) were preliminarily considered as imported cases based solely on their travel history.The studied population included 106 glurp RII repeat region, coded as Genotypes I–IV. Fragment size ranged from 800 to 1100 bp. Genotypes I (800 bp) and IV (1100 bp) were the most common, present in 49.4% and 35.8%, respectively, of the samples analyzed for this marker. The other two genotypes (II and III) were identified in 12 (14.8%) samples, mostly related to imported cases  harbored msp-1 Genotype I (500 bp), and only five samples from suspected imported cases displayed Genotype II (600 bp)  .msp-2 gene revealed that both allelic families were present in the studied samples. A high proportion of samples carried the 3D7 allelic family (87.1%). Ten samples (9.9%) harbored the FC27 family, from which seven were of imported origin. Three samples carried both allelic families .Restriction profile analysis of the families . In the P. falciparum endemic regions in Panamá, as well as in imported cases from different continents . Haplotype 1  and Haplotype 3  were the most prevalent and widespread. Haplotype 3 was detected in indigenous patients from the three ntinents . Two hapThe MCA analysis showed that high parasitic loads in samples from male adults and without gametocytes were evenly distributed across the studied samples . Haplotymsp-2 gene were further analyzed by sequencing the nested PCR products [msp-2 block 3 region was successful in 42 samples from this study: 33 considered indigenous and 9 imported based on epidemiological data from the DCV-MoH. The size of the sequenced fragments ranged between 400 and 600 bp. Initial phylogenetic analysis clearly separated both msp-2 allelic families: 9 samples for FC27 and 33 samples for 3D7. Phylogenetic tress for FC27 and 3D7 were constructed and analyzed separately.Allelic frequencies for the products . DNA seqP. falciparum samples evaluated in this study, the msp-2 partial sequences were aligned and compared with previously published msp-2 sequences from Latin America and from various geographic localities where the presumed imported cases originated. The final phylogenetic analysis contained 63 sequences, including 42 from this study and 21 reference sequences accessed from GenBank  grouped together with other reference sequences from Asia, Africa and South America  were closely related to the only reference sequences representative for the Mesoamerican region , suggesting that some P. falciparum populations circulating in Panamá might have different genetic structures compared to the rest of the region. Within the FC27 analysis, local cases  grouped together with South American reference samples from Colombia and Brazil and apart from branches containing samples from India and Myanmar . However, when these samples were disaggregated based on the travel history of the patients, imported samples accounted for most of the observed genetic diversity. In fact, when combining the three genes, most indigenous samples grouped in only two of the 12 haplotypes identified in this study (H1 and H3)—a finding consonant with the low transmission of malaria in the country [In this context, the present study has identified a relatively high diversity in Panamanian samples using three conventional molecular genes recommended to genotype  country  and the  country . ImportaP. falciparum parasites circulating in Panamá have been subjected to different drug pressures mostly because of changes in treatment policies based on clinical and molecular findings [P. falciparum in the country, and might explain why, except for Panama, Mesoamerica is remarkably the only region in the world were autochthonous P. falciparum parasites are still sensitive to CQ. Not surprisingly, the phylogenetic analysis did not group the Panamanian P. falciparum samples with other Mesoamerican reference isolates  have been identified by the WHO as having the potential to soon eliminate malaria . In thesP. falciparum samples, echoing the low transmission intensity of this parasite observed in Panamá, a pattern likely to be extended across Mesoamerica. In addition, several imported cases, as expected, were genetically dissimilar to local infections and representative of more diverse extra-continental lineages.This study has shown few multiclonal infections and low genetic diversity among locally transmitted


Twelve-week LC n-3 PUFA supplementation significantly decreased PWV  and heart rate  in patients with AAA. No change was observed for patients receiving placebo capsules. While this raises the possibility that LC n-3 PUFAs provide improvements in aortic stiffness in patients with AAA, the clinical implications remain to be fully elucidated.Abdominal aortic aneurysm (AAA) is a vascular disease involving permanent focal dilation of the abdominal aorta (≥30 mm) that can lead to catastrophic rupture. Destructive remodeling of aortic connective tissue in AAA contributes to wall stiffening, a mechanical parameter of the arterial system linked to a heightened risk of cardiovascular morbidity and mortality. Since aortic stiffening is associated with AAA progression, treatment options that target vascular inflammation would appear prudent. Given this, and growing evidence indicating robust anti-inflammatory and vasoprotective properties for long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs), this study evaluated the impact of these nutrients (1.8 g/day for 12 weeks) on indices of vascular stiffness in patients with AAA. At baseline, pulse wave velocity (PWV) and augmentation index normalized to a heart rate of 75 bpm (AIx75) were significantly higher in patients with AAA compared to control participants (PWV: 14.2 ± 0.4 m.s Abdominal aortic aneurysm (AAA) is a clinically silent cardiovascular disease characterized by permanent and progressive full thickness dilation of the abdominal aortic segment . AAA affFactors contributing to vascular stiffening include increasing age, elevated arterial pressure and asymmetry in the occurrence of the major scaffolding proteins collagen and elastin . In patiThe association between vascular stiffening and inflammation involves (i) impaired vasodilatory responses by inflammatory cytokines , (ii) veImproving vascular stiffness through pharma-nutritional supplementation is a growing area of interest. Our group recently reported a significant decrease in RDW in patients with AAA following 12-week supplementation with 1.8 g long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) . LC n-3 n = 30), recruited for an observational (case-control) study [n = 20) involved in the same study (n = 15) or placebo capsules , for 12 weeks  and a previously described protocol [C·T diff™ Analyzer, as previously described [t-test and are presented as mean ± SD. Categorical demographic variables were compared using a Fisher’s exact test. Experimental data are presented as mean ± SEM and between group differences were examined by student’s t-test analysis. The association between PWV and AAA was assessed using linear regression analysis with adjustment for covariates shown to be imbalanced between groups . Group size estimates for the observational study were based on PWV values reported in healthy controls  and patients with AAA  [p < 0.05.Vascular stiffness was evaluated in men with AAA (l) study  and compme study . The impnce, see ). Patienescribed . The omeescribed . Continu−1 vs. 12.6 ± 0.4 m.s−1, p = 0.014; AIx75: 26.4 ± 1.7% vs. 17.3 ± 2.7%, p = 0.005).PWV and AIx75 measurements were significantly higher in patients with AAA compared to the healthy control cohort (PWV: 14.2 ± 0.4 m.sp = 0.012) following adjustment for covariates shown to be imbalanced between the control and AAA cohorts .Linear regression analysis highlighted a significant association between PWV and AAA  and a significant positive correlation was observed between PWV and RDW  in patients with AAA at baseline. A significant negative correlation was observed between PWV and omega-3 index (p = 0.014) and heart rate (p = 0.009) in patients with AAA . Participants did not receive LC n-3 PUFA supplementation, however, differences in background fatty acid levels were noted through assessment of the omega-3 index. The results indicated that higher omega-3 index values were associated with lower carotid-femoral PWV following age-and sex-adjusted analyses  and following multivariable adjusted analyses . This is concordant with the current study where a significant negative correlation was observed between PWV and omega-3 index in patients with AAA. In this study, twelve-week supplementation with LC n-3 PUFAs  significantly decreased PWV in patients with AAA. This finding is consistent with a large community-based study involving similarly aged individuals without AAA . In thatAlthough the precise mechanisms underlying LC n-3 PUFA-mediated improvements in vascular stiffness remain to be established, their beneficial effects on cardiovascular health are reported to be a consequence of abundant accumulation in the sn-2 position of membrane phospholipids. In this location, LC n-3 PUFAs compete with arachidonic acid (an omega-6 fatty acid) for enzymes involved in the biosynthesis of pro-inflammatory mediator molecules . The subAge, sex, systolic blood pressure and heart rate are determinants of PWV. An effect of heart rate on PWV has been described previously in Sprague-Dawley rats that were fitted with a right atrial pacing electrode . The stu−1 increase in PWV reported for each mmHg increase in SBP [Older populations have a higher PWV than younger populations ,40,41 anDiscrepancies exist in the literature regarding the effect of LC n-3 PUFAs on PWV. Where an effect of LC n-3 PUFAs has been reported, participant age was found to be a determinant of efficacy. In a study recruiting healthy participants, daily intake of 1.86 g EPA and 1.5 g DHA for 12 weeks caused a significant (−9%) reduction in carotid-femoral PWV in older participants (60–80 years), but provided no benefit in a younger cohort (21–35 years) . A trendThis study observed elevated PWV values in patients with AAA compared to a healthy control cohort. A twelve-week LC n-3 PUFA supplementation regimen lowered AAA patient aortic stiffness and resting heart rate to levels that were comparable to the control cohort. While these improvements are likely to be beneficial in terms of risks associated with cardiovascular morbidity and mortality, the clinical implications remain to be fully elucidated.


Plasmodium falciparum had been the main parasite responsible for indigenous cases and primaquine is the first line treatment of cases and for radical cure. However, the lack of knowledge of the national prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency may be one of the constraints to the malaria elimination process. Hence, this first study determines the prevalence of G6PD deficiency (G6PDd) in the archipelago. Blood samples were collected from patients who voluntarily agreed to participate in the study, in the health facilities of eight municipalities on four islands, tested with G6PD CareStart ™ deficiency Rapid Diagnosis Test (RDT). All subjects found to be G6PDd by RDT then underwent enzyme quantification by spectrophotometry. Descriptive statistics and inferences were done using SPSS 22.0 software. A total of 5.062 blood samples were collected, in majority from female patients (78.0%) and in Praia (35.6%). The RDT revealed the prevalence of G6PD deficiency in 2.5% (125/5062) of the general population, being higher in males (5.6%) than in females . The highest G6PDd prevalence was recorded in São Filipe, Fogo, (5.4%), while in Boavista no case was detected. The G6PDd activity quantification shown a higher number of partially deficient and deficient males (respectively n = 26 and n = 22) compared to females (respectively n = 18 and n = 7), but more normal females (n = 35) than males (n = 11). According to the WHO classification, most of the G6PDd cases belongs to the class V (34.5%), while the Classes II and I were the less represented with respectively 5.8% and zero cases. This study in Cabo Verde determined the G6PDd prevalence in the population, relatively low compared to other African countries. Further studies are needed to characterize and genotyping the G6PD variants in the country.Cabo Verde aims to eliminate malaria by 2020. In the country,  Malaria remains one of the major public health problem worldwide, despite the unprecedented gains made so far, with the reduction of the morbidity and mortality thus allowing its elimination in some countries ,2. Basedth century during the settlement of the islands, and with some efforts, the disease has been eliminated twice from the archipelago. In the last years, the malaria epidemiological situation has become unstable with the sporadic seasonal transmission, and local transmission exclusively occurring in Santiago and Boavista islands [Historically, malaria has been the first vector-borne disease in Cabo Verde since the 16 islands ,5.Plasmodium falciparum malaria cases are treated with Artemether + Lumefantrine (AL) and a single dose of primaquine (0.25 mg/kg body weight) in the first day of treatment. While all the P. vivax and P. ovale malaria cases receive the primaquine with the aim to eliminate the hypnozoites. The schizontocidal treatment for non–falciparum malaria is done with the AL or Artesunate + Amodiaquine, following the WHO guidelines [Despite the low malaria incidence, the archipelago is still prone to outbreaks, as the most recent one recorded in 2017 with 423 indigenous cases . This shidelines –12.The G6PD deficiency (G6PDd) is a X-chromosomally transmitted disorder, affecting both men and women, and individuals with G6PD deficiency may develop potentially fatal anaemia during food-induced oxidative stress, medication or infection by microorganisms . The G6PP. falciparum malaria [Studies carried out have identified so far approximately 220 genetic variants causing clinical deficiency linked to the G6PDd –20. The The high prevalence of G6PDd occurs in Sub-Saharan Africa , 17, 22 This was a cross sectional prevalence study to determine the G6PDd prevalence in Cape Verde conducted from September to December 2018 in four out of the nine inhabited islands of the country. During the study period, a total of 5062 volunteers were enrolled to estimate the prevalence of the G6PD deficiency. The technical staff consisted of 50 volunteers, including laboratory technicians, mobilizers and coordination members were involved in collection and analysis of this study.2 with about half millions of residents. The study was conducted on four islands and 8 municipalities based on the risk of malaria in 2017. On the one hand, the islands of Santiago  and Boavista where indigenous malaria cases were reported the last year. While the islands of Fogo (São Filipe) and São Vicente were selected as areas without any local cases during the last century. Noteworthy, Boavista and São Vicente islands consist each of only one municipality of the same name, where the study was conducted.The study was carried out in the archipelago of Cape Verde, which consists of ten islands . The couConsenting informed volunteers residing in the four selected islands were included in the study. The sample size for each study population was determined using data of the last Cape Verdean population census from the National Statistics Institute , which e™ RDTs of , following the manufacturer's instructions. The test has a sensitivity (95% CL) 100% and specificity 97%. Approximately 5 ml of venous blood samples were collected by properly trained laboratory technicians from all G6PD deficient volunteers using sterile venepuncture device and stored in individual tubes containing an Ethylene Diamine Tetra-Acetic Acid (EDTA). Samples were then sent to and stored at +4°C in the Laboratory of Clinical Analyses of the Regional Hospital of Santiago Norte, in Santiago island, for the further analysis to confirm and quantify G6PD the deficiency. People refusing to sign the terms of the consent form or withdrawing the engagement were excluded. Three individuals from which insufficient blood samples were collected for the qualitative and quantitative analysis of the G6PD and samples which did not meet the qualities for the subsequent processessing were excluded.Sample collection was carried randomly from patients attending the health structures in Santiago island, namely, Praia, Santa Catarina, Santa Cruz, São Miguel and Tarrafal municipalities, in Boavista, São Filipe in Fogo and in São Vicente. Individuals of both sexes aged of at least 3 years, who agreed personally, or on the behalf of the authorized person, to participate in the study were enrolled then screened using the G6PD deficiency CareStart In addition to the blood samples collected from each participant, supplement information including the sex, age, residence, citizenship, education, marital status, were also collected, using a pre-conceived data collection sheet.™ RDTs of  were used to confirm the previous results. To crosscheck the enzyme sensitivity, all the cases classified as G6PD deficient or indeterminate were confirmed by quantitative enzymatic analysis spectrophotometry using the RANDOX G-6-PDH KIT, performed on the AU480 automatic analyser , with temperature controlled, at 37ºC.Labelled EDTA-stored venous blood samples were received and individually verified by the quality controller and properly stored in the laboratory at +4°C and analysed within the next three days upon reception. The same G6PD deficiency CareStart The Unicell DxH 800 Beck Coulter was used to count the Red blood cells of each enrolled subject according to their G6PD status as G6PD deficient or intermediate. Samples collected from the Santa Catarina and São Miguel Health centres, both in Santiago Island, were analysed within 24 hours after the collection. While those from the other municipalities of Santiago as well as from the other islands were transported refrigerated (+4°C) and processed within 72 hours in the Santiago Norte Regional Hospital Laboratory. All the equipment were daily controlled and calibrated before carrying out the tests.The enzymatic activity the G6PD deficiency was quantified on the Beckman Coulter AU480 equipment from the whole blood of each G6PD deficient volunteers using the RANDOX G-6-PDH reagent kit, following the manufacturer's instructions. The lyophilized controls were diluted with 0.5 ml of distilled water (Cat.NO.PD 2617 and PD 2618) then stored at +4°C for use within 120 hours. The control samples were used according to the manufacturer’s criteria to enzymatic each test and control the quality of G6PD enzyme activity; with the values 1298mU/ml and 214mU/ml corresponding respectively to a normal and poor enzymatic activity.The results of the quantitative analysis of the G6PD deficiency were generated using a formula pre-incorporated in the software associated with the automatic analyser AU480. The results of the G6PD activity were interpreted according to the manufacturer's guidelines, as i) deficient for an activity <2.5 U / g Hb; ii) partially deficient for activity between 2.5 to 7.8 U / g Hb; and iii) normal for an activity ≥ 7.9 U / g Hb. The observed G6PD variants were also classified according to the WHO guideline .All the demographic data of the volunteers , and the G6PDd test results were recorded in Microsoft Excel database and analysed using the SPSS 22.0 software version. Descriptive statistics and inferences to verify the association between independent variables and dependent variables were performed using the chi-square test. All variables were inserted into the binary logistic model to evaluate the adjusted odds ratio. Statistical significance was set at p < 0.05.rd Ordinary Meeting, held on September 27, 2018, according to the Resolution No. 53/2018.The study was approved by the National Committee for Ethics in Research for Health, of the Ministry of Health and Social Security, during its 863Children and minors were included in the study after obtaining the signed authorization from parents or the legal guardian. Participation in the study was free and on a voluntary basis with a signed informed consent agreement from each participant or the legal guardian for a minor. All the individuals were recruited among those present within the health structures  of the selected study sites during the study period. Potential participants present at the health structure received full details about the project objectives and the study protocol. All participants were free to leave the study or consult the information at any time point of the study.Each volunteer was provided with an individual consent form for signing. Then a socio-epidemiological questionnaire was applied to each of the interviewees, using an accessible language.A total of 5062 blood samples were collected from the eight municipalities of four Cape Verdean islands . The majOverall, of the 5062 samples collected, 125 (2.5%) was G6PD deficient result RDT. No G6PD deficiency was found in Boavista, which prevalence varied spatially between other islands and municipalities. Between islands, it was the highest in Fogo 5.4%) where the study was conducted in only one municipality (namely São Filipe), followed by the Island of São Vicente (3.3%). The overall G6PDd prevalence was 1.7% in the island of Santiago, being the highest in the municipality of São Miguel (3.3%) and the lowest in Santa Cruz (0.4%) . In the % where tThe prevalence of G6PDd in the Cape Verdean population was higher in males (5.7%) than females (1.6%) . One indTM RDT tests, analyses were performed to quantify the deficiency. Therefore, 119 G6PD deficient samples out of the 125 samples tested, were subsequently processed and quantified using the Randox G-6-PDH kit, adapted to the Beckman Coulter AU480. Six samples, one undetermined case and 5 cases of enrolled patients who left the study after the RDT tests, were not processed.After the confirmation of the G6PDd obtained from Carestart™ RDTs, resulted in different results between male and female population , higher than the female (18 and 7). The opposite is verified in the samples normal, 35 in female and 11 in males. Doing the analysis of the G6PD deficient cases, the result is presented in the For the screening test and taking into account its distribution of individuals by enzymatic activity, in according with WHO classification –27, it wMalaria is considered as the greatest source of selective pressure known in mankind's recent history. The presence and the distribution of various polymorphisms associated with the surface antigens of the erythrocytes, including the globin genes  Thalassemias, oxidative stress (G6PD), cytoadherence and the immune system have been associated with a certain level of a protective effect against malaria .G6PDd is considered one of the most common genetic disorders worldwide, but prevalence estimates are highest in Africa, Asia, the Mediterranean, and the Middle East , 29–31. P. falciparum malaria, excepted for pregnant women, women breastfeeding a child under 6 months of age and children under 6 months of age. With the use of primaquine, it is crucial to evaluate the prevalence of G6PDd, especially in the current context of the ongoing malaria elimination programme which requires better management of all confirmed malaria cases to stop transmission. To our knowledge, this study is the first at national level, undertaken to provide such information to support the National Malaria Control Programme to achieve the elimination goal by implementing evidence-based control interventions. Indeed, despite being preliminary and partial the results reported here provide important data on the current prevalence of the G6PDd in Cape Verde. This is especially crucial in the islands with ongoing indigenous malaria and where primaquine is used to stop the transmission. Our study has shown the presence of the G6PDd, thus providing updated data for evidence-based intervention in the areas under consideration for better management of the use of primaquine to prevent its adverse effects.In Cape Verde, the malaria treatment protocol relies on the WHO guidelines for the low-transmission area , 35. TheThis study determined the prevalence of total and partial G6PD deficiency among malaria affected and non-affected groups in Cape Verde. Among the Cape Verdean study population, the overall prevalence of G6PDd was 2.5%. This value is more than three times higher in males (5.7%) than in females (1.6%). These results are consistent with results from a preliminary study in the country in 2010 , and simOur results showed that G6PDd affects more males than females in the country. In fact, the locus of the G6PDd recessive gene has been mapped into the X chromosome. Consequently, a male with the mutation will be hemizygote and will have full expression of the gene causing G6PD deficiency . In contStudies demonstrate that the distributions of G6PD deficient in males is predominantly lie under the 30% G6PD activity thresholds and heterozygous females contribute predominantly to the intermediate activity ranges of less than 80% activity . Our resP. falciparum malaria [P. vivax. Additionally, recent studies in West African countries [P. falciparum malaria but also with severe malaria. Similarly, the same study concluded, no significant association between G6PD deficiency and other species of malaria, including P. vivax and P. malariae or combinations of any two or all the three species, and not evident conclusion on the exact relationship. Another conclusion is that G6PDd is endemic in Africa, and the African G6PDd patients have relatively higher enzyme activity and milder consequences than Mediterranean or Asian, which could be one possible explanation the observed G6PD associated protect from P. falciparum malaria in Africa [Several studies have also suggested that G6PD has a protective effect against  malaria ,49, withountries , 39, 50,ountries  investigountries  to assesn Africa , 28.The WHO (1989)  classifi™ G6PD rapid test [Although in some study using the CareStartpid test –53, had pid test .Some studies report inconsistency in the definition of normal G6PD activity as well as the definition of test sensitivity and specificity used, what can justify the difference in the classification of our qualitative results , 53. OthThis study carried out in Cabo Verde to estimate and quantify the prevalence of G6PD deficiency, concluded that, despite relatively low, the presence the G6PD deficiency, a greatest concern in an area where primaquine is used for malaria treatment in the ongoing elimination programme. The evidence reported here provide imperative information to the NMCP. However, data are partial thus complementary nationwide studies are necessary to map the distribution of the G6PDd across the archipelago, to perform the techinique and test used and to better support decision-making in malaria case management in the context of elimination in Cabo Verde.S1 Appendix(DOCX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.