Plasmodium spp. The aim of this study was to assess the prevalence of submicroscopic malaria in afebrile immigrants living in a non-endemic area.The importance of submicroscopic malaria infections in high-transmission areas could contribute to maintain the parasite cycle. Regarding non-endemic areas, its importance remains barely understood because parasitaemia in these afebrile patients is usually below the detection limits for microscopy, hence molecular techniques are often needed for its diagnosis. In addition to this, the lack of standardized protocols for the screening of submicroscopic malaria in immigrants from endemic areas may underestimate the infection with Plasmodium spp. were performed. Patients with submicroscopic malaria were defined as patients with negative blood smears and detection of DNA of Plasmodium spp. with one or both molecular techniques. Demographic, clinical, analytical and microbiological features were recorded and univariate analysis by subgroups was carried out with STATA v15.A prospective, observational, multicentre study was conducted. Afebrile immigrants were included, microscopic observation of Giemsa-stained thin and thick blood smears, and two different molecular techniques detecting Plasmodium falciparum alone was the main detected species (10 out of the 14 patients) and in 4 cases (28.6%) Plasmodium vivax or Plasmodium ovale were detected. One patient had a mixed infection including three different species.A total of 244 afebrile immigrants were included in the study. Of them, 14 had a submicroscopic malaria infection, yielding a prevalence of 5.7% . In 71.4% of the positive PCR/negative microscopy cases, Plasmodium vivax and P. ovale were detected in almost a third of the submicroscopic infections. Screening protocols for afebrile immigrants with molecular techniques could be useful for a proper management of these patients.The prevalence of submicroscopic malaria in afebrile immigrants was similar to that previously described in Spain. The online version of this article (10.1186/s12936-019-2870-3) contains supplementary material, which is available to authorized users. Plasmodium infections [Plasmodium infections, and especially those caused by Plasmodium falciparum are still one of the most important tropical health problems.Malaria remains the most important parasitic infection for humans, causing about 2000 deaths per day, especially in African children younger than 5 years old . An estiOutside endemic areas, imported clinical malaria is a well-known disease; in 2017 more than 8000 imported malaria cases were reported in immigrants and travellers in European Union countries , of whicPlasmodium spp. [In endemic countries, including high-level and low-level transmission areas, low-density infections seems to be important for the maintenance of malaria transmission –12, as tium spp. –15. Usinium spp. . Some auium spp. , but wheium spp. .Plasmodium vivax and Plasmodium ovale, some studies suggest that submicroscopic cases are more likely to have lower levels of platelets and haemoglobin than non-infected patients, as well as higher rates of malnutrition, anaemia and thrombocytopaenia [Furthermore, in addition to the importance of diagnosing mixed infections with potentially latent species like topaenia –21.Plasmodium species observed in microscopy smears but also to diagnose the above-mentioned patients in recent and long-term immigrants [As for laboratory diagnostic methods, while microscopic observation of thick and thin blood smears and rapid diagnostic tests (RDTs) remain the most extended diagnostic techniques, they are insufficiently sensitive in patients with a low level parasitaemia. Hence, polymerase chain reaction (PCR) has proved to be an important tool not only to differentiate migrants .The aim of this study was to estimate the prevalence of submicroscopic malaria infection among afebrile immigrants in Spain as well as to describe the demographical, clinical and analytical features of these patients.A prospective, observational, multicentre study was conducted. Patients were enrolled at different settings in Madrid  and La Rioja, Spain  between January 2015 and February 2018.Patients from endemic malaria countries . In addition to this, blood samples were sent to the National Centre of Microbiology  where a multiplex nested PCR detecting the four Plasmodium species was performed to validate the commercial PCR  [A total volume of 5 ml venipuncture blood was collected from all included patients and routinely processed at each setting. Microscopic observation of Giemsa-stained thin and thick blood smears were routinely performed by well-trained staff at each centre. All samples were also sent to the Microbiology Service of Hospital Universitario 12 de Octubre where thick and thin smears were observed by the same microscopist for all samples and a commercial real-time PCR  detecting blished) .Plasmodium spp. with one or both molecular techniques. When discordant results were obtained with both PCRs, patients with a positive result in at least one PCR were considered cases of submicroscopic malaria. Anti-malarial treatment was offered to all submicroscopic malaria cases. Malaria was ruled out when both molecular techniques were negative.Patients were classified according to a composite gold-standard reference method . PatientSocio-demographical, epidemiological, analytical and clinical features were recorded using Redcap (Research Electronic Data Capture) software. One clinician from each hospital had access to the Redcap and was in charge of the database.3 leukocytes/mm3, thrombocytopaenia was defined as < 150 × 103 platelets/mm3 and anaemia was defined as haemoglobin level below 13 mg/dl for men and below 12 mg/dl for women. Submicroscopic malaria prevalence was calculated as number of patients with submicroscopic malaria/total number of patients included in the study. 95% confidence interval for uncertainty was calculated.Leukopaenia was defined as < 3 × 10Analysis by subgroups was carried out following a univariate analysis to evaluate association of socio-demographical, clinical and analytical variables. Kolmogorov–Smirnov and Shapiro–Wilk tests were performed to assess the normal distribution of the analysed variables. Student’s t-test and Chi-square test were performed for continuous and categorical variables with normal distribution. Mann Whitney U-test and Fisher’s exact tests were performed when variables did not fit normal distribution.Data analysis was performed with STATA v15 for macOS .Of the 367 initially evaluated patients, 109 29.7%) had a positive blood smear and 24/109 (22.0%) were afebrile. These 109 patients were excluded from the present study, but their socio-demographic features are detailed in Additional file 9.7% had P = 0.163) as well as shorter time since last travel to endemic area . Immigrant screening  and myalgia  were the reasons for consultation associated to submicroscopic malaria cases (P = 0.013 and P = 0.040). Filariasis was also associated with submicroscopic malaria patients . Higher percentages of strongyloidiasis and schistosomiasis were found in submicroscopic malaria patients although these associations were not significant .Fourteen patients were defined as submicroscopic malaria cases by PCR, yielding a prevalence of 5.7% . In 230 patients (94.3%) all microbiological tests for malaria were negative. Although the associations were not statistically significant, submicroscopic malaria cases had shorter time of residence in Spain than negative patients  of all patients attending the immigrant screening consultation. Among women, there was one who was pregnant. Socio-demographic, epidemiological, analytical and microbiological features of the 14 patients with submicroscopic malaria are shown in Table P. falciparum submicroscopic malaria, 2 (14.3%) patients had mixed infections  and 2 (14.3%) patients had Plasmodium vivax infection.Ten patients (71.4%) had In Spain, scarce studies have evaluated the prevalence of submicroscopic malaria in afebrile immigrants , 27. TheP. falciparum, could develop clinical malaria [P. falciparum was detected in three patients who had not returned to endemic area in the year before consultation, noting the possibility of being pauci or asymptomatic for a long period, as described by other authors [P. falciparum infection and mixed or non-falciparum infections were not evaluated.According to some studies from endemic areas up to 50% of sub-clinical children carrying  malaria . Current malaria . In this authors . UnfortuRegarding analytical features, evidence from studies developed in sub-Saharan countries associates anaemia and thrombocytopaenia not only with symptomatic but also with afebrile patients with malaria . In the Anopheles maculipennis complex (Anopheles atroparvus being the most frequent species) is widely distributed throughout Europe and especially Spain [P. vivax , cannotP. ovale , 27. AltP. ovale and P. vivax remains a potential problem, and noting that, as in this cohort, an important percentage of afebrile infections can be caused by these species. In this study, 15% of asymptomatic immigrants attending an immigrant screening consultation had submicroscopic malaria, the revision and actualization of these protocols should be a priority, mainly for those with anaemia and thrombocytopaenia and for those asymptomatic or with atypical symptoms like myalgias.Treatment protocols for asymptomatic patients before arriving to non-endemic countries have been developed in some countries, including the USA . The US The main limitation of this study was the low number of submicroscopic cases detected, limiting the possibility of statistical analysis, and the lack of epidemiological information in some of the cases because of the existing sociocultural and language barrier. It is surprising that only 43% of submicroscopic cases reported having previous malaria infections, suggesting that it could probably be due to the difficulty in communication or due to a lack of awareness of the disease. The population included in the study represents a wide range of the immigrant population living in Madrid, as it is a multicentre study with a large number of hospitals participating in it. Although, only one hospital outside Madrid is included, so maybe a broader study could recall more reliable data.P. vivax and P. ovale were involved in an important percentage of these infections. Screening protocols for afebrile immigrants with molecular techniques could be useful not only for a proper management and treatment of submicroscopic infections but also to prevent the potential reintroduction of malaria in areas where the vector is present.The prevalence of submicroscopic imported malaria found in this study was similar to that previously described by other authors in non-endemic areas in afebrile immigrants, namely Additional file 1: Table S1. Socio-demographic features of the total 109 microscopy positive patients.Additional file 2: Table S2. Laboratory test results of the 109 patients with microscopic malaria.Additional file 3: Table S3. Reason for consultation of the total 109 patients with microscopic malaria.Additional file 4: Table S4. Co-morbidities and other infectious diseases of the 109 patients with microscopic malaria.


Arterial stiffness is evaluated with the measurement of pulse wave velocity (PWV), while overhydration (OH) and nutritional status are evaluated with bioimpedance spectroscopy (BIS). In this study, we investigated the effect of a single dialysis session on arterial stiffness, hydration status, and laboratory parameters. The observational, cross-sectional, cohort study included 71 HD patients with mean age 64 ± 16 yrs. A Complior device was used to perform PWV measurements. The patients were examined immediately before and 15 min after a mid-week hemodialysis session. Body fluids and nutritional status were studied using a Body Composition Monitor (BCM), Fresenius Medical Care. Clinical and laboratory data were also analyzed. Multivariate regression analysis of PWV before HD showed that an OH increase of 1 L relate to a PWV parameter rise before HD of 0.523 m/s. Multivariate regression analysis of PWV after HD showed that a rise of central SBP after HD of 10 mmHg relate to a PWV increase after HD of 0.707 m/s. Our data indicate that hydration status and blood pressure may be major determinants of PWV in HD patients. The mechanisms underpinning increased arterial stiffness in ESRD patients are not fully defined, but major contributors identified include vascular calcification, chronic volume overload, chronic microinflammation, lipid peroxidation, oxidant stress, sympathetic overactivity, and activation of the renin–angiotensin system.In this study, we investigated the effect of a single dialysis session on arterial stiffness, hydration status, and laboratory parameters.The observational, cohort study was carried out in May–July 2015. The 71 patients (mean age 64 ± 16 yrs) who qualified for the study were being hemodialyzed in the Department of Nephrology, Dialysotherapy and Internal Medicine, Medical University of Warsaw, Warsaw, Poland, and the Dialysis Unit of the Infant Jesus Teaching Hospital in Warsaw, Poland. With written and oral instructions, patients were informed about the rules, aims, and benefits of the study and gave their written consent to participation. The inclusion criteria specified that each patient should be tested prior to the midweek HD session, have a stable clinical status, receive standard HD with three dialysis sessions per week, be on HD for at least 3 months prior to study enrollment, be free of overt CVD, and be more than 18 years of age. Excluded from the study were patients who: had atrial fibrillation, since this condition makes pulse wave velocity testing uninterpretable; had an active inflammatory process; had peripheral artery disease; had stage III–IV congestive heart failure according to the New York Heart Association classification; had regular cardiovascular instability on dialysis ; were pregnant; had residual diuresis >500 mL/24 h; had active arteriovenous fistulas on both upper extremities; had metallic implants ; and had undergone amputation of lower or upper limbs.www.clinicaltrials.gov, identifier NCT02443376). The investigation conformed to the principles outlined in the Declaration of Helsinki. PWV and BIS measurements were performed during midweek HD sessions by a trained researcher. Body mass, height, waist circumference, and body mass index (BMI) were assessed. BMI was calculated as body weight (kg) divided by the square of body height (m). Abdominal obesity was defined as a waist circumference of 102 cm in men and 88 cm in women.The study protocol was approved by the local Research Ethics Committee at the Medical University of Warsaw  and registered at the Clinical Trials register (The results of laboratory investigations were measured on the same day as PWV and BIS measurements and were obtained from the patient’s medical records. All laboratory parameters were measured using automated and standardized methods.European Society of Hypertension (ESH). This measurement allowed brachial systolic blood pressure (bSBP) and brachial diastolic blood pressure (bDBP) to be obtained. The values were reported as the mean of two stable readings.Brachial blood pressure was recorded in the dominant or non-fistula arm using a validated oscillometric device , recommended by the t) between the appearance of the pulse wave on the carotid and femoral artery was measured automatically in 10 subsequent cycles (successive beats) and was averaged. Once the result of the measurement of the distance between the sensors (m) was complete, PWV was calculated by the device and expressed in (m/s) according to the equation: PWV = m/t. According to recent recommendation, when measuring PWV the distance between the sensors was automatically multiplied by coefficient 0.8 by software Complior.PWV measurements were performed with the use of a Complior device . Patients were examined in a quiet, air-conditioned room after a 15-min rest in a supine position, directly prior to and 15 min after the commencement of the mid-week HD session. In brief, one of the device’s two sensors was placed on the site of palpable pulse on the carotid artery, and the other on the site of palpable pulse on the femoral artery. The time (2)), Fat Tissue Index (FTI (kg/m2)), Total Body Water (TBW (L)), ECW (L), ICW (L), and OH (L). Two volume ratios were quantified: ECW/ICW and ECW/TBW.,The study of body fluids and nutritional status was performed using a bioimpedance monitor  Body Composition Monitor software version: 3.3.0.1637) that measures 50 different frequencies ranging from 5 to 1000 kHz, thereby analyzing the whole-body bioimpedance. The BCM is commercially available modern device that can classify ESRD subjects in terms of volume status. The bioimpedance measurement was performed directly before the commencement of HD, with the patient in a supine position and with the use of single-use disposable electrodes placed in doubles on the subject’s wrist and ankle on the side opposite to the arteriovenous fistula. The BCM discriminates fluid of the intracellular (ICW) and extracellular (ECW) water content of lean tissue mass (LTM) and overhydration (OH). LTM and OH are obtained from measurements of body weight, height, and whole-body ICW and ECW determined by BIS. OH represents the excess fluid (fluid overload). The following parameters were recorded in each patient: Lean Tissue Index  were presented as quantity (n) and percentage values of the whole group (%), while proportions in groups were assessed with the Chi-squared test. Statistica 12 software  was used in the statistical analysis. p < 0.05 was adopted as the significance level.Results concerning quantitative variables were presented as average values ± standard deviation (SD). In the comparative characteristics of PWV, the n = 27; 38%), followed by diabetes mellitus , hypertensive nephropathy , polycystic kidney disease , and other causes . There were 36 male and 35 female patients. The mean HD time in these patients was 84 ± 71 months. All patients underwent regular dialysis for 4–5 h and were dialyzed with standard bicarbonate HD solutions and synthetic dialyzers with blood flow rates of 200–250 mL/min, aiming at a dialysis dose of Kt/V > 1.2 with mean URR 74.1 ± 4.9%. Mean ultrafiltration (UF) during HD was 2245 ± 659 mL and bodyweight decreased by a mean of 2.3 ± 0.9 kg.Among the causes of ESRD, the most frequent was glomerulonephritis . This testifies to a different arterial elasticity behavior in the patients studied depending on the excess fluid status and that OH is an independent risk factor in arterial distension. The treatment of hypertension and fluid overload are issues of major importance in HD patients.The present study provides new information on the relationship between nutritional status and hydration status, blood pressure, and PWV in HD patients during an intra-dialysis session. The main finding of this study was that in HD patients the PWV correlated with OH, a marker of the fluid status representing body fluid distribution. Our own study findings show that when PWV was increased following HD the mean OH was 2.63 ± 0.9 [L], while when PWV was decreased following HD the mean OH was 1.74 ± 1.1 [L]  are quite low when compared with other studies in HD patient. Too aggressive a rate of ultrafiltration for dialysis lasting 3.5–4.0 h may result in inadequate dialysis, which in turn can lead to OH and the occurrence of volume-dependent hypertension with higher values of PWV. In the present study, the length of dialysis sessions varies on average between 4 and 5 h with low blood flow:- 200–250 mL/h . In the study, after HD if central blood pressure increase, results as increased the PWV, while the decrease, decreased the PWV. PWV increased group have more OH be evocative of paradoxical hypertension. Paradoxical hypertension is defined by BP values during and at the end of the dialysis session exceeding BP values at dialysis onset. It occurs in around 10% of HD patients.p = 0.739). For the second HD session: PWV before HD= 9.58 ± 0.3, PWV after HD = 9.48 ± 0.3 m/s (p = 0.830).In the study by Georgianos et al.The study carried out by Su et al.r = 0.465; p < 0.001) and with after dialysis PWV values . Blood pressure changes during dialysis were weakly correlated with post-dialysis PWV , but not with PWV changes during dialysis.In the study by Di Iorio et al.,p < 0.004). In addition, an HD session significantly reduced AIx from pre-HD 27.9 ± 11.9% to 18.2 ± 18.3% (p < 0.05). With provocative pharmacological use of inhaled salbutamol and sublingual nitroglycerin testing, Covic et al. revealed that an HD session led to a non-significant improvement in the endothelium-dependent vasomotor function, while correcting the endothelium-independent dysfunction to a level comparable to that in a control group of essentially hypertensive patients.The study by Covic et al.p = 0.019). In their study, Yoon et al.Among others, RDW is a relatively new prognostic parameter in heart failure.This is a cross-sectional study in design and cannot provide cause-and-effect associations. The study measurements may be susceptible to biases related to the peridialytic setting of recordings. Data on wave reflection indices were not assessed. To find the most representative time point for PWV measurements during HD, our study design did not include patient evaluation on all three dialysis sessions of the week and inter-dialysis days.Our data indicate that hydration status and blood pressure may be major determinants of PWV in HD patients.


Analysis of anti-malarial antibody responses has the potential to improve characterization of the variation in exposure to infection in low transmission settings, where conventional measures, such as entomological estimates and parasitaemia point prevalence become less sensitive and expensive to measure. This study evaluates the use of sero-epidemiological analysis to investigate heterogeneity of transmission in area conducting elimination in Indonesia.19) and apical membrane antigen 1 (AMA-1) for Plasmodium falciparum and Plasmodium vivax were measured using indirect enzyme-linked immunosorbent assay (ELISA). Seroconversion rates (SCR) were estimated by fitting a simple reversible catalytic model to seroprevalence data for each antibody. Spatial analysis was performed using a Normal model (SaTScan v.9.4.2) to identify the clustering of higher values of household antibody responses. Multiple logistic regression was used to investigate factors associated with exposure.Filter paper bloodspots and epidemiological data were collected through a community-based cross-sectional study conducted in two sub-districts in Sabang municipality, Aceh province, Indonesia in 2013. Antibody responses to merozoite surface protein 1  vs 2.0% (95% CI 1.4–2.8). SCR estimates suggest that there was a significant change in P. falciparum transmission with no exposure seen in children under 5 years old. Plasmodium falciparum SCR in over 5 years old was 0.008 (95% CI 0.003–0.017) and 0.012 (95% CI 0.005–0.030) in Sukakarya and Sukajaya sub-districts, respectively. Clusters of exposure were detected for both P. falciparum and P. vivax, most of them in Sukajaya sub-district. Higher age, P. vivax seropositivity and use of long-lasting insecticide-treated bed net (LLIN) were associated with higher P. falciparum exposure.1624 samples were collected from 605 households. Seroprevalence to any Analysis of community-based serological data helps describe the level of transmission, heterogeneity and factors associated with malaria transmission in Sabang. This approach could be an important additional tool for malaria monitoring and surveillance in low transmission settings in Indonesia.The online version of this article (10.1186/s12936-019-2866-z) contains supplementary material, which is available to authorized users. In recent years, there has been a decline in malaria transmission in many regions, leading to optimism that malaria elimination might be achieved in numerous countries –8. As trAn additional approach to measure malaria transmission is to detect anti-malarial antibodies, which provide a marker for exposure to malaria . MalariaSeroconversion rate (the proportion of people in the population who are expected to seroconvert each year) is a serological parameter used to understand malaria transmission dynamics. Previous studies found that seroconversion rate (SCR) provides a proxy measure for estimating the transmission intensity in a community as it was strongly correlated with the EIR and annual parasite incidence collected by the malaria surveillance programme , 14. Ser19) and Apical Membrane Antigen 1 (PfAMA-1) as serological parameters to assess malaria transmission intensity in Indonesia. First, a population-based cross-sectional study conducted in three different endemicity areas showed the potential application of these methods for detecting changes in transmission exposure, particularly in lower transmission settings and with less immunogenic antigens (such as PfMSP-119) [19 antigens have been reported to persist for several years after infection and in the absence of reinfection, any antibodies detected in younger children would reflect more recent infection in low transmission settings [Recent studies have reported the potential use of recombinant Merozoite Surface Protein 1 (PfMSP-1MSP-119) . Second,MSP-119) . These ssettings . Therefo2 covering five islands but only the largest island, Weh, is permanently inhabited. The population on Weh island is approximately 30,000 and it is divided administratively into two sub-districts (Sukakarya and Sukajaya) with 18 villages. Sabang has a very low-level annual parasite incidence, 0.13 per thousand population in 2011. Based on its geographic position at the western end of the archipelago, its diverse mosquito fauna, the presence of both major malaria parasites, and its strong local government, Sabang municipality was considered as an appropriate place to pilot malaria elimination in Indonesia [The study was conducted in Sabang municipality, Aceh province, Indonesia Fig. . The munndonesia .Fig. 1MaCommunity-based cross-sectional surveys were performed during the malaria transmission season between October and December 2013. Households list were obtained from local authorities and were arbitrarily assigned numbers according to their geographic location. Households were randomly selected and invited to participate in the study. Households with no adult present were excluded from the survey and were replaced by the neighbouring households. Individual signed consent was obtained from all adults or guardians of household member under 18 years of age. Samples were collected from all household members present aged over 6 months. The minimum sample size of 439 individuals per sub-district was met to ensure the antibody SCR of 0.0036 could be estimated with a precision level of ± 0.0013 . StandarPlasmodium falciparum MSP-119, P. falciparum AMA-1, Plasmodium vivax MSP-119 and P. vivax AMA-1 were used as antigens in indirect enzyme-linked immunosorbent assay (ELISA) as described in [2SO4). The optical density was read using ELISA reader at 450 nm. All serology was performed by trained laboratory technicians at the Department of Parasitology, UGM, Yogyakarta.Giemsa-stained thick and thin malaria films reading was performed by trained laboratory technicians to identify active infections. For serological assays, the recombinant proteins P. falciparum if their antibody responses were above the cut-off for PfAMA-1 and/or PfMSP-119 and seropositive for P. vivax if their antibody responses were above the cut-off for PvAMA-1 and/or PvMSP-119. Seroconversion rates were estimated by fitting a simple reversible catalytic model to seroprevalence data for each antibody [P. falciparum (and P. vivax) exposure among study participants. Logistic regression models were performed to estimate odds ratios (ORs) of factors associated with being seropositive to P. falciparum or P. vivax, respectively. Adjusted odds ratios (aORs) were obtained using a multivariable model, including the following covariates: age, gender, seropositivity to P. vivax, education status, employment status, LLIN use, IRS in last 12 months, fever status, and altitude. Samples from participants aged under 18 years old were excluded from analysis of education and employment status. Statistically significant variables (p < 0.05) detected in bivariate analysis were included in a multivariable model. The final model was developed using the forward stepwise approach which compared multivariable models to the most significant bivariate model using p-values calculated from likelihood ratio tests. Scatter plots matrix and coefficient correlation analysis were done to assess potential cross-reactivity between P. falciparum and P. vivax antigens.All statistical analyses were conducted in Stata IC 15 . Infants under 1 year of age were excluded from each dataset to remove any influence of maternally derived antibodies . Raw OD antibody . Models 19, PvAMA-1 and PvMSP-119. The Normal model was used to detect clusters of households with higher than average age-adjusted antibody responses to PfAMA-1, PfMSP-119, PvAMA-1, PvMSP-119 antibody responses. This method has been previously utilized in several studies investigating malaria transmission heterogeneity in low endemic setting [The spatial software SaTScan (v.9.4.2) was used to detect clusters of higher than average age-adjusted antibody responses to PfAMA-1, PfMSP-1 setting , 37. AntGeneral characteristics of the sampled population is presented in Table P. falciparum antigens was higher than seropositivity to P. vivax antigens, with seroprevalence 6.89% (95% CI 5.76–8.24) and 1.97% (95% CI 1.39–2.77), respectively. Seroprevalence ranged from 1.2 to 11.4% for P. falciparum and 0.5 to 2.8% for P. vivax across age groups. Notably, there were no seropositive individuals aged under 5 years old identified for either P. falciparum or P. vivax (0/210). Seroprevalence to each antigen can be found in Additional file Seropositivity to P. vivax and use of LLINs were significantly associated with P. falciparum seropositivity, after controlling for other covariates. As would be expected, seroprevalence increased with age. Adults were more likely to be seropositive compared to children under 15 years old, with adjusted OR 5.69 (95% CI 2.43–13.37), 12.05 (95% CI 5.59–25.94) and 10.27 (95% CI 4.74–22.27) for age group 16–24, 25–40 and over 40 years old, respectively. Seropositivity to P. falciparum was also significantly associated with higher proportion of LLIN use, with adjusted OR 1.80 (95%: 1.20–2.72). In addition, people who were seropositive to any P. vivax antigen were 3 times more likely to be seropositive for P. falciparum, with adjusted OR 3.47, (95% CI 1.48–8.12). Other factors such as gender, residence, education, employment, IRS, fever and altitude were not significantly associated with P. falciparum seropositivity. Multivariable logistic regression revealed that there were no factors significantly associated with P. vivax seropositivity  and SCR 0.012 (95% CI 0.005–0.030) in Sukakarya and Sukajaya, respectively. The SCR estimates for P. vivax  was very low 0.07%  in the region [This study describes the analysis of community-based serological data to investigate malaria transmission dynamics in a low transmission setting, Sabang, Indonesia. The seroprevalence and SCR data represent exposure to infection and demonstrate that the population level of transmission intensity were similarly very low for both ndonesia . Moreovee region .P. falciparum SCR estimates suggest that there was no exposure seen in children under 5 years old in both sub-districts in Sabang municipality. These results could represent a step change in P. falciparum transmission due to the successful impact of malaria control programme implemented in the study area, evidenced by lower antibody prevalence in children born after the intervention scale-up. This finding was supported by a previous study documenting a significant drop in malaria cases after the launch of the control program in 2004. Malaria cases in Sabang declined from 88 cases per 1000 population in 2004 to 1 per 1000 by 2010. The decline in malaria transmission in Sabang is likely related to an extensive IRS programme immediately following the tsunami in 2004, large scale LLIN distribution, and a change in malaria treatment policy to artemisinin-based combined therapy as first-line treatment for uncomplicated malaria [P. vivax, 4 P. falciparum and 2 mixed P. vivax and P. falciparum infections from 2011 to 2013, with no local transmission. However, the surveillance system detected 15 PCR confirmed Plasmodium knowlesi infections that classified as an outbreak in 2014 [The  malaria . Sabang  in 2014 .P. falciparum SCR estimates in people over 5 years old, multivariable analysis revealed that adults were more likely to be seropositive compared to children under 15 years old. This is likely the result of higher exposure by staying overnight in high-risk areas. A recent study revealed that the clusters of malaria (P. knowlesi) infections in Sabang was associated with people who had a history of staying overnight in the forest, without protection from mosquitoes, in an area where macaques are common [P. falciparum seroprevalence was higher. Consistent with previous report suggesting high coverage of LLINs (over 75%) in six malaria focal villages in Sabang, this finding suggests that people living in higher risk of exposure were aware of the importance of LLIN to prevent malaria transmission in those areas [Consistent with the higher e common . Unfortuse areas .P. vivax transmission in either sub-district in Sabang. Plasmodium vivax seroprevalence was very low (2.0%) and, therefore, the absence of any associations is likely due to the statistical limitations of the low number of seropositive samples. The other possible explanation is that P. vivax infections may induce lower antibody responses or shorter-lived responses which the current assay may miss. Work is ongoing to identify P. vivax antigens that elicit short-term responses for easy identification of very recent exposure [P. vivax antigens is supported by a previous study showing that the number of P. vivax cases tend to be higher than the number of P. falciparum cases in Sabang [The estimated age-seroprevalence curves and SCR value suggested that age was not associated with exposure , 40. Then Sabang .19) identified significant clusters of higher exposure (hotspots) for both P. falciparum and P. vivax exposure across the study areas. Although multivariable risk factors analysis found there was no significant association between residence and higher seroprevalence to P. falciparum and P. vivax, the spatial analysis suggested that the risk of malaria transmission in the study setting is heterogeneous with people experiencing higher exposure in Sukajaya sub-district. The spatial analysis also suggest that the clusters identified for P. falciparum and P. vivax were seen in the same areas. Being able to characterize the micro-epidemiology of malaria exposure could assist malaria control programme to better allocate resources and target the intervention to achieve their goal of elimination. Targeting hotspots could be a highly efficient way to reduce malaria transmission at all levels of transmission intensity [P. knowlesi infections in Sabang were identified in similar high-risk areas identified in this study [The spatial analysis of age-adjusted antibody responses to either antigen  to P. falciparum and to P. vivax antigens tested in the study describing the absence of cross-reactivity between the P. falciparum and P. vivax antigens.


This study sought to determine the prevalence of malaria and LF in bateyes of the DR and to describe related risk factors for disease.The island of Hispaniola, shared by Haiti and the Dominican Republic (DR), is the only remaining malaria-endemic island in the Caribbean and accounts for 95% of the lymphatic filariasis (LF) burden in the Americas. Both countries aim to eliminate the diseases by 2020. Migration from Haiti, where both diseases are more prevalent, may promote transmission in the DR. Historically, Haitian migrant labourers live in rural Dominican agricultural ‘company towns’ called bateyes stratified into three regions: southwest, north and east. A household questionnaire (n = 776), captured demographics, ethnic origin, mobility patterns, malaria intervention coverage, and knowledge, and recent fever and treatment-seeking. Two individuals per household (n = 1418) were tested for malaria parasites by microscopy and rapid diagnostic test (RDT) and LF antigen by filariasis test strip (FTS). Population-level estimates and confidence intervals (CI) were computed adjusting for the survey design. Two-sided t-tests compared differences in knowledge scores.From March to April 2016, a cross-sectional, cluster survey was conducted across Dominican Plasmodium-positive by microscopy or RDT. Six individuals were FTS-positive , but none (0%) of these were microfilariae-positive. Most batey residents were born in the DR (57.8%), documented (85.0%), and permanent residents (85.1%). Very few respondents (9.4%) reported travel to Haiti in the past year. Overall, half (53.8%) of respondents owned a bed net, and 82.3% of net owners reported using it the previous night. Indoor residual spraying (IRS) differed by region (range: 4.7%–61.2%). Most of those with recent fever sought care (56.0%), yet only 30.5% of those seeking care were tested for malaria. Compared to Dominican-born populations, Haitian-born respondents more frequently reported recent fever, did not seek care for the fever, had not heard of malaria, and could not name symptoms or prevention methods.No (0%) blood sample was bateyes, which are a mixture of Haitian and Dominican residents. Travel to Haiti is rare, meaning risk of malaria and LF importation is low. Addressing identified gaps in intervention coverage, malaria knowledge, treatment seeking and service delivery will improve the quality of surveillance for these diseases, particularly among marginalized populations and promote island-wide elimination.Malaria and LF transmission appear absent or extremely low in Dominican The online version of this article (10.1186/s40249-019-0547-3) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium falciparum transmitted via Anopheles albimanus mosquitoes. The parasite remains chloroquine sensitive [Wuchereria bancrofti, thin parasitic worms that inhabit the afferent lymphatic vessels and that are transmitted by Culex quinquefasciatus mosquitoes [Malaria and lymphatic filariasis (LF) are mosquito-borne, parasitic diseases responsible for significant morbidity and mortality globally and disproportionately affect the poor –3. The iensitive , with chsquitoes . Infectisquitoes —the samecommunes) were considered endemic [Programa de Eliminación de la Filariasis Linfática, PELF) sequentially scaled-up MDA in each foci beginning in 2002, with implementation units (IU) differing across each region due to administrative and endemicity boundaries: the Southwest focus IUs  received five rounds of annual DEC-albendazole MDA between 2002−2007, La Ciénaga IUs (sub-barrios) received three rounds between 2004−2006, and the East IUs (bateyes) received three rounds between 2014−2017. As of 2018, all three areas stopped MDA for LF and are in post-treatment surveillance.The strong association between poverty and these diseases is apparent when considering that Haiti, one of the poorest countries in the world , bears t endemic . The Min endemic . As of 2 endemic . In the  endemic  identifi endemic . The natth century, Haitian labourers were recruited en masse to work on sugar plantations, living in adjacent ‘company towns’ called bateyes [batey populations became a complex admixture of Haitian migrants, Dominican-born children of the migrants, and Dominicans searching for employment in the sugar industry. A porous border and discriminatory policies in the DR leave migrants and their descendants vulnerable to human rights abuses, particularly in regards to obtaining authorized status [Sentencia effectively stripped entire generations of mostly Haitian-descended Dominicans of their citizenship [bateyes are currently some of the poorest communities in the DR [Historically, malaria and LF in the DR have occurred along the border with Haiti and in agricultural regions populated by migrant labourers, suggesting importation from Haiti , 18. Sin bateyes . Over tid status . For exaizenship . With thn the DR .The distribution of malaria and LF varies considerably not just between the two countries but within them. In low-transmission settings such as the DR, remaining parasite reservoirs tend to be clustered among marginalized social groups such as migrants and the rural poor; reaching these groups and maintaining ongoing surveillance is crucial to elimination , 24. Howbateyes, and to describe batey residents’ demographics, mobility patterns, disease-specific knowledge and prevention practices, healthcare utilization, and barriers to care. This information is critical to the design of interventions to eliminate these two diseases in the DR and across Hispaniola.The purpose of this study was to assess malaria and LF prevalence in Dominican zafra, or annual sugar cane harvest season, when the migrant labour population is thought to be largest.The study design was a cross-sectional multi-stage cluster survey conducted from 9 March to 24 April, 2016. This period roughly corresponds to the end of the bateyes, which tend to be concentrated in four regions: Southwest, East, North, and greater capital area [A target sample size of 1446 individual blood samples was sufficient to detect a 5% prevalence of malaria and LF with absolute precision of ± 2.5% at the 95% two-sided significance level, a design effect of 1.5, and a 10% non-response rate. To obtain this target sample size, a sampling frame was generated using a 2012 nationwide census of tal area . After ebateyes), or 17 bateyes within each region, were selected from a random start using probability of selection proportional to population size (PPS). Several larger bateyes were selected more than once, in which case the batey comprised two  clusters. Within each cluster, 15 households were systematically selected from a random start using a sketch map of households made by field staff before surveying activities.Across the three regions, a total of 51 clusters  head-of-household or his/her spouse was asked to complete a survey questionnaire. The questionnaire included ethnicity , basic demographics, mobility patterns, fever and illness experiences, treatment-seeking behaviour, knowledge and practices regarding malaria, LF, and diabetes, and perceptions of interpersonal discrimination. The findings from the diabetes and discrimination modules will be reported elsewhere (unpublished observations). The original English version of the questionnaire was translated into Haitian Kreyòl and Spanish and then back-translated into English to compare to the original. After piloting, some basic changes were made to the questionnaire to ensure comprehension and comfort of participants. Household questionnaire responses were collected using Eagle Survey  software on Samsung GalaxyTab3 tablets .Ministerio de Salud Pública) and National Center for Control of Tropical Diseases (CENCET).Survey teams were composed of questionnaire administrators and laboratory technicians. Questionnaire administrators were Haitian-born and bilingual . Laboratory technicians were affiliated with the Dominican Ministry of Public Health  and blood smear microscopy, which is the gold standard diagnostic procedure in the DR. LF antigen was detected by filarial test strips . Follow-up confirmatory night blood testing for microscopic detection of microfilariae (mf) was conducted for all FTS-positive individuals. Any malaria- or LF-positive participant was offered treatment based on national guidelines.Survey respondents and one other randomly selected individual of any age within the household were asked to provide a finger-prick blood sample for malaria and LF testing. Blood was collected in heparin-coated microtainer tubes and transported to local laboratory facilities for diagnostic testing the day of collection by CENCET scientists. Malaria was diagnosed by multi-species bateyes were sampled across the three geographic strata. The raw dataset contained 1439 individuals from 780 distinct households. Electronic household questionnaire data were aggregated, exported to Excel format, and merged with laboratory results. Discrepancies between household and laboratory identification codes were reconciled by reviewing the original, paper data forms used by laboratory technicians. Twenty one laboratory samples and four household questionnaire observations could not be reconciled and were excluded from analysis. After data cleaning, a total of 1418 blood samples and 776 household questionnaires were included in the analysis.A total of 51 clusters in 44 unique svy routine to account for the survey’s sampling weights, clustering effects, and stratification. To compare malaria knowledge between groups, a continuous outcome variable was created based on summation of correct responses to questions on transmission (range: 0−1), symptoms (0−6), and prevention behaviours (0−4); incorrect responses did not subtract from total score [P < 0.05) in malaria knowledge scores were computed using t-tests. All analyses were done in Stata v.14.2 . The subpop command was used in analyses restricted to sub-groups.Population-level estimates were calculated using Stata’s al score . SignifiPlasmodium infection by RDT or microscopy. Six individuals tested positive for LF antigen by FTS, resulting in a prevalence estimate of 0.5%  across all three strata (Table batey for at least nine consecutive months in the previous year), with four of the five born in the DR. None of the FTS positive individuals reported travel to Haiti within the past 12 months.Of the 1418 individuals providing blood samples, 59% were female. Median age was 34 years (range 2−96). None (0%) of the samples were positive for batey estimates. The majority (84.1%) of respondents identified as the head-of-household. Mean respondent age across the three geographic strata was 44.4 years. Overall, a majority of respondents (54.1%) were female, with greater proportions of female than male respondents in the Southwest (66.0%) and North (70.3%) regions.Table bateyes of the East had the highest proportion of Haitian-born individuals (50.9%), while the North had the largest proportion of ethnic Dominicans (i.e. non-Haitian born or Haitian descended). In line with Haitian birth or descent, most participants (58.2%) completed the survey in Haitian Kreyòl rather than Spanish (41.8%).Overall, a majority of respondents (57.5%) were born in the DR, which included those of Haitian- (25.5%) and non-Haitian descent (32.0%). Those born in Haiti therefore comprised the single largest ethnic group (42.5%). Some differences between survey regions were observed: Most respondents (85.0%) reported having some form of official documentation. When stratified by ethnicity, 73.2% of Haitian-born were documented compared to 85.8% of Dominican-born with Haitian descent and 99.5% of Dominican-born without Haitian descent.bateyes (having lived in the batey for at least 9 consecutive months in the previous year). Among ethnic groups, 23.2% of Haitian-born respondents were non-permanent (migrant) residents, compared to 14.1% of Dominican-born with Haitian descent and 4.8% of those without Haitian descent. Only a small percentage of respondents (9.4%) reported traveling to Haiti in the previous year.Most respondents (85.1%) were considered permanent residents of their Finally, respondents tended to be unemployed (36.4%), work in agriculture/farming (29.9%), do domestic work or live as homemakers (10.5%), or work as market vendors (12.0%). Differences in occupations were noted across the three regions. For example, 37.4% of respondents in the East described working in agriculture, compared to 10.4% in the North and 13.6% in the Southwest. Other occupations included moto taxi drivers, teachers, health or social service workers, and mechanics/technicians.Nearly one-third (29.8%) of respondents overall said that they had been tested for malaria at home, though rates were higher in the Southwest (46.8%) and the North (49.8%) Table . ApproxiOverall, less than half (44%) of the population had heard of malaria, with awareness among Haitian-born respondents (33.0%) notably lower than among Dominican-born without Haitian descent (57.8%). Of those who had heard of malaria, the most commonly reported symptom among all three groups was fever (57.8%), although 28.5% could not name a symptom. Most respondents (55.8%) cited mosquito bites as the primary cause of malaria, while 36.5% did not name a cause. Differences were not significant across ethnic groups in regards to reporting malaria symptoms or transmission source.Sleeping under a mosquito net was the most commonly cited malaria prevention method (31.5%), followed by filling in puddles (9.6%) and avoiding mosquito bites (9.1%). Differences were not statistically significant across ethnic groups, though Haitian-born individuals and those of Haitian descent tended to cite sleeping under a net, filling in puddles/stagnant water and taking preventative medicine more than Dominicans without Haitian descent, whereas other methods tended to be more common among the latter group.Working at night, which can lead to increased exposure to bites from malaria and LF-carrying mosquitos, was 16.0% overall and did not appear to differ significantly across ethnic groups, although working at night was less common among Dominican-born persons without Haitian descent.n = 368). Mean scores were similar between male and female respondents. Those born in Haiti had the lowest mean score (2.4) followed by those born in the DR of Haitian descent (2.7) and Dominican-born without Haitian descent (2.9) (P < 0.001). Higher scores among those born in the DR—whether of Haitian descent or not (mean: 2.9)—were likely related to ability to read in Spanish, as scores were significantly higher among those who could read an entire sentence in Spanish (mean score: 3.0) vs. those who could not (mean score: 2.3) (P < 0.001). Malaria knowledge scores tended to be higher among permanent residents (2.8) versus non-permanent residents (2.1) (P = 0.06). Higher scores were also noted among those who do not work at night (2.8) versus those who do (2.0) (P = 0.014). Borderline differences were found between those who sought care for recent fever (2.9) versus those who did not seek care (1.8) (P = 0.066), while significant differences were not found between those who own mosquito net (2.7) versus those who do not (2.7) (P = 0.784) or between those who reported using the mosquito net last night (2.7) versus those who did not report using the net last night (2.7)  (P = 0.913).Table Table The most commonly cited reason for not seeking care for the recent fever was that the illness was not considered serious (61.8%). To note, only Haitian-born (6.0%) and Haitian-descended (6.5%) respondents cited cost as a barrier to care; only Haitian-born respondents reported that, 'Medical personnel do not treat me with respect' (18.3%); and only Haitian-born individuals endorsed lack of documents as a reason for not seeking care.The publicly-funded health system appeared to be the most preferred source for fever care. Of those who sought care for a recent fever, nearly three-quarters (73.4%) attended a public hospital, and another 11.9% went to a public primary care health centre. However, only 30.5% of those that sought care for a fever said that they received a blood test for malaria. A majority (69.2%) of those with a recent fever reported taking some type of medicine for the fever.CI: 4.2–11.6%) reported having lymphedema within the past three months and 6.2% (95% CI: 2.4–14.8%) of men reported having an enlarged testicle. Lymphedema prevalence was similar across regions, while hydrocele was lowest in the North and highest in the East, but differences were not statistically significant.Survey respondents also were asked to self-report LF morbidity. Overall, 7.1%  bed net usage and low IRS coverage in the Southwest and the North highlight gaps in intervention coverage. Additional laboratory testing of filter paper specimens collected from survey participants is underway that includes polymerase chain reaction (PCR) analysis to detect low density Plasmodium infections, and serological analysis to determine evidence and timing of past exposure to malaria and LF among this population.Reported cases of confirmed malaria in the DR have declined 70% since 2010 to only 755 cases in 2016 . Despitesmission . The estsmission . Howeversmission . The decbateyes in the three western-most provinces of Bahoruco, Barahona, and Independencia) and the East . Following the halt of MDA in the Southwest focus after 2007, post-treatment surveillance (PTS) surveys conducted in 2009 and 2012 revealed low (< 0.2%) antigen prevalence across all-age groups, with most positive individuals identified as non-permanent residents of the area [CI: 0.07−1.9%). The two individuals were older (age 46−66 years), amicrofilaremic, and permanent residents of near-by, but separate, survey clusters in different provinces, suggesting that these cases represent residual antigen persistence from past infections and that transmission likely remains interrupted in the Southwest focus. In the East LF focus, baseline prevalence was 1.4% in 2011 and one round of MDA (in 2014) had taken place by the time of this survey. Despite this, only one FTS positive individual was identified across all age groups, resulting in a prevalence estimate of 0.3% (95% CI: 0.03−2.2%) among the five-province LF East focus. Since then, two additional rounds of MDA have occurred, and a recent TAS-1 survey confirmed that prevalence among school-children 6−7 years old was significantly below the 2% prevalence threshold for MDA to stop (unpublished observations).This survey also provides important information for LF elimination efforts in the DR by assessing prevalence across all age groups in both historically LF-‘endemic’ and ‘non-endemic’ areas. Two of the three LF-endemic foci in the country were included in this survey: the Southwest foci , less than one-third of them reported being tested for malaria at their care source, which was typically a public hospital or clinic. These findings suggest a need to improve diagnostic testing for malaria at the most commonly used points of care.In addition to gaps in net coverage and indoor residual spraying (IRS), this survey identified other gaps in vector-borne disease interventions. Nearly 70% of the Sentencia as well as poverty, discrimination, and limited social and economic mobility contributed to a diminished sense of social standing among batey residents [bateyes may be due to the survey’s timing, which came nearly three years after the verdict. In the interim between the Sentencia and the survey, some undocumented residents may have moved elsewhere—certainly Haiti [Historic associations of malaria and LF with areas of high concentration of Haitian migrant labourers have resulted in longstanding narratives of blame directed against Haitian and Haitian-descended people in the DR . Culturaesidents . High lely Haiti —while otly Haiti . In a poSentencia. In essence, those most at-risk for vector-borne disease in the DR are also those who face greater degrees of structural violence in everyday life. Reaching this population, gaining their trust, and maintaining surveillance in their communities will be a challenge.In addition to higher levels of perceived discrimination, there appeared to be a consistent trend of increased risk for vector-borne disease among those with Haitian ancestry. Proportionally more Haitian-born and Dominican-born, Haitian-descended people reported recent fever compared to those without Haitian descent; proportionally more Haitian-born respondents did not seek care for their fever compared to the other two groups. While most agreed that not seeking care for recent fever was because, ‘The illness was not serious enough,’ it was nonetheless striking that nearly 20% of Haitian-born respondents said that they did not seek care because ‘Medical personnel do not treat me with respect,’ while no one from the other two groups cited that reason. Cost as a barrier to seeking care was cited only by Haitian-born and Haitian-descended people. Proportionally more Haitian-born respondents had never heard of malaria, did not know a symptom of malaria, did not know a prevention method, and had never received an at-home blood test for malaria compared to the other two groups. More Haitian-born individuals did not have a mosquito net for personal use compared to those born in the DR with and without Haitian descent, and more Haitian-born respondents did not sleep under their nets the night before the survey followed by those with Haitian descent and lastly those without Haitian descent. These differences in knowledge of vector-borne disease and preventive behaviours suggest different ways in which these groups understand malaria and varying degrees of exposure to public health campaigns in both Haiti and DR. Of course, these differences are also reflective of a larger pattern of health and socioeconomic disparities. Haitian migrants are likely undocumented, less educated, live and work in arduous conditions, and face a language barrier in the DR. Persons of Haitian descent born in the DR also face hurdles accessing educational and work opportunities, especially in the aftermath of the bateyes. The survey was intentionally conducted during the zafra harvest period to provide the best opportunity of including migrant labourers. It is not known whether the household-based survey failed to capture this population or whether the role of migrant labourers simply has diminished with the decline of the sugar industry. Anecdotal reports indicate that migrants now mainly work in construction and other non-agricultural industries throughout the country. This suggests the present study results are credible and also highlight the need for additional surveys in other geographic areas to assess disease status among present day migrants. Third, despite the study team intentionally conducting survey activities during weekend evenings, when most household members were reported to be available, more females than males completed the household interview. It is possible that men were under-represented, perhaps because they were working at the time or had migrated elsewhere at the time of the survey. Finally, there is always potential for response bias among questionnaire participants due to nuances of language, power dynamics and rapport between respondents and interviewers, and other factors.There are important limitations to this study, including the potential underestimation of malaria infection prevalence due to limited sensitivity of RDTs, which will be addressed through subsequent confirmatory polymerase chain reaction PCR testing. Secondly, despite the motivation to understand the risk of malaria among mobile populations within the DR, the survey surprisingly found a highly settled population currently living in bateyes and other rural areas, along with urban and peri-urban areas of Santo Domingo given the historic LF transmission and recent malaria outbreaks there [bateyes, ongoing surveillance will be crucial to rapid detection of new cases, response to outbreaks, and prompt treatment to prevent transmission. Surveillance will in turn depend on strengthening relationships between community health workers, clinical care sites, and the general population. Some findings from this survey provide substance for reinforcing or developing new health promotion educative talks in the community. For example, given that the most common reason for not seeking care for recent fever was that, ‘The illness was not serious enough,’ health messages in higher-risk areas could underscore the importance of reporting fever to community health workers or otherwise seeking formal care, so that blood testing can detect possible infection.Going forward, the final push towards elimination of both malaria and LF in the DR will focus increasingly on at-risk populations such as Haitian migrants, those living in ks there , 28. Whibateyes across the country, collaborating with local authority structures, recruiting and training community health volunteers, and achieving high levels of participation in mass drug administration (MDA) campaigns [The migrant population deserves special consideration for community engagement given their comparatively low level of knowledge about malaria and preventive behaviour, continued higher risk of importing malaria into the DR, and adverse social and political conditions that affect them. Furthermore, both Haitian migrants and Dominican-born persons of Haitian descent may harbour feelings of mistrust and disempowerment towards those in authority, given festering problems of discrimination and poverty. The national PELF represents one source of effective community engagement. Since 2002, this community-level program has worked extensively in ampaigns , 41.Finally, in the clinical setting, there is also room for improvement. While most of those with recent fever sought care (56%), most of them (67%) did not get tested for malaria at those care sites , suggesting diagnostic and/or clinical limitations in care settings. Improving diagnostic capacity and clinical training, especially at sites that serve at-risk populations, will also be needed.Additional file 1:Multilingual abstract in the five official working languanges of the United Nations. (PDF 484 kb)


Plasmodium falciparum malaria infection, which can be lethal if not treated. A decrease in blood sugar (BS) level has been correlated with disease severity, parasitaemia and the use of certain antimalarial drugs. This study explored the relationship between pancreatic pathology, including the expressions of insulin and glucagon in the islets of Langerhans, and the BS levels in P. falciparum malaria patients. Pancreatic tissues from malaria patients were divided into three groups, namely those with BS < 40 mg/dl, BS = 40–120 mg/dl, and BS > 120 mg/dl. In P. falciparum malaria, pancreatic tissues showed numerous parasitised red blood cells (PRBCs) in the capillaries, oedema, acinar necrosis and the presence of inflammatory cells. The islet size and the expression of insulin were significantly increased in P. falciparum malaria patients with hypoglycaemia. In addition, insulin expression was positively correlated with islet size and negatively correlated with BS levels. This pioneer study documents an increase in insulin expression and an increase in islet size in hypoglycaemic patients with P. falciparum malaria. This could contribute to the pathogenesis of hypoglycaemia and provides evidence for the potential need to effectively manage the hypoglycaemia seen in malaria infection.Hypoglycaemia is an important complication of  Plasmodium falciparum is still a widespread parasitic infection causing high morbidity and mortality. The main clinical presentations of severe malaria include cerebral malaria, metabolic acidosis, anaemia, acute kidney injury, pulmonary oedema, renal failure, hypoglycaemia, hypertension and shock1. Of these severe manifestations, clinical hypoglycaemia occurs in 8–30% of all severe P. falciparum malaria cases3. Children and pregnant women are prone to develop hypoglycaemia in severe malaria possibly linked to lowered immunity. The clinical symptoms of severe hypoglycaemia include hunger, sweating, palpitation, dyspnoea, tachycardia, confusion, anxiety, somnolence, seizure, breathlessness, coma, and conditions may progress to death if not properly treated1. The presentation of hypoglycaemia can mimic clinical cerebral malaria but can be differentiated by blood sugar (BS) determination.Malaria caused by 1. The causes of hypoglycaemia in severe P. falciparum malaria had been reported to be due to the consumption of glucose by malaria parasites4, the effect of antimalarials such as quinine5, malarial hepatitis6, defects in the gluconeogenesis pathway7, the effect of tumor necrosis factor release and the influence of other cytokines produced during malaria infection8.Insulin stimulates cells to take up glucose from blood resulting in lower BS levels. On the other hand, glucagon stimulates hepatocytes to release glucose into the blood. Hypoglycaemia in malaria is diagnosed when BS levels are below 2.2 mmol/l or 40 mg/dl9. In human pancreatic tissues with leptospirosis, the histopathological changes include oedema, mild inflammatory infiltrate of lymphocytes, haemorrhages, congestion, fat necrosis and calcification10. Oedema and pancreatitis were histopathological changes noted in dengue fever11. The histopathology of pancreatic tissues with P. falciparum malaria infection is still incompletely understood and only limited information is available. This study aimed to explore pancreatic pathology in P. falciparum malaria and to investigate the expressions of insulin and glucagon in the islets of Langerhans in parallel.Various studies have described histological changes in the pancreas with infectious diseases. In the study of canine babesia, pancreatic tissues show interstitial and stromal oedema, parenchymal haemorrhages, parenchymal necrosis, fat necrosis and infiltration of a large number of neutrophilsP. falciparum malaria, comprising of 8 cases defined with hypoglycaemia (BS < 40 mg/dl), 9 cases with BS = 40–120 mg/dl, and 10 cases defined with hyperglycaemia (BS > 120 mg/dl). Eight normal pancreas from non-malaria patients were used as controls. No difference in clinical parameters was observed across the groups, including: mean age (years) , sex ratio (M:F) , number of days of fever , haemoglobin level (g/dl) , and white blood cell count (×103/μl)  . There was a significant negative correlation between parasitaemia and BS levels .Pancreatic tissues were obtained from 27 patients who died from P. falciparum malaria patients usually showed oedema, acinar necrosis and inflammatory reactions, in addition to the presence of parasitised red blood cells (PRBCs). However, there was no difference in the overall total score of histopathological changes between malaria patients and control group (P = 0.392). The histopathology of pancreatic tissues from malaria patients, compared to normal pancreatic tissues is illustrated in Fig. 2). Numerous PRBCs were frequently seen in the capillaries of pancreas from severe malaria patients , especially in malaria patients with BS < 40 mg/dl (P = 0.004) and those with BS = 40–120 mg/dl (P = 0.004). Necrosis of acinar cells is considered in pale staining cells with the loss of nuclei and visible cellular distortion, causing deformation in pancreatic parenchyma . The presence of inflammatory cells, particularly lymphocytes, neutrophils and occasionally eosinophils in the interlobular septum and interglandular areas were significantly higher in all malaria patients compared to the control group (P = 0.011) . The quantification of total histopathological changes in the pancreatic tissues of malaria patients compared with the control group are shown in Table Pancreatic tissues from nts Fig. . Oedema nts Fig. . There wyma Fig. . Acinar 11) Fig. . FibrosiP. falciparum malaria with hypoglycaemia (135.80 ± 10.23 µm), compared to P. falciparum malaria with BS = 40–120 mg/dl , P. falciparum malaria with hyperglycaemia , and the control group . The calculated area of a pancreatic islet was 15,136.99 ± 2,625.02 µm2, 6,702.34 ± 458.21 µm2 and 3,922.37 ± 200.76 µm2, respectively, as compared to control group . There was a significant negative correlation between the size of pancreatic islets and BS levels  in malaria patients .The comparative size of islets of Langerhans is depicted in Fig. nts Fig. . No sign). In this study, insulin expression was prominent in the group of P. falciparum malaria patients with hypoglycaemia , and those with BS = 40–120 mg/dl . A trend towards a reduction in the expression of insulin was revealed in malaria with hyperglycaemia  and the hyperglycaemic group . The quantified total expression scores of insulin and glucagon are shown in Fig. rs = −0.728; P = 0.002)   Fig. . In addi04) Fig. .Figure 3P. falciparum malaria patients include oedema in the interlobular and interglandular spaces, acinar necrosis and inflammatory reactions. Although these changes were frequently found in P. falciparum malaria patients with hypoglycaemia (oedema and necrosis), similar histopathological changes were reported in other infectious diseases including dengue infection11, leptospirosis10 and babesia9. Limited studies have been reported on the pathology of pancreatic tissues in severe P. falciparum malaria. Previous rare autopsy findings showed an enlarged pancreas with minimal fluid in the peritoneal cavity, related clinically to pancreatitis12, and necrotic pancreatic tissue13. Our findings are similar to reports in animals infected by other plasmodium species. In a study of P. berghei in mice, pancreatic tissues revealed acute inflammatory reactions of acini, islets cells and interlobular duct14. Another study of Macaca mulatta monkeys infected with P. knowlesi showed acinar necrosis and the disappearance of pancreatic islets15. In addition, hypoglycaemia was observed in animal models infected with P. knowlesi16. The inflammatory cells such as lymphocytes and neutrophils are crucial in the control of malarial parasite replication, which contribute to the subsequent elimination and resolution of the infection through the process of phagocytosis and/or production of inflammatory mediators17. Fibrosis, areas of haemorrhages and fat necrosis in the pancreatic tissues were not features of P. falciparum malaria patients.Histopathological changes documented in pancreatic tissues of 18. Fibrosis is characterized by the accumulation of extracellular matrix and an increase in fibroblastic reaction. Though fibrosis is not significantly present in P. falciparum malaria patients, possibly due to the acute nature of malaria infection, a previous study has reported that high BS levels can stimulate the proliferation of pancreatic stellate cells and subsequent increased in fibroblast production19.The presence of PRBCs within the capillaries can cause an increase in blood viscosity. In addition, malaria parasites in the red blood cells (RBCs) can cause decrease in RBC membrane deformity. These circulatory disturbances can lead to tissue hypoxia, release of pancreatic acinar enzymes, impaired capillary and venous drainage, which in turn can lead to haemorrhagic pancreatic necrosisP. falciparum malaria patients.Whether the increase in islet size is caused by malaria infection per se, or is secondary to antimalarial drugs, needs to be further investigated. Nevertheless, the enlarged pancreatic islets can contribute to hyperinsulinemia and subsequent hypoglycaemia seen in P. falciparum malaria patients. Similar to previous reports, insulin immunopositive cells were found in greater density in the central part of the islets of Langerhans, compared to islet cells expressing glucagon, which are frequently detected around the periphery of pancreatic islets20.The present study used immunohistochemical methods to demonstrate the expressions of insulin and glucagon in terms of distribution and intensity in the pancreatic tissues of P. falciparum malaria patients with hypoglycaemia can be secondary to the malaria parasite itself, the use of antimalarial drugs, alteration in the gluconeogenesis pathway, the effects of cytokines during malaria infection and renal injury6. Malaria parasites in the circulation need glucose from the host in order to produce energy, mainly via aerobic glycolysis, for their survival4. A previous report showed that high parasitaemia is associated with high glucose requirements by malaria parasites, which can lead to clinical hypoglycaemia21. Antimalarial drugs, such as quinine and quinidine have an effect on glucose metabolism in malaria patients, causing a decrease in BS level5. Most of our malaria patients were treated with quinine. Quinine has been reported to be a more potent stimulant of insulin secretion than quinidine5. These drugs act similarly to glucose on potassium permeability of the beta cells membrane, causing calcium influx and subsequent release of insulin to reduce BS levels5. We accept that quinine/quinidine have an effect in lowering insulin level and is still used in endemic areas and in places where artesunate is not available. Our data suggests the need for precautious use of quinine/quinidine in these circumstances, and recommend closed monitoring of the BS level of malaria patients. Other antimalarial drugs, such as chloroquine, amodiaquine, mefloquine and halofantrine have no direct effect on insulin secretion22. Interestingly, the gluconeogenesis pathway is abnormal in severe P. falciparum malaria patients due to insufficient galactose substrate, resulting to low BS levels7. The effect of cytokines released during P. falciparum malaria infection, such as tumour necrosis factor (TNF), interleukin (IL)- 1 and 6 can inhibit the activity of phosphoenolpyruvate carboxykinase in the gluconeogenesis pathway and also mediate a reduction in hepatic glycogen content, hence the lower BS levels24. In addition, IL-1 and 6 have been reported to stimulate islet cell hyperplasia25. It can be hypothesised that IL-1 and 6 released during malaria infection can cause islet cell hyperplasia resulting to enlargement of pancreatic islets and subsequent increase in insulin expression. Furthermore, the kidney is an important organ involved in glucose regulation. Occurrence of acute kidney injury in severe P. falciparum malaria can affect the net glucose release and reabsorption, resulting in hypoglycaemia26.The increased expression of insulin in 27 and Chagas’s disease in a murine model28. Although these studies have shown that hypoglycaemia is not a major cause of death, in patients with severe P. falciparum malaria it is an important clinical manifestation that may well contribute significantly to high mortality rates. This pioneer study documents an increase in insulin expression and an increase in islet size in P. falciparum malaria patients with hypoglycaemia. The study will be useful for clinicians interested in understanding and managing the process of hypoglycaemia in severe P. falciparum malaria. The paper sets out recognizable histopathologic and immunologic changes associated with malarial pancreatic tissues.The maintenance of a normal BS level is a critically important process in the metabolism in living organisms. Other parasitic infections have been reported to cause hypoglycaemia, e.g. trypanosomiasis in both human and animalsP. falciparum malaria patients and control pancreas were obtained from the Department of Tropical pathology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. For malarial cases, pancreatic tissues were divided into three groups based on the BS levels before death, namely BS < 40 mg/dl, BS between 40–120 mg/dl, and BS > 120 mg/dl. Control pancreatic tissues were obtained from accidental death cases, which showed normal islets of Langerhans, secretory acini, blood vessels, ducts and interstitial area. The study protocol was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand (MUTM 2015–041–01 and MUTM 2015-041-02). Methods were performed in accordance with the relevant guidelines and regulations of the above committee. Written informed consent from closest relatives was obtained for all malaria patients. The Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand approved the use of control pancreatic tissues obtained from discarded autopsy specimen.Pancreatic tissues from 30, with minor modifications . To study the average size of islets of Langerhans, islets were measured and evaluated by randomisation of 40 pancreatic islets under ×100 magnification using the “ImageJ” software program, developed by the National Institutes of Health, Bethesda, Maryland, USA. In addition, the pancreatic tissues were interpreted based on seven histological criteria, namely presence of parasitised red blood cells (PRBCs), oedema, haemorrhage, inflammatory infiltration, acinar necrosis, fat necrosis and fibrosis, under ×200 magnification. The changes were graded on a scale of 0–3. All criteria were assessed in the lobule, interlobular and intergladular areas, except for fat necrosis where the occurrence was at the peripancreatic tissue level. The grading criteria were based on previously described studies2O2 for 3 minutes, resulting in a brown coloration, which indicated the target antigen. Subsequently, pancreatic sections were counterstained with Mayer’s haematoxylin and mounted with a coverslip for microscopic evaluations.The expressions of insulin and glucagon were detected by immunohistochemical staining, using an avidin-biotin peroxidase complex method . Primary antibodies used were rabbit polyclonal anti-insulin and anti-glucagon . Pancreatic tissues at 4 µm thickness were placed on an adhesive poly-L-lysine slide and heated in a hot oven at 56 °C for 30 minutes. Tissue sections were de-paraffinised and rehydrated in xylene and a decreasing series of graded alcohol, respectively. Then, the pancreatic tissue sections were incubated in 3% hydrogen peroxide diluted in distilled water for 30 minutes at 37 °C in order to block the endogenous peroxidase activity in the tissue. To retrieve the antigen, tissue sections were incubated with 0.1 M sodium citrate buffer at pH 6.0 in a 800 W microwave for 20 minutes. When the slides cooled down to room temperature (RT), the sections were washed in PBS buffer at pH 7.4. To reduce the non-specific background, the sections were incubated with diluted normal goat serum for 30 minutes. Then the pancreatic sections were incubated with a primary antibody  in a moisture chamber overnight at 4 °C. The following day, sections of pancreas were washed with PBS buffer at pH 7.4 for three time then incubated with diluted biotinylated goat anti-rabbit Ig G for 30 minutes at RT and treated with avidin-biotin peroxidase complex (ABC) conjugated with horseradish peroxidase (HRP) for 30 minutes. After all sections were washed, the tissue sections were reacted with 0.05% 3,3′ diaminobenzidine (DAB) and 0.01% H31.The expressions of insulin and glucagon were evaluated in the cytoplasm of the islet cells in the pancreatic islets. Twenty islets from each pancreatic section were randomly observed at ×400 magnification. The percentages of insulin- and glucagon- expressing cells were determined by counting the number of positive cells over the total number of cells in each islet, multiplied by 100. The intensity of staining with both antibodies was subjectively scored as follows: 0 = no staining, 1 = weakly positive, 2 = moderately positive, and 3 = strongly positive. The total score (TS) was calculated by multiplying the percentage of positive cells (%) and staining intensity (I), according to previous studyrs). Mean differences in the total scores of histopathological and immunohistochemical studies between experimental groups were analysed by Mann-Whitney U test. The P-value < 0.05 was considered to be statistically significant.Data were recorded into a computer database and analysed with SPSS software version 18.0. All quantitative data were represented as mean ± standard error of mean (SEM). The test for normality of distribution was calculated using Kolmogorov-Smirnov test. Comparisons of the difference in the size of pancreatic islets between groups were analysed by one way ANOVA. Correlations between variables of histological and immunohistochemical analyses and clinical data were analysed by Spearman’s rank correlation (


It remains unclear whether improving iron status increases malaria risk, and few studies have looked at the effect of host iron status on subsequent malaria infection. We therefore aimed to determine whether a child’s iron status influences their subsequent risk of malaria infection in sub-Saharan Africa.We assayed iron and inflammatory biomarkers from community-based cohorts of 1309 Kenyan and 1374 Ugandan children aged 0–7 years and conducted prospective surveillance for episodes of malaria. Poisson regression models were fitted to determine the effect of iron status on the incidence rate ratio (IRR) of malaria using longitudinal data covering a period of 6 months. Models were adjusted for age, sex, parasitemia, inflammation, and study site.P < .001) and IDA . Low transferrin saturation (<10%) was similarly associated with lower malaria risk . However, variation in hepcidin, soluble transferrin receptors (sTfR), and hemoglobin/anemia was not associated with altered malaria risk.At baseline, the prevalence of iron deficiency (ID) was 36.9% and 34.6% in Kenyan and Ugandan children, respectively. ID anemia (IDA) affected 23.6% of Kenyan and 17.6% of Ugandan children. Malaria risk was lower in children with ID (IRR, 0.7; 95% confidence interval [CI], 0.6, 0.8; ID appears to protect against malaria infection in African children when defined using ferritin and transferrin saturation, but not when defined by hepcidin, sTfR, or hemoglobin. Additional research is required to determine causality.ISRCTN32849447 Decreased ferritin and transferrin saturation are associated with protection against malaria in African children. Hepcidin, soluble transferrin receptor, and hemoglobin concentrations are not associated with malaria protection. These findings may reflect differences in parasite iron acquisition. Iron deficiency (ID) and malaria remain important public health problems in African children , 2. ID, The safety of iron supplementation has been a long-standing concern among policy makers and clinicians in malaria-endemic areas , 7. In tPlasmodium falciparum malaria in African children [Plasmodium liver stage of iron [Few observational studies have investigated the effect of iron status on malaria risk. These studies indicate that ID is associated with a reduced risk of both mild and severe children  but have of iron , but stu of iron , 18. Two of iron , 20, whi of iron . There aIn this study, our aim was to determine whether iron status influences the subsequent risk of malaria infection in 2683 Kenyan and Ugandan children, thus making this the largest observational study on iron status and risk of malaria to date with the most comprehensive range of iron markers.Ethical approval was provided by the Scientific Ethics Review Unit of the Kenya Medical Research Institute for the Kenyan cohort and the Uganda Virus Research Institute and the London School of Hygiene and Tropical Medicine for the Ugandan cohort.This study used data from 2 African community-based cohorts of children in Kilifi, Kenya, and Entebbe, Uganda.The Kenya cohort included 3 community cohorts: Junju, Ngerenya, and malaria RTS,S vaccine. Junju and Ngerenya are ongoing rolling cohorts evaluating malaria immunity as described elsewhere . The malThe Entebbe Mother and Baby Study is a prospective birth cohort study that was originally designed as a randomized, double-blind, placebo-controlled trial (ISRCTN32849447) to determine whether anti-helminthic treatment during pregnancy and early childhood was associated with differential responses to vaccination or incidence of infections such as pneumonia, diarrhea, or malaria . Blood sP. falciparum parasitemia and temperature. Genotyping of hemoglobin types was conducted using polymerase chain reaction assay [Longitudinal parasitemia data were obtained from active surveillance during the 6 months following measurement of iron biomarkers. For the study, 94% of Kenyan and 90% of Ugandan children were followed for 6 months, while the length of follow-up for the remainder ranged from 1 to 5 months. We chose a follow-up period of not more than 6 months since iron status may change over a longer follow-up period. Secondary analyses included a 1-year follow-up period. Clinical malaria data included microscopy-confirmed density of asexual on assay  with DNAThe assayed biomarkers of iron status included plasma ferritin , hepcidin , sTfR , iron , transferrin , and hemoglobin (Medonic CA 530 hemoglobinometer). Since biomarkers of iron are influenced by inflammation, C-reactive protein (CRP)  was assayed to adjust for inflammation .The following 2 definitions of ID were used: based on low ferritin defined as plasma ferritin <12 µg/L or <30 µg/L in the presence of inflammation (CRP >5 mg/L) in children aged <5 years or <15 µg/L in children aged ≥5 years , and TSAe-transformed to normalize their distributions. Geometric means of iron biomarkers and proportions of ID and anemia were computed. Two-tailed Student t tests were used to test for difference in means between groups. Poisson regression models of counts of malaria episodes were fitted as predicted by iron status  and were adjusted for age, sex, parasitemia, inflammation, and study site. Difference in individual length of follow-up was accounted for in the model by including the length of follow-up as “exposure” in the model. We accounted for multiple episodes using robust cluster variance estimation, which takes into account correlations between multiple events. Secondary analyses involved excluding children with parasitemia or inflammation at baseline to mitigate the effects of concurrent infection on iron status [P value of <.05 was considered significant.All analyses were conducted using STATA 13.0 . Iron biomarkers (except hemoglobin) were logn status . We usedor ferritin or hepcidin or sTfR or TSAT or hemoglobin or anemia and malaria children.” We found 5 longitudinal studies that investigated the effect of ID on malaria risk. A metaanalysis of the current study and 4 previous longitudinal studies that reported risk ratios of the relationship between ID and malaria risk was performed using the “metan” command in STATA.We searched the PubMed and Google Scholar databases with search terms that included “ID A total of 1309 Kenyan and 1374 Ugandan children aged 0–7 years and 1–5 years, respectively, were included in the analyses. P < .001) and in each cohort individually (P = .009). However, hepcidin, sTfR, and hemoglobin concentrations were not associated with subsequent risk of malaria . These findings were consistent for the individual cohorts (P = .016). However, anemia itself was not significantly associated with variation in malaria risk , hepcidin, and sTfR were not significantly associated with variation in malaria risk.Consistent with our findings, previous observational studies have reported that ID based on low ferritin concentrations confers protection against malaria infection in African children . NyakeriP. falciparum has been demonstrated to obtain iron from transferrin using in vitro parasite culture [Also, we found that ID defined by TSAT was associated with a 20% reduction in the subsequent risk of malaria and that TSAT was positively associated with malaria incidence. Using a combination of low TSAT and low ferritin, Nyakeriga et al reported a 30% reduction in clinical malaria among iron-deficient children . In supp culture . These sP. falciparum compared to those that are iron replete, and this protective effect is reversed by iron supplementation [Plasmodium [So, how might ID protect against malaria infection? In in vitro parasite cultures, iron-deficient human erythrocytes are poorly infected by entation . In mousentation . Furtherasmodium . Anotherasmodium .P. falciparum [We also report that anemia (or hemoglobin concentration) does not influence subsequent malaria risk. Similar observations from 2 previous studies have been reported. A recent longitudinal study in Papua New Guinean infants aged 3 months and followed for 1 year reported a nonsignificant association between lower hemoglobin concentrations and subsequent malaria infection . Ghosh elciparum . Howeverlciparum  and the lciparum . It remalciparum .Plasmodium [We hypothesized that increased hepcidin concentrations may reduce malaria risk through sequestration of iron within macrophages and enterocytes , therebyasmodium . Since tasmodium . Furtherasmodium . Howeverasmodium , we founPlasmodium parasites preferentially infect young red blood cells [Our data indicate that a child’s erythropoietic drive (as measured by sTfR) does not influence their subsequent risk of malaria infection. The expression of sTfR increases with both ID and expanded erythropoiesis  , factorsod cells , whereasod cells , 41.A major challenge in this study is that iron biomarkers are influenced by infections and inflammatory processes that may confound the effect of iron status on malaria infection . To mitiOur findings, which are in agreement with other studies, suggest that ID protects children against malaria infection and thus that improving iron status may predispose African children to infection. Interestingly, of the iron biomarkers, only higher concentrations of ferritin and TSAT were predictive of increased rates of subsequent malaria, perhaps reflecting differences in their relationship to parasite mechanisms of iron acquisition. Although WHO recommends iron supplementation coupled with malaria treatment and prevention strategies in malaria-endemic areas , these sClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciy791_suppl_Supplementary_MaterialClick here for additional data file.


Plasmodium lactate dehydrogenase (pLDH) based RDTs. To improve the diagnosis of malaria in febrile children, two diagnostic algorithms, based on sequential interpretation of a malaria rapid diagnostic test detecting two different targets of Plasmodium species and followed by expert microscopy, were evaluated.Malaria rapid diagnostic tests (RDT) have limitations due to the persistence of histidine-rich protein 2 (HRP2) antigen after treatment and low sensitivity of pLDH. If T2-pLDH test was negative, the malaria diagnosis was considered as “inconclusive” and microscopy was performed; Algorithm 2: The result of line T2-pLDH were read first; if positive, malaria infection was considered to be present. If negative, confirmation was done with the line T1-HRP2. If T1-HRP2 was positive the malaria diagnosis was considered as “inconclusive” and microscopy was performed. In absence of malaria microscopy, a malaria infection was ruled out in children with an inconclusive diagnostic test result when previous antimalarial treatment was reported.Two diagnostic algorithms were evaluated using 407 blood samples collected between April and October 2016 from febrile children and the diagnostic accuracy of both algorithms was determined. Algorithm 1: The result of line T1-HRP2 were read first; if negative, malaria infection was considered to be absent. If positive, confirmation was done with the line T2-PfHRP2 was 98.4% and the specificity was 74.2%, and for the pLDH test the sensitivity was 89.3% and the specificity was 98.8%. Malaria was accurately diagnosed using both algorithms in 84.5% children. The algorithms with the two-line malaria RDT classified the test results into two groups: conclusive and inconclusive results. The diagnostic accuracy for conclusive results was 98.3% using diagnostic algorithm 1 and 98.6% using algorithm 2. The sensitivity and specificity for the conclusive results were 98.2% and 98.4% for algorithm 1, and 98.6% and 98.4% for algorithm 2, respectively. There were 63 (15.5%) children who had an “inconclusive” result for whom expert microscopy was needed. In children with inconclusive results (PfHRP2+/pLDH- only) previous antimalarial treatment was reported in 16 children with malaria negative microscopy  and 1 child with malaria positive microscopy .For single interpretation, the sensitivity of The strategy of sequential interpretation of two-line malaria RDT can improve the diagnosis of malaria. However, some cases will still require confirmative testing with microscopy or additional investigations on previous antimalarial treatment. Plasmodium falciparum histidine-rich protein 2 (PfHRP2) based RDT, which is specific for Plasmodium falciparum only, and the Plasmodium lactate dehydrogenase (pLDH) detecting RDT, which can detect 4 human Plasmodium species  or a combination test, which has two test lines .Nowadays, malaria parasite specific antigen-detecting rapid diagnostic tests (RDTs) are considered essential tools in routine practice for the management of febrile illness in malaria endemic settings  2] 3]. [3].2] [. [3].2] PfHRP2 and pLDH-based RDTs using microscopy as the gold standard  6]  [6] PfHR  [6] PfHstandard . Most ofollected  [5] 7]  PfHRP2 aollected  9] 10] [10]PfHR [10]PfHRollected . On the sitaemia . As a co general .pLDH, specific of Plasmodium species, has been proposed.To circumvent this dilemma, sequential reading/interpretation of the two-line RDT detecting HRP2 and PfHRP2 . The management of uncomplicated medical cases, including uncomplicated malaria , is done at the health facilities by nurses according to the national guideline of integrated management of childhood illness .Consequently, inadequate prescription of antimalarial observed with pLDH and the high sensitivity of PfHRP2 leads to malaria false negative and positive results respectively. Moreover, after successful antimalarial treatments, pLDH antigen is metabolised within one week, but HRP2 antigens can persist in the blood up to 4 weeks and over. It is evident that low malaria parasitemia and persistence of HRP2 antigen after successful antimalarial treatment could lead to inconclusive diagnostic results (HRP2+/pLDH-) following the algorithms. A previous study reported that in a malaria endemic area, almost 50% of non-malaria infection due to bacteria and virus can be hide under a false positive HRP2 results  [22 [2221] [Finally, most likely the implementation of the proposed algorithms would be by sequentially using two different RDTs. However, this approach can double the time needed to confirm the diagnosis of a malaria infection as the result of the second test needs to be available too, and thismay also require taking a second blood sample. Research is needed towards patient and health care provider acceptability of such an approach.The strategy of sequential interpretation of two-line malaria RDT can improve the diagnosis of malaria by grouping the low malaria parasitemia and false HRP2 positive children in inconclusive test for which additional investigations on previous antimalarial treatments are required.