I2 = 88.6%), a statistically significant increase in systolic blood pressure , I2 = 87.2%), a nonsignificant increase in diastolic blood pressure , I2 = 73.1%), and a nonsignificant increase in mean arterial pressure , I2 = 69.9%) compared to other agents. The risk of bias was high and the certainty of evidence was low. In conclusion, among patients undergoing airway manipulation and needing sedation, the use of a ketamine/propofol admixture may be associated with better hemodynamics compared to nonketamine/propofol sedation. This trial is registered with CRD42019125725.To evaluate the effectiveness of an admixture of ketamine and propofol on peri-induction hemodynamics during airway manipulation, we searched electronic databases of randomized controlled trials from January 1, 2000, to October 17, 2018. Trial screening, selection, and data extraction were done independently by two reviewers with outcomes pooled across included trials using the random-effects model. We included 10 randomized trials . American Society of Anesthesiologists physical status was reported in 9 trials with classes I and II representing the majority. Ketamine/propofol admixture was associated with a nonsignificant increase in heart rate ,  Peri-intubation hypotension, defined by either systolic blood pressure or mean arterial pressure (MAP) below a certain threshold  or the introduction of vasopressors, has been recognized as a potential target area for research given its association with patient-centered outcomes. For example, peri-intubation hypotension has been associated with both increased length of stay and mortality –3. FurthSeveral putative risk factors have been implicated in the pathway to peri-intubation hypotension. Age, illness severity, and preintubation hemodynamic derangement have consistently been implicated in the development of peri-intubation hypotension , 3, 8, 9The majority of studies on ketamine/propofol admixture have evaluated critically ill patients in the emergency department with the evidence demonstrating a potential sparing effect on hemodynamics along with improved pain relief and sedation quality. These studies have evaluated ketamine/propofol admixture from the standpoint of a continuous infusion for procedural sedation and analgesia –16. TherCRD42019125725).We used the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) 2015 statement to report the trial –22. All Randomized controlled trials published in peer-reviewed journals and written in English were eligible if they included the following: (1) adult patients who received ketamine/propofol admixture as an induction agent in procedural areas and underwent airway manipulation such as endotracheal intubation or supraglottic device placement; (2) a comparison to other induction agents including propofol, ketamine, etomidate, and sodium thiopental or any other combination; and (3) a report of hemodynamic effects  during the first 10 minutes after the induction of general anesthesia in American Society of Anesthesiologists I II, III, and IV patients. The intervention must have equal or similar doses . No restrictions were placed on trial location, clinical procedure, or patient severity. We excluded pediatric patients, patients who did not undergo induction of anesthesia, or patients who did not undergo airway manipulation. We also excluded observational studies, review articles, erratum, letters, and notes.A comprehensive search of several databases was conducted by a medical reference librarian. The databases included Ovid MEDLINE, Epub Ahead of Print, Ovid Medline In-Process and Other Non-Indexed Citations, Ovid MEDLINE, Ovid EMBASE, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Database of Systematic Reviews, and Scopus. Controlled vocabulary supplemented with keywords was used to search for the trials. We limited our search from January 1, 2000, to October 17, 2018. The search strategy is listed in Reviewers, working independently, screened abstracts and titles for eligibility using the above inclusion and exclusion criteria. Full-text articles were then further screened using the same criteria. At the level of full-text screening, any disagreements were resolved by consensus between the two reviewers (MS and AM) or by consulting a third reviewer (NJS).The primary outcomes were hemodynamics  at 5 and 10 minutes following intravenous anesthetic administration. The secondary outcome of interest was pain score as assessed by the visual analog scale (VAS) during the 24 hours postdrug administration.The risk of bias was assessed using the Cochrane Collaboration's tool for randomized clinical trials. We assessed random sequence generation, allocation concealment, blinding of providers, outcome assessors, and patients, incomplete outcome data, selective outcome reporting , and other sources of bias . Disagreements were resolved by consensus between two reviewers (MS and AM). The overall certainty across trials for each outcome was appraised by discussion between the two reviewers using the grading of recommendations assessment, development, and evaluation (GRADE) approach. Using the grading of recommendations assessment, development, and evaluation approach, randomized trials, as in this systematic review, would provide a starting level of certainty that is high. This level can be downgraded based on the risk of bias of the individual trials, inconsistency in the results, indirectness, imprecision, and other considerations to provide a global assessment of the certainty warranted by the body of evidence .Two reviewers (MS and AM) independently extracted the following information from each trial: author, publication year, patient characteristics, intervention, comparison, and outcomes.P value of less than or equal to 0.05 was used to determine significance for the secondary outcome. To evaluate heterogeneity, we calculated the I2 statistic [P values, where I2 more than 50% or a P value of less than 0.05 suggests high heterogeneity. All statistical analyses were performed using Stata, version 15.0 .We extracted or calculated the weighted mean difference (WMD) for continuous outcomes and relative risk for binary outcomes with associated 95% confidence intervals (CIs) from the included studies. Outcomes without measures of variations  were not included in the meta-analysis. The DerSimonian and Laird random-effects models were used to generate combined effects . A two-statistic , 26 and n = 722)  met all criteria and were included in data analysis (n = 722) . All theThere were a total of 10 trials with 722 patients included. The number of patients in each trial ranged from 40 to 100  with a mean age of 53.99 years. The proportion of participants who were female ranged from 0 to 100% . The American Society of Anesthesiologists physiologic status among participants ranged from I to III. Publication dates ranged from 2000 to 2018 ). The included trials were conducted in several different countries . The characteristics of the included trials with dosing are summarized in Overall, the risk of bias of included trials was high due to inadequate allocation concealment , partial blinding of outcome assessor , and selective reporting . The risk of summary bias is provided in n = 465) [n = 244) [I2 = 88.6%) and 10 minutes , I2 = 69.9%) compared to patients in the nonketamine/propofol admixture group  –31, 34, n = 244) , 31, 34 re group .n = 385) [n = 164) [I2 = 87.2%). At 10 minutes, patients in the ketamine/propofol admixture group had a nonsignificant increase in systolic blood pressure compared to patients in the nonketamine/propofol admixture group , I2 = 0.0%)  , 28–31 an = 164) , 31 repo = 0.0%) .n = 305) [n = 84) [I2 = 73.1%) compared to patients in the nonketamine/propofol admixture group. At 10 minutes, there was a statistically significant increase in diastolic blood pressure in the ketamine/propofol admixture group compared to the nonketamine/propofol admixture group , I2 = not applicable)  , 28–30 a5 , 28–30[n = 84)  reportedlicable) .n = 345) [n = 164) [I2 = 69.9%) and 10 minutes , I2 = 41.4%) compared to patients in the nonketamine/propofol admixture group  , 30, 34 5 , 30, 3n = 164) , 34 repore group .n = 60) [P=0.12). Four patients in the propofol plus dexmedetomidine group had higher pain scores (VAS 3–5) whereas no patients in the ketamine/propofol admixture group had high pain scores .Only one trial assessed postprocedural pain by VAS (n = 60) . Twenty-The present systematic review and meta-analysis demonstrate that ketamine/propofol admixture results in a potentially better hemodynamic profile as compared to other agents used for induction of anesthesia. In particular, systolic blood pressure was significantly higher with ketamine/propofol admixture sedation as compared to nonketamine/propofol admixture sedation when induction of anesthesia is required for airway manipulation. Although not statistically significant, heart rate, diastolic blood pressure, and MAP were higher when ketamine/propofol admixture-based sedation was employed versus nonketamine/propofol admixture based-sedation for induction of anesthesia. Furthermore, one study indicated a possible beneficial effect on pain scores in the immediate 24 hours postdrug administration .P=0.045) and increased odds of experiencing intensive care unit length of stay greater than 14 days, duration of mechanical ventilation longer than 7 days, and requiring renal replacement therapy  [P=0.004) [P ≤ 0.001) [Evidence has demonstrated that maintaining hemodynamics around the time of airway instrumentation such as endotracheal intubation is vitally important, in both the critically ill and noncritically ill. For example, in the critically ill, peri-intubation hypotension was associated with increased odds of dying  . In noncP=0.004) . MoreoveP=0.004) . LikewisA recent systematic review suggested poor outcomes with increased end-organ injury in patients undergoing noncardiac surgery who experience MAP decreases <80 mm Hg for ≥10 minutes . By the Given that periprocedure  hemodynamics are influenced by sedation and the preponderance of evidence demonstrating that preventing hypotensive episodes, especially with MAPs less than 65 mmHg or SBPs less than 90 mmHg, and maintaining perfusion pressure near baseline improves outcomes, the selection of an intravenous anesthetic agent during a procedure  ought to have this goal in mind. To this end, a recent editorial questioned the current blood pressure parameter of MAP 65 mmHg in the intensive care unit, which largely comes from two retrospective studies . The autP=0.04; relative risk for bradycardia 0.47, 95% CI: 0.28–0.72, P=0.008). The authors also demonstrated similar rates of psychotomimetic complications and nausea-vomiting when compared to propofol [P=0.04); however, recovery times were similar in both groups. Hemodynamic thresholds were not assessed in this review [A agonist activity [The current systematic review and meta-analysis are consistent with prior reviews of ketamine/propofol admixture administration for procedural sedation. Prior reviews have demonstrated better hemodynamics with ketamine/propofol admixture-based procedural sedation than with nonketamine/propofol admixture-based procedural sedation. For example, a recent review demonstrated that ketamine/propofol admixture-based procedural sedation was effective in reducing cardiovascular complications (relative risk for hypotension 0.11, 95% CI: 0.17–0.97, propofol . A secons review . Althougactivity . This maactivity , 14, 31.Our study has several benefits. First, we focused on a theoretical advantage of ketamine/propofol admixture over other agents used for induction of anesthesia during procedures such as endotracheal intubation, namely, hemodynamic preservation. Furthermore, we extracted hemodynamic data in the immediate period after drug administration, thereby limiting the effects of other cointerventions. Second, we extracted data on all drug comparisons to ketamine/propofol admixture. Third, we included trials that had either similar or near equivalent dosing of ketamine/propofol admixture.Similar to other systematic reviews and meta-analyses, our results are limited by clinical trial quality. Although our search strategy was comprehensive, we included only English-language articles and published articles after 2000 and thus may have missed trials not published in English or published before the year 2000. Hemodynamic data were not recorded similarly in all trials nor was the frequency of hemodynamic data collected. Furthermore, 5 and 10 minutes may be an oversimplification of hemodynamic representation as intravenous anesthetics used for induction of anesthesia have a short initial elimination half-life and, therefore, collecting hemodynamic data every minute would have been ideal , 33, 35.We demonstrated a potentially better hemodynamic profile with ketamine/propofol admixture-based induction of anesthesia versus nonketamine/propofol admixture-based induction of anesthesia for procedures requiring airway manipulation such as endotracheal intubation. However, given moderate to high heterogeneity of trials included in the current meta-analysis, our results should be interpreted with caution.


Congenital malaria is a serious and common infection in tropical Africa. It has multiple consequences on the newborn and the mother. Methodology. It is a cross-sectional and prospective study, conducted at Issaka Gazobi Maternity of Niamey, from June 1 to November 30, 2017. The diagnosis was made by microscopy of a thick and thin blood smear of mother, newborn, and umbilical cord.  The objective of this study is to calculate the prevalence of congenital malaria, describe its clinical signs, and analyze its associated factors. μl . The prevalence of congenital malaria disease was 14.06% (35/249) with a parasite density of 108 P/μl  of neonates had hyperthermia and did not have a sucking reflex, 8.5% (3/35) were anaemic, 11.42% (4/35) had convulsed, 20% (7/35) had a coma, and 45.71% (16/35) had a low birth weight. No deaths were recorded, and only the nonuse of bed nets was significantly associated with congenital malaria (p = 0.04).  Two hundred and forty-nine (249) consecutive newborn/mother pairs were included. The prevalence of congenital malaria infection was 26.51% (66/249) with a parasite density of 101 P/Plasmodium. Infection can progress to congenital malaria disease. The use of mosquito nets and intermittent preventive treatment would reduce the incidence of congenital malaria. In Niger, one out of four newborns is infected with  This contamination of the foetus is explained by the fact that the parasite can cross the placental barrier [Congenital malaria is the ildbirth . Biologi barrier , followi barrier .Plasmodium falciparum especially. This infection is a major cause of anaemia in pregnant women [Plasmodium. This infection can progress to clinical malaria known as congenital malaria disease.In malaria-endemic areas, congenital malaria is a major public health problem according to the World Health Organization . In tropnt women , 8. In fnt women . This font women . Newbornnt women . Placentnt women  and evennt women . Childrent women . HoweverThe clinical manifestations of congenital malaria aren't most often specific . The maiCongenital malaria infection is not uncommon in sub-Saharan Africa, and its prevalence varies from one site to another depending on the intensity of transmission . This prIt is in this context that we conducted a cross-sectional, prospective study to calculate the prevalence of congenital malaria, describe its clinical signs, and analyze its associated factors at Issaka Gazobi Maternity of Niamey-Niger.It is a cross-sectional and prospective study to calculate the prevalence of congenital malaria, describe its clinical manifestations, and identify risk factors.Anopheles gambiae and Anopheles coluzzi. Plasmodium falciparum is the main transmitted parasite [The study was conducted at Issaka Gazobi Maternity of Niamey, which is the National Reference Center (CNR) for Reproductive Health (RH), in collaboration with the Malaria Unit of the Center for Medical and Health Research, (CERMES) of Niamey. This site is located in a mesoendemic malaria transmission zone. This transmission is seasonal. It lasts 3 to 4 months, from July to October. The main vectors of malaria are parasite . The stuThe study population consisted of 249 consecutive mother/newborn pairs.Were included in this study all term neonates, hospitalized at Issaka Gazobi Maternity neonal department and their mothers. They are 0 to 7 days old.Were not included in this study, all newborns without a health record, those who received a blood transfusion, premature babies and those whose parents did not consent.We used the simple probabilistic method. The inclusion of newborns followed the pace of hospitalization. With an estimated prevalence of 20% congenital malaria, a 95% confidence interval, and an accuracy of 5%, the sample size was 246 subjects.Data were collected from an individual clinical record to trace the history of each patient. The variables collected on mothers are the identifier, the age, the origin of the patient, the number of prenatal consultations, the notion of malaria or a notion of treatment of malaria during pregnancy, the observance of intermittent preventive treatment (IPT), the use of a long-lasting insecticide-treated net (LLIN), and a biological examination to evaluate the plasmodial infection.The variables collected on the newborn were gestational age, birth weight, delivery route, newborn Apgar at birth, amniotic fluid colour, general condition of newborns , convulsive number, thick blood smear (TBS), thin smear (TS), blood count, Protein C Reactive (CRP), thick blood smear and thin smear cord blood, parasite density, plasmodial species, evolution of clinical signs, thick control after treatment, duration of hospitalization, and finally the way out of newborns.Microscopy, which is the WHO reference method for diagnosing malaria, was used. Each recruited newborn received a thick blood test (TBT) and thin smear (TS) of peripheral blood and umbilical cord blood by layer. If he is positive at birth, a TBT and TS control were performed at 48 hours.Each recruited mother had received a thick blood test and thin smear of peripheral blood from her fingertips.The blades of each patient are identified with the number of his medical file. The preparation of thick blood test and thin smears was made in accordance with WHO protocol .The dried slides were stained with May Grunwald Giemsa diluted 1/10 for 15 minutes. They are then rinsed and left in the open to dry.The slides were read under an optical microscope at 100X magnification. The thick drop was used to search and count the parasite. The parasite density was calculated by counting the number of parasites in parallel with 200 leucocytes. The parasite density is determined by the following formula: PD = Number of parasite counted × 8000/40. The thin smear was used to identify the plasmodial species.All haematological and biochemical examinations were performed at Issaka Gazobi Maternity Biology Laboratory using an automatic device (Reference: MSLAB O7 Plus). The parameters measured are red blood cell count, white blood cell count, hematocrit, platelets, blood sugar, and CRP.This study was authorized by the Faculty of Health Sciences of the University of Niamey as part of its doctoral dissertation work for 2017-2018. The anonymous nature of the questionnaire makes it possible to guarantee the confidentiality of the information collected. The informed consent of all mothers was obtained before their inclusion.An entry mask was created using Epi Info 7.0 software. All data has been entered and processed with this software. Chi-square Pearson and Fisher tests were used to compare percentages. A threshold of significance of 5% was retained.Two hundred and forty-nine (249) mothers were included in the study. The mean age of mothers was 27 years . 92.77% (240/249) of women came from the Urban Community of Niamey (UCN), and 7.23% (9/249) came from rural areas. 96.79% (241/249) had given birth to Issaka Gazobi Maternity, and 3.21% (8/249) had been referred.Two hundred and forty-nine (249) newborns were also included in the study. The sex ratio of newborns was 0.9. They were all born to terms. The age of newborns at inclusion varies from zero to seven days. Their average weight was 2754 g . Their average size was 47.23 cm . The characteristics of the population are summarised in μl . All infections (100%) were Plasmodium falciparum.26.51% (66/249) of newborns had a positive tick drop. The mean parasite density was 101 P/μl , confirming a persistent infection two days after birth.Forty-eight hours after the first biological examination, the thick blood drop remained positive in 14.06% (35/249) of neonates with a parasite density of 108 P/60% (21/35) of newborns had at least one general sign after delivery. 51.43% 18/35) of newborns had a temperature above 37.5°C. 42.86% (15/35) of newborns had an Apgar at eight [/35 of ne97.14% (34/35) of neonates were treated with artemether-lumefantrin in oral suspension for three days and 2.86% (1/35) with injectable artesunate. All the thick blood smear of the treatment control was negative. There was no relapse after treatment. The average duration of treatment was three days. All newborns were cured, and no deaths were recorded.μl . The average temperature was 37.6°C . The mean heart rate was 155 beats per minute . The mean respiration rate was 49 per minute . The mean haemoglobin level was 14.8 g/dl . The mean haematocrit was 40.7% . The platelet average was 259.4 . The average number of white blood cells was 14057 . The mean glucose level was 0.79 g/L . The average of C-Reactive Protein (CRP) was 8.7 .The mean parasite density of neonates with congenital malaria disease was 108 P/95.18% of pregnant women had prenatal consultations. Twenty-six point ten percent (65/249) of the mothers had three antenatal visits during pregnancy. 24.10% (60/249) completed four prenatal consultations, and 4.82% (4/35) did not attend prenatal consultations. Prenatal consultation is not a protective factor against congenital malaria. 88.57% (31/35) of neonates from prenatal mothers have had congenital malaria compared to 11.43% (4/35) of nonattenders. There is no difference in congenital malaria between the groups of women who consult and those who do not consult.Twenty-seven point thirty-one percent (68/249) of pregnant women received no dose of sulfadoxine-pyrimethamine (SP) during pregnancy. 24.9% (62/249) received one and two IPT, respectively. 22.09% (55/249) received three intermittent preventive treatments. Newborns of mothers who do not take IPT are more exposed to congenital malaria. In fact, 63% (22/35) of newborns of mothers who did not take IPT made congenital malaria compared to 37% (13/35). Children of mothers who did three IPTs had less congenital malaria (5.71%) than mothers who did two IPTs (28.57%) and those who did not receive IPT (37.14%).p = 0.04). Long-lasting insecticidal mosquito net is a protective factor against congenital malaria.Sixty-eight point sixty-seven percent (171/249) of women did not sleep under a long-lasting insecticide-treated bed net. Newborns of women who use LLINs have had less congenital malaria than women who do not use it. There is a statistically significant difference in congenital malaria between the two groups (Twenty-four point one percent (60/249) of women had fever during pregnancy. Newborns of women who have had a fever during pregnancy are half likely to be exposed to congenital malaria.th of June to 30th of November 2017 at the Neonatology Department of Issaka Gazobi Maternity in Niamey, describes the epidemiological, clinical, and biological characteristics of congenital malaria, analyses the factors of its transmission, and finally proposes preventative measures.This cross-sectional and prospective study, conducted from 1μl . Congenital malaria infection is common in sub-Saharan Africa [μL [Plasmodium from mother to child by the placenta is accidental because the foetuses are normally protected by the maternal antibodies, foetal haemoglobin, and colostrum that the newborn receives [The study was conducted on 249 pairs of mothers and newborns. The prevalence of congenital malaria infection, objectified by a positive thick blood slide, was 26.51% (66/249) with an average parasite density of 101 P/n Africa . Its preD: 47.3; 0; 320). n Africa . It was rica [μL , 10% in rica [μL , 0.64% irica [μL , 0.03% irica [μL , and 0% rica [μL . In uterμl  in Tanzania [P. falciparum and P. ovale were found in Mali [The incriminated plasmodium species are most often in Ghana . PlasmodTanzania , and fin in Mali .Clinical manifestations of congenital malaria are most often not specific . The maip = 0.04). Newborns of mothers with fever during pregnancy are at least twice at risk of congenital malaria. Fever is a bad prognostic factor for congenital malaria. Intermittent preventive treatment tends to show a protective effect against congenital malaria: Only thirty-seven percent of newborns in IPT mothers have had congenital malaria compared to 63% of those who do not. The effect of IPT also increases with the dose. Only 5.71% of newborns of mothers who did three IPTs had congenital malaria compared to 28% of those who had one dose of IPT.Only the nonuse of long-lasting insecticidal nets was significantly associated with congenital malaria (Several recommendations are made to diagnose and treat congenital malaria. Francisca et al. believes that in malaria-endemic areas, any newborn with a sepsis chart should be investigated for possible congenital malaria. This recommendation will be very strong if the mother has not correctly taken antimalaria prophylaxis . Other aThe main limitation of this study is the lack of a serological assessment to confirm the exposure and use of PCR to highlight submicroscopic infections.The frequency of congenital malaria infection is very high in Niamey where transmission is permanent with a strong recrudescence in the rainy season. On the other hand, the prevalence of congenital malaria disease is lower. The main risk factor is the lack of use of mosquito nets. Intermittent preventive treatment and distribution of long-lasting insecticide-treated mosquito nets should be provided to all pregnant women to prevent congenital malaria.


The spatial distribution and burden of dengue in sub-Saharan Africa remains highly uncertain, despite high levels of ecological suitability. The goal of this study was to describe the epidemiology of dengue among a cohort of febrile children presenting to outpatient facilities located in areas of western Uganda with differing levels of urbanicity and malaria transmission intensity.Eligible children were first screened for malaria using rapid diagnostic tests. Children with a negative malaria result were tested for dengue using a combination NS1/IgM/IgG rapid test (SD Bioline Dengue Duo). Confirmatory testing by RT-PCR was performed in a subset of participants. Antigen-capture ELISA was performed to estimate seroprevalence.p < 0.001).Only 6 of 1416 (0.42%) children had a positive dengue rapid test, while none of the RT-PCR results were positive. ELISA testing demonstrated reactive IgG antibodies in 28 (2.2%) participants with the highest prevalence seen at the urban site in Mbarara (19 of 392, 4.9%, Overall, these findings suggest that dengue, while present, is an uncommon cause of non-malarial, pediatric febrile illness in western Uganda. Further investigation into the eocological factors that sustain low-level transmission in urban settings are urgently needed to reduce the risk of epidemics.The online version contains supplementary material available at 10.1186/s12879-020-05568-5. Dengue is a mosquito-borne viral disease that is estimated to cause upwards of 400 million infections each year , 2. WhilAedes aegypti mosquito - the primary vector of dengue, along with yellow fever and Rift Valley fever - is ubiquitous in Uganda. Dengue outbreaks and transmission have been documented in neighboring countries, and cases have been reported among travelers returning from Uganda ) or a reported history of fever within the last 7 days were eligible. Children presenting without a parent or guardian to provide consent were excluded. After consent was provided, study personnel recorded demographic information, vital signs, and administered a brief health questionnaire  presenting to one of three outpatient clinics with documented fever   and collected dried blood spots (DBS) on filter paper . DBS sampling was chosen as a pragmatic but effective option for sampling children across the different field conditions at our study sites . The relResults of the dRDT were provided to the responsible clinician for the purposes of counseling and case management. Treatment and disposition (i.e. admission versus discharge) plans, as determined by the clinician, were recorded. DBS were stored at room temperature under desiccation and serum samples were stored at − 20 °C until analysis.All RDTs for the study were obtained directly from the manufacturer, stored in the original packaging at room temperature, and used in accordance with the manufacturer’s instructions prior to the expiration date. In brief, whole blood, either capillary (i.e. via finger prick) or venous was applied directly into the specimen wells of the malaria and/or dengue RDT. Assay diluent was added into the round well of the malaria and dengue IgM/IgG cassettes and the test was allowed to run for 15–20 min. Two staff members recorded the RDT results. A third, senior staff member adjudicated any discrepancies between the initial reads.Further description of specific procedures is provided below.Plasma proteins were eluted from DBS as previously described –20. One p-nitrophenyl phosphate substrate. Absorbance at 405 nm  was measured by spectrophotometry on a plate reader. ELISA data are reported as OD values that are the average of technical replicates. The average OD for technical replicates using DBS eluate obtained from flavivirus-naïve individuals (NHS) served as the negative control in ELISA assays. The cut off for positivity was calculated for each plate as the average OD of NHS = standard deviations + 0.1 [Binding IgG to DENV or ZIKV was measured by antigen capture ELISA as previously described . Brieflyns + 0.1 –20.Testing for anti-DENV IgM was only performed on participant samples with either a positive dRDT or IgG ELISA per CDC MAC ELISA protocol after titrating individual reagents per instructions . DBS eluNeutralization titers were determined by 96-well microFRNT , 25. DueWhole blood was drawn into serum serparator tubes and centrifuged. Viral RNA was extracted from 140 μL of serum using the QIAamp extraction kit  and eluted in 60 μL. Real time PCR was performed on the Rotor-Gene Q  platform using the RealStar® kits for Dengue, V2.0, Chikungunya, V2.0 and Zika V.1.0  in accordance with manufacturer’s instructions.p-value of < 0.25 were included in the subsequent multivariate analysis [p-value of < 0.05 was considered statistically significant in the final models.Data was collected in a REDCap database and analyzed with Stata 15.1  . We summanalysis . A resulp < 0.0001), likely attributable to an existing village-based, community health worker led fever management intervention targeting children less than 5 years of age and reducing the number of these patients that presented to our study site [A total of 1702 children met the eligibility criteria and were screened for malaria with a mRDT. Complete laboratory results were available for 1693 (99.5%) particpants, who were subsequently included in the analysis. Characteristics of the cohort are summarized in Table udy site .Table 2p = 0.006), but when the analysis was limited only to the high-transmission areas with higher risk, the protective effect was no longer significant .Overall, 369 children (21.8%) had a positive mRDT result. The lowest rate was observed at MRRH , whereas much higher rates were seen in Kasese  and Bugoye . Self-reported ITN use was associated with a protective effect against malaria  had an evaluable dRDT result, of which 5 (0.39%) had a positive result, defined as a visible NS1, IgM, or IgG band. Individual tests results include one child with positive IgM bands, three with positive IgG bands, and one with positive IgM and IgG bands. There were three positive dRDT results in Bugoye, two in Mbarara, and and one in Kasese . The Mbarara study site was the only significant predictor of a dengue infection in the multivariable analysis when using Kasese as the reference, although there was a statistical trend towards males being at higher risk of infection (p = 0.10)  DBS samples from participants with a negative mRDT test result identified reactive IgG antibodies in 28 2.3%) participants. Demographic and clinical characteristics are summarized in Table % particiOf the 28 participants with a reactive IgG antibodies by ELISA, only 1 (3.6%) had a positive IgG band on the dRDT. This occurred in the one patient with both IgM and IgG bands positive on the dRDT. Overall, the correlation in IgG results between the dRDT and ELISA was poor .p = 0.73). The vast majority of children  were discharged from the outpatient clinics, while 35 (2.7%) were admitted. Children with a anti-dengue IgG were admitted from the outpatient clinic at a higher rate, although the absolute number of admissions was low . There were no significant differences in admission rates by sex, age category, or clinical site.Among those with a negative mRDT result, 1297 of 1324 (98.0%) received treatment with an antibiotic, including five who also received treatment with artemether/lumefantrine (AL). An additional 17 (1.3%) received treatment with AL (and no antibiotic) despite negative mRDT test results. There were no differences in the rate of antibiotic administration between children with a reactive IgG antibody result and those without  samples tested , as wellchildren –33. The Given the low number of positive cases, a rigorous assessment of the performance of the dengue rapid diagnostic tests was not possible. The relatively poor correlation in IgG results between the dRDT and the ELISA is consistent with a study of febrile outpatients conducted in the Democratic Republic of Congo, which showed 2.5% IgG positivity by dRDT and 34.0% positivity by ELISA with an estimated sensitivity of 7.6% . In primOur results do highlight the urgent need for better diagnostics and management algorithms for children with acute febrile illness. Malaria was identified as the cause of fever in only 22% of participants, and among participants with a negative malaria test, nearly 80% reported cough or rhinorrhea with more than half (51.1%) reporting both symptoms. While not confirmed by microbiologic or molecular methods, many of these children likely had viral infections, including other arboviral diseases such as West Nile or Chikungunya , that wop = 0.53). Second, our facility-based and temporally-limited enrollment strategy may have missed focal outbreaks of dengue in the community. Dengue is well-known to emerge rapidly in non-immune populations then quickly disappear. Studies from neighboring countries and other surveillance methods do, however, support the validity of our findings. Third, we did experience some attrition of samples, due to improper labeling, inadequate specimen, or loss/damage in transport. We were, however, able to complete dRDT and ELISA testing in more than 97 and 94% of eligible participants, respectively. Fourth, IgG ELISA may overestimate dengue prevalence as this assay may also detect cross-reactive antibodies elicited by infection by related flaviviruses, though neutralization testing also supported prior infection by dengue for some subjects in this population [To our knowledge, our study is one of the first to examine the burden of dengue among children presenting to outpatient clinics in western Uganda with undifferentiated febrile illness. Strengths of our approach include the diverse geography of the clinical sites, large sample size, and different diagnostic testing strategies . The study also has a number of limitations. First, we excluded children with a positive mRDT from further consideration. While dengue-malaria coinfections have been reported in other countries , 38, 39,pulation , 41. LasDengue does not account for a large proportion of non-malarial, acute febrile illness presentations in western Uganda. However, the presence of the vector and the pathogen, along with a large susceptible population make sporadic outbreaks a major concern. Further investigation into the ecological factors that sustain low-level transmission in urban centers such as our Mbarara site, are urgently needed.Additional file 1.


Here we use a simple molecular epidemiology framework to examine the transmission dynamics of Plasmodium vivax in Mâncio Lima, the Amazonian municipality with the highest malaria incidence rate in Brazil. We prospectively genotyped 177 P. vivax infections diagnosed in urban residents between June 2014 and July 2015 and showed that local parasites are structured into several lineages of closely related microsatellite haplotypes, with the largest genetic cluster comprising 32% of all infections. These findings are very unlikely under the hypothesis of multiple independent imports of parasite strains from the rural surroundings. Instead, the presence of an endemic near-clonal parasite lineage circulating over 13 consecutive months is consistent with a local P. vivax transmission chain in the town, with major implications for malaria elimination efforts in this and similar urban environments across the Amazon.The relative contribution of imported  The epidemiology of urban malaria remains little explored in Latin America, the most urbanised region in the developing world . MalariaUrban malaria is affected by human mobility patterns, as parasites from the rural communities of high endemicity may be introduced into urbanised spaces and may be locally transmitted, leading to micro-epidemics or sustained endemic propagation in cities and towns , 10, 14.Plasmodium vivax [Brazil has experienced a dramatic decline in malaria incidence over the past three decades, but residual transmission remains entrenched in the Amazon Basin . This reum vivax .P. vivax lineage, consistent with sustained local malaria transmission.Here we introduce a simple molecular epidemiology framework  to explo2 in north-western Brazil ; no minimal parasite density threshold was set. Only urban residents were enrolled because directly observed therapy and post-treatment follow-up would be impractical in remote rural sites. Exclusion criteria were severe or complicated malaria, pregnancy or lactation, severe malnutrition, severe anaemia and other serious chronic clinical conditions, glucose-6-phosphate dehydrogenase deficiency, antimalarial use in the preceding 2 weeks and known hypersensitivity or allergy to study drugs. Spatial analyses considered only subjects whose houses had GPS coordinates determined. We used 200-μl of anticoagulated blood drawn before treatment to isolate parasite's DNA, using QIAamp DNA blood kits . A single pre-treatment sample was analysed for each participant.A reatment . EligiblWe typed 14 single-copy microsatellite loci that map to 10 different chromosomes ; 13 of tHE) as a measure of overall genetic diversity, defined as n is the number of isolates analysed and pi is the frequency of the i-th allele in the population. HE gives the average probability that a pair of alleles randomly obtained from the population is different and ranges between 0 and 1. We next calculated the pairwise number of different alleles or mismatches, out of 14 loci analysed, as an estimate of genetic distance between isolates. The observed mismatch distribution was compared with that expected under random association of alleles in a panmictic population, which was generated by using 50 000 simulated datasets in which alleles were randomly reshuffled among haplotypes using the randperm function of MATLAB . Finally, we calculated the distance between each haplotype and the nearest haplotype found in the population, here termed ‘distance to nearest', which ranges from 0  to 14 mismatches . Similarly, we estimated the distance-to-nearest distribution that is expected under panmixia by randomly reshuffling alleles among haplotypes as above.We used Arlequin 3.5 software  to calcuVD) of the number of alleles at which each pair of haplotypes differ in the population (the mismatch distribution as above) with the variance expected under random association of alleles (VE) as follows: l is the number of loci analysed [VE was derived from 10 000 random permutations. LD is expected to increase VD relative to VE [VD was greater than 95% of VE values. Data were analysed with LIAN 3.5 software [The standardised index of association analysed . VE was ve to VE ; signifisoftware .We used the goeBURST algorithm  implemenThis study follows the STROBE guidelines for observational studies and its extension STROME-ID for molecular epidemiological studies.The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Study protocols have been approved by the Institutional Review Board of the University Hospital of the University of São Paulo (550.565). Written informed consent was obtained from all study participants or their parents/guardians.P. vivax infections were laboratory confirmed among urban residents in Mâncio Lima. DNA samples from 177 of them were completely genotyped at all 14 microsatellite loci . Further analyses only considered fully typed isolates. The temporal distribution of genotyped P. vivax infections followed closely that of all P. vivax infections diagnosed in Mâncio Lima throughout the study period . The urban population of P. vivax was nearly as diverse  as its rural counterparts across the Brazilian Amazon (HE ranging from 0.68 to 0.74) [Between June 2014 and July 2015, 1487 to 0.74) . Overallwithin the same clonal lineages or haplogroups (see from different haplogroups (see vs. <0.1% expected in a randomly mating population) had their nearest haplotype ⩽2 mismatches apart  identified 21 haplogroups in the population, each comprising between 2 and 57 isolates . Only 53We found highly significant LD analyses . We firsanalyses , our finP. vivax lineage over the study period. Interestingly, if urban malaria transmission does occur, visitors from the town can also introduce locally circulating parasites into neighbouring rural villages, indicating that the urban centre may be both a source and sink of parasites  comprised 57 isolates  and 35 unique haplotypes  that are represented in red in arasites .Fig. 5.The second largest clusters, haplogroup 2  are theWe examined the genetic connectivity of parasites circulating in the urban population over 14 months. Although completely identical haplotypes were relatively infrequent in unrelated infections, 70% of isolates shared ≥12 alleles (out of 14 loci typed) with one or more samples, clearly deviating from expectations for a randomly mating population. Significantly, nearly one-third of genotyped isolates clustered into a single endemic haplogroup, which was widely distributed over space and time. This finding would be extremely unlikely in a scenario where all infections among urban residents are independent imports from different rural villages . In contOne can argue, however, that a ubiquitous parasite lineage might have been imported from multiple source villages surrounding the urban centre. The source–sink model shown in P. vivax carriers are representative of all parasites circulating in the rural Amazon [Studies of source–sink dynamics should ideally include samples from all relevant source and sink communities. Nevertheless, this and previous molecular epidemiology studies of urban malaria in South America (e.g. ) were rel Amazon , we recoDespite these limitations, we interpret the presence of an endemic near-clonal parasite lineage circulating in symptomatic infections in the town over 13 consecutive months as a strong evidence of self-sustained urban malaria transmission in an Amazonian setting. Therefore, we suggest that interventions targeting urban dwellings are critical to eliminate malaria transmission in the main hotspot of Brazil and similar urbanised settings in South America where vector proliferation has been favoured by man-made larval habitats such as fish-farming ponds .


People with multiple sclerosis (pwMS) have increased comorbid disease (CMD) risk. Most previous studies have not considered overall CMD burden.To describe lifetime CMD burden among pwMS.n = 25,476) were matched by sex, age, and county of residence with general-population comparators . Prevalence, prevalence ratios (PRs), survival functions, and hazard ratios by MS status, age, and time period compared seven CMD: autoimmune, cardiovascular, depression, diabetes, respiratory, renal, and seizures.PwMS identified using Swedish registers between 1968 and 2012 (The magnitude of the PRs for each CMD and age group decreased across time, with higher PRs in earlier time periods. Before 1990, younger age groups had higher PRs, and after 1990, older age groups had higher PRs. Male pwMS had higher burden compared with females. Overall, renal, respiratory, and seizures had the highest PRs. Before 2001, 50% of pwMS received a first/additional CMD diagnosis 20 years prior to people without MS, which reduced to 4 years after 2001. PwMS had four times higher rates of first/additional diagnoses in earlier time periods, which reduced to less than two times higher in recent time periods compared to people without MS.Swedish pwMS have increased CMD burden compared with the general population, but this has reduced over time. Increased surveillance for common CMD may also reduce additional burden faced by health-care systems as multi-morbidity care has substantial medical and financial costs.12People with multiple sclerosis (pwMS) have increased risk of additional diseases including cardiovascular, respiratory, autoimmune, and mood disorders such as depression, among others.Despite studies demonstrating increased risk of CMD among pwMS, the prevalence of overall comorbid burden remains largely unknown or is known for specific diseases rather than multiple conditions. To date, no study has provided comprehensive estimates of CMD prevalence by age, sex, and time period of pwMS compared to the general population in Sweden. Therefore, this study aims to describe the lifetime burden of CMD in pwMS compared with a sample of the general population and determine whether pwMS has an overall increased prevalence or risk of CMD diagnoses.13 Diagnostic accuracy of the NPR for diseases that are not rare such as the disease groups studied here varies between 85% and 95% as described elsewhere.14 The MSR introduced in 1996 provides a high degree of diagnostic accuracy and coverage.16This cohort study identified pwMS in Sweden recorded from either the Swedish Multiple Sclerosis Register (MSR) or National Patient Register (NPR) with a primary or secondary diagnosis using International Classification of Diseases (ICD) codes  between 1968 and 2012. The NPR includes inpatient hospital diagnoses since 1964 and outpatient diagnoses since 2001.n = 29,310) were individually matched by birth year, sex, region, and vital status at earliest date of MS diagnosis (index date) from either the NPR or MSR with up to 10 randomly selected general-population comparators  using the Total Population Register, which includes information on vital status and region of residence of all Swedish residents from 1968 onward. Highest attained education level was obtained from the Longitudinal Integrated Database for Health Insurance and Labour Market Studies (LISA).17 Data linkage was achieved using the unique personal-identity number issued to all Swedish residents.PwMS (Study entry began from index date (date of first MS diagnosis or the same date for the matched comparators). Individuals were included if alive, had not emigrated from Sweden prior to study entry, and were 6–100 years of age. Matched individuals without MS were excluded if they did not meet the above inclusion criteria. Therefore, 25,476 pwMS and 251,170 matched individuals without MS were included.119 For the general-population cohort, diagnoses may have been a first or a comorbid diagnosis. A CMD diagnosis was defined as a record of any ICD code in the NPR (Supplement 1). Diagnoses prior to and post index date were identified. If individuals had more than one disease diagnosis in the same disease category, they were counted only once using the earliest date of diagnosis and were considered affected for all subsequent years.Seven chronic CMD categories were considered: autoimmune, cardiovascular, depression, diabetes, respiratory, renal, and seizures. These disease categories were previously identified as being overrepresented in pwMS.Approval was given by the Regional Ethical Review Board at Karolinska Institutet (2013/1156-31/5). Informed consent is required for inclusion in the MSR.Prevalence over four time periods, 1968–1980, 1981–1990, 1991–2000, and 2001–2012, was determined in age groups of 6–18, 19–40, 41–60, 61–80, and 81–100 years and stratified by MS status and sex. These time periods were chosen to identify the earliest years of the NPR; the years before (DMT) were available; the years in which the first-line DMT became available; and finally, availability of the second-line DMT in conjunction with the addition of outpatient hospital diagnoses to the NPR. PwMS and the general-population cohort entered the denominator of the age group in each time period provided they had not emigrated or died for all attained age groups within the time period. Individuals could contribute to multiple age groups and time periods. The prevalence numerator was the total number of individuals in the age group and time period with the specific CMD calculated with 95% confidence intervals (CIs). Prevalence ratios (PRs) with 95% CIs comparing pwMS with the general population were calculated.To determine age at diagnosis and rate of a first/additional CMD by MS status, survival probabilities and hazard ratios were calculated stratified by both time period and baseline CMD status. Individuals entered at index date and were at risk until occurrence of their first/additional CMD diagnosis in any of the disease categories: death, emigration, age 100 years, or end of the time period, whichever came first. Provided the individual had valid entry characteristics by the beginning of the next time period, individuals were included again and stratified by their new baseline CMD status in the time period.20 Four-knot models placed according to default locations20 modeled age as the underlying timescale. Due to violations in the proportional hazards assumption assessed by the Schoenfeld residuals, two internal knots were used to model the (time-varying) effect of MS status by age and baseline CMD status. CMD status was categorized as 0, 1, and 2+ as few individuals had more than two CMD. Visual inspection of hazard functions derived from models and minimization of the Akaike and Bayes Information Criteria determined number of knots.Survival probabilities and hazard ratios were predicted using flexible non-parametric survival (FNPS) models with age as the underlying timescale using restricted cubic splines. FNPS allows greater flexibility in the shape of hazard functions compared with other non-parametric models and easily accommodates multiple timescale and time-dependent effects.21 Potential confounders included sex, county of residence, educational level , and overall follow-up time split by 2-year intervals.Standardized survival curves and hazard ratios (HRs) were predicted taking into account differences in characteristics between people with and without MS to ensure comparability of estimates.Supplement 4. Statistical analysis was performed using SAS software version 9.422 for prevalence estimates and the command stpm220 and stpm2_standsurv were used for the FNPS in Stata 15 software.Two sensitivity analyses were conducted to define possible seizure etiology and type of diabetes. For people classified with seizures in each time period, the proportion of them with one primary diagnosis or two primary diagnoses at two separate occasions were determined as this gives a crude indication of possible chronic seizures. To determine the proportion of people with type 1 and 2 diabetes within each time period, their age at first diabetes diagnosis was used for classification and is further described in The proportion of pwMS with incident diagnosis of MS was greater than prevalent diagnosis over several time periods, with exception of 1991–2000 . The majSupplement 2). Seizure and respiratory disease prevalence increased moderately over time, while cardiovascular disease (CVD) and diabetes remained stable. Renal disease steeply decreased over time. Similar trends were observed among male and female pwMS (Supplement 4).The prevalence of autoimmune disease and depression increased steeply from 1968–1980 to 2001–2012 in the same age groups . Lowest prevalence was for diabetes and CVD, but autoimmune disease was higher among female pwMS. For both sexes with MS, 41–60 years had highest prevalence for respiratory and renal disease (in earlier time periods) and CVD. Lowest prevalence among female pwMS was seizures and diabetes and autoimmune disease and seizures among male pwMS. Male pwMS had higher prevalence for renal, respiratory, and CVD than females as females had higher prevalence of autoimmune disease and depression. Both sexes aged 61–80 years had highest prevalence of respiratory disease, CVD, and renal disease in earlier time periods, although all were higher among male pwMS. Lowest prevalence for both sexes with MS was autoimmune  and seizures. Depression was higher among females. Both sexes with MS 81–100 years had highest prevalence for renal disease (in earlier time periods), CVD, and respiratory disease with men having a much higher prevalence for all of them, including diabetes. Lowest prevalence was autoimmune disease and seizures for both sexes, although women had higher depression prevalence than male pwMS.Few pwMS between 6–18 years were diagnosed with CMD: highest prevalence among both sexes was respiratory disease and lowest prevalence was for depression and CVD irrespective of time period and is included in tables in ale pwMS , and autale pwMS . Among bOverall  and for Supplement 3). In the 81–100 age group, PRs are smaller with larger CIs than other age groups.Between 1968–1980 and 1981–1990, pwMS in younger age groups (<41 years) compared to the general population had higher PRs than older age groups with the exception of respiratory and renal diseases –6. BetweOf the individuals included with seizures, the proportion of them with one or two primary diagnoses of seizures was reduced; however, comparing the PRs of individuals with one or two primary diagnosis remained similar and were similar to the main seizure burden analysis (results not shown). This indicates that the possible seizure etiology is more representative of individuals with repeated seizures (epilepsy) than single episodes.The age at diagnosis for 50% of pwMS for a first/additional CMD diagnosis in all time periods but 2001–2012 was approximately 20 years before the general population irrespective of initial CMD status . In 2001PwMS had higher CMD burden than the general population. This shifted from younger to older age groups over time, but still 50% of pwMS were diagnosed with a first/additional CMD diagnosis 20 years before the general population in all time periods except 2001–2012. Male pwMS had higher burden than females, but for both sexes highest disease burden was for renal, respiratory, and seizure diseases, although there was slight variation by age group. Female pwMS had higher burden of autoimmune disease and depression.3 depression,2 various cardiovascular diseases,1 diabetes,1 epilepsy/seizures,23 and lung disease,4 consistent with overall trends reported here. However, this study found that PRs of renal disease was higher among pwMS than the general population, despite their prevalence drastically decreasing over time for only pwMS, in contrast to PRs of renal disease reported in other studies.4 The most common CMD previously reported among pwMS were depression and chronic lung diseases;18 however, this study found that CVD and respiratory and renal diseases were most prevalent, with depression only most common among females.PwMS are known to be at higher risk of CMD. Previously, no studies (we know of) have determined disease burden prevalence or PRs among different age groups, by sex, time period and over a wide variety of disease categories in Sweden using national-level data. Several meta-analyses of CMD in pwMS were recently published indicating a higher proportion of autoimmune diseases,The magnitude of PRs were lower in 81–100 year olds. This is due to pwMS experiencing shortened life spans, an effect most pronounced in this study’s male MS population. In addition, people with and without MS who live longer may have fewer CMD than people with shorter lives. In addition, CIs were very wide among 6–18 year olds as pediatric MS is uncommon, and they had few CMD. Point estimates among this group may be unreliable and should be interpreted with caution; however; they still provide an indication of disease burden.24 Higher rates in males could be due to the increased severity of their MS requiring hospital care25 in particular for respiratory disease as pwMS tend to have more respiratory dysfunction.26 MS has a long and insidious latency period, with appearance of symptoms occurring over years. Therefore, pre-clinical symptoms of MS could contribute to development or diagnosis of another chronic disease as pwMS have been shown to have increased health-care utilization 5 years prior to MS onset.27 Awareness of CMD is especially important for pwMS, they may interpret symptoms as part of their MS, leading to synergistic negative effects of multiple conditions having an impact on disability,28 quality of life,29 and risk of hospital admission.11Male pwMS had greater disease burden than female, especially for renal, respiratory, and CVD. Females had higher burden of depression and autoimmune disease, although both sexes had a higher burden of CMD than the general population. Seizures had some of the highest PR especially among females with MS, but their prevalence was much lower than other CMD. Females may seek health care at an earlier time possibly reducing the need for hospital care as women have been shown to be older at first hospital admission.MS treatment typically takes a multi-disciplinary approach to manage the wide breadth of symptoms. A patient’s clinical complexity can be influenced by disease course, also by age, sex and CMD; all of which could influence disease outcomes. This was demonstrated here by the observed sex and age differences for people with and without MS in prevalence and risk of first/additional CMD, although differences over time became less pronounced especially with transition of disease burden from younger to older ages over time. There was also a marked decrease in PRs over time showing a reduced burden among pwMS compared to non-MS. This suggests better management of MS and use of more effective DMT, possibly slowing the progression of MS and accumulation of CMD. Many CMD occurred in pwMS 20 years prior to the general population, but possibly due to effective treatments and more awareness of MS among clinicians and pwMS, the age difference reduced to 4 years in 2001–2012. Although when comparing higher levels of baseline CMD  in 2001–2012, the age difference was 8 years, which suggests the possibility of improvement in screening and monitoring for CMD and counseling on modifiable risk factors in pwMS. This may not only reduce the risk of CMD but also have implications for MS and severity due to influence of CMD, particularly among male pwMS.Advantages of this study include use of both the MSR and NPR to ensure the widest capture of MS patients, with no loss to follow-up in Sweden between 1968 and 2012. Using both registers allowed use of the earliest possible recorded date of MS diagnosis. Due to the wide-inclusion criteria, the results of this study may be generalizable to other Nordic countries, or other high-income countries with similar standards of hospital-care and patient treatment.30 Another limitation includes possible misclassification of CMD as the first diagnosis of a CMD was used to identify affected individuals. However, using hospital diagnoses may reduce the number of misclassifications versus primary-care records as hospital diagnoses indicate more severe conditions requiring medical care: this will apply equally to those with and without MS. Although the NPR has good diagnostic accuracy for more common diseases such as CVD and diabetes and some autoimmune diseases like rheumatoid arthritis,14 it may be lower for less common diseases or diseases that are less severe not requiring a hospital admission. The specificity of some ICD codes was limited, such as for type 1 and 2 diabetes and chronic or isolated seizures. For diabetes, age at first diagnosis was used to broadly differentiate between the disease types and for seizures we examined single or multiple hospital contacts with seizures as the primary discharge or outpatient diagnosis. While neither of these approaches will be completely accurate, they largely indicate the proportion of disease types and how this differs between people with and without MS. Furthermore, the use of broad disease categories examined the overall disease profile rather than a specific disease, providing an overview of CMD and reducing misclassification. Some CMD may have been underestimated, such as for depression, as inpatient/outpatient hospital diagnoses were used as primary-care records were unavailable. This may mostly affect the non-MS individuals as they may require less hospital care if they do not have any chronic disease. This is relevant to risk of surveillance bias as pwMS tend to encounter medical professionals more frequently than the general population. Stratifying the survival/hazard models by baseline CMD provided control for regular hospital-care contacts, thus reducing the likelihood of differential surveillance bias. Individuals without MS, but with a chronic disease (1 or 2+ CMD), may also require increased contact with health care increasing comparability with pwMS. The estimates are still representative of conditions warranting a hospital diagnosis, demonstrating the greater burden of disease among pwMS. As pwMS generally have increased contact with health-care services, this may provide a unique opportunity for increased screening for common CMD in order to reduce disease burden and further reduce the differences between pwMS and the general population.Potential limitations include not being able to control for lifestyle risk factors such as smoking.In conclusion, this study provides a comprehensive overview of age-specific and sex-specific CMD burden pwMS experience over their entire life course. Clinicians managing pwMS should be aware of this increased disease burden, and efforts for early screening and prevention may be key in reducing CMD later in life.Click here for additional data file.Supplemental material, MSJ910497_supplement_1 for Comorbid disease burden among MS patients 1968–2012: A Swedish register–based cohort study by Kelsi A Smith, Sarah Burkill, Ayako Hiyoshi, Tomas Olsson, Shahram Bahmanyar, David Wormser, Yvonne Geissbühler, Alan Moore, Vineetkumar Kharat and Scott Montgomery in Multiple Sclerosis JournalClick here for additional data file.Supplemental material, MSJ910497_supplement_2 for Comorbid disease burden among MS patients 1968–2012: A Swedish register–based cohort study by Kelsi A Smith, Sarah Burkill, Ayako Hiyoshi, Tomas Olsson, Shahram Bahmanyar, David Wormser, Yvonne Geissbühler, Alan Moore, Vineetkumar Kharat and Scott Montgomery in Multiple Sclerosis JournalClick here for additional data file.Supplemental material, MSJ910497_supplement_3 for Comorbid disease burden among MS patients 1968–2012: A Swedish register–based cohort study by Kelsi A Smith, Sarah Burkill, Ayako Hiyoshi, Tomas Olsson, Shahram Bahmanyar, David Wormser, Yvonne Geissbühler, Alan Moore, Vineetkumar Kharat and Scott Montgomery in Multiple Sclerosis JournalClick here for additional data file.Supplemental material, MSJ910497_supplement_4 for Comorbid disease burden among MS patients 1968–2012: A Swedish register–based cohort study by Kelsi A Smith, Sarah Burkill, Ayako Hiyoshi, Tomas Olsson, Shahram Bahmanyar, David Wormser, Yvonne Geissbühler, Alan Moore, Vineetkumar Kharat and Scott Montgomery in Multiple Sclerosis Journal


Leishmania parasites. The aim of this study was to check if Plasmodium falciparum  DNA remains stable in blood samples deposited in Whatman paper after suffering different transport and storage conditions, and to compare the sensitivity of these results with those offered by thick a smear and Rapid Diagnostic Test, and besides to examine whether P. falciparum DNA would be detected and quantified by Real time quantitative PCR (qPCR) from hairs of people with different types of malaria. P. falciparum Histidine Repeat Protein II (pHRP-II) antigen detection and P. falciparum DNA were detected in 18 of 19 dry blood samples adhered to Whatman paper (94.74%), besides, Plasmodium DNA was also detected in seven out of 19 hair samples analyzed (36.84%), remaining stable until analysis for several months under the exposure to different environmental conditions. Although the sensitivity of PCR for the diagnosis of malaria in hair samples is not as high as blood analysis, the study of Plasmodium DNA presence in blood and hair could constitute a complementary tool with numerous advantages in sample collection, transport and storage. We suggest that the method could be also applied to medical, forensic and paleo-parasitological diagnosis, not only for malaria but also for searching many other pathogens in hair samples.Malaria has been for millennia one of the best known and most destructive diseases affecting humans. Its high impact has aroused great interest for the development of new effective and reliable diagnostic techniques. Recently it has been recently published that hairs from mammal hosts are able to capture, hold and finally remove foreign DNA sequences of  Anopheles mosquito and caused by protozoan parasites of the genus Plasmodium. Five species of this genus are known to cause infections in humans—Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi, with P.falciparum being the most virulent and deadly species.Malaria is one of the best known and most destructive disease affecting humans. It is transmitted by the female Plasmodium in asymptomatic patients or with unspecific symptoms [The 2019 World Health Organization report stated that there were about 228 million cases worldwide, compared with 251 million cases in 2010 and 231 million cases in 2017. Despite deaths from malaria have been reduced from 400,000 in 2010 to 260,000 in 2018, the sub-Saharan area of Africa and India remain the most affected areas in the world [Plasmodium species, but also for the quantification of parasite load in blood. However, detection can be limited in those cases with low parasite loads, in addition to having other drawbacks such as the need for an optical microscope, clean glass slides, immersion oil, reagents for staining, and most importantly, a skilled microscopist [Currently, diagnostic methods implemented in most surveillance programs include microscopy and rapid diagnostic tests (RDTs), both recommended by WHO . Microscoscopist ,5. On the other hand, immunological techniques and the development of various RDTs have allowed quick diagnosis in the field, although with rather high economic costs . HoweverPlasmodium species. Although this technique is more expensive than traditional thick smear, it has advantages such as its high precision and sensitivity [Plasmodium [Molecular methods are a suitable alternative, although costs and technical requirements hamper currently its implementation in areas with limited resources . PCR metsitivity ,11,12. Basmodium , mainly asmodium ,14. Plasmodium DNA has been also described in urine and saliva [Leishmania infantum and Leishmania major parasites, respectively. Dielectrophoretic and magnetophoretic approaches have been recently proposed such as promising new techniques due to their high specificity for red blood cells infected with the malaria parasite [Nowadays, in rural areas of developing countries, the accurate diagnosis based on non-invasive samples is very necessary and demanded, especially in cases where sampling must be repeated . Despited saliva ,17,18,19d saliva  and wildd saliva , as welld saliva  are ableparasite .Plasmodium falciparum  DNA remains stable in blood samples deposited in Whatman paper after suffering different transport and storage conditions, and to compare the sensitivity of these results with those offered by thick smear and RDTs. Taking these into account, one of the aims of this study was to check if P. falciparum DNA could be detected and quantified by Real time quantitative PCR (qPCR) from hairs of people with different types of malaria, and if it would be possible to apply this method to diagnose the disease.On the other hand, we wanted to examine whether We think that this sampling method may help in malaria diagnosis, especially in situations where is difficult to find laboratory facilities, as in rural areas. We consider that countries with greater economic resources must collaborate on the eradication of malaria by detecting asymptomatic patients, involving themselves in the WHO universal access strategy for the malaria diagnosis, and providing the diagnosis in other countries with imported malaria due to migratory movements and international travelling.P. falciparum .A transversal descriptive study was conducted with 19 patients attended in Nemba District Hospital (Rwanda) selected in a consecutive way. All of them came from the district of Gakenke  with confirmed malaria caused by Several types of malaria (uncomplicated (U), severe (S) and cerebral (C)) and different grades of severity and range of symptoms were present in these patients. Malaria was diagnosed by thick blood smear, and parasite load was assessed using the plus system , as follThe remaining volume of blood from routine thick blood smear diagnosis was deposited in two 20 mm diameter circles of 3MM Whatman  filter paper (100 µL of blood/ circle) and dried at room temperature (18–22 °C). Besides, head hairs without roots (about 50 units) were obtained from all patients. Hairs were cut closest to the bulb with sterile scissors. As all patients had short hair (1–4 cm), we obtained approximately 25 cm/hair per patient. All samples were introduced in sterile resealable plastic bags and were properly identified, including the clinical history of each person. Samples were shipped to LeishmanCeres laboratory (Spain) and then maintained in different storage conditions until analysis to check the stability of samples: at room temperature (4 months at 18–24 °C), refrigeration (3 months at 2–6 °C) and freezing (1 month at −20 °C and 1 month at −80 °C). P. falciparum Histidine Repeat Protein II (pHRP-II), and also against Plasmodium Lactate Dehydrogenase (pLDH), a Pan antigen common for the four malaria species . A positive result only for pHRP-II band indicates an uncomplicated infection by P. falciparum. Positivity results only for the pLDH band shows the presence of infection caused by other species different from P. falciparum. The reaction against the two bands could be interpreted as positive against P. falciparum, without discarding the possibility of a mixed infection by other species.To confirm the microscopic diagnosis of malaria, a rapid diagnostic test was performed. For this purpose, from each patient 50µL of blood  was incubated in 50µL of PBS + 0.05% Tween-20 for 30 min at 37 °C. Approximately, 5 µL of the supernatant was used to conduct an immunochromatographic analysis using the On Site Pf/Pan Malaria Ag Rapid Test , following the manufacturer’s instructions. This kit detects antibodies against specific P. falciparum DNA could be detected and quantified in head hairs. For these purposes, one blood disk and about 25 cm of hair per patient were incubated separately for 2 h at 37 °C in 250 µL (hair) or 500 µL (blood) of lysis buffer  with 100 µg/µL of proteinase K . After an overnight incubation at 56 °C, DNA from all samples was obtained with Ultra Clean Blood Spin kit  according to the manufacturer’s instructions. The detection and quantification of parasite DNA were performed using the Primer Design™ Quantification of P. falciparum Advanced Kit , designed to target a sequence within the plasmeps in 4 genes of P. falciparum that has a very low homology with other species of plasmodia that cause malaria. PCR reactions were carried out in 96-well PCR plates in a final volume of 20 µL (5 µL of DNA + 15 µL of Reaction Mix), containing 1 µL of P. falciparum Primer/Probe Mix, 1 µL of internal extraction control primer/probe Mix, 3 µL of RNAse/DNAse free water and 10 µL of iTaq™ Universal Probes Supermix . The thermal cycling profile was: incubation at 50 °C for 2 min, initial denaturation step at 95 °C for 10 min, 50 cycles of denaturation at 95 °C for 10 s and finally an annealing-extension at 60 °C for 1 min. Positive  and negative (RNAse/DNAse free water) controls and an internal DNA extraction control (used to detect the presence of PCR inhibitors) were included in the assay. All PCR analyses were performed in a Step One Plus Real Time PCR System . To detect and quantify parasite DNA, a standard curve was carried out using 4 quantities  of the P. falciparum DNA included in the kit, analyzed together with the samples in duplicate. Samples were considered positive when the threshold cycle (Ct) of almost one of the wells was lower than the Y-intercept value (the expected Ct for 1 copy of the target gene).These analyses were conducted to evaluate the sensitivity of blood-qPCR, to compare the results with those obtained by microscopy and with the RDT and also to examine whether The study was designed according to the Declaration of Helsinki, and the protocol was approved by the Nemba District Hospital ethics committee (REF: 252-16). All subjects were previously informed, they understood the objectives and practices involved in the study and they signed an informed consent document. In terms of minor patients, the collection of samples was done in the presence of their parents or guardians after they have signed the informed consent.Blood samples were obtained as part of routine clinical procedure for the diagnosis of malaria. Furthermore, at that moment, hair samples were obtained to be studied in this work. These samples were collected by the medical personnel involved in the study, who coded and anonymized them.P. falciparum by microscopic thick blood smear technique, with different degrees of parasite load (from slight to very high), and some of them received treatment against malaria . All of them were diagnosed as positive to  malaria .P. falciparum (positive results in pHRP II band), in 18 out of 19 patients (94.74%)  , with 12P. falciparum DNA was detected in 18 out of 19 patients (94.74%), with copies of the target gene ranging from 11.68 to 171,040.16  ranging the no. of DNA copies from 3.8 to 177.73 , were found in people with cerebral malaria treated with quinine, who also showed very high parasite load by thick smear .P. falciparum and other malaria-causing species are mainly present in Africa and parts of Rwanda [f Rwanda , the stuf Rwanda . For theP. falciparum, except in one patient with low parasitemia. These are similar results to those were reported by other RDTs applied to fresh blood samples that show a sensitivity of around 95% of the cases confirmed by microscopy [P. falciparum infections. The Pan-malarial pLDH antigen has been described also as a useful tool to monitor success of antimalarial therapy, as the recognition of the pLDH band turns negative after a successful treatment [Immune chromatography-based rapid diagnostic tests have been used since 1990, and nowadays are very common in field studies, using blood to diagnose this disease. In our work we used an RDT which has been evaluated by the WHO ,26, and croscopy ,28,29,30croscopy . These rcroscopy , which ireatment ,33. It sreatment ,14. The P. falciparum DNA by qPCR in 18 out of 19 patients (94.74%), with confirmed malaria. According to the results, there seems to be a certain correlation between the parasitic load present in blood by microscopy with the another one detected by quantitative PCR, as previously reported [However, the quality of the DNA recovered from dried blood spots decreases with storage time . In our reported ,34,35.Plasmodium DNA in other samples besides blood, such as urine and saliva, was recently demonstrated [Leishmania-infected animals opened a new suitable scientific field to study the physiology of these epithelial cells during several diseases, acting as a very specialized tissue to sequestrate and eliminate foreign genetic material [P. falciparum DNA in malaria patients’ hair confirms again the excretion and purification function of this tissue. It also allows the development of a new tool for epidemiological testing, having significant methodological advantages since the samples are obtained by a noninvasive way. In addition, the foreign DNA is highly stable inside the hair [In relation to malaria diagnosis by qPCR, the presence of nstrated ,17,18,19material ,22,36,37the hair  due to tthe hair .Plasmodium DNA incorporation in the hair of malaria patients. According to the data presented, positive hair samples corresponded to patients with either uncomplicated or cerebral malaria. The negative data, that constitute 63.16% of the total analyzed samples, cannot be determined. The sensitivity observed in hair is lower than that previously reported (70%) for Leishmania-infected dogs [Leishmania causes skin disorders [Leishmania DNA was present in the order of 10,000–20,000 copies/parasite [Plasmodium was present in a single copy/parasite. In our opinion we could improve the detection of Plasmodium DNA in hair using another target gene, for example the mitochondrial genome, considered to be present in a quantity from about 20 to 200 copies/parasite [It is true that there are still many questions which need to be further assessed, about the chronology and mechanism for ted dogs . This faisorders ,39, and parasite , whereasparasite ,13.Plasmodium DNA could be incorporated into follicular cells by migration from the bloodstream, but this mechanism is still unclear. Therefore, further research taking this into account is needed to improve the sensitivity of the technique. The DNA presence seems to be strongly associated with the parasite load, since positive results belonged to patients with high amounts of DNA copies detected in blood and high blood thick smear parasitemia. However, the absence of DNA in hair samples could be due to the clinical stage  of the patient at the moment of sampling. The sensitivity of the hair-qPCR method for malaria diagnosis must be the aim of wider molecular and clinical future studies as stated before. Despite the fact that the sensitivity of the hair-PCR method obtained in Plasmodium was too low to apply it as a medical diagnosis method, we think that it would have important implications in forensic science and also in paleoparasitology studies [In addition, we believe that since malaria is a hematic parasitic infection, obtaining hair with bulbs could increase the sensitivity of the technique, as  studies .Despite the limitations of the study, mainly due to the sample size, we consider that this new finding has a notorious impact in clinical and care practice, not only for patients with malaria, but also for patients with other infectious diseases. Moreover, hair samples have numerous advantages. The collection is noninvasive, and transport and storage do not require special conditions.Finally, we believe that countries with a greater socioeconomic level could collaborate in eradication of malaria becoming involved in the WHO universal access strategy for the malaria diagnosis.P. falciparum DNA was successfully detected by qPCR in blood samples conserved in Whatman paper. After being shipped from Rwanda and submitted to several storage conditions, blood and plasmodial DNA remained stable, providing high sensitivity results in qPCR, compared to thick blood smear and RDT and confirming malaria diagnosis.P. falciparum DNA in human hair detected by qPCR, supporting previous studies in animals infected with Leishmania spp. Our results might help to understand the role of hair and its physiological functions. Despite this, we cannot propose this method as a diagnostic tool until we conduct more studies, necessary to know the mechanism of the incorporation of DNA of P. falciparum in hair, which is unclear. However, we suggest that the findings revealed in our study could have several important practical applications. It is a noninvasive collection of samples, which avoid the suffer and the pain of the patient, transport and storage do not require special conditions, so it facilitates the universal access to the diagnosis and the detection of cases in the early stages of the disease.We must highlight that this study shows for the first time the presence of


Ethiopia has set a goal for malaria elimination by 2030. Low parasite density infections may go undetected by conventional diagnostic methods (microscopy and rapid diagnostic tests) and their contribution to malaria transmission varies by transmission settings. This study quantified the burden of subpatent infections from samples collected from three regions of northwest Ethiopia.Plasmodium infection.Sub-samples of dried blood spots from the Ethiopian Malaria Indicator Survey 2015 (EMIS-2015) were tested and compared using microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (nPCR) to determine the prevalence of subpatent infection. Paired seroprevalence results previously reported along with gender, age, and elevation of residence were explored as risk factors for Plasmodium infection was found with nPCR 3.3% (95% CI 2.7–4.1) compared with RDT 2.8% (95% CI 2.2–3.5) and microscopy 1.2% (95% CI 0.8–1.7). Of the nPCR positive cases, Plasmodium falciparum accounted for 3.1% (95% CI 2.5–3.8), Plasmodium vivax 0.4% (95% CI 0.2–0.7), mixed P. falciparum and P. vivax 0.1% (95% CI 0.0–0.4), and mixed P. falciparum and Plasmodium malariae 0.1% (95% CI 0.0–0.3). nPCR detected an additional 30 samples that had not been detected by conventional methods. The majority of the nPCR positive cases (61% (53/87)) were from the Benishangul-Gumuz Region. Malaria seropositivity had significant association with nPCR positivity .Of the 2608 samples collected, the highest positive rate for Using nPCR the detection rate of malaria parasites increased by nearly threefold over rates based on microscopy in samples collected during a national cross-sectional survey in 2015 in Ethiopia. Such subpatent infections might contribute to malaria transmission. In addition to strengthening routine surveillance systems, malaria programmes may need to consider low-density, subpatent infections in order to accelerate malaria elimination efforts. The Global Technical Strategy for Malaria of the World Health Organization (WHO) calls for a world free of malaria . To contPlasmodium infections. A relatively high prevalence of subpatent or low parasite density infections, that are often missed by conventional diagnostic methods (microscopy and rapid diagnostic tests) are being reported especially from low transmission settings [Malaria elimination requires the detection and clearing of all settings –14. Suchsettings . Even insettings .Plasmodium falciparum and Plasmodium vivax are the major reported malaria parasite species with rare reports of Plasmodium malariae and Plasmodium ovale. In 2017, out of a total confirmed malaria cases of 1,530,739, P. falciparum accounted for 1,059,847 cases and P. vivax for 470,892 cases [In Ethiopia, The current study assessed the magnitude of subpatent infections in northwestern Ethiopia, using samples collected in 2015 from a nationwide household survey of malaria endemic areas.The study used Dried blood spot (DBS) samples collected as part of the National Ethiopian Malaria Indicator Survey conducted in 2015 (EMIS-2015)  also fouPlasmodium infection identification by RDT and microscopy, haemoglobin testing  and for collection of DBS samples. Whatman 903  filter paper cards were used for DBS collection. These were air dried, individually packed in a plastic bag together with a desiccant and stored at − 20 °C at EPHI before they were sent to the U.S. Centers for Disease Control and Prevention (CDC) in, Atlanta, Georgia, for processing.EMIS-2015 was a cross-sectional survey, which used a two-stage cluster sampling methodology [Serology methods and results for these samples have been previously reported . BrieflyPlasmodium antigens: four human malaria species-specific merozoite surface protein-1 19 kD antigens (MSP-1) and Apical membrane antigen-1 (AMA-1) for P. falciparum and P. vivax.P. falciparum and pan-Plasmodium lactose dehydrogenase (LDH) for P. falciparum, P. vivax, P. ovale and P. malariae.The CareStart™ Malaria HRP2/pLDH (Pf/PAN) Combo RDT were used to detect malaria in the field. CareStart™ tests for histidine-rich protein 2 (HRP2) to detect plasmodial infections. Microscopists were WHO certified and were blinded to the RDT and survey results. A slide was classified as negative if no Plasmodium asexual forms or gametocytes were found after viewing 100 fields. Any positive slide was confirmed by two other microscopists.Thick and thin blood smears were made on the same slide, air-dried, and transported to EPHI. The slides were stained with 3% Giemsa for 10 min and screened for the presence of The DBSs were shipped to CDC at ambient temperature. A total of 2533 RDT-negative and 2 samples without RDT results available from the three regions were pooled and analysed  with slight modification. Samples were left for an overnight incubation in ATL buffer plus Proteinase K to ensure proper lysis during pooling, and the DNA was then extracted following a normal extraction protocol. The purified DNA was tested using nested genus PCR (nPCR). For any pool with a positive PCR result, individual DBSs were extracted following the same protocol as for the pooled samples and species-specific PCR assays were performed.Purified DNA templates were used for amplification of the 18S rRNA gene subunit using a modified method as previously described . BrieflyData were analysed using Stata 13 . Descriptive statistics with 95% confidence interval were estimated for demographic characteristics. Multiple logistic regression was used to assess the association of risk factors  with nPCR parasitaemia results. Percent relative difference and concordance for microscopy and RDTs were calculated against PCR results as the reference standard. Values were considered significant at P < 0.05. A Venn diagram was constructed using STATA to visualize the relationship amongst the four diagnostic methods: microscopy, RDTs, nPCR and serology. QGIS 2.18 was used to construct a map of the study area.P. falciparum (MSP-1 or AMA-1 antigen responses) and 34.0% (95% CI 31.6–36.6) seropositive for P. vivax (MSP-1 or AMA-1). Additionally, 7.0% (95% CI 5.7–8.4) and 4.9% (95% CI 3.8–6.2) of participants were seropositive for P. malariae and P. ovale MSP-1 antigen responses, respectively. Overall, 59.6% (95% CI 57.3–61.9) of respondents providing samples reported having slept under a bed net in the previous night  due to P. falciparum (only HRP2 positive), 0.4% (95% CI 0.2–0.7) due to P. falciparum infection with or without other Plasmodium species (HRP2 and panLDH positive) and 0.5% (95% CI 0.3–0.5) were P. vivax, P. ovale or P. malariae infection (only panLDH positive). Using microscopy, 1.2% (95% CI 0.8–1.7) of the samples were found to be positive for Plasmodium, of which 1.0% (95% CI 0.6–1.4) were due to P. falciparum infection, 0.2% (95% CI 0.0–0.4) were due to P. vivax infection and 0.08% (95% CI 0.0–0.3) were due to mixed P. falciparum and P. vivax infection. RDT positivity was over twofold higher than microscopy positivity  of infection, P. vivax accounted for 0.4% (95% CI 0.2–0.7) of infection, mixed P. falciparum and P. vivax 0.1% (95% CI 0.0–0.4), and mixed P. falciparum and P. malariae 0.1% (95% CI 0.0–0.3). PCR detected 64.4% and 16.1% more positives compared to microscopy and RDT, respectively (P < 0.05). In addition, PCR detected two P. malariae cases mixed with P. falciparum cases. By RDT, these were both appropriately positive for HRP2 and panLDH antigens. Using microscopy, one of the P. malariae positive infection was missed and the other was identified as mixed infection. No P. ovale infection was identified by any diagnostic method  positivity for any malaria species. Of this, .7–4.1 poP. falciparum infection, five were due to P. vivax infection, and two were due to a mixed infection of P. falciparum and P. malariae. Half of the additional positive samples (14 of 30), and mixed P. falciparum and P. malariae cases were identified from samples collected in Benishangul-Gumuz, a region of higher malaria transmission in Ethiopia.PCR identified 30 additional samples as positive that were negative by RDT Table . Of thesP. falciparum is predominant. In the Amhara and Tigray regions agreement with nPCR diagnosis was lower at 64.3% and 68.8%, respectively.The percent concordance of RDTs and microscopy with nPCR detection on sample positivity varied between the three regions Table . OverallPlasmodium infection in malaria endemic settings in northwest Ethiopia. Nested PCR identified 30 additional positive malaria infections that were missed by RDTs in the field, and thus not provided treatment at the time of the survey. The prevalence reported by nPCR was 3.3%, 16.1% higher than that detected by RDTs and 64.4% higher than from microscopy. More than 60% of the nPCR positive cases were identified from Benishangul-Gumuz Region, which is one of the higher malaria transmission region in Ethiopia [In this study, results of microscopy, RDT and nPCR detection methods were used to examine the prevalence of Ethiopia . The regEthiopia .Plasmodium infection in areas reporting low incidence.The study compared microscopy and RDT detection methods with a pooling nPCR approach that can detect parasite density as low as 0.1–10 parasites/µl of blood –26. ClosP. falciparum infections has been raised in Ethiopia with alarming reports originating from Eritrea [Although the lower RDT positivity is likely due to low-density infections, the possibility of hrp2/3 gene deleted  Eritrea –33. Alth Eritrea , a studyPlasmodium infections in low transmission settings using PCR from a range of countries [Several studies have reported asymptomatic ountries , 35, 36.ountries  and Sri ountries  reportedountries  and Tadeountries  reportedSubpatent infections, despite the low parasite density, could be infectious to mosquitoes , 19, 39.Several new tools are being developed with higher limits of parasite detection in field settings, such as, ultrasensitive RDTs and loop-mediated isothermal amplification (LAMP) , 42. TheThe current study reports the presence of subpatent infection in the samples collected as part of a national cross-sectional malaria survey in 2015. Using nPCR, the detection of malaria parasites increased by nearly threefold over rates compared to microscopy. Subpatent infections could potentially be infective to mosquitoes contributing to ongoing malaria transmission. Malaria elimination efforts may need to consider low-density, subpatent infections in managing sources of infectious reservoirs or hotspots, in addition to strengthening routine surveillance and response systems.