Valid results were obtained from 652 participants. Mean (±SD) PWV and C-SBP for the SPRINT cohort were 10.7 ± 2.7 m/s and 132.0 ± 17.9 mm Hg respectively. Linear regression analyses for PWV and C-SBP results adjusted for age, sex, and race/ethnicity in relation to several markers of glucose homeostasis and CKD did not identify any significant associations with the exception of a marginally statistically significant and modest association between PWV and urine albumin-to-creatinine ratio . In a subset of SPRINT participants, PWV was significantly higher than in prior studies of normotensive persons, as expected. For older age groups in the SPRINT cohort (age > 60 years), PWV was compared with a reference population of hypertensive individuals. There were no compelling associations noted between PWV or C-SBP and markers of glucose homeostasis or CKD.Arterial stiffness, typically assessed as the aortic pulse wave velocity (PWV), and central blood pressure levels may be indicators of cardiovascular disease (CVD) risk. This ancillary study to the Systolic Blood Pressure Intervention Trial (SPRINT) obtained baseline assessments (at randomization) of PWV and central systolic blood pressure (C-SBP) to: 1) characterize these vascular measurements in the SPRINT cohort, and 2) test the hypotheses that PWV and C-SBP are associated with glucose homeostasis and markers of chronic kidney disease (CKD). The SphygmoCorClinical Trial Registration: NCT01206062. Arterial stiffness–typically assessed as central (aortic) carotid-femoral pulse wave velocity (PWV)–is an indicator of cardiovascular disease (CVD) risk independent of both the peripheral  BP and pulse pressure.–3 Recent1C) renin, aldosterone, and markers associated with CKD ).The Systolic Blood Pressure Intervention Trial  was designed to test the hypothesis that treatment to a more intensive systolic blood pressure (SBP) target of < 120 mm Hg would reduce cardiovascular morbidity and mortality versus a standard SBP target of <140 mm Hg among non-diabetic adults. This SPR2, and persons age 75 years and older. For participants younger than 75 years old, either pre-existing CVD, CKD, or a Framingham risk score calculated to be above 15% was required. Notable exclusions included a history of diabetes mellitus, stroke, congestive heart failure. Additional exclusion criteria for the Senior subgroup included: a clinical diagnosis of or treatment for dementia, an expected survival of less than 3 years, unintentional weight loss (>10% of body weight) during the preceding 6 months, an SBP of less than 110 mm Hg following 1 minute of standing, or residing in a nursing home. The study interventions ended earlier than planned when the Data Safety Monitoring Board (DSMB) noted that the primary study outcome–a composite CVD outcome comprised of myocardial infarction, acute coronary syndrome, stroke, heart failure, or CVD death–and all-cause mortality were significantly lower in the intensive blood pressure treatment group. / 22.5]–was used as a measure of insulin sensitivity.th, 75th percentile range, and categorical variables are presented as number with percent. Baseline characteristics were compared among participants who were or were not in this ancillary study, with the use of the chi-square test, Wilcoxon rank-sum test, and two-sample Student t-test where appropriate.All statistical analyses were conducted at the SPRINT Coordinating Center with the use of SAS software, version 9.4 . Continuous variables are presented as mean and standard deviation (SD) or median and 25Baseline PWV in the SPRINT-PWV cohort was compared to Grade 1 Hypertension levels from reference populations reported by The Reference Values for Arterial Stiffness Collaboration, using t2) and less likely to be in the group with prevalent clinical or subclinical CVD. The number of CVD risk factors was lower in the PWV cohort since some participants were included because of low eGFR without other CVD risk factors. The mean (± SD) baseline PWV was 10.7 ± 2.7 m/s and C-SBP was 132.0 ± 17.9 mm Hg.Valid PWV results could not be obtained in 123 (15.4%) participants because of either atrial arrhythmias or obesity, the latter of which precluded obtaining an accurate femoral artery wave form. Demographic and clinical characteristics for the remaining 652 participants who participated in this SPRINT ancillary study and for whom a valid PWV measure was obtained are provided in As expected, there were direct correlations between PWV and age , peripheral SBP , and C-SBP . C-SBP was weakly associated with age . However, there was no significant correlation between PWV and BMI . There was an inverse correlation between C-SBP and BMI .The age distribution of PWV results by decade are presented in The SPRINT study population provides an extremely well characterized participant cohort in whom additional information derived from measures of PWV and central blood pressure were evaluated. These results from a subgroup of the SPRINT study cohort demonstrate that their measures of vascular stiffness are lower than a Grade I Hypertension population for persons age 60 years and older. Furthermore, adjusted linear regression analyses for PWV and C-SBP results identified a marginally statistically significant and modest association between PWV and UACR.SPRINT was designed to recruit “a diverse population with hypertension and existing cardiovascular disease, existing chronic kidney disease, or an elevated estimated risk for cardiovascular disease based on age and other risk factors.” As shownWith respect to demographics, there was only one significant finding: C-SBP was higher in women than men in SPRINT, in parallel with the sex difference in brachial, peripheral BP. The generalized linear model relating C-SBP to BMI was of borderline statistical significance, but in the same direction identified in the unadjusted correlation. The unexpected lack of (for PWV) and inverse (for C-SBP) relation with BMI may be explained by the selection criteria that were used for SPRINT as a whole, where persons with diabetes were excluded, and for this ancillary study in particular, since participants with higher BMI were more likely to have PWV studies that could not be conducted or that failed to meet quality control parameters.Based on prior investigations that have identified higher PWV in persons with diabetes –21 or wiSeveral limitations inherent to this study’s design merit consideration. The SPRINT cohort is not a population-based sample. The randomized clinical trial participant population had several exclusions–notably diabetes–and may not be generalizable to a general hypertensive population. The age distribution of the population–age 50 years to 90s –may represent another challenge to its generalizability.In a subset of SPRINT participants, PWV was significantly higher than in prior studies of normotensive persons, as expected. For older age groups in the SPRINT cohort (age > 60 years), PWV was lower than a reference comparison population of hypertensive individuals. There were no compelling associations noted between PWV or C-SBP and markers of glucose homeostasis or CKD.S1 Table(DOCX)Click here for additional data file.


Rapid diagnostic test (RDT) kits have been useful tools to screen for the presence of infection with malaria parasites. Despite the improvement, false results from RDTs present a greater challenge. The need for quality test kits is desirable. We evaluated the diagnostic accuracy of three malaria RDTs.The team consented and enrolled 754 participants from the two major public hospitals in Kintampo districts of Ghana from June 2014 to August 2014. Venous blood samples were obtained by trained personnel and samples were screened for malaria using CareStart and SD Bioline HRP2 and HRP2 with pLDH based RDTs with blood slides for malaria microscopy as “gold standard”. Geometric mean parasite densities were estimated and parasite densities were used to estimate the quantitative limits of the RDTs. The sensitivities, specificities and other performance criteria were calculated using statistical analytical software.The median age of participants was 21 (range 5–31) years. There were 28.6% (215/752) were males and 71.4% (537/752) were females. Comparing with microscopy, the sensitivity, specificity, positive predictive value, negative predictive value and the ROC were for CareStart (HRP2), 98.2%, 66.5%, 82.6%, 95.6%, 0.82; for CareStart (HRP2/pLDH) 98.2%, 66.5%, 82.6%, 95.6%, 0.82 and for SD-Bioline (HRP2/pLDH) RDTs 98.2%, 69.2%, 84.2%, 96.0%, 0.84 respectively. The performance for all the kits were acceptable at a cut-off of 25 or more parasites/μl of blood.The diagnostic performance of the three malaria RDTs was acceptable, according to the World Health Organisation criteria, to detect densities ≥25 parasite/μl of blood. The RDTs with HRP2/pLDH targets were comparable to those with only HRP2 and could successfully substitute current and commonly used HRP2-based RDTs. Plasmodium falciparum infection [Plasmodium falciparum (Pf) is responsible for the high cause of deaths in children who are 5 years of age or less [Malaria parasite infection contributes greatly to morbidity and mortality in the developing part of the world . A largenfection , 3. Plas or less , 5. Mala or less –8. A num or less  and the  or less , 10.Plasmodium falciparum hrp2 gene [hrp2 deletions is reported to occur as was seen by Koita et al, 2012 and that P. falciparum parasites with such deletions cannot be detectable [hrp2-deleted gene pose, combination of multiple protein targets is suggested in the development of malaria RDTs. One of such parasite protein is the parasite Lactate Dehydrogenase (pLDH). It is believed that addition of pLDH to HRP2 in RDTs can improve their performance by identifying malaria parasites with hrp2-gene deletion [Artemisinin-based Combination Therapy (ACT) use, as recommended by the World Health Organisation (WHO), is restricted to patients with confirmed malaria parasite infections , 12. Witrp2 gene . Spontantectable , 17 usindeletion , help todeletion , 21, 22.The team presented all information including the objectives and procedures of the study to every participant. The team sought and obtained written informed consent from all adult participants and from the parents of all minors to voluntarily participate in the study. Care-takers were invited to give their written informed consent on behalf of participating minors. Children aged 10 to 17 years were invited to give their written informed assent for consenting in addition to their parental consent. The Kintampo Health Research Centre Institutional Ethics Committee (KHRC-IEC) (FWA No. 00011103) gave ethical approval of the protocol before the study commenced.The team adopted a cross-sectional study design. The study was conducted in Kintampo North Municipal and Kintampo South District all in the Brong Ahafo Region of Ghana, West Africa. The study area is estimated to cover about 7162 sqkm with a resident population of approximately 140,000 . The locA minimum sample size of 350 was determined, based on our sample size calculation. We assumed 0.98 expected Area under ROC curve compared to 0.95 of a Null hypothesis value , 1:2 ratPersonnel from Kintampo Health Research Centre (KHRC) and the two major public hospitals carried out the study activities to recruit the number of participants needed from the health facilities from June 2014 to August 2014. Patients that had requests on their forms for malaria tests to be done in the hospitals’ laboratories were contacted to seek their voluntary written informed consent to be recruited to participate in the study. The study required participants to donate 2mls of venous blood samples for evaluation of the test kits. The study information, activities and procedures were explained in turn to each participant; allowing each of them time to decide whether to participate or not. The study team obtained written informed consent from each participant enrolled in the study period from the two major public hospitals. Demographic data such as age and sex were collected. The blood samples were collected into EDTA test tubes by trained study laboratory personnel and transported to the KHRC laboratory for analysis according to the Standard Operating Procedures for sample collection, transport and analysis. Ten percent (10%) of the samples were retested as quality assurance measure for the RDT methods. This was done by selecting every 10th sample for RDT retesting.Blood samples taken from the two major health facilities and received in KHRC laboratory were used to screen for malaria parasites using the three malaria RDTs , CareStart Malaria (HRP2/pLDH), SD Bioline Malaria (HRP2/pLDH)) and also using routine microscopy as the “gold standard”.Thick and thin blood slides films were prepared from each sample received in the KHRC laboratory and used for malaria parasite identification as described previously , 29. In Pf (HRP2/pLDH), there were three bands representing a control line (“C”) and two separate test lines (labelled as “T1” and “T2”). The antigens that were targeted were Pf-HRP2 (T1) and Plasmodium vivax (Pv)-, Plasmodium ovale (Po)-, Plasmodium malariae (Pm)-or Pf-pLDH (T2) put on the same strip [Pf positive while “C” and “T2” indicated positive test for other Plasmodium species . With three colour bands, positive “C”, “T2” and “T1” lines were interpreted as mixed infection of Pf and any of the other Plasmodium species. There was no case where “T1” was negative and “T2” was positive. The study strictly adhered to the manufacturers’ instructions to obtain good results from the RDTs. Results from two independent observers that were concordant were used as the final results for the RDT tests. One general challenge that was identified with both RDTs was the fact that extra caution needed to be taken to be able to fill the volume of blood needed for testing to the mark (5 μl). Improving in the process of filling the capillary tubes would improve in the test performance time.Participants’ samples were also screened for malaria parasites using 5 μl with each of the RDTs from CareStart Malaria (HRP2), CareStart Malaria (HRP2/pLDH)  and SD Bioline Malaria Antigen Pf (HRP2/pLDH) . Each of the CareStart malaria RDTs (both HRP2 and HRP2/pLDH) had only two bands consisting of a control (“C”) and test (“T”) lines each. With the CareStart RDTs, a test is positive for malaria parasite when there is a reaction in the control and test lines. The control band must at all times be positive before a test could be classified as valid (Manufacturer’s instructions). For SD Bioline Malaria Antigen me strip . For theFull Blood Count of haematological parameters was determined with ABX Micros 60 analyzers . The validated instrument was used to obtain the counts of blood cells of interest to estimate the malaria parasite densities , 29–31 nSupervisors checked for data completeness and consistency. Data were then double-entered using Microsoft Access software 2010. All queries were resolved after data entry. Stata Software version 13  and MedCalc software version 12.5  were used on the cleaned data for analysis. The calculated sensitivity (SE), specificity (SP), negative and positive predictive values (PPV and NPV) and the Area under the Receiver Operator Characteristics (ROC) curve using malaria microscopy results as “gold standard” was used to estimate the diagnostic performance of the malaria RDTs. In estimating SE, we calculated the proportion of positive test results identified by the test kit compared to the positives given by the “gold standard”. With SP of the RDT, the proportion of negative test results was compared to the true negatives given by the “gold standard” (microscopy). The PPVs and the NPVs were calculated as described by Endeshaw et al, 2012 . All repThe formulae below were employed in our calculations.All personnel that were involved in the study and testing processes before the start of the study were trained on the protocol and procedures. According to, and using the manufacturers’ instructions, the training included handling the RDTs, picking blood with the pipette for testing on the RDTs, use of the buffer for RDT and the reading and interpretation of test from the RDTs. The methods for preparing the blood films (thin and thick), staining and species identification were also included in the training schedule , 29. TheA total of 453 out of 754 samples from participants recruited from the two major public hospitals in the Kintampo North and South districts were used to evaluate the rapid diagnostic test kits. Of these, 28.7% (130/453) were males and 71.3% (323/453) were females. The demographic profile of the participants are shown in P. falciparum ranged between 25 and 903120 parasites/μl of blood (IQR: 589–88580) with median density of 9695 parasite/μl.The parasite density for CareStart malaria (HRP2) RDT and Malaria microscopy: The SE, SP, PPV and NPV with their confidence limits, of CareStart malaria (HRP2) RDT method compared to the “gold standard” were 98.2% , 66.3% , 82.6%  and 95.8%  respectively  RDT method compared to the “gold standard” were 98.2% , 66.3% , 82.6%  and 95.8%  respectively . The ROCThe SE, SP, PPV and NPV with their confidence limits, of SD Bioline malaria (HRP2/pLDH) RDT method compared to the “gold standard” were 98.2% , 69.2% , 83.9%  and 90.4%  . The ROCIn 5 samples, all three RDTs read negative compared to the “gold standard”. The parasite densities in these 5 samples were 36, 44, 36, 73 and 81 parasites/μl of blood. However, the RDTs could identify positive cases compared to microscopy for 19 other samples with parasite densities from 25 parasites/μl of blood to 96 parasites/μl of blood. This is suggestive of something else other than the low parasite density which might have led to the 5 negative results in Considering the performance of the kit at a cut-off criteria of 25, 50, 100, 200, 1000, 5000, 10000, 50000 and 1000000 parasites/μl of blood, a ROC of 0.97 was obtained for all the three malaria RDTs as presented in Pf (HRP2/pLDH) RDTs evaluated in this study were acceptable with sensitivities higher than 98% and varied specificity from 66.3% to 69.2% (Rapid Diagnostic Tests (RDTs) of different makes have played significant roles in the management of malaria. As new formulations emerge, they need to be assessed to determine their roles in malaria diagnosis. The diagnostic performances of CareStart Malaria (HRP2) RDTs compared to malaria microscopy, of CareStart Malaria (HRP2/pLDH) RDTs, and of SD Bioline Malaria Antigen to 69.2% . The accThe diagnosis, treatment and overall management of malaria adopt several guidelines using clinical judgement together with the parasitological detection of malaria parasites based on available accurate, validated and accepted standards for testing. Malaria which is known to be a leading cause of morbidity and mortality in the developing world  is prefehrp2 gene deletion have brought up the discussion of “false negative reporting” when RDTs solely developed with HRP2 based proteins are used for testing [hrp2 gene deletion in some strains of malaria parasites [hrp2 gene deletion are expected to produce pLDH which would be detected when the parasite is present considering the time of sample analysis [hrp2 gene deletion needs to be explored. This is because, the RDTs could not identify the infection that was identified by microscopy in 5 samples. In these samples it is unlikely that the RDTs failed to identify parasites because they had low parasite density since other samples had comparable parasite densities but were correctly identified by the RDTs. A thorough review of HRP2-based RDTs is required given the reports of hrp2 gene deletion infections in Mali [The technique employed in developing these RDTs is based on detecting the HRP2 or HRP2 with pLDH proteins of malaria parasites if present. Recent reports of human infections with malaria parasites with  testing , 17. It arasites . This isanalysis , 22. How in Mali  and potePlasmodium falciparum. The kits therefore have the potential to be used for effective malaria diagnosis that will prevent over prescription of antimalarials.The three RDTs evaluated in this study had acceptable performance in samples that had parasite densities from 25 to 200 parasites/μl of blood. It is assuring to note that the kits with targets to pLDH detecting antigens in addition to HRP2 performed well compared with only HRP2 antigen detecting kit at low parasite densities. The RDT kits particularly with pLDH detecting antigens can therefore be considered for use to screen and detect infections with These malaria RDTs which are all packaged ready for use require less skills and complex equipment to perform. The malaria RDTs evaluated in this study like all others used approximately 15 minutes to give reliable results without any equipment that require electricity. This will effectively improve early detection and diagnosis of malaria parasite infection for prompt treatment especially in endemic region.The study was carried out in three months (June-August), corresponding to the rainy season where malaria transmission is very high. However, malaria transmission and infection is perennial with peaks and troughs in the study area and the range of parasite densities recorded in this study cut across low to high densities. It is assumed that results obtained from the rainy season would apply in the dry season as well, which may not be true. However, the sample size considered adequate number of positive and negative cases in their correct ratios and that in our view would not be significantly affected by season in validating the results and performance of the kits.The diagnostic performance of the HRP2 and HRP2 with pLDH based RDTs assessed in this study was acceptable and could detect parasitaemia as low as 25 parasite/μl of blood. The HRP2 RDTs combined with pLDH targets be considered for malaria control programmes. There is however a decrease in specificity which could result from the persistence of HRP2 proteins even weeks after effective treatment of malaria.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Dear Sir,1 This discusses the state of anti-disease resistance that may eventually be achieved by a person if he/she is repeatedly exposed to infection with Plasmodium falciparum, assuming that no infectious episode has a fatal outcome. This “steady state” between parasite dispersal and host survival can be observed in those adult residents of P. falciparum–endemic areas who have not succumbed to exposure during childhood. The author makes a compelling case for using the term “malaria-tolerant” in preference to “malaria-immune,” which is found far more frequently in the published literature. The reason for this is that such individuals often harbor blood-stage parasites below a threshold of parasitemia required for them to show clinical manifestations of malarial infection, and thus they are apparently tolerant to the residual but persistent presence of parasites in their peripheral circulation. Hence, their naturally acquired protective immune response, a form of premunition, reduces infection for the most part to subclinical levels, but does not eradicate all parasites. It is likely that these asymptomatic yet chronic carriers of malaria parasites provide a reservoir of infection for local transmission if and when an Anopheles mosquito takes a blood meal from them, especially if this bite coincides with a recrudescence of patent infection.I read with interest the recent thought-provoking article by Dennis Shanks.P. falciparum infections. Continuing this vein of academic pedantry, I wish to raise a point that some may consider to be trivial in the context of the important underlying message that is conveyed by this article. Nevertheless, in my opinion, there is an ambiguity that should be clarified. The first three sentences read as follows: “Adults who have not grown up in a malaria-endemic area may experience severe malaria soon after entering a malarious area. Such mortality is usually limited to a short period of time (months), after which they are thought to be ‘immune’. Such anti-disease immunity may be more accurately considered as tolerance.” Drawing attention to the second sentence, to my mind if mortality is experienced by a person, the only reason they will be rendered immune to further malarial infection is because they will in fact be dead. Although it may be argued, tongue planted firmly in cheek, that host mortality is the ultimate expression of non-protective immunity, it is not a connotation that I think the author means to convey. As such, Shanks appears inadvertently to confuse mortality with morbidity, or perhaps the duration of risk of mortality. It would be better to write the following, or a similar form of words: “Such potentially life-threatening clinical manifestations are usually limited to a short period of time (months), after which, if the person survives, they are thought to be ‘immune’.” Of course, given the tenet of his argument, the author may prefer “Such potentially life-threatening clinical manifestations are usually limited to a short period of time (months), after which, if the person survives, they are thought to be ‘tolerant’.”Shanks is being pedantic, in the very best sense of the word, in choosing “tolerant” over “immune” in relation to chronic


Patients with knowlesi malaria were older and had lower parasitemia compared to human-only species. Severe knowlesi malaria occurred only in adults. Anemia was common in children despite lower parasitemia. Parasitemia predicted disease severity: Intravenous artesunate is warranted initially for parasitemia>15000/μL. Plasmodium knowlesi is increasingly reported in Southeast Asia, but prospective studies of its clinical spectrum in children and comparison with autochthonous human-only Plasmodium species are lacking.Plasmodium monoinfection presenting to 3 district hospitals in Sabah, Malaysia.Over 3.5 years, we prospectively assessed patients of any age with molecularly–confirmed P < .001) and vivax malaria. In P. knowlesi, World Health Organization–defined anemia was present in 82%  of children vs 36%  of adults. Severe knowlesi malaria occurred in 6.4%  of adults but not in children; the commenst severity criterion was acute kideny injury. No patient had coma. Age, parasitemia, schizont proportion, abdominal pain, and dyspnea were independently associated with severe knowlesi malaria, with parasitemia >15000/μL the best predictor . Two knowlesi-related adult deaths occurred .Of 481 knowlesi, 172 vivax, and 96 falciparum malaria cases enrolled, 44 (9%), 71 (41%), and 31 (32%) children aged ≤12 years. Median parasitemia was lower in knowlesi malaria  than in falciparum (9600/μL; Age distribution and parasitemia differed markedly in knowlesi malaria compared to human-only species, with both uncomplicated and severe disease occurring at low parasitemia. Severe knowlesi malaria occurred only in adults; however, anemia was more common in children despite lower parasitemia. Parasitemia independently predicted knowlesi disease severity: Intravenous artesunate is warranted initially for those with parasitemia >15000/μL. Plasmodium knowlesi human cases in Sarawak, Malaysia, in 2004 [P. knowlesi now accounts for >90% of all government-notified malaria cases [Plasmodium knowlesi is also increasingly reported in areas of western Indonesia [P. knowlesi cases in Southeast Asia threaten regional malaria elimination. Conventional public health measures are unable to target zoonotic transmission to humans from the P. knowlesi reservoir in monkey hosts, particularly outdoors in agricultural or forest areas [Since the initial description of a large focus of zoonotic  in 2004 , knowles in 2004 . In Malaia cases , with >9ia cases . Plasmodndonesia . Difficundonesia  have limndonesia , 12. Desndonesia , increasst areas .Plasmodium falciparum [Prospective studies have described the clinical spectrum of naturally acquired adult knowlesi malaria , 18. Sevlciparum , 19–21.P. falciparum and Plasmodium vivax [P. knowlesi now accounts for around 49% of all reported pediatric malaria cases in Sabah [P. falciparum and P. vivax in district settings.Malaria notification data in knowlesi-endemic areas show a median age of 31 years, much higher than that seen with um vivax , althougum vivax . With thin Sabah . Despitein Sabah , 22, or P. knowlesi or other Plasmodium species infection, and evaluated predictors of disease severity in a coendemic primary care setting.In this study, we compared the predefined clinical spectrum between children and adults with malaria due to 2 and with a total growth-rate adjusted Malaysian census–estimated population in 2016 of 199600 people. Each of the 3 districts in this division has a central referral hospital and subdistrict health clinics, consistent with other districts in Sabah. Malaysian Ministry of Health guidelines stipulate that all patients with fever receive microscopic blood slide screening for malaria parasites, with mandatory hospital admission, free treatment, and notification of positive cases [This study was conducted in Kudat Division, northwest Sabah, Malaysia, covering an area of 4623 kmPlasmodium malariae infection on polymerase chain reaction (PCR), if Plasmodium species PCR was not confirmed, or if cross-check research microscopy was negative. A subset of patients with uncomplicated P. knowlesi and P. vivax malaria was also enrolled in previously reported randomized controlled treatment trials [Patients of all ages presenting to study hospitals with microscopy-diagnosed malaria were enrolled following written informed consent. Children were predefined as age ≤12 years, consistent with Malaysian Ministry of Health pediatric ward admission. Patients were not included in the final analysis if they were pregnant or had t trials .P. knowlesi: hyperparasitemia threshold of 100000/μL, and jaundice defined as bilirubin >50 μmol/L with parasite count >20000/μL and/or creatinine >132 μmol/L [Baseline and longitudinal clinical, laboratory, and epidemiological data were entered using standardized case record forms. Venous blood was taken for baseline investigations and then daily for microscopy and hematology during hospital admission and at the follow-up visit 28 days after treatment initiation. Severe malaria was defined using World Health Organization (WHO) 2014 research criteria , includi2 μmol/L . Nonseve2 μmol/L . AKI wasPlasmodium species confirmation was done using PCR [Microscopic asexual parasite and gametocyte counts were calculated by research microscopists using thick blood smears and quantitated leukocyte count. Standard hospital automated hematology, biochemistry, and microbiology laboratory results were used. Final sing PCR , 30.t test or the Wilcoxon–Mann-Whitney test for 2-group comparisons according to distribution. For categorical variables, χ2 or Fisher’s exact test was used. Logistic regression models were fitted to determine a priori predictors of severe malaria based on standard clinical and laboratory WHO 2014 research criteria [e parasitemia; patients with hyperparasitemia as a sole severity criterion were considered nonsevere.We compared between-group differences with analysis of variance or Kruskal-Wallis testing for continuous variables, and Student criteria  evaluablThis study was approved by the medical research ethics committees of the Ministry of Health, Malaysia; London School of Hygiene and Tropical Medicine, United Kingdom; and Menzies School of Health Research, Australia.P. knowlesi, 172 P. vivax, and 96 P. falciparum malaria cases included in the final analysis. From 2014 to 2015, the estimated minimum yearly malaria incidence in Kudat Division  for P. knowlesi, P. vivax, and P. falciparum was 0.79, 0.40, and 0.19 cases per 1000 people per year, respectively. Patients with knowlesi malaria had a median age of 33 years , higher than those with vivax  and falciparum  malaria . A bimodal age distribution was seen for females with both P. knowlesi and P. falciparum infection. Of P. knowlesi cases, 44 (9%) were children, compared to 71 (41%) of P. vivax cases and 31 (32%) of those with P. falciparum malaria (P < .001). Only 6 (1.3%) knowlesi cases were <5 years of age, with only 1 infant (<1 year), a 6-week-old with no travel history or forest or plantation exposure. Compared to children with P. knowlesi malaria, adults were more likely to be male , with this relationship also evident for P. vivax cases .From October 2012 until April 2016, 811 malaria patients were enrolled . There wP < .001), although vomiting occurred more often in those with P. vivax (P = .033)  and P. falciparum . The highest parasite count recorded for a child with knowlesi malaria was 74365/μL, in an 11-year-old boy with uncomplicated disease. There were 36 (84%) children with knowlesi malaria with nonsevere anemia at presentation, comparable to children with other Plasmodium species infection, with no relationship to parasitemia demonstrated. The lowest hemoglobin level of 5.1 g/dL was seen in a 4-year-old child with knowlesi malaria 2 days after treatment, with 2 other children having minimum hemoglobin levels of 7.0 g/dL, all of whom had a parasite count <1000/μL at presentation. Children with knowlesi malaria had lower neutrophil and lymphocyte counts on presentation compared to those with other Plasmodium species (P = .002). Thirty (68%) children with knowlesi malaria had thrombocytopenia (platelet count <150 × 103/μL), including 4 (9%) with a platelet count <50 × 103 cells/μL. Frequency of thrombocytopenia in children with knowlesi malaria was comparable to those with P. vivax but more common than with P. falciparum malaria . Children with knowlesi malaria were more likely to develop mild to moderate AKI compared to those with P. vivax .Abdominal pain was more common in children with knowlesi compared to vivax malaria  . ChildreP. knowlesi–infected adults was comparable to both children with P. knowlesi and adults with malaria due to other Plasmodium species  or falciparum  malaria , but not P. falciparum or P. vivax infection , and children with knowlesi malaria . As with children, in adult knowlesi malaria parasitemia was not associated with anemia at enrollment after controlling for age. Adults with knowlesi malaria had lower platelet counts than those with other Plasmodium species (P < .001), with thrombocytopenia more common in adults compared to children with knowlesi malaria . The risk of AKI was higher in adult P. knowlesi patients compared to P. vivax , although this did not remain statistically significant after controlling for age; and was also comparable to that seen in both adults with P. falciparum and children with knowlesi malaria. Liver aminotransferases were higher in adults with knowlesi malaria compared to those with P. vivax including after controlling for age (P = .001). Of the 322 adults with knowlesi malaria who had blood cultures, only 1 grew a noncontaminant isolate, a 14-year-old with Neisseria meningitidis.Duration of fever for  species . Adult P malaria . Age wasnfection . Adult PPlasmodium knowlesi was the most common cause of severe malaria, with 28 of 481 (5.8%) knowlesi cases having severe disease (P < .001). The platelet count was lower in severe vs uncomplicated knowlesi malaria , neutrophil count was higher , and proportion of patients with hyponatremia was higher . Five (18%) knowlesi patients had documented hypotension, all of whom had other severity criteria including 2 patients with respiratory distress. Empiric antibiotic treatment was given to 9 (32%) patients with severe knowlesi malaria. Of the 4 severe P. vivax cases, 2 were children with hyperbilirubinemia and a parasite count >20000/μL, both with moderate anemia . The other 2 patients with severe vivax malaria were adults, including a 17-year-old female with severe anemia, and a 53-year-old man with hypotension and respiratory distress. There were 5 patients with severe falciparum malaria (5.2%), including 2 children. No patient with malaria from any Plasmodium species had coma. disease , all of  disease . The mosP = .048). Adults with knowlesi malaria had a higher risk of severe disease compared to adult patients with vivax malaria , and a comparable risk to falciparum malaria.On multivariate logistic regression controlling for age and parasitemia, independent clinical and parasitological predictors of severe disease in knowlesi malaria included schizont proportion >10%, abdominal pain, and dyspnea . Among WP. knowlesi, giving an overall P. knowlesi case fatality risk of 2 of 481 , or 2 of 437  in adults. The P. knowlesi–related deaths were a 62-year-old woman with hyperparasitemia (263772/μL) and moderate AKI (creatinine: 224 μmol/L), who developed hypotension and acute respiratory distress [There were 2 deaths attributed to malaria, both P. knowlesi malaria cases to date, and the first to prospectively compare the clinical spectrum of disease between adults and children. Although 91% of knowlesi malaria cases were adults, morbidity in children was also demonstrated, with an 11-fold higher risk of anemia at presentation and a similar risk of mild to moderate AKI compared to adults [P. knowlesi infection compared with the human-only Plasmodium species may indicate a lower pyrogenic threshold and greater inflammatory response, consistent with poor adaptation of this zoonotic parasite to the human host. Although P. knowlesi has a 24-hour blood-stage life cycle in humans, the low parasitemia in most infections may indicate variable efficiency in human red blood cell (RBC) invasion [This study is the largest series of o adults . The majo adults , 18. Theinvasion . Only a invasion , 18, 32.invasion .P. knowlesi infections were in children compared to those with P. vivax or P. falciparum. The lower incidence of clinical disease from P. knowlesi infection in infants and also older children has been attributed to epidemiological factors such as lower forest exposure [P. vivax or P. falciparum infection, in addition to a lower neutrophil count compared to P. vivax. However, there was a comparable risk of nonsevere anemia and AKI seen in P. knowlesi–infected children as in those with P. vivax or P. falciparum.A lower proportion of exposure , 13, 18,exposure , and asyexposure . Most chexposure . Althougexposure . ChildreP. knowlesi infection was comparable to that seen in P. falciparum. The risk of severe knowlesi malaria in this primary referral setting in Sabah, 6.2% in adults, was similar to district hospital presentations in Sarawak (9.3%) [Plasmodium knowlesi parasitemia [The proportion of adults with severe disease from k (9.3%) , and lowk (9.3%) . Severe k (9.3%) , 18, 32.asitemia  independasitemia . With agasitemia , 36 may P. falciparum, with coma remaining unreported and a lack of the retinal microcirculatory changes found in severe falciparum malaria [P. knowlesi as in P. falciparum infection [Plasmodium transmission may also modulate disease severity.The pathophysiological mechanisms in severe knowlesi malaria are not well understood but likely differ from  malaria . With ennfection , 36, thenfection , requirenfection , howevernfection , requirenfection . Other hPlasmodium species, these management guidelines have contributed to a decline in reported malaria case-fatality rate [Current knowlesi malaria management guidelines in Sabah recommend referral for tertiary care and initial treatment with intravenous artesunate for any patients >50 years of age or with a parasitemia >20000/μL , 40. Aloity rate , 18, 20.P. knowlesi infection causes morbidity at comparatively low parasitemia in both adults and children. Adults are at risk of severe and fatal disease, in contrast to children, among whom this was not demonstrated. A conservative treatment approach utilizing parasite counts to predict severe disease is warranted.In conclusion, although the majority of cases are uncomplicated, Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.


Plasmodium falciparum pre-erythrocytic antigens and their potential association with protection against clinical malaria. Antigen-specific antibody levels in plasma collected at six time points from a longitudinal cohort of one-to-five year old children resident in a seasonal malaria transmission area of northern Ghana were assessed by ELISA. Antibody levels were compared between parasite-positive and parasite-negative individuals and the association of antibody levels with malaria risk assessed using a regression model. Plasma antibody levels against five of the seven antigens were significantly higher in parasite-positive children compared to parasite-negative children, especially during low transmission periods. None of the antigen-specific antibodies showed an association with protection against clinical malaria. The study identified five of the seven antigens as markers of exposure to malaria, and these will have relevance for the development of disease diagnostic and monitoring tools. The vaccine potential of these antigens requires further assessment.The current global malaria control and elimination agenda requires development of additional effective disease intervention tools. Discovery and characterization of relevant parasite antigens is important for the development of new diagnostics and transmission monitoring tools and for subunit vaccine development. This study assessed the natural antibody response profile of seven novel  Plasmodium parasite proteins becomes more essential to the development of integrated tools for control and prevention. To date, only a fraction of the over 5,500 proteins encoded by the P. falciparum genome during its multistage life cycle have been described and immunologically evaluated1. There is an urgent need to enrich the pipeline of vaccine development with novel candidates since an effective vaccine is one of the integrated tools that is needed to potentially ensure eradication2. Additionally, further characterization and understanding of human immune responses to malarial antigens will enhance eradication efforts through the development of more sensitive diagnostic3 and transmission monitoring7 tools. Naturally induced antigen-specific antibody levels are generally known to be influenced by such factors as host age, frequency of exposure to parasites as well as the extent of disease transmission8. Naturally acquired immunity to malaria has mostly been associated with antibody levels against certain parasite antigens, especially in adults repeatedly exposed to parasites10. It is also known that individuals living in areas of high malaria transmission usually have high levels of malaria antigen-specific antibodies compared to those in low transmission areas11. These assertions collectively point to the need for repeated or persistent parasite exposure in order to achieve the semi-immune status that has been well described among adults living in malaria endemic areas. In many instances, levels of the same antigen-specific antibodies in children have been identified to be mere markers of exposure to the parasite12 and not a marker for immunity. The dynamics of antibody levels, longevity and specificity based on the above parameters determines the usefulness and applicability of the corresponding antigen targets, either for vaccine development, diagnostics or as biomarkers for monitoring disease transmission.As malaria cases continue to decline globally, identification and characterization of 13. PE antigens however remain very important candidates for vaccine development15 and transmission monitoring tools16. Additionally, parasite antigens that occur in both the pre-erythrocytic and erythrocytic parasite stages are ideal multi-stage options for vaccine development. On-going work at the Naval Medical Research Center (NMRC) Malaria Department seeks to identify novel PE antigens using samples from subjects and animals immunized with radiation attenuated sporozoites (RAS) and assessing their immunogenicity for the purpose of sub-unit vaccine development. A number of such antigens have been identified and successfully expressed as recombinant proteins using a cell-free wheat-germ system19. The antigens are recognized by sera and T cells from RAS-immunized subjects17. Although the antigens were selected on the basis of being expressed in sporozoite and liver PE stages, localization studies with polyclonal sera induced in either rabbits or mice show that some of the antigens are concurrently expressed in asexual blood stages of the parasite17, suggesting that they might serve as targets of multi-stage anti-malarial immunity. The aim of this study was to assess the levels and kinetics of naturally induced antibodies to seven of these antigens in plasma from children living in an area of northern Ghana with marked seasonal malaria transmission. The seven selected antigens have been putatively described and include a glideosome-associated protein (Pf02), a transcription initiation factor TFIID subunit (Pf26), a GPI-anchored micronemal antigen (Pf56), a conserved Plasmodium protein with unknown function (Pf61), a cysteine protease inhibitor (Pf106), a subpellicular microtubule protein (Pf116) and a gamete egress and sporozoite traversal protein (Pf144). These were selected from a panel of 21 proteins previously expressed and characterized using sera from RAS-immunized subjects and from animal immunizations17.While many asexual blood stage parasite antigens have been described and immunologically characterized, only a handful of pre-erythrocytic (PE) stage antigens have been identified and similarly characterizedA total of 288 plasma samples from 48 child subjects (six plasma samples per child) were included in this study. Children at each sampling time point were categorized either on the basis of having blood film parasites or having suffered a clinical malaria episode Table . Over thA good proportion of children had either mild (Haemoglobin (Hb) concentration 10.0–10.9 g/dl) or moderate (Hb concentration 7.0–9.9 g/dl) anaemia over the study period, and only three cases of severe anaemia (Hb concentration <7.0 g/dl) were identified, occurring in September and November 2004  were however statistically significantly higher in subjects who had blood film parasites compared to levels in those without parasites, especially over the dry season when malaria transmission intensity was lowest Fig. . AntibodA correlation matrix was created in order to assess the relationships between the different antigen-specific antibody levels Fig. . There wAntibody data was fitted using a mixed effects model to identify potential antibody correlates of protection against malaria Table . None of21. Antigens that are expressed in multiple parasite stages are however ideal since immune responses against such single targets could be effective against the multiple stages.Antigen discovery and the identification of immunologically relevant antigens from multiple parasite stages are crucial for designing effective diagnostics and disease monitoring tools as well as for anti-malarial vaccine development. For the latter application, it is currently believed that an effective vaccine must include multiple antigens from different parasite stagesP. falciparum PE antigens using plasma samples from a longitudinal cohort of young children living in a malaria endemic area of Ghana with seasonal disease transmission. The majority of children were positive for blood film parasites over the entire period of sample collection, but only a limited number of clinical malaria cases were observed. Anaemia was the only condition identified, with the majority of cases occurring during the rainy season  and the merozoite surface proteins (MSPs)12. The long term availability of these proteins from the cyclic asexual blood stages result in prolonged stimulation of the immune system to elicit antigen-specific antibodies. This may also be true for the pre-erythrocytic antigen Pf116, as it is localized to the sporozoite surface like CSP. Conversely, the absence of re-stimulation of the immune system in parasite-negative children will result in a decrease in malaria antigen-specific antibody levels, as has been previously observed25. The potential of the five antigens to discriminate between parasite-positive and parasite-negative children during periods of low transmission makes them very essential markers for transmission monitoring. This holds even more relevance for settings where malaria transmission intensity has reduced significantly to the extent that conventional methods of transmission monitoring, such as entomological inoculation rate estimation, have become less sensitive26.Levels of antibodies against five  of the seven antigens were however significantly higher in parasite-positive children, especially during periods of low disease transmission Fig. . The fivOnly anti-Pf116 antibody levels showed a moderate negative correlation with parasitaemia at a single time point (November 2004), and the significance of this is not currently apparent. No direct relationship was identified between specific antibody levels on the one hand and the parasite developmental stage specificity, antigen localization or function of the seven putative proteins.27.Most of the sampling time point-specific differences in antibody levels between parasitaemic and non-parasitaemic children occurred between January 2005 and March 2005, which coincided with the dry season when malaria transmission was low. Differences in antibody levels between parasite-positive and parasite-negative children were however not generally seen during the major transmission period, especially between July and November 2004 Fig. . These m29. Antibodies in this age group appear to be markers of exposure rather than correlates of protection because their levels are probably below the threshold required to achieve clinical immunity31. This threshold may most likely be achieved with age and after several repeat infections12. The direct association of parasite presence with these antigen-specific antibody levels suggests a potential for these antigens to be used as malaria transmission monitoring biomarkers.The observations collectively suggest that the measured antibodies are potential markers of the extent of exposure to infectious bites, and this has previously been described for both pre-erythrocytic and erythrocytic stage parasites in this age group of children32. Subsequent analysis of data after 24 months of follow up showed association of antibody levels against some erythrocytic stage antigens  with a reduced risk of clinical malaria. This protective effect was however only detected after accounting for heterogeneity in exposure to parasites by including only parasite infected children and considering differences in exposure levels based on clinical, serological and demographic data in Cox regression models32. These conditions were met in the current study as two parasite exposure determinants  as well as age were considered as potential confounders but these did not significantly influence or improve the assessment models. Additionally, assessment of correlates of protection in this study included only data from children who had demonstrable exposure to malaria parasites as assessed by blood film examination. These notwithstanding, it is important to note that while a greater proportion of children were blood film parasite-positive over the six sampling time points, very few children suffered clinical malaria episodes over the same period. This could therefore influence the output from our regression analysis. Some of these antigens are being further investigated as potential vaccine candidates33.The observation that antigen-specific antibodies measured in this study were exposure biomarkers rather than correlates of clinical protection was confirmed by data from regression analysis, which showed no association between any of the antigen-specific antibody responses and association with clinical protection from either clinical malaria or parasite infection Table . A birthOur inability to detect sub-microscopic parasitaemia due to the unavailability of corresponding DNA samples, and the uneven numbers of parasitaemic and non-parasitaemic children from one sampling time point to another are important limitations to the study and our analysis. These notwithstanding, we present very relevant information that adds to the literature on malaria parasite antigens and their potential applications for disease control.In summary, the data shows the potential of at least five of the seven antigens as biomarkers for monitoring and diagnostics purposes. These antigens may also need to be further investigated for their vaccine potential using a well-defined case-control design in a naturally exposed population.Approvals for this study were obtained from the Institutional Review Boards of the Noguchi Memorial Institute for Medical Research  and the Navrongo Health Research Centre  in Ghana, as well as from the Ethics Review Committee at the Naval Medical Research Unit 3  in Cairo, Egypt. All three ethics bodies have a United States Government Federal Wide Assurance from the Office for Human Research Protections. The original study that collected plasma samples also obtained approvals from the NMIMR and NHRC Ethics Committees stated above. The study was conducted in accordance with the principles in The Belmont Report and federal regulations regarding the protection of human subjects in research. Written informed consent was obtained from parents/guardians of all child subjects before their recruitment and inclusion in the study.35. Malaria transmission in this region has been seasonal and overlaps with the rainy and dry seasons. A schematic of the current study design and sampling time points is presented in Fig. This study utilized stored plasma samples 48 children collected at six time points per child  in a previous study in the Kassena-Nankana District of the Upper East Region of Ghana. The dry season in the region spanned between November and April of the following year, and the rainy season was between May and October36. Samples originally came from a cohort of 325 children between 1 and 5 years old who were enrolled in May 2004 and followed up every two months until May 2005. Finger-prick blood samples (0.5–1 ml) were collected at the bi-monthly follow-up times for the preparation of plasma. Also at these sampling time points, body temperature was measured and parasite infection status was determined by microscopic examination of thick and thin blood smears. Blood haemoglobin (Hb) levels were measured using a Hemocue® device. Demographic information and data relating to the subjects’ malaria exposure during the transmission season was captured by a study questionnaire. Clinical malaria, for the purposes of this analysis, was defined as having a febrile episode (temperature >37.5 °C), parasite positivity by microscopy and at least one other common symptom of malaria that included malaise, headache, chills, diarrhoea and vomiting. Children diagnosed with clinical malaria were referred to the nearest designated health facility for appropriate treatment as specified in the study protocol. Children who were included in the current study were randomly selected on the basis of having at least one parasite-positive blood smear at any of the six follow up time points and on the availability of samples from the six study time points. For the current study, anaemia was defined as either mild, moderate or severe based on WHO’s standard definitions37.The malaria attack rate in the district during the study period was approximately 3.5 attacks per child per yearP. falciparum antigens were used for the measurement of naturally induced antigen-specific antibody levels. Selection of these antigens was on the basis of their expression in more than one developmental stage of the parasite, and on their recognition by antibodies from RAS-immunized subjects as well as specific antibodies raised in mice and rabbits17.Seven 17. The known characteristics of each antigen, including their putative description, the parasite stage specificity and localization within the parasite are presented in Table 17.All antigens were expressed in a cell-free wheat germ system as GST-tagged proteins and purified as previously described® ELISA plates  with the respective recombinant proteins and incubating overnight at 4 °C. After plates were blocked, plasma samples, diluted 1:100 were incubated and antibody reactivity detected by horseradish peroxidase-conjugated anti-human IgG, followed by incubation with the 3, 3′, 5, 5′-Tetramethylbenzidine (TMB). The color reaction was terminated by addition of 0.2 N H2SO4 and absorbance read at 450 nm. Absorbance data was converted into titers, expressed in arbitrary units (AU), using the four-parameter logistic curve fitting program known as ADAMSEL (Edmond J. Remarque®).Antigen-specific antibody levels were measured using an enzyme-linked immunosorbent assay (ELISA) approach that involved coating of MaxiSorpFor quality control, a pool of hyperimmune sera (positive control) from malaria exposed adults were run in duplicate on each antigen specific plate and the resulting titer plotted on a Levey-Jennings chart that was generated from previous repeated runs of the same pooled hyperimmune sera. Assay data on any plate was passed only when the positive control on that plate fell within two standard deviations of the mean on the Levey-Jennings chart.lme4. For this analysis, the data was categorized into two groups; samples from children who experienced at least one clinical episode over the study period and those from children who remained asymptomatic despite evidence of malaria infection by microscopy at any of the six sampling time points. Initially, antigen-specific antibody data was entered into the model as fixed effects with age as an interaction term, while sampling time point, study subject and bednet usage were entered as random effects. This model was compared to other models that either dropped the subject age interaction term or one of the random effects, and the best model, based on the lowest Akaike Information Criterion (AIC) values was selected for further analysis. The best model had log2–transformed antibody level as the fixed effect and the study subject variable as a random effect since the interaction term (age), bednet usage and sampling time point did not significantly contribute to the models. P-values for the intercept and fixed effect were obtained by likelihood ratio tests (with Laplace Approximation).Antigen-specific antibody levels are presented as box and whisker plots and comparison of antibody levels between parasitaemic and non-parasitaemic children at each sampling time point was performed using the Mann-Whitney U test. The relationship between antigen-specific antibody levels and parameters such as Hb levels and parasitaemia were assessed by Spearman correlation tests. Differences were considered to be statistically significant when P values were less than 0.05. For the assessment of potential antibody correlates of protection against clinical malaria, a generalized mixed effects regression model for repeated measurements was fitted to antigen specific antibody data using the R package Supplementary file


Plasmodium falciparum antigens. However, the effect of that exposure to subsequent immune responses has not been fully elucidated. This study aimed at determining the effect of prenatal exposure to P. falciparum on Interleukin-10 and Interferon-γ responses during clinical malaria episodes in the first 24 months of life. Infants born to mothers with placental malaria are prenatally exposed toγ in infants and detect IgM in cord blood. Data were analyzed using SPSS version 20. This prospective cohort study involved 215 infants aged 0-2 years born to mothers with or without placental malaria. Enzyme-linked immunosorbent assay (ELISA) was used to determine levels of IL-10 and IFN-γ in exposed infants was 557.9 pg/ml (95% CI: 511.6-604.1) and in unexposed infants it was 634.4 pg/ml (95% CI: 618.2-668.5) (P=0.02). Mean IL-10 was 22.4 pg/ml (95% CI: 19.4-28.4) and 15.1 pg/ml (95%CI: 12.4-17.6), respectively (P=0.01). Geometric mean for IFN- P. falciparum antigens significantly affects IL-10 and IFN-γ responses during clinical malaria episodes in the first two years of life. Prenatal exposure to Plasmodium falciparum malaria is a disease of public health importance and causes mortality of at least one million children below five years of age annually [ in utero effects on the immune cells due to early exposure to the parasite antigen may persist in childhood, leading to subsequent hyporesponsiveness or immunosuppression to the same parasite antigen and hence increased susceptibility to malaria infection [ in utero exposure to P. falciparum may modulate the development of fetal protective immune cells culminating into disruption in the balancing of T helper 1 (Th1) and T helper 2 (Th2) immune responses as reported elsewhere [γ (IFN-γ) is a Th1 proinflammatory cytokine that has been demonstrated to have antiparasitic effect [ P. falciparum antigens on selected cytokines (IL-10 and IFN-γ) response during clinical malaria episodes in infants aged zero to two years. The two cytokines IFN-γ and IL-10 were selected since IFN-γ is a key Th1-derived inflammatory cytokine that is important for clearance of intracellular parasites like P. falciparum, while its biological activity can be inhibited by Th2-derived IL-10 that inhibits natural killer cells and macrophage activity and has been demonstrated to be an important anti-inflammatory cytokine [ P. falciparum on cytokine responses such as season of birth, infant birth weight, use of Insecticide Treated bed Nets (ITNs), gravidity, parasite density, and timing of malaria episodes were also assessed.annually –4. Infanannually , 6. The nfection . This sulsewhere . Interfec effect , 10, whic effect . Althougcytokine . Other fDetailed description of the study area appears elsewhere . Briefly P. falciparum, while 165 infants born to mothers without placental malaria were categorized as unexposed. The infants were followed at three monthly intervals (regular scheduled visits) and whenever they fell sick up (irregular unscheduled visits) to the age of two years. During the follow-up, all clinical malaria episodes were recorded and respective blood samples were collected for determination of IL-10 and IFN-γ.Detailed description of the study design and participants recruitment process was described in an earlier report . Brieflyγ.Detailed procedures for collection of placental tissues, storage of placental tissues, histopathology, and follow-ups were reported elsewhere . Brieflyμl and 100 μl each, respectively. Aliquot sera were initially stored at −20°c at the field site and later transported in a cool box packed with frozen ice to −80°c freezers at Muhimbili University of Health and Allied Sciences (MUHAS) Immunology Laboratory for determination of IL-10 and IFN-γ.During follow-ups, recruited infants were examined for clinical signs at the health facilities and a clinical malaria episode was described as fever (≥37.5°C) plus parasitaemia established through microscopic examination of 10% Giemsa stained smears. All clinical malaria episodes encountered by recruited infants born to mothers with placental malaria and without placental malaria were recorded, respective blood samples were collected, and the infants were managed at the health facilities. Following clotting of collected cord and infant peripheral blood samples, sera were separated in aliquots of 200 γ and IL-10 levels were quantified in cord blood sera as baseline and in peripheral blood sera collected during clinical malaria episodes using ELISA in separate plates as per manufacturer's instructions . All materials and prepared reagents were equilibrated at room temperature prior to use in the assay. 100 μl of IL-10 and IFN-γ standards and test sera were added to appropriate wells and were covered well and incubated for two and a half hours at room temperature. The solutions were discarded and the plate was washed four times with washing buffer. The complete removal of liquid at each step was essential to ensure good performance of the assay. After the last wash, any remaining buffer solution was removed by aspiration and the plate was inverted and blotted against a paper towel. 100 μL of working biotinylated IL-10 and IFN-γ detection antibodies was added to respective wells and incubated for one hour at room temperature while shaking gently. This was followed by discarding the solution and washing three times. 100 μl of working horseradish peroxidase streptavidin solution was added to each well and was incubated for 45 minutes at room temperature while shaking gently. The solution was then discarded and washed three times. 100 μL of Tetramethylbenzidine (TMB) one-step substrate reagent was added to each well and incubated for 30 minutes at room temperature. Finally, 50 μl of stop solution was added to each well and optical density (OD) was read at 450 nm. The serial diluted concentrations of standard and their corresponding OD were used to plot a standard curve with standard concentrations on x-axis and mean absorbance on y-axis. The best fit straight line was drawn and the concentrations of IL-10 and IFN-γ in test samples were obtained and recorded in pg/ml. Determinations of IgM specific to PfMSP1-19 and PfMSPII antigens were done in cord blood sera samples. Presence of IgM in cord blood sera specific for PfMSP1-19 and PfMSPII antigens defined in utero sensitization while their absence defined in utero nonsensitization. The detailed ELISA procedures have been reported elsewhere [IFN-lsewhere . The posγ during clinical malaria episodes between exposed and unexposed infants were carried out using independent-samples t-test. ANOVA was used to assess the mean difference in levels of IL-10 and IFN-γ in exposed sensitized, exposed nonsensitized, and unexposed infants. Multiple linear regression model was used to assess the effect of prenatal exposure to P. falciparum and other factors  on levels of IL-10 and IFN-γ. Significance of difference was judged at 95% confidence level and a P value < 0.05.Data were analyzed using IBM SPSS version 20. Analysis was done on log transformed data in order to use parametric tests in the analysis. Comparisons of mean levels of IL-10 and IFN-Detailed description of the social demographic characteristics of the study population was reported elsewhere . Brieflyγ. P. falciparum was found to be the only Plasmodium species diagnosed using light microscopic technique. The details of timing and number of clinical malaria episodes were reported elsewhere [γ in cord blood for exposed and unexposed were 407.1 pg/ml (95% CI: 366.5-447.6) and 390.2 pg/ml (95% CI: 355.5-424.9), respectively, and the difference was not statistically significant  and 634.4 pg/ml (95% CI: 618.2-668.5), respectively, and the difference was statistically significant (P=0.02). Out of 50 infants who were born to mothers with placental malaria, 5 (10%) had specific IgM to PfMSP1-19 and PfMSP2 and were categorized as sensitized to P. falciparum antigens and the rest (90%) were nonsensitized. All infants born to mothers without placental malaria were not prenatally exposed to P. falciparum and had no detectable IgM against PfMSP1-19 and PfMSPII in cord blood sera. The ANOVA for levels of IL-10 and IFN-γ in the three subgroups of infants, exposed sensitized, exposed nonsensitized, and unexposed infants, indicated that the cytokine response for the three groups was different and this difference was statistically significant  during clinical malaria episodes, while other factors did not significantly associate with IL-10 and IFN-γ responses  and IFN-esponses .γ which may mitigate parasite development and stimulate monocyte phagocytosis towards clearance of parasitized red blood cells [γ and it is a major mediator for the production of acute phase reactants in the pathogenesis of malaria [Immunopathology of malaria disease involves proinflammatory cytokines such as tumor necrosis factor-alpha , Interleukin-12 (IL-12), and IFN-od cells , 17. IL- malaria . This im malaria . P. falciparum antigens has been demonstrated to affect the development of fetal immune cells [ in vitro studies have demonstrated that cord blood lymphocytes from malaria exposed placenta are nonresponsive when stimulated with similar parasite antigens. This nonresponsiveness has been ascribed to early exposure to the parasite antigens and is implicated to play a negative role in subsequent development of protective immune responses against the same parasite antigen. This phenomenon of prenatal sensitization has been demonstrated in filarial and other helminthes infections [Prenatal exposure tone cells  and affene cells , 19–22. fections –26. P. falciparum antigens on cytokine response during clinical malaria episodes focusing on IL-10 and IFN-γ, which are anti-inflammatory and pro-inflammatory cytokines, respectively [ P. falciparum antigens is significantly associated with low IFN-γ and high IL-10 responses during clinical malaria episodes in the first two years of life.On the basis of these findings, this study was designed to further characterize the effect of prenatal exposure toectively , 27, 28.γ response during clinical malaria episodes in exposed infants in this study may partly explain the effect of prenatal exposure to P. falciparum on antimalarial immunity in infants prenatally exposed to P. falciparum antigens, since IFN-γ has been demonstrated to effectively induce parasite killing by monocytes and neutrophils [γ during clinical malaria episodes may negatively affect the infant's ability to clear the malaria parasites through the cytokine responses and indicates a possible disruption of the biological roles of IFN-γ, including but not limited to activation of macrophages for phagocytosis and major histocompatibility complex class II (MHCII) molecule expression, which are important in antimalarial immunity [The low IFN-trophils . This obimmunity .γ responses during clinical malaria episodes observed in this study corroborate previous findings that demonstrated that prenatal exposure to P. falciparum downregulated Th1 responses in utero and that possibly later in life could contribute to susceptibility of infants to malaria infection in early childhood [The observations in this study may be supported by previous finding that indicated increased susceptibility of infants born to mothers with placental malaria when naturally challenged with malaria infection. These infants succumbed to clinical malaria infection at a shorter time from birth and experienced more attacks of clinical malaria episodes in their first two years of life, which may suggest compromised protective immunity development in these infants . Furtherhildhood . P. falciparum tends to suppress fetal expression of Th1 and CD8+ T cells as demonstrated by a study conducted in Gabon [γ and high IL-10) in Gabon's in vitro study with cord blood cells and the present in vivo study in infants' peripheral blood plasma suggests that the effect of fetal T cell exposure to the parasite antigens persists to the first two years of life.Early fetal exposure to parasite antigens modulates the development of fetal T cells leading to fetal immunosensitization or immunological tolerance which is indicated by hyporesponsiveness. The effect of immunological tolerance following in utero exposure toin Gabon . The simγ and IL-10 responses. The geometric mean level of IFN-γ during clinical malaria episodes in the first two years of life in exposed infants was significantly lower than that in unexposed infants. The low IFN-γ responses in exposed infants indicate the effect of prenatal exposure to P. falciparum on antimalarial immunity in infants prenatally exposed to P. falciparum antigens. IFN-γ has been demonstrated to effectively induce parasite killing by monocytes and neutrophils [γ during clinical malaria episodes may negatively affect the infant's ability to clear the malaria parasites, a finding that may be corroborated by an earlier study [In a multivariate analysis, after adjusting for other factors , which may influence acquisition of malaria infection and subsequent host immune response, the results showed that prenatal exposure was the only factor that was significantly associated with IFN-trophils ; therefoer study  that demγ production observed in this study are likely to create a conducive host environment for the survival of P. falciparum. In terms of malaria pathogenesis, it may be hypothesized that P. falciparum parasites evade parasite clearance by modulating the host immune system such that IL-10 that has been associated with susceptibility to malaria [γ that has antiparasitic effect becomes downregulated, thus creating a host environment that may favor propagation of P. falciparum. The high IL-10 response during clinical malaria episodes demonstrated in infants born to mothers with placental malaria in this study may also indicate a possible inhibition of Th2 cytokine response accompanied with high production of IL-10, leading to disruption of Th1/Th2 balance, curtailing the inhibition of P. falciparum replication, a role played by IFN-γ [The high levels of IL-10 during clinical malaria episodes accompanied with downregulated IFN- malaria  becomes by IFN-γ . P. falciparum malaria. In this study, the majority of infants who were born to mothers with placental malaria were nonsensitized to P. falciparum antigens. This observation may possibly explain the downregulation of IFN-γ during clinical malaria episodes, which potentially lowers antiparasitic effects and makes the infants more vulnerable to malaria infection. Furthermore, this study shows that cytokine responses are an important component of immunity against P. falciparum infection in childhood. Therefore, strategies towards design of malaria vaccine candidates may among other aspects consider a vaccine candidate that will restore the optimal Th1/Th2 balance that will be able to potentiate the host antiparasitic effects, restrain the propagation of parasites, and mitigate the occurrence of clinical malaria episodes.The results of this study therefore shade more light on the effects of prenatal exposure to the parasite antigen on host susceptibility and development of protective immunity against P. falciparum significantly affects IFN-γ and IL-10 cytokine response during clinical malaria episodes in infants aged zero to two years.Prenatal exposure to


Plasmodium knowlesi, a parasite of long-tailed macaques (Macaca fascicularis), is an important cause of human malaria. Plasmodium cynomolgi also commonly infects these monkeys, but only one naturally acquired symptomatic human case has been reported previously.In Southeast Asia, Malariometric studies involving 5422 subjects (aged 6 months to 65 years) were conducted in 23 villages in Pailin and Battambang, western Cambodia. Parasite detection and genotyping was conducted on blood samples, using high-volume quantitative PCR (uPCR).P. cynomolgi was found in 11, P. knowlesi was found in 8, and P. vivax and P. cynomolgi were both found in 2. Only 2 subjects were female, and 14 were men aged 20–40 years. Geometric mean parasite densities were 3604 parasites/mL in P. cynomolgi infections and 52488 parasites/mL in P. knowlesi infections. All P. cynomolgi isolates had wild-type dihydrofolate reductase genes, in contrast to the very high prevalence of mutations in the human malaria parasites. Asymptomatic reappearance of P. cynomolgi occurred in 2 subjects 3 months after the first infection.Asymptomatic malaria parasite infections were detected in 1361 of 14732 samples (9.2%). Asymptomatic infections with nonhuman primate malaria parasites were found in 21 individuals living close to forested areas; P. cynomolgi and P. knowlesi infections can both occur in humans.Asymptomatic naturally acquired NCT01872702. P. knowlesi infection and 13 with P. cynomolgi infection). These parasites represented 1.9% of all identified malaria parasites.Epidemiological studies in western Cambodia identified 21 subjects with asymptomatic monkey malaria parasite infections (8 with  The excMalaysia . Laboratescribed , 3, 4, bescribed .P. knowlesi in the monkey population as a source of human infections [P. knowlesi and P. cynomolgi and present here a molecular evaluation of these infections.A malaria elimination agenda has been endorsed by all countries of the Greater Mekong Subregion in Southeast Asia . Howeverfections . Submicrfections  are impofections , 9. In tMalariometric surveys were conducted in 23 villages in the Pailin and Battambang provinces of western Cambodian as described previously , 10, 11 Plasmodium genus–specific 18S ribosomal RNA (rRNA) as described previously. This has a lower limit of accurate quantification of 22 genomes/mL [Of 6100 invited participants, 5422 provided a blood sample at least once. A total of 14732 samples were sent to the reference laboratory in Bangkok for testing by high-volume quantitative PCR (uPCR) targeting the gene encoding nomes/mL . Ethics Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and the 2 Plasmodium ovale species. A subset of samples containing Plasmodium DNA did not amplify with these species-specific probes and were investigated further.Samples testing positive by uPCR were then assessed by real-time PCR, using species-specific probes for detection of the 5 human malaria parasites: P. cynomolgi, P. knowlesi, Plasmodium coatneyi, Plasmodium brasilianum, Plasmodium inui, Plasmodium simium, Plasmodium semiovale, Plasmodium fieldi, Plasmodium fragile, Plasmodium vinckei, Plasmodium yoeli, Plasmodium chabaudi, Plasmodium berghei, and Plasmodium adleria . Two pairs of oligonucleotide primers were designed to amplify both the A and S types of the genes encoding 18S rRNA  and P. knowlesi (n = 3), we performed whole-genome amplification, using multiple displacement amplification by Phi29  according to the manufacturer’s instructions. Following collection, white blood cells were depleted from blood samples so that contaminating human DNA was less likely to limit the amplification efficiency in this system. To rule out cross-contamination during processing, 1 negative control consisting of only water was added for every 3 samples. We evaluated and compared DNA sequences and microsatellites before and after amplification in 14 samples, of which 9 were positive for P. cynomolgi, and 5 were positive for P. knowlesi. Findings were 100% concordant.For further analysis of very-low-density infections with P. cynomolgi and P. knowlesi dhfr-ts were assessed by nested PCR amplification covering the full length of dhfr-ts , followed by sequencing of the gene in an ABI Sequencer . The sequences were then aligned against the dhfr-ts of reference Cambodian strain (PCYB_053150) of P. cynomolgi and strain H (PKNH_0509600) of P. knowlesi and were analyzed using Bioedit software .Point mutations in P. cynomolgi, 8 microsatellite markers of di/tri/tetra-nucleotide repeats distributed across chromosomes 1–12, named 1.307 (TCT), 2.36 (AT), 3.574(GTGA), 4.41(TCT), 4.462(TCC), 5.956(AAG), 6.455(AGG), 7.1006(ATAC), 10.179(TA), 10.621(TGTA), 11.1096(TGTA), and 12.286(AAT), were assessed using seminested PCR as described previously [P. knowlesi, 10 microsatellite markers of trinucleotide repeats spanning chromosomes 3–13, named NC03_2(AAG), CD05_06(TAA), CD08_61(TAC), NC09_1(GAA), NC10_1(TTA), CD11_157(GAG), NC12_2(AAT), NC12_4(GAA), CD13_61(AAC), and CD13_107(AGG), were assessed using seminested PCR as described previously [To assess genetic relatedness between infecting nonhuman primate malaria parasites and between initial and recurrent infections, microsatellite genotyping was performed. For eviously . For P. eviously . The lenGenetic similarity between isolates was analyzed and a dendrogram constructed on the basis of the microsatellite markers, using an unweighted pair group method with arithmetic mean (UPGMA), performed with BioNumerics software, version 7.5 .This trial was registered on 4 June 2013 at Clinicaltrials.gov (NCT01872702).P. falciparum was detected in 114 (10.1%), P. vivax was detected in 976 (86.8%), and both species were detected in 14 (1.2%) [P. vivax species, using a less sensitive and specific real-time PCR protocol [P. vivax DNA. However, because of the 98% similarity between the genes encoding the 18S RNA of P. vivax and P. cynomolgi, which raised the possibility of misidentification, we subsequently retested the same set of samples, using the specific primate nested PCR and sequencing protocol. Three of 853 specimens (0.3%) originally identified as containing P. vivax actually contained P. cynomolgi only.Of the 14732 samples collected (7320 from Battambang and 7412 from Pailin), uPCR indicated that 1361  contained malaria parasite DNA. Of these, DNA in 1125 could be identified to the species level: d in 114 .1%, P. vprotocol , 853 samP. cynomolgi monoinfections were found in asymptomatic afebrile residents from 5 villages located close to the forest. All individuals were male; the median age was 28 years . The 5 villages comprised Chakrya, Ou Treng, Tik Mong, Veal Roleum, Phnom Rey, in Battambang, and Krong Pailin, in Pailin, Western Cambodia , and 14 were men aged 20–40 years, reflecting the demographic group most likely to enter and spend time in or near the forest.Eight dhfr-ts gene (approximately 1884 bp) was sequenced in all P. cynomolgi infections. No nonsynonymous mutations were found. Thus, 0 of the 7 drug resistance point mutations in the dhfr drug binding pocket, which are highly prevalent in P. vivax isolates in this area [P. cynomolgi contained identical repeats within the Pcdhfr of (SGDTH)(SGDNTNG)(SGDAVGT). This contrasts with P. vivax, in which the number of intragenic repeats is highly variable. In the 2 P. cynomolgi and P. vivax mixed infections, only the P. vivax dhfr-ts was amplified by PCR, presumably because P. cynomolgi was the minority population. The P. vivax dhfr-ts had the common double resistance mutations at S58R and S117N with the (GGDNTS)(GGDNADK) repeat.The full length his area , were fodhfr-ts gene (approximately 1881-bp) sequences of the 8 P. knowlesi isolates had no nonsynonymous mutations, although there was 1 deletion at codon 105 without any interruption of protein translation. There was also 1 nonsynonymous mutation (G257S) in the TS gene.The full length of P. cynomolgi isolates, 8 tandem repeat loci bearing 2–4 bp specific for P. cynomolgi were tested (n = 13). The mean number (±SE) of alleles per locus was 6.37 ± 0.29  presented with recurrent P. knowlesi parasite isolates was assessed using 7 microsatellite markers. The mean number (±SE) of alleles per locus was 2.85 ± 0.17 . P. cynomolgi in rhesus monkeys was recognized as behaving similarly to P. vivax, with frequent relapses from an exoerythrocytic source (later shown to be the dormant “hypnozoites” in the liver), and so this infection became the animal model for relapsing malaria. The potential for mosquito-to-human transmission of P. cynomolgi was illustrated first by the renowned entomologist Don Eyles when he and then his assistant became ill with malaria while studying mosquito transmission in laboratory primates [P. cynomolgi is very similar to P. vivax, although the incubation period of sporozoite-induced infections in humans is significantly longer [P. cynomolgi has not caused severe malaria in experimentally infected humans. Studies of untreated infections with both the M and B strains by Coatney et al documented persistence of the initially symptomatic P. cynomolgi human infections for as long as 58 days [P. vivax results in P. cynomolgi premunition, or vice versa, is not known. Important anopheline vectors of human malaria in Cambodia and Vietnam (Anopheles dirus and Anopheles maculatus) have been shown to carry P. cynomolgi and P. knowlesi sporozoites [P. cynomolgi infections is likely to be the highly effective forest vector A. dirus.This is the first report from a malaria-endemic area of asymptomatic infection with the nonhuman-primate malaria parasite primates . Before primates , 4. Morp 58 days , 4. Thusrozoites , 22. TheP. cynomolgi and P. vivax probably contribute to underrecognition of the monkey parasite in human infections in the Greater Mekong Subregion. Symptomatic patients in whom malaria is diagnosed either by microscopy or a genus-specific rapid diagnostic test will probably be labeled as having vivax malaria. In malariometric surveys, cross-reactivity in some of the PCR methods used may also lead to misdiagnosis, as happened early in this study. The specificity of the PCR method finally used in this study is supported by the analysis of the dhfr-ts genes. Their protein product is the target of antifolate drugs. Nonsynonymous drug resistance mutations are found in nearly all P. vivax  isolates in Cambodia and adjoining Thailand [P. cynomolgi isolates contained the corresponding Pcdhfr mutations. This is explained by the absence of drug pressure in the primate population and the likely very small contribution of human infection to P. cynomolgi transmission and epidemiology.The morphological and genetic similarities of Thailand , yet nonP. cynomolgi was recovered twice, with an intervening interval of approximately 3 months. In one case genetically distinct parasites were identified, and in the other the paired parasites were related, indicating that they were siblings derived from the same mosquito inoculum. Whether these were relapses, reinfections, or persistent infections with blood-stage parasites, with a mixed infection, cannot be determined with certainty, although reinfection is highly unlikely in the latter case. The genetic diversity of the P. cynomolgi and P. knowlesi isolates was low. The multiplicity of P. cynomolgi infections  was lower than observed in P. vivax infections from Thailand , Myanmar , and India , which may reflect the relative rarity of successful transmission to humans [P. cynomolgi human red blood cell invasion is restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC) or CD234 [P. cynomolgi–infected individuals possess a facilitatory genetic polymorphism affecting these receptors also cannot be excluded.In 2 subjects, o humans . These sor CD234 , so the P. knowlesi infections are increasingly recognized in Southeast Asia—particularly in Malaysia [P. knowlesi is the main cause of human malaria, yet P. inui and P. cynomolgi are the more prevalent malaria parasites in long-tailed macaques, which are the main local P. knowlesi reservoir [P. cynomolgi infections have not been found there to date [P. knowlesi by the same anophelines. P. knowlesi is a quotidian parasite that may reach high densities in humans and cause potentially lethal infections, although the majority of presentations are associated with uncomplicated malaria and low-density infections [P. knowlesi may be mistaken for P. falciparum at the young ring stage and with P. malariae at the trophozoite stage. P. knowlesi is detected by pan-species rapid tests, although the limit of detection is higher than for the human malaria parasites. In early PCR protocols, there was cross-reactivity with P. vivax, its closest phylogenetic relative among the human malaria parasites, but more-recent methods such as those used here are highly specific. As with P. cynomolgi, P. knowlesi is not under antimalarial drug selection pressure and so remains fully susceptible to all of the antimalarial drugs [P. cynomolgi carried at lower parasitemia levels. Such densities, if maintained, might be too low to infect anopheline mosquitoes.Naturally acquired Malaysia , 25–30. Malaysia , 22. Inteservoir , 32. Asy to date . This maal drugs . Althougdhfr in an area where nearly all human malaria parasites are mutated at this locus provides strong supportive evidence.In malariometric surveys that employ microscopy, rapid diagnostic tests or non–species-specific PCR methods of detection, the nonhuman primate malaria parasites will not be recognized. With species-specific PCR protocols, positive samples that are not recognized by the human malaria parasite–specific probes are likely to carry nonhuman-primate malaria parasites. If there is insufficient DNA for accurate characterization, whole-genome amplification protocols may be used successfully, as in this study. If there is any uncertainty, then genes other than that encoding 18S RNA can be assessed. In this study, the finding of a “wild-type” As human malaria is controlled and the prevalence falls, primate malaria parasites will comprise an increasing proportion of cases in areas where monkeys live in close proximity to people. In this study, conducted in western Cambodia, nonhuman-primate malaria parasites comprised 1.9% of identified parasites. Regional malaria elimination assessments must recognize the propensity of these primate malaria parasites to cause asymptomatic infection, as well as symptomatic infection.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table1-5Click here for additional data file.


Malaria and leishmaniasis are the two largest parasitic killers in the world. Due togeographical overlap of these diseases, malaria-visceral leishmaniasis co-infections occur in large populations and exist in different areas even if they have been poorly investigated. The aim of this study was to determine malaria-visceral leishmaniasis co-infection and their associated factors among migrant laborers.Plasmodium parasites. Recombinant kinensin (rk39) antigen test was performed to detect anti-leishmania donovani antibody. Data was coded, entered, checked for completeness and analyzed using SPSS version-20 statistical software. Chi-square test was applied to show a significant association between variables. P-value < 0.05 was considered as statistically significant.Community based cross-sectional study was conducted from October–December 2016 on migrant laborers who are residents of rural agricultural camp in West Armachiho district and involved in sesame and sorghum harvesting. Standardized questionnaire was used to collect socio-demographic data and risk factors. Capillary blood was collected for giemsa stained blood film examination to detect and identify . All variables were not significantly associated with malaria-visceral leishmaniasis co-infection (P > 0.05).A total of 178 migrant laborers were included in this study. Of these, 74.2% belong to the age group 15–29; 61.2% come from lowland areas and 51.6% visit the area more than four times. Seroprevalence of visceral leishmaniasis was 9.6% (17/178); and 22.4% (40/178) of tested migrant laborers were found malaria infected. The overall prevalence of malaria-visceral leishmaniasis co-infection was 2.8%. Of the total migrant laborer, 47.8% used bed nets, of them 1.2% were malaria-visceral leishmaniasis co-infected; 72.5% used outdoor sites as usual sleeping site, among them 3.1% were malaria-visceral leishmaniasis co-infected; 60.1% were migrants, of which 2.8% were malaria-visceral leishmaniasis co-infectedPrevalence of malaria-visceral leishmaniasis co-infection was low and it is not significantly associated with residence, number of visits, bed net utilization and outdoor sleeping habit even if both diseases are prevalent in the study area. Plasmodium  considering 95% CI and taking prevalence (P) 0.12 from previous study conducted in the same study area [The required sample size was calculated using a single population proportion statistical formula [n = udy area  and 10% Proportionate two stage cluster sampling method was used to select study participants. First farm sites were grouped by the name of the owner of farm site. Thirty (30) farm sites were enrolled in the study and among them 11 farm sites were selected by simple random sampling technique using lottery method. One hundred seventy eight (178) study subjects were selected from 11 farm sites proportionally.Information on demographic and risk factors was collected using Amharic version standardized questionnaire. Required information was gathered from labour and social affairs office, health offices, and environmental protection, land administration and use authority at woreda and zonal level. Before the actual data collection, site visit to the enrolled farm sites and discussion with managers was held. Finally, interview was conducted by trained health workers and field research assistants at selected farm sites.After interview, blood film examination was performed based on the standard and well accepted guideline . CapillaA single finger prick peripheral blood specimen (8-12 μL) was collected from each study participants and tested by immuno-chromatographic technique (ICT) using rk39 antigen test kit , a 39 amino acid repeat more suitable for field use, having high sensitivity and specificity. Finally the result was interpreted accordingly. To avoid reader variability, it is checked by three senior laboratory professionals.Giemsa stock solution was filtered during preparation of working solution, the quality of each batch of working giemsa stain solution was then checked by using known negative and positive blood smear before use. Stained slides were re-checked by two experienced laboratory technologists blindly. To maintain the quality of rk39 test kit, appropriate storage condition was met. Generated data were checked for completeness and cleanness before starting the actual analysis.P-value < 0.05 was considered as statistically significant.Data were entered and analyzed using SPSS version 20.0  software. Then, the study findings were explained in words, tables and graphs. Chi-square test was used for statistical analysis to show presence of significant association between variables. A total of 178  migrant laborers selected from 11 farm sites were included in this study. Their mean age was 26.1 (range 15–65 years) with standard deviation of 8.6. Among these, 91.6% were male and 74.2% were in the age range of 15–29. Of them, 92.7% were from Amhara region and 60.1% were migrants. About 36.5% of study participants have completed elementary school and 51.6% of migrant laborers visit the area more than four times (Table n = 40), 9.6% (n = 17), 2.8% (n = 5) were infected with malaria, visceral leishmaniasis, and both malaria and visceral leishmaniasis respectively ,Plasmodium falciparum, Plasmodium vivax and mixed infection accounts 72.5% (n = 29), 10% (n = 4) and 17.5% (n = 7) respectively  but there was no significant association between malaria-visceral leishmaniasis co-infection and age group  were migrants, of which 3 (2.8%) were malaria-visceral leishmaniasis co-infected. All variables were not significantly associated with malaria-visceral leishmaniasis co-infection (P > 0.05)  used bed nets. Of these, 1 (1.2%) migrant laborer were malaria-visceral leishmaniasis co-infected. Majority of the migrant laborers; 129 (72.5%) used outdoor sites as usual sleeping accommodation, of which 4 3.1%) were malaria-visceral leishmaniasis co-infected. Among the tested migrant laborers, 60.1%  and Metema Hospital [In this study the prevalence of malaria was 22.4% which is higher than a study conducted among migrant laborers in two districts of North Gondar Zone (Metema and West Armachiho) which is 12%  and amonHospital , 29.The study was conducted in a very remote area (hard to reach population) where no prevention measure is applied to reduce the burden of both malaria and visceral leishmaniasis in such high risk group of population; and our finding may alert stakeholders to design timely and appropriate intervention strategy. However, we are unable to use molecular technique for parasite detection; and to calculate malaria parasitaemia.The prevalence of malaria-visceral leishmaniasis co-infection was low even if the area is endemic for both diseases and it is not associated with residence, number of visits, bed net utilization and outdoor sleeping habit of migrant laborers. Regular health education programs should be organized and implemented for such high risk population to create awareness.