The purpose of this study was to determine the prevalence of coinfection with malaria and intestinal parasites, as well as to determine its association with anaemia in children aged 10 years and below in Muyuka, Cameroon.This was a cross-sectional study. Participants were febrile children who were admitted to the Muyuka district hospital between April and October 2012. Blood and stool samples were collected from those participants who gave consent to take part in the study. Haemoglobin concentration (Hb) and complete blood count (CBC) were performed using an automated haematology analyser . Giemsa-stained blood film was examined to detect malaria parasites, while the formol-ether concentration technique was used to detect intestinal parasitic infections (IPIs). The Pearson’s chi-square, Student’s T-test and correlation analysis were all performed as part of the statistical analyses.p = 0.000), whereas IPIs were more prevalent in children aged between five and 10 years (p = 0.006). The parasite species isolated included Ascaris lumbricoides (36 [73.5 %]), Entamoeba histolytica/dispar (9 [18.4 %]) and hookworm (4 [8.2 %]). The mean Hb observed was 10.64 g/dl (±1.82). A significant negative correlation was observed between malaria parasite density and Hb. There was no significant difference in the prevalence of anaemia among children infected with malaria, IPIs, or malaria and IPI coinfection, or among non-infected children. Similarly, the mean Hb did not differ among infected and non-infected children.Four hundred and eleven (411) children successfully took part in this study. The prevalence of malaria, IPIs, malaria and IPI coinfection, and anaemia observed were 98.5 %, 11.9 %, 11.9 % and 44.8 %, respectively. Anaemia and IPIs were significantly associated with age; anaemia was more prevalent in children under five years of age (This study showed that malaria and IPIs still constitute a major public health problem in the study area despite a lack of any significant association between these infections and anaemia. The findings suggest that there is a need for the implementation of control measures to curb the rate of malaria and IPIs in the study area. Plasmodium vivax, P. ovale, P. malariae, P. knowlesi and P. falciparum, cause malaria, with the latter being the most virulent and accounting for the majority of malaria deaths ) compared to children below five years of age (290/295 [98.3 %]). Again, no significant difference was observed between the prevalence of malaria and age . The parasite density ranged between 65 and 160,523 .Four hundred and five (405) participants were positive for malaria, resulting in a prevalence of 98.5 % (95 % CI: 97.4–99.7). The prevalence was higher among males (196/197 [99.5 %]) than females (209/214 [97.7 %]), however, no significant association was observed between the prevalence of malaria and gender  in children aged between five and 10 years (22/116 [19.0 %]) compared to children below five years of age (27/295 [9.2 %]). The prevalence of infection was higher in males (27/197 [13.7 %]) than in females (22/214 [10.3 %]), however, no significant association was observed between the prevalence of IPIs and gender . Infection with the Ascaris lumbricoides species was the most common IPI identified (36 [73.5 %]), followed by Entamoeba histolytica/dispar (9 [18.4 %]) and hookworm (4 [8.2 %]). No significant association was observed between the prevalence of parasite species and age . Infection with more than one species of intestinal parasites (polyparasitism) was not observed in this study.Among the 411 participants, 49 were positive for intestinal parasites, resulting in a prevalence of 11.9 % (95 % CI: 8.78–15.1). Infections with helminths were more common than those with protozoa (81.6 % vs. 18.4 %). As well as that, IPIs were more prevalent  in children aged five years or below (151/295 [51.2 %]) compared to children between five and 10 years of age (33/116 [28.5 %]). The prevalence of anaemia was higher in females (101/214 [47.2 %]) compared to males (83/197 [42.1 %]), however, no significant association was observed between prevalence of anaemia and gender .In this study, 184 of the 411 participants were anaemic, resulting in a prevalence of 44.8 % (95 % CI: 39.9–49.7). The prevalence rates of mild, moderate and severe anaemia were 69.6 % (128), 17.4 % (32) and 13.0 % (24), respectively. Anaemia was more prevalent . Among the participants with IPIs, 27 (55.1 %) were anaemic. Again, no significant association was observed between the prevalence of IPIs and anaemia . Species-specific analysis didn’t reveal a significant association between the different species of intestinal parasites and anaemia   were anaemic, however, no significant association was observed between the prevalence of malaria and anaemia .The prevalence of severe anaemia was 13.1 %, 14.8 % and 14.8 % among the participants with malaria, IPIs, and malaria and IPI coinfection, respectively (see Table p = 0.127), or between children with IPIs and those without (p = 0.14) (see Table p = 0.000)  Hb was 10.64 g/dl (±1.82) range: 3.5–13.7). The mean Hb did not differ significantly among children who were infected with malaria and those who were not , which conforms to studies done elsewhere [Ascaris lumbricoides (73.5 %), Entamoeba histolytica/dispar (18.4 %) and hookworm (8.2 %). Ascaris lumbricoides species being the predominant parasite species causing infection in children is in line with other studies conducted in other areas of Cameroon [E. histolytica from E. dispar.In the present study, the prevalence of IPIs was 11.9 %, which is lower compared to the 34.7 % reported in communities around Dschang, in the West Region of Cameroon , 22.7 % lsewhere , 32. ThiCameroon , 37, 38,Cameroon . InfectiAll the children with IPIs in the present study also had malaria, resulting in a prevalence of 11.9 % for coinfection. This figure is low compared to the 26.1 % reported by Makoge et al. , but higp = 0.000), but no significant association was observed between the prevalence of anaemia and gender. Young children are more vulnerable to anaemia, a situation complicated by the presence of infections with bacteria, malaria and intestinal parasites. The association between anaemia and age aligns with some studies [The prevalence of anaemia was 44.8 % in this study. This is high and may have consequences on learning and development of children if not addressed rapidly. The prevalence of anaemia observed in this study is lower compared to the 57.6 % reported in Mbonge , but hig studies , 17, but studies , in whic studies , 29, 31  studies . No sign studies  also fou studies  likewise studies –44. Mala studies . Not obs studies .Entamoeba histolytica can also lead to anaemia through blood loss in diarrhoea. In this study, species-specific analysis didn’t reveal a significant association between the different parasite species and anaemia. This differs from the study conducted by Osazuwa et al. [Ascaris lumbricoides, and anaemia.The association between intestinal parasites, especially hookworm, and the development of anaemia is well known, however, infection with other parasites such as a et al. , in whicp = 0.127), or between participants infected with intestinal parasites and those not infected (p = 0.14). The lack of a significant association between malaria and Hb could be attributed to the very high prevalence of malaria in this study. In contrast, a study conducted by Yentür et al. [The mean (±SD) Hb of participants in this study was 10.64±1.82. No significant difference in the mean Hb was observed between malaria infected and non-malaria infected children . This conforms to a study done in Nigeria by Achidi et al. [A significant negative correlation between malaria parasite density and Hb was observed in this study  and anthelminthic medication should be encouraged.This study revealed a very high prevalence of malaria and anaemia, and a lower prevalence of IPIs, in the study area.


To assess the use of mid-upper arm circumference (MUAC) for identification of overweight and overfatness in rural South African children and adolescents.Anthropometric data  from a cross-sectional sample of 978 black South African 5–14-year-olds were analysed. Receiver operating characteristic (ROC) curve analysis determined the validity of MUAC as a proxy for determining overweight and overfatness.Area under the curve (AUC) results were generally high. Boys and girls aged 10–14 years had ROC-AUC for overfatness classed as ‘excellent’, 0.97 and 0.98 respectively. Cut-points in the MUAC distribution which optimised the ROC-AUC for identification of overfatness and obesity were determined for boys and girls aged 5–9 and 10–14 years, and had high sensitivity and specificity.MUAC may have potential for clinical and surveillance applications as an accurate yet simple and widely available indicator of overweight and overfatness in children and adolescents in resource-poor settings. Mid-upper arm circumference (MUAC) is widely used as an indicator of severe and moderately acute undernutrition, with simple guidelines available for children aged up to 5 years.It is not known if MUAC is helpful as an indicator of overweight or overfatness in older children and adolescents.MUAC can identify overweight and overfatness with reasonable accuracy in school-age children and adolescents.MUAC may have potential as a clinical and surveillance indicator of obesity in children and adolescents.Childhood obesity has become pandemic, and has short-term and long-term adverse impacts on health.3Mid-upper arm circumference (MUAC) is an easy, quick and inexpensive field measure most commonly used for identification of severe acute undernutrition in young children (6–60 months of age) in resource-limited settings. There is a dearth of research on the potential for MUAC as a screening tool for obesity, or its use in older children and adolescents.The present study aimed to determine the accuracy of MUAC for the assessment of overweight (as defined on the basis of BMI-for-age) and overfatness (as defined by bioimpedance estimates of body fatness) in children and adolescents.Study participants were black Zulu children and adolescents from school grades 1, 5 and 9  living within the Africa Centre Demographic Surveillance Area (DSA) in KwaZulu–Natal, South Africa, who were enrolled in a larger cross-sectional study which aimed to determine prevalence of overweight and obesity in this area.5All statistical analysis was carried out using STATA V.11.0.Receiver operating characteristic (ROC) analysis was used to test the ability of MUAC to determine those children and adolescents identified as overweight by BMI-for-age and overfat by bioelectrical impedance. For the analysis, participants were divided into sex-specific  and age-specific child (5–9 years) and adolescent (10–14 years) groups, these age groups were chosen based on previously published MUAC guidelines.The area under the curve (AUC) is a measure of accuracy and is indexed from 0 to 1 where 1 indicates a perfect test and ≤0.5 a worthless test.A probability curve was plotted to determine the probability of being overweight or overfat at each MUAC reading. The point at which the probability curve showed a marked increase was selected as a possible indicator. Based on the results of the ROC curve and the probability graph, two potential optimal operating points (OOP) were determined, and their accompanying data with regards to probability, sensitivity, specificity and classification accuracy was calculated.Two potential cut points were chosen for each age-specific and gender-specific group, these potentially OOPs aimed to maximise sensitivity and specificity, while also taking into account results of the ROC curve, probability curve and level of classification accuracy, with the intention that future studies would be able to verify which of the two cut-points is most suitable, and how generalisable they might be to different populations.A total of 978 participants were included in this analysis  and 497 aged 10–14 years  see .AUC results from the ROC curve were ‘excellent’ for girls and boys 10–14 years of age ; for girls and boys aged 5–9 years classification accuracy was ‘good’ (0.88) for girls and ‘poor’ (0.66) for boys.5In general, sensitivity and specificity were relatively high for the cut-points chosen for all age and gender groups . The excBased on the ROC analysis and probability curve, the following optimal MUAC cut-points to identify overfatness were identified: 5–9 years 19.2 cm/19.5 cm and 18.4 cm/18.7 cm for girls and boys, respectively; at ages 10–14 years, 22.0 cm/22.6 cm and 23.2/23.6 cm for girls and boys, respectively.The ROC–AUC for MUAC versus overweight was ‘excellent’ for girls and boys in both age groups .Based on ROC analysis and probability curve, the following potentially optimal MUAC cut-offs to identify overweight are proposed: ages 5–9 years, 18.3 cm/18.9 cm and 18.4 cm/18.6 cm for girls and boys, respectively; at ages 10–14 years, 22.5 cm/22.8 cm and 22.2 cm/23.2 cm for girls and boys, respectively.In the present study, the classification accuracy of MUAC was high for overweight (defined by BMI), and for overfatness , although accuracy was higher for BMI (a weight-based measure) than for body fatness. Sensitivity, specificity and AUC were generally high relative to similar studies of the classification accuracy of BMI and waist circumference in children and adolescents as summarised in two recent systematic reviewsThe present study provides a ‘proof of concept’ of the potential for MUAC to define overnutrition. The present study should not provide the basis of a recommendation to use MUAC to define overnutrition, and the cut-points identified as potentially optimal should only be used with caution. Ideally, the potentially optimal MUAC cut-points from the present study should be tested in an independent sample in order to provide confidence in the ability of MUAC to detect overweight and overfatness and analysis of alternative age groupings or even single ages may also be necessary. Adjustments may also be necessary for stunting status and pubertal development given that these factors have been found to impact BMI and body fat status and, therefore, may impact interpretation of results. Future research should also investigate the functional outcomes of MUAC, that is, the extent to which a high MUAC predicts the development of the comorbidities of obesity, such as diabetes and cardiovascular disease.Our findings suggest that MUAC has potential as a proxy measure for overweight and overfatness in children and adolescents in rural South Africa and probably other resource-poor settings. Measurement of MUAC is a simple, practical proxy for undernutrition,et al definitions of overfatness, based on a European population, to other populations is unclear at present, but we note that these definitions use what would generally be accepted to be very high levels of body fatness in all populations. An alternative approach would have been to use a criterion method of body fatness measurement (a 3 or 4 component model) to define overfatness, but the criterion methods are not practical for use in large samples in resource-limited settings.Few studies have measured MUAC in large samples; the majority of research, to date, has focussed on children under 5 years of age and predominantly on the use of MUAC for the assessment of undernutrition. One limitation of the present study is that the optimal cut-offs in the MUAC distribution were not cross-validated in an independent sample which would be necessary to establish the value of MUAC for the assessment of overweight and overfatness, The present study used the widely accepted and recommended BMI-for-age cut-offs to define overweight (including obesity), but for overfatness, the McCarthy body fat reference curve was used.In summary, the present study provides novel evidence that MUAC has the potential to be used in the screening of overnutrition, and calls for further studies to investigate this concept in other populations. Future research may also seek to determine the effect puberty and different obesity phenotypes have on MUAC, as well as assessing functional outcomes of a high MUAC.


Plasmodium infections or asymptomatic Plasmodium carriers in endemic areas. Thus, highly sensitive detection methods are needed to understand the precise malaria situation in these areas.Although the malaria burden in the Lao PDR has gradually decreased, the elimination of malaria by 2030 presents many challenges. Microscopy and malaria rapid diagnostic tests (RDTs) are used to diagnose malaria in the Lao PDR; however, some studies have reported the prevalence of sub-microscopic Plasmodium infections and their results were compared. A questionnaire survey of all participants was also conducted to estimate risk factors of Plasmodium infection. Numbers of infections detected by the three methods were microscopy: P. falciparum (n = 1), P. vivax (n = 2); RDTs: P. falciparum (n = 2), P. vivax (n = 3); PCR: Plasmodium . Using PCR as a reference, the sensitivity and specificity of microscopy were 33.3% and 100.0%, respectively, for detecting P. falciparum infection, and 7.0% and 100.0%, for detecting P. vivax infection. Among the 47 participants with parasitemia, only one had a fever (≥37.5°C) and 31 (66.0%) were adult males. Risk factors of Plasmodium infection were males and soldiers, whereas a risk factor of asymptomatic Plasmodium infection was a history of ≥3 malaria episodes.A cross-sectional malaria field survey was conducted in 3 highly endemic malaria districts  in Attapeu province, Lao PDR in 2015, to investigate the precise malaria endemicity in the area; 719 volunteers from these villages participated in the survey. Microscopy, RDTs and a real-time nested PCR were used to detect Plasmodium carriers in the study areas of Attapeu province in 2015. Adult males, probably soldiers, were at high risk for malaria infection. P. vivax, the dominant species, accounted for 87.2% of the Plasmodium infections among the participants. To achieve malaria elimination in the Lao PDR, highly sensitive diagnostic tests, including PCR-based diagnostic methods should be used, and plans targeting high-risk populations and elimination of P. vivax should be designed and implemented.There were many asymptomatic  Plasmodium vivax . Males and soldiers tended to be infected with malaria parasites whereas those with a history of multiple malaria episodes (≥3 times) tended to be asymptomatic malaria carriers. These findings suggest the need to implement targeted elimination plans for vivax malaria that include measures to protect males and soldiers, who are at high risk of malaria infection.The Lao People’s Democratic Republic (Laos) is a country in the Greater Mekong Subregion. In Laos, the numbers of reported cases of malaria and deaths due to malaria have been gradually decreasing. Recently, the Lao government adopted a goal of eliminating malaria by 2030. To achieve this goal, we must understand the precise situation in each endemic area. With this background, we conducted a field survey in Attapeu, one highly endemic province, in 2015. We collected blood samples from 719 villagers, and most  had no fever (<37.5°C) at the time of survey. The samples were examined using three diagnostic methods: microscopy, rapid diagnostic test (RDT) and polymerase chain reaction (PCR). PCR revealed parasitemia in 6.5% of the villagers (47/719), and only one showed a fever (≥37.5°C) at the time of survey. The dominant species was  The malaria burden in the Lao People’s Democratic Republic (PDR) has gradually decreased thanks to the efforts of the Lao government and the support of partners such as the World Health Organization (WHO), and the Global Fund to Fight AIDS, Tuberculosis and Malaria . In 2015Plasmodium carriers in the endemic areas.In central, provincial and district hospitals in the Lao PDR, malaria is typically diagnosed by microscopy, whereas rapid diagnostic tests (RDTs) are used as a sub-standard diagnostic method at locations in rural areas, such as health centers and selected villages with high malaria endemicity (API ≥10). Recently, highly sensitive methods, such as polymerase chain reaction (PCR) , ultra-sPlasmodium falciparum carriers who could be considered to be parasite reservoirs or a cryptic malaria group. In most cases, the parasite densities among the villagers were below the microscopic threshold, i.e., sub-microscopic malaria cases. Interestingly, these parasite carriers were likely to be grouped within a family ) from the dried blood spot on the filter paper were used for DNA extraction, which was equivalent to 30–40 μL of whole blood. The extracted DNA was eluted with 50 μL of elution buffer in the kit and preserved until use at -30°C. To identify malaria parasite infection, a real-time nested PCR was conducted using a primer set that was reported in a previous study  12, 13], 1312, 1P. falciparum and P. vivax were calculated using the results of the real-time nested PCR as a reference.The sensitivity and specificity of microscopy and the malaria RDTs for the diagnosis of Plasmodium infection. A P value less than 0.05 was considered statistically significant. Multivariate logistic regression analyses that adjusted for the effects of other variables were conducted to estimate the association between variables and Plasmodium infection and asymptomatic Plasmodium infection using SPSS version 18.0 . All variables with a P-value of 0.20 from univariate analysis were entered into a multivariate logistic regression analysis. Multicollinearity among all independent variables was tested before logistic regression.For bivariate analyses, the Chi-square test and Fisher’s exact test were used to evaluate an association between variables and Plasmodium infection was defined by the following criteria: Plasmodium DNA was positive by PCR, tympanic temperature was less than 37.5°C (no fever) at the time of the survey and no history of any subjective symptoms and signs at the time of the survey and in the preceding 14 days.Asymptomatic A total of 719 villagers  participated in this survey . The socPlasmodium DNA was detected in 47 (6.5%) participants by the PCR  and P. vivax , and 5 of them were also detected by the RDTs: P. falciparum  and P. vivax  including the individuals who were microscopy-positive. Including all diagnostic tests, 5 of the 47 participants were P. falciparum, 41 of the 47 participants were P. vivax and 1 of the 47 participants was mixed infection with P. falciparum and P. vivax. Using the results of the real-time PCR as a reference, the sensitivity of microscopy for detecting P. falciparum and P. vivax was calculated as 16.7% (95% CI -13.2–46.5) and 4.7% (95% CI -1.6–10.9), respectively  and 7.0% (95% CI -0.6–14.6), respectively. The specificity of microscopy and the RDTs was 100.0% for both P. falciparum and P. vivax. With regard to gender, 41 of the 336 male participants were Plasmodium-positive, whereas only 6 of the 383 female participants were Plasmodium-positive . The positive rate of soldiers (50%) was the highest among the occupations (Plasmodium infections was heterogeneous among the districts: Xaysettha 0.40 (95% CI -0.38–1.18), Phouvong 7.11 (95% CI 3.61–10.60) and Sanamxay 12.11 (95% CI 8.09–16.13)  was significantly higher than that of the female participants (1.6%) (upations . The pre9–16.13) .P. vivax using the real-time nested PCR. The fever started 2 days before the survey. This girl had used an insecticide-treated bet net while sleeping. The average age of all participants was 26.4 years , whereas that of the 47 PCR-positive participants was 29.0 years . Fifteen of the 47 PCR-positive participants answered that they had some symptoms or signs of health problems, such as fever, shivering and nausea, but only one participant had a fever (>37.5°C) at the time of the survey , Soldier , and being Lao Loum (AOR: 2.71 95% CI: 1.12–6.59)    . The ris–132.00) .Plasmodium infections among the participants (6.5%), most  of whom were adult males (≥18 years). The national malaria data, which were summarized by the CMPE in 2015, revealed that P. falciparum, P. vivax and mixed infection accounted for 40%, 58% and 2% of malaria infections, respectively. The national data were based on the results obtained by microscopy  and RDTs  through the passive detection of symptomatic cases. In the present study, the proportions of P. falciparum  and P. vivax  infections detected by the RDTs were consistent with the national data from 2015.In the present study, all 719 participants resided in villages in the districts of Attapeu province that showed high malaria endemicity in the Lao PDR. We found 47 PCR-positive P. vivax infections  among the participants. This finding suggests that malaria infections, particularly P. vivax infections, which can only be detected by microscopy or RDTs, represent the “tip of the iceberg”. Thus, a large portion of the malaria parasite population cannot be detected by the standard (microscopy) or sub-standard (RDTs) methods. To achieve the goal of eliminating malaria in the Lao PDR by 2030, a highly sensitive malaria detection method, such as PCR, should be implemented to detect the large portion of hidden malaria infections to prevent them from acting as reservoirs for the next outbreak of malaria.However, the PCR analysis revealed that there were many Plasmodium infections were, being male, Soldier, and Lao Loum (Plasmodium infections were asymptomatic or cryptic Plasmodium carriers (75% (3/4) of the P. falciparum infections, 65.9% (27/41) of the P. vivax infections, 100.0% (2/2) of the mixed infections with P. falciparum and P. vivax) in Attapeu province. The risk factor of cryptic Plasmodium infections was a history of 3 or more malaria episodes  or bias of the sample collection in this study (not a random sampling but a convenience sampling). Therefore, further investigations are requisite to understand the risk factor of soldier and male in Lao PDR. The prevalence of Plasmodium infection in Som Poi village was the highest among the 6 villages. Som Poi village is located near the border between the Lao PDR and Cambodia  was significantly associated with the cryptic Plasmodium infections  in the present study were farmers. Both male farmers (205) and female farmers (267) worked in the fields, but only the males worked and sometimes stayed overnight in the forests with/without bed nets for the purpose of collecting food and logging. Another high-risk population was soldiers, who also work and stay overnight in the forest while training or on patrols, especially along the border of the country. In this sense, soldiers can be categorized as mobile and migrant populations that are considered to be high-risk populations for malaria [P. vivax, and 4 of the 5 soldiers had no symptoms at the time of the survey or in the preceding few days before the survey. Staying in the forest in the Greater Mekong Subregion is also considered to be associated with a high risk of malaria infection [In the present study, 12.2% (41/336) of the males and 1.6% (6/383) of the females were infected with  malaria . In factnfection –21. The nfection –24.P. vivax and P. falciparum is not understood [P. vivax infections in low-transmission settings, such as Cambodia, the Thailand-Myanmar border, Vietnam [According to previous studies in low-transmission settings, the protective natural immunity to infection by derstood . However Vietnam , the Chi Vietnam , Indones Vietnam , Colombi Vietnam , Solomon Vietnam  and Ethi Vietnam . Our finP. vivax infections involves the treatment regimen that is administered for vivax malaria in the Lao PDR. The current guidelines recommend Coartem (artemether + lumefantrine) as the first-line drug for the treatment of vivax malaria, whereas previously, chloroquine had been recommended [P. vivax from the bloodstream of a patient. However, neither Coartem nor chloroquine can kill hypnozoites (a dormant stage of P. vivax) in human liver cells, which cause relapse. Presently, 8-aminoquinoline (or primaquine) is the only drug that shows efficacy against hypnozoites in the liver cells. A complete radical cure of vivax infection requires a 14-day course of primaquine [Another possible reason for the many ommended . These aimaquine . Howeverimaquine –35. If tAlthough the prevalence of G6PD deficiency among populations in several countries—including the Greater Mekong Subregion—has been reported, the prevalence of G6PD deficiency among the Lao population is unknown . RecentlP. vivax infection among the participants might have been influenced by this study period. According to a report from the CMPE in 2009–2015, the peak malaria incidence occurred between July to October in the Lao PDR. Generally, the prevalence of P. falciparum would be lower at the beginning of the rainy season because it is directly associated with the population density of Anopheles mosquitoes. If our field survey were conducted during July to October (the middle to the end of the rainy season), a greater number of P. falciparum-positive samples would have been detected in the same areas. In fact, in a previous study in Xepon district, Savannakhet province during August to September 2013, 78.8% (41/52) of Plasmodium infections comprised P. falciparum mono-infection [Plasmodium infections were P. falciparum mono-infection and 56.6% (99/175) were P. vivax mono-infection [The present study was conducted in May, which is the beginning of the rainy season in the Lao PDR. The predominance of Plasmodium infections can transmit malaria [Plasmodium carriers can contribute to malaria transmission [The current malaria elimination program in the Lao PDR only targets symptomatic cases. However, it has been shown that mosquitoes that feed on blood from people with sub-microscopic  malaria , 38. Thusmission , 40. ThiFever is one of the major symptoms of malaria. However, in the present study, only one of the 47 PCR-positive participants had a fever (>37.5°C) at the time of the survey. A similar result was reported from Africa. In low and unstable malaria transmission areas in Kenya, fever was a sensitive indicator of malaria only in children (<5 years) but not in older children and adults . Thus, fPlasmodium infections. However, the villagers’ answers regarding the question about bed net use might be biased because some of our interviewers were the ones who distributed the bed nets to the villagers and asked them to use the nets when they sleep. Thus, some (or many) villagers were likely to answer “yes” to the interviewers even though they did not use the bed net at all or did not always use it.In the present study, there was no association between the use of insecticide-treated bed nets and Plasmodium infections [Plasmodium infection might be preventable in the Lao PDR [One limitation of the present study was that we did not collect a history of sleeping away from home  or in the forest or of working in the forest. Previous studies in the Greater Mekong Subregion including the Lao PDR showed that a history of sleeping in a farming hut or in the forest was associated with fections , 42, 43.fections –21. Neve Lao PDR .Plasmodium carriers because in such villages, it is difficult for the villagers to access health care services even they have health problems. Thus, if there is a malaria patient or asymptomatic Plasmodium carrier and Anopheles mosquitos in one of these villages, the transmission would continue within the village, especially in the patient’s household and among the neighbors.Another limitation of the present study was the exclusion of hard-to-access villages from the study area. Therefore, the villages selected in this study might not represent a complete picture of the districts. If a malaria survey is conducted in such hard-to-access villages in Attapeu province, there might be many malaria patients and asymptomatic Plasmodium infections (6.5%) among the 719 voluntary participants from malaria-endemic villages in Attapeu province in May 2015. Most of them  were adult males (≥18 years old), and the dominant parasite species was P. vivax. Males and soldiers were associated with Plasmodium infections whereas a history of 3 or more malaria episodes was associated with cryptic Plasmodium infections. Of the 32 asymptomatic Plasmodium carriers, 30 (93.8%) had infections that were below the detection threshold of microscopy, and 28 (87.5%) had infections that were below the detection threshold of the RDTs. To achieve the goal of malaria elimination in the Lao PDR by 2030, sensitive diagnostic methods—such as a PCR- or LAMP-based method—should be utilized, and plans targeting high-risk populations  and the elimination especially of P. vivax should be designed and implemented.The present PCR examination revealed 47 asymptomatic S1 Checklist(DOC)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Plasmodium falciparum-infected patients. We characterized associations among the helminth parasite infra-community, and then tested for independent (direct) and co-infection dependent (indirect) effects of helminths on cerebral malaria risk. We found that infection by Ascaris lumbricoides and Trichuris trichiura were both associated with direct reductions in cerebral malaria risk. However, the benefit of T. trichiura infection was halved in the presence of hookworm, revealing a strong indirect effect. Our study suggests that the outcome of interactions between two parasite species can be significantly modified by a third, emphasizing the critical role that parasite community interactions play in shaping infection outcomes.Multi-species interactions can often have non-intuitive consequences. However, the study of parasite interactions has rarely gone beyond the effects of pairwise combinations of species, and the outcomes of multi-parasite interactions are poorly understood. We investigated the effects of co-infection by four gastrointestinal helminth species on the development of cerebral malaria among  Ascaris lumbricoides and Trichuris trichiura were both associated with direct reductions in cerebral malaria risk. However, the benefit of T. trichiura infection was halved in the presence of hookworm, revealing that interactions among helminth species can jeopardize the beneficial role of individual helminths. Our study shows that the outcome of interactions between two parasite species can be significantly modified by a third, emphasizing the critical role that parasite community interactions play in shaping infection outcomes and their impact on public health. These insights are especially relevant for neglected tropical diseases that occur in areas of high pathogen diversity.Over the last decade, an increasing number of studies have shown that co-infected individuals can show strikingly different disease symptoms than singly infected individuals. Among these studies, the potential protective effect of some helminth species on malaria severity has attracted considerable attention because of the possible public health impact. However, despite the fact that helminth species frequently co-occur together within an individual, no studies have investigated the role of within-host helminth community interactions on malaria severity. Taking the advantage of a cohort study in Thailand, we found that infection by  Most hosts are co-infected by a range of pathogenic organisms , and inThe majority of ecological studies on co-infection have focused on understanding the ecological, epidemiological, and evolutionary consequences of pairwise parasite interactions e.g., –12). How. How12])Plasmodium falciparum) are among the most well-studied pairwise parasite interactions [Plasmodium spp., resulting in increased malaria incidence and severity, while co-infection with other helminth species is associated with protective effects against cerebral malaria, the most lethal form of malaria  in experimentally co-infected lab mice [A. lumbricoides and hookworm, two soil-transmitted intestinal helminths known to interact with Plasmodium, may themselves interact [Interactions between helminths and human malaria . How another ,26. For lab mice . In humainteract . Thus, iPlasmodium system, we tested the hypothesis that interactions between helminth species can modify the impact of single-species helminth infections on malaria severity. Taking advantage of a dataset on 283 Plasmodium-infected patients from western Thailand, who were screened for helminth infections and monitored for the development of cerebral malaria [Given the possible web of complex interactions that can occur in the helminth- malaria ,30, we c malaria ), to chaP. falciparum biomass, defined as parasitemia >5% or >200,000/μl, or presence of schizonts in a peripheral bloodsmear) were screened for the development of cerebral malaria  and the presence of four helminth species: Ascaris lumbricoides , Trichuris trichuira , hookworm , and Strongyloides stercoralis  in fecal samples. Experienced microscopists were responsible for screening for both Plasmodium (using thick and thin blood smears stained with 10% Giemsa and read at 1000x magnification) and gastrointestinal nematode species via egg morphology . Sex and age class  were also recorded. Out of the 283 hyperparasitemic cases, 67 developed cerebral malaria, while the remaining 216 did not. The demographic composition of the study population is provided in Passive surveillance data on malaria patients were collected from the Hospital for Tropical Diseases, Bangkok, Thailand, and are described in detail elsewhere ,30. The Given that co-infection by multiple helminth species commonly occurs, univariate analysis of each species’ impacts on the development of cerebral malaria may be confounded by the patient’s co-infection status. Furthermore, facilitative or competitive interactions among helminth species would mean that occurrences of interacting parasites in any given patient are not independent of one-another. To address this issue, we first identified co-infection frequencies and quantified associations between helminth species using association screening (SCN) analysis, a method recently developed to identify complex association patterns in large parasite assemblages , combineNP combinations, where NP = the number of parasite species) under the null hypothesis of random parasite associations. Observed co-infection combinations falling above or below this envelope are considered to occur more or less frequently, respectively, than in 95% of the random simulations. Significance of the association is given as a p-value, calculated as the number of instances in which the simulated co-infection frequency differed  from the observed frequency divided by the total number of simulations, and is thus dictated by the variance in the simulated frequencies. Power tests on this method revealed that there should be at least as many host individuals in the dataset as the number of possible parasite species combinations (2NP), and that stronger associations had lower p-values [To identify potential interactions occurring between all possible combinations of the four helminth species in our dataset, we used SCN analysis  to quantp-values .p-value was below 0.05 in at least 95% of the runs. A robustness score (RR) was given to each species combination, ranging from 0 to 10, representing how much data sub-setting an association could tolerate before it was no longer detectable. A score of RR = 1 indicates that 100% of the data are needed, RR = 10 indicates that the association was detected in all 10 partitions. Stronger associations will have higher RR scores; thus, RR scores provide a quantitative measure of the relative strength of significant associations which often have similar p-values.We tested the robustness of each association to sampling biases by rarefaction analysis, where the SCN analysis was performed on progressive sub-sets of the data: 10 partitions sampling from 10% to 100% of the data .Direct effects of infection by each helminth species on the development of cerebral malaria, and indirect effects of interactions between helminth species, were tested using logistic regression with backwards stepwise removal of higher-order interaction terms. Analyses were implemented using the ‘glm’ function from the age in R  with a bOut of the 283 hyperparasitemic malaria patients screened, 139 (49.1%) were infected with at least one helminth species, including 20 (29.8%) of 67 that developed cerebral malaria. The most prevalent helminth infection was HW , followed by TT (24.7%), AL (19.1%), and SS (10.6%). Half (49.6%) of all patients infected with helminths were infected with more than one helminth species, including 4 individuals who were co-infected with all four species . The disp<0.00001, RR = 7), indicating a strong positive 3-way association between these species , while AL, TT, and HW were all found to occur alone significantly less often than expected by chance , it is possible this result simply indicates that the mechanism responsible for the 3-way association between AL, TT, and HW operates whether or not SS is present. Moreover, the low observed frequency of 4-way co-infections (n = 4) accounts for the low robustness score, further indicating a lack of strong support for this result. Restricting the analysis to males, females, or the most co-infected age group  and interaction (indirect) effects of helminths on cerebral malaria. We evaluated the main effects of all four helminth species, as well as all 2- and 3-way interactions for AL, TT, and HW. We excluded SS from interaction terms due to its low overall prevalence and low occurrence in helminth co-infections  and Trichuris trichiura (TT) were both independently associated with significant reductions in cerebral malaria risk, with TT conferring an estimated 90% protection from severe malaria. However, in addition to these direct effects we found that the interaction between two species, TT and hookworm (HW), also had consequences for the development of cerebral malaria. Specifically, the presence of HW nearly halved the protective effect of TT, revealing an intriguing indirect effect of HW on cerebral malaria potentially manifest through its interaction with TT. While the protective effects of single helminth species on cerebral malaria have been previously described [We found that infection by escribed ,24,30,33Ascaris lumbricoides on cerebral malaria is in agreement with previous reports for this helminth species [T. trichiura has not been previously reported. The protection conferred by some helminths on severe malaria disease has been widely attributed to helminth-mediated reductions in immunopathology [A. lumbricoides and T. trichiura confer protection from cerebral malaria are still not fully understood, the effect likely relates to a shift towards regulatory (Treg) and Th2-type lymphocytes which dampen the immune hyper-activation that underlies cerebral malaria [Schistosoma mansoni in baboons was shown to protect against severe malaria outcomes following inoculation with Plasmodium knowlesi, and immunological assays revealed that helminth-infected individuals showed fewer signs of overactive inflammatory responses [Our observation of a direct protective effect of  species ,30. Howeathology ,35. Alth malaria . For exaesponses .Trichuris trichiura on cerebral malaria was nearly halved in the presence of hookworm. Unlike many other helminth species, the human hookworm, Necator americanus, does not stimulate Th2-type immunity, rather it down-regulates the Th2 response [N. americanus was likely the most common hookworm in our dataset, it is possible that concomitant hookworm infection dampened the immune-regulatory response triggered by T. trichiura thereby reversing some of the protection conferred by this species. Although the mechanistic details by which Trichuris and hookworm interact to affect cerebral malaria are largely speculative at this point, our result indicates that the co-occurrence of multiple helminth species can have cryptic and profound implications for disease severity.Intriguingly, the protective effect of response . Since NThe direct and indirect associations we found between helminth infections and malaria severity may have been influenced by a number of other risk factors that our data could not address. For example, variance among patients in malaria exposure history, socio-economic status, education level, or environmental factors that influence risk for both helminth infection and development of cerebral malaria could all conceivably change the interpretation of our results. Further research will be needed to understand how such factors, particularly immunological heterogeneity due to previous malaria exposure, might affect the patterns we describe here. Additionally, further studies in populations from different geographic regions could help elucidate how generalizable these associations are across diverse human and pathogen populations.A. lumbricoides, T. trichiura, and hookworm, in which co-infection with all three species occurred more frequently than expected by random chance. This observation is in line with results from many other human populations [Trichuris trichura and hookworm, can strongly shape the outcome of malaria infection. Given the commonness of multi-species helminth infections, these types of interactive effects of co-infecting helminths on microparasite infections may be more important than is currently recognized.Co-infection with multiple helminth species is the norm rather than the exception in most hosts . In our ulations –43. Suchulations ,45. IrreEscherichia coli strains in sterile culture were significantly altered when the experiments were conducted in the presence of natural human gut microbes. Taken together, these studies highlight the importance of moving from a pairwise to a multi-species perspective for understanding co-infection outcomes. Despite growing acceptance of the need for this host-as-habitat approach [Indirect, or “context-dependent”, effects of environment, resources, or host traits have been repeatedly shown to impact parasite interactions ,47, inclapproach , fueled approach  and [53,approach ) continuAscaris) and those with strong effects on blood loss (e.g. hookworm) [The hypothetical framework used to explain interactions between gastrointestinal helminths and malaria has shifted over time from a nutritional one , to one ookworm) . Howeverookworm) , the preThe datasets supporting this article have been uploaded as part of the supplementary material.S1 Table(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.


Central arterial stiffness has been shown to play a key role in cardiovascular disease. However, evidence regarding such arterial stiffness from various arterial segments in relation to B-type natriuretic peptide (BNP) remains elusive.A total of 1255 participants  with preserved left ventricular function (ejection fraction ≥50%) and ≥1 risk factors were consecutively studied. Arterial pulse wave velocity (PWV) by automatic device  for heart-femoral (hf-PWV), brachial-ankle (ba-PWV), and heart-carotid (hc-PWV) segments were obtained and related to BNP concentrations .Subjects in the highest hf-PWV quartile were older and had worse renal function and higher blood pressure . Elevated PWV (m/s) was correlated with elevated BNP (pg/ml) . After accounting for clinical co-variates and left ventricle mass index (LVMI), both hf-PWV and ba-PWV were correlated with higher BNP . Adding both hf-PWV and ba-PWV to LVMI significantly expanded ROC in predicting abnormal BNP>100 pg/ml (both P < 0.01), but only hf-PWV presented significant integrated discrimination improvement to predict risk for BNP concentrations .A significant segmental PWV associated with biomarker BNP concentrations suggests that arterial stiffness is associated with myocardial damage. Heart failure continues to be a major and growing public health issue and remains the leading cause of hospitalization; it is associated with an approximately 45% post-discharge mortality and readmission rate within 3 months.,2 DespitPulse pressure (PP) has emerged as a predictor for heart failure in the elderly population. ,11 GreatCardiac dysfunction can be caused by several factors including arterial stiffness, systemic inflammation, accumulation of adiposity, and vascular-ventricular overload, and uncoupling. –20 Tarti2. Coronary artery disease was defined by known myocardial infarction history or significant coronary stenosis requiring percutaneous intervention, including stent deployment. Patients with a known history of cardiovascular surgery or the presence of rheumatic heart disease, atrial fibrillation, previous implantation of a pacemaker, and overt renal insufficiency (creatinine > 3.0 mg/dl) or with dialysis history were all excluded from the study.All participants in the tertiary center study were recruited from cardiovascular outpatient clinics at Mackay Memorial Hospital from August 2009 to July 2011. A cross-sectional study was conducted in patients with preserved left ventricular systolic function (LVEF ≥50%) and at least 1 risk factor  from the cardiovascular outpatient department. This study was approved by local ethical institutional committee  for retrospective data analysis without informed consent of study participants. Data security was guaranteed, and all authors had no access to patient identifying information before and after data analysis. Study participants involved in this study were not under clinical service of current study physicians or researchers. In brief, 1500 consecutive patients were enrolled who presented at outpatient clinics with intermediate risk and preserved heart systolic function. A total of 11 patients did not have blood test data, 66 patients did not perform the complete pulse wave velocity exam, 12 patients did not receive clear echocardiography images, and 154 patients did not consent to the BNP blood test. One patient with extremely high BNP concentration (5000 pg/ml) and another with extremely low pulse wave velocity (hf-PWV = 1.19 m/s) were excluded. A total of 1255 patients with complete arterial stiffness exams and BNP data were enrolled in the study. Of the 1255 participants with complete arterial pulse wave velocity exams, 47.8% patients were male with a mean age of 62.6 ± 12.3 (±SD) years. Demographic data and medical history were collected by three independent cardiologists during face-to-face interviews. Anthropometric parameters including height, body weight, and waist circumference were measured by an experienced study nurse. The risk factors were age , high-density lipoprotein cholesterol , hypertension, hypercholesterolemia, diabetes mellitus (DM), chronic renal insufficiency (creatinine>1.3 mg/dl), smoking history, and obesity. Hypertension was defined by usage of anti-hypertensive agents or systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg. Hypercholesterolemia was defined by usage of lipid-lowering medicine (statin or ezetamide) or total cholesterol ≥200 mg/dl or low-density lipoprotein cholesterol ≥130 mg/dl. Diabetes was defined by known diabetes history, fasting blood glucose >126 mg/dl or any current usage of DM medication. Smoking history was defined as being an ex-smoker or with current tobacco usage. Patients were considered obese with a body mass index (BMI) > 30 kg/m2) was defined as the ratio of LV mass to body surface area (BSA). Gender-specific LV hypertrophy (LVH) was defined by LVMI >95gm/m2 for women and > 115gm/m2 for men. [Transthoracic echocardiography in two dimensions and Doppler mode examinations were performed on 1371 participants using a commercially available ultrasound system  equipped with a 2–4 MHz transducer. All reported echocardiography measurements are the average value of three successive heart cycles. We assessed parameters of left atrial (LA) diameter, interventricular septal thickness, posterior ventricular wall thickness, left ventricular diameter of both end-diastolic and end-systolic phase, and subsequent LV mass (g) determined from linear M-mode method according to the recommended method established by the American Society of Echocardiography.  LV mass for men. Arterial stiffness was measured using pulse wave velocity. After resting for 5 min in a supine position, bilateral ba-PWV, systolic blood pressure, and diastolic blood pressure from four limbs were measured simultaneously with an automated machine . Carotid artery and femoral artery pulse waveforms were measured via manual recording by a single experienced technician. The central aortic hf-PWV and carotid hc-PWV were measured with a time delay between the rapid upstroke of the heart-femoral artery and the heart-carotid artery and then calculated by an automatic device and manual measurement . PWV was calculated as the distance between the two arterial sites divided by the time delay between the two arterial point sites and presented as centimeter per seconds. [2) = 175 × PCr  −1.234 × age −0.179 × (0.79 if woman). [Overnight fasting blood serum and plasma samples were collected to determine glucose and lipid profiles . Some biochemical measurements, including renal function, were also obtained. Serum samples were collected using standard sampling tubes or tubes containing separating gel. After ensuring individualized patient samples, calibrators and controls were set at ambient temperature (20°C–25°C), and the measurement was taken within 2 h. Hs-CRP level was determined by a highly sensitive, latex particle-enhanced immunoassay using Elecsys 2010 . Serum BNP concentrations were measured using a fluorescence immunoassay microtiter plate with a coefficient of variation of 10.4% . Renal function was determined by estimating the glomerular filtration rate (eGFR), which was calculated using the Modification of Diet in Renal Disease formula in a Chinese population: eGFR  was used to test for continuous variables among the quartile and chi square or Fisher’s exact test method were used to compare categorical variables. The relationship between BNP and PWV was examined by age- and gender-adjusted Spearman’s partial correlation coefficients. Multiple logistic regression analyses were conducted to measure different PWVs associated with BNP concentrations independent of other clinical parameters. A receiver operating characteristic (ROC) curve was used to evaluate the area under the curves to identify abnormally high BNP (BNP>100 pg/ml) with c-statistics used for comparison between different models. For reclassification analysis, cross-classification was measured separately in participants with higher BNP concentrations (>100 pg/ml) and its lower parts (≤100 pg/ml). Integrated discrimination improvement is equal to the difference in discrimination slopes between the old model and a new PWV predicting model. The reproducibility of PWV measurement was presented using the Bland–Altman plot. All statistical tests were tow-tailed, and P < 0.05 was considered statistically significant. Analyses were performed with Stata version 11  and SAS 9.2 version .X2 = 0.002).All 1409  participants were stratified by hf-PWV level. Participants in the highest quartile were older, had worse renal function, and had higher blood pressure; their serum BNP levels were comparable with those patients in the lowest quartile , in predicting high-risk heart failure  may provide additional information beyond degree of ventricular remodeling. Both high degree of LV remodeling in terms of large LVMI and large LA diameter were linked to elevated BNP, thereby reflecting the LV remodeling process and diastolic dysfunction. Moreover, aortic arterial stiffness (hf-PWV) had an additional risk reclassification effect in identifying the cardiac damage risk model, and it was defined by an abnormally high BNP level (>100 pg/ml). In general, central aortic elastic compliance supplied a cushioning effect during heart diastole and maintained the pulse processing from central to downstream sites. High baby birth weight is positively associated with greater central aortic stiffness, implying that impaired glucose metabolism may play an important role. The carotid artery remodeling proximal to the heart from adaptative remodeling of increased wall tension in greater arteries is tightly linked to load status, such as blood pressure or PP.  The diam1. The coupling of ventricular arterial stiffness with mechanical interaction of systemic and coronary flow balance, regulation of endothelial function, and smooth muscle tone implies the importance of vascular pressure load to heart function. [Increasing rates of preserved EF heart failure within the aging society is an important problem that will become an economic burden in the near futureunction. ,41 In eaunction. ,42 A recunction.  In our sArterial stiffness was the consequence of progressive arterial dilation and degeneration of the arterial wall, and it occurs alongside aging and increased systolic pressure. Furthermore, peripheral brachial systolic pressure impaired systolic pressure in the aorta and left ventricle, thereby inhibiting the vessel dilation effect of anti-hypertensive drugs.  In additIn this study, we determined the central aortic and peripheral muscular arterial stiffness indicators (hf-PWV and ba-PWV). Our current study simultaneously measured central aortic (hf-PWV), heart carotid (hc-PWV), and peripheral muscular (ba-PWV) arterial stiffness. Results demonstrated a relationship between hf-PWV and BNP levels even after adjusting for clinical and epidemiologic factors. Although these results create a large database for the use of arterial stiffness in predicting BNP concentration, the study’s generalizability was limited to outpatient clinic patients, so it may not be applicable to community-based populations. Furthermore, we used indirect methods to evaluate arterial stiffness, and data were restricted to a single center. Finally, this work was a cross-sectional study, and further research to confirm this conclusion in longitudinal outcome-driven follow up may be warranted in the future.Our study showed a significant segmental PWV associated with BNP concentrations, thereby implying that arterial stiffness was related to cardiac damage. Adding on geometric LVMI, hf-PWV, and ba-PWV significantly increased our ability to predict heart damage. Only central aortic hf-PWV significantly improved our ability to predict BNP concentrations, and this parameter was correlated with aortic arterial stiffness and worsening heart function. Further study and evaluations are warranted.S1 Table(DOC)Click here for additional data file.


Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria.In severe  P. falciparum [P. falciparum-infected red blood cells (Pf-iRBCs) to vascular endothelium (cytoadherence) [P. falciparum malaria. The severity of clinical infection is proportional to the degree of microvascular obstruction in vital organs .There were an estimated 198 million cases and 584,000 deaths from malaria in 2013 [herence) , and to herence) –9 are ceP. falciparum [P. falciparum erythrocyte membrane protein-1 (PfEMP-1) [var gene family of P. falciparum (60 var-genes) [in vitro [in vivo [falciparum malaria . In this study, we investigated ex-vivo the effects of sevuparin on rosette formation and on adhesion of Pf-iRBCs to endothelial cells under static and flow conditions. The results provide a basis for assessing sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria patients.Major endothelial cell receptors identified for cytoadherence include CD36 (platelet glycoprotein IV) , TSP (thlciparum –18. ThisPfEMP-1) , a polypr-genes) . PfEMP-1r-genes) , 22. Sper-genes) , 24 by vr-genes)  and A, Br-genes) . Some ror-genes)  and alphr-genes) . Antibodr-genes) , 29–31. r-genes) . The cytr-genes) . Previour-genes) –37. Hower-genes) ,39, howein vitro  and in r[in vivo ,41 are rPlasmodium falciparum malaria and parasite densities >10,000 Pf-iRBCs/μL. The mean  age of patients was 30.23 (9.11) years and the mean (SD) haematocrit was 38.1% (4.7%). The geometric mean (95% CI) parasite density was 29,518 . 50 samples were cultivated successfully. The three isolates which could not be assessed had very low parasitaemia, grew slowly and developed gametocytes. Of 50 samples 47 (97%) developed to trophozoites and rosetting assays were performed and 49 (98%) samples from cryopreserved parasites grew and static binding assays were performed.Blood samples were obtained from 53 patients aged between 18–58 years old with P. falciparum isolates from uncomplicated malaria patients (n = 47) formed rosettes. The median  percentage of Pf-iRBCs forming rosettes was 12% (10.0–13.0). The median (IQR) proportion of Pf-iRBCs forming rosettes in blood group A (n = 21) was 12% (11.0–14.5), blood group B (n = 10) was 10% (8.0–12.5), blood group AB (n = 3) was 12% (8.0–13.0), and blood group O (n = 13) was 11% (9.5–13.0). There were no differences between the proportions of rosettes in the different blood groups (p = 0.205 degrees of freedom (df) = 3) and no correlation was found between numbers of rosettes and age (Spearman's (rs) = 0.072, p = 0.632), haematocrit  and parasite density . Rosetting decreased with increasing sevuparin concentration in vitro (p < 0.001) (All < 0.001) . In 16 o< 0.001) .P. falciparum malaria which bound to human dermal microvascular endothelial cells (HDMECs) (28 of 49) was 57%. The overall median (IQR) number of Pf-iRBCs bound to HDMECs (n = 28) was 8.5 (3.0–38.0) IRBCs/1000 HDMECs, The overall proportion of parasites from uncomplicated p = 0.040), 400 μg/mL (p < 0.001) and 800 μg/mL (p < 0.001), The inhibition of adhesion was calculated from the number of adherent parasites in control minus adhesion number of parasites in sample/adhesion number of parasite in control x100. There was no inhibition effect on the adhesion of parasites to endothelial cells at a sevuparin concentration of 50 μg/mL, but significant reductions were observed at higher concentrations when compared with controls (untreated or no sevuparin); 100 μg/mL (p = 0.028), 200 μg/mL , haematocrit  or parasite density  of patients. There was no correlation between the number of rosettes and the number of Pf-iRBCs binding to HDMECs , The median (IQR) number of Pf-iRBCs binding on HDMECs (/1000 HDMECs) in blood group A (n = 13) was 9.0 (4.0–25.5), blood group B (n = 5) was 41.0 (5.5–51.5), blood group AB (n = 3) was 81.0 (9.0–181.0) and blood group O (n = 13) was 3.0 (1.0–4.0) /1000 HDMECs). There was more adhesion of Pf-iRBCs in blood group AB when compared with the other blood groups . There was no correlation between numbers of IRBCs binding and age , age , haematocrit  or parasite density .Ten parasite isolates were treated with sevuparin at concentration 50, 200 and 800 μg/mL and assessed under shear stress at 0.05 Pa, 0.5% parasitaemia and 1% haematocrit. The median (IQR) number of Pf-iRBCs adhering to HDMECs  was 24.5 (18.0–37.5) Pf-iRBCs/6 minutes. Following sevuparin treatment, as concentrations increased from 50, 200 to 800 μg/mL, the median (IQR) number of parasites adhering to HDMECs progressively decreased 12.4 (9.8–20.5), 14.2 (8.8–18.5) and 6.7 (4.7–10.8) Pf-iRBCs/6 minutes, respectively , the oves = 0.175, p = 0.707), age , hematocrit  or parasite density . Moreover, there was a borderline association between adherent Pf-iRBC adherence and the number of rolling Pf-iRBCs .The median (IQR) number of Pf-iRBCs rolling on HDMECs  was 1 (1.0–2.3) Pf-iRBCs/6 minutes. The median (IQR) rolling were 1 (1.0–3.2), 1 (1.0–1.0), and 0.4 (0.0–4.0) Pf-iRBCs/6 minutes at concentrations 50 (n = 6), 200 (n = 5) and 800 (n = 3) μg/mL (Pf-iRBCs/6 minutes), respectively. The median (IQR) of Pf-iRBCs rolling was not different across the sevuparin concentrations . There was no correlation between number of Pf-iRBCs rolling on HDMECs with blood group of Pf-iRBCs  to adhere to vascular endothelium and other RBCs (rosette formation) is thought to be a major factor accounting for the virulence of this parasite [In vivo studies of parasite development indicate that the prognosis of severe malaria is related to the sequestered malaria parasite biomass [in vivo in rats and monkeys [in vitro [The propensity of parasite . Both ofparasite , 43 and parasite , rosettiparasite , 46. In  biomass . Severel biomass . Sevupar monkeys . Sevuparin vitro .P. falciparum isolates from uncomplicated malaria patients. In the current study, sevuparin concentrations ranged from 62.5–1000 μg/mL in the rosetting experiments and from 50–800 μg/mL in the HDMEC cytoadherence experiments, which were based on in vitro concentration ranges published in the literature [We investigated the effect of sevuparin on rosette formation and cytoadherence of terature ,48,49. Sterature , 38, 48.terature . Howeverterature , 37, 52.From previous studies, Leitgeb and colleague  reportedP. falciparum isolates obtained freshly from malaria patients. In the current in vitro study, 43% of fresh parasite isolates did not cytoadhere to our HDMEC cell culture expressing CD36 and the level of binding was low, varying between 1–181 infected red blood cells/1000 HDMECs. This is comparable with Udomsangpetch et al, 1996 (over 60% of isolates did not adhere to CD36) [P. falciparum VAR genes [CD36 binding is a property of almost all to CD36) . Part ofAR genes . StudiesAR genes , 55. MorAR genes . In ThaiAR genes , 57. A sAR genes . Not allBinding of PfEMP-1 to endothelium is promiscuous and mediated by a range of membrane receptors, including Endothelial Protein-C Receptor (EPCR), thrombospondin, intercellular adhesion molecule-1 (ICAM-1), heparan sulphate (HS) and CD36. There is a wide range of literature on the importance of different receptors in mediating severe disease , 59. MorP. falciparum rosette formation and cytoadherence to HDMECs in a dose dependent manner and therefore might be a candidate for adjunctive therapy in severe falciparum malaria.Pf-iRBCs tethered, rolled, and adhered on human microvascular endothelial cells. Under shear stress conditions, sevuparin significantly inhibited cytoadherence to HDMECs. A previous study reported that Pf-iRBCs interaction was synergistic with multiple adhesion molecules on vascular endothelial cells whereas adhesion of Pf-iRBCs to HDMECs was largely CD36 dependent, while the rolling could be mediated by any of the adhesion molecules . Pf-iRBCP. falciparum infections admitted to Mae Sot and Mae Ramat Hospital, Tak province, Thailand. Written informed consent was obtained from all patients before blood sampling. Ethical approval was obtained from the Ethics Committee of Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand and the Ministry of Public Health of the Royal Government of Thailand, and from the Oxford Tropical Research Ethics Committee (OxTREC) of Oxford University, Oxford, UK as part of a larger clinical study . Human dermal microvascular endothelial cells (HDMECs) were harvested from discarded neonatal human foreskins. The protocol was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University and Queen Sirikit National Institute of Child Health, Bangkok. Thailand.Blood samples were obtained from patients with uncomplicated P. falciparum malaria were considered for enrollment. Uncomplicated malaria defined as febrile disease with a positive peripheral blood malaria slide. Patients with any sign of severe malaria, determined on the basis of World Health Organization criteria [Adult male and female (18–65 years old) presenting with acute uncomplicated criteria  were notin vitro parasite growth. For the rosetting assay, all assays were done on fresh samples and 47 of 50 samples could be evaluated. Out of these, 28 cryopreserved isolates showed cytoadherence in the static assay and were further assessed for sevuparin. Out of these 28, 10 strains with most prominent binding were selected for the flow based endothelial cells adhesion assays.Five millilitres of citrate anticoagulated blood samples were collected prior to the start of antimalarial drug treatment for microscopy malaria parasite identification in thick and thin blood smears and blood group assessment. Samples were then centrifuged at 2,500 rpm at 4°C for 5 minutes and plasma was stored at -80°C. The packed red blood cells were suspended and washed three times with RPMI-1640. Then Pf-iRBCs were cultured and the parasites cryopreserved. All experiments were performed during the first cycle of P. falciparum parasites were grown in tissue culture flasks  in complete malaria culture medium (MCM) containing RPMI-1640 medium  supplemented with 25 mM HEPES , 2 g/L NaHCO3 , 4.5 g/L D-glucose , 0.1 mM hypoxanthine , 40 mg/L gentamicin  and 0.5% Albumax ® . Parasite cultures were incubated at 37°C with 5% CO2. The culture medium was changed daily. Thin blood smears were prepared, fixed with absolute methanol , and stained with Field’s stain . Pf-iRBCs were harvested for the experiment when the parasites had developed to the trophozoite stage (34–36) hours, 80% synchronization. Staging was done by light microscopy of 100 Pf-IRBCs, using published criteria for staging [ staging . Synchro2) segments, and digested at 4°C for 16 hours in supplemented M199 containing 0.5 mg/mL collagenase type 1A . Microvessels were released from digested tissue segments by compressing the tissue segments gently with a spatula. To remove large debris, vessel preparations were passed through a 100 mm nylon mesh . The cells were collected by centrifugation at 2000 rpm for 10 minutes, and the cell pellets were resuspended in endothelial basal medium (EBM-2) with supplements supplied by the manufacturer , and then seeded in gelatin-coated tissue culture dishes or on cover slips. Finally, The HDMECs culture dishes were incubated at 37°C, 5% CO2. The experiments used HDMECs from passage 3 to 6 obtained from 5 different foreskin donors. HDMECs cultures were assessed for the endothelial marker CD31 and the parasite cytoadhesion receptor CD36 by an indirect immunofluorescence assay using FITC-mouse antihuman CD31  and PE-mouse antihuman CD36   were harvested from discarded neonatal human foreskins as described previously . In brie 555455) . 5 diffeSevuparin was provided by Dilaforette AB, Sweden. Sevuparin is a compound derived from heparin. Sevuparin was diluted in RPMI-1640 to 2 mg/mL as stock solution and kept at 4°C until used. Sevuparin was diluted 2-fold for the experiments (range 62.5–1000 μg/mL).P. falciparum (0.5% parasitaemia and 1% haematocrit) were treated with sevuparin (62.5 μg/mL—1000 μg/mL) at 37°C for 30 minutes. The cell suspension alone was used as control . Rosette formation was then examined under light microscopy. The number of rosettes was counted per one hundred infected RBC under high magnification (100X objective lens). A rosette was scored if two or more uninfected red blood cells were bound to a single infected red blood cell. All samples were done in duplicate.Trophozoite stages of P. falciparum (0.5% parasitaemia and 1% haematocrit) were treated with sevuparin  were used. Trophozoite stages of controls , 63, 66.P. falciparum (at 0.5% parasitaemia and 1% haematocrit) were preincubated with sevuparin at 50, 200 and 800 μg/mL of seveuparin for 1 hour, after which the drug was removed by washing 1 time by centrifucation at 2500 rpm, for 5 minutes and resuspension in RPMI-1640 to an haematocrit of 1%, after which the Pf-iRBCs suspension was used immediately.Five to one hundred thousand HDMECs in 1 ml of supplemented medium were plated on 35mm glass coverslips  in 6 well plates. The cells were allowed to adhere for 2 h at 37°C, after which 3 ml of supplemented medium were added to each well. After 40 to 48 h of culture, confluent monolayers were rinsed in HBSS (Life Technologies) and used without delay in the laminar flow studies. In the adhesion assys, trophozoite stage 2. A recording video was obtained of Pf-iRBCs binding over 6 separate fields; 6 minutes/field. The Pf-iRBCs suspension was passed over the endothelial cells coated cover glass slip for 6 minutes. Cell free medium was then flushed through the chamber at the same shear stress for 2 minutes to wash away non-adherent cells. Stationary adherent parasites were counted in at least six fields along the slide (both rolling and static) and the results were expressed as the number of adherent Pf-iRBCs/6 minute [The method was modified from that previously reported , 66, 67.6 minute , 65, 66.U- test was used for non-normally distributed data to compare different levels of drug concentration against the controls. Comparisons across more than two groups were made using the Kruskal-Wallis test. Associations were assessed using the Spearman's rank correlation coefficient.All statistical analyses were performed using SPSS 18 for Windows. The data were tested for distributional assumptions by the Kolmogorov-Smirnov test. The Mann-Whitney S1 FigThe figures show the expression of CD36 and CD31 on HDMECs monolayer. Magnification:200X.(TIF)Click here for additional data file.


Plasmodium infection, exposure of human host immune cells to both the asexual and the sexual stages of the parasite elicit immune responses. These responses may be protective and prevent the development of high parasitaemia and its associated clinical symptoms, or block the transmission of malaria to an uninfected person. This study aimed at examining the dynamics of naturally acquired immune responses against the asexual and sexual forms of Plasmodium falciparum as well as assessing differences in the multiplicity of infection (MOI) in asymptomatic Ghanaian children living in two communities with varying malaria transmission intensities.During a Plasmodium falciparum parasite prevalence was determined by microscopy and PCR. Serum eluted from the DBS were used to assess anti-Pfs230  and anti-MSP3  antibody levels using indirect ELISA and DNA extracted from the DBS used to assess MOI.School children aged between 6 and 12 years were recruited from Obom, a high malaria prevalence setting and Abura, a low malaria prevalence setting and enrolled in monthly multiple cross sectional surveys between February and May 2015. Filter paper blood blots (DBS) as well as thick and thin blood smears were made from finger-pricked blood at each visit. Malaria parasite point prevalence and MOI throughout the study was higher in Obom than Abura. The trend of parasite prevalence estimated by microscopy was similar to that determined by PCR in Obom but not in Abura. The trend of MSP3 antibody seroprevalence followed that of PCR-estimated parasite prevalence in Obom, while in Abura the trend of Pfs230 antibody seroprevalence followed that of PCR-estimated parasite prevalence.P. falciparum parasite prevalence can predict antibody seroprevalence to MSP3, whilst in low transmission settings, seroprevalence against Pfs230 may be a useful predictor of parasite prevalence.Microscopy can more accurately predict changes in parasite prevalence in high transmission settings than low transmission settings. In high transmission settings, The online version of this article (10.1186/s13071-017-2607-5) contains supplementary material, which is available to authorized users. P. falciparum parasites may constitute a challenge for malaria control as such individuals can serve as reservoirs for transmission [Asymptomatic carriage of smission .P. falciparum [Individuals living in malaria endemic regions develop partial immunity against clinical malaria after repeated exposure to lciparum , 3. Thislciparum . Althouglciparum . Howeverlciparum  and in tlciparum . WhereasSome studies have shown that individuals living in areas of seasonal malaria transmission naturally acquire antibodies against multiple stages of the parasite , 9. AntiP. falciaprum, known as gametocytes, are the transmissible forms of the parasite. It is currently known that the sexual stage parasites are formed during every asexual blood stage cycle [The sexual stages of ge cycle  and presge cycle –21. Althge cycle –24, the ge cycle , 26.The dynamics of the naturally acquired antibodies against a merozoite surface antigen (MSP3) and the sexual stage antigen (Pfs230), has not been well studied in asymptomatic individuals living in different transmission settings in Ghana. This study aimed to assess the acquisition of antibodies against MSP3 and Pfs230 in two asymptomatic populations living in the southern zone of Ghana in the transition period from the dry to the major rainy season.P. falciparum. Obom is within the Ga South Municipality of the Greater Accra Region of Ghana and Abura is in the Abura municipality of the Central Region of Ghana . During each visit, 100 μl of finger-pricked blood was collected and used to prepare thick and thin blood smears on microscope slides as well as spot on Whatman filter paper . The filter paper blood blot (DBS) was air-dried and stored desiccated at room temperature for less than a month.P. falciparum infected red blood cells (iRBCs) observed after counting 200 white blood cells (WBCs) was recorded. The number of iRBCs was converted into parasite density/μl blood by multiplying the value by 40 (based on the assumption that there are 8000 WBCs/μl of blood) [Each thick blood smear was air-dried and stained in 10% Giemsa for 15 min and then air-dried again . The smef blood) . Two indDNA extraction was carried out by the Saponin-Chelex extraction method as previously described . Brieflymsp2 and glurp genotyping reactions were performed using a protocol adapted from [msp1 and 2 genes using a nested PCR as previously described [2, 133 nM of each forward and reverse primer, and 0.5 units of One Taq DNA polymerase  in addition to 4 μl of genomic DNA (gDNA) template. The secondary reaction mixture contained 200 nM dNTP, 1.8 mM MgCl2, 200 nM of each forward (S1fw) and either of the allele specific reverse primers (M5rev and N5rev) and 0.5 unit of One Taq DNA polymerase supplemented with 0.5 μl of the primary PCR product. A semi-nested PCR reaction was used to amplify the glurp gene using a reaction mixture containing 200 nM dNTP, 2 mM MgCl2, 200 nM of each forward and reverse primer and 1 unit of One Taq DNA polymerase. The primary and semi-nested reaction mixtures for glurp differed only in the amount of template used: 4 μl of gDNA for the primary and 1 μl of primary PCR product for the semi nested reaction. Each PCR reaction contained positive controls: MRA-102G (3D7) for msp2 3D7 alleles, MRA-159G (KI) for msp1 KI and msp2 FC27 alleles, MRA-155G (HB3) for msp1 MAD20/msp2 FC27 alleles and MRA-200G (R033) for msp1 RO33/msp2 3D7 alleles and a no template negative control. All primers used and their annealing temperatures are listed in Additional file msp2 allele specific PCR products from some samples collected from Obom.The ted from , 32 to described . BrieflyC0Ll in carbonate buffer, pH 9.0 [IgG antibodies against the antigens were quantified by indirect ELISA as previously described  with sli, pH 9.0  and MSP3, pH 9.0  were use, pH 9.0 . A calib, pH 9.0 . Plates P. falciparum (uninfected). ADAMSEL  was used to convert OD values into concentration. Individuals with IgG concentrations higher than mean value for the negative  control plus two standard deviations were rated seropositive. A smear was considered positive when at least one P. falciparum parasite was observed after counting at least 200 white blood cells.Data were entered into Excel and analysed using GraphPad Prism v.5. Data were tested for normality with the Shapiro-Wilk normality test. Mann-Whitney U-test and Kruskal-Wallis test were used to compare the differences between groups. Statistical significance was set at 0.05 unless otherwise stated. Samples that produced more than a single product after the nested or semi-nested PCR reaction were considered to harbor multiclonal infections. When no PCR product was obtained after a repeat gDNA extraction and subsequent PCR amplification, the sample was considered negative for The proportion of males to females during the entire sampling period was essentially the same for the two study sites ranging between 41.7–46.7% in Obom and between 42.4–46.3% in Abura  but decreased significantly in May  = 34.5, P = 0.001). Statistical comparisons of PD in Abura could not be done due to the low prevalence of microscopic parasites  = 102.0, P = 0.0019). Seroprevalence of antibodies to MSP3  = 1399, P = 0.0002 and U = 830.0, P < 0.0001 respectively) than those in children from Abura during the same months . In Obom, the geometric mean of IgG concentrations showed significant  sequential monthly variations with IgG concentrations in February and April being high whereas those of March and May were low. IgG concentration of antibodies against Pfs230 in children from Obom were significantly higher than those in children from Abura during the first three months, February through April  = 1291, P < 0.0001; U = 232.0, P < 0.0001; U = 625.0, P < 0.0001, respectively), however during the last month (May) antibody concentrations were significantly higher in Abura than Obom  = 1309, P = 0.031)  = 783, P < 0.0001)  Fig. . Antibod01) Fig. . The conIn areas of seasonal malaria transmission, the highest prevalence of malaria parasites occurs at the peak of the season which follows soon after the peak of the rainy season , 37. ThiP. falciparum among the enrolled children revealed that the parasites were present as asymptomatic infections in these children before the peak transmission season Additional file 2:Figure S1. Representative agarose gel images of msp2 amplified products. Identification of the FC27 msp2 allelic family members using the S1fw and M5 primer set (a) and the 3D7 msp2 allelic family members using S1fw and N5 primer set (b). Lane N: no template (water) negative control; Lane C1: 3D7 positive control sample; Lanes C2: FC27 positive control sample: Lanes 1–14: samples collected in April from children living in Obom. The samples were scored as clonal or multiclonal for msp2 depending on the number of PCR products per sample. (DOCX 499 kb)