Despite high prevalence of visceral leishmaniasis and malaria in the study area, their coinfection remains unknown. Therefore, this study was aimed to document VL-malaria coinfections and their associated factors. A cross-sectional study was conducted among clinical suspected VL patients attending Metema hospital, Northwest Ethiopia, from January 2014 to June 2014. Blood sample was tested by rk39 antigen-based DiaMed IT-Leish dipstick and Giemsa stain microscopic examination of thick and thin blood smears for malaria detection was performed. P. falciparum and 5 (11%) positive for P. vivax. The overall prevalence of VL-malaria coinfection was 16 (4.2%). One-hundred eighty (46.9%) study participants have history of travel. Of these, 10 (5.6%) have VL-malaria coinfections. Age less than 5 years was associated with VL-malaria coinfection. A total of 384 VL suspected patients were included in the study. Out of these, the prevalence of VL was 83 (21.6%) while the prevalence of malaria was 45 (11.7%). Of malaria cases, 40 (89%) were positive for This study highlights the importance of performing malaria screening amongst VL patients living in malaria-endemic areas, particularly in patients under five years. Plasmodium falciparum, and 200–300 children are dying every hour from this disease [Leishmaniasis and malaria are among the most important six diseases on the World Health Organization (WHO) or Tropical Disease Research list. There are 2 million new cases of Leishmaniasis diagnosed every year . As for  disease . Leishmania donovani complex [ Anopheles mosquito borne disease caused by an intracellular protozoan parasite of Plasmodium species [Visceral leishmaniasis (VL) is the phlebotomine sand flies borne disease caused by an intracellular protozoan parasite of the complex , while m species . Usually species .Visceral leishmaniasis and malaria are the two major life-threatening parasitic diseases that still remain a serious public health problem, particularly in endemic areas , 9. DiffVisceral leishmaniasis and malaria coinfections have been reported across various African and Asian countries, with the prevalence among VL patients ranging from 20.8% and 6.4% in Uganda , 13 to 1This study was conducted at Metema hospital from January 2014 to June 2014. Metema is a town in Northwestern Ethiopia, on the border with Sudan. This town is located in the North Gondar Administrative Zone, Amhara region, 897 km North of Addis Ababa and 197 km from the ancient city of Gondar, and it has a latitude and longitude of 12°58′N 36°12′E with an elevation of 685 meters above sea level. This area is malarious and has cases of VL.A cross-sectional study was carried out at Metema hospital to determine VL-malaria coinfection among all clinically suspected VL patients. Visceral leishmaniasis suspected patients who have taken treatment for malaria and/or VL for the last two weeks were excluded from the study. Visceral leishmaniasis suspects are defined as those individuals who develop clinical evidence of infection usually have fever, loss of appetite (anorexia), weight loss, fatigue, anemia, enlargement (swelling) of the spleen and liver, and abnormal blood tests .Well-structured questionnaire was prepared in English version by the research team. It was translated later into the local language, Amharic. The questionnaire addresses patients' sociodemographic information and associated factors. A pretest was conducted among five percent of the total sample size by trained data collectors and any ambiguous questions and repetitive ideas were corrected. Additional response categories were added based on the pretest findings.After interviewing the patients, fresh peripheral whole blood sample was tested by the rk39 ICT with sensitivity of 100% and specificity of 98%  for diagP value < 0.05 was considered statistically significant.Data were entered and analyzed using SPSS version 20 software. The Pearson Chi-square test was employed to examine associated factors with the coinfection. Ethical clearance was obtained from Institutional Review Board of University of Gondar. Written informed consent was obtained from each of the volunteer study subjects or guardian of children. Patient information was anonymized and deidentified prior to analysis. Positive results were given for physicians working in the hospital for treatment according to the national treatment guideline.A total of 384 VL suspected participants were included in this study. Out of these, 334 (87%) were males. The mean (standard deviation) age was 28.1 (11.8) years (ranging from 2 to 78 years). Majority 227 (59.1%) of the study participants were in the age group of 15–29 years old. Among the study groups, 230 (59.9%) were illiterate and 267 (69.5%) were farmers followed by 54 (14.1%) daily laborers. The majority of study participants 234 (60.9%) were from rural residents .P = 0.004). Visceral leishmaniasis-malaria coinfection by gender showed that 15 (4.5%) of males were coinfected by VL-malaria. Individuals who live in the urban 8 (5.3%) and rural 8 (3.4%) were coinfected with VL-malaria. 5 daily laborers (9.3%) were coinfected with VL-malaria  VL-malaria coinfections were found in the age groups of <5 years followed by 9 (4%) within 15–29 years old  were positive for P. vivax . The prevalence of VL was 83 (21.6%) while the prevalence of malaria was 45 (11.7%) . Of the P. vivax .From the total study participants, 292 (76%) were used bed nets. Of these, 13 (4.5%) patients were VL-malaria coinfected. Most of the study participants, 217 (56.5%) sleep inside home. One hundred eighty (46.9%) study participants have history of travel from Metema to any region of Ethiopia and vice versa. Of these, 10 (5.6%) of them have VL-malaria coinfection. All study variables did not show statistical significance .Visceral leishmaniasis and malaria coinfection may exist in endemic areas due to similarity in clinical manifestations of the two diseases but their coinfection has been poorly investigated. In the present study, the overall prevalence of VL-malaria coinfection was 4.2%. This is in agreement with the study which is conducted in India 5.9% among Indian patients with fever and splenomegaly . HoweverIn this study, prevalence of VL-malaria coinfection was nonsignificantly higher in males than females. A similar finding was also reported in other studies –27. The Visceral leishmaniasis has been a major health problem in North Gondar, especially in lowlands of Metema and Humera plains, which are important endemic foci in Northwest of Ethiopia . In the  Plasmodium species detected was P. falciparum. This was in agreement with other previous studies [Malaria is also one of the most public health problems in Ethiopia. The total land mass of the country is regarded as malarious and about 68% of the total population is at risk of malaria infection . In this studies , 32, 33.P = 0.004) followed by patients in the age groups of fifteen to twenty-nine years. This indicates that children less than five years old are prone to acquire VL-malaria coinfections which might be associated with their low immune system. On the other hand, high prevalence of VL-malaria coinfection within fifteen to twenty-nine years old might be associated with their daily activities. Farming is extensive in Metema due to the fact that young daily laborers move to Metema from different areas for application of herbicide and for gathering of crops. Because of high temperature in this area, daily activities are accomplished especially during night. This may expose them to the bite of mosquitoes and sand fly which means a more concentrated effort should be given to educate these age groups about this disease and its vector.Visceral leishmaniasis-malaria coinfection was significantly higher in patients with the age groups of less than five years (In this study, majority of study subjects use their bed nets. However, the prevalence of VL-malaria coinfection was high in these subjects. This might be due to improper and infrequent use of their bed net or it might be due to lack of impregnating their bed nets regularly. In the study area most of study participants who were coinfected with VL-malaria were daily laborers followed by merchants. This might be associated with their daily activities which may expose them for vector bite.This study is limited to the data obtained from the VL suspected patients, which may reduce the prevalence of malaria. Moreover, this study for VL diagnosis depends only on serological tests. There is no microscopically confirmed parasitological data for VL. To fill all these gaps, there is a need for further study including all febrile patients with different study design.In conclusion, this study tries to see VL-malaria coinfection for the first time and found 4.2% coinfection rate. A significantly higher number of VL-malaria coinfections were showed in study subjects whose age was less than five years old. Based on these findings, we recommend that malaria screening be implemented for all VL patients who live in malaria-endemic areas in order to promptly initiate antimalarial drug treatment. Moreover, to minimize the disease burden, health planners and administrators need to give intensive health education to increase the community awareness about the two diseases' prevention and control strategies.


Plasmodium parasites in the infected host. Naturally acquired antibodies against gametocytes exist and may play a role in controlling transmission by limiting the gametocyte development in the circulation or by interrupting gamete development and fertilization in the mosquito following ingestion. So far, most studies on antibody responses to sexual stage antigens have focused on a subset of gametocyte-surface antigens, even though inhibitory Ab responses to other gametocyte antigens might also play a role in controlling gametocyte density and fertility. Limited information is available on natural antibody response to the surfaces of gametocyte-infected erythrocytes.Transmission of malaria from man to mosquito depends on the presence of gametocytes, the sexual stage of Plasmodium falciparum mature gametocytes were investigated in sera of semi-immune adults and malaria-exposed children. In addition, the effect of immunization with GMZ2, a blood stage malaria vaccine candidate, and the effect of intestinal helminth infection on the development of immunity to gametocytes of P. falciparum was evaluated in malaria-exposed children and adults from Gabon. Serum samples from two Phase I clinical trials conducted in Gabon were analysed by microscopic and flow-cytometric immunofluorescence assay.Ab responses to surface antigens of erythrocytes infected by in vitro differentiated Ascaris lumbricoides had a significantly higher anti-gametocyte Ab response compared to non-infected children.Adults had a higher Ab response compared to children. Ab reactivity was significantly higher after fixation and permeabilization of parasitized erythrocytes. Following vaccination with the malaria vaccine candidate GMZ2, anti-gametocyte Ab concentration decreased in adults compared to baseline. Ab response to whole asexual stage antigens had a significant but weak positive correlation to anti-gametocyte Ab responses in adults, but not in children. Children infected with The current data suggest that antigens exposed on the gametocyte-infected red blood cells are recognized by serum antibodies from malaria-exposed children and semi-immune adults. This anti-gametocyte immune response may be influenced by natural exposure and vaccination. Modulation of the natural immune response to gametocytes by co-infecting parasites should be investigated further and may have an important impact on malaria control strategies. Malaria remains a major global public health problem affecting hundreds of millions of people annually, mainly in sub-Saharan Africa. Each year approximately half a million people die, mostly children younger than 5 years . GametocTransmission blocking interventions that target gametocyte development and gamete fertilization are considered an essential part of malaria control, especially if containment or eradication of the disease is the aim. Transmission blocking vaccines (TBVs) would have a great public benefit in malaria-endemic countries by breaking the life cycle and decreasing the number of new infections. In addition, it is assumed that TBVs could help in containing the spread of parasites resistant to drugs or malaria vaccine components directed against asexual blood stage or pre-erythrocytic stages , 7. To bPlasmodium and other apicomplexan parasites [Antibodies (Abs) are important mediators of sexual stage immunity against arasites –12. Sucharasites  or by diarasites –15. The arasites –18, e.g.arasites , 20 is marasites .Indirectly, immune responses to asexual stage antigens may decrease transmission by limiting the number of asexual parasites that develop to gametocytes , similarNaturally acquired sexual-stage antibodies are produced against gametocyte-infected erythrocyte surface antigens or gamete-specific antigens in the circulation and also against mosquito-stage parasites that act following ingestion of the parasite . There aPlasmodium falciparum clinical isolate and one laboratory strain (NF54) was measured by flow cytometric immunofluorescence assay (IFA) in sera from malaria-exposed children and semi-immune adults. Since exposure to asexual blood stage antigens and co-infection with other highly prevalent parasites may modulate immune responses [P. falciparum glutamate-rich protein (GLURP) and merozoite surface protein 3 (MSP3) [In the present study, the concentration of anti-gametocyte Abs against in vitro differentiated mature gametocytes of one esponses , 34, her3 (MSP3) . TherefoGametocyte recognition of antibodies were investigated in adults and children who had a different history of exposures to malaria and infection status with intestinal helminths. The serum samples analysed in the current investigation are from two studies , 37 condP. falciparum. During the GMZ2 trials, participants received three doses of either 30 or 100 μg GMZ2, adjuvanted with aluminum hydroxide or a control vaccine (Verorab) and were followed for 1 year. Further details of the studies are published elsewhere [As part of the GMZ2 trials, samples were collected from 30 children aged 1–5 years  and 40 slsewhere , 37. SerP. falciparum was part of the respective study protocols.The studies were conducted according to the principles of the Declaration of Helsinki and good clinical practice guidelines. The respective competent ethics committees: the Comité d’Ethique Régional Indépendant de Lambaréné, CERIL and the Gabonese Ministry of Health reviewed and approved the studies. A signed informed consent was obtained from each participant or parent/guardian of participant aged less than 18 years. Analysis of Ab levels against P. falciparum parasites as described earlier [2 and 5% O2 with weekly sorbitol synchronization and daily change of medium ; medium of the clinical isolate contained in addition 5% human serum. The P. falciparum laboratory strain NF54  and a laboratory adapted clinical isolate from Lambaréné, Gabon where the samples had been collected were used for the experiments. The clinical isolate (JH013) was cultivated from a blood sample collected in 2009 from an individual with P. falciparum mono-infection and cryopreserved at −150 °C in glycerolyte as reported previously [Gametocyte culture was initiated from a continuous culture of asexual  earlier . Briefly2/CO2 at 37 °C with daily change of medium without parasite dilution. On Day 7 the haematocrit was lowered to 4.5% by doubling the volume of medium. Beginning with Day 11 the parasites were treated with 50 mM N-acetyl-d-glucosamine  for 4 days to eliminate asexual stage parasites.Gametocyte culture was performed as described earlier , 42 withg for 20 min) on 33% NycoPrep 1.077 cushions  followed by magnetic separation with LD-MACS magnetic columns  was performed.To enrich stage V gametocytes from approximately 2% to more than 90% on Day 15 a density gradient centrifugation . Fluorescence of each event was analysed and the result expressed as percentage of positive fluorescent cells (PPFC) based on 20,000 erythrocytes (events) acquired.For comparison, a subset of previously reported GMZ2-specific IgG ELISA values (Days 0 and 84) of clinical trial participants were used for the current analyses , 37. ELIDuring follow up of study participants enrolled in the Phase I paediatric clinical trial of GMZ2 , stool sGraphPad Prism version 5.0  and SPSS version 16 were used for statistical analysis. Nonparametric tests were used for statistical testing. The significance level was set to a two-sided alpha of 0.05 and corrected using the Bonferroni method where applicable. Paired samples were tested with the Wilcoxon signed-rank test whereas the Mann–Whitney test was applied in case of unpaired data. Spearman correlation between Ab responses to whole sexual and asexual stage (including recombinant GMZ2) antigens were calculated and presented with 95% confidence intervals . A samplP. falciparum mature gametocytes was evaluated in sera from adults (n = 36) and children (n = 30) from the two GMZ2 trials  and NF54 were compared to identify the strain that is best recognized by participants’ Abs . There w and NF54The effect of GMZ2 vaccination on the development of anti-gametocyte immune response in adults and children was assessed. It was hypothesized that immune responses generated by the GMZ2 vaccine may divert the immune response towards the vaccine or lead to a lower gametocyte density, despite its relatively low efficacy 11–14%) in children between 1 and 5 years . Interes% in chilSince sexual stage immunity may be developed independently from asexual-stage immunity , 15, posP. falciparum with other infectious agents including helminths [Trichuris trichiura and Ascaris lumbricoides were present in a relative high proportion on enrolment  and on Day 84 , respectively. There was also a low rate of infections (<7%) by Strongyloides, Schistosoma, and Ancylostoma. Interestingly, children infected with Ascaris lumbricoides had significantly higher anti-gametocyte Ab response compared to uninfected children when using live non-permeabilized gametocytes . The difference of the correlation results between adults and children might be due to the very low level of anti-gametocyte immune response in children complicating to see differences between groups. Additionally, Ab response to matured gametocytes did not change in children following vaccination with GMZ2 or the rabies control vaccine. Unfortunately our analysis has been underpowered to detect subtle differences in the study populations. Our result shall be confirmed with a bigger sample size.As expected, antigen recognition by immune sera was significantly enhanced after fixation and permeabilization of gametocyte-infected erythrocytes as reported earlier . This efP. falciparum gametocytes of the laboratory strain NF54 and a clinical isolate by serum antibodies of semi-immune adults was assessed. Both lab strain and clinical isolate have been detected by the serum antibodies and no significant difference in the level of Ab recognition of the two strains could be seen. This was unexpected as the clinical isolate was collected from the area where the study participants were recruited. However, similar to the finding presented here, it has been shown earlier by Dinko et al. that the plasma antibodies from Ghanian participants recognizing the laboratory strain 3D7 and a clinical isolate from Kenia [Recognition of om Kenia .Trichuris trichiura infected children when the assay was done with permeabilized gametocytes. However this might not represent the suppression of the immune response to gametocytes but rather the response to the asexual or both stages of the parasite. This corroborates earlier findings in the same population, which showed that Trichuris trichiura infection is associated with 3.4-fold reduced Ab levels to the blood-stage asexual GMZ2 vaccine antigen while the response was increased in Ascaris lumbricoides-infected participants [Trichuris trichiura infection could not be seen in the assay using non-permeabilized gametocyte-infected erythrocytes. In line with this, a cross-sectional study has reported a significant reduction of gametocyte specific (Pfs48/45) antibody titers in individuals infected with Schistosoma haematobium, though this effect is not seen when comparing another gametocyte specific antigen (Pfs230) [The immune response to malaria is modulated by co-infection with other infectious agents . There wicipants . However(Pfs230) .The response to any gamete-specific antigens was not measured and therefore activation of non-fixed gametocytes cannot be ruled out. Mature gametocytes should be handled with care and a drop in temperature could provoke exflagellation. Therefore, the small fraction of recognized erythrocytes with gametocytes could also be a small fraction that was accidentally activated. However, we tried to ensure that non-fixed and non-permeabilized erythrocytes in the assay were intact and controlled for exflagellation by microscopy. Therefore, the measured response most probably represents largely the antibody response to antigens displayed on the surface of gametocyte-infected erythrocytes. Another explanation for the low response could be that only a fraction of gametocytes transport proteins to the outside and express surface proteins on the surface of erythrocytes. A reduced transport of proteins to the erythrocyte surface is known for asexual parasites of laboratory strains in long-term culture. To check this, the prevalence of positive cells was tested also for one clinical isolate (JH013), but did not find a difference in the percentage of recognized cells when compared to NF54.Ascaris lumbricoides resulted in an increased anti-gametocyte immune response compared to the uninfected participants. This was surprising but similar effects on the anti-malarial immune response have been observed earlier [Infection with  earlier . HoweverP. falciparum parasitaemia. This may have a significant impact on malaria control strategies with the aim to reduce transmission of malaria.The level of Ab responses to mature gametocytes can be measured using flow cytometry. Adults show higher Ab responses when compared to children. Anti-gametocyte Ab responses are enhanced following permeabilization but may include responses to asexual antigens. Helminth infections and anti-malarial interventions modulate the humoral immune response to asexual and sexual blood stage


Entamoeba histolytica has been an important public health problem in Jaffna district, northern Sri Lanka for last three decades. In order to draw up a control strategy for elimination of this condition, knowledge of its epidemiology and factors associated with this condition in the local context is vital.Clinically diagnosed amoebic liver abscess (ALA) caused by Entamoeba histolytica in the sample. Climatic data and the total toddy sales in the district were obtained from the Meteorological and Excise Departments respectively. A sub group of randomly selected 100 patients were compared with 100 toddy consumers who were negative for E. histolytica antibody to explore the potential risk factors.All clinically diagnosed ALA patients admitted to the Teaching Hospital, Jaffna during the study period were included in the study and the data were collected using an interviewer administered questionnaire. One hundred blood samples from randomly selected toddy  consumers and 200 toddy samples were collected. Toddy samples were cultured in Robinson’s medium to establish the presence of E. histolytica. The monthly incidence of disease peaked in the dry season, matching the total toddy sales in the district. Age, type of alcohol and frequency of drinking were identified as potential risk factors whereas frequency of alcohol consumption and type of alcohol (consuming toddy and arrack) were identified as the independent risk factors. Moreover, the knowledge, attitude and practices towards ALA were poor among participants and the control group.Between July 2012 and July 2015, 346 of 367 ALA patients were enrolled in this study. Almost all patients (98.6%) were males with a history of heavy consumption of alcohol (100%). Almost all (94.2%) were within the age group 31–50 years. None of the cultured toddy samples grew E. histolytica leading to ALA has to be further explored. Moreover, greater awareness among the public who are at risk would be beneficial in order to eliminate the disease.Though the number of cases has declined in recent years, ALA still remains as an important public health problem in Jaffna district. The transmission route of The online version of this article (10.1186/s12889-018-5036-2) contains supplementary material, which is available to authorized users. Entamoeba histolytica, affects at least 50 million people globally and killing 100,000 individuals every year [Entamoeba histolytica in 12.5% of cultured toddy in vitro [Amoebiasis, a parasitic condition, caused by ery year –6. Recenery year , 5 and fery year , 7–9 thaIn addition to the consumption of alcohol, poor living conditions, overcrowding, unhygienic practices , age, seAlthough malaria has been under control in Jaffna district since 2002, cases of clinically diagnosed ALA have been continuously reported to the Teaching Hospital (TH), Jaffna. In order to draw up a control strategy for elimination of the condition, knowledge about disease burden, its local epidemiology, associated risk factors, seasonality of the condition in the local context and the knowledge, attitude and practice (KAP) towards the disease among those who are at risk, is essential.Entamoeba histolytica in Jaffna district, northern Sri Lanka.Therefore, this study was carried out to examine the above factors in order to propose a control plan which is expected to prevent or minimize the transmission of E. histolytica antibody.A longitudinal study was conducted among the clinically diagnosed ALA patients admitted to Jaffna TH, during the period from July 2012 to July 2015. Clinicians arrived at a clinical diagnosis mainly based on the patient’s history, clinical features and other investigations such as haematological parameters and mainly with the aid of ultrasonography. Out of 367 patients admitted during the study period, 346 consented for study (response rate - 94%). Further, to explore the associated factors, a sub study was conducted by comparing 100 randomly-selected, male patients with sex-matched controls of toddy consumers who were negative for Relevant data were collected from the patients and the control group using a pretested interviewer administered questionnaire. The questionnaire contained both open and close ended questions in the native language (Tamil). The questionnaire included the following information, a) general socio demographic data b) personal hygienic practices and alcohol consumption history and c) the knowledge, attitude and practices in relation to ALA.Five taverns were selected based on where most of the patients consumed toddy. Twenty toddy samples (250 ml each) from each tavern were collected on different days of the month. Further, 100 samples of fresh toddy (250 ml each), were also obtained just after collection from the palmyrah tree, at each particular location. All samples were brought to the laboratory and filtered through a sieve, centrifuged at the rate of 2500 rpm for 10 min and the supernatant was discarded. The pellet was used for further investigation.Records of monthly toddy sales (in bottles) in the entire district throughout the study period was obtained from the Excise Department, Jaffna.Records of monthly rainfall, temperature and the relative humidity of Jaffna district for the study period (2012–2015) were also obtained from the Meteorological Department, Thirunelvely, Jaffna.Pellets obtained from centrifuged toddy samples were used for microscopic examination and cultured in Robinson’s medium . BrieflyE. histolytica serum antibody to explore the risk factors. Pearson’s, Chi –squared with Yates correction statistical tests were used and odds ratios were calculated to identify the potential risk factors.Data were analyzed using SPSS statistical software version 16 and Win Pepi software version 11.62. In addition to describing the characteristics and hygienic practices of the patients admitted to TH Jaffna, randomly selected one hundred male patients were compared in the sub study with 100 male participants who were negative for In 2012, there were 9 ALA cases per 10,000 total hospital admissions which gradually declined to 3 cases per 10,000 hospital admissions in 2015 . The large majority of patients (94.2%) were men of the age group 31–50 years. Most of them (77.6%) had studied up to G.C.E. Ordinary Level (secondary level of education) and the majority (64.5%) was labourers. Most of them (86%) stated that they earned a monthly income of LKR 5000–10,000 (USD 32.74–65.49)  in Jaffna district and the majority (75%) of them was from the rural area.The majority (93%) stated that they lived in households with water sealed toilets and most of them (88.4%) revealed that the distance between the toilet and the drinking water source was roughly about 50 feet Table . HoweverThe comparison of hygienic practices among the patients and the control group showed no statistical differences as shown in the Table None of the patients nor the controls had the knowledge of the name of the disease condition, aetiological agent, transmission route, predisposing factors and prevention and control measures. Both the patients and control group (70% and 80% respectively) believed that eating well was the way to prevent any liver disease caused by alcohol consumption. Similarly, both groups did not adopt any particular preventive measures in order to prevent the condition, ALA.The monthly incidence of ALA showed the same pattern as the monthly average temperature Fig.  during tAs shown in Fig. n = 245, 70.8%) stated that they consumed alcohol daily (Table All patients (100%) had a positive history of consumption of alcohol. The majority (79.2%) were in the habit of consuming toddy and arrack, depending on the money availability. Those who were consuming toddy, consumed palmyrah toddy (100%). The majority , northern Sri Lanka. We found that the annual admission rate of ALA in 2012, 2013, 2014, 2015 were 9.0, 7.0, 4.0, and 3.0 per 10,000 total admissions at TH, Jaffna, respectively. Even though a decreasing trend was seen, ALA is still responsible for significant morbidity.In 1985, the reported rate of ALA at TH, Jaffna was 5.9% of total hospital admissions . Taken tMarked male predominance 98.4%) was observed in our study as well as in other surveys carried out in Sri Lanka and elsewhere 8.4% was , 14–28. Similarly, the age distribution 31–50) also tallies with reports around the world 1–50 also–28, 30. It has been suggested, based on animal models (hamster), that the male hormone testosterone could be a host factor that favours the development of ALA . The posE. histolytica enzymes [E. histolytica [Further, it is a well-known fact that iron is a crucial component of  enzymes . In vitrtolytica . Regulartolytica  which artolytica . Furthertolytica  have alsMakkar et al.  have sugApart from age and sex, the other associated risk factor identified in our study was consumption of alcohol 100%). A majority of the researchers working on ALA have reported the habit of alcohol consumption, particularly in endemic areas 0%. A maj–21.The percentage of patients who consumed alcohol varied from 50% in Bangladesh  to 100% Ghosh et al.  have repE. histolytica is facilitated by alcohol which is harmful to the liver and by nutritional deficiencies in alcoholics, thus leading to a higher incidence of ALA in alcoholics. Further, they reported that duration and quality of alcohol consumed also play an important role in the higher incidence of liver abscess and therefore alcoholism is the most important predisposing factor [Further, Raja and Karthick  suggesteg factor .E. histolytica.Meanwhile, Mukhopadhyay et al.  suggesteAt the same time, Raja and Karthick  further E. histolytica ingested with the drink”. They argued that ALA seems to be common in alcoholics irrespective of the type of alcohol used.Further, Makkar et al.  also disE. histolytica from toddy. Further, we found that 79% of the patients consumed toddy as well as arrack depending on the money availability which was found as an independent risk factor.In our study too, we were unable to demonstrate the infective stage of We found that the peak of the disease was correlated with the dry seasons Fig. . The numThere is a belief among the consumers that the toddy has a cooling effect on the body during the hot dry season. Therefore, this may be the reason for increased intake of toddy during the dry season and for the increasing rate of the disease which was further supported by the increased sales of toddy during the dry season.Entamoeba histolytica cysts. Hence, establishing the route of transmission of the parasite is of great importance. Moreover, all patients and controls were found lacking in knowledge, attitude and practice towards ALA. Therefore, conducting awareness programs through mass media and community level to the public who are at risk is essential, in order to eliminate the disease.Though the number of cases has declined in recent years, amoebic liver abscess still remains an important public health problem in Jaffna district, northern Sri Lanka. Middle aged males who reported heavy consumption of alcohol, and lived mainly in rural areas, were mostly affected by this condition. The incidence was highest in the dry season. Statistical analysis showed the type of alcohol and the frequency of consumption to be the potential risk factors. However, none of the toddy samples demonstrated


Congenital malaria, defined as the presence of asexual forms of malaria parasites in the peripheral blood during the first 7 days of life, remains a neglected area of research. Knowledge gaps exist about prevalence and management of malaria in this age group. The objective of this study was to evaluate the prevalence of congenital malaria and the validity of a rapid diagnostic test (RDT) for its diagnosis in rural Burundi.A cross-sectional study was conducted in a meso-endemic malaria context in Burundi among 290 mothers, and their newborns (n = 303), who delivered at the maternity departments of Kirundo and Mukenke Hospitals during March and April 2014. Peripheral blood samples were collected from all mothers/newborns pairs in order to examine the presence of malaria parasites with two RDT (SD-Bioline HRP2 and Carestart pan-pLDH) and a blood slide. In addition, quantitative real-time polymerase chain reaction (PCR) was performed from the newborn peripheral sample. Frequencies and proportions were calculated for categorical variables. Sensitivity and specificity were calculated with a 95 % confidence interval (CI).None of the newborns were found positive by PCR . The prevalence in newborns born from microscopy-positive mothers was 0 % . Two newborns were positive with SD-Bioline HRP2  but none with Carestart pan-pLDH or microscopy. Sensitivity of the diagnostic tests could not be evaluated as no congenital malaria was detected. Specificity of SD-Bioline HRP2, Carestart pan-pLDH and microscopy to detect congenital malaria was 99.3 % (95 % CI 97.6–99.8), 100.0 % (95 % CI 98.3–100.0) and 100.0 % (95 % CI 98.8–100.0), respectively.In Burundi or the Central African region, no recent prevalence studies for congenital malaria have been carried out. This study found that the prevalence of congenital malaria in two hospitals in Kirundo province is zero. RDT showed to have an excellent specificity and, therefore, can be used to rule out congenital malaria: the risk of overtreatment is low. However, as no cases of congenital malaria were detected, the study was not able to draw conclusions about the sensitivity of the RDT, nor about risk factors for congenital malaria. Further studies evaluating the sensitivity of RDT for diagnosis of congenital malaria are needed. Congenital malaria is defined as the presence of asexual parasites in the cord blood or peripheral blood during the first week of life, due to the transmission of parasites through the placenta just before or during the delivery . It remaThe prevalence of congenital malaria in sub-Saharan Africa is estimated between 0 and 54 % , 4: the Because of the effectiveness of the placenta as a barrier, the presence of maternal antibodies and the protective effect of foetal haemoglobin (Hb F), congenital malaria has long been considered as a rare event . Its truNeonates usually present with a low to very low parasitaemia: a study carried-out in four meso- and hyper-endemic regions in Nigeria found a mean parasite density of 48 parasites/µL (range: 8–200), with 62 % of the neonates spontaneously clearing infection within 2 days .Microscopy is the main diagnostic tool currently used for the diagnosis of congenital malaria but access to quality microscopy is often problematic in low-income contexts, even in hospital settings. PCR is considered as the most sensitive tool but is mainly used for research purposes. Rapid diagnostic tests (RDT) are the most widely used diagnostic tests for malaria; they are easy to use, cheap and reliable and can, therefore, be used at peripheral levels. However evidence is lacking about the use and validity of RDT for the diagnosis of congenital malaria; to our knowledge only one study compared the use of PCR, microscopy and RDT (HRP2/pLDH and First Response Combo) . No caseIn Burundi, a sub-Saharan African country known to have a very high malaria burden , MédecinThe main objectives of our study were to define the prevalence of congenital malaria among newborns in Kirundo Province, Burundi, and to assess the validity of RDT for the diagnosis of congenital malaria.All mothers with their newborns who were born in Kirundo and Mukenke district hospitals between March and April 2014.Burundi is a Central African country with a population of approximately 10.5 million people. The Northern part is considered as a meso-endemic zone for malaria with transmission throughout the year and peaks during the two rainy seasons (February–May and October–December). Since 2013 MSF provides support to the MSPLS in the Province Sanitaire de Kirundo, with a main focus on severe malaria.This was an observational cross-sectional study carried out in the maternity and paediatric services of Kirundo and Mukenke district hospitals in Kirundo-Province, Burundi.Plasmodium falciparum; 14.3 % P. falciparum and/or mixed infections and 0 % non-P. falciparum) and at 23.8 % using microscopy  [P. falciparum with a sensitivity of 90 %, 138 PCR(+) samples were needed . Similar calculations applied for the specificity: a number of 138 PCR(−) samples were required.The prevalence of malaria among children 6–59 months is estimated at 30.6 % using RDT (16.3 % ciparum) . Data coInclusion criteria were: 1/mothers and their newborns of 0–7 days, born in the maternity-department 2/children 0–7 days old admitted in the paediatric department and their mothers. Excluded were children of 0–7 days having received anti-malaria treatment since birth.Approximately 100 µL of capillary blood was collected from mothers and newborns through finger-prick or heel-prick, within 18 h after delivery, for testing with RDT, thick smear/thin smear examination and PCR.The performance of two different types of RDT was evaluated: a histidine rich protein (HRP2)-based test and a parasite lactate dehydrogenase (pLDH)-based test. The HRP2-test was the SD Bioline Malaria Antigen Pf (HRP2) (Manufacturer: Standard Diagnostics Inc.), currently used in Burundi as well as in many other contexts. The pLDH-test evaluated was the Carestart Malaria pLDH (pan) (Manufacturer: Access Bio Inc.). Quantitative PCR (qPCR) was used as gold standard.Thick and thin smears were prepared at the bedside from two small drops of blood applied on the same glass slide directly from the finger- or heel prick. Each thick smear was analysed with light microscopy during 6 min to determine parasitaemia. For positive thick smears, the corresponding thin smear was analysed to determine the Plasmodium species. All smears were analysed by two readers. In case of a discordant result, a third reader analysed the slide to obtain a final result. An expert microscopist reread 10 % of the samples as a quality control.Plasmodium falciparum qPCR targeting 18S gene was performed as described elsewhere [For the molecular diagnosis (qPCR), filter papers with dried blood samples were punched, and one circle of 5 mm in diameter was used for DNA extraction with a QIAamp DNA blood extraction kit (Qiagen) following the manufacturer’s recommendations. Extracted DNA was eluted in 150 µL water and used for quantitative real time PCR (qPCR). lsewhere . The lowData were collected about the clinical condition and the outcome of the newborns, the clinical condition of the mothers, the history of malaria during the last trimester and the use of long-lasting insecticide-treated nets (LLIN) during pregnancy.Individual consent was obtained from the mother or in absence of the mother the representative of the eligible children. Separate information sheets and consent forms were used for the prevalence and the validity study and were piloted beforehand to ensure they were easy to understand by the patients. The information sheet was read and explained to each eligible mother or to the representative of each eligible child. Informed consent was in writing, through signature on the consent declaration or, in case the study participant or legal representative was illiterate, through a mark. The study participant or representative was given a copy of the information sheet and consent form.All participant records were given an identification code. Only the anonymous codes were entered in a dedicated Microsoft Excel (version 2010) database, thus participants could not be identified. Patient records and dedicated electronic database were only accessible to the principal investigator and data encoder. All encoded data related to the study objectives were crosschecked for validation with the questionnaires and laboratory registers. Data analysis were performed by using Epidata analysis software . Baseline characteristics were described using medians and interquartile ranges (IQRs) for continuous variables and counts and percentages for categorical data. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) variables were calculated with a 95 % confidence interval.P. falciparum with a mean parasitaemia of 483 parasites/µL (ranges: 64–490667) during their delivery, only 6 % had a fever (>38.2 °C). Among the newborns, 85 (28 %) had a low birthweight (<2500 g) and 2 % were observed with a fever. No newborns suspected with congenital malaria were admitted in the paediatric service during the study.During the study period a total of 290 mothers delivered in Kirundo and Mukenke maternity wards of a total of 303 newborns. All 290 mothers were willing to participate, thus 290 mothers and 303 newborns were included in the study. The characteristics of the study participants are summarized in Table None of the newborns were found positive by PCR  Table . The preAs no congenital malaria was detected with qPCR it was not possible to evaluate the sensitivity of the diagnostic tests. However, the specificity of the SD-Bioline HRP2, the Carestart pan-pLDH and microscopy to detect congenital malaria was 99.3 % (95 % CI 97.6–99.8), 100.0 % (95 % CI 98.3–100.0) and 100.0 % (95 % CI 98.8–100.0), respectively  is not transmitted. This study could not confirm that newborns from mothers with high parasitaemia are more likely to have parasites .Third, the measures to prevent malaria in pregnancy are poorly implemented. A nationwide survey carried-out in 2012 reported that only 52 % of the pregnant women in the Northern Region slept under a LN the night before the survey . The useSince the year 2000, studies on congenital malaria have been carried-out mainly in western Africa and recent data about the occurrence of congenital malaria in the Central African region are not available. The prevalence found in this study is similar to the low prevalence of 1.4 % reported in Burkina Faso  or 2.2 %This study also had some limitations. First, it was not feasible to collect samples from placental and cord blood. Other studies reported various results depending on the type of sample collected, and in studies collecting both cord blood and peripheral blood the frequency or level of parasitaemia was lower in peripheral blood than in cord blood , 21–23. Secondly, mothers in Burundi leave the hospital within a maximum of 24 h after birth, often between 6 and 12 h, therefore, only one sample could be taken at birth. Several authors recommend repeated sampling to evaluate the presence of congenital malaria; the prevalence was found lower in samples collected within a few hours after birth, compared to sampling on consecutive days , 19. OthThird, no sick newborns suspected of congenital malaria were admitted in the paediatric ward during the whole study period, and thus the prevalence of congenital malaria among symptomatic newborns could not be evaluated.Fourth, this study only assessed the prevalence among newborns born in Kirundo and Mukenke Hospitals. From all expected deliveries, about one out of four still take place at home  and, theFinally, the fact that the same laboratory technicians performed the RDT as well as the microscopy analysis might have introduced a certain bias.This is the first study which has evaluated the prevalence of congenital malaria in Burundi, and no studies have been carried-out recently in the Central African region. The prevalence of congenital malaria was found to be zero. It was also one of the first studies evaluating the performance of RDT for the diagnosis of congenital malaria. As their specificity was very high, RDT can be used to rule out congenital malaria; the risk of giving unnecessary treatment is low. However further research is needed for evaluating the sensitivity of the RDT or the risk factors for congenital malaria.


Health clinics in rural Africa are typically resource-limited. As a result, many patients presenting with fever are treated with anti-malarial drugs based only on clinical presentation. This is a considerable issue in Uganda, where malaria is routinely over-diagnosed and over-treated, constituting a wastage of resources and an elevated risk of mortality in wrongly diagnosed patients. However, rapid diagnostic tests (RDTs) for malaria are increasingly being used in health facilities. Being fast, easy and inexpensive, RDTs offer the opportunity for feasible diagnostic capacity in resource-limited areas. This study evaluated the rate of malaria misdiagnosis and the accuracy of RDTs in rural Uganda, where presumptive diagnosis still predominates. Specifically, the diagnostic accuracy of “gold standard” methods, microscopy and PCR, were compared to the most feasible method, RDTs.Patients presenting with fever at one of two health clinics in the Kabarole District of Uganda were enrolled in this study. Blood was collected by finger prick and used to administer RDTs, make blood smears for microscopy, and blot Whatman FTA cards for DNA extraction, polymerase chain reaction (PCR) amplification, and sequencing. The accuracy of RDTs and microscopy were assessed relative to PCR, considered the new standard of malaria diagnosis.Plasmodium infection by at least one method. Comparing diagnostic pairs determined that RDTs and microscopy performed similarly, being 92.6 and 92.0 % sensitive and 95.5 and 94.4 % specific, respectively. Combining both methods resulted in a sensitivity of 96.0 % and specificity of 100 %. However, both RDTs and microscopy missed one case of non-falciparum malaria  that was identified and characterized by PCR and sequencing. In total, based on PCR, 62.0 % of patients would have been misdiagnosed with malaria if symptomatic diagnosis was used.A total of 78 patients were enrolled, and 31 were diagnosed with P. falciparum species, which highlights an important limitation of this test and a treatment concern for non-falciparum malaria patients. Nevertheless, RDTs appear the only feasible method in rural or resource-limited areas, and therefore offer the best way forward in malaria management in endemic countries.Results suggest that diagnosis of malaria based on symptoms alone appears to be highly inaccurate in this setting. Furthermore, RDTs were very effective at diagnosing malaria, performing as well or better than microscopy. However, only PCR and DNA sequencing detected non- In Africa, 70 % of fevers are initially managed at home, with traditional remedies and bed rest used to alleviate symptoms , 2. OnlyIn many malaria endemic regions, safe, inexpensive anti-malarials such as chloroquine have become ineffective due to the emergence of drug-resistant parasites. These drugs are being replaced by more toxic and expensive alternatives, including sulfadoxine-pyrimethamine . PrescriUganda is currently among the few countries where cases of malaria have recently increased . Indeed,Plasmodium falciparum, which is the recommended RDT for malaria diagnosis in Uganda. Health practitioners read, interpreted, and recorded the result of the test after 15 min. Results were recorded as positive or negative, with faint lines being interpreted as positive. An additional two drops of blood were used to make thick and thin blood smears for microscopic diagnosis. Finally, one to five additional blood drops were collected on Whatman FTA Classic Cards for subsequent molecular analyses. All patients were offered complimentary treatment based on RDT results and the health practitioner’s diagnosis. Blood samples were shipped to North America for microscopic examination and molecular diagnostics.Two rural health clinics located within the Kabarole District of Uganda were enlisted in this study. Patients visiting these clinics in June and July 2011 with a fever (an axillary temperature of 37.5 °C or higher), who were not pregnant, and who had not treated their symptoms with standard anti-malarials, were asked to participate. Following informed consent, blood samples were collected via finger prick by the clinic’s health practitioner. One drop of blood was used in an RDT that detected histidine-rich protein II (HRP-II) antigen of Plasmodium. Parasitaemia of positive smears was calculated following a previously described formula [Thin and thick smears were stained with Giemsa and viewed under 100× oil immersion objective magnification. Each slide was read by two independent microscopists; in the event of a discrepancy, the slides were read by a third microscopist. Thick smears were used to confirm the presence of blood parasites, while thin smears were used to confirm speciation and determine infection intensity (parasitaemia). For each thin smear, a field containing a representative monolayer of red blood cells (RBCs) was identified and all RBCs in that field were counted. This was repeated five times to calculate an average number of RBCs per field. One hundred fields, or the maximum number of viewable fields on the slide, were scanned for intra-erythrocytic parasites morphologically consistent with  formula .prepGEM  or QIAamp DNA Investigator  kits.DNA was extracted from Whatman FTA Classic Cards following modified manufacturer’s instructions. Briefly, a 4-mm sample disc was punched from each FTA card and placed in a PCR amplification tube. FTA purification reagent (200 µl) was added to each tube and incubated for 5 min at room temperature. The FTA purification agent was removed by pipette, and this process repeated an additional three times. 200 µl of TE buffer  was then added to each PCR tube and incubated for 5 min at room temperature. TE buffer was removed by pipette and this process was repeated once more. The sample disc was allowed to dry completely at room temperature before the disc was used as template in PCR amplification. This method resulted in consistently higher, better quality DNA yields than extraction methods that eluted DNA, such as Plasmodium was conducted using primers CytB3384F (5′-GTAATGCCTAGACGTATTCCTG-3′) and CytB4595R (5′-GTTTGCTTGGGAGCTGTAATC-3′) in the external reaction, and CytB3706F (5′-GTTTGCTTGGGAGCTGTAATC-3′) and CytB4595R in the internal reaction. This procedure generated amplicons of 1254 bp  and 932 bp  predicted size [Plasmodium gallinaceum-positive sample that underwent the same extraction method was used as a positive control; exogenous DNA added post-DNA extraction was used as internal negative control . All samples were run in duplicate at separate intervals to ensure diagnostic accuracy.A semi-nested PCR targeting the cytochrome b (cytb) gene of ted size . Both exted size . A PlasmPlasmodium by PCR but negative by microscopy and/or RDT, amplicons were Sanger sequenced in both directions using internal primers CytB3706F and CytB4595R on ABI 3730xl DNA Analyzers  at the University of Wisconsin Biotechnology Center DNA Sequence Facility. Sequences were hand-edited using Sequencher v4.9 . To identify parasite species, newly generated DNA sequences were compared to reference sequences in Genbank using BLASTn [For samples identified as positive for g BLASTn .The sensitivity and specificity of RDTs and microscopy were compared to PCR, considered the primary reference standard because it is established to outperform microscopy in sensitivity and specificity –24. ParaPlasmodium-positive samples (as determined by microscopy). Average parasitaemia was 0.45 % (±1.00 SD) in females and 0.31 % (±0.43 SD) in males, and was 1.26 % (±1.43 SD) in children aged five or younger and 0.16 % (±0.31 SD) in patients older than five. Females presenting with fever were more frequently positive for malaria than males . All patients were tested by RDT. Blood samples for PCR were collected from 71 of these patients, and blood samples for microscopy were collected from 67 patients. Parasitaemia ranged from 0.003 to 3.399 % of total RBCs in Plasmodium. Microscopic examination of peripheral blood smears identified 28 patients (out of 67) as positive for P. falciparum, and RDTs identified 32 patients as positive  as positive for  as positPlasmodium malariae cytochrome b sequences (Genbank Accession Numbers AB489194 (unpublished) and AB354570 [P. falciparum cytochrome b sequences from India [P. falciparum, sharing 100 % sequence identity with sequences from the aforementioned Indian population set [RDTs and microscopy both missed one patient sample that was positive by PCR. Interestingly, DNA sequencing revealed that this sample was 100 % identical to two published AB354570 ; BLASTn)om India . Similartion set . Lastly,Plasmodium. Thus, the majority of patients (62.0 %) were misdiagnosed and would have been treated with anti-malarials if not for diagnostic intervention. These results corroborate findings of malaria over-diagnosis elsewhere. For example, a cross-sectional study evaluating Uganda’s policy of treating febrile illness with anti-malarials reported rates of over-diagnosis ranging from 45.3 to 80.9 % [This study evaluated the rate of malaria misdiagnosis occurring in rural Uganda, and assessed the accuracy of RDTs, the most promising diagnostic method for limiting over-diagnosis in resource-limited settings. All individuals in this study were symptomatically diagnosed with malaria by health practitioners, based on the presence of fever. However, results of the gold standard diagnostic test suggest that only 38.0 % of patients were positive for o 80.9 % . Indeed,o 80.9 % .Results indicate that RDTs performed as well as the traditional gold standard, microscopy. Both were over 90 % sensitive and specific, although RDTs were marginally better at detecting infection when present and returning negative results when the parasite was absent. That RDTs are as effective or better than microscopy is consistent with the findings of others . HoweverP. malariae. In this study, RDTs specific for P. falciparum were selected based on this species’ ubiquity in the study area, and their recommended usage for malaria diagnosis in Uganda [P. malariae and Plasmodium vivax, <1 % for Plasmodium ovale) based on surveys published in the 1960s [Combining RDT and microscopic diagnosis resulted in perfect specificity, which indicates that malaria-negative patients are unlikely to be misdiagnosed as positive by combining these methods. However, one malaria-positive patient was still missed, even when RDTs and microscopy were combined. This patient was only detected by PCR, and sequencing revealed infection with n Uganda . Howevern Uganda , this sthe 1960s . NeedlesA surprising result in this study was that PCR failed to detect two samples that were confirmed positive by RDT and microscopy. Other studies have suggested that PCR false positives can be the result of recently cured malaria . HoweverDespite limitations in detecting non-falciparum malaria, the new generation of RDTs satisfy all criteria required for implementation in rural settings, being easy to use and durable over the long-term in tropical conditions . FurtherDespite global efforts to eradicate malaria, the burden of this disease is still high. In rural Uganda, 38.0 % of patients visiting peripheral health clinics with fever were positive for malaria. However, this means that 62.0 % of patients symptomatically diagnosed with malaria were negative for the parasite, suggesting that over-diagnosis of this disease remains a critical problem. Combining both microscopy and RDT testing yielded high sensitivity and specificity. However, only PCR detected non-falciparum parasites, and is the only method that offers species-level diagnoses. Nevertheless, the cost and expertise required for this method render it impractical in nearly all developing healthcare settings. On the other hand, the ease and rapidity of RDTs confirm this method is perhaps the best approach for reducing malaria over-diagnosis, even in resource-limited settings.


Plasmodium species are indigenous, there are multiple etiologies of fever leading to misdiagnoses, especially in populations where HIV is prevalent and children. To determine the frequency of malaria infection in febrile patients over an 8-month period at the Regional Hospital in Bamenda, Cameroon, we evaluated the clinical efficacy of the Flourescence and Staining Technology (FAST) Malaria stain and ParaLens AdvanceTM microscopy system (FM) and compared it with conventional bright field microscopy and Giemsa stain (GS).Malaria is a major world health issue and its continued burden is due, in part, to difficulties in the diagnosis of the illness. The World Health Organization recommends confirmatory testing using microscopy-based techniques or rapid diagnostic tests (RDT) for all cases of suspected malaria. In regions where Peripheral blood samples from 522 patients with a clinical diagnosis of “suspected malaria” were evaluated using GS and FM methods. A nested PCR assay was the gold standard to compare the two methods. PCR positivity, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined.Four hundred ninety nine samples were included in the final analysis. Of these, 30 were positive via PCR (6.01%) with a mean PPV of 19.62% and 27.99% for GS and FM, respectively. The mean NPV was 95.01% and 95.28% for GS and FM, respectively. Sensitivity was 26.67% in both groups and specificity was 92.78% and 96.21% for GS and FM, respectively. An increased level of diagnostic discrepancy was observed between technicians based upon skill level using GS, which was not seen with FM.The frequency of malarial infections confirmed via PCR among patients presenting with fever and other symptoms of malaria was dramatically lower than that anticipated based upon physicians’ clinical suspicions. A correlation between technician skill and accuracy of malaria diagnosis using GS was observed that was less pronounced using FM. Additionally, FM increased the specificity and improved the PPV, suggesting this relatively low cost approach could be useful in resource-limited environments. Anecdotally, physicians were reluctant to not treat all patients symptomatically before results were known and in spite of a negative microscopic diagnosis, highlighting the need for further physician education to avoid this practice of overtreatment. A larger study in an area with a known high prevalence is being planned to compare the two microscopy methods against available RDTs.The online version of this article (doi:10.1186/s40249-017-0251-0) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium falciparum [Malaria is an ongoing threat to world health, leading to over 600 000 deaths each year despite increasing awareness and public health efforts directed at controlling the disease. In Cameroon, malaria is perennial, rainfall-dependent and the prevalence is 42.5% in children under 5 years of age, 31.5% in those between 5 and 15 years of age, and 10.5% in those older than 15 years. It causes 50% morbidity in children under 5 years old, and is implicated in 40% to 50% of medical consultations [Plasmodium parasite [The current World Health Organization (WHO) recommendation is detection of parasites using bright field microscopy or rapid diagnostic tests (RDTs) before making a diagnosis and implementing treatment . Despiteparasite , 7–12. Tparasite . Due to parasite . In 2014parasite .Currently, the gold standard for malaria diagnosis is the use of microscopy-based methods such as bright field microscopy with Giemsa-stained specimens (GS) or by validated RDT when available. However, the lack of clear policies within some institutions, varied behaviour of individual clinicians, lack of quality control, and inadequate resources may result in poor outcomes . In ordeMycobacterium tuberculosis [TM light microscope attachment  since it was a system we recently evaluated against a standard light microscope for diagnosis of Mycobacterium infections and observed 100% agreement between both methods [Mycobacterium species and have shown positive results and increased sensitivity in comparison to standard methods [Plasmodium organisms in capillary tubes and is, therefore, more expensive. The main advantages of the FAST Malaria stain over GS are that it is presumably less technically complex, faster to perform, can be used with a relatively inexpensive LED microscopy attachment, obviates the need for a halogen lamp or disposing of the heavy metals, and may require less training and experience to make an accurate diagnosis. Because of these advantages, such a technique could be employed in developing countries where malaria is endemic in order to improve diagnostic accuracy without a large increase in costs.The use of fluorescent-based microscopy techniques can be superior to GS in terms of sensitivity and have been successful in detection of rculosis . Howeverrculosis –21. In l methods . The use methods , 23. A sTM microscope system (FM) in comparison to GS in a prospective study in Cameroon. Polymerase chain reaction (PCR) was used to confirm the presence of blood-borne Plasmodium species and as the standard for comparison between the two microscopy methods. The objectives of this study were to determine the frequency of malaria in patients presenting with fever and to compare the sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the two basic staining methods.In the current study, we evaluated the performance of the FAST Malaria stain and ParaLens AdvanceThis study was conducted in accordance with the principles outlined in the Helsinki Declaration and ethical approval was received in February 2011 from the University of Missouri-Kansas City Institutional Review Board (IRB) and the Cameroon Ministry of Health Regional Hospital IRB in Bamenda, Cameroon. Training of the technicians in the FM technique and reagent quality control was executed the week prior to the initiation of the study until there was less than 10% difference in skill between results using known semi-quantitative 1+, 2+, and 3++ malaria control slides. The collection and staining of slides and samples for PCR were in parallel to routine standard of care (Giemsa) at the Regional Hospital over an 8-month period between January and August 2011. Samples spotted in quadruplicate onto filter paper cards for PCR analysis were transported by air to the United States in 2012 and analysed in a blinded fashion at Kansas City University of Medicine and Bioscience between 2013 and 2014.Peripheral blood samples were obtained sequentially from voluntary participants with a clinical diagnosis of “suspected malaria infection” at the Ministry of Heath Regional Hospital in Bamenda, Cameroon. Patients were referred for phlebotomy if they exhibited the following clinical signs and symptoms of a malarial infection: fever (temperature > 38.0 °C) with associated arthralgia, myalgia, headache, abdominal pain, and malaise, or recent diagnosis of malaria within the past 6 weeks. No changes occurred in the standard of care prior to the study. The blood specimens were collected and slides were prepared for thin and thick smears, de-identified, and assigned a sequential identification number. Two additional sets of thin and thick blood smears were made for each patient and a drop of blood was collected on FTA™ filter paper cards  to be used for confirmatory PCR analysis in our research laboratory in the United States. Positive controls from malaria-positive participants were prepared for further standardization and training.TM microscope attachment  at 1000X magnification. The stain is proprietary and developed specifically to enhance Plasmodium species and the microscope LED attachment can be charged using sunlight. The second set of thin and thick smears were stained with the traditional Giemsa stain  and evaluated with bright field microscopy for the presence of parasites. Thick smears were used for screening first at 1000X magnification. If parasites were detected using the thick smears, there was confirmatory examination of the thin smears at 1000X to detect parasites and life cycle stage. These two sets of samples were randomized to prevent sequential reading of the same sample by the same technologist. The samples were divided randomly into three unique sets, which were read blind by three different technicians with differing training and experience levels . There was no cross examination of samples between the technicians. Additionally, a randomly selected set of samples was analysed blind by a highly skilled technician (technician D) using the GS and FM system at the Mezam Polyclinic HIV/AIDS Treatment Center. To minimize any variability due to potential fluorescence stain fading, all slides stained with FM were read within 48 h of staining. All parties were blinded to the results of the microscopy studies and PCR analysis that was carried out on a later date. Figure The first set of thin and thick smears were stained with the FAST Malaria stain kit  and evaluated with the ParaLens AdvancePlasmodium.A two-step nested PCR assay was used to confirm the results of the microscopy studies. PCR has been shown to have high sensitivity in the detection of malarial species and was, therefore, chosen to compare the microscopy results , 25. ThiTM filter paper. A 1 mm punch was taken from the filter paper and washed in 150 μL nuclease- free water for 5 min at room temperature. The PCR assay was performed within 3 h of washing. The first nest reaction was performed in a 50 μL reaction vessel using a Phusion Blood Direct PCR Kit  and the primers rPLU1 and rPLU5, which produce a 1 640 bp amplicon and serve as the template DNA for the second nest reaction. The second nest was performed at a total reaction volume of 25 μL using a Phusion® High-Fidelity DNA Polymerase kit  and the primers rPLU3 and rPLU4, which produced a 240 bp amplicon. Previous studies have shown reliable results using these primer sets [The assay was performed directly from the blood samples preserved on the FTAmer sets –28.P < 0.05 was considered to indicate a statistical significance. Statistical Package for the Social Science (SPSS) version 23  was used for the analysis.Four technicians analysed the blood smears from 499 patients. The accuracy measures from each technician for both GS and FM including PPV, NPV, sensitivity, specificity, and error rate are summarized. Descriptive statistics such as mean and standard deviation were obtained. A one-tail proportional z-score test was employed to determine whether PPV, NPV, and error rates produced by FM were significantly lower than GS. A Plasmodium falciparum is indigenous to this area and the fact that all samples confirmed by PCR were reviewed by two clinical pathologists and determined to be P. falciparum based upon microscopy, it was not considered cost-effective to further validate the species by additional PCR. To our knowledge, P. vivax, P. ovale, and P. malariae have not been identified in this area of Cameroon, as in the South Western coastal region where more than 85% are P. falciparum [p = 0.0259 (z = 1.95). Both one- and two-tailed proportional z-score tests were applied showing significance, though due to our prior publication for a similar study using one-tail z-score, we chose to use this for reporting statistical significance [There were 522 patients with a presumptive diagnosis of malaria presented for the study and all provided blood samples. The ages ranged between 1 month to 92 years with a mean age of 35.35 years (SD = 19.44). Of the 522 samples, 23 were excluded from the final analysis due to either poor staining (20 slides), damage , or mislabelling (1 with unclear label). Two patients were excluded due to concomitant lymphoma, which could not be excluded as the source of the fever. In this study, 469 patients received therapy irrespective of the laboratory diagnosis (the PCR results were not known at that time). After final analysis, 30 samples (6.01%) were positive via PCR as determined by a 240 bp region corresponding to our primer length. Five of the 30 positive samples (16.67%) were specimens from individuals under the age of 18. Given that P = 0.001). There was no significant difference of NPV between GS (95.01%) and FM (95.28%) methods . Both microscopy methods had identical sensitivities (26.67%); however, there was an improved specificity using the FM method . The data, including individual technician breakdowns, are summarized in Table Using the PCR analysis to confirm the results using each method, the PPV and NPV were determined for both methods. The mean of PPV from Giemsa 19.62%) and FM (27.99%) methods were significantly different (z = −3.10, 9.62% andLaboratory accuracy is a necessity in the diagnosis of malaria, as there is currently a high incidence of misdiagnosis leading to unnecessary or non-beneficial treatment of other infections , 8, 29. The WHO has expressed concern over the lack of quality laboratory diagnostic tests in regions where malaria is endemic, which leads to increased financial burden and waste of resources . WhetherThere are many factors affecting the laboratory diagnosis of malaria using traditional methods that must be taken into consideration when interpreting a diagnostic report. In the case of GS, lack of appropriate resources and training are a key feature , 32. AddPlasmodium species [The detection threshold for GS thick blood smears increases with decreasing technician skill level and quality of equipment . Even wi species .Plasmodium infection could enhance clinician confidence in laboratory data and reduce reliance on clinical symptomatology, which is inherently nonspecific [Our data indicate an increase in the PPV and specificity in the diagnosis of malaria using FM. We found that FM was faster than GS and that there was significantly less artefactual staining, which could explain why some of the technicians were able to recognize the parasites more easily. Our data suggest this increased specificity is less sensitive to the length of training. The latter finding is important in addressing problems associated with the diagnosis of malaria, as the level of technician training is highly correlated with the use of GS. However, given the low number of confirmed malaria cases in this study, it is important to evaluate the impact of skill and training on the GS method in an area with a much higher incidence of malaria since conventional staining techniques such as GM and FM are likely to be in use for long into the future. While technician concordance was not assessed in this study, there appeared to be increased reliability with FM when compared with the PCR results. This improved accuracy despite variability in technician experience using the FM system, if substantiated, could yield improvement in laboratory testing, reduce cost, and reduce the misuse of medications leading to increased drug resistance. Employing FM technique to increase the PPV and specificity in the detection of a specific –9.Interestingly, despite the increased sensitivity of FM in the diagnosis of tuberculosis in a similar format, this was not observed for malaria in this study . We postWhile studies have suggested a prevalence of malaria in Cameroon ranging from 17.2% to 53.21% over the past 9 years , 9. FurtAnecdotally, physicians were reluctant not to treat all patients symptomatically before results were known, even with the negative test results. Despite this, the preliminary findings in our study led to a policy change at the Cameroon Ministry of Heath Regional Hospital in Bamenda in an effort to mitigate these unnecessary treatments. However, as shown elsewhere, physicians and allied health personnel practices are resistant to change . This poThis study showed that there was a low number of malaria-positive patients of those presenting with a presumptive diagnosis of malaria at the Bamenda Regional Hospital in 2011. We also showed a correlation between technician training and the accuracy of malaria diagnosis using bright field microscopy with GS. Differences in skill level diminished when using the FM system, which is faster, slightly more specific, had an increased PPV, and reduced staining artifact compared to standard GS. However, a significantly low sensitivity of both GS and FM was present even with highly experienced technicians. This study highlights the need for an inexpensive diagnostic method with improved reliability and acknowledges the potential for emergence of drug resistance exacerbated by the widespread practice of empirical treatment for malaria. Public health systems should be vigilant to encourage improvement in physician prescribing practices. Continued education of technical personnel is important, as is encouragement in the use of validated RDTs when resources are available. Finally, education of the public as to the causes of drug resistance enhanced by self-medication for malaria is important. A larger study to determine and compare the impact of technical expertise using the two methods in an area with a higher prevalence of malaria is planned with the additional objective to compare these methods against available RDTs and validate our current findings. Furthermore, it would be worth assessing the concordance in diagnostic accuracy between technicians of varying skill levels on the same samples.


P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3β1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5β1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5β1 significantly increased with age. P. falciparum-infected erythrocytes (IE) underlie cerebral malaria1 and maternal malaria2. Sequestration confers a survival advantage on the parasite: mature stage IE accumulate in post-capillary venules and placental intervillous spaces, where they avoid immunologic surveillance in the spleen, and where low oxygen tension favors the growth of P. falciparum. In vitro studies have demonstrated that IE can bind to several endothelial surface molecules, including thrombospondin (TSP), CD36, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, P-selectin, chondroitin sulfate A (CSA), PECAM-1/CD31, gC1qR/HABP1/p32, endothelial protein C receptor (EPCR), integrin α5β1, and possibly the integrin molecule αVβ315. Parasite adhesion to the endothelium is mediated by a family of proteins expressed on the surface of infected erythrocytes (IE) named Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)16.Adhesion and sequestration of 17. Earlier studies that examined the relationship between specific binding interactions and severe malaria have yielded inconsistent results. Studies in Thailand, Kenya and Malawi failed to associate ICAM-1- binding parasites with severe malaria21, while studies in Kenya22 and Tanzania15 found a significant association. Recent studies have implicated novel receptors, such as endothelial protein C receptor (EPCR) related to severe malaria in Tanzania15, and the receptor gC1qR/HABP1/p32 related to seizures in Mozambique23. In Kenya and Tanzania, parasites collected from children with severe malaria were more likely to bind to multiple receptors than other parasites24, possibly confounding the effort to associate individual receptors with disease.An understanding of the binding receptors involved in severe malaria syndromes will guide the development of anti-adhesion therapies and vaccines. Naturally occurring red cell variants, such as hemoglobins C and S, modify PfEMP1 expression as well as parasite adhesion to endothelial cells, suggesting a possible mechanism for their protective effects25. Separately, host genetic factors like hemoglobin AS and AC reduce the risk of severe malaria. In this study we quantified the level of IE adhesion to a panel of endothelial receptors and analyzed the relationship to host risk factors.Age is a key risk factor for severe malaria: children in malaria endemic areas develop immunity to severe malaria more rapidly than immunity that reduces parasite burdenThe analysis of IE binding included 2904 parasite isolates collected from 1089 children aged 1–60 months (mean (SD) 24.9 (15.4)). 59 of the 2904 parasite isolates were collected from 52 children presenting with severe malaria. Severe malaria presentations included severe malaria anemia (n = 26), prostration (n = 23), repeated convulsions (n = 9), and coma (n = 1). There were no differences in age between children with severe and non-severe malaria. Severe malaria incidence rate analysis of the whole cohort showed that the rate of severe malaria increased in children aged >12 months  families, VE-cadherin, vitronectin and ICAM-2.27. Further, it has been reported that laboratory parasite line FCR3 binds cellular fibronectin28. Other receptors were selected based on their known role in leukocyte adhesion like junctional adhesion molecules (JAM)29, ICAM-2, VE cadherin, integrin types β1 and β3 constitutively expressed in vascular endothelium including brain microvessels30.The selection of candidate novel receptors for the study was based on the following observations: laminin plasma levels are increased during acute malaria infection, suggesting vascular injury that may expose extracellular matrix molecules (ECM) like laminin and possibly other ECM like cellular fibronectin that can then mediate IE adhesionEach individual receptor, including all novel receptors selected for study, supported the binding of a subset of clinical samples Table . The larPlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s have been grouped according to upstream sequences and further subgrouped based on domain types and domain combinations called “domain cassettes”31. We hypothesized that IE adhesion to different receptors will correlate if the adhesion interactions are mediated by different domains of the same PfEMP1. We therefore examined the correlations between IE binding levels to the various receptors, and found a large cluster of correlated interactions with the receptors integrin α3β1, integrin α5β1, integrin αvβ3, P-selectin, E-selectin and PECAM-1  was 10.7% and HbAS (including 3 children with HbSS and 2 children with HbSC types) was 7.6%. Sickle cell trait did not significantly reduce IE adhesion to any of the receptors. Hemoglobin AC significantly reduced IE binding to the receptors CD36 and integrin α5β1, and its effect to reduce binding to integrin α3β1 approached significance Age affected the binding patterns to the various receptors in one of 3 ways: binding decreased as children aged; binding increased as children aged; binding was stable across the early years of life. IE binding to the receptor JAM-B significantly decreased with age. Conversely, IE binding to the receptors CD36 and integrin α5β1 significantly increased with age. IE binding to the receptors ICAM-1, ICAM-2 and integrin α3β1 also increased with age, and these relationships approached statistical significance. IE adhesion to the receptors TSP, E-selectin, PECAM-1, VCAM-1, P-selectin, CSA, EPCR, vitronectin, cellular fibronectin, laminin, integrin αvβ3, JAM-A and VE-cadherin did not change with age . Some of the novel receptors described here might similarly be part of a multi-receptor adhesion process that involves both rolling and firm adhesion types, rather than unrelated adhesion events. Further, IE binding levels to members of the integrin family, PECAM-1, P-selectin and E-selectin correlated, which could be related to the polyclonality of clinical isolates, multi-receptor binding of individual parasites, or both.IE adhesion under flow was described as a multistep process that includes tethering and rolling followed by firm adhesion, for example rolling on the receptors P-selectin or ICAM-1 and then firm adhesion to CD36 , and to purified CD36 and ICAM-1, although in one study adhesion to HUVECs  was unchanged47.We examined hemoglobin variants for their effects on IE adhesion. While hemoglobin AS has been consistently associated with protection from severe malaria, earlier studies have differed as to whether AS also reduces the risk of infection or the density of parasitemia48, while HbAC might confer protection from severe malaria by reducing parasite adhesion.In the current study, hemoglobin AC significantly reduced the level of IE binding to endothelial receptors CD36 and integrin α5β1, while sickle cell trait did not significantly reduce the level of IE binding to any of the endothelial receptors. In this cohort of children, parasite densities were significantly lower among children with hemoglobin AS (p < 0.0001). These results suggest that HbAS confers protection from severe malaria and reduces parasite densities by other mechanisms such as enhanced phagocytosis of ring-stage parasites37. We found that IE from children with blood group O bind at significantly higher levels to ICAM-1, an unexpected pattern for a host factor associated with protection from disease. These findings could be explained if the binding interaction involved is unrelated to severe malaria, and/or that the protective effect of blood group O is not mediated by cytoadhesion to endothelial receptors but by other adhesion mechanisms like decreased IE rosetting39.Blood groups A, AB and B were associated with a significant reduction of IE binding to the receptor ICAM-1 compared to blood group O. The protective effect of blood group O on severe malaria has been inconsistent in earlier studies, although recent genome-wide association studies observed that blood group O reduced the risk of severe malaria23, but no consistent pattern has emerged between studies. Severe malaria was related to ICAM-1-binding parasites in some but not other studies22, and ICAM-1 polymorphisms did not correlate with severe malaria risk49. Clinical isolates from children with severe malaria bind EPCR (and ICAM-1) in Tanzania at higher levels15, and in Mozambique a higher proportion of IEs from children presenting with seizures bound to the receptor gC1qR/HABP1/p3223. In the current study, severe malaria IE adhered at similar levels to all the receptors.A number of receptors have been proposed to mediate severe malaria IE binding52. An overlap in the PfEMP1 repertoire among young children including those with severe malaria could yield similar IE adhesion properties, such as we have observed. Furthermore, age should be assessed for its confounding effects in studies to associate parasite adhesion to malaria severity. Of note, median age did not differ between children with or without severe malaria in the current study, and the rates of severe malaria remained stable post-infancy, such that age should not be a confounding factor in the results reported here.We propose two explanations for the lack of a clear distinction in binding levels to the different receptors between IEs from children with severe versus non-severe malaria. First, earlier studies demonstrated that IEs from children with severe malaria as well as IEs from young children in general are more commonly recognized by plasma from the community, suggesting that these IEs may surface-display a shared repertoire of PfEMP1Second, peripheral blood isolates are likely to be a mixed pool of parasites, which might variably derive from those causing and those not causing a specific disease presentation. For example, in our studies of pregnancy malaria, we showed that placental parasites have a unique binding phenotype and uniformly bind to the receptor CSA. However, peripheral blood parasites from the same women sometimes bind to CD36 as well as CSA, or to CD36 alone, demonstrating that peripheral samples do not always reflect the phenotype of the sequestered parasites causing a specific syndrome. Notably, parasites selected to bind CSA lose the ability to bind CD36, and hence the peripheral blood samples that yield evidence of binding to both CD36 and CSA, are likely to represent mixed pools of parasites, rather than a single pool of parasites that bind multiple receptors.In summary, the current study has identified several novel receptors for IE adhesion such as JAM-B, cellular fibronectin and laminin, and described distinct binding interactions that are modified by red cell variants, blood group and host age. This large scale study demonstrates the complexity of associating IE binding phenotypes with disease severity. The lack of a significant association between severe malaria and adhesion to any of the receptors suggests that either IE associated with severe malaria do not have a distinct adhesion phenotype compared to IE from young children without severe malaria, or severe malaria IE adhere to a discrete endothelial receptor not included in the panel evaluated in this study.Children participating in this study were enrolled between August 2010 and December 2014 into two longitudinal cohorts conducted as part of the Immuno-Epidemiology (IMEP) project in Ouelessebougou, Mali: 1. A birth cohort of pregnant women and their children; 2. Children aged 0–3 years. A parent or guardian gave informed consent for their child’s participation in the study, after receiving a study explanation form and an oral explanation from the study clinicians in their native language. The protocol and study procedures were approved by the institutional review board of the National Institute of Allergy and Infectious Diseases at the National Institutes of Health , and the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Bamako, Mali. All study methods were performed in accordance with the relevant guidelines and regulations of the institutional review boards.Active follow-up included clinical examination and blood smear microscopy for detecting malaria parasites, monthly during the rainy season (July-December), every two months during the dry season (January-June), and at any time the child was sick, up to the age of 5 years.Severe malaria was defined as parasitemia together with at least one of the following WHO criteria for severe malaria: >2 convulsions in the past 24 hr; prostration (inability to sit unaided or in younger infants inability to move/feed); hemoglobin <5 g/dl; respiratory distress ; coma (Blantyre score  <=2).in vitro culture for 16–20 hr. IE were enriched using the gelatin flotation method and adjusted to 0.2–20% parasitemia before binding assay. IE binding to specific receptors immobilized on Petri dishes was determined in a static binding assay. The panel of purified receptors included the following human endothelial molecules: CD36, ICAM-1, VCAM, PECAM-1, E-selectin, P-selectin, integrin α3β1, integrin α5β1, integrin αvβ3, JAM-A, JAM-B, VE-cadherin, vitronectin, ICAM-2 (R&D); TSP, CSA, cellular fibronectin, and laminin from placenta ; EPCR (My BioSource); EPCR produced in eukaryotic cells53 that was provided by Dr. Benoît Gamain. Bovine serum albumin (BSA)  was used as a negative control. 20 μl of recombinant proteins (10 μg/ml) were applied to a petri dish , and incubated overnight at 4 °C in a humid chamber. Prior to the adhesion assay, receptor solutions were removed and 20 μl of blocking solution (3% BSA in PBS) added and incubated for 30–60 minutes at room temperature. 20 μl of parasite suspension were applied, and after 30 minutes at room temperature, unbound IE and erythrocytes were removed by 3 washes with PBS. Bound IE were fixed with 0.5% glutaradehyde in PBS for 10 minutes and stained with 1% Giemsa for 2 minutes. Bound erythrocytes and IE were counted under 1000x magnification, and 20 fields with the highest IE counts were recorded. Raw binding data is included in Supplementary Table Blood samples were collected every 3 months during the first 2 years of life, every 6 months thereafter, and during every symptomatic malaria infection diagnosed at an unscheduled visit. Samples from malaria-infected children (defined by blood smear microscopy) were used in adhesion assays. Ring stage parasites in blood samples collected from children were allowed to mature to the trophozoite/schizont stages in Hemoglobin types  were determined using Titan III® Cellulose Acetate Plate according to the manufacturer’s instructions .Data were collected in standardized forms, entered and verified using DataFax .specific receptor − Especific receptor − IEBSA), and negative values were converted to 0. The relationships between adhesion to endothelial receptors and severe malaria, age, hemoglobin type, and blood group, were assessed using multivariate linear regression models adjusted for parasite density in the assay. P values of 0.0026 were considered significant to account for the comparison of 19 receptors. The analyses were carried out in R (version 3.3.2).The level of IE binding was adjusted for the background by subtracting RBC (E) count for the specific receptor and IE count for BSA (adjusted IE binding = (IEIncidence rates of severe malaria by age category were compared by Poisson regression model via GEE to account for correlation due to repeated measures for each child. At the individual level, number of severe malaria events were regressed on indicator variables of the age strata along with a log-offset of time at risk in that strata.The analyses included binding data from freshly collected parasites to relate to WHO defined severe malaria, hemoglobin type, blood group and age for study participants. All data analyzed during this study are included in the article (Supplementary Table Supplementary Information