The interaction between intestinal parasites and malaria is still not clear. Data in published literature are conflicting. We studied the effect of intestinal parasitic infection (IPI) on the clinical outcome of malaria in coinfected children.In a cross sectional study performed between October 2014 and September 2015, children infected with malaria, as demonstrated by the presence of asexual parasites in Giemsa stained blood films, were enrolled. Stool samples were obtained from participants and subjected to the formol-ether concentration technique for the detection of intestinal parasites. The Complete blood count was performed using an automated haematology analyser . The risk ratio, Pearson’s chi-square and the student T test were all performed as part of the statistical analyses. Statistical significance was set at p < 0.05.In all, 405 children successfully took part in the study. The children were between 1 week and 120 months of age (mean ± SD = 41.5 ± 33.5). Coinfection with intestinal parasites was observed in 11.6%. The rate of severe malaria (SM) attack in this study was 10.9%. SM was not observed to be associated with age (p = 0.377) or gender (p = 0.387), meanwhile coinfection with intestinal parasites was associated with age (p = 0.003). Among SM cases, IPI prevalence was higher in children with mild (WHO group 3) severe malaria (p = 0.027). Overall, IPI was not observed to be associated with SM (p = 0.656) or malaria parasite density (p = 0.185) or haemoglobin concentration (p = 0.205). The main clinical features of SM observed were hyperpyrexia (68.2%), severe malarial anaemia (61.4%), and multiple convulsion (52.3%).IPI was not observed to be associated with the severity of malaria, the malaria parasite density, and the haemoglobin concentration in coinfected children in Cameroon. The clinical outcome of malaria in children coinfected with intestinal parasites may depend on the geographical setting after all. Ascaris lumbricoides, may attenuate the severity of malaria in the presence of coinfection. In this cross-sectional study, we evaluated the effect of intestinal parasitic infection on the severity of malaria, malaria parasite density and the haemoglobin concentration in children coinfected with malaria and intestinal parasites in Cameroon. We did not observe any significant association between intestinal parasitic infection and severe malaria or malaria parasite density or haemoglobin concentration. Stratification of severe malaria according to the degree of severity revealed a significant association with intestinal parasitic infection, in which prevalence of intestinal parasites was higher in children with mild severe malaria. Analyzing the different species of intestinal parasite did not yield any significant association either. These findings are contrary to many research publication on the subject. Several factors could have contributed to our observation, including the regular deworming campaign organized by the Cameroon Ministry of Public health, accounting for the lower prevalence of intestinal parasitic infection, and also the geographical setting.Coinfection with malaria and intestinal parasites are common in Sub-Saharan Africa, particularly in impoverished and poor sanitary settings. The interaction between intestinal parasites and malaria in coinfected children is still not clear. Some published papers suggest intestinal parasites, especially  Plasmodium are known to cause malaria in humans including P. falciparum, P. ovale, P. vivax, P. malariae, and P. knowlesi. Among them, P. falciparum is the most virulent species responsible for a majority of cases and almost all malaria associated deaths [Malaria and intestinal parasitic infections (IPIs) are parasitic diseases that are highly endemic in Sub-Saharan Africa, especially in impoverished and poor sanitary settings. In 2013, there were 198 million cases and 584000 deaths as a result of malaria worldwide [Plasmodium and other confounding factors could equally contribute to the varying results observed in these studies [Infection with intestinal parasites especially helminths is strongly suspected to influence the incidence, parasite density and the clinical outcome of malaria in endemic areas. Earlier studies have reported increased susceptibility to malaria –8, incre studies –24.In Cameroon, like most Sub-Saharan African countries, coinfection between malaria and intestinal parasites is common in children –28. AcroThis was a cross sectional study performed between October 2014 and September 2015.Anopheles mosquitoes can be seen around homes. Buea has two seasons; the dry season (between October and March), and the rainy season (between April and September). In Buea, human malaria can be described as mesoendemic in the dry season and hyperendemic in the rainy season, with peaks at the beginning and towards the end of the rainy season. The population of Buea experiences an estimated 3.93 infective bites/person/night [Plasmodium falciparum accounts for up to 96% of malaria infections in this area [Buea (4°10′0″N 9°14′0″E) with an elevation of 870m  is located in the eastern slopes of Mount Cameroon. Buea is the capital of the Southwest Region of Cameroon. The population of Buea is estimated at 200,000  and it ion/night . Plasmodhis area . In addihis area ,34. Schohis area  to curb Febrile children (≤10 years) who came to consult in the outpatient department or emergency unit of the Regional Hospital of Buea were considered. The vital signs of the patients were taken and they were examined by the consulting physicians. Patients that were positive for malaria parasites by light microscopy were enrolled.Excluded from the study were patients with a history of anti-malarial treatment within one week prior to admission. Patients were also excluded from the study based on evidence of other infectious disease, such as typhoid, gastroenteritis, meningitis, malnutrition, upper respiratory tract infections or any other identified cause of anaemia other than malaria.The study protocol was approved by the Institutional Review Board (IRB) of the Faculty of Health Sciences, University of Buea, Cameroon. Administrative clearance was obtained from the delegation of public health in the South West region of Cameroon. Written informed consent was obtained from the parents or guardians of the patients prior to their inclusion into the study.Blood and stool specimen were collected from the participants. About 3ml of blood specimen was collected into test tubes containing EDTA anticoagulant following aseptic techniques. The parents or guardians were instructed to provide a teaspoon full of their child’s stool into a sterile leak-proof wide open neck stool container.Thick and thin blood films were prepared and stained with 10% Giemsa and examined using methods previously described . If paraThe haemoglobin concentration (Hb) was obtained from the complete blood count (CBC) results of the patient. The CBC was performed using the Mindray Auto haematology analyzer .The formol ether concentration technique (FECT) was used for the detection and quantification of intestinal parasites in stool samples from participants. Since hookworm eggs clear very rapidly, the FECT was performed within 30mins upon receipt of stool specimen following the proceedings described by Cheesbrough . BrieflySevere malaria (SM) was defined and categorized base on the WHO  criteriaSM was further classified according to the degree of severity using the WHO scheme  into 3 gData collected were keyed into Excel spreadsheets and analysed using Stata version 12.1 (StataCorp LP). Statistical tests performed included the risk ratio, and the Pearson’s Chi square test for group comparison, student T test for the comparison of group means. Statistical significance was set at p < 0.05.Plasmodium falciparum, 5 (1.2%) had mixed infection with P. falciparum + P. malariae, and 2 (0.5%) had mixed infection with P. falciparum + P. ovale.Out of the 637 children who were approached, 405 children met the inclusion criteria and were therefore enrolled. The ages of the children ranged between 1 week and 120 months (mean±SD = 41.5±33.5). Among the participants were 209 (51.6%) females and 196 (48.4%) males. Among the participants, 398 (98.3%) had single infection with Plasmodium falciparum. No significant association was observed between severe malaria and age (p = 0.377) nor was there any significant association between SM and gender (p = 0.387)  was observed in 44 (10.9%) of the 405 participants. All (100%) SM cases had single infection with = 0.387) .Stratified according to the degree of severity of SM, the distribution of the cases in decreasing order were: 26 (55.3%) for group 1; 17 (36.2%) for group 2; and 4 (8.5%) for group 3.The main clinical presentation of SM in decreasing frequency were hyperpyrexia 30 (68.2%), severe malarial anaemia 27 (61.4%), multiple convulsion 23 (52.3%), circulatory collapse 19 (43.2%), respiratory distress 16 (36.4%), jaundice 15 (34.1%), protraction 11 (25%), hyperparasitaemia 7 (15.9%), impaired consciousness 7 (15.9%), hypoglycaemia 6 (13.6%), cerebral malaria (BCS≤2) 5 (11.4%), frequent vomiting 4 (9.1%), and coma 2 (4.6%).The overlap of severe malaria anaemia with cerebral malaria and respiratory distress were observed in 4.6% (2/44) and 15.9% (7/44) participants respectively . One of Ascaris lumbricoides 36 (69.2%), Entamoeba spp. 12 (23.1%), and Hookworm 4 (7.7%).Among the 405 participants, 47 were coinfected with intestinal parasites giving a prevalence of 11.6% (95% CI: 8.7–15.1). No significant association was observed between the prevalence of coinfection and gender (p = 0.484) . On the Among the coinfected children, 6/47 (12.8%) had SM meanwhile among non coinfected children, 38/358 had SM (10.6%). However no significant association was observed between the presence of coinfection and SM  .Stratified according to the degree of severity, the prevalence of coinfection was highest among cases of group 3 (mild SM) of the WHO classification . A signiSpecies-specific analysis revealed no significant association between the different species of intestinal parasites and SM . Furthermore, the risk of developing SM was similar between all intestinal parasite species .The geometric mean parasite density (GMPD) in this study was 10332.7 (range: 65–160523). The GMPD was higher in children without IPI 10732.1 (range: 65–160523) compared to coinfected children 7290.6 (range: 171–82956). However this difference was not observed to be significant statistically (p = 0.185).Entamoeba spp. was generally rare across all groups (The average intensity (epg) of intestinal helminth was 28.2 (range: 2–134). The average intensity of intestinal helminths was higher in SM cases 36.8 (range: 8–105) compared to UM cases 26.9 (range: 2–134) . Howeverl groups .The mean Hb observed in this study was 10.6 (±1.8). The mean Hb was lower in children with IPI (10.4g/dl) compared to children without IPI (10.7g/dl) . HoweverA. lumbricoides, hookworm, Entamoeba spp. and mixed A. lumbricoides/Entamoeba spp. respectively. However no significant association was observed between anaemia and the infecting parasite species .The prevalence of anaemia were 44.4% (16/36), 50.0% (2/4), 57.1% (4/7) and 60.0% (3/5) in participants infected with Ascaris lumbricoides (69.2%). Other studies have also shown that Ascaris lumbricoides is the most prevalent species of intestinal parasites isolated from children [In this study, 11.6% of the participants were coinfected with intestinal parasites. These findings are similar to that reported by Njunda et al. . The prechildren ,46–49. HP. falciparum was the only parasite species observed to be associated with SM. P. falciparum is well recognised as the main cause of SM in endemic areas. Although there are reports of P. vivax also causing SM [P. vivax was not identified as a species causing malaria in this study. The main clinical features of SM in this study were hyperpyrexia, severe malaria anaemia and convulsion, which is similar to studies performed elsewhere [In the current study, it was observed that 10.86% of the participants had severe malaria (SM) based on the case definition of severe malaria by the WHO . The preusing SM , P. vivalsewhere ,56.Ascaris lumbricoides was observed to be associated with increased attack of SM. This could be attributed to the differences in study design—our study was a cross sectional study involving children infected with malaria whereas theirs was a case-control study. Furthermore, no significant association was observed between the intensity of helminthic infection and SM in this study (p = 0.272). In this study, coinfection with malaria and intestinal parasites was significantly higher in group 3 (i.e. mild SM) of the WHO classification of SM (p = 0.027). The main phenotype of SM in this group is persistent vomiting; infection with malaria parasites or intestinal parasites are associated with gastrointestinal disturbances which may results in nausea and vomiting. Concomitant infection with both parasites may increase the progression of the gastrointestinal disturbances leading to increase in the frequency of vomiting. This observation should however be interpreted with caution as just a few (6) cases of IPI was observed among participants with SM. Larger studies will therefore be required to confirmed this association.In the current study, no significant difference was observed in the prevalence of severe malaria in children coinfected with intestinal parasites 12.8%) compared to malaria monoinfected children (10.6%). This is contrary to studies associating IPI and protection from severe malaria ,20, as w.8% compaAscaris lumbricoides was observed to be associated with a 2-fold increase in malaria parasitaemia (suggesting a protective effect). The failure to observe any significant difference in the mean malaria parasite density between children with IPI and those without could be attributed to the study design. The cross sectional design of the present study, cannot differentiate between present and past infection; the Th2 polarization of immune responses to intestinal helminths due to previous exposure may still be maintained through certain period although the infection itself has been cleared at present.In the present study, the geometric mean parasite density was higher in malaria monoinfected children (10732.1 parasites/ul) compared to children coinfected with intestinal parasites (7290.6 parasites/ul). The interaction between intestinal parasites and malaria is not well understood. IPI especially helminths have been shown to have an immunomodulatory effect by inducing a Th2 host response. The host response will depend on the stage and intensity of helminth infection ,58. CleaTrichuris trichiura, cause anaemia by increasing blood and iron loss in the intestinal tract. However the difference in the mean Hb between IPI coinfected children and children without IPI was not observed to be significant (p = 0.205), which is contrary to the study by Nacher et al. [The mean Hb observed in this study was low in children coinfected with IPI compared to children without IPI. IPI, especially infection with hookworm and r et al. . MoreoveThe finding of no significant association between IPI and the clinical outcome of malaria in this study could be attributed to the low prevalence of IPI in the study area in addition to the other factors highlighted above. Studies involving larger number of cases with IPI are therefore required.In conclusion, 11.6% of the participants were coinfected with intestinal parasites. The rate of severe malaria attack observed in the study was 10.9%. Severe malaria was not observed to be associated with age or gender. The prevalence of IPI was observed to be significantly higher in children with mild severe malaria. The main clinical presentations of severe malaria were hyperpyrexia, severe malarial anaemia and multiple convulsion. IPI was not observed to influence the severity of malaria, the malaria parasite density and the haemoglobin concentration in children in the study area, which is contrary to many studies published in the scientific literature. As it may appear, the clinical outcome of malaria in children coinfected with intestinal parasites may depend on the geographical setting.S1 Table(DOCX)Click here for additional data file.


Further, there is no information on the influence of the sickle gene on the parasitic diversity of P. falciparum population in severe symptomatic malaria. This study was undertaken to assess the effect of the sickle gene on the parasite densities and diversities in hospitalized adult patients with severe falciparum malaria. The study was carried out in 166 adults hospitalized subjects with severe falciparum malaria at Sickle Cell Clinic and Molecular Biology Laboratory, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, Odisha, India. They were divided into three groups on the basis of hemoglobin variants HbAA (n=104), HbAS (n=30) and HbSS (n=32). The msp-1 loci were genotyped using a PCR-based methodology. The parasite densities were significantly high in HbAA compared to HbAS and HbSS. The multiplicity of infection (MOI) and multi-clonality for msp-1 were significantly low in HbSS and HbAS compared to HbAA. The prevalence of K1 (p<0 .0001) and MAD20 (p=0.0003) alleles were significantly high in HbAA. The RO33 allele was detected at a higher frequency in HbSS and HbAS, compared to K1 and MAD20. Sickle gene was found to reduce both the parasite densities and diversity of P. falciparum in adults with severe malaria.Although several studies have supported that sickle cell trait (HbAS) protects against  Malaria is the major public health problem in India and accounting 1.1 million of reported cases in the year 2011. The number of malaria deaths in subjects aged 5 years or older was higher compared to children younger than 5 years,3P. falciparum. msp-1 has extensive genetic diversityP. falciparum, notably in the genes of P. falciparum those affect the success and virulence of infection.11The merozoite surface protein 1 (MSP-1) is a leading vaccine candidate antigen and the most abundant surface protein on the blood stage of P. falciparum population in severe symptomatic malaria in children or adults. Moreover HbSS, a severe form of chronic hemolytic anemia remains a source of great suffering to patients, especially in a developing country like India where the numbers are staggering. Furthermore, when the patients with HbSS get malaria, there will be a superimposed acute hemolytic anemia that become a major cause of death in these patients. Therefore, there is an urgent need to investigate the association, outcomes and mechanism of interaction between HbSS and malaria to provide suitable protection against the potentially fatal threat of P. falciparum malaria. Given this we undertook this study to find out the influence of sickle cell gene on the parasitic diversity in the Block 2 region of the msp-1 in adult subjects with severe malaria.Surprisingly there is no information on the influence of sickle cell gene on the parasitic diversity of P. falciparum cases and 15% of malaria-related deaths in India.P. falciparum accounted for 87.8% of malarial infections.14The study was undertaken at the Sickle Cell Clinic and Molecular Biology Laboratory of Veer Surendra Sai Institute of Medical Sciences and Research, Burla in the state of Odisha, India. This hospital caters the population residing in the western part of Odisha state and the eastern part of Chhattisgarh state. This region has low perennial transmission of malaria with a high frequency of sickle cell gene (21%)P. falciparum malaria, were included in the study. The severity of P. falciparum malaria was defined as per WHO criteria.16Subjects aged 15 years and above, hospitalized in the Department of Medicine, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, between July 2007 to September 2008 and diagnosed to have severe Subjects with the following conditions were excluded from the study:Plasmodium species;subjects co-infected with other subjects with other sickle cell syndromes like HbSβ-thalassemia, HbSE, HbSC, HbSD-Punjab;children <15 years of age;pregnant women;subjects who refused to consent.P. falciparum examination was made by light microscopy (100 X) of thick blood smears stained by Giemsa. Parasite densities were counted against 200 leukocytes in thick blood films. All the subjects were screened for sickle cell gene by sickling slide test. Those found positive were subjected to agarose gel Hb electrophoresis (pH 8.6) and high performance liquid chromatography (HPLC) using the VARIANT™ Hemoglobin Testing System  according to the manufacturer’s guidelines. A complete blood count was done on an automated hematology analyzer . Biochemical parameters such as serum bilirubin, creatinine, urea, aspartate transaminase (AST) and alanine transaminase (ALT) were done in a semi autoanalyzer  as per the manufacturer’s instructions.P. falciparum infection was done by single step polymerase chain reaction (PCR).msp-1 located on chromosome 9 was analyzed by nested PCR for genotypingmsp-1 gene, three distinct allelic families have been described, namely K1, MAD20 and RO33.DNA was extracted from 5ml of blood by the standard phenol-chloroform method.msp-1. Family specific primers were used for the secondary reactions with 1μl of the primary PCR product. For msp-1, primer pairs specific for each allelic family  were used. The primer sequences and PCR conditions were as described by Zwetyenga et al.The primary PCR was done with 2μL of DNA as a template, using conserved primers for msp-1.The multiplicity of infection (MOI) was calculated by dividing the total number of fragments detected in the individual system by the number of samples positive in the particular system.The study was approved by the Institutional Ethical Committee.msp-1 family that is  in the three hemoglobin variants  subjects were made using the Kruskal-Wallis tests. The χ2 test was done to compare the prevalence of msp-1 alleles in the hemoglobin variants and the incidence of various subphenotypes in severe malaria subjects. P <0.05 was considered statistically significant.Statistical analysis was done using GraphPad InStat Version 3.00 for Windows. The difference in the MOI in the P. falciparum infection, 166 were finally included in the study  are provided in p <0.0001) and did not change with the age of the subjects  were observed among the isolates in the three groups studied. The length variations of msp-1 amplified products in the three groups have been depicted in p=0.03) in HbAS and HbSS subjects in comparison to HbAA  and MAD20  alleles were significantly high in HbAA in comparison to HbAS and HbSS subjects, while the prevalence of RO33 was comparable  in the three groups  and HbSS . However, it was similar when compared between HbAS and HbSS . The combination of clones detected in the P. falciparum isolate has been illustrated in The proportion of multiclonal isolates in the three groups HbAA, HbAS, and HbSS were 84.6%, 40%, and 37.5% respectively. This multi-clonality was significantly high in HbAA compared to both HbAS  were over-represented in any of the severe malaria sub-phenotypes  in the three groups of subjects .None of the P. falciparum malaria . This could be due to two reasons that are, the prevalence of the sickle gene in the western belt of Odisha state is 21%12We undertook this hospital-based study on 166 subjects in a tertiary care medical center located in western Odisha. The sizable number of adults with severe malaria could be due to the low and markedly seasonal transmission of malaria in this area. In a population with low, erratic and seasonal parasitic transmission most of the people lack acquired antiparasitic immunity (premunition), and symptomatic and severe malaria is found in all age groups including adultsria 30, 1.07%. ThiP. falciparum malaria, one of which could be increased the parasitic virulence of individual strains reflected by distinct msp alleles.22The age, sex distribution and baseline laboratory parameters like Hb, TLC and PLT were similar amongst the three groups. The death rate in HbSS was higher in comparison to other two groups, although it did not reach statistical significance. There are several causes of increased mortality in HbSS subjects infected with P. falciparum in post-capillary microvessels of the brain and other organs, the clearing effect of fever on parasitaemia and the increased level of parasitized HbAS red cells by macrophages.32The mean parasite densities of microscopically positive samples in the present study were significantly lower in HbAS adults when compared to HbAA. Although several authors have reported low parasite densities in HbAS during episodes of asymptomatic parasitaemia,In the four different African studies, undertaken in children with uncomplicated malaria, the parasite densities in HbSS were significantly lower in comparison to that of HbAA and HbAS.P. falciparum malaria that may overwhelm hosts’ immune systems, leading to resource competition amongst the parasite clones that might affect the host morbidity.P. falciparum population are associated with more virulent infection.P. falciparum. MOI has been shown to be reduced in clinical malaria due to the anti-parasitic properties of fever and cytokines.P. falciparum and found that multiclonal infections were lower in HbAS and HbSS subjects compared to HbAA.Multiple-strain infections are common in P. falciparum parasite.44 The lower MOI and clonality in HbAS and HbSS subjects in our study could be due to low parasite densities due to the inhibitory effect of HbS. Besides this other factors including Hb variants that can influence the parasite diversity are pre-hospital treatment status, age, transmission intensity, genotyping methods, phenotypes of malaria, pregnancy and parity, and immune status. In our study, 90% of the subjects had received pre-hospital treatment with anti-malarial drugs. The mean age was similar, and all the three groups of subjects came from areas with similar transmission pattern. We used the same genotyping method for all the subjects, and none of the subjects was pregnant. All the subjects in the three groups had severe malaria, and the subphenotypes of severe malaria were similar. So the only variable in the three groups which has influenced the parasite diversity is the presence of sickle cell gene in the HbAS and HbSS state.Several authors have reported a positive association between parasite diversity with densities of msp-1 allelic family distribution in African children with asymptomatic parasitemia in HbAA and HbAS subjects have reported inconsistent results.msp alleles family. In the present study, the presence of K1 and MAD20 was significantly higher in HbAA in comparison to HbAS and HbSS subjects.msp-1 allelic families was similar. However, the RO33 allele was over-represented in HbAS (73.3%) and HbSS (93.8%). The predominance of the certain allelic family in Hb variants could be due to reduced fitness of certain P. falciparum strains in erythrocytes containing HbS.28Studies of msp-1 allelic families was over-represented in any of the severe malaria subphenotypes in the three groups of subjects . Although some studies have reported over-representation of particular strain/strains of parasites in subphenotypes of severe malaria, these results could not be substantiated further in most of the studies.39We studied the association of the various allelic families with phenotypes of severe malaria in the three groups of subjects. None of the msp-1) may underestimate the genetic diversity of infection.P. falciparum population.This study has certain limitations. The use of a single genetic marker (In conclusion parasite densities and all the three parameters of parasite diversity namely MOI, clonality and allelic family distribution were significantly reduced in HbAS and HbSS subjects compared to HbAA. Given this provision of early effective anti-malarial treatment during severe illness and chemoprophylaxis of HbSS subjects in malaria endemic area will save valuable lives.


Plasmodium vivax AMA-1 and MSP-119 in a rural population of the Brazilian Amazon.Polyparasitism is a common condition in humans but its impact on the host immune system and clinical diseases is still poorly understood. There are few studies of the prevalence and the effect of malaria-intestinal parasite co-infections in the immune response to malaria vaccine candidates. The present study determines whether the presence of malaria and intestinal parasites co-infection is associated with impaired IgG responses to Plasmodium and intestinal parasites were detected by microscopy and molecular tests. Blood cell count and haemoglobin were also tested and antibody response specific to P. vivax AMA-1 and MSP-119 was measured in plasma by ELISA. The participants were grouped according to their infection status: singly infected with Plasmodium (M); co-infected with Plasmodium and intestinal parasites (CI); singly infected with intestinal parasites (IP) and negative (N) for both malaria and intestinal parasites.A cross-sectional survey was performed in a rural area of Rondonia State and 279 individuals were included in the present study. At recruitment, whole blood was collected and 19 were detected in 74 % of the population. The prevalence of specific IgG was similar for both proteins in all four groups and among the groups the lowest prevalence was in IP group. The cytophilic sub-classes IgG1 and IgG3 were predominant in all groups for PvAMA-1 and IgG1, IgG3 and IgG4 for PvMSP-119. In the case of non-cytophilic antibodies to PvAMA-1, IgG2 was significantly higher in IP and N group when compared to M and CI while IgG4 was higher in IP group.The prevalence of intestinal parasites was significantly higher in individuals with malaria and protozoan infections were more prevalent. IgG antibodies to PvAMA-1 and/or PvMSP-1P. vivax AMA-1 and MSP-119. However, IgG response to both AMA1 and MSP1 were lower in individuals with intestinal parasites.The presence of intestinal parasites, mainly protozoans, in malaria co-infected individuals does not seem to alter the antibody immune responses to  Plasmodium, etiological agents of malaria, and many species of intestinal parasites (protozoa and helminths) share the same geographic distribution area and both types of parasites can infect the same population of hosts.It is well known that polyparasitism is a common condition in humans, however, little is known about the interaction between parasites and its impact on the host immune system and clinical diseases . MalariaAscaris lumbricoides infection may protect against cerebral malaria [Schistosoma haematobium has a protective effect on the density of the Plasmodium falciparum infection, [Ascaris lumbricoides were more susceptive to either P. falciparum infection or acute malaria attacks [Plasmodium-specific antibody responses was observed [The implication of concomitant infection in humans has been evaluated mainly regarding the effects of intestinal helminth infections on falciparum malaria, obtaining conflicting results. While  malaria , 4 and Sfection, , 6 child attacks –9. In ot attacks –13. In r attacks –17. In h attacks , 19. So  attacks –26. In s attacks , 25 whilobserved , 23. Simobserved , 26.Plasmodium vivax [P. vivax is the most prevalent malaria species corresponding to 83.7 % of the 134,907 cases registered in 2014 and it is concentrated in the Amazon region where intestinal parasites infections are prevalent [The effects of helminths on falciparum malaria in humans remain uncertain and few data are available about the interaction between intestinal parasites and um vivax . In Brazrevalent , 29.19) [P. falciparum and P. vivax and they are involved in erythrocyte invasion [AMA-1 is expressed on merozoites and sporozoites as a type I integral membrane protein and MSP-1 is expressed abundantly on the merozoite surface and synthesized as a 195-kDa protein and sequentially processed into a cysteine-rich 19-kDa fragment (MSP-119) , 31. Theinvasion , 33.P. vivax proteins, apical membrane antigen-1 (AMA-1) and merozoite surface protein (MSP-119) in individuals co-infected with Plasmodium and intestinal parasites and in individuals with single infections.Therefore, the aim of the study was to determine the prevalence of co-infection of malaria and intestinal parasites and whether the presence of co-infection was associated with impaired IgG responses against The individuals who took part in this study were part of a previous study investigating the effect of intestinal parasites on the circulating levels of cytokines and inflammatory markers . The stuP. vivax and/or P. falciparum at the time of blood collection were subsequently treated using the chemotherapeutic regimen recommended by the Brazilian Ministry of Health. Participants positive for intestinal parasites were also treated.Ethical approval was given by the Fundação Oswaldo Cruz Ethical Committee . Informed and written consents were obtained from all participants. For all eligible participants a clinical examination was performed. Donors positive for Plasmodium. Parasitaemia was expressed as the number of parasites/µL of blood in the thick blood smear. To confirm the parasitological diagnoses, molecular analyses of all samples using primers specific for genus (Plasmodium sp.) and species  were done. PCR amplification and detection and the PCR primers used have been previously described [After written informed consent and an epidemiological survey from all adult donors or from parents of donors in the case of minors, blood samples were collected by venipuncture for serological assay and a thick and thin blood smear was prepared for microscopic detection of escribed . SubjectFor parasitological examinations, participants were requested to provide a morning faecal sample and a labelled screw-capped plastic container was provided. A single stool sample was collected from each subject on the following day and samples were screened for intestinal parasites and examined at the same day by direct wet mount microscopic and concentration techniques by a technician with expertise in intestinal parasites identification.19 and PvAMA-1 in plasma were determined by enzyme-linked immunosorbent assays (ELISA). The expression and purification of the recombinant proteins were performed as previously described [o-phenylenediamine and hydrogen peroxide. The absorbance was read at 492 nm using an ELISA reader . The results for total IgG were expressed as reactivity indices (RI), which were calculated by dividing the mean optical density (OD) values of tested samples by the mean OD values plus three standard deviations (SD) of 24 non-exposed control individuals living in non-endemic areas of malaria . As positive controls, five plasma samples from exposed native individuals with high antibodies OD levels for both proteins were used. Subjects were considered as positive to the corresponding antigen if the RI was higher than 1. An ELISA to detect the IgG sub-classes was also performed for positive responders. Plates were coated with antigen, blocked and incubated with plasma diluted 1:100 as in the ELISA for total IgG. After washing, plates were incubated for 1 h at 22 °C with mouse mAbs to human IgG sub-classes diluted in blocking buffer according to the manufacturer’s specifications. The mAbs were from clones HP-6001 for IgG1, HP-6002 for IgG2, HP-6050 for IgG3, and HP-6023 for IgG4 (Sigma) and have been used previously to characterize IgG subclass reactivity. After incubation, plates were washed and incubated for 1 h at 22 °C with peroxidase-labelled goat anti-mouse antibody (KPL) diluted 1:1,000 in blocking buffer.Specific IgG antibodies to PvMSP-1escribed , 36. The19 were: IgG1 = 0.1885, IgG2 = 0.2718, IgG3 = 0.1579, IgG4 = 0.1462.Plates were washed, incubated with ABTS  substrate solution, and the (OD) measured as described above. Sub-class-specific prevalence for each antigen was determined from OD values using three standard deviation (SD) above the appropriate mean OD of 24 non-exposed controls as the cut-off for positivity. The corresponding cut-offs for PvAMA-1 were: IgG1 = 0.1910, IgG2 = 0.2401, IgG3 = 0.1558, IgG4 = 0.1828, and for PvMSP-12) with Yates correction was used to calculate differences in seroprevalence between groups. Wilcoxon-Mann–Whitney rank sum test was used for comparison between M and CI groups. Differences of reactivity index values were calculated with a pairwise test for multiple comparisons of mean rank sums (Nemenyi-Tests) [Epidemiological and experimental data were stored in Epi- Info 3.5.1 . Statistical analysis were performed using the R statistical environment and all p values were adjusted with false discovery rate method . The risi-Tests) . P valuePlasmodium only ; individuals co-infected with Plasmodium and intestinal parasites ; individuals infected with intestinal parasites only  and individuals negative . The group N was defined as subjects with no symptoms, negative for Plasmodium by thick blood smear and PCR and negative for intestinal parasites by direct wet mount microscopic and concentration techniques. CI and IP groups were composed of individuals positive for intestinal parasite infection: helminths only (H), protozoa only (P), and both helminths and protozoa (P+H).Combining the results across all tests Table , 279 indP. vivax was the most prevalent species . The prevalence of intestinal parasites was significantly higher in individuals infected with malaria (75 %) than with those who were not infected (45 %). Of note, all study participants with malaria presented clinical symptoms, such as history of fever and headache, and no difference was observed in the parasitaemia levels between malaria and co-infected groups. Among the 146 individuals infected with intestinal parasites, ten different parasite species were detected  and six helminths . Giardia intestinalis and Entamoeba coli were the most prevalent protozoa, found in single infections or associated with other species of protozoa and helminths . The prevalence of protozoan in CI group (81.25 %) was not statistically different from IP (70.4 %). In both, CI and IP groups, Giardia intestinalis and Entamoeba coli were the most prevalent protozoan and Ancylostoma duodenale, Ascaris lumbrocoides the most prevalent helminths.In both M and CI groups, Table 19 is presented in Fig. 19 were detected in 74 % of the population. The prevalence of individuals that recognize both proteins (55 %) was higher than those that recognize a single protein (8 % PvAMA-1 and 11 % PvMSP-119). To determine whether the presence of co-infection was associated with impaired IgG responses, the prevalence of specific IgG directed to PvAMA-1 and PvMSP-119 between groups were compared  reaching almost 100 % in the group of individuals singly infected with malaria (M group). This confirms that PvAMA-1 and PvMSP-119 are immunogenic molecules during natural malaria infections and that the presence of intestinal parasites does not interfere in the antibody response to both antigens. However, it cannot be ignored the reduced IgG prevalence and RI to both proteins in the group of individuals singly infected with intestinal parasites (IP), even when compared with the Negative Group. These results cannot be compared to those that reported reduced levels of IgG directed to P. falciparum antigens in helminths and P. falciparum co-infection, since helminth infections counted for only 19.7 % of the intestinal parasites detected in IP and 29.6 % in CI groups [Plasmodium in the immune response. It seems that Plasmodium have some effect on intestinal protozoa and not the contrary since lower antibodies response is only observed in IP group. Chronic protozoan infections have previously been suggested to be associated with type 1-regulatory immune response and combined with the induced pro-inflammatory response induced by the Plasmodium could balance the anti-inflammatory effect of the response [The study of antibody responses to nfection –44. In m protein , PvMSP-1 protein , MSP-9 [ protein , and PvA protein , 48, 49.I groups , 24. On response . Studies19 antigens is important for evaluating protective activity as IgG subclasses differ in their immune effector functions and having such knowledge is important for understanding the immunity to vaccine development. The results of the present study confirmed studies that showed IgG1 and IgG3 isotypes, previously identified as protective to malaria, were the predominant subclasses in response to both antigens in all groups [19 in malaria and intestinal parasites co-infected individuals. However, in the IP group IgG1 reactivity index to MSP-1 were lower when compared to individuals from N. These results suggest that the presence of intestinal parasites can induce non-cytophilic antibodies that could counterbalance the production of cytophilic antibodies in IP group. The increased prevalence observed for non-cytophilic IgG2 response to PvAMA-1 in IP group does not seem to be due to intestinal parasites since N group also presented increased IgG2. In this group IgG4 RI to AMA-1 were also increased when compared to CI and N groups. Studies that investigate malaria and helminths co-infection reported a decrease of cytophilic IgG1 and IgG3 responses to MSP-1 and an increase in non-cytophilic IgG4 response to MSP-3 in individuals infected by Ascaris lumbricoides, Strongyloides stercolaris, Trichuris trichiura and Hymenolepis nana [Schistosoma infection and IgG1 and IgG3 directed to anti-malarial total extract was reported in individuals infected with Schistosomahaematobium and malaria [Schistosomahaematobium co-infection was also reported [Plasmodium, due to the anti-inflammatory effect of cytokines induced by helminths. However, the cytokine profile of this population have been published and for malaria-infected individuals (M and CI groups) the profile showed high levels of IL-1, IL-6, TNF, IL-10, and CRP and decreased levels of IL-17A while for malaria-negative individuals (IP and N) the profile was high levels of IL-17A, NO and decreased levels of IL-10 and CRP [Analysis of IgG isotypes response to PvAMA-1 and PvMSP-1l groups , 46, 51.pis nana . Similar malaria . In contreported . Althoug and CRP . TherefoP. vivax AMA-1 and MSP-1 proteins in all groups indicated that the participants had been exposed to malaria infection and the IgG subclass responses were largely in agreement with previously published results. Although in the present work there were changes in total IgG directed to PvAMA1 and PvMSP-119 in Intestinal parasites group, a decrease in IgG and in cytophilic responses associated to co-infections was not observed. These responses might perhaps relate to other factors such as antigen properties, number and time of exposure, host age and genetic determinants. Further studies should be conducted to determine the effect of intestinal protozoa in the immune response to malaria antigens.The presence of antibody responses to both


Cytokines play an important role in human immune responses to malaria and variation in their production may influence the course of infection and determine the outcome of the disease. The differential production of cytokines has been linked to single nucleotide polymorphisms in gene promoter regions, signal sequences, and gene introns. Although some polymorphisms play significant roles in susceptibility to malaria, gene polymorphism studies in Brazil are scarce.IFNG + 874 T/A, IL10A-1082G/A, IL10A-592A/C, IL10A-819 T/C and NOS2A-954G/C. Specific DNA fragments were amplified by polymerase chain reaction, allowing the detection of the polymorphism genotypes. The polymorphisms IL10A-592A/C and IL10A-819 T/C were estimated by a single analysis due to the complete linkage disequilibrium between the two SNPs with D’ = 0.99. Plasma was used to measure the levels of IFN-γ and IL-10 cytokines by Luminex and nitrogen radicals by Griess reaction.A population of 267 individuals from Brazilian Amazon exposed to malaria was genotyped for five single nucleotide polymorphisms (SNPs), IFNG + 874 T/A and NOS2A-954G/C between positive and negative subjects for malaria infection. Interesting, the genotype NOS2A-954C/C was not identified in the study population. Significant differences were found in IL10A-592A/C and IL10A-819 T/C genotypes distribution, carriers of IL10A -592A/-819 T alleles (genotypes AA/TT + AC/TC) were more frequent among subjects with malaria than in negative subjects that presented a higher frequency of the variant C allele (p < 0.0001). The presence of the allele C was associated with low producer of IL-10 and low parasitaemia. In addition, the GTA haplotypes formed from combinations of investigated polymorphisms in IL10A were significantly associated with malaria (+) and the CCA haplotype with malaria (−) groups. The IL10A-1082G/A polymorphism showed high frequency of heterozygous AG genotype in the population, but it was not possible to infer any association of the polymorphism because their distribution was not in Hardy Weinberg equilibrium.No differences were observed in genotype and allelic frequency of IL10A-592A/C and IL10A-819 T/C polymorphisms were associated with malaria and decreased IL-10 levels and low parasite density suggesting that this polymorphism influence IL-10 levels and may influence in the susceptibility to clinical malaria.This study shows that the  Plasmodium that affect humans have different pathogenic potential. However, beyond the pathogenic potential of the parasite, there are environmental factors, host genetics and parasite virulence associated to susceptibility and resistance to malaria  H, Sigma, UK) and NADPH β-nicotinamide adenine dinucleotide phosphate . Griess reagent  was added and proteins were subsequently precipitated by trichloroacetic acid . The tube contents were mixed and centrifuged ; two samples of each supernatant were transferred to a flat-bottomed microplate and their absorbances were read at 520 nm using a microplate reader . NO values were calculated from standard calibration plots.A modified Griess reaction was used to detect nitrite and nitrate ,37. The 2 test. The risk of malaria associated with polymorphisms was estimated using odds ratios (OR) and confidence interval of 95% (CI) with and without adjustment by age, gender, length of residence in an endemic area and number of previous episodes of malaria. Differences between malaria (+) and malaria (−) groups were estimated by Mann–Whitney and t tests. Relationships between epidemiological factors and cytokine levels were assessed by Spearman’s correlation The Hardy-Weinberg equilibrium (HWE) was assessed by χ2 test and Fisher Person. The genetic analyses correspond to codominant logistic regression model . The linkage disequilibrium was calculated by D statistic and frequency haplotypes by calculating the maximum likelihood estimator via the EM algorithm (Expectation Maximization) using two-step, step “E” (Hope) and step “M” (max) until convergence is achieved. Statistical analyses were performed using Graph Pad Prism software and SNPStats 5.0 . Statistically significant values of p < 0.05 was considered.Epidemiological and experimental data were stored in the Epi- Info 3.5.1 . Allele and genotype frequencies were estimated by gene counting and differences between groups by χP. vivax and/or P. falciparum indicating that the majority of the individuals were exposed to malaria parasite throughout the years. Among studied participants, 55.4% were male and the mean age was 30.99 ± 14.37 years. Comparing both groups, a higher frequency of males was observed in malaria (+) group. However, no statistical differences were identified regarding age, the length of residence in an endemic area and number of previous episodes of malaria between groups. Yet, the time since last infection was higher in malaria (−) group (Table P. vivax and 20 (27.4%) with P. falciparum, consistent with Plasmodium species distribution reported by the Brazilian Ministry of Health [The epidemiological surveillance shows that a significant proportion 93.16%) of the inhabitants reported a prior infection with 6% of thef Health . No caseP. vivax (n = 53) infected individuals when compared with the levels of IFN-γ (98.40 pg/mL [2.00-548.15]), IL-10 (1149.88 pg/mL [3.02-6540.45]) and NO levels (28.49 μM [12.99-40.31]) in P. falciparum (n = 20) infected individuals  and IL-10 (795.34 pg/mL [1.15-16782.93]), were higher in plasma of malaria (+) when compared with malaria (−) group (IFN-γ 19.44 pg/mL [1.46-3919.00] and IL-10 1.29 pg/mL [0.40-989.00]). The levels of NO did not differ significantly when the groups were compared  25.49 μM [11.65-75.85] and malaria (−) 35.41 μM [0.52-110.95])  a2 test for the genotype observed and expected for all polymorphism except for the IL10A-1082G/A . The IFNG + 874 T/A genotype and allelic frequencies did not differ between malaria (+) and malaria (−) groups. In both groups, the most frequent genotype for IFNG + 874 T/A was the homozygous mutant AA followed by AT heterozygous and homozygous wild T allele. The variant allele +874 A was more frequent than wild T. In contrast, the distribution of the IL10A-1082G/A genotype differed between malaria (+) and malaria (−) groups while allelic frequencies were similar in both groups. Although the heterozygous AG genotype were more frequent in both groups, this genotype was significantly higher in malaria (+) (p < 0.001). The polymorphisms IL10A-592A/C and IL10A-819 T/C were estimated by a single analysis due to the complete linkage disequilibrium between the two SNPs with D’ = 0.99. The A allele of IL10A-592A/C was always linked with the T allele of and IL10A-819 T/C and C with C. IL10A-592A/A and IL10A-819 T/T genotypes were more frequently observed in malaria (+) compared with malaria (−) group (p < 0.001), even when adjusted by sex and age. Similarly the wild alleles A/T were more frequent in malaria (+) than malaria (−) groups . Stratification of the individuals into haplotypic groups based on the tree promoter IL10A polymorphisms at positions −592/-819/-1082 yielded the following haplotypes distribution: Hap1 (CCG), Hap2 (ATA), Hap3 (CCA) and Hap4 (ATG) with frequencies that varied from 7% to 38% in both malaria (+) and malaria (−) groups. As shown in Table NOS2A-954G/C genotype in the population showed that the homozygous GG was more frequent in both groups and the genotype NOS2A-954C/C was not identified in the study population. The G allele was also the most frequent allele with similar distribution in both groups.The SNPs investigated, their location in the gene, the genotype and allele frequencies observed in malaria (+) and malaria (−) groups are presented in Table IFNG-874 T/A, NOS2A-954G/C and IL10A-1082G/A SNPs and the levels of their products in plasma  compared to subjects with IL10A-592 AC and -819TC (2.3 pg/mL [0.4-11233]) and wild homozygous individuals with IL10A-592AA/-819TT (18.4 pg/mL [0.4-16782]) (p < 0.0001). Wild homozygous individuals with IL10A-592AA/-819TT presented IL-10 levels three times higher than individuals carrying variant allele C, indicating an association between the AA/TT genotypes with clinical malaria risk  parasites/μL for heterozygous genotype AT, 11.72 (199–6518) parasites/μL for homozygous wild TT and 1861 (52–11546) parasites/μL for homozygous variant AA  parasites/μL for GC genotype and 2120 (50–17933) parasites/μL for homozygous GG  parasites/μL for the homozygous AA variant, 2200 (50–17933) parasites/μL for the heterozygous AG and 1906 (84–6000) parasites/μL for homozygous GG wild , IL10A  and NOS2A (−954G/C). Firstly, genotypic and allelic frequencies of the IFNG + 874 T/A polymorphism showed that the variant allele A was more common in the population as well as the corresponding homozygous AA genotype. The frequencies found in the study population, are in agreement with previous studies conducted in other Brazilian [The SNPs investigated include razilian  and Colorazilian . Howeverrazilian ,13.IFNG + 874 T/A polymorphism was observed between malaria (+) or malaria (−) groups. Few studies find associations between severe malaria and IFNG + 874 T/A polymorphism and these associations were weak and not significant after correction for multiple comparisons [IFNG + 874 T/A polymorphism and IFN-γ serum levels were not observed in this study, although high levels of IFN-γ were detected in malaria (+) subjects. The results of this study were different from Medina et al. that reported in a population of an endemic area in Brazil, a concentration of IFN-γ significantly lower in the serum of patients with AA individuals compared with T wild allele carriers [IFNG polymorphisms that were not effectively tagged by this marker cannot be excluded, and further studies of the locus are warranted.In the study population, no difference in the allelic and genotypic distribution of parisons ,43. In mcarriers . The posIL10A gene, investigation of three polymorphisms at positions −592, −819 and 1082 of the promoter region, were associated with the actual production of IL-10, which has an important role in the immune response to malaria infection [IL10A gene polymorphism at position −1082, have been associated with decreased production of IL-10 and clinical and severe malaria [et al. [IL-10A-1082G/A polymorphism with reduced risk to clinical malaria [IL10A-592A/C and -819 T/C polymorphisms, it was observed association with IL-10 production and parasite density. Interestingly, a high prevalence of the C alleles and homozygous variant -592CC/-819CC was found in malaria (−) group while a higher prevalence of wild alleles A/T and homozygous genotype -592AA/-819TT in malaria (+), suggesting that the C allele were at lower risk to have malaria due to its prevalence in the malaria (−) group. The homozygous genotype -592CC/-819CC was associated with a reduced IL-10 levels and low parasite density compared to other genotypes. Indeed, carriers of the C allele variant were low producers of IL-10 and presented low parasite density while carriers of A/T wild alleles were high producer of IL-10 and presented high parasite density. The presence of the CC genotype may create site for enhanced binding of repressor that favor reduced IL-10 production.In the nfection ,44,45. T malaria ,20,46. H [et al.  reported [et al. , while a malaria . Some st malaria ,20,47. IIL10A at positions −592/-819/-1082, no association was observed between the most frequent haplotypes CCG and ATA with the risk of having malaria. However, carriers of the less frequent haplotype GTA were more prevalent in malaria (+) group while carriers of the ACC haplotype were more prevalent in malaria (−) group. The haplotype analysis of these three polymorphism in the Amazonas state, the GCT allelic combination were associated with low risk of any form of malaria, this haplotype was not present in the study population in Rondônia State neither in Para State, both Amazonian endemic areas [IL10A promoter variants and protection against severe malarial anaemia and increased production of IL-10 [IL10A gene associated with malaria infection, IL-10 levels and parasite density. Similar IL10A haplotypes distribution were reported in a population from the State of Para another malaria endemic area in Brazil. However, the authors did not evaluate the IL-10 levels and did not find any influence of these haplotypes in susceptibility to malaria [Considering the haplotypes of ic areas . The dific areas . Study iof IL-10 . Howeverof IL-10 . A studyof IL-10 . In Braz malaria .NOS2A-954G/C polymorphism and susceptibility to malaria, NO levels or parasitaemia. Indeed, the G-954-C C allele was present in less than 4% of the study population, it is absent in Caucasian and is found at low frequency in Asia. [NOS2A-954G/C polymorphism have been reported in studies with tuberculosis and leprosy. In both studies, the allelic and genotypic frequencies were similar to the one found in this study, even though their population were from South and Southeast region [NOS2A-954G/C polymorphism and NO levels in malaria exposed individuals in endemic region of Brazil. Indeed, the NOS2A gene polymorphism have been associated with susceptibility to P. falciparum malaria and conflicting results have been obtained in studies that associate the presence of the G-954-C C allele and either risk of cerebral malaria or NO production [Finally, it was not found association between in Asia. . In contin Asia. . In Brazt region ,51. Althoduction ,52. It sIFNG + 874 T/A, IL10A-1082G/A and NOS2A-954G/C polymorphisms was not associated with the occurrence of malaria or with the production of its respective cytokine and nitric oxide products. The IL10A-592A/C and IL10A-819 T/C polymorphisms were associated with malaria and decreased IL-10 levels and low parasite density suggesting that this polymorphism influence IL-10 levels and may influence in the susceptibility to clinical malaria in Amazonian population.This study shows that


Plasmodium falciparum transmission, most of the childhood anaemia disease burden is being attributed to malaria. The pathogenesis of malaria anaemia, which may well be the single most frequent cause for childhood mortality worldwide, has been debated for many decades.Anaemia is a major health burden in sub-Saharan Africa. With an estimated two-thirds of children being affected, childhood anaemia causes poor cognitive development, reduced growth and impaired immune functions associated with increased mortality. In malaria-endemic regions, particularly in those with perennial In order to identify underlying mechanisms for malaria anaemia two studies were evaluated: a cohort study including 465 children that were followed weekly over eight months and a severe malaria case-control study including >4,500 children, both carried out in a hyperendemic malaria area of Ghana, West Africa. Human genetic polymorphisms of candidate genes were genotyped and tested for association with malaria anaemia in both studies.We found several genotype-phenotype associations, which had differing effects after stratifying for anaemia with and without complications. For instance, uncomplicated anaemia was found associated with the A allele of rs196854, which was associated previously with an increased expression of interleukin-6, a key mediator of the systemic inflammatory response.Our results suggest that there are at least two different underlying mechanisms of malaria anaemia, uncomplicated anaemia characterized by a slow and continuous development towards a deficit of red blood cells, and anaemia with complications characterized by a rapid and substantial loss of red blood cells. The uncomplicated form appears to be caused by chronic systemic inflammatory processes rather than malaria episodes or parasite densities, similar to what is found in other forms of anaemia of chronic disease (ACD).


Severe falciparum malaria may be complicated by prolonged haemolysis and recurrent fever after parasite clearance. However, their respective etiologies are unclear and challenging to diagnose. We report the first case of severe falciparum malaria followed by prolonged haemolytic anaemia and rhinomaxillary mucormycosis in a previously healthy adult male.A 30-year old Bangladeshi man was admitted with severe falciparum malaria complicated by hyperlactataemia and haemoglobinuria. Prior to admission he was treated with intravenous quinine and upon admission received intravenous artesunate and empiric ceftriaxone. Thirty hours later the peripheral parasitaemia cleared with resolution of fever and haemoglobinuria. Despite parasite clearance, on day 3 the patient developed recurrent fever and acute haemolytic anaemia requiring seven blood transfusions over six days with no improvement of his haemoglobin or haemoglobinuria. On day 10, he was treated with high-dose dexamethasone and meropenem with discontinuation of the ceftriaxone. Two days later the haemoglobinuria resolved. Ceftriaxone-induced haemolysis was the suspected final diagnosis. On day 16, the patient had progressively worsening right-sided facial pain and swelling; a necrotic ulceration of the hard palate was observed. Rhinomaxillary mucormycosis was diagnosed supported by microscopy findings. The patient initially responded to treatment with urgent surgical debridement, itraconazole, followed by two weeks of amphotericin B deoxycholate, however was subsequently lost to follow up.This case highlights the range of potential alternative aetiologies of acute, prolonged haemolysis and recurrent fever following parasite clearance in severe falciparum malaria. It emphasizes the importance of a high degree of suspicion for alternative causes of haemolysis in order to avoid unnecessary treatments, including blood transfusion and steroids. It is critical to consider and identify common invasive bacterial and rare opportunistic co-infections as a cause of fever in severe malaria patients remaining febrile after parasite clearance to promote antimicrobial stewardship and prompt emergency care. The mortality rate of adult severe malaria remains high at between 10 % and 30 %. In surviving patients recovery is usually complete, and neurological sequelae occur in less than 1 % , 3. ProlPlasmodium falciparum infection complicated by prolonged haemolysis after parasite clearance.Invasive fungal infections complicating severe falciparum malaria infections are rare, particularly mucormycosis –27. ZygoA 30-year-old previously well Bangladeshi male was admitted to a local tertiary care hospital with a 20-day history of fever, chills, rigors, headache, myalgias, and anorexia. He had been suffering from nausea, vomiting, diarrhoea, jaundice with scleral icterus and dark urine for five days. One day prior to admission, he was diagnosed with malaria on thick smear at a private hospital, however parasite density result was unavailable. There he was treated with intravenous quinine (750 mg 8-hourly x five doses) and azithromycin, due to limited availability of first line therapy of artesunate, before being referred for further management.Past medical history was significant for three prior malaria infections. His most recent episode of suspected vivax malaria was 30 days prior to admission, which was partially treated with an incomplete course of chloroquine. He had no history of jaundice, liver disease, transfusions, malignancy, or autoimmune disease. There was no family history of jaundice or iron treatments. He did not take any regular or traditional medicines. There was no history of betel nut consumption.oC, Glasgow Coma Score (GCS) 15/15, blood pressure (BP) 127/60 mmHg, heart rate (HR) 127 beats per minute, a respiratory rate (RR) of 24 breaths per minute with oxygen saturation (SaO2) of 93 % on room air, and body weight of 65 kg. He appeared unwell with pronounced scleral icterus, black urine, hepatomegaly and splenomegaly . There was no conjunctival suffusion or lymphadenopathy.On admission, he had a temperature of 39.7 Admission peripheral blood smear confirmed a diagnosis of falciparum malaria (360 parasites/μl). Severity criteria included hyperlactataemia (lactate 6.8 mmol/L), hyperbilirubinaemia , and 4+ haemoglobinuria (blackwater fever); hence the case was classified as severe malaria Table . He was 2 90 %), tachycardia (HR 106), hypotension (BP 100/80 mmHg), recurrent 4+ haemoglobinuria with severe microcytic anaemia (haemoglobin of 32 g/L) accompanied by elevated lactate dehydrogenase and bilirubin, consistent with acute haemolytic anaemia  was empirically substituted for amikacin and ceftriaxone given his renal dysfunction to cover potential extended-spectrum β-lactamase producers, and dexamethasone 5 mg 6-hourly was initiated for suspected autoimmune haemolytic anaemia. The haemoglobinuria resolved two days after steroid treatment and ceftriaxone discontinuation. His haemoglobin slowly recovered, reaching 76 g/L by day 20.Mucor,  was observed on his initial presentation with echanism , howeverechanism , and theechanism . In patiechanism . The onsechanism , 34. A dechanism . Since sThe differential diagnosis for acute onset haemolytic anaemia with positive IAT and negative DAT following parasite clearance includes alloantibody mediated from previous transfusion, drug-induced haemolytic anaemia, microangiopathic haemolytic anaemia, an intrinsic RBC abnormality, or may be a non-specific finding , 33. DelMucor morphology on slide microscopy . The liver dysfunction was likely due to ischaemia during hypotensive episodes.In this case it was difficult to definitively exclude a predisposing immunocompromising condition. However, bone marrow biopsy did not suggest an underlying haematologic malignancy, HIV serology was negative, and random glucose was normal in the setting of acute illness, although haemoglobin A1C testing was unavailable to definitely rule out diabetes. It is likely that malaria related immune suppression augmented by the administration of steroids and multiple blood transfusions with massive haemolysis (iron overload) contributed. The initial low parasitaemia is consistent with post-treatment presentation and the severity criteria present on admission are consistent with a delayed time to recovery in severe malaria patients from low transmission settings [Mucorales. Although the patient follow up was truncated there was a favorable in-hospital clinical response to itraconazole, which is in keeping with other reports of successful treatment with itraconazole monotherapy [In addition to urgent surgical debridement, the recommended first line anti-fungal therapy for the treatment for mucormycosis is amphotericin B deoxycholate or liposomal amphotericin B. Unfortunately, the availability of these medications is often inadequate in resource-limited settings. In this case, surgical debridement was successful but itraconazole was the only available anti-fungal with activity against otherapy , 45.Mucor from aspergillus as broad, aseptate hyphae at 90° angles were observed.The oral ulceration differential diagnosis includes squamous cell carcinoma, deep fungal infection, tertiary syphilis and acute necrotizing ulcerative gingivitis –49. In oThis case illustrates that both prolonged haemolysis and recurrent fever occurring after parasite clearance in falciparum malaria are challenging to diagnose. A systematic approach to the differential diagnoses is vital as management can be critically different. The potential diagnosis of ceftriaxone-induced haemolytic anaemia is important as this drug is widely used empirically. Although the incidence of ceftriaxone-induced haemolytic anaemia is low, this diagnosis should be considered and ceftriaxone discontinued to prevent unnecessary blood transfusions, steroid treatment and mortality , 33. StePotentially reversible drug-induced haemolytic anaemia may rarely complicate severe malaria following parasite clearance but needs to be considered to avoid unnecessary blood transfusion or steroid treatment.Ceftriaxone is a commonly used empiric antibiotic, however rarely (<0.1 % incidence) it may cause life threatening haemolysis. This diagnosis must be considered early as stopping the drug may be the only treatment needed and the haematologic response can be rapid.Caution must be exercised when considering steroid use in severe falciparum malaria, since no benefit has been shown, whereas there are potential adverse effects, such as in the present case where it may contribute to immunosuppression facilitating fungal infection.Recurrent fever after parasite clearance should prompt a thorough septic evaluation to avoid delays in diagnosis and overuse of empiric antimicrobials.Invasive fungal infections are a rare complication (<0.1 % incidence) of severe malaria but should be considered in individuals with prolonged fever after parasite clearance.Mucormycosis infection is a medical emergency requiring urgent surgical and medical management and carries high morbidity and mortality.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


The efficacy of mother-to-child placental transfer of antibodies specific to malaria blood stage antigens was investigated in the context of placental malaria infection, taking into account IgG specificity and maternal hypergammaglobulinemia. The impact of the resulting maternal antibody transfer on infections in infants up to the age of 6 months was also explored. This study showed that i) placental malaria was associated with a reduced placental transfer of total and specific IgG, ii) antibody placental transfer varied according to IgG specificity and iii) cord blood malaria IgG levels were similar in infants born to mothers with or without placental malaria. The number of malaria infections was negatively associated with maternal age, whereas it was not associated with the transfer of any malaria-specific IgG from the mother to the fetus. These results suggest that i) malaria-specific IgG may serve as a marker of maternal exposure but not as a useful marker of infant protection from malaria and ii) increasing maternal age contributes to diminishing febrile infections diagnosed in infants, perhaps by means of the transmission of an effective antibody response. Plasmodium falciparum-infected erythrocytes in the placenta is known to decrease maternofetal antibody exchange. Several studies have demonstrated an association between placental malaria (PM) and decreased maternal antibody transfer to the fetus, taking into account maternal antibody levels at delivery ). Briefly, standard curves were established using purified human IgG  to determine the concentration of specific antibodies. Each point was tested in duplicate.Enzyme-Linked ImmunoSorbent Assay (ELISA) ,16 was pSpecific and total IgG were measured using recombinant proteins diluted in phosphate-buffered saline (PBS) for specific IgG or an anti-human IgG  diluted in carbonate buffer for total IgG, both were coated at 0.1-μg/well on MaxiSorp Nunc plates  and blocked with 3% powdered milk, 0.1% Tween, 20 PBS. Maternal and cord blood samples were diluted at 1:200 for all IgG-specific, recombinant proteins except for AMA1  and total IgG . An anti-human IgG coupled with peroxidase  was used to reveal the reaction with TMB One . Plates were read at 450 nm.http://www.emvda.org/portfolio/project-index/optimalvac-project-completed), to determine antibody concentrations (μg/mL). Discordant duplicates (with a variation coefficient >15%) were re-tested.ELISA optical densities (OD) were analyzed with ADAMSEL FLP b039 software ). Placental antibody transfer was defined as the natural logarithm of the ratio between neonatal and maternal antibody levels expressed in μg/mL (ln (CORD/CIRC)).t-test. A chi-squared test was used to compare categorical variables .For quantitative variables, means were compared using a Student unpaired ariables . Linear bles (−1 ) and theThis study protocol was approved by the institutional Ethics Committee of the Faculté des Sciences de la Santé from the Université d’Abomey-Calavi in Benin and the Comité Consultatif de Déontologie et d’Ethique from the French Institut de Recherche pour le Développement. The nature of the project was explained in detail to the participants and informed consent was obtained from the women included in the study. All women signed informed consent before enrollment  and could withdraw from the study at any time .n = 54). Maternal hypergammaglobulinemia, maternal weight, infant gestational age and low infant birth weight were not affected by PM. The risk of PM was higher in primigravid mothers and in younger mothers  and lower total IgG levels (p = 0.026) in PM infection  for all antigens except MSP2-FC27 and total IgG  and respiratory (65.2%) febrile syndromes [Maternal age was negatively associated with the presence of a febrile infection  in 6-month-old infants . Given tyndromes ) and malThis study aimed to describe the neonatal transfer of maternal antibodies specific to malaria blood-stage antigens. It showed that i) PM was associated with a lower CORD/CIRC ratio of malaria-specific IgG, ii) maternal IgG transfer differed depending on malaria antibody specificity, iii) cord blood antimalarial IgG levels were equivalent after adjustment for hypergammaglobulinemia and PM and iv) older maternal age was associated with fewer malaria infections in infants.One limitation of this study may be the definition of PM based on light microscopy. As antibody transfer increases at the end of pregnancy, an active placental infection could reinforce the strong association with lower antibody transfer. Using more sensitive methods such as histopathology or molecular biology, which can determine low parasitemia in placental infections, could refine the results. However, a recent study in which placental infection was defined by light microscopy, quantitative PCR or histological examination provided results comparable to those in this study .The present study confirmed that hypergammaglobulinemia or high levels of specific antibodies in maternal blood were associated with a decrease in the CORD/CIRC ratio after adjustment for PM. Regarding the mechanism of placental antibody transfer, it has been suggested that the FcRn expressed by syncytiotrophoblasts in placenta binds IgG with subsequent internalization of the FcRn-IgG complex in endosomal compartments and release of maternal IgG in fetal circulation ,21. The Several studies, including one that uses the same cohort as the current study –29, estast year of life was also observed in another study [These results show that maternal IgG to MSP2-FC27 tended to be positively related to the number of non-malaria infections in infants under 6 months old, independently of PM. Given the few tests performed, these results should be interpreted with caution. However, a positive association between the transfer of IgG1 to AMA1 as well as IgG to parasite lysate and malaria incidence during the 1er study . Similarer study . Lastly,er study .P. falciparum. Several studies have already shown that maternal antibodies can inhibit infant responses to different diseases, such as measles or tetanus [Based on these studies and the present work, it seems that maternal IgG specific to malaria is a marker of maternal exposure to  tetanus –34. How et al. as “the concept that during pregnancy, significant cross-talk occurs between the maternal and fetal immune system with potential long-term effects for both the mother and child” [Interestingly, we found that the older a pregnant woman was, lower the number of malaria infections of her infant was, suggesting that effective maternal immunity acquired with age can be transmitted to offspring. This raises questions on how the immune response evolves in pregnancy and we speculate on the role of one or several maternal molecules. These molecules must i) be produced differently with age and ii) have the property of either crossing the placental barrier or inducing, by their presence at the maternal—fetal interface, tolerance/protection against malaria infections during the first 6 months of life. Cytokines and antibodies are two types of molecules with these properties, in agreement with a definition of transplacental immune regulation formulated by Santner d child” . Few clud child” . Newbornd child” . Similard child” , which md child” . Lastly,d child” . Based op = 0.598). Transfer of functional malaria antibodies to the fetus has been demonstrated by measuring their ability to inhibit parasite growth in cord blood and infant blood, but the experiment did not consider maternal blood or maternal age [P. falciparum erythrocyte membrane protein 1 (PfEMP1) antigen [This study did not explore the functionality of maternal antibodies, possibly one of the immune parameters evolving with maternal age (in that the total IgG quantity was not associated with maternal age resulting from a linear regression coefficient = −0.0038; rnal age . In fact antigen –41. MateP. falciparum in placental malaria. Why the degree of maternal antimalarial IgG transfer was not related to the incidence of febrile infections in infants’ first 6 months of life, but instead maternal age, needs further investigation.In conclusion, This study confirmed lower maternal transfer of IgG directed to several asexual stages of


Plasmodium falciparum malaria and endemic Burkitt’s lymphoma (eBL) has been an enigma for more than 50 years, since it was first observed that the occurrence of the two coincided [P. falciparum) and a mammalian virus (EBV) combine to cause cancer. While the mechanisms linking EBV to lymphoma development are becoming better understood, the link between P. falciparum malaria and eBL has remained completely unexplained until now. Finally, we have a breakthrough in this longstanding issue from two separate studies: one through investigation of human tissues and one in a mouse model.The link between oincided ,2. So cooincided ,4. EBV ioincided  and is coincided ,8. The soincided . The eniP. falciparum induces the DNA-mutating and double-strand-breaking enzyme activation-induced cytidine deaminase (AID) [P. falciparum. This enabled them to isolate GC B cells directly from individuals at risk for developing eBL. They went on to show that individuals chronically infected with P. falciparum malaria had higher numbers of GC B cells that expressed elevated levels of AID and, furthermore, had an extremely high level of GC B cells latently infected with EBV [P. falciparum-infected red blood cells directly caused a strong activation of AID in tonsil B cells in vitro. They additionally showed that this was due, at least in part, to the action of hemozoin, the metabolic product of hemoglobin digestion by P. falciparum parasites. Together, these results allowed the investigators to conclude that P. falciparum infection increases two major risk factors for lymphoma development. The first is induction of AID in GC B cells, putting them at increased risk for a translocation; the second is a much higher frequency of EBV-infected cells in the GC, increasing the chances that the translocation will occur in a cell that will tolerate it , the origins of sporadic BL , why eBL is predominantly a cancer of children, and why the tumor is located in very specific anatomical regions that tend to change with age (the jaw in children and the abdomen in adults). Nevertheless, these experiments represent an important breakthrough in our understanding of how P. falciparum and EBV interact to cause eBL. However, the limitations inherent in working with human populations and the lack of an experimental animal model of the disease has hindered a more detailed investigation.The first breakthrough occurred with the demonstration that se (AID) . This isse (AID) . Howeverse (AID) . Usuallyse (AID) . The stuwith EBV . To explerate it . The incP. falciparum infection would lead directly to AID-dependent DNA damage and translocations in the GC, ultimately producing lymphoma—a prediction impossible to test in an actual human infection. This prediction has now been confirmed by employing the P. chabaudi mouse model of malaria [P. falciparum. These authors went on to show that this resulted in the accumulation of widespread DNA damage in the GC cells, including translocations, which was associated with subsequent lymphoma development, but only when expressed on a p53-null background. One might speculate that the p53 knockout essentially complemented the lack of EBV infection in the mice and in the GC B cells (but see caveat below). What was lacking in this study was evidence that the rodent parasite P. chabaudi was in any way an accurate model for P. falciparum and, specifically, that the rapid expansion of GC B cells and induction of AID expression are actually seen in the human disease. This was conveniently provided by the Torgbor et al. study.The study described by Torgbor et al. predicted that  malaria . In thisP. falciparum and is primarily observed in regions of the world endemic for this species of parasite, namely sub-Saharan Africa and Papua New Guinea. While P. falciparum generally causes a more severe form of malaria, all species can cause chronic infections, and, thus, the reason for its specific association with eBL remains unanswered. Given this specificity, it is reasonable to question if P. chabaudi is an accurate model of P. falciparum infection. The Torgbor et al. study implicated hemozoin as playing a role in inducing AID expression in GC B cells through Toll-like receptor (TLR) signaling, but this mechanism was not tested in the mouse study. Furthermore, hemozoin alone was not as effective as complete extracts obtained from P. falciparum-infected red blood cells, suggesting other components also play a role.While the mouse studies provide a potential animal model for eBL, there remain serious concerns, most notably with respect to specificity. Human malaria is caused by five different species of Plasmodium; however, eBL is specifically associated with P. falciparum-specific protein PfEMP1 in polyclonal B cell activation and increased survival, leading the authors to propose a possible link to eBL [P. falciparum; thus, if PfEMP1 plays a key role in eBL, it could explain the specificity of the disease to P. falciparum. Like all Plasmodium strains, the rodent parasites, including P. chabaudi, express antigenic proteins on the infected red blood cell surface, but they are unrelated to PfEMP1. If it can be shown that PfEMP1 does indeed play an important role in the development of eBL, it would raise significant questions about the specificity of the mouse model.Recent studies have implicated the k to eBL . PfEMP1 P. chabaudi did not increase the incidence of lymphoma but only altered the tumors to a more mature phenotype. This is in contrast with P. falciparum, for which compelling epidemiological evidence has demonstrated that it dramatically increases the occurrence of eBL. Taken together, these observations suggest the mouse model may mimic only part of the human disease and lacks the specificity associated with P. falciparum.A second concern with the mouse model is that it was necessary to perform the infection on a p53-/- background. The p53-/- strain alone produces lymphoma, and infection with P. falciparum malaria and the development of eBL. The first paper [P. falciparum malaria is a risk factor for eBL because it drives a high throughput of EBV-infected cells through the GC, where it also deregulates AID, leading to DNA damage, c-myc translocations, and lymphoma (In summary, therefore, two recent, very complementary papers finally provide the missing link between infection with st paper  demonstrst paper  extendedlymphoma .


Haem oxygenase-1 (HMOX1) gene encoding HO-1 have been associated with several diseases including severe malaria. The objective of this study was to determine the allele and genotype frequencies of two single nucleotide polymorphisms; A(−413)T and G(−1135)A, and a (GT)n repeat length polymorphism in the HMOX1 promoter in paediatric malaria patients and controls to determine possible associations with malaria disease severity.Haem oxygenase-1 (HO-1) catabolizes haem and has both cytotoxic and cytoprotective effects. Polymorphisms in the promoter of the Plasmodium infection or asymptomatic carriers of the parasite were enrolled as controls. Blood samples from participants were extracted for DNA and allele and genotype frequencies were determined with allele-specific PCR, restriction fragment length analysis and microsatellite analysis.Study participants were Ghanaian children (n=296) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as having uncomplicated malaria (n=101) or severe malaria  or cerebral malaria (n=132)). Furthermore, 287 individuals without a detectable n repeats in the study participants varied between 21 and 46 with the majority of alleles having lengths of 26 (8.1%), 29/30 (13.2/17.9%) and 39/40 (8.0/13.8%) repeats, and was categorized into short, medium and long repeats. The (−413)T allele was very common (69.8%), while the (−1135)A allele was present in only 17.4% of the Ghanaian population. The G(−1135)A locus was excluded from further analysis after failing the Hardy-Weinberg equilibrium test. No significant differences in allele or genotype distribution of the A(−413)T and (GT)n repeat polymorphisms were found between the controls and the malaria patients, or between the disease groups, for any of the analysed polymorphisms and no associations with malaria severity were found.The number of (GT)HMOX1/HO-1. This current study did not find any evidence of association between HMOX1 promoter polymorphisms and malaria susceptibility or severe malaria and hence contradicts previous findings. Further studies are needed to fully elucidate the relationship between HMOX1 polymorphisms and malarial disease.These results contribute to the understanding of the role of  HMOX1 is located on chromosome 22q12 [HMOX1 is governed by multiple regulatory elements localized in the promoter of the gene, as well as by enhancers, responsible for HMOX1 induction in response to increased haem concentration [In malaria patients, a large number of infected erythrocytes rupture in the bloodstream, releasing considerable amounts of erythrocyte haemoglobin , which ime 22q12 , is apprme 22q12 . Transcrntration ,11. HO-1ntration . All of ntration  and ferrntration ,15, wherntration -18, and ntration . Howeverntration -22. Althntration -25.HMOX1/HO-1 activity due to polymorphisms in the HMOX1 promoter [n repeat length polymorphism in the HMOX1 promoter have been described [in vitro compared to the T allele [n repeat length polymorphism has been described in several studies in various ethnic populations with repeat size varying from 13 to 45 repeats and main alleles at 23, 30 and 39 repeats [n repeat length polymorphisms have been associated with many different diseases, including diabetes, cardiovascular, pulmonary, and neurological disease as reviewed by Exner et al.) and by Garcia-Santos & Chies, where long (GT)n repeats, associated with lower HO-1 expression, were identified as risk factors [Humans have been shown to differ quantitatively in their promoter -29. Two escribed ,27,28. TT allele . Finally repeats -35. This repeats ,37. (GT) factors ,39.Plasmodium falciparum infections and have demonstrated both increased expression of HO-1 during malaria infection [HMOX1 promoter polymorphisms and malaria disease severity [n repeat alleles and risk of severe malaria have been shown in human studies in both The Gambia, Myanmar, and Angola [Several studies have investigated a possible association between HO-1 and nfection -42 and aseverity -35,43,44d Angola -34. A lan repeat were assessed in 583 Ghanaian children with malaria from 0–15 years of age to search for possible associations with malaria disease susceptibility and severity.In the present study, the presence of the two single-nucleotide polymorphisms (T(−413)A and G(−1135)A) and the length of the (GT)P. falciparum parasitaemia of more than 10,000 parasites/μl, and an axillary temperature of more than 37.5°C. A total of 296 malaria patients were enrolled; 101 with uncomplicated malaria and 195 with severe malaria (defined as severe anaemia (n=63) or cerebral malaria (n=132)). Patients with a positive sickling test or any other disease than malaria were excluded as a criterion used in the study the samples were originally collected for. Blood samples from healthy sickle cell-negative children between 0 and 15 years of age were collected as control samples from Dodowa, a nearby community (n=287). Both patients and controls were included after signed consent from patients or guardians after receiving standardized information in local language. Both patient and control population is a mixture of several ethnic groups, possibly with a slightly more uniform population in Dodowa. However, Ga-Adangme is the dominating ethnic group in both populations. The study population is well described with very thorough patient characterization and has been studied extensively, among other things, with regards to mannose-binding lectin genotypes [The study population consisted of patients admitted to the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season (June to August) in 1995, 1996 and 1997, as described in detail in earlier publications ,46. All enotypes  and compenotypes . The stuUpon admission, venous blood samples were collected in EDTA-containing test tubes. Within two hours after collection, plasma was separated, and pellets frozen at −20°C. Genomic DNA was extracted as described previously . Whole gHMOX1 promoter; the T(−413)A and G(−1135)A. A simple allele-specific PCR was developed to detect the T(−413)A SNP. Two primer-pairs were designed to amplify a 307-bp target sequence based on the nucleotide sequence of Genbank S58267, sharing the same forward primer, and only differing in the 3’-nucleotide end of the reverse primers, making them allele specific. Furthermore, to increase the specificity of the allele-specific primers, a mismatch near the 3’ends were introduced , two bands of 23 and 202 bp  or all three bands (heterozygote).A restriction fragment length polymorphism (RFLP) analysis was used to detect the G−1135)A SNP. Primers, as described elsewhere , contain135A SNP.2O, 5.0 μl 2.0 μM primermix and 10.0 μl TEMPase Hot Start . The reactions were performed in a 96-well PCR plate  in a VWRi Duo Cycler . Conditions for amplification are provided in Table For both PCR protocols, one μl DNA extract was amplified in a 20 μl reaction mix consisting of 4.0 μl Hn repeat was amplified with a fluorescein-conjugated forward primer and an unlabelled reverse primer designed by Takeda et al. [2O, 2.5 μl 1.0 μM primermix and 5.0 μl TEMPase Hot Start DNA Polymerase . The reactions were performed in a 96-well PCR plate  in a VWRi Duo Cycler . Primer sequences and amplification conditions are provided in Table TM  and 0.25 μl GeneScanTM 500 LIZTM Dye Size Standard  in a 96 well MicroAmp® Optical Reaction Plate  and denatured for 3 minutes at 95°C before analysis with an ABI 3730 XL Genetic Analyzer . Subsequent allele scoring of the microsatellites was performed using GeneMapper version 4.1 . Alleles were divided into short repeats “S” (<27 repeats), medium “M” (27–32 repeats) and long “L” (>32 repeats) based on earlier classifications [A PCR product containing the (GT)a et al.  One μl Dications ,34,49. SDeviations from the Hardy-Weinberg equilibrium at the two loci; T(−413)A and G(−1135)A were tested using The Court Lab Calculator . Cut-offIn total, blood samples were collected from 287 controls and 296 patients; 101 with uncomplicated malaria, and 195 with severe malaria (defined as severe anaemia (n=63) or cerebral malaria (n=132)). All patients were children 0–15 years of age , whereas, the G(−1135)A genotype distribution was not in equilibrium,  and this SNP was therefore excluded from further analysis. Analysis of the T(−413)A alleles and genotype frequencies showed no significant differences between the control, uncomplicated or severe malaria  groups . Furthermore, logistic regression, adjusted for age and sex, showed no significant association between the T(−413)A alleles or genotypes and severity of malaria .The allele-specific PCR and RFLP analysis successfully determined the allele distribution of the T−413)A SNP in 556 of 583 samples (95.4%) and the G(−1135)A SNP in 478 of 583 samples (82.0%), respectively. Results were confirmed by sequencing of selected samples representing the six possible genotypes. As can be seen in Table A SNP in n repeat length polymorphism were successfully genotyped in 572 of 583 samples (98.1%). Sequencing of selected samples was performed to define the size of the repeats (data not shown). The distributions in the control, uncomplicated malaria, severe anaemia and cerebral malaria groups are shown in Figure n alleles were identified, ranging from 21 to 46 repeats. The majority of alleles had lengths of 26 (8.1%), 29/30 (13.2/17.0%) or 39/40 (8.0/13.8%) GT repeats. The alleles were categorized into: short “S” (<27 repeats), medium “M” (27–32 repeats), or long “L” (>32 repeats). Based on these three categories, the patients were classified as having a S/S, S/M, S/L, M/M, M/L or L/L genotype. The allele and genotype frequencies are shown in Table The (GT)mples 98.%. Sequen29, (−413)A/(GT)30 and T(−413)/(GT)40 were the most common combinations with frequencies of 22.4%, 30.4% and 19.1%, respectively. It seems that the longer repeats are more often present with a T(−413) allele than the shorter repeats (p < 0.001), however, no linkage disequilibrium (LD) was found (data not shown). In Table n repeat genotypes and the T(−413)A genotypes are shown. The most prevalent combinations were ML/TA (16.7%), LL/TT (14.9%), SL/TT (12.1%), ML/TT (10.6%) and SM/TA (10.0%), whereas the combination SS/AA was not found. A hypothesis based on earlier human studies was that the combination LL/TT should confer protection against severe malaria. However, analysis with the logistic regression model showed that no combination were more likely to develop severe malaria compared to LL/TT (p > 0.05).The T(−413)A and (GT)n repeat alleles were next considered together, and the frequencies are shown in Figure HMOX1 polymorphisms and severity of malaria. Based on earlier studies, it was hypothesized that certain polymorphisms in the promoter of the HMOX1 gene encoding HO-1 could confer protection against severe malaria. However, this study could not provide evidence for such an association.This study investigated the possible association between n repeat length polymorphism have been studied extensively in the past decade, relating short or long repeat alleles to the risk of many different diseases [n nucleotide repeats. However, this did not affect the length-category analysis since both the 29 and 30 repeat alleles were categorized as medium-sized and 38–40 as large. Studies outside Africa have found much lower frequencies of the 39-repeat allele than the current study of less than 3% in Japan [The (GT)diseases ,39. In tdiseases ,34. Howediseases . The majin Japan , Thailanin Japan  and Greein Japan , and absin Japan ). In conin Japan ,31,33,35in Japan .n repeats were associated with symptomatic malaria, however, the patients were mainly infected with P. vivax and only five severe cases were included [n repeats and severity of malaria was found, however, limited sample size in some groups might have influenced results and furthermore, the study group consisted of both P. falciparum and P. vivax infected patients [n repeat polymorphism and severity of malaria has been shown in two other African populations, this study could not confirm such association. In The Gambia, the allelic and genotypic distributions were different from this current study Thus, the severe malaria patients with short repeat alleles were more prevalent in The Gambia than in Ghana (50% vs 19%), and the long repeat alleles more prevalent in Ghana (43% vs 26%) [In both the Myanmar study and the two African studies in Angola and The Gambia, short repeat alleles were positively correlated with severity of malaria. Studies in Thailand and Brazil have shown conflicting results ,44. In tpatients . Althoug vs 26%) . This waHMOX1 has been associated with several diseases [HMOX1.As with pro-inflammatory cytokines for example, excessive levels are cytotoxic  and the diseases ,39, whicHMOX1 were also determined. No analysis was done regarding the G(−1135)A SNP, since none of the groups were in Hardy-Weinberg equilibrium. The T(−413)A SNP has been associated with differences in promoter activity, however, it has not been studied as extensively as the (GT)n repeat length polymorphism. The frequency distribution of the T(−413)A SNP reported here is similar to previous findings in a Chinese population [Two single nucleotide polymorphisms (SNPs) in pulation  and a stpulation . Howeverpulation . In contn repeat alleles were considered together, long repeats were mostly found with the T(−413) allele, which is consistent with the findings of the Japanese study [n repeat category genotypes were combined; no association with severity of malaria was found. Thus in summary, based on the findings of this study together with the fact that previously found associations between malaria and HMOX1 have shown effects in opposing directions suggest that malaria does not seem to be a major selective force on the polymorphisms of the HMOX1 promoter.When alleles of the T(−413)A SNP and (GT)se study . Howevern repeat allele frequencies found in this study are similar to those of other African studies. However, the association between the (GT)n repeat alleles and severity of malaria was not confirmed in this well-characterized Ghanaian population [n repeat alleles. In this population, the polymorphisms of the HMOX1 promoter are not associated with severity of malaria, and another selective force may be influencing these alleles.The GT)pulation . Further