Individuals living in malaria endemic areas generally harbour multiple parasite strains. Multiplicity of infection (MOI) can be an indicator of immune status. However, whether this is good or bad for the development of immunity to malaria, is still a matter of debate. This study aimed to examine the MOI in asymptomatic children between two and ten years of age and to relate it to erythrocyte variants, clinical attacks, transmission levels and other parasitological indexes.+-thalassaemia (-α3.7 type) were determined using PCR. Multiplicity of Plasmodium falciparum infection, i.e. number of concurrent clones, was defined by PCR-based genotyping of the merozoite surface protein-2 (msp2), before and at the end of the malaria transmission season. The χ2-test, ANOVA, multivariate linear regression and logistic regression statistical tests were used for data analysis.Study took place in Niakhar area in Senegal, where malaria is mesoendemic and seasonal. Three hundred and seventy two asymptomatic children were included. Sickle-cell trait, G6PD deficiency (A- and Santamaria) and αMOI was significantly higher at the end of transmission season. The majority of PCR positive subjects had multiple infections at both time points (64% before and 87% after the transmission season). MOI did not increase in α-thalassaemic and G6PD mutated children. The ABO system and HbAS did not affect MOI at any time points. No association between MOI and clinical attack was observed. MOI did not vary over age at any time points. There was a significant correlation between MOI and parasite density, as the higher parasite counts increases the probability of having multiple infections.msp2 genotyping method.Taken together our data revealed that α-thalassaemia may have a role in protection against certain parasite strains. The protection against the increase in MOI after the transmission season conferred by G6PD deficiency is probably due to clearance of the malaria parasite at early stages of infection. The ABO system and HbAS are involved in the severity of the disease but do not affect asymptomatic infections. MOI was not age-dependent, in the range of two to ten years, but was correlated with parasite density. However some of these observations need to be confirmed including larger sample size with broader age range and using other  Plasmodium falciparum strains co-infecting a single host. In malaria endemic areas, MOI can be a useful indicator of the transmission level. Data suggest that the average number of malaria parasite strains in an individual is well correlated to the transmission level /100 leukocytes) than after (7.6 [1.3–33.1]/100 leukocytes) the transmission season  when subjects were grouped into 2, 3, 4, 5 and 6–10 years, whereas the effect of age on PD was no more significant in January (P > 0.18).Before the transmission season, parasite density decreased significantly with age  were noted at any time points.P < 0.0001)  was significantly higher after the transmission season  of parasites from 3D7, 25% (24%) of parasites from FC27 and 40% (43%) of clones from both allelic families in June (January).The distribution of P > 0.1) or in January (P > 0.7). MOI was positively related to parasite density at both time points (P = 0.004 in June and P < 10-3 in January). However, as age was negatively related to PD, a multiple linear regression including age as an explicative variable was performed. Age remained no significant (P > 0.5) and the same pattern of results was obtained at each time points consistent with a positive association between MOI and PD (P = 0.008 in June and P < 10-3 in January).There was no significant association between age and MOI in June  had significantly lower P = 0.05) and number of concurrent clones (P = 0.05) were to borderline to be significantly lower, in alpha-thalassaemic children with sickle cell trait (n = 10) as compared to the others (n = 298).Parasite prevalence (P > 0.7).MOI has been shown to influence clinical manifestation; hence the association between MOI and malaria attacks was examined in present study. Seventy (45%) out of 154 children for whom the clinical survey was performed had a malaria attack. Of children who had at least one malaria attack, 57 and 84 percent had multiple infections in June and January, respectively. However, no significant association was observed between MOI and clinical presentation  and after (43/44 percent) the transmission season was similar. These figures are very close to those reported in Gambia [P. falciparum infection may differ according to geographical location, transmission intensity and season of sampling.The distribution of n Gambia , Papua Nn Gambia  and Senen Gambia  but diffn Gambia ,30. ThisAs has been proposed by previous studies ,32, highWhether or not MOI is influenced by RBC variants was examined by present study. ABO blood group had no impact on MOI at any time points. This probably reflects the fact that the ABO system does not affect the parasite densities rather the clinical outcome of the disease, where blood group O protects against cerebral malaria .et al [Previous studies on the association between sickle cell trait and MOI are contradictory ,15. No eet al . The reamsp2 variants, and that could eventually influence the MOI. In accordance with this, no significant increase in the MOI, after the transmission, was seen in the α-thalassaemic children. This suggests that α-thalassaemic RBCs may protect against invasion by certain parasite strains. However, these data contradicts findings reported by Mockenhaupt et al [P. falciparum infection. It is now evident that α-thalassaemia does not affect parasite density in individuals that go on to present clinical malaria [et al studied symptomatic patients, while present study comprised asymptomatic children. Differences in sample sizes of the two studies could also be a reason for contradictory observations. In any case these data indicate that α-thalassaemic individuals may be less susceptible to infection by certain parasite strains but once they get infected they are not protected against clinical malaria.Alpha-thalassaemic red blood cells have altered membrane properties ,34, whicpt et al , who did malaria -37. The et al [Similar to α-thalassaemic, G6PD mutated children did not show a significant increase in the MOI after the transmission season. This may be explained by early phagocytosis of infected G6PD deficient RBCs. Cappadoro et al  have shoPrevious studies regarding the variation of MOI over age have suggested that the influence of age on the multiplicity of infection is highly affected by endemicity of malaria ,9-11. ThReports regarding the relation of MOI and malaria morbidity are contradictory. A number of studies has suggested that high MOI may confer protection from subsequent clinical malaria -6. A fewP. falciparum infection in relation to other parasitological indexes and/or host factors, performed in different areas, methodology used by these studies should be taken into consideration.However, to be able to compare findings by different studies on diversity of The genotyping results generated by PCR may differ between different laboratories because of applying variable reagents and protocols or even handling . In addiP. falciparum strains, the protection conferred by G6PD deficiency is probably through clearance of the malaria parasite at early stages of infection, and that the ABO system and HbAS do not affect asymptomatic infection. These data also revealed that the multiplicity of P. falciparum infection in asymptomatic children living in a mesoendemic area was correlated with parasite density but not age, in the range between two to ten years. MOI did not influence the risk of clinical attack. However, to be able to draw a firm conclusion, this needs to be repeated and confirmed by others and/or using other methodology. Larger sample size and broader age range are also required to confirm observations by this study.In summary, results of this study suggest that; α-thalassaemia may protect against infection by certain MV performed parasite genotyping, participated in DNA extraction from filter papers and drafted the manuscript. MTB participated in study design and coordination and revised the manuscript. JA contributed to DNA extraction for parasite genotyping. AG performed the statistical analysis, helped to draft the results and revised the manuscript. FMN conceived of the study, participated in study design and coordination, carried out human genotyping, helped in statistical analysis and revised the manuscript. All authors read and approved the manuscript.


Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory.Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6–1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56–1.76 and 0.69–1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5–1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5–1.4 and 0.6–1.5).In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken.There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to  Malaria infection and other parasitic infections are widespread in developing countries. There is evidence from some studies that intestinal worm infections may increase the risk of developing febrile malaria. However, the evidence is mixed, and some studies have found no effect or even protective effects. A vaccine trial was recently conducted to assess the efficacy of a candidate malaria vaccine. Episodes of malaria were monitored. The vaccine was not protective, but data was also recorded on the prevalence of worm infections. The rates of febrile malaria did not seem to vary according to worm infection in this study. However, because of the relatively low prevalence of worm infection, the study did not have high power. Given the conflicting findings in the literature, and the potential for the effect of worm infection to vary geographically, it is important that larger, definitive studies are conducted, since even quite small effects might be important for global public health. Helminth and malaria endemic areas frequently coincide, and an interaction between the two would be of considerable public health importance The Th2 cytokine milieu induced by helminth infection is thought to drive the antibody response of malaria co-infected individuals towards the production of non-cytophilic subclasses , whereas protection against malaria is associated with the presence of the IgG1 and IgG3 cytophilic subclasses We recently conducted a randomized controlled trial of a T cell inducing vaccine in a malaria endemic area. 387 immunised children were monitored for episodes of malaria for 9 months after receiving either an experimental T cell inducing vaccine or a control (rabies) vaccination http://www.controlled-trials.com/isrctn/trial/. The study was performed with the permission of KEMRI National Ethics Committees, and COREC, the NHS Central Office for Research Ethics Committees. The children were healthy, and resident in the study area. The study area was limited to Junju sub-location in Kilifi district, Kenya. The entomological inoculation rate (EIR) is 22–53 infective bites/person/year Plasmodium falciparum malaria. Treatment with mebendazole and praziquantel was available in local dispensaries, but drug use was monitored among the study participants for the duration of the study by locally-based field workers.405 children, aged 1 to 6 years (inclusive), were randomized for either an experimental prime boost malaria vaccine or control vaccination (rabies). The trial was assigned registration number ISRCTN88335123 with the International Standard Randomized Controlled Trial Number Register (Eosinophilia was measured in blood taken one week post-vaccination (early May 2005). Single urine and stool specimens collected for microscopy in January 2006. Full blood counts were successfully analysed on 347 of the 387 children, and stool and urine samples collected for 315 and 294 children, respectively.P falciparum per µl was used to determine febrile malaria. This threshold was derived in previous studies in Kilifi for use in children above one year of age Children were seen weekly by field workers, and blood films made when the temperature was ≥37.5°C. Field workers lived in the study area, and parents brought their children for assessment between regular weekly visits if the child developed fever. Treatment for episodes of malaria was with the Government of Kenya recommended first line treatment, artemether-lumefantrine. In analysis, a threshold of 2,500 asexual parasites of Blood films were examined in duplicate by two microscopists, and examined a third time if there was a discrepancy. Eosinophil counts were measured by a COULTER® Ac·T™ 5diff CP. Wet microscopy was conducted on mid-morning terminal urine samples and stool.Children who were positive for schistosomiasis or gastrointestinal helminth infection were given praziquantel (40 mg per kg) or mebendazole (100mg twice daily for 3 days), respectively. Vaccination was complete by day 90, and treatment was given on day 150. All analysis is conducted on pre-treatment data.The primary analysis was a log rank test comparing the time to the first or only episode of malaria between worm infected and uninfected children (unadjusted). The hazard ratio and 95% confidence interval was also estimated by Cox's regression, adjusted for age group, ITN (insecticide treated net) use and village. Age group was a categorical variable with three levels . Village had 5 levels. ITN use was defined as sleeping every night under a treated net, which had less than three holes into which a finger could comfortably fit. The vaccination was not efficacious Poisson regression was used to estimate the incidence rate ratio taking into account all malaria episodes, adjusted for the same covariates. A period of 28 days after each malaria episode was deducted from the person time at risk.Ascaris lumbricoides (18%), Trichuris trichiura (4.5%) and hookworm ova (3%).Stool samples were examined from 326 children. 25% were positive for ova indicating gastrointestinal worm infection. The three most common infections were The Kaplan Meier (KM) plot  of cliniA. lumbricoides infection alone was also considered. The HR for first episode of malaria was 0.65  and in Poisson regression, the IRR was 0.67 .Further modelling of the hazard and of multiple episodes  excludedSchistosoma haematobium in urine, as identified by microscopy. The confidence intervals of estimates of clinical malaria risk among children with urinary schistosomiasis are therefore wide, and so data on the survival function is not shown in detail. The adjusted HR was 0.55  and the IRR was 0.69  had eggs of 6/ml) and 31% had mildly elevated counts (>0.5×106/ml). Peripheral blood eosinophil counts were conducted as a more sensitive marker of parasitic infection than microscopy. Neither Kaplan Meier plots , and gastrointestinal worm infections were seen in 11% of 1–2 year old, 25% of 2–5 year olds and 36% of 5–7 year olds (p = 0.003). Interestingly, eosinophilia became less frequent with age, from 63% of 1–2 year olds to 50% of 2–5 year olds and 42% of 5–7 year olds (p = 0.03).The average haemoglobin among children with gastrointestinal helminth infection was 10.47 g/dl (95% CI 10.2–10.8) and 10.21 g/dl (95% CI 10–10.4) among uninfected children, p = 0.23. For children with schistovuria the average haemoglobin was 10.83 g/dl (95% CI 10.3–11.4) and 10.33 g/dl (95% CI 10.1–10.5) among uninfected children, p = 0.16.The data presented here were acquired during a trial designed primarily to examine vaccine efficacy. Children with gastrointestinal parasites or urinary schistosomiasis were treated with mebendazole or praziquantel, respectively. However, treatment was delayed for 150 days after screening, and the majority of malaria episodes (25% of children) occurred before the children were treated. Only 6% of children had a malaria episode after treatment (which coincided with the end of the high transmission season). We did not identify altered susceptibility to malaria associated with helminth infection, determined either by direct microscopy or inferred by peripheral eosinophilia.A. lumbricoides infection are risk factors for hospital admission with malaria There were similar findings in Uganda A. lumbricoides infection did not show a tendency towards increased risk of malaria in infected children either.Microscopy was only performed on a single specimen in our study, and we did not use concentration techniques. This would have missed lighter infections. This misclassification may have introduced bias against detecting an association. In previous studies that identified an increased risk of malaria in schistosome-infected children, multiple specimens were examined, although the greatest increase in risk was associated with heavier infections A. lumbricoides infection alone, with CIs for Schistosoma haematobium the widest. The tendency is in the direction of decreased rather than increased susceptibility, so the possible increases in risk that could have been missed are not large for gastrointestinal helminth infections and eosinophilia, but quite large effects of schistosome infection might have been missed. The primary analysis was log rank testing of time to event. Assuming a constant effect over time, with the prevalence of gastrointestinal helminth infection at 25%, then a log rank analysis had 80% power to detect a 70% increase or 50% decrease in risk due to helminth infection. Power was very low for schistosomiasis, with 80% power to detect a 110% increase or 90% decrease, but 80% power to detect an 80% increase or 60% decrease for A. lumbricoides infection alone. However, as it turned out, the upper limits of the confidence intervals measured suggest that 60–80% increases in risk are unlikely, although greater decreases in risk have not been ruled out. Previous studies have suggested a doubling in risk of febrile malaria in schistosome-infected children The low prevalence of helminth infection limits the power of the study, but by how much? This can be judged by the confidence intervals associated with the estimates of efficacy, from power calculations, and from inspecting the KM plots. The CIs around the estimates of risk are narrower than a halving or doubling of risk in each analysis conducted. The narrowest CIs are those derived for all gastrointestinal helminth infections and eosinophilia, followed by On inspection of the KM plots, there is no marked separation of the helminth-infected and uninfected at any stage. Between 50 and 125 days, helminth-infected children have acquired slightly fewer cases of febrile malaria, but this difference is slight, and the lines converge by 150 days. After 150 days there is a slight reduction in the number of children with febrile malaria among those with moderate and high eosinophil counts. This suggests that further studies to examine the impact of helminth infection in this population would need to be considerably larger.The results of individual studies may be dependant on the transmission intensity of both worm and Plasmoidium infections. In our study, the moderately high malaria transmission meant that significant immunity had been acquired by children aged 5–7 compared with 1–2 year olds . However, worm infection is more frequent in the older age groups, and the interaction between worms and malaria may depend critically on being exposed to helminth infections while immunity to malaria is being acquired. We did not measure intensity of infection in this study, and it is possible that among older children, with more intense worm infections, there might have been a relationship between helminth infection and malaria. However, these children would have a low incidence of malaria. Previous studies identifying increased susceptibility to malaria among helminth infection have been conducted in settings of more prevalent helminth infection.Strongyloides stercoralis infection is also associated with eosinophilia, and would not have been identified by the stool examination conducted in this study. Eosinophilia is closely associated with the TH2 cytokine responses which might result in the increased susceptibility to malaria 50% of children had either mild or high eosinophilia, compared with a prevalence of 7% for urinary schistosomiasis and 25% for gastrointestinal helminth infection. The higher prevalence may reflect infections missed by microscopy, but also could be due to filarial infection (which we did not examine for). This study suggests that in a cohort of one to six year old children in a moderately high malaria transmission area are not at increased risk of malaria from concurrent helminth infection, as evidenced either by microscopy or by eosinophilia. In our study, the prevalence on helminth infection was relatively low, and heavy infections were probably infrequent. This reflects the age of the children enrolled. However, it would not have been practical to study older children at this transmission intensity, since immunity against febrile disease is acquired rapidly (the HR among five to six year old children was 0.29 (95% CI 0.15–0.58). In areas with lower malaria transmission, older children may retain susceptibility to malaria.Strongyloides stercoralis and eosinophilia. Only a large and detailed study of this nature will satisfy concerns about multiple infections, changing infection status of individual children, and increased susceptibility depending critically on worm burden and age of acquisition of immunity to malaria.What further studies should be conducted? An interaction between helminth infection and malaria is of considerable public health importance. If present only at lower transmission intensities, this would still account for a very significant number of infections globally


Malaria carries high case fatality among children with sickle cell anaemia. In Uganda, chloroquine is used for prophylaxis in these children despite unacceptably high levels of resistance. Intermittent presumptive treatment with sulphadoxine-pyrimethamine (SP) has shown great potential for reducing prevalence of malaria and anaemia among pregnant women and infants.To compare the efficacy of monthly SP presumptive treatment, versus weekly chloroquine for malaria prophylaxis in children attending the Sickle Cell Clinic, Mulago Hospital.Two hundred and forty two children with sickle cell anaemia were randomized to presumptive treatment with SP or weekly chloroquine for malaria prophylaxis. Active detection of malaria was made at each weekly visit to the clinic over one month. The primary outcome measure was the proportion of children with one malaria episode at one month follow-up. The secondary outcome measures included malaria-related admissions and adverse effects of the drugs.Ninety-three percent (114/122) of the children in the chloroquine group and 94% (113/120) in the SP group completed one month follow up. SP reduced prevalence of malaria by 50% compared to chloroquine ; p = 0.042. Six percent (7/122) of the children receiving weekly chloroquine had malaria related admissions compared to 2.5% (3/120) on presumptive treatment with SP. No serious drug effects were reported in both treatment groupsPresumptive treatment with SP was more efficacious than weekly chloroquine in reducing prevalence of malaria in children with sickle cell anaemia. Continued use of chloroquine for malaria chemoprophylaxis in children with sickle cell anaemia in Uganda does not seem to be justified.ClinicalTrials.gov Identifier: NCTOO124267 Sickle cell anaemia (SCA) is a major health problem in Uganda with an average of 25,000 babies born annually ,2. EightPersons with SCA are four times more susceptible to malaria than those with sickle cell trait. Malaria is a major contributor to morbidity and mortality in these children -7. It prIn Uganda, chloroquine chemoprophylaxis was first used in 1962 where it significantly reduced malaria incidence by 43% ,10. WhilIntermittent presumptive treatment is a new approach to malaria prevention. This strategy was first used among pregnant women among whom it was found to be very effective. It has been successfully used in Tanzania, Ghana and Mozambique among infants . The mosThe study was carried out at the Sickle cell Clinic situated in Mulago, Uganda's national referral hospital in Kampala, from October 2006 to February 2007. Malaria transmission in this area is mesoendemic and perennial but peaks during the rainy seasons  . The annChildren between six months and 12 years of age attending the sickle cell clinic were consecutively selected and enrolled if they met the inclusion criteria, that is: 1) living within 10 kilometers from the Hospital; 2) having a negative malaria smear; 3) parents or guardians consented to participation. Children with a history of allergy to sulphonamides were excluded; those on co-trimoxazole prophylaxis or those with any severe illnesses needing admission. The study was approved by Makerere University Faculty of Medicine Ethics and Research Committee and Uganda National Council for Science and Technology.This was a double-blind randomized controlled trial, where children between six months and 12 years of age attending the sickle cell clinic were randomized to receive either monthly SP or weekly chloroquine. The sample size of 242 was based on the assumption that the prevalence of malaria would 29.4% in the chloroquine group and 11.1% in the SP group ,19 with A paediatrician not involved in the study generated a set of 242 random numbers. These numbers were then assigned to two codes in blocks of 4 to 12. Each participant was assigned a serial enrolment number and a random number corresponding to the treatment code. The pills were packed in an opaque envelope labeled with both the study serial number and a random number corresponding to the intervention. The drugs were packed in envelopes and released to the study team only after an eligible participant had been enrolled. The drugs and placebo were similar in colour and shape. They were manufactured by the same company  and labeled using two codes. Each (CQ) sugar coated tablet contained 150 mg chloroquine base and SP tablet contained sulphadoxine 500 mg and pyrimethamine 25 mg. The treatment code was concealed to the study participants, the treatment nurse and the investigators. The randomization code was only released to the investigators following completion of data analysis.The drug were administered by a treatment nurse according to the body weight of the patients as follows: chloroquine 5 mg/kg and sulphadoxine(25 mg/kg)-pyrimethamine (1.25 mg/kg). Participants were observed for 30 minutes after drug administration and treatment re-administered if vomiting occurred.Plasmodium falciparum parasite. Children who developed malaria were treated according to the national treatment guidelines at the time .Patients were followed up weekly for one month. At each visit, a finger prick for malaria parasites and parasite density estimation was done, in addition to haemoglobin estimation by Drabkin's method. Parents were requested to report any adverse events to the investigators. A malaria episode was considered if the study participant had fever documented by a temperature of ≥ 37.5°C and presence of any Analysis was by intention to treat: that is all patients randomized were analysed.The occurrence of at least one episode of malaria in the two treatment groups was compared using Odds Ratios and 95% confidence interval. The student's t-test was used for continuous variables. Logistic regression analysis was used to predict variables affecting the occurrence of malaria.During this period 278 patients were screened and 36 patients did not fulfill the inclusion criteria. Two hundred and forty two were randomized to either SP (monthly) or chloroquine weekly (120 and 122 respectively). Seven patients were lost to follow up from the SP arm and 8 from the CQ arm. All these patients were lost follow up during the first week after enrollment. Five patients from each group that were lost to follow-up relocated to other places during the study period. The rest had different reasons such as a guardian's busy work schedule or having a sick guardian. Two hundred and twenty seven patients were followed up for one month (113 SP arm and 114 CQ arm). Baseline and laboratory characteristics (Table Only 14% (17/120) of the children in the SP arm contracted malaria compared to 24.6% (30/122) in the CQ arm by one month follow-up. Children receiving chloroquine were almost two times more likely to have malaria compared with those receiving SP (Odd's Ratio 1.98 95% CI 1.023 - 3.82).A higher proportion of children had malaria related admissions in the CQ arm, compared to those in the SP arm (5.7% versus 2.5%). Children receiving chloroquine were almost two and half times more likely to be admitted than those receiving SP.  though this was not statistically significant, p = 0.223.Reasons for admissions were as follows; SP group: malaria (2), malaria and anaemia (1), anaemia (2) septicaemia and anaemia (1). Chloroquine group: lobar pneumonia (1), malaria and anaemia (2), septicaemia (3), malaria with painful crisis (3), and malaria (2). Even though there were more all cause admissions in the chloroquine group (11/122) 9% than in the SP group (6/120) 5%, the difference was not statistically significant (p = 0.22)Side effects were assessed for on a weekly basis as children actively reported to the clinic. The proportion of children that vomited after administration of the drugs was 6.6% (SP) versus 11.5% . None of the skin rashes documented were suggestive of a drug reaction. No serious adverse drug reactions were reported in any of the treatment arms Table .Children receiving weekly chloroquine were two times more likely to get malaria compared to those on SP. This shows that SP was more protective, reducing the prevalence of malaria by 50% compared to chloroquine. This difference in efficacy between the two drugs could be explained by the fact that resistance to therapeutic treatment of chloroquine (60%-80%) is higher compared to SP (18% - 25%) in Uganda ,20.The other reason is that SP has a longer half life and its terminal elimination phase normally exceeds the minimum parasiticidal concentrations (lowest concentrations that give maximum effect). In contrast this does not occur for chloroquine .While SP may have failed as a treatment drug it is still effective for prophylaxis . SeveralIn the current study, 24.6% of children receiving chloroquine contracted at least one episode of malaria compared to 40% reported in the Ethiopian study . This isIn our study, SP reduced malaria related admissions by 50% compared to chloroquine, but this was not statistically significant. This suggests that SP may have a role in reducing malaria related admissions. This is in contrast to results from Ghana, where the reduction in malaria related admissions by SP was 39% in comparison to placebo among children without sickle anaemia.Although all cause admissions varied, the percentage of admissions was higher in the chloroquine group (9% versus 5%). Of note, admissions due to lobar pneumonia (1), painful crisis (3) only occurred in those receiving chloroquine. This might be because sulphonamides may offer an anti-bacterial effect against the diseases as has been reported among children with HIV infection . This imDrug side-effects such as vomiting, skin rash, pruritus, blurred vision were documented in a small proportion of the children (range 0.8% to 11.5%) ,20. No sMonthly presumptive treatment with SP was more efficacious than weekly chloroquine in reducing prevalence of malaria among children with sickle cell anaemia. Minor side effects such as pruritus, vomiting, blurred vision and skin rash were reported in a small proportion of patients while no serious adverse events wee documented. Monthly SP should be considered for malaria prophylaxis in children with sickle cell anaemia. Continued use of chloroquine for malaria chemoprophylaxis in children with sickle cell anaemia in Uganda does not seem to be justified.The authors declare that they have no competing interests.VN, GN, CM, DN and JKT designed the study. VN was responsible for patient recruitment. GN, DN, CM and JKT supervised the study. VN, GN and JKT participated in the data analysis and interpretation of the results.VN, DN and JKT wrote the paper with major contributions of the other authors. All authors have seen and approved the final draft of the manuscript.


Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis.Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II.In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of  Sox9 [Sox9 - positive chondroprogenitor cells produce cartilage-specific extracellular matrix (ECM) components like Aggrecan and Collagen II and undergo terminal differentiation into chondrocytes. Later in development, the cartilaginous matrices of the skeletal elements become vascularized and undergo ossification.The limbs of tetrapods form as mesenchymal protrusions of the somatic lateral plate mesoderm covered by an ectodermal sheath. The cartilaginous anlagen of the limb skeletal elements form from the centralmost region of the limb mesenchyme in a process called chondrogenesis (reviewed in ). ChondrSox9 -4. SubseInteraction of limb mesenchyme and ectoderm is important in the regulation of patterning, morphogenesis and differentiation of the limb bud ,6. The lA major signaling center in this process is the ectoderm. The subectodermal mesenchyme, which will form the dermal layer of the skin, retains its non-condensed mesenchymal morphology, whereas in deeper cell layers premyogenic and prechondrogenic condensations occur. This regionalization of condensed and non-condensed mesoderm appears to be under the control of the ectoderm, since factors produced by the ectoderm prevent differentiation of nearby mesoderm to cartilage -11. ClasIn the paraxial mesoderm, Wnt6 is known to act via the canonical Wnt pathway, in which, upon Wnt ligand binding, a transmembranous Frizzled receptor triggers an intracellular signaling cascade leading to the accumulation of cytoplasmic beta-catenin and the activation of the LEF/TCF-transcription complex to promote target gene expression ,17. A nuTo date, the molecular nature of the cartilage-inhibiting signals from the ectoderm, and the role of these signals during the molecular regulation of cartilage formation in the avian limbs, have not been established. Recently, it has been shown that Wnt3a along with FGF signaling is able to inhibit limb chondrogenesis . In thisSox9  and ColIIA   and ColIIA , after Wnt6-cell-injection at 24 h and 48 h post surgery. Conversely to the ectoderm ablation results shown above, we observed a severe downregulation of both, Sox9 and ColIIA expression, in the chondrogenic regions of the manipulated wing buds  was peeled from the mesenchyme on the dorsal side of the forelimb bud as far as possible towards the edges without disturbing the apical ectodermal edge ,15. Aftein situ hybridization.Transfection and processing of CHO cells producing Wnt-6 protein were done as described earlier . CHO conin situ hybridization was performed as previously described [Sox9 , chicken Collagen IIA .Embryos were washed in PBS and fixed overnight in 4% paraformaldehyde at 4°C, washed twice in PBT, dehydrated in methanol and stored at -20°C. Whole-mount escribed . SelecteWhole-mount alcian blue staining was performed to visualize the skeletal phenotype of the manipulated embryos. Specimens were firstly stained with 0.015% alcian blue in 80% ethanol and 20% acetic acid for 1-3 days, fixed and dehydrated in ethanol for 1 day, and cleared and stored in 100% methylsalicylate .PG-L performed the largest part of the experiments and participated in the design of the study and in the writing of the manuscript. SN participated in the performance of the experiments and documentation of data. RH and BC participated in the design of the study, evaluation of results and interpretation of data. MS participated in the design of the study, evaluation of results and interpretation of data, and wrote the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum malaria occurred in a UK airport among 30 travelers returning to the United States from East Africa. Molecular genotyping analysis indicated that all had been exposed to different parasites. The travelers’ use of chemoprophylaxis was poor; their perception of risk was limited.A cluster of 6 cases of  Although most malaria occurs in Africa and Asia, it is also found in travelers returning from malaria-endemic regions  within 6 months of return. The high attack rate in this group of students and the close temporal clustering of cases led us to examine the possibility that the group had experienced a mini-outbreak as a result of a point-source exposure.P. falciparum merozoite surface protein 1 (Pfmsp1) and Pfmsp2 loci as previously described . Many had little protective clothing with them and while waiting until dusk that evening, noticed a large number of mosquitoes of unknown species around them. This event occurred 20 days before first onset of symptoms. After visiting Uganda, the group returned to Nairobi on July 11, arrived in London on July 13, and went to London’s Heathrow Airport on July 20 for the flight back to the United States.Pfmsp1 and Pfmsp2 loci  is cause for concern  would have been on a 9-hour flight to the United States; consequences for the patients could have been fatal. These cases highlight the benefits of vigilant staff at airports, even for passengers boarding from non–disease-endemic areas. Prompt contact tracing enabled a missed diagnosis of malaria to be rectified at a US facility and highlights the advantage of actively seeking all those who might have shared the same exposure.The clustering of the onset of symptoms for patients 1–6 suggested a common timing of infection, although other unproven factors, such as the effect of atmospheric pressure changes during air travel, may have led to the synchronous onset of the cases. Molecular evidence suggests that despite synchrony of symptoms, the genetically heterogeneous parasite variants were unlikely to have come from 1 infectious mosquito. We estimate that at least 12 distinct haploid parasite genotypes were circulating among the 6 patients. Our data also demonstrate, for patient 2, that circulating parasite genotypes can change profoundly immediately after treatment, as previously observed for quinine-treated persons (Anopheles spp. peak abundance (Anopheles, and although the students did not recall being bitten by mosquitoes on other occasions, we cannot rule out infection later in the journey, which would be more probable given the date of onset of symptoms in nonimmune persons.The only reported intense exposure to mosquitoes was during the travel delay in Mwea. The main rice-growing season starts in June, which is also the time of This mini-outbreak demonstrates the need to encourage travelers to take malaria prophylaxis and to improve their knowledge about the risk of malaria in their area of proposed travel. It also underscores the value of alerting fellow travelers in the same party to the possibility of contracting malaria.


Malaria and anaemia are the leading causes of morbidity and mortality in children in sub-Saharan Africa. We have investigated the effect of intermittent preventive treatment with sulphadoxine-pyrimethamine or artesunate plus amodiaquine on anaemia and malaria in children in an area of intense, prolonged, seasonal malaria transmission in Ghana.2451 children aged 3–59 months from 30 villages were individually randomised to receive placebo or artesunate plus amodiaquine (AS+AQ) monthly or bimonthly, or sulphadoxine-pyrimethamine (SP) bimonthly over a period of six months. The primary outcome measures were episodes of anaemia (Hb<8.0 g/dl) or malaria detected through passive surveillance.Monthly artesunate plus amodiaquine reduced the incidence of malaria by 69%  and anaemia by 45% , bimonthly sulphadoxine-pyrimethamine reduced the incidence of malaria by 24%  and anaemia by 30%  and bimonthly artesunate plus amodiaquine reduced the incidence of malaria by 17%  and anaemia by 32%  compared to placebo. There were no statistically significant reductions in the episodes of all cause or malaria specific hospital admissions in any of the intervention groups compared to the placebo group. There was no significant increase in the incidence of clinical malaria in the post intervention period in children who were >1 year old when they received IPTc compared to the placebo group. However the incidence of malaria in the post intervention period was higher in children who were <1 year old when they received AS+AQ monthly compared to the placebo group.IPTc is safe and efficacious in reducing the burden of malaria in an area of Ghana with a prolonged, intense malaria transmission season.NCT00119132ClinicalTrials.gov  In Africa, malaria causes over a million deaths and many millions of episodes of illness in children less than five years of age each year The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Plasmodium falciparum is the dominant malaria parasite in the area and remains sensitive to SP and AQ. A study undertaken in 480 children aged 3–59 months with symptomatic malaria at the time of the trial, described in another publication, showed that the drugs used in this trial of IPTc were efficacious. The polymerase chain reaction (PCR) corrected parasitological failure rate by day 28 was 2.3% for AS+AQ and 6.8% for SP. However, the re-infection rate by day 28 was slightly higher: 16% for AS+AQ and 19% for SP This study was carried out in Hohoe district, Ghana. The transmission of malaria in the study area is intense with two seasonal peaks. The major wet season lasts from April to July and the minor one from September to November. The entomological inoculation rate during the study period was ∼65 infective bites per person per year (unpublished data). From a sampling frame of all villages and communities in the district, the number of children living in each community was determined from the 2000 census. Thirty communities with the required number of children for the trial (2125) were randomly selected by probability proportional to the number of children living in each community. Several meetings were held with the health authorities and with community leaders and care givers in each selected community to explain the study objectives and procedures. Caretakers of all children aged 3 to 59 months in the selected communities were invited to a central point in the community to allow screening and enumeration of their children.Each of the four treatment groups was sub-divided into three sub-groups to reduce the chance of accidental unblinding through recognition by trial staff of outcomes in one particular group. Thus, eligible children were randomized into 12 groups. The number of children to be allocated to each of the 12 subgroups was determined in each village/cluster by dividing the total number of eligible children living in each village (rounded to multiples of 12) by 12. For each village, the required number of tokens, marked with a study code, was prepared and mixed in a large container. Caretakers of eligible children who reported on the enrolment day picked a token from the container and their child was then allocated to the treatment group indicated by the token.After obtaining written, informed consent, demographic information and data on socio-economic status, including the use of bednets, were collected. A clinical examination was conducted and anthropometric measurements were made. A study identity card was given to the parents of each child and they were advised to bring their child to a study health facility if the child became ill.Sample size was estimated on the basis of the following assumptions: (1) the incidence of anaemia (Hb<8.0 g/dl) would be 0.28/child/year in the SP bimonthly group, 0.23/child/year in the AS+AQ bimonthly group and 0.18/child/year in the AS+AQ monthly group respectively ; (2) loss to follow up would be 10%; (3) the study needed to have 90% power to detect a 33% difference between the SP bimonthly and AS+AQ monthly groups (likely to be the least and most effective interventions respectively) at a 5% significance level. The estimated sample size needed to meet these requirements was 2125 children (531 per arm). We also assumed that the incidence of clinical malaria would be 0.5 /child/year in the placebo group, 0.35/child/year in the SP bimonthly group, 0.25/child/year in the AS+AQ bimonthly group and 0.20/child/year in the AS+AQ monthly group. A sample size of 2125 children would have 99% power at a 5% level of significance to detect a 43% difference in the incidence of malaria between the SP bimonthly and AS+AQ monthly groups. Since more children were identified in the selected villages than needed to meet the required sample size and because the caretakers were eager to enroll their children in the study, a total of 2451 children (613 per arm) were enrolled.Study drugs and matching placebos were supplied by Kinapharma Limited, Accra, Ghana. Their quality was confirmed at the London School of Hygiene & Tropical Medicine by dissolution and content assays for AQ and content assays for AS and SP. One of the investigator (MK) and six “drug-packers” packed the study drugs for each child in well labeled envelopes according to age and study group using the randomisation list for each village. MK and the “drug-packers” did not take any part in assessing adverse events or in measuring study outcomes.All doses of the study drugs were administered to children by community-based volunteers. The volunteers were selected by the mothers/guardians of the children in each community to administer drugs and collect information on morbidity and adverse events and were paid an allowance of $10 a month for the periods during which they worked. Tablets were crushed and mixed with water. Children aged 3–5 months received a quarter of a tablet , those aged 6–11 months half a tablet, those aged 12–23 months three quarters of a tablet (3/4) and those aged 24 months and above received one tablet each of SP, co-formulated AS+AQ or placebo. Children received study drugs every 28 days on six occasions.Field workers visited study children to solicit any adverse events 7 to 10 days after administration of study drugs. Reported adverse events were investigated and managed by a study clinician. Field workers visited the children once a week during the period of drug administration to enquire about their health and completed a morbidity form if a child had any illness. If a child had a history of fever or vomiting within the past 48 hours the parents were advised to take their child to the nearest health facility for examination and treatment.A passive surveillance system to monitor malaria and anaemia in study children throughout the study period was set up in the district hospital and in 21 health centres in the study area. If a child who presented at one of these facilities had fever or any features suggestive of malaria, a finger prick blood sample was collected for malaria parasite examination before treatment was given. Blood slides were read at the respective health facility to decide on treatment and read again in a central laboratory to confirm the diagnosis. Children with proven or presumptive malaria were treated with oral quinine according to the Ministry of Health (MOH) treatment guidelines.Cross sectional surveys were carried out at the end of the intervention period (November 2005), at the end of the following dry season (April 2006) and at the end of the following rainy season (November 2006). During these surveys, temperature, weight, height and mid upper arm circumference were measured and a finger prick blood sample was collected for determination of malaria parasitaemia, haemoglobin (Hb), and markers of SP resistance. Venous blood samples were collected from a sub population of enrolled children for determination of G-6-PD deficiency and the presence of haemoglobin C and S. This was done by sampling children who were due for blood donation at the time the clinician arrived at the meeting place for the cross sectional survey. A household survey was carried out in August 2006 to determine ITN use.Hb was measured for children attending a clinic with an axillary temperature of > = 37.5°C or a history of fever or vomiting during the past two days. Children found to have an Hb<6.0 g/dl were referred to the district hospital for management.Laboratory assistants, masked to study group, examined thick blood films for parasitaemia. A sample was considered negative only after 200 high power fields had been read. Parasite counts were converted to parasites per micro liter (µl), assuming a white blood cell count of 8000 leukocytes per µl of blood. If there was a discrepancy in the findings in a slide between the two initial technicians (positive or negative or a 50% or more difference in parasite density) a third, more senior microscopist read the slide and his reading was deemed to be the correct reading. A senior microscopist from the Noguchi Memorial Institute of Medical Research  examined all the positive blood films and a 20% random sample of negative blood slides for quality control. Haemoglobin was measured using Hemocue ® Photometer .Phenotypic evidence for Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency was determined by the methaemoglobin reduction method . Sickle cell genotyping was done by haemoglobin electrophoresis.Data from participants were recorded on specified forms and were checked by field supervisors and data manager for consistency and accuracy. All data were entered twice into a database using EPI Data software. The accuracy of data input was checked and validated using customized validation programmes. The cleaned data were converted to Stata version 8 file by a statistician  prior to analysis.P. falciparum at any level of parasitaemia. An additional case definition for malaria of fever or history of fever plus a parasite density of greater than 7000/µl was also used. Severe malaria was defined as a hospital admission which met the above definitions of malaria. Any symptom or sign that first appeared or increased in severity within seven days of drug administration was defined as an adverse event. Time at risk of primary and secondary endpoints started at the time of enrollment. Person time at risk for anaemia and malaria was calculated by subtracting the date of enrolment from the date of exit from the study. The date of exit for children who died was the date of death. The date of exit for children who were lost to follow up  was the last date of contact with the passive or active surveillance systems. Children who moved temporarily out of the study area but returned to take some of the courses of treatment remained as part of the study cohort. Four weeks per episode was subtracted from the person time at risk for children who had an episode of malaria that was treated with an effective antimalarial. Children who had an episode of anaemia were not sensored from the person time at risk because it is not clear how long a child will be risk free after an episode of anaemia.The primary trial endpoints were the incidences of anaemia, defined as an Hb<8.0 g/dl and severe anaemia, defined as an Hb<5.0 g/dl. Malaria anaemia was defined as anaemia, defined as above, plus malaria parasitaemia. Clinical malaria was defined as a reported history of fever within 48 hours or a measured temperature of 37.5°C or greater with a positive blood slide showing asexual forms of Analysis of the primary and secondary outcomes was carried out on an intention-to-treat basis in which all randomised who received the first dose of IPTc children were included until completion or exit from the study due to withdrawal of consent, loss to follow up or death. Proportions of outcomes were analysed using chi-square test. Differences in means were calculated using t test for comparison of two means. All analyses were done with STATA software version 8.2. The incidence rate ratio (IRR) was estimated using the placebo group as the comparator. Protective efficacy was calculated as (1−IRR)×100. The Kaplan- Meier method was used to plot the time to the first episode of anaemia and malaria attack after the start of the intervention. Poisson regression was used to estimate the protective efficacy of the IPTc drugs adjusted for the effect of Hb type, G6PD deficiency and bednet use in a subset of children who had data on Hb type and G6PD status.The study was approved by ethical committees of the Ghana Health Service/Ministry of Health (GHS/MOH) and the London School of Hygiene & Tropical Medicine. A Data Safety and Monitoring Board (DSMB) was established to monitor the trial. The DSMB approved the study protocol and its analytical plan.The sponsor had no role in the study design, data collection, data analysis, data interpretation or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Two thousand, seven hundred and fifty-five children were screened in May 2005 and 2602 (94%) were eligible for inclusion in the trial. However, only 2451 (89%) reported for enrolment and received the first IPTc dose. The number of children assigned to each group  and the follow up rates achieved in each group are shown in During the six months of the intervention period, the incidence of anaemia, the primary trial endpoint, was significantly lower in all IPTc groups compared to the placebo group . ProtectThe incidences of clinical malaria in children attending an OPD with any parasitaemia or high density parasitaemia during the intervention period were significantly lower in all three IPTc groups compared to the placebo group . During The differences in OPD attendance, all cause and malaria specific admissions to the hospital between the intervention groups and the placebo group were not statistically significant. In the subset of 529 children for whom there were data on Hb genotype and G6PD status, the protective effect against malaria and anaemia adjusted for the effect of age, bednet use, Hb type and G6PD status was statistically significant in the AS+AQ monthly group compared to the placebo group .There were very few cases of anaemia or malaria in children in any of the groups during the post-intervention dry season and there were no statistically significant differences between the groups . During The prevalence of malaria parasitaemia was significantly lower in the AS+AQ monthly group at the end of the intervention period but it was significantly higher at the end of the dry season following the intervention period compared to the placebo group . There wAdverse events were reported slightly less frequently in each of the three IPTc groups compared to the placebo group throughout the intervention period (5.6 % vs 5.9%). Diarrhoea, vomiting, drowsiness and abdominal pains were the most frequently reported symptoms in both IPTc and placebo groups. The number of children who reported at least one adverse event or any specific adverse event did not differ significantly between the study groups. Ten deaths  occurred during the intervention period and 16 deaths  occurred during the 12 month post intervention period. There was no significant difference in the numbers of deaths between the IPTc and placebo groups  respectively.The protective effect of IPTc against malaria and anaemia observed in our study is consistent with findings from Senegal and Mali. Monthly SP+AS IPTc had a protective efficacy against clinical attacks of malaria of 86% (95% CI: 81%–90%) in Senegal Our study has shown that even in an area with up to six months of intense transmission, monthly IPTc with AS+AQ has substantial benefits in reducing the incidence of malaria and anaemia. The combined results of studies of IPTc suggests that for drugs such as SP and AQ monthly treatment is probably necessary to achieve a high level of protection. Although IPTc was effective in reducing episodes of anaemia detected at a clinic we did not find a significant reduction in the prevalence of anaemia at the end of the IPTc intervention period. This is consistent with the observation in Senegal and with the findings of IPTi studies in Tanzania We found that the prevalences of G-6-PD deficiency and haemoglobin S and C in the study area were similar to those reported from the northern and central parts of Ghana We did not find any significant increase in the incidence of clinical episodes of malaria associated with parasitaemia at any density or at high density parasitaemia in the rainy season following IPTc intervention period in older children (12–59 months), consistent with the results of studies done in Mali and Senegal. However, we found a slight increase in the incidence of clinical malaria associated with parasitaemia of any density or high density parasitaemia in infants aged 3–11 months in the IPTc treatment groups during the rainy season following the IPTc intervention period. The overall incidence of malaria was lower during the rainy season following the intervention than during the intervention period . The reaSP, AS and AQ were well tolerated and no serious adverse events attributable to the study drugs were reported during the study period. The incidence of mild adverse events such as fever, general weakness, vomiting, diarrhea, abdominal pain and cough were similar in the placebo and IPTc groups. These symptoms and signs were probably due to in a large part to common childhood illnesses and not related to the administration of the IPTc drugs. Nevertheless, some mothers/guardians withdrew their children because of perceived adverse events associated with drug administration. This is a concern if IPTc is to be rolled out on a national scale. Mothers' main concerns were vomiting and drowsiness after drug administration. Clear health education messages, including information on potential adverse events and the means to overcome these events, would need to be delivered to IPTc providers and the target population in order to achieve an effective coverage of IPTc in a routine programme setting.A recent multicentre pharmacokinetics study showed that children aged 2–5 years should be treated with 1 g sulphadoxine and 50 mg pyrimethamine to achieve drug concentrations equivalent to those needed in adults for treatment of uncomplicated malaria Potential weaknesses of this study are that there was an imbalance in the number of children allocated to one study group for reasons that we have not been able to explain. We have explored all possible reasons as to why this might have occurred but have not been able to find any rational explanation other than chance. Since we have adjusted the protective efficacies for the effect of age at enrolment and bednet use, the potential effect modifiers, it is unlikely that this imbalance in the number of children between the groups would have biased our observation. During the intervention period, losses to follow up were very low. Because there was no active follow up during the post intervention period we cannot be sure how many children had moved out for substantial periods during this period and we had, therefore, to assume that those who took part in the last cross sectional survey had remained in the study area throughout the follow up period. Approximately 40% of children enrolled in each group did not attend the last cross sectional survey but there was no difference in the response rate between the four study groups and this loss to follow up is unlikely to have biased our observation.We found that adherence to IPTc drugs delivered through community-based volunteers was similar to that achieved by project staff in Senegal (88% versus 91%). Delivering IPTc through community-based volunteers has advantages. They are resident in the community, making drug administration easy, they can give a repeat dose if a child vomits and can remind mothers/guardians to complete the course of treatment. However, sustaining the motivation of the volunteers for long periods is difficult. Delivering IPTc through the routine health delivery system is an attractive option because medically qualified staff are available to monitor any adverse events. However, in Ghana, only 50% of children aged between 12 and 23 months use growth monitoring services at established health posts. This means that it would be difficult to achieve a high coverage of IPTc using this route. A comparison these two modes of delivery has recently been undertaken in the Jasikan region of Ghana, an area adjacent to the one where the current study was done, and the results of this trial will be reported shortly.Checklist S1CONSORT Checklist(0.05 MB DOC)Click here for additional data file.Protocol S1Trial Protocol(0.93 MB DOC)Click here for additional data file.


Plasmodium species and Stool samples prepared using Kato-Katz technique were used to examine for intestinal helminths. Haemoglobin concentration was measured using a portable spectrophotometer (Hemocue HB 201). Anthropometry-based nutritional assessment of the study participants was done by measuring body weight to the nearest 0.1 kg and height to the nearest 0.1 cm.Distribution of malaria and intestinal helminths is known to overlap in developing tropical countries of the world. Co-infections with helminth and malaria parasites cause a significant and additive problem against the host. The aim of this study was to asses the prevalence of malaria/helminth co-infection and the associated problems among febrile outpatients that attended Alaba Kulito Health Center, southern Ethiopia November and December 2007. A total of 1802 acute febrile patients were diagnosed for malaria. 458 Giemsa-stained thick and thin blood films were used for identification of Plasmodium and helminth parasites is associated with significantly (p < 0.001) higher anaemia prevalence than single infection with Plasmodium parasites. And this difference was also significant for haemoglobin concentration , in which patients co infected with Plasmodium and helminth parasites showed lower mean haemoglobin concentration. More than one-third of the infected cases in both malaria infections and malaria/helminth co infections are undernourished. However the statistics for the difference is not significant.458 of the total febrile patients were positive for malaria. Co infection with Malaria and soil-transmitted helminthiasis obviously contribute to anaemia and low weight status and these conditions are more pronounced in individuals concurrently infected with malaria and soil-transmitted helminths. Hence, simultaneous combat against the two parasitic infections is very crucial to improve health of the affected communities. Overlapping distribution of intestinal helminths and malaria results in a high rate of co-infection ,2, whichIn addition, co-infections with helminth and malaria parasites have negative impact upon host nutrition through a number of mechanisms which may have additive or multiplicative impacts, especially in childhood . HelmintThe study was conducted at Alaba Kulito Health Center in Alaba Woreda (administrative unit), located 313 km south of Addis Ababa. The Woreda is semi-arid with annual temperature ranging from 18 to 23°C and mean annual rainfall from 100 to 120 millimeter. Malaria and intestinal parasites are the most prevalent public health problems in the area. Malaria transmission in Alaba is unstable, seasonal and depends on altitude and rainfall. There are two main seasons for transmission of the disease; September to December, after the heavy summer rains, and March to May, after the light rains.Plasmodium species and older than one year, had no history of anti-malarial drug administration in the two weeks prior to screening, absence of any other serious chronic infection, had ability to give blood and stool samples were included in the study. However, pregnant women, children younger than 1 year and individuals with known concomitant chronic infection were excluded from the study, and this work has been published elsewhere [The study was conducted on acute malaria patients that attended Alaba Kulito Health Center in November and December 2007. Cases positive for lsewhere .2). In classifying the weight status of the study participants the standard weight status categories associated with BMI ranges were used for overweight and obesity in adult [2), grade-2 thinness (BMI = 16-16.9 Kg/m2) and grade-3 thinness (BMI = 17-18.4 kg/m2) were used [Anthropometry-based nutritional assessment of the study participants was done by measuring body weight to the nearest 0.1 kg and height to the nearest 0.1 cm. Then the body mass index (BMI) was calculated to determine the weight status of the study participants using the formula  and specially designed microcuvette . Then, the haemoglobin values were used to assess the status of anaemia. For haemoglobin, the cut-off criterion levels below which indicating anemia was the WHO cut-off of 110 g/L for children 6-59 months; 115 g/L for children 5-11 years; 120 g/L for children 12-14 years; 120 g/L for non-pregnant women above 15 years of age and 130 g/L for men above 15 years of age .Data entry and validation was performed in excel, and statistical analysis was done using Statistical Package for Social Science version 13.0. Mantel Heinzel Chi-square test including odd ratios at 95% CI and one way ANOVA were used to test for differences in proportions and means, respectively. Values were considered statistically significant when p-values were less than 0.05.Plasmodium falciparum were treated with coartem, for P.vivax with chloroquine, for mixed infection with P. falciparum and P. vivax with coartem and chloroquine and for soil-transmitted helminthiasis with albendazole.This study was part of the ongoing project on efficacy of anti-malarial drugs against malaria at the Akililu Lemma Institute of Pathobiology, Addis Ababa University. The study was conducted after ethical clearance was obtained from the Institute and the Health Bureau of South Nations Nationalities and Peoples Region (SNNPR). Patients were involved in the study after obtaining informed consent. Consent was also obtained from guardians and/or parents for children under 18 years of age. All positive subjects for Plasmodium parasites and 458 subjects  fulfilled the inclusion criteria and enrolled in the co-infection study. From the 458 malaria infected patients, 255 (55.7%) were positive for one or more STHs. The most prevalent helminth was hookworm (37.8%) followed by A. lumbricoides (24.7%) and T. trichiura (8.3%) [Out of the 1802 acute febrile patients, 502 (27.9%) were positive for a (8.3%) .A. lumbricoides and < 5 years old for T. trichiura with a mean EPG of 542.72, 6249.9 and 699.33, respectively.The overall intensity of infection of intestinal helminthiasis expressed as geometric mean among the study subjects for children younger than 5 years of age, children 5-14 years old and adults ≥ 15 years old was, 955.33 , 2309  and 1045.8, respectively. The intensity of infection tends to reach its peak in the age group ≥ 15 years old for hookworm, 5-14 years old for Plasmodium alone or both Plasmodium and helminth infected females were found to have significantly lower mean haemoglobin concentration than males. Children younger than 5 years were found to have significantly the lowest mean haemoglobin concentration as compared to the older age groups.Mean haemoglobin concentration of the study participants was, 13.1 g/dl (ranging from 5.2 g/dl to 21.4 g/dl) with standard deviation of 2.37. Based on the WHO cut off values of haemoglobin concentration, a total of 144 anaemic cases were found, making an overall prevalence of anaemia among the study subjects 31.4%. Severe anaemia (haemoglobin concentration ≤ 7 g/dl) was very rarely seen (1.7%) and most of these were young children.P. vivax, P. falciparum and mixed infected subjects were anaemic, respectively. Similarly, the prevalence of anaemia was higher in malaria and intestinal helminth co-infected patients. The prevalence of anaemia was 43.3%, 29.2%, 35.7% and 42.3% for hookworm, A. lumbricoides, T. trichiura and multiple intestinal helminth and malaria co infected subjects, respectively  higher anaemia prevalence than single infection with Plasmodium parasites  than in males 29.6%) and highest in children younger than 5 years (46.3%) (p = 0.003). It was observed that 18.7%, 40.7% and 50% of 9.6% and Plasmodium and helminth parasites was significantly (p ≤ 0.001) associated with high prevalence of anaemia than single infection with Plasmodium parasites in children younger than 5 years and adults ≥ 15 years. And this difference was also significant for haemoglobin concentration , in which patients co infected with Plasmodium and helminth parasites showed lower mean haemoglobin concentration. Similarly, the prevalence of anaemia in males was higher (p < 0.001) in malaria and intestinal helminth co infected patients than those single infected with malaria. In contrast, no significance difference was observed both among females and children, 5-14 years , 5.9% (27/458), 13.5% (62/458), 47.6%(218/458), 10.3%(48/458) and 3.6%(16/442) of the cases are grouped under grade-3 thinness, grade -2 thinness, grade-1 thinness, normal, overweight and obese categories, respectively.A. lumbricoides, malaria/T. trichiura and malaria/multiple intestinal helminth infected patients were in underweight (thin) status category, respectively. And the statistics for this difference is not significant. Similarly the statistics for underweight prevalence difference between malaria infection  and malaria/helminth co infection in general  is not significant . Similarly, high anaemia prevalence for malaria infection , malaria/hookworm infections  and malaria/A. lumbricoides infections  was observed in patients with underweight status when compared with those with normal and overweight status  and malaria/hookworm infected patients was observed in those grouped under Grade-3 thinness. And the highest proportion of anaemia for malaria infected patients was observed in those grouped under Grade 1 thinness categories  than those in the normal plus overweight  group. This difference was significant statistically (p < 0.001, Odds Ratio = 2.62, XPlasmodium falciparum infection [P. falciparum cases than those with P. vivax. A relatively lower mean haemoglobin concentration was also observed in those having mixed infections than those with single Plasmodium infected patients.Haematological abnormalities are considered a hallmark of malaria, especially in nfection . The prePlasmodium parasites and helminths in Kenya. In addition similar to previous reports [This study has demonstrated that cases with malaria/helminth co-infections had significantly higher prevalence of anaemia and lower mean haemoglobin concentration when compared with malaria infection cases. And this is in agreement with previous report , which o reports  this stu reports ,27 in ch reports . However reports . ConsequAscaris and hookworm secret potent inhibitors of pancreatic enzymes, which may block host nutrient absorption in the small intestine directly [Although the statistics for underweight prevalence difference is not significant between malaria infections and malaria/helminth co infections, more than one-third of the infected cases in both groups are undernourished. Several studies have shown that malaria has a negative effect on the nutritional status of under 5 children and individuals older than 5 years -29. Maladirectly . Furtherdirectly .A. lumbricoides. This seems to support the effect of A. lumbricoides on aggravating preexisting anaemia by decreasing appetite and thus food and iron intake [The high prevalence of anaemia in malaria patients of the underweight status groups than those in the normal weight and overweight groups suggests malnutrition as a risk factor for anaemia besides malaria and helminth infections. Anaemia prevalence was highest in the grade-3 thinness grouped subjects compared to those in other groups when the co-infection was malaria/n intake .Plasmodium parasitaemia level to make comparisons valid. Hence this study did not assess association between helminth intensity and Plasmodium parasiteamia with anaemia and weight status. These are some of the limitations of the study.As indicated above malaria and helminth co infections have impact on anaemia prevalence and weight status in infected patients. However other factors like diet and socio economic status which may have impact on haemoglobin levels and nutrition status are not considered in this study. And also, for each blood and stool specimens from participants only one slide was read to detect malaria and/or helminth infection and identify the type of species. In addition, there were small sample sizes in each infection types of different helminth intensity and Generally, malaria and soil-transmitted helminthiasis obviously contribute to anaemia and low weight status and these conditions are more pronounced in individuals concurrently infected with malaria and soil-transmitted helminths. Hence, simultaneous combat against the two parasitic infections is very crucial to improve health of the affected communities in economically developing countries.The authors declare that they have no competing interests.ML and AA conceived the project idea. ML, AD and AA with further input from BE designed the project. AD colleted the data and ML, and AA supervised data collection and assisted in the analysis. AD with the assistance of BE prepared the first draft of the paper and all authors reviewed and approved it.


Plasmodium vivax and Plasmodium falciparum and yet the comparative epidemiology of these infections is poorly defined.Multidrug resistance has emerged to both All laboratory-confirmed episodes of malaria in Timika, Papua, Indonesia, presenting to community primary care clinics and an inpatient facility were reviewed over a two-year period. In addition information was gathered from a house-to-house survey to quantify the prevalence of malaria and treatment-seeking behaviour of people with fever.P. falciparum and 37%  to P. vivax. Malaria was most likely to be attributable to pure P. vivax in children under one year of age . In the household survey, the prevalence of asexual parasitaemia was 7.5%  for P. falciparum and 6.4%  for P. vivax. The prevalence of P. falciparum infection peaked in young adults aged 15–25 years (9.8% 69/707), compared to P. vivax infection which peaked in children aged 1 to 4 years (9.5% 61/642). Overall 35%  of people questioned reported a febrile episode in the preceding month. Of the 60% of people who were estimated to have had malaria, only 39% would have been detected by the surveillance network. The overall incidence of malaria was therefore estimated as 876 per 1,000 per year (Range: 711–906).Between January 2004 and December 2005, 99,158 laboratory-confirmed episodes of malaria were reported, of which 58%  were attributable to P. vivax and P. falciparum, both species are associated with substantial morbidity, but with significant differences in the age-related risk of infection.In this region of multidrug-resistant  Plasmodium falciparum and Plasmodium vivax infection almost equal. According to the National Health Household Survey in 2001, approximately 15 million people with clinical malaria sought treatment , with 17% (870) children aged less than 5 years , P. vivax infection in 248 (39%), P. malariae in 24 (4%) and mixed infections in 72 (11%)  in young adults aged 15–24 years. In contrast, the median age of those with P. vivax infection was significantly lower  with a peak of 9.5% (61/642) in children aged one to four years  falling to 47% (61/130) in children aged one to four years, with no change thereafter ; p = 0.004  with ) Figure . The pres Figure . The pro4 figure . The ove4 figure .P. falciparum, P. vivax, or mixed infections, asymptomatic carriage was significantly more likely in people with pure P malariae infections (91.7% (22/24); Odds Ratio = 5.3 [95%CI 1.2–33]), p = 0.023. Patients with P. vivax parasitaemia were significantly less likely to be afebrile if they were less than 1 year of age (33% 4/12) compared to 75% (85/113) of older children and 70% (86/123) of adults (p = 0.004 and 0.02 respectively), although this age difference was not significant for the other species  of respondents with asexual parasitaemia were asymptomatic (afebrile with no history of fever in preceding 24 hours). Although this proportion did not differ for s Figure . The risP. falciparum parasitaemia above 1,734 μl-1 had a 57% (57/100) sensitivity for predicting fever and a positive predictive value of 61% (57/93). The corresponding threshold for P. vivax was 310 μl-1, with parasitaemia above this level having a sensitivity of 77% (56/73) and associated with fever in 46% (56/122) of cases.The density of asexual parasitaemia of any species was significantly higher if the patients were febrile Table . A P. faIn 2005, a review of 13,766 laboratory records revealed that 96.6% of patients with malaria had one blood film for each episode of malaria, 3.25% had two blood films and 0.13% had three films. The total number of cases treated as community outpatients were therefore multiplied by 0.967 to account for multiple sampling. Hence the total number of patients with confirmed malaria reported by the surveillance network during the two-year period was 95,886.P. falciparum, 322 (Range: 262–333) for P. vivax, 15.7 (Range: 12.7–16.2) for P. malariae, and 26.3 (Range: 21.3–27.2) for mixed infections.In the household survey, a history of fever within the last month was reported in 35% (1813/5255) of household members, of whom treatment seeking behaviour could be assessed in 1015 (56%). Of these respondents, 60% (607) were estimated to have had malaria (based upon their treatment and subsequent microscopy at the time of the survey) of whom 39% (238/607) would have been diagnosed and thereby picked up by the surveillance network. The sensitivity analysis defined the lower and upper estimates as 37.7% (261/693) and 48% (206/426) respectively. After correcting for multiple sampling at the community clinics, the surveillance network identified a total 47,943 reports of malaria per annum for a population of 140,4000 people, representing 39% of the total burden of malaria. The overall incidence of malaria, therefore, was estimated as 876 per 1,000 per year (Range: 711–906). The corresponding breakdown was 512 (Range: 416–529) for P. vivax malaria from Asia has been under appreciated, despite the region contributing almost 40% of the world's malaria [Plasmodium falciparum and P. vivax are equally prevalent over much of the continent, however P. vivax is usually assumed to be benign and its associated morbidity often overlooked [P. falciparum. The latter have subsequently spread to almost all regions of the malarious world, undermining control programmes and significantly exacerbating the burden of malaria [P. vivax has taken longer to emerge, the first reports of chloroquine resistance coming from the island of New Guinea in 1989 [P. vivax has become increasingly prevalent in Papua province, with reports of declining efficacy also documented across the Indonesian archipelago as well as in Myanmar, India and South America [P. vivax and P. falciparum.The burden of  malaria . Plasmoderlooked ,8. The s malaria . Antimal America ,16-18. DP. falciparum and P. vivax, with high grade resistance a significant problem [P. vivax was responsible for 42% of malaria treated in the outpatients and 19% of patients admitted to hospital with malaria. In the household surveys, the overall prevalence of malaria was 16%, lower than that reported in West Sumba, Indonesia (31%) [P. vivax was higher: 39% in Timika compared to 32% in West Sumba and 19% in PNG.The study presented reviews the epidemiology of malaria in patients attending community clinics and the only referral hospital in southern Papua. In this region cure rates following chloroquine plus sulphadoxine-pyrimethamine have fallen below 50% for both  problem . The resia (31%) , althougP. falciparum. In contrast, the burden and proportion of P. vivax peaked in early childhood with more than half of malaria in infants attributable to P. vivax irrespective of healthcare facility. These findings concur with studies from PNG, Vanuatu and Thailand [P. vivax infection is the presence of the hypnozoite stage which in equatorial regions can result in frequent relapse and recurrent symptomatic infection. The predominance of P. vivax in infancy presumably reflects early exposure to infection, high rates of subsequent recurrence and the rapid acquisition of immunity. The additional impact of chloroquine resistance is difficult to gauge, but likely to further exacerbate the recurrence of malaria following partially effective treatment. In contrast to the PNG study, in which children had acquired almost complete immunity by the age of 9 years old [P. vivax infections [There were significant differences in the age-stratified rates of infection Figure . The worThailand -22. A noears old , both thP. falciparum and P. vivax infections, the pyrogenic thresholds differed considerably: 310 μl-1 for P. vivax and 1,734 μl-1 for P. falciparum. The ability of P. vivax to induce fever at lower levels of parasitemia than P. falciparum is well described [P. vivax, as evidenced by higher plasma concentration of fever-inducing cytokines, such as TNF, in vivax malaria compared to P. falciparum infections with similar parasitaemia [P. falciparum carriage did not change with age, there was a significant reduction in symptomatic carriage with P. vivax, falling from 67% in infants to 28% in adults (Figure In the prevalence survey, approximately a third of patients with peripheral parasitaemia reported an associated fever. Although there was no difference in this proportion between escribed ,23 and isitaemia ,25. Whers Figure . All patP. falciparum and P. vivax, respectively.Since the surveillance system did not include a prospective cohort an estimate of the incidence of clinical infection was derived from the clinic workload and a treatment seeking behaviour survey. The latter will be reported more fully in a separate paper, however the results highlight that only 39% with symptomatic malaria would have sought treatment at a facility in the surveillance network, the majority either staying at home or seeking treatment through the private sector or pharmacies. Extrapolating the total number of reported malaria cases we estimated an incidence of malaria of 512 and 322 per 1,000 population per year for P. falciparum and P. vivax are associated with significant morbidity. P. vivax infection predominated in infancy with symptomatic infections reducing with age, however even in adults this species is responsible for a third of malaria treatments. The presence of high grade chloroquine resistance in both major Plasmodium species in this region, is likely to exacerbate the burden of disease due to the impact of partial eradication and the inability to suppress or delay the relapses from the liver stage of P. vivax.In conclusion, in Papua Indonesia both The authors declare that they have no competing interests.MK, SY, RV, PMP, PS, PE, NMA, ET and RNP designed and supervised the study. LB, EK, NW, RM, KD, collected the data and carried out the preliminary analysis. MK, SY and RNP analysed the data and wrote the first draft of the manuscript. All authors contributed to drafting and approving the final manuscript.