A randomized trial reported by Alassane Dicko and colleagues shows that intermittent preventive treatment for malaria in children who are protected from mosquitoes by insecticide-treated bednets provides substantial protection from malaria. Previous studies have shown that in areas of seasonal malaria transmission, intermittent preventive treatment of malaria in children (IPTc), targeting the transmission season, reduces the incidence of clinical malaria. However, these studies were conducted in communities with low coverage with insecticide-treated nets (ITNs). Whether IPTc provides additional protection to children sleeping under an ITN has not been established.p = 0.45). A total of 672 episodes of clinical malaria defined as fever or a history of fever and the presence of at least 5,000 asexual forms of Plasmodium falciparum per microlitre  were observed in the control arm versus 126  in the intervention arm, indicating a protective effect (PE) of 82% (95% CI 78%–85%) (p<0.001) on the primary endpoint. There were 15 episodes of severe malaria in children in the control arm compared to two in children in the intervention group giving a PE of 87% (95% CI 42%–99%) (p = 0.001). IPTc reduced the prevalence of malaria infection by 85% (95% CI 73%–92%) (p<0.001) during the intervention period and by 46% (95% CI 31%–68%) (p<0.001) at the end of the intervention period. The prevalence of moderate anaemia (haemoglobin [Hb] <8 g/dl) was reduced by 47% (95% CI 15%–67%) (p<0.007) at the end of intervention period. The frequencies of adverse events were similar between the two arms. There was no drug-related serious adverse event.To assess whether IPTc provides additional protection to children sleeping under an ITN, we conducted a randomised, double-blind, placebo-controlled trial of IPTc with sulphadoxine pyrimethamine (SP) plus amodiaquine (AQ) in three localities in Kati, Mali. After screening, eligible children aged 3–59 mo were given a long-lasting insecticide-treated net (LLIN) and randomised to receive three rounds of active drugs or placebos. Treatments were administered under observation at monthly intervals during the high malaria transmission season in August, September, and October 2008. Adverse events were monitored immediately after the administration of each course of IPTc and throughout the follow-up period. The primary endpoint was clinical episodes of malaria recorded through passive surveillance by study clinicians available at all times during the follow-up. Cross-sectional surveys were conducted in 150 randomly selected children weekly and in all children at the end of the malaria transmission season to assess usage of ITNs and the impact of IPTc on the prevalence of malaria, anaemia, and malnutrition. Cox regression was used to compare incidence rates between intervention and control arms. The effects of IPTc on the prevalence of malaria infection and anaemia were estimated using logistic regression. 3,065 children were screened and 3,017  were enrolled in the study. 1,485 children (98.5%) in the control arm and 1,481 (98.1%) in the intervention arm completed follow-up. During the intervention period, the proportion of children reported to have slept under an ITN was 99.7% in the control and 99.3% in intervention arm  of malaria.Malaria accounts for one in five of all childhood deaths in Africa and of the one million annual malarial deaths world-wide, over 75% occur in African children <5 years old infected with IPT involves administration of antimalarial drugs at defined time intervals to individuals, regardless of whether they are known to be infected with malaria, to prevent morbidity and mortality. IPT was initially recommended for pregnant women and recently this strategy was extended to include infants (IPTi). Now, there is also intermittent preventive treatment of malaria in children (IPTc), which is designed to protect against seasonal malaria transmission including those above one year of age.Large clinical trials have shown that IPTc involving the administration of two to three doses of an antimalarial drug (sulphadoxine pyrimethamine [SP] and artesunate [AS] or amodiaquine [AQ]) during the high malaria transmission season effectively reduces the incidence of malaria. However, these studies were conducted in countries where the use of insecticide-treated bednets—an intervention that provides at least 50% protection against morbidity from malaria and is the main tool used for malaria control in most of sub-Saharan Africa—was relatively low. Therefore, it is unclear whether IPTc will be as effective in children who sleep under insecticide-treated bednets as has been previously shown in communities where insecticide-treated bednet usage is low. So to determine the answer to this important question, the researchers conducted a randomized, placebo controlled trial of IPTc with SP+AQ (chosen because of the effectiveness of this combination in a pilot study) in children who slept under an insecticide-treated bednet in an area of seasonal malaria transmission in Mali.The researchers enrolled 3,017 eligible children aged 3–59 months into a randomized double-blind, placebo-controlled trial during the 2008 malaria transmission season in Mali. All children were given a long-lasting insecticide-treated bednet at the start of the study with instructions to their family on the correct use of the net. Children were then randomized into two arms—1,509 were allocated to the intervention group and 1,508 to the control group—to receive three courses of IPTc with SP plus AQ or placebos given at monthly intervals during the peak malaria transmission season. The researchers monitored the incidence of malaria throughout the malaria season and also monitored the use of long-lasting insecticide-treated bednets throughout the study period. In addition, researchers conducted a cross-sectional survey in 150 randomly selected children every week and in every child enrolled in the trial 6 weeks after the last course of IPTc, to measure their temperature, height and weight, and blood hemoglobin and parasite level.Plasmodium falciparum per microliter) in the control arm versus 126 episodes in the intervention arm, which is an incidence rate of 1.90 episodes per person year in the control arm versus 0.34 in the interventions arm—giving a protective efficacy of 87%. IPTc reduced the prevalence of malaria infection during the intervention period by 85% and by 46% at the end of the intervention period. The prevalence of moderate anemia was also reduced (by 47%) at the end of intervention period. The frequencies of adverse events were similar between the two arms and there were no drug-related serious adverse events.The number of children who slept under their long-lasting insecticide-treated bednet was similar in both arms. During the intervention period, the researchers observed a total of 672 episodes of clinical malaria  provides information on  An estimated 863 million people live in sub-Saharan Africa of whom 16.2% are under 5 y of age Plasmodium falciparum to these drugs in the proposed study areas. Very similar protocols were used for the studies conducted in Burkina Faso and Mali.Intermittent preventive treatment (IPT) is a new approach in the prevention of malaria in infants and older children. Several randomised controlled trials have demonstrated that IPT of malaria in infants (IPTi) with sulphadoxine pyrimethamine (SP) given during routine vaccinations at approximately 2, 3, and 9 mo of age, reduces the incidence of clinical malaria by 22% to 59% The protocol of the trial , protocoThe primary objective of the study was to determine the degree to which IPTc given during the malaria transmission season reduces the incidence of clinical malaria in children who sleep under a long-lasting insecticide-treated net (LLIN). Secondary objectives were determination of the impact of this strategy on severe malaria, all cause hospital admissions, anaemia, nutrition , malaria infection, and molecular markers of resistance to SP and AQ.The study was conducted in two rural villages, Djoliba and Siby, and the small town of Ouelessebougou situated in the district of Kati in the savannah region of Mali. Djoliba and Siby are located 40 and 30 km south west of the capital city Bamako, respectively, and Ouelessebougou is located 80 km south of Bamako. In Djoliba and Siby, community health centres are staffed with a physician and nurses. In Ouelessebougou, the community health centre is staffed by an assistant physician and nurses, but located less than 100 m from a district health centre staffed by four physicians and six nurses. A research team composed of physicians and medical residents was established in each of the three sites to follow up and provide health care to the study participants.Malaria transmission in the study area is highly seasonal and 80%–90% of malaria cases occur between August and November. The entomological inoculation rate (EIR) was 9.4 and 6.6 infective bites per person per season, respectively, in Siby and in Ouelessebougou, two localities far from any river and 37.3 infective bites per person per season in Djoliba located on the bank of the Niger River . The cov. Children aged 3–59 mo were enumerated and given a census identification number including a house number to facilitate their identification at screening, enrolment, and follow-up. Recruitment was started in Djoliba followed by Siby. In these communities all available children in the target age group who were not selected for the baseline survey of drug resistance were screened and enrolled if they met the inclusion criteria. In the larger community of Ouelessebougou, children were screened for enrolment on a first-come first-served basis until the required sample size was met. Children were eligible to join the study if they were aged 3–59 mo at the time of enrolment and permanent residents of the study area with no intention of leaving during the study period. Exclusion criteria were the presence of a severe, chronic illness, such as severe malnutrition or AIDS, and a history of a significant adverse reaction to SP or AQ. Cases of an acute illness, such as malaria, were not excluded. Such cases were treated appropriately and the child randomised and retained in the trial.The study was designed as an individually randomised, placebo-controlled trial of IPTc with SP+AQ in children who received a LLINhttp://www.pharmaclin.com).The study protocol was reviewed and approved by the Ethical Committee of the Faculty of Medicine, Pharmacy and Dentistry, University of Bamako, Mali and by the Ethics Committee of the London School of Hygiene and Tropical Medicine. Community consent was obtained at meetings with leaders, heads of families, and other community members of each locality prior to the start of the study. Individual, written, informed consent was obtained from a parent or guardian of each child prior to screening and enrolment. A Data and Safety Monitoring Board (DSMB) was established and monitored the trial with the support of a local medical safety monitor. Current good clinical practices (cGCP) monitoring of the trial was performed by PharmaClin  that was marked with the child's identification number regardless of whether or not the child was enrolled. Instructions were given to the parent or guardian on how to use the net and the importance of using the net regularly was emphasized. Monitoring of utilisation of ITNs by study participants was made in 150 randomly selected children each week and in all study children during the cross-sectional survey conducted at the end of the malaria transmission season.Eligible children were treated with a course of SP+AQ or matching placebos on three occasions at monthly intervals during the malaria transmission season, starting in August 2008. SP and AQ were manufactured by Kinapharma Limited and quality control checks on the drugs for solubility and content were performed at the London School of Tropical Medicine and Hygiene, prior to their use in the trial. Tablets met internal standards for drug solubility and content. Doses of SP and AQ were based on weight with children stratified into one of the three weight categories . SP was given at a dose of 175/8.75 mg to children 5–9 kg, 350/17.5 mg to children 10–18 kg, and 550/26.25 mg to those who weighed ≥19 kg. The corresponding doses for AQ were 70 mg, 140 mg, and 220 mg, respectively. AQ was given over 3 d. Drugs were prepackaged to facilitate administration and put in envelopes with colour codes, one for each weight group. Within each weight stratum, children were individually randomised using a computer-generated random number sequence and blocks of varying length. Treatment allocations were provided within sealed, opaque envelopes.Drugs were given under direct observation at a research clinic by study staff. Children were observed for 30 min after drug administration. If vomiting occurred during this 30-min period, drugs were readministered. If vomiting occurred on a second occasion, this was noted but the drugs were not given again. Such children were not excluded from the trial and they were eligible to receive drugs on the subsequent 2 d and during subsequent monthly IPT rounds. If a child missed the day set for treatment, a home visit was made to enquire why the child had not been brought for treatment and the reason was recorded. If the family wished to continue with treatment but was unable to attend on the specified day, then treatment was reoffered within an interval of 7 d of the designated date. Children with an acute malaria episode were treated with artemether-lumefantrine (AL) and did not receive IPT with SP+AQ if the treatment for acute malaria was received within 7 d of the scheduled date of IPT. Such children were eligible for treatment in future treatment roundsP. falciparum asexual parasitaemia at a density greater or equal to 5,000 parasites per microlitre. Secondary endpoints were: (i) the incidence of clinical malaria defined as the presence of fever or a history of fever in the past 24 h and the presence of P. falciparum asexual parasitaemia at any density; (ii) incidence of severe malaria defined according to the WHO criteria The primary endpoint of the study was the incidence of clinical malaria; this was defined as the presence of fever (axillary temperature ≥37.5°C) or a history of fever in the past 24 h and the presence of http://www.indepth-network.org/index.php?option=com_content&task=view&id=95&Itemid=183).Passive surveillance for clinical malaria started at the time of the administration of the first dose of IPTc in August 2008 and continued until the end of the malaria transmission season in November/December 2008, 6–7 wk after the last round of IPTc. Parents were encouraged to bring their child to a study health centre, where medical staff were available 24 h a day and 7 d a week, if the child became unwell. A finger prick blood sample was be obtained from all study children with fever (an axillary temperature of 37.5°C or higher) or a history of fever within the previous 24 h for preparation of a blood film, measurement of Hb concentration, and for a rapid diagnostic test (RDT) OPTIMAL_IT (Diamed AG) for malaria. Children who had a positive RDT for malaria were treated immediately with AL. Severe cases were admitted to the health centre or referred to the paediatric ward of the Gabriel Touré Hospital in Bamako. Causes of death were assessed within a month of death using a modified version of the INDEPTH post mortem questionnaire  and 1 d after the last dose of treatment (day 3) at each round.Monitoring of the frequency of molecular markers of resistance to sulphadoxine, pyrimethamine, and AQ was performed in two cross-sectional surveys, the first at baseline in August 2008 and the second during the survey undertaken at the end of malaria transmission season. The baseline survey was conducted in 256 children randomly selected from the screening list. These children were not enrolled in the placebo control trial. Participants enrolled in the placebo control trial were surveyed about 6 wk after the third course of IPTc, at the end of malaria transmission season, to assess whether administration of IPT with SP+AQ had lead to an increase in molecular markers of resistance to these drugs. Thick and thin blood smears and blood blotted onto filter papers were collected during both surveys for molecular analysis as described below.Thick blood films were air dried, stained with Giemsa, and examined for malaria parasites by two well-trained technicians. 100 high power fields were counted before a film was declared negative. Parasite density was determined by counting the number of parasites present per white blood cell (WBC) on a thick smear and assuming a WBC count of 8,000 per µl. In the case of a discrepancy (positive/negative or a difference in parasite density greater than 30%), a third reading was done. The median parasite density of two or three readings was used. An external quality control of slide reading performed by the Malaria Diagnosis Centre of Excellence (MDCoE) of the Walter Reed/Kenya Medical Research Institute, in Kisumu, Kenya, showed an overall concordance of more than 90% on parasite detection and 100% on species identification . Hb concP. falciparum on blood smears were analysed by nested PCR for mutations at codons 51, 59, and 108 of the dhfr gene, 437 and 540 of the dhps gene, 76 of mutations in the P. falciparum chloroquine transporter gene (pfcrt), and 86 of the P. falciparum multidrug resistance gene one (pfmdr1) according to published methods Filter paper samples from children with a mono-infection of Calculation of sample size was based on the assumptions that the clinical attack rate measured by passive surveillance would be 1.0–2.0 attacks per child per year in unprotected children aged 3–59 mo living in the study areas and that sleeping under an LLIN would reduce this attack rate by half to 0.5 to 1.0 clinical episode per child per year. Assuming that children experienced an average of 0.5 clinical episodes per child per year of sufficient severity to present to a health facility, to detect a 20% reduction in this incidence  in children who receive IPTc, the smallest reduction that would be likely to make IPTc a worthwhile investment, and allowing for a 20% loss to follow-up, we estimated that approximately 2,000 children  were required for a study with 90% power at the two-sided 5% level of significance z-scores using WHO's anthropometric software (www.who.int/childgrowth/software/en). Underweight, stunting, and wasting were defined as z-scores of <−2 for the relevant indicator t test. Frequencies of single mutations as well as the triple mutant (dhfr 51+59+108) and quadruple mutant (triple mutant + dhps 437) genotypes were determined and compared between treatment arms and between the beginning and end of the study. Proportions of children with binary outcomes were compared between the two groups using Pearson's Chi square test or generalized linear models adjusted for age, gender, and locality.Data were collected on standardized forms, double-entered, and verified using MS Access and then exported to Stata (StataCorp) for additional cleaning and analysis. A data analysis plan was written and submitted to the DSMB prior to analysis. The final, cleaned database was locked and a copy sent to the DSMB. An intention-to-treat analysis was performed. Incidence rates of clinical malaria, severe malaria, and hospital admissions were calculated by dividing the number of episodes by the total child days at risk. Children were not considered at risk for 21 d after each type of a malaria episode and these days were not included in the calculation of the child days at risk. The incidence rates in the two treatment groups were compared using Cox regression to estimate the incidence rate ratio, with adjustment for age, gender, and locality, and using a robust standard error to allow for the lack of independence among repeated episodes in the same child. The protective effect (PE) of IPTc was computed as 1 minus the incidence rate ratio. Time to first episode of clinical malaria in the two arms was examined using Kaplan-Meier plots and compared using log rank test. Anthropometric data at enrollment and at the end of season cross-sectional survey were converted into weight-for-age, height-for-age, and weight-for-height n = 29), migration to another location (n = 15), a history of allergy to study drugs (n = 4 with two cases confirmed), and death (n = 3). There were no significant differences between intervention and control groups with regard to their age and gender distribution, nor in the prevalence of fever, wasting, or stunting at the time of enrolment .Usage of LLINs was assessed for 590 children in the control group and for 591 children in the intervention group during weekly home visits, undertaken without prior warning, during the course of the intervention period. Usage of an LLIN was high in each of the three study localities and similar between the two groups . An analysis of time to the first episode of clinical malaria, defined as above, also indicated a strong protective effect of IPTc (p<0.001) (p<0.001). Only 17 cases of severe malaria occurred during the follow-up period, 15 in the control group, and two in the intervention group (p = 0.001). The two cases of severe malaria in the intervention arm, one of whom died, occurred more than 3 wk after the third course of IPT.Among children with fever or history of fever who had an RDT positive result, 8.8%  turned out to have negative parasitaemia after microscopical diagnosis of malaria. The impact of IPTc on episodes of malaria detected through passive surveillance is presented in p<0.001) . The incon group , giving p≤0.001 and p = 0.003, respectively).Incidence rates and the PE of IPTc against clinical malaria by locality and age category are presented in p<0.001). At the end of the transmission season, 13.2%  of children in the control group were parasitaemic compared to 7.2%  in the intervention group, giving a protective efficacy of 46% (95% CI 31%–68%) (p<0.001).The percentage of children with malaria infection detected at weekly active surveillance visits was 13.2% (74/563) in the control group compared to 1.9% (11/575) in the intervention group, giving a protective efficacy of 85%, (95% CI 73%–92%) (p<0.001). The relative difference was larger for moderate anaemia (Hb <8 g/dl) with a prevalence of 3.5%  versus 1.9%  in the control and intervention groups, respectively  (p = 0.007). No cases of severe anaemia (Hb <5 g/dl) were observed in either treatment group at the time of the postintervention survey. However, during the follow-up period, a total of eight cases of severe anaemia occurred, two in the intervention arm and six in the control arm. The two participants in the intervention group who developed severe anaemia had not received a complete course of IPT at the time that they developed their severe anaemia.At the end of the malaria transmission season, the proportion of the children with anaemia (Hb <11 g/dl), was significantly higher in the control group compared to the intervention group    and quadruple dhfr  + dhps 437 mutations were higher in the intervention than in the control group at the end of the surveillance period and, for the dhfr 59, dhps 437, triple and quadruple mutations, differences between groups were statistically significant. Frequencies of the pfcrt 76 and pfmdr1 86 did not change significantly over time and were similar postintervention in the intervention and control groups.The frequencies of molecular markers associated with resistance to SP and AQ in the two groups at baseline and postintervention are presented in p = 0.81). There were five deaths, two in the control arm and three in the intervention arm. Two of the five deaths were due to malaria (one in each group). Both occurred during hospitalisation while the remaining three deaths occurred at home. On the basis of the results of a verbal autopsy, these deaths were thought to be due to poisoning by traditional medicines, meningitis and anaemia, and secondary bleeding following a circumcision, respectively.Hospital admissions and deaths that occurred during the study period are listed in p = 0.06 for vomiting and 1.9% versus 0.8%, p = 0.08 for loss of appetite). Proportions of children with skin rash and itching on at least at one occasion were similar between the two arms. Four participants in the intervention arm were withdrawn from the study because of reactions to study drug versus none in the control arm. Two of these children had a documented skin rash at physical examination (one after the first dose of IPT and the other after the second dose of IPT) and these were assessed as being related to study drugs. Both were moderate in intensity, did not involve bullous eruptions, and resolved within 2 d. The parent of the third participant reported itching. Physical examination was normal but the child was withdrawn from the study on precautionary grounds. The fourth participant had an acute respiratory infection at the time of administration of the first dose of IPT. No adverse event was recorded at the time of routine surveillance but the parents requested withdrawal of their child from the study at the time of the second round of IPTc.There was no serious adverse event related to the study drugs. The frequencies of adverse events following the administration of IPTc with SP+AQ or placebo, using active surveillance are summarized in This study has shown that three doses of IPTc with SP+Q given at monthly intervals during the peak transmission season reduced the incidence of uncomplicated and severe malaria by 80% in children 3–59 mo of age who slept under an ITN in three localities in Mali despite the difference in ITN use at baseline. This level of protective efficacy is similar to that reported in a previous trial conducted in an area of Senegal with a coverage of ITNs of less than 1% Despite the large difference in background incidence of malaria in the three sites, suggesting high variability in transmission intensity, the protective efficacy of IPTc against clinical malaria was high and similar between the three sites. This suggests that similar efficacies of IPTc against clinical malaria can be expected in areas with different transmission intensities and baseline ITN coverage. Surprisingly, Siby and Ouelessebougou, which had a low EIR (less than ten infective bites per person/season), had a higher malaria attack rate than Djoliba, which had a higher EIR (37 infective bites per person/season). High malaria infection and attack rates have been reported previously in the context of a low EIR (3.5 infective bites per person/season) in Mali We observed a 47% reduction in the proportion of children with moderately severe anaemia (Hb <8 g/dl) as a result of administration of IPTc. This impact on anaemia is consistent with the reduction of 45% in incidence of anaemia observed when AS+AQ was given at monthly intervals over 6 mo in Ghana, although in the Ghanaian study there was no difference in the proportion of children with anaemia at the end of the 6-mo intervention SP+AQ was chosen as the drug combination for use in the trial on the basis of the results of previous studies that had shown this to be an effective combination for IPTc. This drug combination was generally well tolerated and no serious adverse event attributable to the study drugs was reported. The proportions of children with mild-to-moderate adverse events using active surveillance were not significantly different between the two arms, although there was a trend towards a higher frequency of vomiting and loss of appetite in the intervention group. In the parallel study in Burkina using the same drugs, a higher frequency of vomiting was found in the intervention arm The efficacy of IPTc against clinical malaria has now been demonstrated in a number of studies, including the current trial and a parallel one conducted in Burkina Faso Unlike the case of IPT in pregnant women and infants, IPT in children has no established delivery system, raising concerns as to whether it could be implemented as a control measure. However, studies conducted in Ghana and The Gambia have shown that high coverage with IPTc can be obtained using community health workers dhfr 59 and dhps 437 mutations associated with pyrimethamine and sulphadoxine resistance, respectively, were found significantly more frequently at the end of the malaria transmission season in parasites obtained from children in the intervention group than in those obtained from children in the control group, and this led to higher frequencies of the triple dhfr mutants and the quadruple mutant (triple dhfr + dhps 437) associated with significant resistance to SP in children who had received IPTc. This increase in the frequency of these mutations is consistent with a previous report in Senegal Another concern over the widespread deployment of IPTc is that this will enhance the spread of drug resistance. Therefore, we studied the presence of molecular markers associated with resistance before and after the intervention in children in the intervention or control group. The As is the case with any successful malaria intervention, administration of IPTc to children during several, successive malaria transmission seasons could interfere with the development of naturally acquired immunity, raising concerns that there would be an increased period of risk  during the period immediately after the intervention was stopped if exposure levels remained high. The risk of malaria for children in this trial in the year after the intervention was stopped has been studied and the results are currently being analysed. However, several years of administration would be needed to define the degree to which acquisition of natural immunity would be impaired. It is very unlikely that this would outbalance the substantial gains made during the period when the drug was given.Our study has several strengths. First, the double-blind, randomised controlled design prevented a number of biases in the selection assignment of the participants to the two arms as well as in assessing the outcomes. A second strength is that this is the largest IPTc efficacy trial done so far, providing a more precise estimation of the outcomes measured. Third, the trial was conducted in three localities with different malaria incidence rates, allowing the efficacy of this strategy under different levels of malaria transmission to be assessed. The design would have been stronger if a factorial design had been used to assess the individual and combined impact of IPTc and ITN, but such a trial would be unethical as the efficacy of ITN is already established In summary, IPTc given during the malaria transmission season, provided substantial additional protection against clinical malaria, infection with malaria, and anaemia to that provided by ITNs. IPTc with SP+AQ was safe and well tolerated. As the international community moves towards the target of malaria elimination, new malaria control tools will be needed Text S1Study protocol: A trial of the combined impact of IPT and ITNs on morbidity from malaria in African children.(0.19 MB PDF)Click here for additional data file.Text S2CONSORT checklist.(0.22 MB DOC)Click here for additional data file.Text S3Entomological investigations.(0.45 MB PDF)Click here for additional data file.Text S4External quality assurance of malaria microscopic diagnosis.(0.10 MB PDF)Click here for additional data file.Text S5Protocol amendment.(0.11 MB PDF)Click here for additional data file.Text S6In vivo efficacy of the SP+AQ combination used for IPTc in the study area.(0.51 MB PDF)Click here for additional data file.


Within sub-Saharan Africa, helminth and malaria infections cause considerable morbidity in HIV-positive pregnant women and their offspring. Helminth infections are also associated with a higher risk of mother-to-child HIV transmission. The aim of this study was to determine the prevalence of, and the protective and risk factors for helminth and malaria infections in pregnant HIV-positive Rwandan women receiving anti-retroviral therapy (ART).Plasmodium falciparum was identified from blood smears. The prevalence of helminth infections was 34.3%; of malaria 13.3%, and of co-infections 6.6%. Helminth infections were more common in rural (43.1%) than peri-urban  sites. A CD4 count ≤350 cells/mm3 was associated with a higher risk of helminth infections  and malaria  whilst helminth infection was a risk factor for malaria infection and vice versa. Education and employment reduced the risk of all types of infection whilst hand washing protected against helminth infection ;). The TDF-3TC-NVP , D4T-3TC-NVP  and AZT-NVP  regimens each yielded higher helminth infection rates than the AZT-3TC-NVP regimen. Anti-retroviral therapy had no effect on the risk of malaria.Pregnant females (n = 980) were recruited from health centres in rural and peri-urban locations in the central and eastern provinces of Rwanda. Helminth infection was diagnosed using the Kato Katz method whilst the presence of HIV-positive pregnant women would benefit from the scaling up of de-worming programs alongside health education and hygiene interventions. The differential effect of certain ART combinations (as observed here most strongly with AZT-3TC-NVP) possibly protecting against helminth infection warrants further investigation. There is an overlap in the worldwide distribution of intestinal worms (helminths), malaria and HIV. Co-infections with helminth and malaria parasites cause a significant problem in the host, particularly in the presence of HIV infection. The aim of this study was to assess the prevalence of intestinal worm and malaria infection and co-infections and the associated risk factors among HIV-positive pregnant women that attended rural and peri-urban health centers in Rwanda. Our findings indicate that intestinal worms were more common among HIV-infected pregnant women in the rural than peri-urban settings. HIV-positive pregnant women who had lower CD4 cell counts were more at risk of being infected by intestinal worms and malaria. Malaria also increased the risk of being infected by intestinal worms and vice versa. Socio-economic factors such as lack of education and unemployment were among the risk factors for intestinal worm infections and malaria. Hand washing was found to reduce the risk for worm infections; whilst one particular ART combination (AZT-3TC-NVP) led to a reduced rate of helminth infections when compared to others. Ascaris lumbricoides, Trichuris trichiura, and the hookworm species Necator americanus and Ancylostoma duodenaleGlobally, the most common nematode species that cause soil-transmitted helminthic diseases are The immunological interplay between helminth infections and HIV is complex, and there are different hypotheses on the influence of the infections on each other; the most important being the Th2 bias induced by helminth infections, suppressing Th1 responses specific to HIV; thus leading to more rapid HIV progression Risk factors for helminth infections depend on the route of transmission and the life cycles of the various helminth species; they are usually related to hygiene and sanitation Plasmodium falciparum, combined with helminthic infections in HIV-positive women is of great concern from a public health perspective P. falciparum infection by intermittent preventive treatment and use of insecticide-treated bed nets is of high importance especially in primigravidae Pregnancy may increase susceptibility to helminths, but this is uncertain. A recent study from Gabon showed increased prevalence in pregnancy This prospective cohort study was designed to assess the major risk factors for helminth and malaria co-infection in HIV-positive pregnant women who participated in an early anti-retroviral therapy (ART) initiation program for the prevention of mother-to-child transmission in Rwanda. We describe the baseline prevalence of malaria and helminth infection in HIV-infected pregnant women on ART, and assess the factors that may increase or decrease rates of both infections.Ethical approval was obtained from the Rwanda National Ethics Committee and the Ethics Committee (Human Research) of the University of the Witwatersrand Medical School, Johannesburg, South Africa. All subjects provided written informed consent at enrolment. Subjects who could only provide oral consent were asked to give thumb marks using indelible ink on the consent form, in case they were illiterate; in accordance with the IRB oral standard ethical consent guidelines of the two ethical committees and in accordance with Helsinki declarations.The study participants were recruited amongst women accessing antenatal care and ART services at rural and peri-urban health centers in the central and eastern provinces of Rwanda, between 02 January 2010 and 29 February 2011. After giving written informed consent, women in the second trimester of pregnancy were enrolled at their fourth, fifth or sixth month of gestation. Additionally, women were enrolled if they lived within walking distance from the study areas, and if they planned to deliver at the registered study health center. Enrolment criteria were HIV infection; pregnancy (in the second trimester); use of ART; and willingness to provide three stool samples on consecutive days. Women were excluded if they were diagnosed with tuberculosis, or if they had taken any anti-helminthic drugs at any time point prior to entry into the study. Those who had been enrolled in other research projects during the study period were also excluded. Participants provided blood and stool samples before being treated in accordance with the study protocol.On enrolment, participants were interviewed to obtain demographic and socio-economic information pertaining to the relevant environmental risk factors for helminth infections and malaria. Subjects were asked whether they had attended school, whether they were employed, their source of water (river or piped), if they wore shoes, whether they washed their hands after using the toilet, and whether they used dietary supplements. Study participants were also asked about their number of previous pregnancies.Plasmodium falciparum was identified by light microscopic examination of Giemsa stained thick and thin blood smears, and screening for malaria was also performed by detection of the P. falciparum histidine rich protein 2 (HRP-2) antigen using a rapid diagnostic test kit . In the case of discordant results, expert light microscopy results were considered as gold standard. Participants received treatment for helminth infections and malaria in accordance with the study protocol. Artemether/lumefantrine (120/20 mg) was given as standard falciparum malaria treatment, administered orally in four doses for three days. Deworming was performed every 12 weeks with 400 mg albendazole given to women with helminth infections only/not to those negative in the ‘targeted treatment’arm; whereas all women received 400 mg albendazole irrespective of infection status in the ‘untargeted treatment’arm.Intestinal helminths were identified by the Kato Katz method Women received nevirapine for prevention of mother-to-child HIV transmission and subsequent combination ART, irrespective of CD4 cell levels in accordance with the latest (2010) Rwandan Ministry of Health treatment guidelines Data analysis was performed using Stata version 11.0  and Statistica version 9.1 . Data that was not normally distributed was log transformed to normality before analysis.2 test.The prevalence of helminth, malaria and helminth-malaria co-infections were determined in population sub-groups e.g. in employed and unemployed subjects, and differences between the groups were assessed using the χA. lumbricoides, T. trichiura and hookworms, and a combined model for infection with any helminth species. The independent variables included in the initial logistic regression models were, for helminth infections: location, month of year, ART, gravidity, education, employment, water source, use of shoes, hand washing, dietary supplement use, HIV viral load, presence/absence of malaria, age, height, gestational age and CD4 counts. With malaria as the dependent, dichotomous variable, the same list of independent variables was used. The presence/absence of helminth infection was included as an additional independent variable whilst presence/absence of malaria was removed. With co-infection  as the dependent variable, both malaria and helminth presence/absence were removed from the model. In all the logistic regression models, the independent variable with the highest p-value was removed at iteration until only variables with a p<0.05 were left in the model.Backward, stepwise multiple logistic regression analysis was used to determine the principal factors associated with helminth and malaria infections and helminth-malaria co-infections. Models were constructed for infection with each individual helminth species, i.e. Backward, stepwise multiple regression analysis was used to identify the principal determinants of fecal helminth egg counts and blood hemoglobin levels. Univariate analyses were initially performed, and any variable with p<0.50 was included as an independent variable in the multiple regression models. The same procedure as described for the logistic regression models was then followed.A sample size calculation was not performed for this study. N was chosen based on logistical factors, taking into account future follow-up studies. However, if one performs a post-study sample size calculation based on the logistic regression model for identifying the principal determinants of helminth infection and using the equation N = (10*k)/p A. lumbricoides being the most commonest. The presence of a malaria infection (in the presence or absence of helminths) was more frequent in peri-urban than in rural subjects (p<0.05). This trend was mirrored by malaria-only infections, with a prevalence of 4.39% in rural and 10.7% in peri-urban females (p<0.0005). The prevalence of helminth-malaria co-infection was similar in both environments  had higher prevalence levels of malaria and co-infections compared to those at a later stage of pregnancy (5–6 months) . If this3 were both associated with higher levels of all infections. Subjects with asymptomatic malaria had a higher prevalence of helminth infections, and vice versa . A detectable viral load and a CD4 count ≤350 cells/mmersa see .A. lumbricoides and hookworm compared to AZT-3TC-NVP. The same applied with the AZT-NVP and TDF-3TC-NVP regimens regarding T. trichiura prevalence when compared to the AZT-3TC-NVP therapy.There were lower rates of hookworm infestation in subjects who were screened for infections during January and February compared to those screened later in the year, whilst subjects who were older than 30 years, or who were multigravid had a lower risk of any helminth infection when compared, respectively, to those 30 and younger, or primigravidae.A. lumbricoides, hookworm or any helminth infection in comparison to those from a rural environment. Educated study participants and those who used piped water were at a lower risk of A. lumbricoides or any helminth infection when compared, respectively, to subjects with no formal education and who used river water. Furthermore, pregnant women who were employed or who regularly washed their hands were at a lower risk for A. lumbricoides, T. trichiura or any helminth infection in comparison to subjects who, were employed or did not wash their hands regularly, respectively.Subjects who were residents of a peri-urban location had a lower risk of A. lumbricoides, and subjects with a CD4 count at or below 350 cells/mm3 were at a higher risk for all kinds of helminth infections when compared to those with CD4 counts above 350 cells/mm3. The presence of malaria was associated with a higher risk of any helminth infection.Pregnant females who had a detectable viral load when compared to those who did not, were at a higher risk for The risk for malaria was higher in the months of January and February than from March to May . Risk waBackward, stepwise multiple regression analysis demonstrated that fecal helminth egg counts were highest in females who were multigravid; who did not wear shoes and who had low CD4 counts . HemogloP. falciparum infection, whilst hand washing protected against worm infections. HIV treatment with AZT-3TC-NVP was associated with a lower prevalence of helminth infections. A CD4 count ≤350 cells/mm3 was associated with higher levels of all infections. Multiple linear regression analysis demonstrated that helminth egg counts (EPG) were highest in females who were multigravid and hemoglobin levels were lowest in females who had helminth or malaria infections.In this study population, we determined the prevalence and identified protective and risk factors of helminth, malaria and co-infections in HIV-infected pregnant women on ART in Rwanda. We found that helminth infection was more prevalent in rural than peri-urban settings. Poor education and unemployment were risk factors for both helminth and A. lumbricoides followed by T. trichiura and hook worm species . This is in agreement with previous findings from the same location A. lumbricoides and T. trichiura were more commonly found in Rwanda and Burundi than in most other East African countries The prevalence of helminth infection was higher among the rural than peri-urban populations. Whilst we did not notice general differences between women recruited at the various health centers, this is best explained by variations in lifestyle between both settings. The most prevalent species were P. falciparum infection during pregnancy but this usually remains asymptomatic. In the current study, we found seasonal fluctuation, with the prevalence of asymptomatic malaria being higher in subjects tested in the months of January–February than those tested in March–May.An earlier study of malaria prevalence conducted in HIV-positive pregnant females in Kigali, Rwanda, demonstrated that 8.0% of the study group had malaria In the current study, we report that pregnant females who were older than 30 years at the time of testing had a lower prevalence of helminth infections but higher levels of malaria than younger females. The helminth data is supported by a previous study from Uganda A. lumbricoides infection in primigravid compared to multigravid females Little data exists on the relationship between gravidity and the risk of helminthiasis. One study shows no effect of gravidity on the risk of helminth infection Females who presented for testing at an earlier stage of their pregnancy (4 months) had a higher prevalence of malaria and helminth-malaria co-infection than those at a later stage of pregnancy (5–6 months). This data is supported by findings from previous African studies Education and employment acted as protective factors against both helminth infection and helminth-malaria co-infection. Previous studies have shown similar associations Helminth infection was shown to be more prevalent in subjects who did not wash their hands. Studies have shown that the risk of helminth infection is reduced in subjects who regularly wash their hands, more so in those who use soap Helminth egg counts were highest in multigravidae who did not wear shoes regularly and who had low CD4 counts . HemogloIn the present study the risk of helminth infection was higher in females with a reduced CD4 cell count, and in subjects with a detectable viral load. This is in agreement with previous studies conducted in pregnant females in Uganda vice versa. A study conducted in Ghana on pregnant females also showed that helminth infection increased the risk of malaria The prevalence of helminth infection was increased in subjects with malaria, and in vitro and in vivo investigations indicated that HIV treatment, especially with protease inhibitors (PIs), could have a direct effect in killing of parasites including malaria In our study population all subjects were taking ART irrespective of CD4 counts, as prescribed by the new (2010) Rwandan Ministry of Health guidelines for the prevention of mother to child transmission of HIV The strengths of this study are the screening of a large number of women from eight health centers which caters for women of all socio-economic classes; the screening of three stool samples on three consecutive days for the presence of helminths; the use of a combination of two different screening techniques to increase the sensitivity of helminth diagnosis. However, our study has limitations. Methodologically, its cross-sectional design makes it impossible to determine temporal causality, including the inability of multivariate models to adjust for all confounding factors. All participants in the study were women and as hemoglobin levels differ between men and women, our findings cannot be extrapolated to men. The Kato Katz method used to determine the number of helminth eggs could have underestimated the proportion of women with light hookworm infection. The study had no control group of HIV-positive subjects not receiving ART.In conclusion, we found that the prevalence of helminth infections, malaria and co-infections is common in HIV-positive pregnant women on ART in Rwanda. Helminth and malaria infection in this population are important risk factors for low hemoglobin levels. Subjects with low CD4 counts were at higher risk of infections and helminth infection is a risk factor for malaria. Education and employment were independent protective factors for helminth infection and malaria, whilst hand washing reduced the risk only for helminth infections. The possible antihelminthic effect of some ART combinations warrants further studies.


Endothelium-dependent flow mediated dilation (FMD) and pulse-wave velocity (PWV), are used as measures of vascular health and predictors of cardiovascular risk in clinical studies, and both are age-dependent. Numbers of circulating endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) are also associated with cardiovascular risk, but independent of age in humans. The use of these measurements for pre-clinical assessment of drug cardiovascular safety and efficacy in non-human primates (NHPs) may promote the translation of drug-induced effects on vascular function to clinic outcomes. However, in NHPs, the age effects on the non-invasive measurements of FMD and PWV and the relationship of EMPs/EPCs with FMD are unknown.A non-invasive, clinically-relevant approach to assess FMD and PWV was used to examine their relationship with age and with EMPs/EPCs in NHPs. The effects on FMD of nicotine and rosiglitazone were evaluated in senescent primates in an effort to validate our FMD method for pre-clinical assessment of vascular function.FMD and PWV methods were established in a colony (n = 25) of metabolically healthy, cynomolgus monkeys ranging in age from 6 to 26 years. FMD, defined as the percent change, at 1 min of cuff release, from baseline vascular diameter (0.15 ± 0.03 cm), had a strong, negative correlation with age , ranging from 6% to 33%. PWV positively correlated with age  in the same healthy monkeys. Nicotine and rosiglitazone, were evaluated in subsets of senescent primates (mean age 16.3 ± 1.5[SEM] years). Rosiglitazone significantly improved FMD  without changing baseline diameters, and coincided with a significant increase in circulating numbers of endothelial progenitor cells (CD45-CD31 + CD34 + VEGFR2+ 7.1 ± 1.3 vs. 4.8 ± 1.1 counts/μl) and a decrease in endothelial microparticles (CD45-CD42a-CD54+ 26.7 ± 11.1 vs. 62.2 ± 9.8 counts/μl)(p < 0.05). Conversely, FMD was significantly reduced with nicotine .Adult NHPs demonstrate the characteristic linear relationship between age and vascular function using the non-invasive clinically-related measurements of FMD and PWV. However, numbers of circulating EMPs and EPCs did not correlate with age. Endothelial function assessed with FMD, together with EMPs/EPCs assessment, may serve as a novel approach for translational research and therapeutic discovery. Age should be considered in the study design or data analyses when FMD or PWV is used in NHPs. Clinically, vascular function may be evaluated non-invasively by measuring flow mediated vascular dilation (FMD) , which aNon-human primate (NHP) is an important translational preclinical model for cardiovascular disease, diabetes and other metabolic diseases -16 and lThus, our primary goal was to develop the methods to measure FMD and PWV in NHPs and to examine the effects of age on these clinically relevant assessments of vascular function. In addition, we explored using FMD in senescent NHPs as a translational approach to cardiovascular efficacy/safety assessment. Here, we focus on FMD because there is limited information on using PWV for clinical assessment of drug effects, and FMD has emerged as an important clinical measurement for assessing cardiovascular risk factors and disease therapies ,11,19. FMacaca fascicularis, also known as cynomolgus)  were studied. These monkeys were paired housed, when appropriate, in a room with temperature of 19-25°C, humidity of 30-70%, and 12:45/11:15 light/dark cycles with a 45 minute ramping period to full lights on/off as simulated dawn/dusk. The monkeys were fed a primate chow diet  supplemented with fruits and/or vegetables with the amount adjusted to maintain a normal body weight. Conscious primates, previously conditioned to chair restraint , were seated comfortably in a primate chair for drug administration and blood collection prior to vascular measurements.Studies were conducted with the approval of the Pfizer Institutional Animal Care and Use Committee. Twenty-five healthy monkeys . A minimum of 2 measurements were used to calculate average PWV. Brachial artery measurements were taken at the axilla and femoral artery measurements were taken mid-thigh for PWV. The average distal distance  was 338 ± 5.4 mm and average proximal distance  was 75 ± 3.1 mm (n = 25). The distal vs. proximal distance allowed sufficient difference to calculate the PWV. Average detection variation for PWV was 10.6%.Electrocardiography (ECG) electrodes were placed, to collect a lead II ECG signal. Pulse waves from the brachial and femoral arteries, were recorded using a SphygmoCor  PWV probe that was associated with a modified SphygmoCor program . Distal-proximal distances were measured and entered into the program. PWV was calculated by the program using the equation PWV = ΔFMD in the NHPs was measured using ultrasonography, as illustrated in the Figure Six fasted monkeys (17 ± 1 years of age) were treated orally with both vehicle and rosiglitazone  ≥2 weeks apart in a 2 × 2 crossover design (2 in vehicle-drug sequence and 4 in drug-vehicle sequence). Drug or vehicle was administered at 6:00 AM, and, FMD and PWV were measured ~ six hours later, a time point that showed increased FMD in a human study . Blood sA separate group of monkeys  were sedated, and intravenously infused with saline vehicle, then with 0.844 μg/kg/min nicotine bitartrate , at rate of 0.4 mL/min. FMD was recorded at 5 min of vehicle infusion, and at 15 min of nicotine infusion (while the nicotine infusion was constantly maintained), and the study was completed within 35 min. The nicotine dose used was calculated to approximate the predicted blood nicotine concentration (40 nM) of a human study in which chewing nicotine gum reduced FMD ,27.To measure EPC, whole blood was collected into EDTA tubes from animals fasted overnight and, incubated with anti-human CD34, CD31, CD45  and VEGFR2  antibodies. After lysing the red blood cells, the samples were analyzed using a high performance Canto II flow cytometer (BD) with DIVA software (BD).At the same time, whole blood was collected in sodium citrate tubes and platelet poor plasma (PPP) was prepared to measure EMPs. An aliquot of PPP was added to each of the two panels of antibodies: Panel 1 ; Panel 2 . Cytofix was then added and a 500 uL aliquot was transferred into a Trucount tube (BD) and analyzed by flow cytometry.Venous blood samples were collected in serum separator tubes from untreated, rosiglitazone treated or vehicle treated monkeys fasted overnight, centrifuged at 3000 rpm for 10 min at 4°C, and levels of lipids and glucose measured on Siemens Advia 2400 chemistry analyzer, and levels of insulin measured on the Siemens Centaur Immunoassay, with kits of Advia Chemistry Systems .Lipopolysaccharide  stimulated release of TNFα was carried out, ex vivo, using sodium heparin anti-clotted whole blood that was harvested 6 hours after rosiglitazone dosing. Blood was stimulated with 10 ng/mL LPS for six hours with gentle rocking, plasma separated by 14,000 rpm centrifugation for 20 min at 10-15°C, and TNFα measured by using Linco Luminex®mouse cytokine assay.glc, the reduction of the loge of plasma glucose levels between 5 & 20 min following iv glucose administration and calculated using the formula: Kglc = (logeG1 – logeG2)/(t2 – t1) where G1 and G2 are the glucose levels at time 5 min (t1) and 20 min (t2) [Monkeys fasted overnight were sedated with ketamine for duration of IVGTT. Two iv catheters were placed for blood sampling and glucose administration, respectively. Baseline samples were drawn starting approximately 10 min after initial ketamine dose and catheter placement. A 50% dextrose solution was then administered, iv, at 250 mg/kg over 20 s. Blood samples (~1 mL) for glucose and insulin analysis were collected in serum tubes at 2, 5, 7, 10, 15, 20 and 30 min post dextrose administration, centrifuged and the resulting serum analyzed for glucose/insulin levels. The Area Under the Curve (AUC) for plasma glucose and insulin levels from 0 to 30 min was calculated. Glucose disappearance rate was expressed as Kmin (t2) .t test was used to compare EMPs/EPCs pre- and post- rosiglitazone treatment. Paired t test was applied to analyze the FMD data measured in the nicotine infusion study to evaluate the effect of nicotine on FMD compared to saline vehicle. Results are presented as parameter estimate (Mean) ± SEM. A two-sided p < 0.05 was considered statistically significant.Pearson correlation coefficient was computed for correlation study. Linear regression models were applied to investigate the relationships of age with FMD and PWV and the relationship between FMD and PWV. In order to evaluate the treatment effect of rosiglitazone on the endpoints of interest (FMD and PWV) compared to vehicle, a linear mixed effect model was applied to analyze each of the two endpoints from this 2 × 2 crossover study with age adjustment. Specifically, the model has treatment (drug vs. vehicle), sequence (1 vs. 2), and period (1 vs. 2) as fixed effects, age as a covariate, and subject (monkey) as random effect. Paired To examine age effects on FMD and PWV in NHPs, we established clinically-relevant, non-invasive methods Figure . FMD wasIn these metabolically healthy monkeys, FMD demonstrated a robust dynamic range (6 to 33%) that correlated negatively with age  = -1.119x(year) + 36.00)    and detrimental (nicotine) FMD changes following drug intervention in NHPs Figures  and 4. CDespite the lack of age-related correlation of EPCs and any of the EMPs, acute treatment with rosiglitazone decreased the number of EMP(CD45-CD42a-CD54+) and increased EPC(CD45-CD31 + CD34 + VEGFR2+) . The endothelial function assessment with FMD in NHPs and the associated circulating biomarkers , as described herein, may serve as a novel approach for translational research and therapeutic discovery. Like in humans, age is an important confounder that should be considered when assessing cardiovascular health and interpreting drug actions in NHPs.FMD: Flow mediated dilation; PWV: Pulse-wave velocity; NHP: Non-human primate; EMP: Endothelial microparticle; EPC: Endothelial progenitor cell; PPARγ: Peroxisome proliferator-activated receptor gamma; IVGTT: Intravenous glucose tolerance test.The authors declare that they have no competing interests. DRK’s current address is National Institutes of Health, Department of Health and Human Services. The opinions expressed in this article are the authors’ own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government.DRK designed and supervised the study while employed at Pfizer, Inc., and RLS and AHS performed the in vivo study. MW designed the nicotine study and the cytokine study. DAB and MW assisted with the in vivo study setup and sample processing. SAS and CH designed the EMP and EPC analysis, CH measured the EMPs and EPCs, and SAS analyzed and interpreted the EMP and EPC data. DRK, AHS, and MW analyzed and interpreted the in vivo and in vitro data. MW wrote the manuscript. All authors have read and approved the manuscript for publication.Basal metabolic parameters of the study monkeys. A: Blood lipids profile. B: Glucose, insulin, and IVGTT.Click here for fileFMD or age does not correlate with numbers of EPCs or EMPs in metabolically healthy monkeys. n = 19 and 21 for EPC and EMP, respectively.Click here for fileFigure S2. Rosiglitazone results in favorable changes in circulating numbers of EMPs (CD45-CD42a-CD54+) (A) and EPCs (CD45-CD31 + CD34 + VEGFR2+) (B) in euglycemic, senescent monkeys. n = 5, *: p < 0.05; **: p < 0.01.Rosiglitazone does not affect PWV in NHPs. n = 6, p = 0.22 (age adjusted). NS: non-significant. Click here for fileglc, 5-20: slope of the disappearance of glucose 5-20 min after dosing; AUC0-30: area under the curve for glucose or insulin (0-30 min).Effect of rosiglitazone treatment on blood biochemistry and LPS-stimulated TNFα release. No significant difference between the two groups, n = 5 (vehicle) or 6 (rosiglitazone). KClick here for file


People living with HIV/AIDS (PLWHA) frequently have abnormal blood counts including anemia, leucopenia and thrombocytopenia. The role of infection with plasmodia on these hematological parameters in PLWHA is not well known. In this study we compared selected hematological parameters between malaria positive and negative PLWHA.We conducted a cross-sectional study of PLWHA attending the Douala Laquintinie hospital. After obtaining consent, demographic and clinical data were obtained via a standardized questionnaire. Blood samples collected for hematological assays were run using an automated full blood counter. Malaria parasitaemia was determined by blood smear microscopy.A total of 238 adult PLWHA were enrolled, 48.3% of who were on antiretroviral therapy and 24.8% of whom had malaria parasitaemia. The respective mean (±SD) of hemoglobin level, RBC count, WBC count, platelet count, lymphocyte count and CD4+ T cell counts in malaria co-infected patients versus non-infected patients were: 10.8(±1.9) g/dl versus 11.4(±2.0)g/dl; 3,745,254 cells/µl versus 3,888,966 cells/µl; 4,403 cells/µl versus 4,920 cells/µl; 216,051 cells/µl versus 226,792 cells/µl; 1,846(±711) cells/µl versus 2,052(±845) cells/µl and 245(±195) cells/µl versus 301(±211) cells/µl. All these means were not statistically significantly different from each other.There was no significant difference in studied hematological parameters between malaria positive and negative PLWHA. These data suggest little or no impact of malaria infection. Hematological anomalies in PLWHA in this area need not be necessarily attributed to malaria. These need to be further investigated to identify and treat other potential causes. Malaria and HIV are two important causes of morbidity and mortality in Africa. Over two-thirds of people living with HIV/AIDS (PLWHA) are found in sub-Saharan, a region in which malaria is endemic Although malaria and HIV infections are highly prevalent and remarkably overlapping in Sub-Saharan Africa, the extent and implications of their interactions are still not completely understood In Cameroon, an estimated 610,000 people were living with HIV in 2009, with an adult prevalence of 5.3% This study aimed at assessing the impact of co-infection with malaria on some hematological parameters in HIV-infected people. Such information is expected to help health care practitioners improve on current diagnostic, therapeutic and follow up guidelines of HIV-infected patients presenting with hematological abnormalities.This was a cross-sectional study carried out between the months of April and June 2010 at the “day care center” of Laquintinie Hospital, Douala, Cameroon.Plasmodium falciparum. Douala therefore, was an ideal site to investigate HIV/malaria co-infection.Douala is situated in the tropical rainforest zone of the Congo basin. As the economic capital of Cameroon, Douala attracts people of all social backgrounds seeking employment, besides business people and tourists. The area has rainy and dry seasons, running from mid-march to October and from November to mid-march respectively. The town has several swampy sites, stagnant water pools around houses in unpopular quarters especially during the rainy season, poor waste disposal in a constantly increasing population; all factors that support breeding of female Anopheles mosquitoes, the vectors of the malaria parasite. Malaria in Douala and most of Cameroon is endemic, with high transmission. Malaria is predominantly due to Included in this study were non-pregnant adult people living with HIV/AIDS (PLWHA) presenting at the Laquintinie Hospital for routine follow up and/or treatment. After providing written consent, participants were interviewed using a standardized structured questionnaire. Blood samples were then collected for thick and thin smear microscopy as well as a standard full blood count. Each participant’s medical records were also reviewed to consider past and present underlying opportunistic infections reported, especially those usually affecting blood cells.Using sterile plastic syringes, 3 mls of venous blood was aseptically collected into sterile EDTA-vacutainer tubes for laboratory investigations. Thick and thin films were prepared from a portion of blood for each participant’s blood sample within 30 minutes of collection. After fixing thin smears with absolute methanol, smears were stained using 10% Giemsa for 10 minutes and read by microscopy Another portion of blood was used for full blood counts using the Hema Screen18 automated full blood counter . CD4+ T cells were enumerated using the Partec CyFlow® automated cell counter . Malaria parasitaemia was measured as parasites per microliter based on the measured white blood cell counts (WBC).Data collected were entered into EPI-INFO version 3.5.1 software  and analyzed using STATA version 10 .Two sample Student t-tests were used to assess differences in means between malaria positive and negative participants. To account for potential confounding, multivariate analyses were conducted using multiple linear regression models. Each model had the specific hematological parameter being analyzed  as dependent variable; and malaria parasitaemia (positive versus negative), age, gender, CD4+ cell count and antiretroviral therapy as independent variables. Pearson correlation coefficients were also used to quantify the relationship between malaria parasitaemia and each of red blood cell count, hemoglobin levels, white blood cell counts, lymphocyte counts, CD4+ cell counts and platelet counts, among malaria positive participants. To assess if there were differences  by potential modifiers , we created product interaction terms between parasitaemia and each of the potential modifiers in multivariate linear regression models. None of these interactions were statistically significant. The statistical significance level was set at 0.05.A total of 238 non-pregnant adults fulfilling the inclusion criteria voluntarily participated in the study. Of these 161 (67.6%) were females. Study participants’ characteristics are summarized in In terms of HIV-related comorbidities, 42 patients had a history of pulmonary tuberculosis while three participants each had a history of toxoplasmosis, Pneumocystis pneumonia, oral candidiasis, and HSV disease; two had a history of cryptococcal meningitis and one had a history of oesophageal candidiasis. The CD4+ cell count was <200 in 99 (41.8%) of participants and 48.3% of all participants were on antiretroviral therapy.Plasmodia were detected in 24.8% of participants. Parasitaemia ranged from 30–26,550 with a median of 3,875  parasites/µL.Hematological parameters in malaria positive and negative participants are compared in Amongst the 59 participants in whom plasmodia were detected, the plasmodium parasitaemia levels were not significantly correlated with each of red blood cell count, hemoglobin levels, white blood cell counts, lymphocyte counts, CD4+ cell counts and platelet counts .Plasmodium falciparum infections et al., et al., Hematological anomalies are considered a hallmark of malaria, and reported to be most pronounced in et al., et al., The mean red blood cell count and hemoglobin concentrations were not significantly lower in malaria parasitaemia positive participants than in malaria negative participants (P>0.05). The insignificant difference in hemoglobin concentrations between these groups was contrary to previous findings by Erhabor et al., The mean lymphocyte and total white cell counts in malaria positive participants were not significantly different from those in malaria negative participants (P>0.05). These observations agreed with previous findings by Erhabor The mean platelet count in malaria positive participants was not significantly different from that in malaria negative participants (P>0.05). This was not in accordance with the previous finding that mean platelet count in plasmodium parasitized individuals HIV/AIDS patients was significantly lower as compared to non-parasitized controls + cell count was not significantly lower in malaria positive participants (245±195) when compared to malaria negative participants (301±211). This was in accordance with findings reported by Audu et al., +T lymphocytes decline temporarily during malaria episodes in HIV-infected and uninfected persons + T lymphocytes over time Lastly, the mean CD4Multiple factors could contribute to haematological parameters in people living with HIV/AIDS. These include nutrition and intake of nutrients such as iron, vitamin B12, folic acid, the use of antiretrovirals some of which may impact haematological parameters, opportunistic infections that may modify the immune response and thus modify WBCs and lymphocyte counts as well as therapy for these other opportunistic infections, latent undetected neoplasia and malaria. The relative role of each of these factors could vary from population to population. If the lack of a difference with respect to malaria parasitaemia in this population was confirmed, this would suggest that other factors may have a predominant role. In the meantime, clinicians need to be aware to provide a wider assessment for PLWHA who present with an anomaly in their haematological parameters.While studying a population living in endemic area, use of an accurate quantification for parasitaemia and the use of multivariate analysis controlling for age, gender and antiretroviral use were strengths of this study, the sample size did not allow for a simultaneous adjustment accounting for many other factors such as cotrimoxazole use, and other comorbidities . The relatively small sample size of malaria co-infected people living with HIV could also have resulted in a lack of statistical significance in the observed differences, thus these findings may need to be confirmed in a study with a larger number of malaria positive participants. Also, the cross-sectional design precludes from inferring a causal role (or lack thereof) of malaria parasitaemia on haematological parameters.In conclusion, this comparison of mean red blood cell counts, mean hemoglobin concentrations, mean lymphocyte count, mean platelet counts, total white cell count and CD4 counts between malaria parasitaemia positive and negative people living with HIV/AIDS in Douala suggests no significant differences. These findings indicate that malaria may not be the primary cause or correlate of hematological disorders in these patients. As such findings of hematological anomalies should not be assumed to be due to malaria – a more specific etiologic investigation is needed to establish the likely etiology. Longitudinal studies, in large cohorts, investigating the medium and short term evolution of these hematological anomalies and the relationship with malaria and malaria treatment are also needed to more accurately aid the investigation and management of hematological anomalies in people living with HIV/AIDS.


Malaria is hyper-endemic and a major public health problem in Sierra Leone. To provide malaria treatment closer to the community, Médecins Sans Frontières (MSF) launched a community-based project where Community Malaria Volunteers (CMVs) tested and treated febrile children and pregnant women for malaria using rapid diagnostic tests (RDTs). RDT-negative patients and severely ill patients were referred to health facilities. This study sought to determine the referral rate and compliance of patients referred by the CMVs.In MSF's operational area in Bo and Pujehun districts, Sierra Leone, a retrospective analysis of referral records was carried out for a period of three months. All referral records from CMVs and referral health structures were reviewed, compared and matched for personal data. The eligible study population included febrile children between three and 59 months and pregnant women in their second or third trimester with fever who were noted as having received a referral advice in the CMV recording form.The study results showed a total referral rate of almost 15%. During the study period 36 out of 2,459 (1.5%) referred patients completed their referral. There was a significant difference in referral compliance between patients with fever but a negative RDT and patients with signs of severe malaria. Less than 1%  of the RDT-negative patients with fever completed their referral compared to 88.2% (15/17) of the patients with severe malaria (RR = 0.010 95% CI 0.006 - 0.015).In this community-based malaria programme, RDT-negative patients with fever were referred to a health structure for further diagnosis and care with a disappointingly low rate of referral completion. This raises concerns whether use of CMVs, with referral as backup in RDT-negative cases, provides adequate care for febrile children and pregnant women. To improve the referral completion in MSF's community-based malaria programme in Sierra Leone, and in similar community-based programmes, a suitable strategy needs to be defined. Malaria is one of the leading causes of mortality and morbidity among children in Sub-Saharan Africa. The disease causes nearly 800,000 deaths per year, most of which are in children under five years of age . Sierra ®), ACT (artesunate-amodiaquine) and paracetamol. They were also trained to recognize and ask for signs of severe malaria . The training was both theoretical and practical, including a session on how to perform and read RDTs.Médecins Sans Frontières (MSF) has been working in Sierra Leone since 1995. In collaboration with the MOHS in Bo and Pujehun districts, MSF provides free primary and secondary health care. In 2008 malaria accounted for over 40% of all consultations in the MSF-supported clinics and health posts in Bo and Pujehun districts. At the By June 2009, a total of 136 CMVs operated in 130 villages within MSF's catchment area treating children between three months and five years of age and pregnant women, except for those in the first trimester of their pregnancy, for malaria. Two CMV supervisors with motorcycles oversaw the CMV activities and carried out random supervision visits and monthly meetings where drugs and RDTs were distributed, statistics were collected and general issues related to the CMVs' work were discussed. The CMVs were meant to refer all cases with signs of severe malaria and those with complaints of fever but with a negative RDT to the nearest health facility, located within a range of three to 18 kilometres from the villages where the CMVs were based.A key component of community-based malaria care, using minimally-trained volunteers, is an effective referral system, to ensure that patients with severe malaria or other illnesses receive treatment. This has been discussed in several studies, describing various referral patterns and with referral completion rates varying from 37.5% to 87% . Before This study took place in MSF's operational area in rural areas of Bo and Pujehun districts, Sierra Leone, with an estimated target population of 302,000. These districts were hyper-endemic malaria zones with high transmission rates all year, especially during the rainy season from June to October. The CMV catchment area consisted of 130 villages. There were one or two CMVs in each village depending on population size. The CMVs were divided into 16 networks, each linked to a health centre or a health post for supervision and referral. These health structures were under the responsibility of the MOHS but jointly managed by MSF. Health care was provided for free, and MSF covered costs for staff salaries, drugs and medical materiel and provided supervision. Health posts were provided with partial support (limited number of drugs for children less than 15 years of age and pregnant women). The distance from the villages to the nearest MSF supported health structure varied from three to over 18 kilometres. Apart from the MSF-supported health structures there were no other major care providers (e.g. private clinics or drug vendors).The study population included all children between three and 59 months of age and pregnant women in their second or third trimester who were recorded in the CMV recording forms as referrals. The CMVs had been trained to refer all patients with signs of severe malaria and a positive RDT and all patients with complaints of fever but with a negative RDT. Children less than three months of age and pregnant women in their first trimester, who would always be referred due to contra-indications of ACT, were excluded. Patients with incomplete or un-interpretable data and repeated referrals were also excluded from the study Figure .Data on patients seen by CMVs were entered in a recording form and included basic demographic information, RDT result, treatment given and date of and indications for referral. The CMVs were instructed about the importance of accurate data recording. In addition, a referral follow-up register was introduced in June 2009 into all 16 MSF-supported health structures linked to a CMV network. The health centre/health post personnel were trained on how to fill it in properly and were instructed to ask each patient arriving at the structure whether or not he/she was referred from a CMV. The health structure register contained basic demographic information as well as final diagnosis and treatment. The CMV recording forms were collected during monthly supervision visits by two CMV-supervisors. All records from CMVs and referral follow-up registers were matched and demographic data, date of arrival, name of health structure and name of the referring CMV were compared. If all the above data matched, the referral was confirmed as being completed. For each completed referral, final diagnosis and time of arrival at the health structure (> or < 24 hrs) were recorded. The data collection was overseen by the field investigator, who was able to give regular support to CMV supervisors, health centre/health post nurses and others involved in this study at field level. All data were routinely collected from the CMVs and the health structures. Raw data were entered, validated and encoded in Sierra Leone by the field investigator and mission epidemiologist, with the help of three data-encoders.st of September to the 30th of November 2009. Every village and CMV had an assigned code that was used when entering and crosschecking the data. All patients included in the study were assigned an identification number to facilitate study-related documentation and ensure confidentiality. The principal investigator, who had previously worked as field coordinator in MSF's project, did the analysis at Karolinska Institutet in Stockholm. All data were coded, entered into the data analysis software and double-checked to ensure consistency and accuracy. Data was analysed for frequencies of referral completion and stratified by sex, age and RDT-status using EpiInfo, version 3.5.1.The analysis of record reviews was carried out over a three months period, from the 1The routinely collected programme data that was used in this study was handled with confidentiality. The study was approved by MSF's Ethics Review Board. Permission to carry out the study had previously been granted from the MOHS in Sierra Leone.During the study period the CMVs saw a total of 17,130 patients with complaints of fever. Out of these patients 12,204 (71%) were eligible children and 4,901 (28%) were pregnant women. Out of 2,554 patients registered as referred, 25 patients (1%) were male adults (19) or children above five years of age (6), not fitting the inclusion criteria. Seventeen patient-entries were excluded from the survey due to incomplete or un-interpretable data and 53 referrals were excluded as they were less than three months of age (51) or pregnant women in their first trimester (2). Out of the 2,459 remaining referrals, 2,042 (83%) were children under five years of age and 417 (17%) were pregnant women  were referred to the nearest MSF-supported health structure. A total of 17%  of the children under five and 8.5% (419) of the pregnant women were given a referral advice. The referral rate varied from 15.7%  to 13.4%  over the three study months. Out of the total referrals, 99.3%  had a negative RDT while 17 (0.7%) tested positive, and were referred with signs of severe malaria. The main referral reason for RDT-negative patients was indicated as "fever" . RDT-positive patients were referred for different reasons, such as "complicated", "fever/vomiting", "fever/cough" and "high fever".During the full study period only 36 out of 2,459 patients completed their referral, indicating a referral completion rate of 1.5%. The referral completion rate varied from 2.3% (19/812) to 0.5% (4/779) by study month. Referral completion rate ranged from 0% (0/21) to 18.8% (12/52) across the 16 different networks. The completion rate for children was 1.6%  whereas only three (0.7%) of the 417 referred pregnant women arrived at a health structure. A total of 51.5% (17/33) of completed referrals among children were female and 48.5% (16/33) were male (RR = 1.247 95% CI 0.634 - 2.454). The completion rate varied from 2.7% (7/252) among children less than one year to 0.8% (4/503) for children between two to three years (RR = 3.490 95% CI 1.030 - 11.820). A total of 88.2% (15/17) of patients who tested positive for malaria and were referred with signs of severe malaria completed their referral, while only 0.9%  of patients with a negative RDT arrived at a health structure (RR = 0.010 95% CI 0.006 - 0.015)  of these patients were diagnosed as "malaria". Others were diagnosed as "malaria/anaemia" 20%, 3/15), "malaria/acute respiratory infection (ARI)" , "malaria/convulsion" , "malaria/vomiting"  and "malaria/skin infection" . Out of the patients referred with a negative RDT, the most common diagnosis was "ARI" , followed by "common cold" , "abscess", "skin infection", "fever of unknown origin", "headache"  and "nephritis" . Out of the 36 patients who completed their referral and arrived at a health structure, 92% (33/36) arrived on the same day or within 24 hours. Three patients (8%) arrived at the health structure after 24 hours , trained to recognize and treat simple malaria at village level, are becoming an integral part of many health systems. Research has shown that community-based malaria control programmes can reduce paediatric hospitalisations , improveAn important factor in the use of CHWs to reduce mortality is completion of their referrals. Referral compliance varies in different contexts and at various levels of health care. In Uganda, 87% of patients referred to health centres by CHWs complied with the referral , while cIt is difficult to understand why the referral completion rate in MSF's CMV programme was so low. There could be several reasons, as described above, but adequate supervision and training could be one way of improving the referral compliance. Several studies have shown that training is essential in order to ensure a successful community-based malaria programme. For example, training of caretakers, as well as of private drug vendors in Nigeria has lead to improved recognition, treatment and referrals of malaria cases ,20. A trOne possible strategy to reduce the poor referral compliance rate is to narrow the diagnostic criteria for referral so that patients could avoid unnecessary costs and time in attending a health structure. CMVs could receive additional training regarding respiratory infections to distinguish between serious signs of pneumonia (e.g. increased respiratory rate) and those of common cold viruses. This strategy has support in the literature where CHWs have been able to diagnose and treat pneumonia in Nepal  and in UThere are some encouraging results from this study. Previous research shows that CHWs are capable of using and interpreting RDTs ,26. The This retrospective study had several limitations. The short study duration may have been influenced by disease patterns, disease variations and seasonal activities such as farming, affecting caretakers' behaviour. The low number of referral completions did not allow for a proper analysis of outcome and timeliness of referrals, as well as for impact of distance from villages to referral structures. As analysis of data was done through record reviews it could not be verified that all referred RDT-positive patients had signs of severe malaria. Analysis of signs of severe disease among RDT-negative patients could also not be carried out. The record review was done in two steps: comparison of the two records  and encoding of this data was done in the field. The data analysis was done at Karolinska Institutet, and there is a possibility of misinterpretation of data. With only two CMV supervisors responsible for data collection and direct CMV supervision, it is possible that referred patients were not properly entered in the CMV recording forms. There is also a risk that patients were missed due to variations in their own or their caretaker's name. Using three different data-clerks in the field for encoding might have increased data errors. Finally, a qualitative, in-depth study, interviewing CMVs, health personnel and patients could give a better picture of the underlying reasons for non-compliance of referrals.This study showed that most RDT-negative patients with fever seen by CMVs in a community-based programme in Sierra Leone did not arrive at a referral structure. This finding has prompted a review of the programme by MSF. To map the underlying reasons for non-completion of referrals and to define a suitable strategy for improving the referral completion in MSF's community-based programme, additional research is needed. Similar community-based malaria programmes should assess their referral-completion rates to ensure that patients with severe malaria or other illnesses receive treatment.The authors declare that they have no competing interests.AT designed the study, analysed the data and wrote the paper with major contributions of the other authors. AM approved the study and initiated discussions around study objectives. TR contributed with the study design and gave input to the study protocol and manuscript. MK entered and analysed the data and supervised the fieldwork. JvS, SP and MdS monitored the study progress and contributed significantly to improve the manuscript. All authors read and approved the final draft.