Population resettlement has been considered among factors that may increase risk of malaria transmission. This study reports, the impact of resettlement on malaria incidence and entomological indices among communities resettled in suburbs of Jimma town, southwestern Ethiopia.Plasmodium infection rates.A cohort of 604 study participants (302 resettlers and 302 non-resettlers) was monthly followed-up from September to November 2013 using active case detection. Moreover, longitudinal entomological study was conducted from June to November 2013. Anopheline mosquitoes were collected using CDC light traps and pyrethrum spray catches. Sporozoite ELISA was performed to determine Plasmodium falciparum incidence from resettlement and non-resettlement villages was 52.5 and 14.5/1,000 person-months at risk, respectively. Resettlement villages were three times at higher risk of Plasmodium infection . Anopheles gambiae s.l. was the predominant (86.6%) of all the collected anopheline mosquito species. Plasmodium sporozoite rate in the resettlement and non-resettlement villages was 2.1 and 0.72%, respectively. Plasmodium falciparum entomological inoculation rate (EIR) for An. gambiae s.l. in the resettlement and non-resettlement villages was 13.1 and 0 infective bites/person/night, respectively. Both sporozoite rate and EIR were significantly higher in the resettlement villages (p < 0.05).Overall, 112 malaria cases were recorded during the three-month follow-up, of which 74.1% of the cases were from resettlement villages. Resettled communities were at higher risk of malaria infection as compared to non-resettled communities. Special attention should be given to malaria control interventions during resettlement programmes. Malaria is a serious problem in Ethiopia, where about 68% of the population lives in malaria-risk areas. It is endemic in Ethiopia with differing intensity of transmission, except in the central highlands, which are malaria-free. In 2009, three million suspected malaria cases were recorded and nearly 2.3 million (77%) were tested. Besides the individual suffering caused by the disease, malaria poses a significant economic burden in Ethiopia ,2.et al. [et al. [Malaria showed a decline in Ethiopia over the last ten years as a result of high coverage of key malaria control interventions. This is attributable to the introduction of artemisinin-based combination therapy (ACT), use of rapid diagnostic tests (RDTs) at the peripheral health facilities, wide-scale distribution of long-lasting insecticidal nets (LLINs) and high coverage of sprayed households through targeted indoor residual spraying since 2004/2005 . The Nat [et al.  also rep [et al. . HoweverIn Ethiopia, information on the impact of resettlement on malaria incidence and transmission is scarce. However, few studies conducted in Pawe area, Northwest Ethiopia documented the emergence of malaria following resettlement schemes -10. CurrThe study was carried out in two resettlement and two control (non-resettlement) villages of Jimma town, southwest Ethiopia Figure . Jimma tThe control villages were selected purposefully based on the following criteria: i) Comparable socio-economic condition between resettlement and non-resettlement villages ii) Similarity in eco-topography between resettlement and non-resettlement villages, iii) Minimum distance of 3km between the two villages considering anopheline mosquito’s flight range, iv) Similarity in climate.Jimma town is characterized by warm climate with a mean annual minimum and maximum temperature of 14°c and 30°c, respectively. The mean annual rainfall ranges from 1,138 to 1,690 mm. Maximum precipitation occurs during the three months (June to August), with minimum precipitation in December and January .For parasitological survey, community-based cohort study was conducted using active case detection from September 1 to November 30, 2013. Moreover, longitudinal entomological study was conducted from June to November, 2013.A cohort of 604 (302 from resettlement and 302 from non-resettlement villages) individuals residing in 202 households was followed-up. The sample size was determined using the formula for two population proportion with the following assumptions: Resettlement was considered as an exposure factor, previous malaria prevalence of 5.2% in Jimma town  was conskebele administration. Finally, simple random sampling was employed to select households from each village. All family members in the selected households fulfilling the inclusion criteria were enrolled in the study. Inclusion criteria of the study participants were: intention to remain in the study area for the duration of study follow-up, willingness to participate in the study and being permanent resident (in non-resettlement villages) and re-settler since 2008 (in resettlement villages). Individuals who were taking anti-malarial drugs at the baseline were excluded from the study.Households were selected from each village by dividing the sample size to the average family size of the town . Census Baseline data on socio-demographic and economic characteristics of each household member, use of malaria preventive measures and ownership of domestic animals were collected using semi-structured questionnaire. Presence and distance of temporary vector breeding sites from each household were recorded monthly.Parasitological data were collected by active case detection. A unique identification card was given to each household member. Each study participant was visited monthly for three months. During monthly house-to-house visit, blood sample was collected from the study participants following standard protocol . Thick aAdult anopheline mosquitoes were collected from both resettlement and non-resettlement villages. For the indoor mosquito sampling, adult anopheline mosquitoes resting inside human habitations were collected monthly from 10 selected houses per village using pyrethrum spray catches (PSCs) and from two houses per village at fortnight interval using Centers for Disease Control and Prevention (CDC) light trap catches (LTCs) .Pyrethrum spray catches were conducted early in the morning from 06:00 to 08:00 hr. White sheets of cloth and Baygon aerosol  were used for the spray. After obtaining consent from the inhabitants, foodstuff and utensils were removed before spraying. Openings and eves which allow mosquito escape from houses were covered and white sheets were spread over the floor. Finally, rooms were sprayed with the aerosol for about 10 minutes. After 15 minutes following spraying, the sheet was removed and taken outside to inspect and collect the knockdown mosquitoes. CDC light trap was set indoor near the bed at a height of 1.5 meter from 18:00 to 06:00 hr in the selected houses to collect endophagic anopheline mosquitoes. For the outdoor mosquito sampling, CDC light trap was also set in the vicinity of sentinel houses from 18:00 to 06:00 hr.The collected mosquitoes were transported to the Vector Biology Laboratory and sorted by genus and sex. The mosquitoes were morphologically identified to species using taxonomic keys ,15. FemaPlasmodium falciparum, Plasmodium vivax 210 and P. vivax 247 circumsporozoite proteins (CSPs) simultaneously using ELISA [Dried head and thorax of the preserved anopheline mosquito specimens were carefully separated from the abdomen and tested for ng ELISA ,17.Plasmodium falciparum incidence rates were estimated as the number of cases per 1000 person-months at risk.Data were coded, checked for completeness and entered into a computer. The data were then analysed using SPSS software package version 16.0 . At baseline, household and individual-level comparison was made in terms of socio-demographic and socio-economic characteristics between resettlement and non-resettlement villages using chi-square test. Plasmodium infection between resettlement and non-resettlement villages. Let Yijk be the kth binary outcome measure  for individual (IND) j living in household (HH) i. The following three-level logistic mixed model was considered which accounted for the clustering of the outcomes within individuals and households.Generalized liner mixed effect model (GLMM) was used to compare the risk of airepresent the fact that some households are more successful in utilizing protective methods for malaria than others. The individual effects bj(i) represent the fact that susceptibility to Plasmodium infection is not the same among individuals. Thus, comparison between resettlement and non-resettlement villages was adjusted for the possible confounding factors such as age, gender, distance from vector breeding site, insecticide-treated nets (ITNs), indoor residual spraying (IRS), presence of domestic animals in or around the houses, family size and type of house.The household effects P. falciparum and P. vivax of the total number of mosquitoes tested. Monthly Plasmodium EIRs of Anopheles gambiae s.l. was calculated from mosquito collections by LTCs using the standard formula, 1.605 × (no. circumsporozoite-positive ELISA results from CDC LTCs/no. mosquitoes tested) × (no. mosquitoes collected from CDC LTCs/no. catches) × No. days per month [The sporozoite rate was estimated as the proportion of mosquitoes positive for er month .An. gambiae s.l. density, sporozoite rate and EIRs. SPSS version 16.0  and SAS version 9.3  statistical software packages were used for the analysis. p < 0.05 was considered significant during the analysis.Analysis of variance was employed to compare resettlement and non-resettlement villages in terms Ethical clearance was obtained from the Research and Ethics Review Committee of Jimma University. Permission was sought from Jimma Town Health Office. Written informed consent was obtained from heads of the households and parents/guardians for children. During parasitological survey, participants found positive were treated according to the national malaria diagnosis and treatment guideline.Table The majority (57.1%) of the participants were females. Fifty seven percent of the study participants were above the age of 14 years followed by age groups 5–14 (30.5%) and < 5 years (12.5%) with mean age of 21 years. The difference in gender and age of the study participants between resettlement and non-resettlement villages was not significant (p > 0.05).Plasmodium falciparum, P. vivax and mixed infections accounted for 58 (51.8%), 52 (46.4%) and 2 (1.8%) of the total cases, respectively.All of the study participants completed the first and second round of parasitological survey while 22 (3.7%) were lost to follow-up during the third round survey. The overall prevalence of malaria was 112 (6.3%). P. falciparum (77.6%) and P. vivax (69.2%) cases and two of the cases due to mixed infections were from resettlement villages. Figure P. falciparum incidence was 33.5/1,000 person-months at risk, with 52.5/1,000 and 14.5/1,000 person-months at risk incidence in resettlement and non-resettlement villages, respectively.Eighty three (74.1%) and 29 (25.9%) of the malaria cases were from the resettlement and non-resettlement villages, respectively. The majority of Plasmodium infection between resettlement and non-resettlement villages. The difference in the risk of Plasmodium infection between resettlement and non-resettlement villages was significant . Individuals living in the resettlement villages were three times at higher risk of malaria infection . House structure and family size were the main predictors of Plasmodium infection. The risk of Plasmodium infection among those who lived in houses with holes on the walls was three times higher as compared to those who lived in houses with no holes on the walls . Family size also showed association with Plasmodium infection. For a decrease in family size by one, the odds of Plasmodium infection decreased by 24% . Other covariates assessed did not show significant effect at 5% level of significance.Table An. gambiae s.l., Anopheles coustani complex, Anopheles pharoensis and Anopheles squamosus) were collected over the study period. Anopheles gambiae s.l. was the most predominant species (86.6%). The majority (76.3%) of anopheline mosquitoes were collected from the resettlement villages.Table Anopheles gambiae s.l. in the two villages is shown in Figure An. gambiae s.l. in the study area ranged from 1.96 to 15 per trap/night with mean density of 8.21 per trap/night. Mean indoor host seeking An. gambiae s.l density in the resettlement and non-resettlement villages was 13.38 and 3.04 per trap/night, respectively. The difference in mean density of host seeking An. gambiae s.l between the two villages was significant . Mean indoor resting density of An. gambiae s.l. was significantly higher in resettlement villages (2.8 per house/day) than non-resettlement villages (0.99 per house/day) . Similarly, significantly higher mean outdoor host seeking density of An. gambiae s.l. was recorded in the resettlement villages .Mean monthly indoor and outdoor density of Plasmodium CSPs. Of these, 23 An. gambiae s.l. specimens were positive for CSPs, of which 19 (82.6%) were positive for P. falciparum and 4 (17.4%) were positive for P. vivax . Overall Plasmodium sporozoite rate for An. gambiae s.l. was 1.8%. The sporozoite rates of P. falciparum and P. vivax for An. gambiae s.l. were 1.5% and 0.3%, respectively. Plasmodium sporozoite rates for An. gambiae s.l. from indoor LTCs, outdoor LTCs and PSCs were 2.0%, 1.9% and 1.5%, respectively. Infectivity rates for An. gambiae s.l. in the resettlement and non-resettlement villages were 2.1% and 0.72%, respectively and the difference was significant .A total of 1,510 anopheline mosquitoes were tested for An. gambiae s.l. in the study area. Overall the EIRs of An. gambiae s.l. for P. falciparum and P. vivax were 6.55 and 1.46 infective bites/person/month, respectively. The EIR of An. gambiae s.l. for P. falciparum in resettlement and non-resettlement villages was 13.1 and 0 infective bites/person/month, respectively, while EIR of An. gambiae s.l. for P. vivax EIRs in resettlement and non-resettlement villages was 2.9 and 0 infective bites/person/month, respectively. The difference in P. falciparum EIRs of An. gambiae s.l. between resettlement and non-resettlement villages was significant .Table P. falciparum infection as compared to individuals living in non-resettlement villages. The higher incidence of P. falciparum infection in resettlement villages might be due to suitable mosquito breeding sites created as a result of the resettlement [P. falciparum malaria incidence. The higher incidence of P. falciparum in the resettlement villages calls for malaria control intervention strategies during resettlement schemes.This study revealed that malaria incidence and transmission intensity was significantly higher in the resettlement villages as compared to the non-resettlement villages. Individuals living in resettlement villages were three times more likely to have ttlement . MoreoveP. falciparum compared to permanent residents which is in agreement with the findings of the current study.Other studies have also reported increase in malaria incidence and an increase in malaria affected people as a result of population resettlement . In studThe socio-demographic and socio-economic data indicated that the resettlement and non-resettlement study communities were similar with respect to age, sex, educational status, occupation of heads of the households, monthly income, family size, presence of domestic animals, ITN ownership and IRS. But, nearly three quarters of the households in the resettlement villages were located less than 1 km from vector-breeding sites. Moreover, 12% and 4% of the houses in the resettlement and non-resettlement villages were with holes on the walls, respectively. The overall malaria prevalence (6.3%) recorded in this study is slightly higher than the previous report from Jimma town, southwestern Ethiopia (5.22%) . This coP. falciparum incidence  is higher than malaria incidence of 3.6/10,000 person-weeks and 14.6 cases/1000 person-months at risk reported from Chano Mille [P. falciparum incidence recorded in this study is lower than the incidence  reported from Adama, central Ethiopia [P. falciparum, P. vivax and mixed infection, which might have contributed to the observed difference.In this study, the no Mille  and Gilgno Mille , respectEthiopia . In the An. gambiae s.l. was the most predominant species in the study setting, accounting for 86.6% of all collected anopheline mosquitoes. Other similar studies also documented that An. gambiae s.l. is the most predominant species in Ethiopia [The entomological survey of this study showed that Ethiopia ,29,30.An. gambiae s.l. was recorded in the resettlement villages as compared to the non-resettlement villages. Indoor host-seeking density of An. gambiae s.l. was four times higher in resettlement villages. In addition, outdoor host-seeking density of An. gambiae s.l. was higher in the resettlement villages. These differences could be due to the presence of abundant temporary vector breeding sites in resettlement villages. Cox et al. [P. falciparum sporozoite rate in this study might have led to the occurrence of higher P. falciparum malaria cases than P. vivax in the study setting. Other studies also documented increased Plasmodium sporozoite rate with increased malaria incidence in southwestern Ethiopia [Higher density of x et al.  also repx et al. . Higher Ethiopia ,29.Plasmodium sporozoite rate and mean monthly EIR of P. falciparum for An. gambiae s.l. were significantly higher in the resettlement villages. The higher EIRs observed in the resettlement villages indicate that malaria transmission intensity was higher in those villages. Heterogeneity in anopheline mosquito infectivity rate among villages was also reported in studies conducted in Ghana [Plasmodium infectivity rates can be explained by the spatial heterogeneity of exposures to infection that is, human vector contacts.in Ghana , Burkinain Ghana  and Sudain Ghana . Small-aAn. gambiae s.l. recorded in the present study is higher than the EIR reported from southern, central and south-central Ethiopia, with annual EIRs of 17.1, 34.8, 3.7 infective bites per year, respectively [The annual EIR of ectively ,34,35. TAn. coustani was positive for P. falciparum CSP (not PCR confirmed). Although this species was not yet incriminated as vector of malaria in Ethiopia, it is a complementary vector elsewhere in Africa [In addition, a single specimen of n Africa ,37. ThisPlasmodium infection as compared to communities from non-resettlement villages. Malaria transmission intensity was also higher in the resettlement villages. Appropriate use of the existing malaria control intervention tools such as ITNs and IRS should be in place and scaled-up during resettlement schemes. Health education and environmental management supported by active community mobilization and participation is recommended to eliminate temporary and/or permanent vector-breeding sites in such new settlement areas. Further studies on the infectivity of An. coustani should be carried out using molecular diagnostics.The study communities from resettlement villages were at higher risk of


Malaria-endemic countries in sub-Saharan Africa are shifting from the presumptive approach that is based on clinical judgement (CJ) to the test-based approach that is based on confirmation through test with rapid diagnostic tests (RDT). It has been suggested that the loss of the prophylactic effect of presumptive-administered ACT in children who do not have malaria will result in increase in their risk of malaria and anaemia.We undertook a cluster-randomized controlled trial to compare the effects of the presumptive approach using clinical judgment (CJ-arm) and the test-based approach using RDTs  living around 32 health centres were enrolled. Nearly half were female (48.7%) and 47.8% were below the age of 12 months as at enrolment. Over 24-months, the incidence of all episodes of malaria following the first febrile illness was 0.64 (95% CI 0.49–0.82) and 0.76 (0.63–0.93) per child per year in the RDT and CJ arms respectively (adjusted rate ratio 1.13 (0.82–1.55). After the first episode of febrile illness, the incidence of severe anaemia was the same in both arms (0.11 per child per year) and that of moderate anaemia was 0.16 (0.13–0.21) vs. 0.17 (0.14–0.21) per child year respectively. The incidence of severe febrile illness was 0.15  in the RDT arm compared to 0.17  per child per year respectively. The proportion of fever cases receiving ACT was lower in the RDT arm .The test-based approach to the management of malaria did not increase the incidence of malaria or anaemia among under-five children in this setting.NCT00832754ClinicalTrials.gov  Although appreciable progress has been made in the control of malaria the disease remains an important cause of morbidity and mortality in endemic countries in sub-Saharan Africa.,2 DiagnoThe two main approaches to the diagnosis of malaria are the presumptive and test-based approaches. The presumptive  approach relies solely on presenting signs and symptoms to establish diagnosis while the test-based approach relies on parasitological confirmation. For many years, the presumptive approach has been used across all age groups and transmission settings. The approach was justified on the basis of the following: (1) A high proportion of febrile illnesses in high-endemic settings was due to malaria, and the disease took a rapidly-deteriorating course in under-five children. Treatment therefore needed to be initiated promptly. (2) Microscopy which was the only practical means for confirming the diagnosis of malaria was technically complex and not available in areas that had the highest burden of the disease. (3) The first-line drugs used to treat malaria before the introduction of artemisinin-combination treatment (chloroquine and sulphadoxine-pyrimethamine) were safe, effective and inexpensive.,6 The prIn early 2010, WHO issued revised guidelines for the management of malaria that called for a shift from the presumptive to the test-based approach, across all transmission settings and in all age groups. The justifications for the shift include: (1) Substantial decline in the levels of malaria transmission in many countries including some areas that were previously high-transmission settings; (2) Availability of reliable malaria rapid diagnostic tests (mRDTs) for use as a point-of-care device; (3) Artemisinin-based combination treatments (ACTs), the current drugs of choice for treatment of malaria, are relatively expensive. Given thThose who opposed the shift to this approach argued that reported reductions in the transmission of malaria were not widespread enough to justify a change in the treatment guidelines. They also hypothesized that reduction in ACT use will remove the protective effect of intermittent preventive treatment (IPT) in childCurrently there are very few diagnostic tools that can be applied to determine the aetiology of non-malarial febrile illnesses at point-of-care, and on that basis decide when to prescribe antibiotics. In the absence of improved diagnostics tools for the management of these illnesses, test-based management of malaria could  also lead to substantial increase in the inappropriate use of antibiotics to manage such cases.We report here on a cluster-randomized, controlled trial we undertook a comparison of the effects of test-based (intervention) versus presumptive (control) approaches to the management of malaria in rural Ghana. We hypothesized that test-based management of malaria will lead to reduction in ACT prescription and use. This will result in an increase in the incidence of malaria and anaemia, as well as in the prescription of antibiotics. The trial was registered in the online clinical trials registry as NCT00832754.The protocol for the study was approved by the Ethical Review Committees of KHRC, the Ghana Health Service (GHS), and the London School of Hygiene and Tropical Medicine (LSHTM). Signed (thumb printed if unable to read or write in English) informed consent was obtained from each caregiver prior to enrolment of the child. A Data Safety Monitoring Board (DSMB) periodically reviewed safety parameters and incidence of malaria and anaemia. The DSMB also approved the final analysis plan.The study was conducted in six districts in Ghana between February 2009 and July 2012. The districts lie within the forest-savannah transition zone which is considered to have perennially-high malaria transmission. According to data from routine health services, malaria is the leading cause of morbidity and mortality in the districts. In 2003–4, the Entomological Inoculation Rate (EIR) was estimated at 231–269 infective bites per person per year.  At the tThe unit of randomisation was health centres. Thirty two health centres were stratified by districts, ranked by the number of Diptheria-Pertussis-Tetanus1 (DPT1) vaccinations given in the facility in the preceding year and paired according to the rank within each district. Within each pair of health facilities one was allocated randomly to implement test-based (RDT-arm) management of malaria, while the other health facility within the pair was allocated to implement the clinical judgement (CJ arm) approach. This was done using STATA software.Between January 2010 and May 2010, we enumerated all households that were located within 2km radius of the selected health centres and enrolled one hundred children per health facility who met the following criteria: (1) age <24 months; (2) caregiver indicated they used the index health centre as first point of call whenever the child was unwell; (3) caregiver was willing to give informed consent for the child to be enrolled in the study.We enrolled children living closest to the selected health centres and extended outwards circumferentially till the target of 100 children was achieved. Enrolled children were given a study identification card with a unique identification code. We recruited thirty-two research fieldworkers and trained them on Good Clinical Practice (GCP) and the standard operating procedures of the study. We also trained them on how to take blood from finger prick to perform malaria RDTs, how to use of the Hemocue™ to measure haemoglobin levels, and how to prepare blood smears for microscopy. One fieldworker was stationed at each health centre. Each district had a research field supervisor who paid daily visits to each of the health centres to review work, collect completed study forms and blood samples.All health workers in the participating facilities who took care of sick children were given refresher training in the management of febrile illnesses according to IMCI guidelines. This training was conducted just before commencement of the study and repeated every 4–6 months throughout the period of the study. Clinicians in all the participating health facilities were taken through the correct use and interpretation of RDT. This training relied on the findings of an earlier study into the accuracy of an HRP-2 RDT in the study area by the research team. In that study, a high rate of false-positive RDT results due to antigen persistence was observed. We used Each blood slide in the study was read independently by two expert microscopists at the reference laboratory at the Kintampo Health Research Centre (KHRC). Discrepancies were resolved through a third reading by a third microscopist.In health facilities that were randomised to the test-based (intervention) approach, all enrolled children who reported with fever were tested with mRDT and received an ACT only when the test was positive. The mRDTs used in the study were procured from the Ghana National Malaria Control Program. Two brands of mRDTs procured through the Ghana National Malaria Control Programme, CareStart™ and First Response™, were used throughout the period of the study. They were also treated for any concurrent illnesses according to IMCI guidelines.In health facilities that were randomised to the clinical judgement (control) approach, enrolled children who presented with fever were not tested for malaria. The treatment for malaria in these facilities was presumptive and based on presenting clinical symptoms and signs. The treatment of all other ailments were based on the IMCI guidelines.The fieldworkers stationed in each (intervention and control) health facility helped to ensure compliance with the protocol by reminding attending clinicians about the protocol of the study, as applicable to the particular health centre. This was reinforced during the 4–6 monthly training sessions that were held for health workers in both intervention and control facilities.All caregivers of children enrolled into the study were advised to bring their sick children to the health centre. To overcome the possible effect of financial barrier, the project paid to have all the children enrolled under the Ghana National Health Insurance Scheme. Whenever a study child presented with axillary temperature of 37.5°C or above, or reported fever in the preceding 72 hours, the fieldworker took blood samples for blood smear microscopy and used the Hemocue® to determine haemoglobin level. The result of the haemoglobin test was made available to the attending health worker together with the result of mRDT if the health centre was an intervention facility. In facilities in the CJ arm, only haemoglobin test and blood smear microscopy were done. The results of the haemoglobin test were also made available to the attending health worker to guide clinical management.As part of active surveillance, field supervisors visited study children at least once every two months and asked the caretakers about any febrile illnesses that may have occurred in the period in between visits. The field supervisors completed morbidity questionnaires during each visit. This process of surveillance was instituted to ensure that all febrile episodes that could have been reported to local pharmacies and chemical shops was captured. If a child was unwell at the time of the home visit or reported having had fever in the preceding 72 hours, the field supervisor issued a referral note and advised the caregiver to send the child to the study health centre.The primary endpoint was incidence of malaria during the 24 months of follow-up after the first episode of febrile illness. It was estimated that to assess the effect of test-based management of malaria on the incidence of malaria in children under 48 months of age would require 30 clusters (15 per arm) and 200 person-years at risk (PYAR) per cluster. This was based on the following expectations: (1) Incidence of malaria in the CJ arm would be 0.8 episode/ 1 per child per year  thea priori that the number of deaths would not be sufficient to detect any significant difference between the two arms.As the total number of children living around some of the selected health facilities was <100 an additional pair of health centres was selected randomly in order to reach the required number of children. Although mortality was measured in the study, it was assumed Data were double-entered using FoxPro version 9. The analysis was conducted using STATA version 12. Intention-to-treat analysis was performed using all enrolled children while per protocol analysis was applied to children who adhered to correct assessment and treatment criteria. Person-time in the study for each child was calculated using dates on which children were seen at home visits or at a health facility. If a child was not seen for more than 3 months, the person-time was censored for this period until they were seen again. Malaria was defined as the presence of fever and a positive blood smear microscopy result. Fever was considered to be severe when it was associated with one of more of the IMCI danger signs of lethargy or drowsiness, vomiting everything, refusing all food including breastfeeding and convulsions.The sociodemographic characteristics of children in the two arms of the enrolled cohort were measured and compared using Pearson’s chi-square corrected for clusters. Similarly fever episodes and corresponding blood test results and ACT prescriptions were compared. A multi-level Poisson model assuming unstructured covariance was used to estimate the incidence of malaria over the 24-month period to account for repeat episodes of malaria within a child, and clustering by heath facility. For the incidence of first or only episodes of malaria, moderate anaemia (haemoglobin between 5-8g/dL), severe anaemia (haemoglobin less than 5g/dL) and severe febrile illness, after the first febrile episode, a random effects Poisson model was applied, adjusting for age, socio economic status, and ITN use. In this report we present the survival analysis (in addition to the Poisson models) to show events over the entire follow-up period and also to offer opportunity for cross-reference with other studies.Potentially eligible children were enumerated between April 2010 and May 2010. Those who were available were consent and enrolled in June 2010 and followed up to June 2012. A total of 3046 eligible children (1527 in the RDT arm and 1519 in the CJ arm) were enrolled around the 32 health centres.  Nearly hTable 1) The mean number of passive health facility visits was 4.1 in the test-based arm and 4.3 in the CJ arm (p = 0.73). Two hundred and seventy five (9.0%) study children never visited the health centre throughout the follow-up period but they were regularly seen at home visits.As part of active surveillance, 46,395 home visits were made over the 24-month follow-up period. The median number of home visits was 15 per child in both arms. 23.1% of children were referred to a health centre at least once following a home visit because they had fever or history of fever in the preceding 72 hours. The number of cases referred after homes visits were similar in both arms of the study . (Table 2).The median time to first episode of fever was similar between the two arms (105 days in RDT versus 111 days in CJ arm). At their first visit to the health facility, 713 (29.1%) children had haemoglobin less than 8gm/dL. The median level of haemoglobin at the first contact was similar in both arms of the study (9.0 versus 9.2 gm/dL). The number of episodes of febrile illness per child over the 2 year study period was also similar between the two groups (3.7 vs 3.8 per child). In the RDT arm, 98.2% of febrile episodes seen at the health centres were tested with RDT and 72.3% were RDT positive. The sensitivity, specificity and positive predictive value for RDT use in the RDT-arm were 95%, 64% and 53% respectively. As a deviation from the study protocol, 8 out of 5799 (0.14%) cases that presented to CJ-arm facilities had RDT performed. . Within the RDT arm, the mean time to an episode of malaria following treatment with ACT for a febrile illness was 309 days compared to 252 days in those who did not receive an ACT for febrile illness, p = 0.009. The mean time to an episode of malaria for children who were RDT negative and therefore did not receive ACT but whose blood smear results were positive is 194 days.The incidence of all episodes (first or repeat) of malaria was 0.99 per child per year  in the RDT arm compared to 1.05 per child per year  in the CJ arm. There was no significant difference in the incidence of all episodes of malaria following the first episode of febrile illness  . The surTable 3)A total of 1200 episodes of severe febrile illness occurred among 760 children during the period of follow-up. The incidence of severe febrile illness after a first episode was 0.15 (0.09–0.24) per child per year in the RDT arm compared with 0.17 (0.11–0.28) among children in the CJ arm (p = 0.78). The incidence of severe febrile illnesses was higher in children aged below 12 months of age at the time of enrolment  compared to those ≥12 months of age . (Table 3).The incidence of moderate or severe anaemia in children following an episode of febrile illness was not significantly different between the two groups; 0.16 (0.13–0.21) vs. 0.17 (0.14–0.21) per child year and 0.11 (0.09–0.14) vs. 0.11 (0.95–0.15) per child year respectively. The adjusted rate ratios were 1.04 (0.76–1.44) and 1.04 (0.76–1.41) for moderate and severe anaemia respectively (There were 21 (1.4%) deaths in the CJ arm compared with 15(1.0%) deaths in the RDT-arm. The difference was not statistically significant (p = 0.31).Table 2).The proportion of fever cases that got treated with ACT was lower in the RDT arm compared to the CJ arm . Although the number of blood smears that were positive was similar in both arms , the number of children who were blood smear positive but did not receive ACT was significantly higher in the CJ arm than in the RDT arm  (Table 3)The proportion of children receiving an antibiotic for a febrile episode was slightly lower in the RDT arm than in the CJ arm this difference was not statistically significant . In the RDT arm the proportion of children receiving an antibiotic was higher among RDT negative compared to RDT positive children . The proportion of children receiving a combination of an ACT and antibiotic was lower in the RDT arm but this was not statistically significant . However the proportion of children who received only antibiotics was significantly higher in the CJ arm compared to the RDT arm (15.6% versus 22.9% p = 0.02). This study has used a cluster-randomised design to compare the effects of test-based management of malaria to the presumptive approach that had been practised for several decades. This study achieved a near-perfect provider adherence to test results or to clinical judgement. The observed incidence of 1.09 episodes per child per year in the CJ arm was higher than the assumed incidence of 0.8 episode per child year used in the sample size estimation. Thus the study was adequately powered to detect the anticipated 20% difference in the incidence of malaria. Given that adherence to the protocol was very good, the diagnosis of malaria was confirmed by microscopy and the study was adequately powered, we believe this study provides substantially robust evidence on the likely effects of test-based management of malaria in high transmission settings.Previous studies that examined the effects of test-based management of malaria had been limited by the lack of appropriate comparator cohort,19,20, rThis main finding of this study is that mRDT-based treatment of febrile illness in <5 year old children did not result in an increase in the incidence of malaria, anaemia, severe febrile illnesses or the use of antibiotics in this high-transmission setting. These findings ran counter to our expectation that the test-based approach would deny a high proportion of children the prophylactic benefit that OPT offers in children whose fever is non-malarial. The likeAnother factor that could explain the lack of significant effect on the risk of malaria was the significant difference in the proportion of children who were blood smear positive but did not receive ACT. This occurred more among children in the CJ-arm than among children in the RDT-arm . Given that previous studies have established that the strongest predictor of a repeat episode of malaria is an index episode of parasitaemia,,26 the hAn important factor that motivates the roll-out of test-based management of malaria is the expectation that it will lead to massive reduction in the wasteful use of ACTs.,6,24 TheAn important finding in this study is the fact that compared with children in the RDT arm, a significantly higher number of smear positive children in the CJ arm did not receive ACT. This finding suggest that the presumptive approach is not an efficient means for reaching children who are parasitaemic and need to be given ACTs. Conversely the use of RDT significantly improves the targeting to ACTs to malaria cases. On this basis, the test-based approach can be said to lead to improvement in the quality of care.In this study we found the use of HRP-2 RDT to have a PPV of 64%. This implies that possibly one in every three RDT positive cases could actually be blood smear negative and is RDT positive, likely on account of the persistence of HRP-2 antigens. This findings has implications for the training of clinicians on the interpretation of RDT results, the need for proper history-taking in the assessment of children and caution against over-reliance on RDT result to form the basis of the diagnosis of clinical malaria. There is also the need for caution in interpreting these findings to suggest a need for antibiotics in all cases where RDT result is positive but blood smear result is negative. In earlier work in the same study area, the positive yield on blood culture among RDT negative under-five children was less than 12%.29-33 The basis of the concern is that restricting ACT to confirmed malaria cases will increase the number of cases subsequently classified as non-malarial febrile illnesses, and in the absence of appropriate diagnostic tools these cases will be presumptively treated with antibiotics. In this study we found that prescription of antibiotics only or in combination with ACT was lower in the RDT arm compared to the CJ arm. However, the proportion of children treated with an antibiotic use was highest (85%) among children in the RDT arm who were RDT-negative. This compares with 44% among children in the RDT arm who tested positive, 51% and 62% among children in the CJ arm who (as later determined) were blood smear positive and negative respectively. The high antibiotic use in the RDT negative children did not however translate into overall significant increase in antibiotic use in the RDT arm  because it was offset by reduction in use (44%) in children who had malaria and got confirmed with RDT. The relatively high background malaria prevalence implied that this reduction was large enough to offset the increased use in the RDT negative group. Our data suggests that the result of RDT (either positive or negative) will influence the decisions of health workers whether to use antibiotics or not. However, in high malaria transmission settings the reduction in antibiotic use in RDT-positive children would result in overall reduction in antibiotic use.A major concern about the shift to test-based approach has been the prospect that it could increase inappropriate use of antibiotics.The pattern in antibiotic use and reduction in ACT use, without adverse effect on treatment outcomes suggest that test-based management of malaria improved the quality of care of both malaria and non-malaria febrile illnesses in this high transmission setting. The findings nevertheless add to available evidence on the need for further work to identify how best to target antibiotics use in the management of childhood febrile illnessesThe extrapolation of these findings to program settings is limited by a number of operational considerations. The level of provider adherence to test based management of malaria observed in this study was very high, good quality mRDT stored in optimum conditions, and the study children were closely monitored. Thus any deterioration in their clinical condition resulting from inadequate treatment was promptly identified and the caregivers brought the children for review as recommended. On the contrary, in routine health systems, the follow up of children is often not optimal, caregivers may not return for reviews, and the accuracy of mRDT may be variable.The application of test-based management of malaria did not increase the risk of malaria or anaemia in children in this setting. It improves the quality of care. It however did not lead to massive reduction in ACT use. The WHO recommendation of test-based management of malaria in children is justified in this setting.S1 Checklist(DOC)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.


Plasmodium naturally infect humans. Some species cause non-specific symptoms that can sometimes progress rapidly to severe and fatal outcomes. Early diagnosis and appropriate treatment interrupts progression and cures disease. Confirmation of diagnosis of malaria currently relies on microscopy, or on application of rapid diagnostic tests (RDTs) that are becoming increasingly widely available and are recommended to confirm infection before treating it. The diagnosis of malaria by itself is not sufficient to optimise individual therapies because there is a growing problem of multidrug resistance in parasites . This limits the use of some drugs or combinations by severely compromising their efficacy.Five species of Diagnostic strategies for management of malaria can therefore be improved in several ways. First by an increase in sensitivity of detection of parasites that should improve of the thresholds for detection by the current generation of RDTs because they cannot identify low parasitaemias. Second, a diagnostic that can differentiate all the naturally infecting species of parasite needs to be developed. Finally, rapid (point-of-care) assessment of the drug resistance status of parasites will add greatly to the treatment strategies available to manage individual patients. Technological platforms that can deliver information that is currently missing for the personalised management of malaria infections are being developed and these advances will be presented and discussed in the context of the global burden of disease caused by malaria.


American Journal of Tropical Medicine and Hygiene adds to the substantial and growing body of evidence that a diagnosis of Plasmodium vivax malaria often occurs with serious and threatening illness. The retrospective study by Quispe and othersP. falciparum by virtue of its absence from the region. About one-quarter of patients were classified as critically ill with severe anemia, shock, lung injury, renal failure, and cerebral syndromes, and two patients  did not survive. Essentially similar rates and findings have come from numerous hospital-based studies from all across the global reach of endemic vivax malaria transmission.The report by Quispe and othersP. vivax require more thorough investigation than has yet been reported.per se. Nonetheless, acceptance of the reality of vivax malaria as often provoking pernicious and threatening illness must not await demonstration of the mechanisms or cofactors. The weight of evidence now available leaves no doubt that vivax malaria in many settings often occurs in association with a pernicious and threatening course of illness, which does not assign cause and effect but instead, acknowledges real consequences without regard to their specific genesis.There is no firm understanding of pathogenesis in vivax malaria. The roles of comorbidities in serious and fatal disease with a diagnosis of P. vivax resulted in a 60-year hiatus in biological and clinical research as well as public health policy and practice.The false aegis of inherent harmlessness in P. falciparum-centric strategic postures in research and practice aimed at malaria. The hypnozoite of P. vivax completely transforms the landscape of scientific and strategic thinking in the epidemiology, prevention, control, diagnosis, and treatment of malaria. An inoculation of P. vivax sporozoites by a single anopheline bite seeds the liver with a brood of hypnozoites. These hypnozoites remain latent for periods ranging from 3 weeks to 3 years. When they do emerge, they tend to do so in rapid succession at intervals of 3 or 4 weeks. At least three relapses are very common, and up to 20 relapses within 2 years have been documented. Among cohorts in Thailand and Indonesia,P. falciparum in the most heavily malarious regions.A single entity called the hypnozoite demands a wholly different approach from conventional The hypnozoite in the liver may thus be recognized as a very significant reservoir of infection of blood. A patient diagnosed with acute vivax malaria stands at very high risk of suffering subsequent attacks in quick succession with deepening risk of serious illness along with opportunities for transmission to others. Among the many therapies applied with good safety and efficacy against the acute attack, none affect the hypnozoite. A single drug kills hypnozoites: primaquine. This fact—and the ability of primaquine to provoke a potentially lethal acute hemolytic anemia in patients having an inborn deficiency of glucose-6-phosphate dehydrogenase (G6PD)—imposes very serious obstacles to successful management of vivax malaria as a clinical and public health problem.P. vivax malaria. The absence of evidence-based guidance for this serious problem should be considered an urgent global health problem in need of immediate attention.Quispe and othersP. vivax occurs in greatest abundance (south and southeast Asia).13Furthermore, most patients diagnosed with G6PD deficiency cannot safely receive primaquine therapy. The World Health Organization (WHO) recommendation to do so with eight weekly doses of 45 mg primaquine base does not come with evidence of safety among the most severe G6PD deficiency variants, which happen to be most common where P. vivax. The work of Quispe and othersAn accompanying editorial by Price


Co-infection with malaria and HIV increases the severity and mortality of both diseases, but the cytokine responses related to this co-infection are only partially characterised. The aim of this study was to explore cytokine responses in relation to severity and mortality in malaria patients with and without HIV co-infection.This was a prospective cross-sectional study. Clinical data and blood samples were collected from adults in Mozambique. Plasma was analysed for 21 classical pro- and anti-inflammatory cytokines, including interleukins, interferons, and chemokines.We included 212 in-patients with fever and/or suspected malaria and 56 healthy controls. Falciparum malaria was diagnosed in 131 patients, of whom 70 were co-infected with HIV-1. The malaria patients had marked increases in their cytokine responses compared with the healthy controls. Some of these changes, particularly interleukin 8 (IL-8) and interferon-γ-inducing protein 10 (IP-10) were strongly associated with falciparum malaria and disease severity. Both these chemokines were markedly increased in patients with falciparum malaria as compared with healthy controls, and raised levels of IL-8 and IP-10 were associated with increased disease severity, even after adjusting for relevant confounders. For IL-8, particularly high levels were found in malaria patients that were co-infected with HIV and in those who died during hospitalization.P. falciparum, and suggest a potential pathogenic role for IL-8 and IP-10. However, the correlations do not necessarily mean any causal relationship, and further both clinical and mechanistic research is necessary to elucidate the role of cytokines in pathogenesis and protection during falciparum malaria.Our findings underscore the complex role of inflammation during infection with  Human Immunodeficiency Virus (HIV) 1 and Plasmodium falciparum malaria are two of the major lethal infectious diseases in sub-Saharan Africa, with 1.47 million and 1.13 million deaths, respectively, in 2010 P.falciparum is often associated with the activation of several inflammatory pathways, resulting in systemic and local inflammatory responses that involve the production of a wide range of cytokines,- from several cell types including leukocyte subsets, endothelial cells and various tissues cells such as macrophages. This inflammatory response during malaria infection could activate anti-microbial responses in macrophages, T cells, granulocytes and endothelial cells and thereby be beneficial for the host Infection with + T cells and the development of severe immunodeficiency, it is also characterised by a state of chronic inflammation P. falciparum. However, in what way co-infection with HIV influences cytokine responses during falciparum malaria is still only partly known. In addition, although there are numerous studies on cytokine response during infection with P. falciparum, less is known about the more detailed cytokine network and the relative importance of different mediators with regard to disease severity and mortality in malaria patients.Although the immunological hallmark of HIV infection is a loss of CD4P. falciparum with and without co-infection with HIV-1, we analysed a wide range of cytokines and related inflammatory mediators in blood samples obtained at admission from adult patients admitted to Central Hospital of Maputo in Mozambique with fever and/or suspected malaria. The levels of the different mediators were related to disease severity, mortality, and co-infection with HIV.Mozambique has one of the highest global incidences of co-infection with HIV-1 and falciparum malaria, with a population HIV-1 prevalence of 11.5% that is 22.5% in Maputo  and a malaria parasite prevalence of 27.7% in small children in Southern Mozambique (2007) P. falciparum accounting for 95-100% of the malaria cases The Central Hospital of Maputo is a public teaching hospital for Maputo's 1.2 million citizens and a national referral hospital for Mozambique's population of 22 million. From 8 January 2011 to 31 March 2011 and from 7 November 2011 to 14 March 2012, we conducted a prospective cohort study in all patients consecutively admitted to the Medical Emergency Department in the Central Hospital in Maputo every week from Sunday to Friday, as previously described All non-pregnant adults ≥18 years with an axillary temperature ≥38.0°C and/or suspected malaria were included, provided written consent. As controls, we included health care workers at the hospital, friends, and family members who reported a subjective feeling of wellbeing and had a healthy appearance, as evaluated by the researchers. Female healthy controls were not included if suspected or confirmed pregnant. We defined “Suspected malaria” as a history of fever, chills, headache, mental confusion, vomiting and/or diarrhoea, dyspnea, myalgia, and/or general malaise, provided that there were no other symptoms, findings on clinical examination, or additional diagnostic tests indicating other infections. Additional diagnostic tests and exams were basic laboratory tests , WBC, differential count, ESR, AST, ALT, ALP, bilirubin, L DH, creatinine); other blood tests ; urine analysis ; Cerebrospinal fluid (CSF) analysis ; sputum analysis for M. Tuberculosis with microscopy (AAFB) and culture; stool analysis ; and cytological/histological and different radiological exams, if indicated. “Malaria positive”  was defined as a patient with a positive malaria PCR test. One patient who died with a positive malaria PCR test was categorised as a non-malarial death, because he had a history and clinical findings more compatible with tuberculous meningoencephalitis as the cause of death. No malaria PCR was performed for two patients: they had a positive HRP2-antigen test and were slide-positive with parasitemia 3+ and 5+, respectively, and were defined as malaria positive. HIV-infected patients were defined by a positive HIV serological test and/or a positive HIV PCR test. Malaria severity was categorised according to the number of criteria fulfilled for severe malaria, as defined by the WHO and adjusted to what could be measured locally, as previously reported We recorded a predefined set of clinical data from the patients' files, which was performed as part of the routine clinical examination on admission and on follow-up during the hospital stay. The details are published elsewhere g for 20 minutes to obtain platelet-poor plasma. The serum tubes were allowed to clot at room temperature prior to being centrifuged at 1000xg for 10 minutes. The serum and plasma samples were stored in multiple aliquots at −20°C for 24 hours before being placed at −80°C. The samples were thawed only once.The EDTA tubes were immediately put on melting ice and centrifuged within 30 minutes at 2000xP. falciparum, Premium Medical Corporation Ltd., Daman, India; 2011–2012 ICT Malaria P.f., ICT Diagnostics Cape Town, South Africa), malaria thick blood slides (Giemsa 20% for 5 minutes), and other routine laboratory tests were performed. Parasitemia was categorised in a thick smear as 1+ when 1–10 parasites/100 fields, 2+ when 11–100 parasites/100 fields, 3+ when 1–10 parasites/field, 4+ when 11–100/field, and 5+ when >100 parasites/field According to the hospital routine and in line with standard procedures in the laboratory of the Central Hospital of Maputo HIV-test , HRP-2 Rapid Diagnostic Test for malaria (RDT) -1β, IL-1 receptor antagonist (IL1-ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, interferon (IFN)-γ and tumour necrosis factor (TNF) as well as chemokines IL-8/CXCL 8, eotaxin 1/CCL11, IFN-γ-inducing protein 10 (IP-10/CXCL10), monocyte chemotactic protein-1 (MCP-1/CCL2), and macrophage inflammatory proteins 1α (MIP-1α/CCL3) and MIP-1β/CCL4 by a multiplex cytokine assay  according to the instructions from the manufacturer. Cytokines that were not detectable  were excluded from further analysis . There were no cytokine measurements above the upper detection limit.th and 75th percentiles. Differences in categorical variables such as sex and HIV-status were tested by chi-square tests. Differences in cytokine distributions between the groups of study participants were tested by Mann-Whitney tests. Spearman rank correlation was used for calculating correlations between cytokines in all malaria patients  using the R-packages “supclust“and “corrplot”, respectively. There is a validation of all statistical methods used in For analyses involving variables for which there were missing observations for certain patients, these subjects were excluded; the number of included patients is indicated in the tables. In the calculation of malaria severity, missing observations were reckoned as “normal” in cases in which the investigation in question, e.g., bilirubin, was performed only on the clinical suspicion of specific organ involvement. Most of the statistical analyses were performed with SPSS-21 , but the Pelora analysis and the correlations plots were performed in R version 3.0.  had malaria, and 70 of these (53%) were co-infected with HIV-1 . All mal4 (range 0–8.3 x 106) HIV RNA copies/mL plasma . Unfortunately, data on the CD4 T-cell counts were lacking in all except 11 patients who had a median of 206 CD4+ T cells/µL (range 14–632).Among the malaria patients and healthy controls, the median ages were 38 years (range 18–79 years) and 26 years (range 18–56 years), respectively: 45% and 41% were women, and 98.5% and 96% were ethnic Mozambicans. There was a significant difference between the malaria patients and healthy controls in relation to age (p = 0.003), but not in relation to sex (p = 0.567). The malaria patients with and without HIV co-infection showed no significant differences in age, sex, and duration of symptoms; 66% of the patients (85/129) had severe malaria defined as one or more severity criteria (missing observations for two patients). The malaria patients with HIV co-infection in general had more severe disease, including in-hospital mortality, as recently reported The cytokine responses were extremely variable both inter- and intra-individually, with levels ranging from below the lower detection limit to a 1000-fold increase above this limit. Within the malaria patient group, several of the cytokines were strongly inter-correlated, irrespective of co-infection with HIV, illustrating the marked changes in and close connections between cytokines during malaria infection .In general, the malaria patients had a pronounced and broad cytokine response compared with the healthy controls . We obseIn the malaria patients with HIV co-infection compared with those without, there was a significant increase in the levels of IL-8, eotaxin, and MIP-1α. . Pelora For patients with severe malaria (n = 84), as defined according to the adjusted WHO criteria Categorising the study population into healthy controls and patients with uncomplicated and severe malaria, there was a steady increase with increasing severity in those cytokines that were associated with severity, i.e., IL-8 and IP-10 .During hospitalisation, ten of the malaria patients died, of which nine had a HIV co-infection. The malaria patients who died had significantly raised levels of IL-6 (p = 0.006), IL-8 (p = 0.001), MCP-1 (p = 0.012), and MIP-1β (p = 0.010) compared with the survivors.P. falciparum were observed in malaria patients compared with healthy controls. Some of these changes, particularly the IL-8 and IP-10 levels, were strongly associated with falciparum malaria and disease severity. Both chemokines were markedly increased in patients with falciparum malaria as compared with healthy controls, and raised levels of IL-8 and IP-10 were together with low MCP-1 levels, associated with increased disease severity, even after adjusting for relevant confounders. For IL-8, particularly high levels were also found in malaria patients that were co-infected with HIV and in those who died during hospitalization.In the present study, marked changes in the cytokine response during infection with Previous studies on the impact of cytokines on clinical outcome in malaria have identified TNF, IFN-γ, IL-1β, IL-6, IL-10, IL-12, transforming growth factor β and IP-10 as important mediators, but data on IL-8in this setting are more limited P. falciparum secretes a functional histamine-releasing factor homolog that induces IL-8 release from eosinophils + T cell cytotoxicity Several cells with relevance to severe falciparum malaria, including tissue macrophages and endothelial cells, can release IL-8 when activated. It is tempting to hypothesize that hemozoin and other parasite-related molecules can induce IL-8 release in these cells. Increased levels of IP-10 have been associated with increased severity and mortality with cerebral malaria and with high viral load in HIV-infection Besides IL-8, MCP-1 and IP-10, several of the cytokines that were associated with disease severity during falciparum infection belong to the CC or CXC chemokine family, i.e., eotaxin, MIP-1α, and MIP-1β. All of these are chemo-attractants for different types of leukocytes, including some with important roles in malaria pathogenesis, as the eosinophil granulocytes. Thus, increased levels of those chemokines have been associated with organ failure, such as in malaria-associated acute respiratory distress syndrome (ARDS) and severe malaria, and occasionally with mortality We have previously shown that co-infection with HIV is significantly associated with increased malaria severity and death The present study has some limitations. First, although most patients were severely ill, relatively few died, which limits conclusions concerning cytokine levels and mortality. Second, even if the WHO HIV clinical staging is considered an acceptable disease severity marker, the lack of information on CD4 and CD8 T cell counts is still an important limitation, in relation to the influence of HIV co-infection on the cytokine response during falciparum malaria. Third, since malaria severity tends to increase in older age groups, at least in non-endemic areas In the present prospectively designed study with consecutive inclusion of falciparum malaria patients with and without HIV co-infection, we found marked changes in the cytokine network during malaria infection. In particular, high plasma levels of IP-10 and IL-8 were associated with falciparum malaria and disease severity in infected individuals, and for IL-8, particularly high levels were found in those that were co-infected with HIV. While several previous studies have appointed IP-10, IL-8 has been less described in relation to severe malaria.P. falciparum. However, the correlations do not necessarily mean any causal relationship, and further clinical and mechanistic research is necessary to elucidate the role of cytokines in pathogenesis and protection during falciparum malaria.Our findings underscore the complex role of inflammation during infection with S1 FigureThe correlation between the different cytokines in all malaria patients. Pairwise Spearman correlation is given between the cytokines in all malaria patients. The darker blue, the higher correlation as seen by the correlation scale at the right. The cytokines are sorted according to groupings of highly correlated cytokines, determined by the angular order of the eigenvector.(TIFF)Click here for additional data file.S1 FileValidation of the statistical methods presented in the manuscript.(DOCX)Click here for additional data file.


The rapid diagnostic test (RDT) has been adopted in contemporary malaria control and management programmes around the world as it represents a fast and apt alternative for malaria diagnosis in a resource-limited setting. This study assessed the performance of a HRP-2/pLDH based RDT (Parascreen® Pan/Pf) in a laboratory setting utilizing clinical samples obtained from the field.Whole blood samples were obtained from febrile patients referred for malaria diagnosis by clinicians from two different Upazila Health Complexes (UHCs) located near the Bangladesh-India and Bangladesh-Myanmar border where malaria is endemic. RDT was performed on archived samples and sensitivity and specificity evaluated with expert microscopy (EM) and quantitative PCR (qPCR).Plasmodium falciparum-positive and 54 were Plasmodium vivax-positive. In comparison with EM, for P. falciparum malaria, the RDT had sensitivity: 96.0%  and specificity: 98.2%  and for P. vivax, sensitivity: 90.7%  and specificity: 98.9% . Comparison with qPCR showed, for P. falciparum malaria, sensitivity: 95.4%  and specificity: 98.8%  and for P. vivax malaria, sensitivity: 89.0%  and specificity: 98.8% . Sensitivity varied according to different parasitaemia for falciparum and vivax malaria diagnosis.A total of 327 clinical samples were made available for the study, of which 153 were Parascreen® Pan/Pf Rapid test for malaria showed acceptable sensitivity and specificity in border belt endemic areas of Bangladesh when compared with EM and qPCR. Malaria is often lethal with high potential expenditure for health if diagnosis is inaccurate . AccuratFor decades, expert microscopy (EM) of peripheral thick and thin blood smears has been the standard diagnostic test for malaria, however, it is time consuming and requires substantial expertise ,5. EnzymPlasmodium falciparum), usually by detecting histidine-rich protein 2 (HRP2), or detect any of the four most common malaria parasites , typically by detecting pan-Plasmodium-specific lactate dehydrogenase (pLDH), while three-band tests detect both the P. falciparum-specific antigen HRP2 and the pLDH or any one species specific LDH (mostly P. vivax). The third band is the test control band [After being introduced in the early 1990s, rapid diagnostic tests (RDTs) have become an attractive alternative to the above-mentioned methods in a resource-limited setting for malaria diagnostics. The antigen-based RDTs detect specific antigens produced by malaria parasites by reaction with bound antibodies on an absorbent nitrocellulose membrane. Among several types of RDTs the two-band tests and three-band tests are most widely used. Two-band tests either detect only one species , hereafter noted as Parascreen, is a RDT that has the capability to detect all types of human malaria, as it detects P. falciparum-specific HRP-2 and pan-Plasmodium-specific LDH. It has been evaluated against microscopy and conventional PCR in field and laboratory settings [Southeast Bangladesh, northeast India and southwest Myanmar are similar in geographical characteristics and endemic for malaria. ountries ,11,12. T country -16. Thessettings -24. HereP. falciparum and P. vivax.Whole blood samples were obtained from febrile patients with clinical symptoms referred for laboratory investigation between May 2009 to December 2010. The represented regions include Matiranga Upazila in Khagrachari district and Ramu Upazila in Cox’s Bazar district, two different subdistricts of the southeastern part of Bangladesh from corresponding UHC. Matiranga borders Tripura state of India and Ramu borders Myanmar, where malaria is endemic ,16 and iAn expert medical technologist collected approximately 5 mL of blood from adult subjects and 3 mL from minor subjects by venipuncture. Thick and thin blood films were prepared in duplicate using two drops of blood for each sample. The remaining blood was preserved at -20°C in EDTA tube and transported to the Parasitology Laboratory, icddr,b in cool boxes maintaining the temperature below 4°C using ice bags.Approval from Research Review Committee (RRC) and Ethical Review Committee (ERC) of icddr,b was obtained for this study. Permission for conducting the study was obtained from the National Malaria Control Programme (NMCP). All participants or legal guardians signed informed consent before participant enrolment and sample collection. Complete anonymity was maintained at each stage of the study.P. falciparum and P. vivax) infection were excluded from the study.Blood smears were stained with Giemsa and screened for parasites under the (100X) oil immersion lens at the field site by experienced microscopists in the corresponding UHC. The microscopy results were confirmed by a second independent, experienced microscopist who was blinded to prior results. Parasite density was determined by both microscopists counting the parasites and leucocytes  and the Plasmodium-specific LDH and anti-HRP2 antibody to detect P. falciparum-specific HRP2. All RDTs were performed on archived blood samples by trained and skilled laboratory personnel at the Parasitology Laboratory, icddr,b following the manufacturer’s instructions. Briefly, one pink-purple line in the proximal area (control line) interprets negative for malaria; one pink-purple line in the middle area, along with the control line, interprets non-P. falciparum infection, exclusively P. vivax in this study; one pink-purple line, along with the previous two bands, interprets P. falciparum infection. If any of the two test lines or control line did not appear, the test was invalid and repeated.Parascreen  is a three-band antigen detection RDT which comes in cassette format. It employs a recombinant antibody against pLDH to detect DNA was isolated using QIAamp DNA blood mini kit  following the manufacturer’s instructions from 200 μL of archived whole blood.et al.[P. falciparum was 97.1 and 97.6%, respectively, while for P. vivax 95.2 and 98.1% [P. falciparum and P. vivax) infection diagnosed by qPCR was not considered in this study.Quantitative PCR (qPCR) was performed on isolated DNA following the method described by Alam et al. with InvAll data were encoded in an Excel data sheet and the performance of RDT was calculated by means of the following indicators: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement (kappa) were calculated with their corresponding 95% confidence intervals (95% CI), using EM and qPCR as reference standards. Sensitivity was calculated as the proportion of positive RDT test results among malaria-positive samples identified by EM and qPCR, while specificity was calculated as the proportion of negative test results among the malaria-negative samples identified by the reference standards. PPV and NPV were obtained as the true positive results among all malaria-positive samples and the true negative results among all negative samples, respectively . AgreemeP. falciparum infection and 54 (26.0%) were P. vivax infection. The parasite density for P. falciparum ranged between 16 and 261,480 parasites/μL  with median value of 19,960 parasites/μL, while the parasite density for P. vivax ranged between 16 and 25,120 parasites/μL  with median value of 1,140 parasites/μL. qPCR confirmed 208 (63.6%) positive malaria cases, of which 154 (74.0%) were P. falciparum and 54 (25.9%) were P. vivax. With Parascreen, there were 202 (61.7%) malaria positive cases, of which 150 (74.2%) were P. falciparum and 52 (25.7%) were P. vivax infection.In total, 327 febrile patients were included in this study from two UHCs. The results of EM, qPCR and Parascreen are provided in Table P. falciparum malaria detection, sensitivity: 96.0%  and specificity: 98.2%  and for P. vivax malaria detection, sensitivity: 90.7%  and specificity: 98.9% . When qPCR was used as the reference standard, Parascreen had the following results for any kind of malaria detection, sensitivity: 97.1%  and specificity: 100% (96.1-100.0); for P. falciparum malaria detection, sensitivity: 95.4%  and specificity: 98.8%  and for P. vivax malaria detection, sensitivity: 89.0%  and specificity: 98.8% .Table P. falciparum and P. vivax, whereas for parasite densities ranging from 1–500 parasites/μL, the sensitivity was low (60.0%-83.3%)  in detecting samples with parasite densities >500 parasites/μL for both 00% in deP. vivax was considered as P. malariae and P. ovale cases were not present in the study samples [P. falciparum and P. vivax) and parasitaemia of infections.Parascreen showed acceptable performance in this study with overall sensitivity and specificity of 97.1 and 99.1%, respectively, when compared to EM, and 97.1 and 100%, respectively, in comparison with qPCR. Parascreen can detect all types of non-falciparum malaria but in this study only  samples ,14. ParaP. falciparum detection it showed almost 100% detection rates while for P. vivax it was approximately 30% [P. falciparum detection, the sensitivity and specificity was also in concordance with the previous findings [P. vivax detection, improved sensitivity and specificity are reported. The improved sensitivity and specificity of P. vivax detection compared to previous findings may be due to the increased release of antigen through parasite lysis in the archived sample [Several evaluation studies of Parascreen in different countries reported overall sensitivity ranging from 47.5 to 95.5% and specificity from 64.3 to 98.5% with varying performance for falciparum and non-falciparum malaria detection -21,23,24tely 30% . Here, ifindings ,20,21, wd sample .P. falciparum and for P. vivax 77.2% sensitivity and 98.1% specificity were recorded when compared with EM and similar values observed when compared with PCR [In a study in India, Parascreen showed 94.0% sensitivity and 72.0% specificity for with PCR .P. falciparum and 79.4% sensitivity and 98.7% specificity for non-falciparum malaria [P. falciparum and non-falciparum malaria, respectively, with specificity of 98.0 and 100%, respectively [In Myanmar, two RDTs with similar detection properties have been evaluated in field settings ,26. The  malaria . The VIKOnsite (Pf/Pan), a RDT with similar detection principle recently evaluated in Bangladesh, reported 94.2% sensitivity and 99.5% specificity for falciparum malaria detection and for vivax malaria detection it showed sensitivity and specificity of 97.3 and 98.7%, respectively [WHO recommends sensitivity ≥95% at ≥100 parasites/uL for RDTs [P. falciparum isolates could also account for false negative tests. The extent of HRP-2 variations in Bangladesh is currently unknown, however variations or deletions in HRP-2 have been reported recently from India, [P. falciparum samples showed no HRP-2 test line but Pan specific test line, thus considered as P. vivax, as other types of malaria were absent in the study samples. The intraspecies variation, mutation or deletions in the HRP-2 gene can cause non-expression of HRP-2 [P. vivax-positive samples with parasitaemia ranging from 16 to 200 parasites/uL as negative. This might be due to low pLDH level, as pLDH level is directly proportional to parasitaemia [In this study, Parascreen was unable to detect three microscopically confirmed falciparum malaria samples with parasitaemia ranging from 112 to 2,600 parasites/uL. This might be caused by the degradation of HRP-2 target antigen as the study was carried out with archived samples. Intraspecies sequence variation , deletiom India,  as well m India, ,32. In tof HRP-2  which masitaemia . In manysitaemia ,6,11,26.As all four malarial parasites co-exist in the Bangladesh-India-Myanmar border area, an important criterion for selection of an appropriate RDT is the capability to detect all types of malaria. It is advantageous to use Pf/Pan RDTs which can do so. The high predictive values for Parascreen indicate that it is able to detect true malaria cases as well as ruling out non-malaria cases. High sensitivity, specificity and predictive values for Parascreen present it as a viable alternative for malaria diagnostics in Bangladesh-India-Myanmar border areas where malaria is endemic.P. malariae and P. ovale samples in this study restricts the findings to the detection performance of falcipaum and vivax malaria. The inclusion of P. malariae and P. ovale in the study samples is needed to assess non-falciparum malaria detection performance.The absence of Parascreen showed acceptable performance for falciparum as well as vivax malaria diagnosis in standard experimental conditions. It can be employed in resource-limited settings to diagnose all types of human malaria.The authors declare that they have no competing interests.RE and MSA conceptualized and designed the study, collected and identified samples, analyzed the data, drafted the manuscript and made final revisions. RE, ANM and MSA did sample analysis. WAK and RH made critical revision of the manuscript. RE and ANM performed laboratory tests. All the authors read the final version of the manuscript and approved.


Plasmodium infection and anaemia among children 6–59 months and individuals aged older than 5 years in Kinshasa, a large city with heterogeneity in malaria prevalence.There is little data on the risk factors for malaria infection in large cities in central Africa and in all age groups. There may be different associations with the risk factors for areas with different malaria transmission intensities such as the effect of fever or age. This study aimed at identifying risk factors associated with This study analysed data from 3342 children aged 6–59 months from 25 non-rural health zones (HZs) and for 816 individuals aged older than 5 years from two HZs in Kinshasa , collected during a cross sectional malaria survey in 2011. Logistic regression with random effects was used to investigate predictors for malaria and anaemia. Differences in risk factors in areas with a prevalence of less than 10 and 10 % or greater were investigated.There was evidence of a different age-pattern in the two transmission settings. For children under 5 years, the highest prevalence of malaria was observed in the 48–59 months group in both transmission settings, but it increased more gently for the lower transmission HZs (p = 0.009). In a separate analysis in children over 5 years in two selected HZs, the peak prevalence was in 5–9 years old in the higher transmission setting and in 15–19 years old in the lower transmission setting. Reported fever was associated with malaria in both transmission strata, with no evidence of a difference in these associations (p = 0.71); however in children older than 5 years there was a significant interaction with a stronger association in the low transmission HZ. Insecticide-treated net (ITN) use was associated with a lower risk of malaria infection in children 6–59 months in the high transmission HZs. Similar estimates were found in children over 5 years and the lower transmission HZ but the associations there were not significant. There was no evidence of a difference in these associations by strata. The risk of anaemia decreased with increasing age in all strata, whereas it increased with malaria infection and reported fever. ITN use did not show evidence of protection against anaemia. Low socio-economic status was associated with malaria in high transmission setting in children 6–59 months and anaemia in low transmission setting.This study shows that in areas of low transmission in Kinshasa, the peak prevalence occurs in older age groups however ITN use was highest in children under 5 years. Targeted distribution of ITN to all age groups should be continued. For most risk factors, there was no evidence of an interaction with transmission intensity however the associations with age and with fever in the last 2 weeks did vary significantly. Urbanization, widespread use of malaria control measures and effective treatment in recent years have had a significant impact in reducing the prevalence of malaria in many African cities, as well as contributing to the more heterogeneous risk in malaria observed in many urban areas –5. In KiIn addition, many studies have shown that control of malaria can reduce the prevalence of childhood anaemia –14. AnaePlasmodium falciparum infection and anaemia in Kinshasa in children aged 6–59 months in 25 non-rural HZs, as well as in individuals aged 5 years and older in a separate analysis in two HZs.The present study investigated individual and household risk factors for Anopheles gambiae s.l., and usually peaks during the long rainy season from September to May [http://www.RBM.org) created with the Build component of the open data kit (ODK) software (University of Washington & Google Foundation). Respondents were asked about demographic information of the residents, educational level, assets owned (such as television and bicycle), presence of insecticide-treated bed net (ITN) and use of ITN the night prior to the survey.This study used cross-sectional data from a survey conducted in 2011 in Kinshasa, the capital city of the DRC, which is described in detail by Ferrari et al. . In summr to May . From eaPlasmodium infection and anaemia were assessed for their association with a number of variables. For individuals aged older than 5 years, data collection took place in only two HZs with different transmission intensities ; these data were analysed separately.The analysis was stratified according to malaria transmission intensity, based on the prevalence of malaria infection measured in 2011 among children 6–59 months [Plasmodium malaria as measured by rapid diagnostic test (RDT) and the anaemia test results. A child aged between 6 and 59 months was defined as anaemic if his/her Hb was below 11.0 g/dl. Therefore, the outcomes variables were dichotomous. Recorded explanatory variables were: age, gender, educational level of the respondent, occupation of the respondent, insecticide mosquito-net use and reported fever during the last 2 weeks and wealth index. A wealth index, calculated according to the method of Filmer et al., was constructed for each household based on ownership of household assets  and house characteristics  [The primary outcomes of the study were the presence or absence of aterial) . Three cThe proportions with malaria infection and with anaemia were analysed using a logistic regression model with random effects to take account clustering by health zone and health area. All analysis were performed separately for children (6–59 months) and individuals older than 5 years since they were sampled from different HZs. The analysis was carried out using STATA version 13 .Data collection took place in 2512 households, in the 25 HZs that were visited. A total of 3342 children aged 6–59 months were included in the analysis, 1118 and 2224 in the low and high transmission settings, respectively. A similar number of males (50 %) and females were included; the median age was 30 months . Table Plasmodium infections in children 6–59 months are shown in Table The risk factors for Treated net use was found to significantly lower malaria infection risk in the higher transmission strata with 38 % protection , however the effect was not significant in the lower transmission strata. Children who reported fever in the last 2 weeks had a significantly elevated risk of malaria infection in both strata.Higher education levels showed a trend towards being protective in both transmission settings Table . HoweverPlasmodium infection in individuals aged older than 5 years are shown in Table The risk factors for ITN use was not found to significantly lower the prevalence of malaria infection, although the estimates were in the direction of being protective. Individuals aged 5 years and older who reported fever in the last 2 weeks had an elevated risk of having malaria infection in both sites, and the association was stronger for the lower transmission: OR = 38.71 (95 % CI 11.08–135.23), and OR = 2.05 (95 % CI 1.07–3.95) in Selembao, with a highly significant interaction term (p < 0.0001). There was no evidence of an effect of higher education levels, occupation of the respondent or socio-economic status.The risk of having anaemia was found to decline progressively with increasing age Table  in both There were some age-specific differences in ITN usage Fig. , with hiPlasmodium infection and anaemia, in a city with diverse malaria transmission patterns. The key results from this analysis are the association between malaria infection and age, with older age groups being exposed to higher risk of malaria in low transmission settings and a lower and more homogeneous risk across all age groups in high transmission settings. Shift in the age of peak prevalence towards the older groups has been described for malaria and other infectious diseases and is consistent with exposure-related acquired immunity [Plasmodium infection seems to occur later in childhood, which is consistent with areas of rather low levels of transmission. The prevalence rates by HZ shown in Table The identification of risk factors for malaria infection and anaemia, provides information on the local malaria epidemiology and has the potential to lead to a more effective and targeted use of malaria control measures. This study presents the results of an analysis of the association of a number of variables that alone or in combination could affect the risk of acquiring immunity , 18. In immunity . ClearlyThe relationship of ITN use by the different age groups could also influence the age pattern of risk that was observed: children in the youngest age group, 6–35 months, were significantly more likely to sleep under an ITN in the low transmission setting, whereas utilization was similar among age groups in high transmission setting. A similar shift in age of peak of prevalence towards the older children has been observed elsewhere with an increase in net coverage . Only 44Higher malaria prevalence in older children has also been attributed to increased use of anti-malarials in early childhood . In casePlasmodium infection by 38 %  among children 6-59 months of age in areas of high transmission, consistent with the vast body of evidence supporting the efficacy and effectiveness of ITN in protecting against malaria [Results indicated that sleeping under an ITN the previous night reduced the risk of  malaria . In low Reported history of fever was associated with malaria infection overall. There was evidence of a difference in this association with transmission level among individuals aged older than 5 years (p < 0.001). The weaker association of reported fever with malaria in areas of high transmission could be explained by differences in the levels of acquired immunity.The data confirm that anaemia is frequent in urban Kinshasa, with 65 % prevalence among children 6–59 months, 30 % moderate (7.0–9.9 g/dl) and 1.9 % severe (<7.0 g/dl). ITN use in Kinshasa did not appear to be associated with benefits in lower anaemia risk, contrary to what has been documented in other settings –14. ThesIn this study, the risk of anaemia was shown to decrease with increasing age in both low and high transmission strata. These results are consistent with studies conducted in West Africa, showing a significant reduction in the mean haemoglobin level in children aged 2–5 years compared to children aged 1–2 .This study also showed differences in the effect in malaria risk or anaemia risk by socioeconomic status, consistent with previous studies carried out in sub-Saharan Africa , 26–28, P. falciparum. The underestimation may have differed with acquired immunity affecting the age pattern [This study however has some limitations. Foremost, the analysis draws on cross-sectional data; hence the causal nature of associations should be viewed with a certain caution. A second most important limitation of the study relates to the smaller sample size for individuals over 5 years compared to that of children 6–59 months, limiting the authors’ ability to potentially detect important differences and interactions between risk factors and transmission. Furthermore, the low proportion of males for this survey (34 %) may have triggered a gender-response bias, with consequences on the prevalence and associations found. The direction and magnitude of a possible bias remain unknown. Lastly, RDTs are limited in sensitivity to detect low density parasitaemia and their use may have led to an underestimation of the true proportion of people infected with  pattern –33.For the most part, there was no evidence of an interaction between malaria infections and the risk factors with transmission intensity; however the associations with age and with fever in the last 2 weeks did vary significantly. The results also show that school-aged children are the least protected with ITN, across the different transmission settings, hence representing an important reservoir for infection. The observation of a shift in the peak age of risk for malaria to older groups is consistent with areas of low transmission and highlights the need for a more equal distribution of ITN in Kinshasa to target all age groups and not only the traditional high-risk group of young children.