Plasmodium falciparum parasite prevalence, seroprevalence, and seroconversion rate to P. falciparum merozoite surface protein-119 (MSP-119) were measured in a cross-sectional survey to determine differences in transmission between altitudinal strata. Clusters of P. falciparum parasite prevalence and high antibody responses to MSP-119 were detected and compared. Results show that P. falciparum prevalence and seroprevalence generally decreased with increasing altitude. However, transmission was heterogeneous with hotspots of prevalence and/or seroprevalence detected in both highland and highland fringe altitudes, including a serological hotspot at 2,200 m. Results demonstrate that seroprevalence can be used as an additional tool to identify hotspots of malaria transmission that might be difficult to detect using traditional cross-sectional parasite surveys or through vector studies. Our study findings identify ways in which malaria prevention and control can be more effectively targeted in highland or low transmission areas via serological measures. These tools will become increasingly important for countries with an elimination agenda and/or where malaria transmission is becoming patchy and focal, but receptivity to malaria transmission remains high.Serological markers, combined with spatial analysis, offer a comparatively more sensitive means by which to measure and detect foci of malaria transmission in highland areas than traditional malariometric indicators.  In the east African highland areas, malaria transmission intensity generally decreases with altitude, often becoming heterogeneous as altitude increases, to a point where malaria is no longer transmitted.18There is no standard definition of a malaria hotspot. The World Health Organization has previously defined foci of malaria as localities with continuous or intermittent epidemiological factors necessary for transmission.15Hotspots are likely to persist in highland areas unless interventions are targeted toward them.22However, identifying hotspots of malaria in highlands is challenging. Standard measures such as entomological inoculation rates (EIRs) or parasite prevalence are more difficult to collect in low transmission areas due to very low numbers of either mosquitoes or infected individuals.19 (MSP-119) antibodies persist for 49.8 years, reflecting cumulative exposure to malaria infection.24Conversely, antibody responses to some malaria parasite antigens have the potential to provide information about malaria transmission intensity over short or long periods of time. Drakeley and others estimated that merozoite surface protein-119.Our study used serological measures to assess malaria transmission at different altitudes in southwest Uganda. In addition, spatial analysis was used to determine whether hotspots of parasite-positive individuals are geographically similar to clusters of high antibody responses to MSP-1The ten villages included in the study were situated in the catchment areas of Kebisoni and Bufundi health facilities that are located in the highland and highland fringe districts of Kabale and Rukungiri in southwest Uganda. Bufundi health center is in a highland area at an altitude of 2,200 m and serves a population of approximately 18,000 that resides over altitudes of 1,700–2,200 m  and April (long rains).Plasmodium falciparum infections and to examine serological responses to P. falciparum MSP-119, in a population residing along a transect of low to relatively high malaria endemicity.In August 2007 (dry season), a cross-sectional survey was implemented in ten villages over five altitudinal strata in southwest Uganda. Altitude was used as a proxy for transmission intensity, reflecting the relationship between altitude and lower temperatures that affects the intrinsic incubation period of the parasite in the mosquito. Two of the study strata were in the highland district of Kabale and three were in the highland fringe district of Rukungiri. The aim was to determine the prevalence of Villages in health facility catchment areas were classified into five strata of malaria endemicity based on malaria incidence recorded by the health facility and using the health facility catchment population estimates. Local health teams verified the village categorizations based on their knowledge of malaria transmission intensity and village altitudes. One to three villages were included in each stratum depending on village size. The primary sampling unit for the study was the household. All households in the selected village that had members present and who consented to the study were sampled.P. falciparum parasite–negative individuals, and were stored and prepared as described previously.25All consenting household members were tested for malaria parasite infection using the Paracheck Pf rapid diagnostic test (RDT) produced by Orchid Biomedical Systems . In addition, filter paper bloodspots were collected from all P. falciparum MSP-119 were detected in the blood eluted from the filter paper blood spots by an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant P. falciparum MSP-119.P. falciparum–positive people from ELISA analysis during this study with the rationale that if those who were parasite positive were more likely to have traveled and of a certain age, that their inclusion into the sample would skew the age prevalence data. Restriction to parasite negatives gave a more normal age distribution for establishing the intensity of any ongoing background transmission.Antibodies to 19 from similar epidemiological settings in Tanzania.25Household and laboratory data were entered using Epi Info 2000 ; internal consistency checks and analyses were performed using STATA software . Raw optical densities (ODs) were converted to percentages of a single point on the positive control curve. The cutoff between negative and positive was determined using maximum likelihood to estimate the parameters for a mixture model as described elsewhere.19 and/or P. falciparum infections were distributed randomly over space, and if not, to identify significant spatial clusters.© TM software (http://SatScan.org/) was used . The spatial scan statistic uses a scanning window that moves across space. For each location and size of the window, the number of observed and expected cases is counted, and the window with the greatest ratio of observed to expected cases is noted. The numbers of expected cases are calculated by considering an even distribution of cases across the population. The statistical significance of the hotspot is evaluated by taking into account multiple tests for the many potential cluster locations and sizes evaluated.P. falciparum parasites and 2) hotspots of MSP-119 seroprevalence or immune responses.To detect spatial clusters, all households included in the study were georeferenced and the Kulldorff spatial scan statistic was used to test whether seroreactivity to MSP-1P. falciparum parasite hotspots, cases were people with P. falciparum parasites and controls were people who were P. falciparum parasite negative. For seroprevalence hotspots, log-transformed age-adjusted OD values were calculated. Adjustment for age was done by first log transforming antibody responses and then fitting Loess lines to determine at which point the relationship between age and log (Ab response) became non-linear. A linear regression model was run for each age group  and residuals were used as age-adjusted antibody response.For P-value of the most likely and secondary clusters were determined by conducting Monte Carlo replications of the data set. To scan for small to large clusters, the maximum cluster size was set to 50% of the total population at risk. All data were examined at district level, that is, strata were split into the two highest altitudes  and three lowest altitudes (Rukungiri). In the two highest strata, spatial analysis could only be undertaken with the serological data because of the very low number of P. falciparum parasite positive (N = 3) individuals detected. Maps were made using ArcGIS version 9 .Secondary clusters were set to not overlap the most significant cluster. For each location and size of the scanning window, a likelihood ratio test was conducted to test the hypothesis that there was an elevated rate of disease (or antibody response) when compared with the distribution outside. The window size and location with the maximum likelihood was defined as the “most likely” cluster . The distribution and Ethical clearance was obtained from the Ugandan National Technology and Science Council (HS-35) and the London School of Hygiene and Tropical Medicine (no. 3053). In addition, the blood sampling was demonstrated to the local chairmen and the executive committee of each village surveyed, and approval for the survey was sought. Informed consent was obtained from the head of the household for all human adult participants as well as any children under 18. Parasite-positive individuals were referred to the local health facility to receive free treatment.19 using ELISA. Overall, the sample comprised 45.9% men and 54.1% women aged between 0 and 99 years . The force of infection, calculated from MSP-119 age–seroprevalence data, was estimated as a SCR of 0.04 per year across the study strata, correlating to an EIR of ∼1.3   compared with the lowest altitude . Seroprevalence to MSP-1altitude . However19 seroprevalence and P. falciparum prevalence amongst men and women. Prevalence of P. falciparum infection was highest in 7- to 10-year olds. As would be expected, seroprevalence to MSP-119 increased with age  in stratum 1  to SCR 0.01 (95% CI: 0.00–0.01) in strata 4 and 5 see P. falciparum seroreactivity was significantly increased in six clusters of households across the study sites: three in the upper two strata  and three in the lower three altitudinal strata (Rukungiri).Age-adjusted P. falciparum infection were detected in the highland fringe strata of Rukungiri. One primary cluster of P. falciparum infection was detected with a radius of 3.1 km consisting of 155 households (P = 0.001) (P = 0.04).Two clusters of P = 0.001). A pond lies in the middle of the primary cluster, which is also flanked (outside the cluster) by two protected springs (0.35 km and 0.46 km from the cluster center) and another pond (0.35 km).A primary serological cluster was detected in Rukungiri, which consisted of 11 households  with a rP = 0.004). The other secondary cluster detected consisted of 16 households and spanned 0.28 km (P = 0.006). The center of the cluster was 0.35 km away from a forested area and within the cluster there were three unprotected wells and a stream.Two secondary serological clusters were also detected across the three lower altitudinal strata, the largest of which were close to the lake and consisted of 93 households and spanned a radius of 1.83 km   (P = 0.001) and four (P = 0.001) households in two different villages. None of these villages were situated close to mapped open sources of water such as lakes or ponds.The primary cluster in the upper two strata included 95 households and had a radius of 2.2 km  or Langi and Lalobo's study in the same district in 1992 (PR: 2.7%).51Our results confirm findings from studies that demonstrate altitude to be highly correlated with malaria infection in Uganda and that serological responses to MSP-1In addition, previous studies used microscopy to detect malaria parasites whereas our study used RDTs. Abeku and others have demonstrated that RDT specificity varies considerably by month of test, age of patient, and presence or absence of fever during consultation in this area of Uganda.P. falciparum parasite prevalence existed along lakeshore areas.Anophelesgambiae s.s., the dominant vector in the area, and Anopheles funestus preferred open water bodies with the latter species playing an important role in transmission around the highland lakes.6Three of the six clusters detected (including the largest clusters) were near open water bodies such as lakes or ponds that confirms findings from other studies in highland settings that showed that clusters of P. falciparum prevalence and seroprevalence, if they were absent because of travel outside the area to places of higher malaria transmission intensity.There are several limitations to the study. First, we stratified villages in the study sites by using incidence rates from health facilities in the area. However, it is possible that villages further away from health facilities were classified as lower endemicity when the lower number of malaria cases was as a result of their distance from the facility rather than true endemicity. This would have resulted in a misclassification of village endemicity relative to others along the altitudinal transect. Our survey undersampled men that could have led to an underestimation of overall Regardless of these limitations, the study findings have multiple implications. First, results confirm the existence of hotspots of malaria transmission at extremely high altitudes in Uganda. Although these were documented previously in the 1940s at slightly lower altitudes in the same area,In an era of malaria elimination and in the context of significantly reduced financial resources, methods to accurately identify and target areas at risk of malaria transmission are crucial. As malaria burden decreases, malaria infections are likely to become more spatially heterogeneous because of differences in acquired immunity related to the clustering of malaria transmission in highland areas, and seasonal expansion of hotspots.Supplemental Figure.


Plasmodium knowlesi infections in the human population. The discovery was made during a molecular investigation of atypical P. malariae cases in the Kapit Health Division, Sarawak, Malaysian Borneo. Patients were more symptomatic with higher parasite counts than expected in P. malariae infections. The investigation found only P. knowlesi DNA present in patient blood samples. Morphological similarity had allowed P. knowlesi to masquerade as P. malariae during routine diagnostic microscopy for malaria. P. knowlesi, a malaria parasite of macaque monkeys, had entered the human population. The subsequent development of P. knowlesi species-specific PCR assays soon demonstrated that the entry was not confined to the Kapit Division but extended across island and mainland Southeast Asia. Relevant clinical descriptions and guidelines for the treatment and management of patents with P. knowlesi malaria were not available. Nor was it clear whether P. knowlesi had undergone a host switch event into the human population or if infections were zoonotic. The outputs of studies on P. knowlesi malaria during the past 10 years will be summarized, highlighting major findings within the context of pathophysiology, virulence, host switch events, treatment, control and importantly malaria elimination.Ten years have passed since the publication of a large focus of  Plasmodium, are blood-borne and pose a substantial risk in blood transfusion related infection . Twenty-eight (12%) of these had complications and four (1·7%) patients died, Table P. knowlesi malaria is relatively new, and markers of severe disease are preliminary but suggest that patients with parasitaemia >35 000 parasites/μl, bilirubin > 43 μmol/l, serum creatinine >256 μmol/l or platelets <45 000/μl are at risk . We found that 44% and 56% of patients were infected with Pknbpxa cluster 1 and Pknbpxa cluster 2, respectively. The clusters contained single nucleotide polymorphisms that allowed for differentiation of haplotype groups with 2 or three alleles among the P. knowlesi isolates. Analysis of the alleles with the clinical and laboratory variables in the patient cohort found that patients infected with particular allelic forms of cluster 2 had increased markers of disease severity including parasitaemia [19]. Not all patients with complications were infected with cluster 2 parasites but there were more patients than expected with complications in this cluster  19, 1. We fouPlasmodium knowlesi infections occur throughout the Southeast Asian region. The region is separated by high mountain ranges and large expanses of water including the South China Sea, the Sulu Sea and Andaman Sea [P. knowlesi is a parasite of non-human primates that do not cross oceans and rural human populations at risk tend to be local. It seems likely that, following the primary emergence of P. knowlesi and ancient dispersal events, P. knowlesi, populations have evolved in local geographical isolation. Each location reporting human infections is most likely dealing with different genetic variants of P. knowlesl [P. knowlesi malaria in humans was in Kapit, Malaysian Borneo, and further study found that naturally acquired P. knowlesi infections occurred throughout Malaysian Borneo, Peninsular Malaysia, all geographically distinct areas, and subsequently in most parts of Southeast Asia [aman Sea . P. knowknowlesl . The recast Asia ,11,21,23P. knowlesi isolated from the natural macaque hosts and human infections in Sarawak did not identify genetic clustering of parasites to either host, strongly suggesting a zoonotic origin for the infections in Sarawak [Anopheles balabacensis is a vector of P. knowlesi in Sabah [An balabacensis is better known as a vector of P. falciparum in Sabah and therefore well placed to channel a host switch event for P. knowlesi from macaque-to-human to human-to-human malaria transmission at this location [P. knowlesi infections mixed with P. falciparum and P. vivax [Anopheles dirus is the main malaria vector in this region of Vietnam and transmits all three parasite species, P. knowlesi, P. falciparum and P. vivax, concurrently [P. knowlesi warrants careful observation to pre-empt or at least contain a host switch event in any number of Southeast Asian locations – quite a monumental task.Even so the multiple entry events recorded across Southeast Asia may have been the result of parasite adaptation to the human host and clonal expansion but the infections are not clonal or related to any one index case or location . A study Sarawak . However Sarawak . Also, ain Sabah . An balalocation ,25. In VP. vivax . Anophelurrently ,27. P. kP. knowlesi responds well to currently available antimalarial compounds including chloroquine [P. knowlesi. Studies designed to properly assess clinical and parasitological responses of P. knowlesi to treatment highlight some important differences in response. In 2005, inherent mefloquine resistance of P. knowlesi in experimentally infected Rhesus macaques was reported [P. knowlesi isolates from human infections responded poorly to mefloquine ex vivo [P. knowlesi malaria during a second infection, increased following mefloquine treatment [P. knowlesi infections responded to mefloquine treatment [Earlier studies reported that oroquine ,12,28. Treported . Also, P ex vivo . Furtherreatment . Nonethereatment ,33.Plasmodium knowlesi responds to chloroquine albeit with a higher IC50ex vivo [P. falciparum, mostly chloroquine resistant, as P. knowlesi would put patients at risk.ex vivo . ChloroqP. knowlesi infections. Patients in Sabah with severe knowlesi malaria were more likely to survive when given intravenous artesunate than quinine [P. knowlesi responded better to artemisinin derivatives than all other compounds ex vivo [P. knowlesi infections can be symptomatic at low parasitaemia, lower than the sensitivity of thick film microscopy [P. knowlesi malaria [The most compelling evidence supports the use of artemisinin derivatives either as intravenous artesunate or as oral combination therapies to treat complicated and uncomplicated  quinine . In anot quinine . Both st ex vivo . In othe ex vivo . It shoucroscopy . Current malaria .P. knowlesi malaria occur across Southeast Asia [P. knowlesi transmission, reservoirs of infection, vectors and risk of human infection [P. knowlesi acquired in the jungle setting, over and beyond use of prophylaxis when spending time in areas where other patients with P. knowlesi have been infected. The pragmatic solution would be to encourage location-specific vector, parasite and natural host monitoring and surveillance. These measures are currently costly, require manpower and are logistically difficult. Perhaps a meeting of minds is required to develop alternative sampling and monitoring methods for forest settings that would capture the required data on parasite prevalence, vector capacity, natural hosts but using minimal manpower. Not an easy problem to solve on the backdrop of competing public health problems at each location. In areas where P. knowlesi is transmitted in human settlements or even human-to-human, existing malaria control measures would be expected to be effective.Human cases of ast Asia . This reast Asia . Apart fnfection ,37,38. Welimination is the interruption of local mosquito-borne malaria transmission, i.e. the reduction to zero of the incidence of malaria infection in a defined geographical area.’ [P. knowlesi. There is some evidence that P. knowlesi enters human populations where national human-host-adapted, malaria control initiatives are successful, a good example of this is Malaysia [P. knowlesi? or (ii) By removing human host challenge, particularly with P. vivax, are humans more susceptible to P. knowlesi through loss of cross protection?According to the World Health Organization ‘Malaria l area.’ . MalaysiMalaysia ,40. ThisP. knowlesi cases was discovered in Malaysia [P. malariae in the area. It is possible that these cases were more visible as cases of P. falciparum and P. vivax were controlled. Or were there more of these cases as immunity to malaria declined along with P. vivax and P. falciparum? Returning to P. knowlesi induced pyretic therapy in tertiary syphilis. Patients who were previously treated with P. vivax were partially resistant to subsequent P. knowlesi infection suggesting a degree cross immunity [Both scenarios are worth taking seriously. The relatively large number of Malaysia . The stuimmunity .P. knowlesi being better recognized because of heightened awareness and PCR identification? If so, then the prevalence of P. knowlesi may not have increased, we are simply looking harder, difficult to argue against. However, the number of cases of P. knowlesi in Sabah do seem to be increasing [There is some debate about the prevalence of zoonotic malaria – Is creasing . Regardleradication is defined as the permanent reduction to zero of the worldwide incidence of malaria infection caused by a particular malaria parasite species.’ [P. falciparum, P. vivax, P. malariae and P. ovale while still tolerating P. knowlesi in Southeast Asia.If the ultimate goal is malaria eradication, the WHO definition is much more specific ‘Malaria pecies.’ . If eradPlasmodium knowlesi has entered the human population in Southeast Asia. People who spend time the sylvan transmission sites are at risk. P. knowlesi remains largely zoonotic, but transmission near human settlements is suspected in Sabah. P. knowlesi is diverse and causes severe disease in at least 10% of those infected in Sabah and Sarawak. Patients with severe and fatal P. knowlesi infections fulfil the WHO criteria for severe falciparum malaria but without coma or severe adult anaemia. Healthcare professionals should suspect P. knowlesi in patients and blood donors with a history of time spent in the jungles of Southeast Asia. Blood donor screening protocols, to detect malaria exposure, use recombinant P. falciparum and P. vivax antigen. While there may be some cross reactivity between P. knowlesi and the phylogenetically related P. vivax, these reagents could not be used reliably to detect antibodies to P. knowlesi. At the moment, the risk of donor blood infection with P. knowlesi would be expected to be low; however, a history of time spent in the jungles of Southeast Asia may warrant donor referral for a 1-year period.Importantly, evidence suggests that rapid diagnosis and immediate treatment with artemisinin derivatives is appropriate for this potentially virulent parasite with a short erythrocytic life cycle.


Plasmodium falciparum malaria. The effects of CTX prophylaxis on the acquisition of P. falciparum antigen specific CD4+ T cells-mediated immunity in HEU children is still not fully understood.Cotrimoxazole (CTX) prophylaxis, recommended in HIV-exposed uninfected (HEU) children primarily against HIV-related opportunistic infections, has been shown to have some efficacy against + T cells subsets were determined by immunophenotyping. P. falciparum antigen-specific CD4+ T cells responses were measured by intracellular cytokine staining assay.Peripheral blood was collected from HEU and HIV-unexposed uninfected (HUU) children at 6, 12 and 18 months of age. Proportion of CD4+ T cell subsets between HEU and HUU children at all ages. There was a trend showing acquisition of P. falciparum-specific IFN-γ and TNF-producing CD4+ T cells with age in both HUU and HEU children. There was, however, lower frequency of P. falciparum-specific IFN-γ-producing CD4+ T cells in HEU compared to HUU at 6 and 12 months, which normalized 6 months after stopping CTX prophylaxis.There were no differences in the proportions of naïve, effector and memory CD4P. falciparum-specific IFN-γ-producing CD4+ T cells in HEU children on daily cotrimoxazole prophylaxis, which is evident at 6 and 12 months of age in comparison to HUU age-matched controls. However, whether this delayed acquisition of P. falciparum-specific IFN-γ-producing CD4+ T cells leads to higher risk to malaria disease remains unknown and warrants further investigation.The results demonstrate that there is delayed acquisition of  Plasmodium falciparum antigens contributes towards the development of effective immunity that controls parasitaemia and prevents severe and life threatening illness [+ and CD8+ T cells play an important role in protective immunity to P. falciparum malaria [Plasmodium stages and also direct the immune responses induced by erythrocytic stages of malaria [+ T cells can restrict growth of the malaria parasites and reduce risk of clinical disease [In children, repeated exposure to  illness , 2. Evid malaria –6. T cel malaria . During  disease . Of note disease . IFN-γ h disease .P. falciparum and subsequent acquisition of malaria specific immunity.Continuous malaria chemoprophylaxis has been shown to slow down the development of clinical immunity in children leading to an increased risk of disease following disruption or cessation of treatment , 16. CotAlthough there is now good evidence to show that CTX prevents clinical malaria in children taking it , 18, limP. falciparum merozoite antigens [P. falciparum antigen specific CD4+ T cells in HEU children during and after prophylaxis, and compared with aged-matched HIV-unexposed uninfected (HUU) children.Since CTX is given to these HEU children primarily as an antibiotic rather malaria chemoprophylaxis, its impact on development of natural immunity to malaria in HEU children needs careful assessment. HEU Malawian children on CTX prophylaxis have reduced magnitude and breadth of IgG antibody responses to distinct antigens  indicatiantigens , 22, thiP. falciparum malaria transmission is stable throughout the year, with P. falciparum prevalence rate of around 27 % [Characteristics of the study area, design and participants have already been described in detail before . Brieflyund 27 % . HEU chiChildren were seen every 6 months and at each visit, a total of 3 ml of venous blood was collected. 2 ml in sodium heparin tube for intracellular cytokine assay and 1 ml of in EDTA tubes for immunophenotyping. All immunological assays were conducted blinded to the children’s study group.P. falciparum (3D7) was kindly provided by ICMER laboratory, Blantyre, Malawi. For recovery, parasite aliquots were thawed using two sodium chloride thawing solutions: (200 µl of 12 % NaCl and 10 ml of 1.6 % NaCl) and washed twice with 25 ml of protein free medium (RPMI-1640 supplemented with 25 mmol/l HEPES and 40 mg/ml gentamycin), as described elsewhere [Cryopreserved laboratory adopted strain of lsewhere . Parasit°C and supernatant carefully removed and transferred to a second tube. The mixture was washed and approximately 1 μl of the pellet was collected and parasitaemia determined as previously described [°C.Level of parasitaemia was estimated based on microscopy of thin smears and parasitaemia level of approximately 5 % was used. Pooled cultures were washed and 1.5 ml of warm protein free medium was added to the red cell pellet followed by 2.5 ml warm gelofosine . The mixture was then allowed to stand for 20 min at 37 escribed . The infescribed . ProteinWhole blood (20 μl) was stained with surface markers , CD45RA FITC  and CCR7 APC  and lysed with 2 ml of 1× FACS lysing solution. After wash, the cells were then acquired on a Cyan ADP flow cytometer (Beckman Coulter) and minimum of 10,000 lymphocytes events was collected per tube during acquisition.P. falciparum 3D7 schizonts lysate (5 µg/ml) and mycobacterium purified protein derivative  in parallel with a non-stimulated background control for 24 h. Harvested cells were stained with surface markersCD3-APC-H7, CD4-PerCP , and CD8-Pacific Orange  and lysed as above. The cells were the permeabilized with 500 μl of 1× BD Cytofix/Cytoperm  and then stained with intracellular markers IFN-γ-FITC  and TNF-PE  for 30 min. The cells were then acquired as above and minimum of 10,000 CD4+ T cell events were collected per sample. Single stained BD comp beads were processed and acquired in parallel to samples each day and used for compensation. Analysis was conducted using FlowJo software version 9.7.5 .Heparinized whole blood (100 μl) was cultured in 96 U bottom culture plate and incubated with PMA/Ionomycin , + T cell proportions and frequencies of cytokine producing cells between the study groups at each visit. Background responses generated in the negative control wells were subtracted from responses generated from antigen-stimulation to obtain antigen-specific T cell responses. Differences with P values less than 0.05 were considered significant.Statistical analysis and graphical presentation were done using GraphPad Prism 6  and STATA . A Shapiro–Wilk normality test revealed that all data were non-normally distributed. Mann–Whitney U test was used for comparison CD4p = 0.21). All mothers of the HEU children were on ART and were taking daily CTX prophylaxis during the entire follow up period.Overall, 33 HEU children and 31 age and location-matched HUU children were recruited in the study and followed through from 6 to 18 months of age , central memory , effector memory , and terminally differentiated (CD45RA+CCR7−) T cells , 12 months (HEU 6.5 vs. HUU 0 %) and 18 months (HEU 6.7 vs. HUU 7.1 %). The number of HEU and HUU children with at least one episode of clinical malaria at any time of the follow up period was similar between HEU and HUU children (9.1 vs. 12.9 %). There was no evidence of increased prevalence of asymptomatic and clinical malaria in HEU children on CTX prophylaxis compared to HUU age-matched controls, but the study was underpowered to conclusively determine this clinical endpoint.To determine whether the delayed acquisition of P. falciparum antigen specific CD4+ T cells in HEU children. The study findings demonstrate that in the context of unaltered proportions of naïve and memory CD4+ T cell subsets and unimpaired CD4+ T cell IFN-γ and TNF producing function, the frequency of P. falciparum antigen-specific IFN-γ-producing CD4+ T cells was lower in HEU children on CTX prophylaxis compared to HUU age-matched controls. However, this normalized to levels seen in HUU children, 6 months upon CTX prophylaxis cessation.This study investigated the effect of CTX prophylaxis on the acquisition of + T cell subsets may affect the capacity of antigen specific CD4+ T cell response to infections [+ T cell subsets in between HEU and HUU children from 6  to 18 months of age indicating that HEU children have normal CD4+ T cell phenotypes, contrary to earlier reports which were done mostly during the pre-ART era [Phenotypic alterations in the proportions of naive and memory T cell subsets have been observed in HEU children during early life , 28. Confections , leadingfections –32. In t-ART era .P. falciparum antigen-specific IFN-γ-producing CD4+ T cells in HEU children during the period on CTX prophylaxis was lower in comparison to HUU age-matched controls. However, there was no differences in the mitogen-stimulation or Mycobacterium-specific CD4+ T cells. IFN-γ plays an important role in regulating the protective immune responses against blood stage malaria infection even in the absence of antibody response [P. falciparum exposure [P. falciparum specific CD4+ T cells were observed in Ugandan under-five children with lower prior malaria incidence compared to children with higher prior incidence[P. falciparum, resulting in a delayed acquisition of cell-mediated immunity against malaria, which ultimately may result in impaired development of P. falciparum antigen-specific antibody-mediated immunity [The frequencies of response  and has exposure . Similarincidence. This obimmunity .P. falciparum antigen-specific IFN-γ-producing CD4+ T cells normalized following cessation of prophylaxis and were comparable to HUU children by 18 months of age. Studies from experimental malaria challenge showed that even a single exposure to malaria induced robust CD4+ T cell responses producing IFN-y that could be detected 14 months post infection in the absence of further exposure[+ T cells in response to P. falciparum in HEU children after CTX prophylaxis suggesting different patterns in acquisition of humoral and T cell mediated immunity to malaria following CTX prophylaxis.In contrast to previous results in the same population , where a exposure. These rP. falciparum antigen-specific IFN-γ-producing CD4+ T cells would lead to increase risk to asymptomatic and clinical malaria. Unfortunately, this current study could not gather enough clinical endpoints, as it was observed that the prevalence of parasitaemia and clinical malaria in both HEU and HUU children in the study was much lower than the estimated 27 %. This underpowered the study to conclusively show any clinical endpoint differences attributed to the CTX induced perturbation in P. falciparum-specific immunity between the two study groups. Nevertheless, the study reports an important observation showing delayed acquisition of P. falciparum-specific immunity in HEU children during CTX prophylaxis, which requires further investigation.Due to the importance of the IFN-γ in immunity against malaria, it would be anticipated that delayed acquisition of + T cells producing other cytokines such IL-2, IL-10 and IL-4 alone or in combination with the studied cytokines have been shown to be associated with protection from malaria [+ T cells immune responses. Secondly, although CD4+ T cells are an important producer of IFN-g, they are not the only cells that produce this important cytokine since macrophages, NK and B cells [The choice of immune responses investigated in the study is not exhaustive, and the authors acknowledge that there are other mechanisms that are equally as important. CD4 malaria , 11, 12. B cells , 37 are P. falciparum antigen-specific CD4+ T cells in HEU children. This has potential to increase the risk of malaria infection following cessation of CTX prophylaxis, but this study was not able to conclusively address this hypothesis due to limitations in sample size and the duration of the follow-up period. Future studies should be based on a larger sample size and longer follow up durations to ascertain definitively the impact of CTX prophylaxis mediated delay in anti-malarial immunity on risk to clinical malaria. This is a public health dilemma that needs to be tackled.In conclusion, the study has shown that CTX prophylaxis is associated with delayed acquisition of


Military conflict has been a major challenge in the detection and control of emerging infectious diseases such as malaria. It poses issues associated with enhancing emergence and transmission of infectious diseases by destroying infrastructure and collapsing healthcare systems. The Orakzai agency in Pakistan has witnessed a series of intense violence and destruction. Military conflicts and instability in Afghanistan have resulted in the migration of refugees into the area and possible introduction of many infectious disease epidemics. Due to the ongoing violence and Talibanization, it has been a challenge to conduct an epidemiological study.Plasmodium species were detected using nested PCR (nPCR) and amplification of the small subunit ribosomal ribonucleic acid (ssrRNA) genes using the primer pairs. We report a clinical assessment of the epidemic situation of malaria caused by Plasmodium vivax (86.5%) and Plasmodium falciparum (11.79%) infections with analysis of complications in patients such as decompensated shock (41%), anemia (8.98%), hypoglycaemia (7.3%), multiple convulsions (6.7%), hyperpyrexia (6.17%), jaundice (5%), and hyperparasitaemia (4.49%).All patients were sampled within the transmission season. After a detailed clinical investigation of patients, data were recorded. Baseline venous blood samples were taken for microscopy and nested polymerase chain reaction (nPCR) analysis. P. vivax should be considered by malaria control strategy makers in the world and by the Government of Pakistan. In our study, children were the most susceptible population to malaria infection while they were the least expected to use satisfactory prevention strategies in such a war-torn deprived region. Local health authorities should initiate malaria awareness programs in schools and malaria-related education should be further promoted at the local level reaching out to both children and parents.This overlooked distribution of  Plasmodium vivax and P. falciparum infections. The nested polymerase chain reaction (nPCR) examination detected 154 (86%) and 21 (12%) P. vivax and P. falciparum infections, respectively. We found worsening hygiene conditions in FATA, likely caused by poor socioeconomics and the collapse of the public health infrastructure. Decompensated shock was a common and prominent clinical feature of malaria among all the clinical presentations caused by both P. vivax (53%) and P. falciparum (42.9%). Our results have significant implications for both public health and malaria control in FATA and Pakistan. Our findings illustrate higher prevalence of malaria in children compared to other age groups. Further research on sensible estimates of refugees is required, as well as resistance to anti-malarials.The malaria epidemic and endemic in Pakistan is a present and ongoing threat to public health which could have an impact in the nearby regions as well. For the first time, we report a clinical assessment of malaria endemicity in the Orakzai Agency, which is Pakistan’s most neglected area due to Talibanization and war in Afghanistan. Febrile patient blood samples of the area were investigated to report the clinical assessment of malaria caused by  Disease emergence is influenced by both natural and human factors. Among human activities, military conflicts characterized by war and regional tribal and/or sectarian strife have had huge impact by destroying infrastructure and collapsing healthcare systems. The affected region and the people therein face diverse short-term and long-term consequences. Large population displacements present higher risks of infectious diseases, lack of resources, which is often the case within crowded refugee camps with sanitation issues, increased exposure of the population to disease vectors, and destruction of healthcare systems lead to negative consequences . MoreoveThe prolonged Soviet war in Afghanistan between 1979 and 1995 annihilated the malaria vector control programs in the nation, which had been implemented in the 1960s and 1970s. The programs were so successful that Afghanistan had virtually eradicated the disease in the late 1970s . The colP. vivax is increasing every year. According to the World Health Organization (WHO) report, approximately 75% (previously 64%) of the infections are transmitted through P. vivax, whereas 25% (previously 36%) are caused by P. falciparum, which are the two most prevailing species in Pakistan [P. vivax malaria is accounted in 70% of the malaria burden in Pakistan, and FATA being the most impoverished and extremely underdeveloped area in the Pakistan has the highest malaria burden due to the large Afghan refugees and IDPs [Different researchers reported that permanent residents have low susceptibility and high immunity against malaria as compared to Afghan refugees . The proand IDPs . Social The Orakzai Agency  has beenP. vivax and P. falciparum malaria among male and female patients of different age groups. We also report the analytical, epidemiological and clinical differences between P. vivax and P. falciparum infections.We report here the first study of epidemiology and clinical burden of malaria in this war-torn and Talibanized region, Orakzai Agency in Pakistan  Kalaya in Orakzai Agency, Pakistan between April 2011 and December 2013. Patients presented with major clinical symptoms  of malaria at different partially functional health care centers were referred to the DHH Kalaya. The major clinical symptoms of malaria were based on WHO criteria , 13. AllAfter a detailed clinical investigation of patients, a standardized case report template was designed to compile the complete clinical data of each patient. For children younger than five years, their parents or relatives were asked of their medical history.Plasmodium spp. infection was made by thick or thin smears. Two slides were made from each patient’s blood and both thick and thin films were prepared on the slides in the DHH Kalaya laboratory. Giemsa-stained thick blood smears of patients were examined using Giemsa stain and the parasitemia quantified independently by two skilled microscopists [Baseline venous blood samples were taken for microscopy and nested polymerase chain reaction (nPCR) analysis. The initial diagnosis of scopists . A thickPlasmodium species were detected using nested PCR (nPCR) and amplification of the small subunit ribosomal ribonucleic acid (ssrRNA) genes using the primer pair set A (5’-TTAAAATTGTTGCAGTTAAAACG-3’ and 3’-CCTGTTGTTGCCTTAAACTTC-5’) for the detection of P. vivax; primer pair set B (5’-CGCTTCTAGCTTAATCCACAT AACTGATAC-3’ and 3’-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-5’)for the detection of P. falciparum; primer pair set C (5’-CTGTTCTTTGCATTCCTTATGC-3’ and 3’-GTATCTGATCGTCTTCACTCCC-5’) for the detection of P. ovale; and primer pair set D (5’-GTTAAGGGAGTGAAGACGA-3’ and 3’- TCAGAAACCCAAAGACTTTGATTTCTCAT-5’) for the detection of P. malariae. PCR reactions were carried out on a thermal cycler (Nyx Technik USA), beginning with 5 minutes at 94°C, followed by 25 cycles of 45 seconds at 94°C, 45 seconds at 58°C, and 5 minutes at 72°C for the first round; 30 cycles of 45 seconds at 94°C, 45 seconds at 65°C, and 2 minutes at 72°C was then performed for the second round. The final cycle was followed by an extension time of 5 minutes at 72°C. The amplified PCR products were analyzed by 2%–2.5% agarose gel electrophoresis stained with ethidium bromide and visualized on the Bio-Rad gel doc system . Due to the absence of well-equipped laboratory facilities in the DHH Kalaya, PCR and biochemical analyses were carried out at the Kohat University of Science and Technology and Kohat hospital.For molecular analysis, the parasite DNA was extracted from filter papers using the Qiagen DNA extraction kit , following to the manufacturer’s protocol. The Statistical analysis was carried out on SPSS version 19. Means, odds ratios with 95% CIs, and χ2 test of independence were calculated when applicable. Statistica (version 12) was used for box-and-whisker plots. In all the studied parameters, a p value of ≤0.05 was considered statistically significant. Patients with prior comorbid conditions were excluded from relevant subanalyses, for example, diabetes mellitus patients were excluded from hypoglycemia analysis. All analyses were repeated after excluding all patients with associated infections and comorbid illnesses.Ethical approval for project activities was provided by the Kohat University of Science and Technology. Written informed consent was obtained from the patients and their parents/guardians before recruitment.P. vivax and P. falciparum, as well as co-infections of both pathogens in diverse age groups  by P. falciparum, and 10 patients (5%) doubly infected by both P. falciparum and P. vivax (P. vivax infections and 21 (12%) P. falciparum infections, respectively. This method detected 3 (2%) double infections. However, P. ovale and P. malariae infections were not identified in any of the investigated samples by both test methods. Noticeable discrepancies between microscopic (216 patients) and nPCR (178 patients) detections were observed  to be infected by P. vivax . In contobserved . Previouoscopic 2 patientsPlasmodium infection in males (70%). The further clinical and biochemical tests of patients with P. vivax and P. falciparum infections : decompensated shock , anemia , jaundice , multiple convulsions , hypoglycaemia , hyperpyrexia , and hyperparasitaemia . The frequency of complications among all patients who tested positive with malaria (n = 178) were as follows: decompensated shock , anemia , hypoglycaemia , multiple convulsions , hyperpyrexia , jaundice , hyperparasitaemia , and mixed complications  more than one criteria. The most common malarial complication caused by P. vivax and P. falciparum was decompensated shock (p = <0.001). Decompensated shock symptom had the highest odds ratio (OR) for being reported in patients affected by both malarial species , which is significantly greater than that of P. falciparum patients . The mean illness duration was 5.2 ± 2.0 days for P. falciparum male patients and 4.4 ± 0.7 days for females. Similarly, the mean illness duration for P. vivax male patients was 5.5 ± 1.6 days and 6.1 ± 1.3 days for female patients. The incidence of both vivax and falciparum malaria gradually increased between the ages of 1–20 years with increasing age . High childhood malaria parasite exposure resulted in children (1–15 years) bearing the brunt of the disease burden . This result agreed with previous studies performed in nearby areas in Pakistan, as it was shown that the prevalence of P. vivax changes from 70%  in mid Pakistan province to 90%  in areas near or inside Afghanistan including FATA [P. ovale and P. malariae infections in our febrile patient samples. This result also agreed with the fact that these two species had been negligible in this region [P. vivax transmission season peaks between April and September, while the P. falciparum peaks between August and December [P. vivax is the most prevalent in hilly areas, while P. falciparum has the lowest prevalence rate [For the first time, we carried out a reliable epidemiological and clinical study with febrile patients. In this study, 86.5% of the malaria cases were attributed to s region , 21. To nce rate ; similarAnopheles bites. Therefore, there has been the disproportionate number of male malaria patients. On the other hand, during this study, we were intrigued to see that poor socioeconomic conditions together with the lack of public health infrastructure in Orakzai Agency might also be the cause of the disproportionateness. In Talibanized regions, women were often banned from traveling to a hospital without an accompanying male. In addition, in extreme poverty, a family may not afford medical attention to a sick woman. Indeed, comparable rate of malaria infections in both genders in other Pakistani provinces was observed in previous studies [We observed a relationship between patient sex and malarial infections. The predominance of malarial infection in male patients between the ages of 10 and 20 years was observed in our studies as well as in earlier investigations in Pakistan , 23. It  studies , 23.In this study, we also observed children being excessively susceptible to malaria. Approximately half of the vivax malaria patients (n = 54) were children (≤20 years old) identified as having severe illness. This is consistent with other studies of hospitalized children in the South Asia region and in India . This unP. vivax infected males , while anemia induced by P. falciparum is considered more recurrent and more severe than anemia induced by P. vivax. Similar findings of prevalent P. vivax-induced anemia have also been reported previously [P. vivax (n = 12) were found to be with severe hypoglycemia. This result agreed with a previous report which had identified 9% of P. vivax malaria patients as having severe hypoglycemia [In our study, clinical and laboratory analysis identified 80% of malaria patients (n = 142) as having severe illness . Anemia eviously –27. In aglycemia , 29.P. vivax infection were more intricate than that of P. falciparum malaria. P. vivax malaria has reportedly caused tertian ague malaria that rarely led to a severe form [P. vivax malaria [P. vivax [P. vivax-infected patients (n = 6). We also found a similar prevalence of hyperpyrexia  among P. vivax-infected patients to that of the previous study [P. vivax infection was observed with multiple convulsions accounting for 8% of severely ill subjects, but not as frequently (14%) as seen in Bikaner, India [In our study, it is interesting to note that the manifestations of severe malaria instigated by ere form . However malaria –32. JaunP. vivax . In thisus study . Cerebrar, India .P. vivax was very high in our studies. The lofty level of parasitic density of P. vivax as well as that of P. falciparum in some patients may presumably reflect the immune tolerance resulting from repeated exposures. Our study also reports the frequency of decompensated shock . This rate is the highest among similar studies reported previously. We found that decompensated shock was a common and prominent clinical feature of malaria caused both by P. vivax  and P. falciparum . Our result corresponds to the findings which also reported that P. vivax malaria could cause decompensated shock in infected individuals [Interestingly, the parasite count of ividuals , 27.Nonetheless, our study has its own limitations including lack of meticulous refugee statistics. Our study failed to estimate the true number of imported malarial cases in Orakzai Agency. Due to the presence of the armed Taliban insurgents in the Upper Orakzai, we were unable to collect the disaggregated data of Orakzai Agency. However, this study is the first report on the epidemic situation and clinical analysis regarding this most neglected region. Furthermore, in our study, children were the most susceptible population to malaria infection whereas they were the least expected to use satisfactory prevention strategies in such a war-torn deprived region. Local health authorities should initiate malaria awareness programs in schools and malaria-related education should be further promoted at the local level reaching out to both children and parents. We conclude that this overlooked distribution of malaria should be considered by malaria control strategy makers in the world and by the Pakistani government.


HIV and malaria exert co-pathogenic effects. Malaria surveillance data are necessary for public health strategies to reduce the burden of disease in high HIV prevalence settings.This was a longitudinal cohort study to assess the burden of malaria in rural Rakai, Uganda. Households were visited monthly for 1 year to identify confirmed clinical malaria (CCM), or parasitaemia with temperature >37.5 °C, and asymptomatic parasitaemia (AP). Interviews of the adult or child’s caregiver and clinical and laboratory assessments were conducted. Rapid diagnostic testing for malaria and anaemia was performed if participants were febrile and anti-malarial treatment given per Uganda Ministry of Health 2010 guidelines. Blood was drawn at every household visit to assess for parasitaemia, and blood smears were assessed at the Rakai Health Science Programme laboratory.Plasmodium falciparum by smear. 994 (61 %) individuals had at least one positive smear; 342 (21 %) had one positive result, 203 (12 %) had two, 115 (7 %) had three, and 334 (21 %) had >3 positive smears during follow-up. Seasonal rates generally followed the rains and peaked during July, then decreased through November before increasing again.A total of 1640 participants were enrolled, including 975 children aged 6 months up to 10 years, 393 adult caregivers, and 272 adolescent/adult household members from 393 randomly selected households in two representative communities. 1459 (89 %) participants completed all study visits. CCM was identified in 304 (19 %) participants, with the highest incidence rate for CCM of 0.38 per person-year (ppy) identified in children <5 years, and rates decreased with age; the rates were 0.27, 0.16, and 0.09 ppy for ages 5–<10 years, 10–<18 years, and adults 18+ years, respectively. AP was identified in 943 (57 %) participants; the incidence rate was 1.99 ppy for <5 years, 2.72 ppy for 5–<10 years, 2.55 ppy for 10–<18 years, and 0.86 ppy among adults, with 92 % of cases being attributed to Plasmodium falciparum infection remains high in rural Uganda. Increased malaria control interventions should be prioritized.Trial registration Clinicaltrials.gov identifier NCT01265407 Malaria is a leading cause of morbidity and mortality in Uganda, accounting for 25–40 % of all outpatient visits at health facilities, 20 % of hospital admissions, and 9–14 % of inpatient deaths . BecauseRakai district is on a plateau at an altitude ranging between 750 and 900 m and has fair rainfall throughout the year, with relatively dry periods during January and February and from June through August. Peak rainfall varies from year to year, but occurs typically in March/April and October/November . MalariaRCCS household enumeration census data were used to identify households with two or more children between the age of 6 months and 10 years. A statistician from RHSP randomly selected eligible households from the census data. A study nurse approached households on each list sequentially to provide information about the surveillance study to the primary caregiver, and then screened the household for eligibility. The study recruited: the primary caregiver of children from each household; all eligible children aged 6 months up to 10 years; and one additional adult or adolescent participant aged 14 years or older. Due to limited resources the study team was unable to recruit every individual in each household; therefore, one additional adult/adolescent participant was randomly selected among eligible adult/adolescents in the household using the Kish grid method  participants. The unit of enrolment was the household; therefore, an adequate number of households were enumerated from RCCS census data using known household age cohort structure within these Rakai communities in order to approximate this sample size. Malaria prevalence is reported as proportion of subjects identified during the study period with CCM or AP and stratified by subject characteristics, including age, sex and community. A Chi square statistic was used to compare prevalence across age and gender strata. Incidence estimates are reported per person-year with 95 % CI. A kappa statistic was calculated to measure the level of agreement between RDT and blood smear findings. The malaria attributable fraction (MAF) among febrile subjects is also reported along with 95 % CI for these estimates to provide a measure of the proportion of fevers identified during this study period which can be attributed to malaria. All analyses were conducted using SAS software version 9.4. Copyright, SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA.This study is a descriptive, non-comparative study to assess the epidemiology of malaria infection in children aged 6 months–<10 years, and adults living in same households as children. The main objective is to estimate the incidence of asymptomatic and symptomatic malaria in the study population in preparation for future malaria studies in Rakai, including malaria vaccine trials. As data was lacking on malaria incidence in Rakai communities, and on the variance in children aged 6 months–<10 years, sample size calculations were based on a method  that app® rapid haemoglobin test for febrile subjects and malaria rapid diagnostic test  for febrile subjects; thick and thin slide malaria smears were obtained from all subjects by finger- or heel-prick and evaluated in the RHSP laboratory. The malaria “Total Quick” RDT tests for both antigens specific to P. falciparum (HRP2) as well as lactate dehydrogenase, which is an antigen shared by other Plasmodium species that infect humans, including P. falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax. Thick smears were used to identify parasites and quantify parasite count. Thin smears were used for parasite speciation. All slides were read in the RHSP Lab by experienced microscopists who were blinded to the history or signs of clinical malaria. Internal quality assurance of smear reads was conducted by preserving slides and having a separate blinded microscopist reread 10 % of all slides with any discrepant resolved by a third microscopist.Laboratory evaluations included the following at all scheduled surveillance visits: HemocueThe study was approved by the Uganda Virus Research Institute Science and Ethics Committee, the Uganda National Council for Science and Technology, and the National Institute of Allergy and Infectious Diseases Intramural Institutional Review Board. All study participants at least 18 years of age provided written informed consent prior to screening. Parents or guardians of minors provided written consent and children aged 8 years and older provided assent to the research.The primary caregivers in 403 households were visited in their homes and provided information about the study, of which 393 (98 %) agreed to participate. Of the ten households not enrolled, four caregivers refused participation, three caregivers were unable to be contacted, two households had all children away at boarding school, and one household had migrated out of the study area. A total of 1640 participants were enrolled, including 393 (24 %) adult caregivers, 975 (59 %) children <10 years of age, and 272 (17 %) other adult or adolescent members of the households. An average of 2.5 child participants, and an average of 4.2 total participants, were enrolled per household. Table Retention in the annual surveillance study was high with 1459 (89 %) of participants completing the final study visit. Visit adherence was also very high, with only 481 (2.2 %) of 21,618 expected study visits missed; 21,137 (97.8 %) participant visits were completed during the surveillance period. 108 (6.6 %) participants were lost to follow-up, twenty-two (1.3 %) participants withdrew consent, and there were four (0.2 %) deaths. Forty-seven (2.9 %) of participants were terminated due to other circumstances.There were 21,128 blood smears obtained during the study period. CCM was identified in 304 (19 %) participants  cases, with both P. falciparum and P. malariae; and one (0.03 %) case with both P. vivax and P. malariae.There were speciation results for 3267 positive smear readings. ps Table . There wFever (temperature of >37.5 °C) was identified at 467 (2.2 %) visits. 468 rapid diagnostic tests for malaria were performed in febrile participants and 347 (74.2 %) RDT samples were positive. Subsequent confirmation of these results by thick smear showed 242 (52 %) smear-positivity. There were concordant positive results for 218 (47 %) samples, concordant negative results for 113 (24 %) samples, and discordant results for 137 (29 %) samples: eight (2 %) samples were RDT-negative but smear-positive, and 129 (28 %) samples were RDT-positive, but smear-negative. The kappa statistic for level of agreement between RDT and smear result was 0.42 .The malaria attributable fraction among febrile episodes was consistent across age groups: 67 % among young children <5 years, 68 % in children 5–<10 years, 66 % in ages 10–<18 years, and 70 % in adults 18+ years. There was a notable difference between genders that was consistent across all age groups, with males demonstrating a higher fraction of fevers attributable to malaria. The MAF for fever among male participants was 76 %, whereas for females it was 58 %.Prevalence of anaemia was determined for all febrile participants and results are presented in Table Self-reported (or caregiver-reported for children) prevention methods used at baseline are summarized in Table This study describes the relatively high rates of malaria in rural Uganda that also has a high burden of HIV . This isFindings from this study are consistent with reports from the Uganda MIS from 2009 to 2014/2015 , which show a downward trend for prevalence of malaria and anaemia in young children age 0–59 months. The current study from 2011/2012 is in between these MIS time points, and prevalence estimates for CCM in the youngest age cohort likewise fall in between . Prevalence of anaemia follows a similar trend, with 8 % of the youngest age cohort from the current study identified with severe anaemia (hemoglobin < 8 g/dl), compared to 11 and 4 % respectively from the MIS 2009 and 2014/2015; and 46 % of children <5 years from this study identified with any level of anaemia (hemoglobin < 11 g/dl) compared to 63 % form the MIS 2009. Reported use of malaria prevention strategies in the current study is also comparable to the Uganda MIS findings. Overall use of any mosquito bed net (40 %) and ITN (29 %) fall in between an apparent increasing trend reported in MIS .P. falciparum. The identification of P. vivax in East Africa is not unexpected, although this identification was done by smear and not by PCR. The presence of P. vivax in Duffy-negative individuals, as would be expected in East Africa, has been previously described in East Africa [As expected, the predominant species identified by microscopy was t Africa  and in tt Africa . It is aP. falciparum, one (12 %) was positive for P. vivax, and one (12 %) was positive for P. malariae. For the 28 % of RDTs that were positive but smear negative, it is possible that microscopy sensitivity was imperfect, or that the RDTs were picking up circulating antigen or parasites (gametocytes) that existed after patients had been recently treated for infection [The discrepancy between RDT and smear positivity is also not unexpected but is higher than has been observed in other studies , 3: for nfection .A higher degree of AP than CCM was observed in this study. If molecular methods had been employed in this study, we likely would have detected higher AP rates. Indeed, using sensitive molecular methods and targeting interventions to those with AP is important to eradication since it likely fuels the cycle of transmission. Community-wide drug treatment, indoor residual spraying, bed net distribution, and mosquito larviciding are all efforts that have been and could be guided by highly sensitive community screening and intervention efforts .It is likely that children who had febrile illnesses who were RDT negative had infections other than malaria, and this has been increasingly described in the literature recently. In fact, recent studies have shown that with the decreasing incidence of malaria as evidenced by data in this study and the Uganda MIS 2009 and 2014/2015 , etiologPlasmodium falciparum infection remains high in rural Rakai, Uganda. Increased malaria interventions should be implemented to reduce the burden of disease.


The objective of this paper was to describe a malaria mass testing and treatment (MTAT) intervention implemented in six kebeles were selected based on incidence of passively detected Plasmodium falciparum and mixed  malaria cases during the 2013 malaria transmission season. All households in intervention kebeles were targeted; consenting residents received a rapid diagnostic test (RDT) and RDT-positive individuals received artemether-lumefantrine for P. falciparum/mixed infections or chloroquine for P. vivax. Data were collected on MTAT participation, sociodemographic characteristics, malaria risk factors, and RDT positivity.Intervention P. vivax, 0.7 % P. falciparum, 0.3 % mixed), ranging from 0.3 to 5.1 % by kebele; 39.4 % of RDT-positive individuals were febrile, 28.5 % resided in the same household with another RDT-positive individual, 23.0 % were not protected by vector control interventions [mosquito net or indoor residual spray (IRS)], and 7.1 % had travel history. For individuals under 10 years of age, the odds of being RDT-positive was significantly higher for those with fever, recent use of anti-malarial drugs or residing in the same household with another RDT-positive individual; 59.0 % of RDT-positive individuals had at least one of these risk factors. For individuals 10 years of age and older, the odds of being RDT positive was significantly higher for those with reported travel, fever, recent use of anti-malarial drugs, no use of vector control, and those residing in the same household as another RDT-positive individual; 71.2 % of RDT-positive individuals had at least one of these risk factors.Of 9162 households targeted, 7974 (87.0 %) participated in the MTAT. Among the 35,389 residents of these households, 30,712 (86.8 %) received an RDT. RDT-positivity was 1.4 %  contains supplementary material, which is available to authorized users. More than 63 million people live in areas at risk of malaria in Ethiopia and more than 3 million presumed and confirmed cases of malaria were reported in 2013 . While tThe Ethiopian Federal Ministry of Health is striving to achieve substantial progress toward malaria elimination in low transmission areas by 2020 . CurrentPlasmodium falciparum and mixed  malaria is artemether-lumefantrine with a regimen of six doses over 3 days, with multiple pills in each dose depending on weight or age [Population-wide or mass approaches that target infected individuals for treatment with anti-malarial drugs regardless of whether they are symptomatic or present-to-care are a means to clear malaria parasites and reduce malaria transmission in this context . Mass tet or age . Adherent or age , 13. GivPublished literature has reported on MTAT approaches to malaria parasite clearance in Burkina Faso, Zambia and Zanzibar –16, but kebeles (villages) were purposively selected from a sample of 209 kebeles in eight districts in Amhara Region, Ethiopia, that collectively span the diverse ecological-epidemiological context across the region. Each of these kebeles is served by a health post. Health posts are primarily located in rural areas, typically staffed by two health extension workers (HEWs) employed by the Federal Ministry of Health, and serve the surrounding kebele with an approximate average population of 5000 people. Low transmission of both P. falciparum and P. vivax occur in the area. At the health posts in these 209 kebeles, community-based surveillance assistants work closely with HEWs, using mobile phones to upload information about malaria morbidity and commodities to the web-based District Health Information Software (DHIS 2) health information management system on a weekly basis.Six P. falciparum and mixed malaria cases passively detected during the 2013 malaria transmission season, as reported in DHIS 2, was used to stratify kebeles. Strata included: (1) no transmission, with no P. falciparum or mixed malaria cases per 1000 population per week, (2) very low transmission, with >0 and <0.3 cases per 1000 population per week, (3) low transmission and high altitude, with ≥0.3 and <1 case per 1000 population per week and an altitude ≥2000 m, (4) low transmission and low altitude, with ≥0.3 and <1 case per 1000 population per week and an altitude <2000 m and, (5) moderate transmission, with ≥1 case per 1000 population per week.Information on population, altitude and the number of RDT-confirmed kebeles were purposively selected from each of the three higher transmission strata (strata 3–5) based on accessibility, population mobility and the quality of the malaria morbidity data reported in DHIS 2. The entire kebele was targeted to receive the MTAT intervention. Sites included two kebeles in Bahir Dar Zuriya District (Dehina Sositu and Yeginid Lomi) and one kebele each in Mecha District (Berhan Chora), Aneded District (Zengoba), Kalu District (Choresa) and Metema District (Kumer Aftit)  Fig. . The pop in 2014 .Fig. 1Makebeles at the beginning of the 2014 malaria transmission season, from late August through early September. Prior to the MTAT implementation, kebele community leaders and members of the Health Development Army conducted sensitization activities to inform the community about the objectives of the MTAT intervention. All households in the selected kebeles were censused (enumerated and geo-referenced). A total of 91 field teams of two surveillance assistants each went door to door to all households over a three-week period. The number of field teams in a given kebele ranged from 9 to 19, depending on the size and topography of the kebele. Field teams were supervised by staff from the district, zonal and regional health offices and the primary health care unit responsible for each health post.One round of MTAT for malaria was conducted in the six kebeles were targeted with the aim to visit and test with RDTs the entire population older than 6 months of age and to provide anti-malarial treatment to all individuals testing positive. All households were approached by the field teams and invited to participate. The purpose of the intervention was explained and oral informed consent was obtained from all participants (or their parents or guardians if younger than 18 years old) prior to enrollment. At each household, the field team administered household and individual questionnaires and collected information on the geographic coordinates of the household (if not previously censused), the number of household members, mosquito net ownership, receipt of IRS in the previous 12 months, sociodemographic characteristics  and individual risk factors for malaria . Axillary temperature was measured using an electronic thermometer and recorded on the questionnaire. Reasons for non-participation were recorded for individuals who elected not to participate or who were not found at home. If any household members were absent at the time of the MTAT visit, the field team returned to the household up to two more times over the following 1 to 3 days to attempt to find the missing household members and conduct testing and treatment.All households in the selected P. falciparum/P. vivax RDT for malaria. Individuals testing positive were treated with artemether-lumefantrine  or chloroquine (P. vivax) according to national guidelines [Available consenting household members older than 6 months of age were then tested with a CareStart™ Malaria Ag idelines  and the Individuals with signs of serious illness, febrile individuals (axillary temperature ≥37.5 °C or reported fever in the last 24 h) who received anti-malarials in the previous 2 weeks and individuals with a positive RDT result who had treatment contra-indications were referred to the nearest health centre for evaluation and treatment according to national guidelines. Contra-indications to artemether-lumefantrine included first trimester of pregnancy, history of treatment with artemether-lumefantrine or oral quinine in the previous 2 weeks, or hypersensitivity to artemether and/or lumefantrine. Contra-indications to chloroquine included hypersensitivity to chloroquine, history of epilepsy and psoriasis.P. falciparum and mixed malaria who received artemether-lumefantrine were followed up by field teams approximately 3 days after the initial MTAT visit to collect information in a standardized questionnaire on self-reported and observed adherence to treatment and the occurrence of adverse events. Any individuals with artemether-lumefantrine treatment remaining at the time of the follow-up visit were advised to complete treatment according to national guidelines.Participants with Data were collected using smart phones equipped with questionnaires developed using Open Data Kit (ODK) and a custom android application used to navigate to households. Completed questionnaires were either uploaded directly from smart phones to an ODK Aggregate server and data repository in real time or manually downloaded from the smart phones after data collection was complete, depending on internet connectivity.kebele and the proportion of individuals tested in each household reached. Descriptive statistics were used to create a profile of sociodemographic characteristics, malaria risk factors, RDT-positivity, anti-malarial treatment administration, and adherence to treatment for individuals participating in the MTAT.Data were analysed in Stata 13.1 . Coverage of the MTAT intervention was estimated by calculating the proportion of all enumerated households reached in each kebele and all the a priori confounders/risk factors to produce adjusted ORs.Risk factors for RDT-positivity were assessed using multivariate logistic regression. Two models were run separately for under 10 years of age and 10 years of age and older because the definition of the education and occupation variables differs in children and adults. In children it is the mother’s or head of household’s education and/or occupation (only the head of household’s occupation was available) rather than the individual’s education and occupation that are relevant, whereas in older children and adults it is pertinent to include the individual’s education and occupation. Effect modification between age group  and the individuals’ education and occupation were evaluated and a significant interaction supported the a priori plan to run the two models separately. A univariate analysis was first run to estimate the odds ratio (OR) for the association between each variable and RDT-positivity. A multivariate model with RDT-positivity as the dependent variable was then run including all variables that had a significant OR in the univariate analysis, the The study protocol was approved by the Amhara National Regional State Health Bureau Research Ethics Review Committee and received a non-research determination from PATH. Oral informed consent was obtained from all study participants or their parent or guardian if the participant was less than 18 years of age.kebeles, 7974 (87.0 %) participated in the MTAT intervention  were tested for malaria. Among the 4677 individuals who were not tested during the MTAT, the majority (84.4 %) were not present in the household at the time of the MTAT; 6.5 % refused to participate, 8.5 % were younger than 6 months of age, and 0.6 % did not participate for an unknown reason.kebele ranged from 3001 to 7765 , 5.6 % resided in a household with at least one individual with a travel history, and 1.0 % resided in a household with more than one RDT-positive individual.The number of individuals tested in each The 7974 participating households were visited by 91 field teams over 19 days. On average, each field team visited 8.4 households per day. The average household had 4.4 individuals. With 87.0 % of households in the intervention areas reached and 86.8 % of individuals in the households reached receiving an RDT, the effective coverage of the MTAT intervention was 75.5 %. Among the non-participating households, 80.7 % were never visited by the field workers, either because they were missed by the field teams, they were duplicated in the census, or they were visited but the field teams did not fill out a questionnaire with the reason for non-participation. The age and sex distribution of the individuals not found at home at the time of the MTAT was different than that of those found at home. The mean age of those absent was 26.4 years, compared to 22.8 years for those present and the percentage of males was 71.3 % and 48.0 % respectively, with 26.1 % of individuals not found at home being males between the ages of 15 and 29 years, compared to 11.9 % of individuals found at home.P. vivax, 0.7 % P. falciparum and 0.3 % mixed)  were RDT-positive  varied by kebele, ranging from 0.3 to 5.1 %, and by other characteristics  are described in Additional file Factors associated with RDT-positivity for any species for individuals under 10 years of age and individuals 10 years of age and older are described in Tables For individuals 10 years of age or older, individuals who had a travel history, were febrile, took anti-malarial drugs in the previous 2 weeks, or resided in a household with at least one other RDT-positive individual were significantly more likely to be RDT-positive for any species when compared to individuals without these characteristics after adjusting for all other factors Table . SleepinOf the 308 individuals receiving artemether-lumefantrine, 253 (82.1 %) received a follow-up visit. Individuals were followed up an average of 3.6 days (range 1 to 11 days) after being tested. At the time of the follow-up visit, 224 individuals (88.5 %) reported having taken at least the first treatment dose. Treatment completion could only be assessed for the 96 individuals who were followed up more than 3 days after administration of the first treatment dose , of which 41 (42.7 %) reported having completed all doses of the treatment and 21 (21.9 %) were observed to have more than one dose of treatment remaining in the blister pack at the time of the follow-up visit. No individuals who received a follow-up visit reported any adverse event after taking any dose of artemether-lumefantrine.The Ethiopian Federal Ministry of Health is striving to achieve substantial progress toward malaria elimination by 2020 . As new The results show that it is feasible to implement a mass malaria parasite clearance intervention in a large geographic area over a short period of time. However, it is also resource intensive and the two-person field teams required additional people and training above and beyond existing human resources within the health system. The coverage of households  and the coverage of individuals within households  was high (87 % for both). The main reason for not including 13 % of the households in the area was that households were not visited (81 %), with refusals (4 %) and absent households 15 %) representing a much lower percentage. In the households that were visited, the main reason for not including 13 % of the members was that they were absent (84 %), while the rest were refusals (7 %) and exclusions based on age . This resulted in an effective coverage of 75.5 % of all individuals in the targeted geographic areas, which was lower than desired. Thus greater efforts should be made to improve both the households’ and individuals’ coverage, that is visit every single household in the targeted areas and, for households that are visited but have absent members, try to track those individuals when they come back. These coverage estimates are comparable to published estimates of coverage for MTAT interventions implemented elsewhere. During an MTAT campaign in Zanzibar with two rounds of testing and treatment, 64 % of the population was reached during one or both rounds . During  % represkebele, ranging from 0.3 to 5.1 %. However, malaria prevalence was probably underestimated due to an imperfect RDT sensitivity. While RDTs generally have a high sensitivity in clinical settings, their detection limit in the field is around 100 parasites per µl [Malaria infection prevalence as measured by RDTs was low overall (1.4 %) and varied widely by s per µl . Evidencs per µl . In a res per µl . Thus wiSeveral risk factors were associated with significantly higher odds of being RDT-positive. However, only 59 % to 71 % (in under ten and over 10 years of age respectively) of RDT-positive individuals had at least one of those risk factors. While information on the presence of these risk factors could be used to target populations at highest risk of malaria infection, screening for these risk factors would leave many infections undetected. These results also confirm that, even in low transmission areas, more than half (60.6 %) of RDT-positive individuals were asymptomatic. Interventions that are based on fever screening will miss many infections in such a setting . Furtherkebeles, the field teams tested 73 RDT-negative individuals for every RDT-positive individual identified. This ratio ranged from 20 to 401 depending on kebele, suggesting that the efficiency of a mass malaria parasite clearance approach varies widely by geography, even within areas with consistently low rates of malaria. Focal malaria parasite clearance approaches that target specific sub-sets of the population based on proximity to passively detected malaria cases may be an efficient alternative to mass malaria parasite clearance approaches in areas with less malaria transmission [versus focal approaches. However, as the risk factor analysis showed, any focal strategy that selects the sub-population to test based on specific characteristics will leave infections undetected in the population.Across all smission . The freP. falciparum alone; 47.3 % had P. vivax or mixed malaria. Mass and focal malaria parasite clearance approaches that target P. falciparum alone will leave many transmissible P. vivax infections untreated. For Ethiopia to move to malaria elimination, strategies for malaria parasite clearance will require drugs that effectively clear both P. falciparum and P. vivax infections, including clearance of the persistent liver stages in P. vivax-infected individuals.Only 52.7 % of RDT-positive individuals were infected by With population-wide treatment approaches using a multi-day and multi-dose drug, adherence to the full course of treatment is important. Low self-reported and observed treatment completion rates were found among individuals who received the six-dose regimen of artemether-lumefantrine and who were followed up more than 3 days after administration of their first dose. This is consistent with findings from a recent prospective observational study in Ethiopia that found only 38.7 % of patients to be ‘probably adherent’ to artemether-lumefantrine following administration of the drug in a routine care setting . Additiokebeles were purposively selected to represent variation in malaria transmission intensity, altitude, accessibility, and population mobility within Amhara Region. While results from a mass malaria parasite clearance approach in these kebeles provides insight into potential expansion into other parts of Ethiopia, these kebeles are not necessarily representative of Amhara Region or Ethiopia as a whole. Second, the effective coverage was lower than desired, with an estimated 24.5 % of the targeted population not reached, which indicates that greater efforts would be needed to target the hard-to-reach subgroups. Third, household enumeration was done for this study both prior to and during the intervention. The household enumeration was not validated using satellite images or other means and it is possible that some households were enumerated more than once and that some households were never enumerated. Thus the coverage estimates should be interpreted with caution.The study had some limitations. First, the six intervention kebele. Population movement is a driver of malaria transmission for older individuals (10 years of age and older) while intra-household transmission is a driver for individuals of all ages. This suggests the importance of strategies that target migrant workers and other travelers as well as strategies to improve mosquito net usage and IRS coverage. While several factors are significantly associated with a higher odds of RDT-positivity, many RDT-positive individuals do not have these risk factors. Parasite clearance strategies that target populations for testing and treatment based on these risk factors alone are likely to leave many infections undetected.A population-wide malaria MTAT intervention is operationally feasible and can be conducted with high participation of those reached, although additional strategies to cover the hard-to-reach groups would be needed. RDT-positivity is low and varies widely by