Pan troglodytes) communities across sub-Saharan Africa show large geographical variation in malaria parasite (Plasmodium spp.) prevalence. The drivers leading to this apparent spatial heterogeneity may also be temporally dynamic but data on prevalence variation over time are missing for wild great apes. This study aims to fill this fundamental gap.Cross-sectional surveys of chimpanzee  across four non-consecutive sampling periods between 2005 and 2015.Overall, 89 samples (13%) were PCR-positive for malaria parasite DNA. The proportion of positive samples ranged from 0 to 43% per month and 4 to 27% per sampling period. Generalized Linear Mixed Models detected significant seasonal and inter-annual variation, with seasonal increases during the wet seasons and apparently stochastic inter-annual variation. Younger individuals were also significantly more likely to test positive.These results highlight strong temporal fluctuations of malaria parasite detection rates in wild chimpanzees. They suggest that the identification of other drivers of malaria parasite prevalence will require longitudinal approaches and caution against purely cross-sectional studies, which may oversimplify the dynamics of this host-parasite system. Plasmodium spp.) have been shown to infect wild chimpanzees (Pan troglodytes): Plasmodium reichenowi, Plasmodium gaboni and Plasmodium billcollinsi (subgenus Laverania), and the less common Plasmodium vivax-like, Plasmodium ovale-like and Plasmodium malariae-like parasites [Laverania species of chimpanzee malaria parasites, Plasmodium billbrayi, is recognized by some, but not all, authors [Plasmodium spp. have been the focus of several extensive studies conducted in the sub-Saharan African range of chimpanzees [Pan troglodytes schweinfurthii  [Pan troglodytes troglodytes across multiple sites [Six malaria parasite species , longitudinal studies have shown that malaria parasite prevalence often varies significantly between seasons and years [Plasmodium spp. detection rate varied in a wild human-habituated community across four non-consecutive sampling periods.However, before the effect of such factors can be explored, clarifying temporal variation in nd years , 9. Suchnd years –7, 10. TPan troglodytes verus) community at Taï National Park, Côte d’Ivoire. The site experiences two annual wet  and dry seasons , with an annual average rainfall of 1800 mm and temperatures between 24 and 30 °C. In this study, 681 samples collected between 2005 and 2015 from 19 males and 29 females aged 1–52 years old were included. Samples were selected across four non-consecutive sampling periods . On average, 14.2 samples were selected per individual (range 3–37). As pregnancy correlates with increased Plasmodium spp. detection, known pregnant individuals were excluded [Faecal samples were collected from known individuals of one habituated chimpanzee (b gene—first round primers Plasmseq 1f 5′-GGATTTAATGTAATGCCTAGACGTA-3′ and Plasmseq 1r 5′-ATCTAAAACACCATCCACTCCAT-3′ and second round primers Pspcytbf1 5′-TGCCTAGACGTATTCCTGATTATCCAG-3′ and Plasmseq 1r [Plasmodium and Hepatocystis [Plasmodium sp. (N = 2) [DNA extracted from pea-sized faecal samples was tested for malaria parasites following a two-step screening process. First, samples were screened with a nested qPCR targeting a 90-bp fragment of a non-coding region of parasite mitochondrial DNA (mtDNA)—first round primers qPlasm1f 5′-CTGACTTCCTGGCTAAACTTCC-3′ and qPlasm1r 5′-CATGTGATCTAATTACAGAAYAGGA-3′ and second round primers qPlasm2f 5′-AGAAAACCGTCTATATTCATGTTTG-3′ and qPlasm2r 5′-ATAGACCGAACCTTGGACTC-3′ , 12. Possmseq 1r , 12. Thetocystis , 12. As tocystis . Therefotocystis . If positocystis . Sequenctocystis . Samples (N = 2) .Plasmodium spp. detection in faeces, a Generalized Linear Mixed Model (GLMM) [To determine factors possibly affecting l (GLMM) , 17 was l (GLMM)  using R l (GLMM) , 19). Thl (GLMM) . Individl (GLMM) . Age, col (GLMM)  lacking l (GLMM) ). To tesl (GLMM)  was not Plasmodium spp. sequences in this study exhibited 97–100% sequence identity to published sequences. The dominant Plasmodium spp. was P. gaboni , followed by P. billcollinsi , P. reichenowi , P. vivax-like , and P. malariae-like . A single mixed infection  was detected. During the study, 27 individuals (56%) were positive at least once . In 16 of the 17 individuals who tested positive more than once different malaria parasite species were detected.Overall, 89 samples 13%) were positive, with proportions of positive samples ranging from 0 to 43% per month and 4 to 27% per sampling period , specifically showing age and sampling period as significant predictors . Seasonality  was significant  would have led to the erroneous conclusion that this chimpanzee community is malaria-free.Plasmodium falciparum has been observed worldwide [While shown for the first time in chimpanzees, such variations are not without precedent. Longitudinal studies of other host populations  showed both high seasonal and inter-annual variation, sometimes providing evidence for cyclical patterns with periods of 1–13 years , 24–30. orldwide . For exaorldwide . Similarorldwide , 24, 28.orldwide , 8, 24. orldwide , 31, 32.P. falciparum  [P. falciparum and P. vivax exhibited seasonal patterns associated with temperature and monthly rainfall [Within a year, detection rates followed seasonal patterns: peaking 3 months after the start of the wet seasons and reaching their lowest levels at the end of dry seasons or beginning of wet seasons. Such seasonal increases in prevalence have been observed for most malaria parasites, including the closest relative of the chimpanzee-adapted Laveranian parasites,  humans) . In Bangrainfall . This marainfall , 24, 25.rainfall , 9, 27, Temporal variation also highlights the complexity of malaria parasite epidemiology in wild chimpanzee communities. This is partly due to the inherently complicated parasite life cycle and further complicated by fluctuating chimpanzee demography, community structure and behaviour . For exaP. falciparum system.This study shows strong temporal fluctuations of malaria parasite prevalence in wild chimpanzees. These findings call for longitudinal approaches to further characterize this host-parasite system and contribute to stress the resemblance with the human-


The hypotheses put forward to explain the malaria transmission cycle in extra-Amazonian Brazil, an area of very low malaria incidence, are based on either a zoonotic scenario involving simian malaria, or a scenario in which asymptomatic carriers play an important role.Plasmodium spp. DNA and its role in residual malaria transmission in a non-Amazonian region of Brazil.To determine the incidence of asymptomatic infection by detecting Upon the report of the first malaria case in 2010 in the Atlantic Forest region of the state of Espírito Santo, inhabitants within a 2 km radius were invited to participate in a follow-up study. After providing signed informed consent forms, inhabitants filled out a questionnaire and gave blood samples for PCR, and thick and thin smears. Follow-up visits were performed every 3 months over a 21 month period, when new samples were collected and information was updated.Plasmodium vivax, one for Plasmodium malariae and one for both P. vivax and P. malariae, corresponding to a prevalence of 3.4% (2.3% for each species). During follow-up, four new PCR-positive cases (two for each species) were recorded, corresponding to an incidence of 2.5 infections per 100 person-years or 1.25 infections per 100 person-years for each species. A mathematical transmission model was applied, using a low frequency of human carriers and the vector density in the region, and calculated based on previous studies in the same locality whose results were subjected to a linear regression. This analysis suggests that the transmission chain is unlikely to be based solely on human carriers, regardless of whether they are symptomatic or not.Ninety-two individuals were initially included for follow-up. At the first collection, all of them were clearly asymptomatic. One individual was positive for The low incidence of cases and the low frequency of asymptomatic malaria carriers investigated make it unlikely that the transmission chain in the region is based solely on human hosts, as cases are isolated one from another by hundreds of kilometers and frequently by long periods of time, reinforcing instead the hypothesis of zoonotic transmission.The online version of this article (10.1186/s12936-018-2263-z) contains supplementary material, which is available to authorized users. With an estimated 212 million new cases in 2015, leading to 429,000 deaths, malaria is an infectious disease which has an enormous global economic and social impact , 4 involPlasmodium, the anopheline mosquito vectors, and the susceptible or infected humans. However, this transmission chain has several complexities that can compromise the effectiveness of control measures. With the development of molecular biology techniques, the number of Plasmodium species known to be able to cause infections in humans has increased from four in the oldest references, to six in the most recent, with the recent split of Plasmodium ovale into two different species [Plasmodium knowlesi, was originally identified as the species that caused simian malaria in Southeast Asia, but is now known to also play an important role in the disease in humans living in the same region [P. knowlesi, other species may act as parasites in humans and non-human primates, forming a zoonotic transmission chain. This makes it necessary to develop more complex measures to either eliminate the disease or to at least impede its spread, as the elimination of diseases with a zoonotic cycle might only be attainable at an unacceptably high cost to the environment.Malaria control strategies target the various components of the transmission chain: the protozoans of the genus  species –9. One oe region –12. LikeAnother factor that makes the malaria transmission chain more complex is the role played by asymptomatic carriers. These are known to exist ubiquitously, and their frequency varies according to the degree of endemicity –32. TheyPlasmodium vivax and Plasmodium malariae [Kerteszia, which breeds in the axils of bromeliads, and therefore has a very close relationship with the Atlantic Forest ecosystem [While more than 99% of malaria cases in Brazil are restricted to the Amazon region , residuamalariae . Howevercosystem , 33–37. cosystem , 38–47. Plasmodium spp. DNA in the blood samples of 48 out of 1527 asymptomatic inhabitants of the region, corresponding to a prevalence of 3.1% [Plasmodium carriers are asymptomatic were considered.A previous study in the malaria transmission area in the state of Espírito Santo detected  of 3.1% . In the P. vivax in 48 out of 51 symptomatic individuals by both microscopic examination of blood films and polymerase chain reaction (PCR). Plasmodium malariae was identified by PCR in one sample, while in two samples blood smear tests revealed parasites morphologically similar to P. vivax, but with a negative PCR result. The cases were detected in nine municipalities located between 19.6° and 20.6° south latitude and 40.6° and 41° west longitude [The state of Espírito Santo is the Brazilian state with the most cases of residual malaria in the Atlantic Forest . The disongitude  Fig. 1)P. vivax Quarterly assessments of inhabitants in the region where the first malaria case detected in 2010 occurred were scheduled, resulting in a total of eight assessments over 21 months. Before the first assessment the participants signed a voluntary informed-consent form. During each assessment, a questionnaire was filled out covering demographic data and any instances since the previous collection of febrile illnesses, as well as thick-smear tests for malaria and trips outside the area where the participant lived. A total of 5 mL of blood was then collected in Vacutainer tubes containing EDTA, thick and thin peripheral blood smears were prepared, and abdominal palpation for splenic enlargement was performed.The blood collected was centrifuged at 300 g for 10 min in the Protozoology Laboratory at the Tropical Medicine Unit, Federal University of Espírito Santo, to separate the plasma from the red blood cells. The latter were then aliquoted and stored at -20 °C in Eppendorf tubes, which were sent to the Protozoology Laboratory at the São Paulo Institute of Tropical Medicine, where DNA was extracted and PCR was performed.Thick and thin blood smears were prepared following the method recommended by the World Health Organization . The smePlasmodium 18S RNA gene subunit. The first step was carried out in a final volume of 20 μL containing 0.8 μL of each primer, P1UP and P2 (10 μM), 0.25 μL of dNTP mix , 2 μL of 10 × PCR buffer, 1 μL of MgCl2 (50 mM), 0.16 μL of Platinum Taq DNA polymerase-Invitrogen (5 U/μL) and 5 μL of DNA. The amplification was run in an Applied Biosystems thermocycler with the following programme: 92 °C for 2 min, 35 cycles of 92 °C for 30 s and 60 °C for 90 s, and one cycle of 60 °C for 5 min.Genomic DNA was extracted from the samples with a NucleoSpin Tissue purification kit (Macherey–Nagel) following the manufacturer’s instructions, and amplified following Win et al. . BrieflyP. vivax, F2—Plasmodium falciparum or M1—P. malariae).The product from the first step was diluted at a ratio of 1:50 in sterile water and used in the second step with the P1 (genus–specific) primer and one of the three reverse species-specific primers , 0.5 μL of dNTP , 2 μL of 10 × PCR buffer, 0.16 μL of Taq DNA polymerase (5 U/μL), and 2 μL of the diluted final product from the first step. The second amplification was carried out using the same equipment as the first, and the cycling parameters were 92 °C for 2 min, followed by 18 cycles of 92 °C for 30 s and 60 °C for 1 min, and one cycle of 60 °C for 5 min. The amplified products of the second step correspond to species–specific fragments of about 100 bp.P1UP (F): 5′ TCC ATT AAT CAA GAA CGA AAG TTA AG 3′P2 (R): 5′ GAA CCC AAA GAC TTT GAT TTC TCA T 3′P1 (F): 5′ ACG ATC AGA TAC CGT CGT AAT CTT 3′V1 (R): 5′ CAA TCT AAG AAT AAA CTC CGA AGA GAA A 3′F2 (R): 5′ CAA TCT AAA AGT CAC CTC GAA AGA TG 3′M1 (R): 5′ GGA AGC TAT CTA AAA GAA ACA CTC ATA T 3′The primers used were:P. vivax malaria patients treated at the São Paulo Superintendency for the Control of Endemic Diseases (SUCEN), from P. falciparum cultures, and from blood smears positive for P. malariae provided by the Centers for Disease Control and Prevention (CDC) was used as a control.The amplified product was run on 2% agarose gel at 80 V for 40 min. The gel was stained using ethidium bromide, and the bands were visualized under a UV transilluminator. DNA extracted from peripheral blood of 0) for the mathematical malaria transmission model proposed by Anderson and May [0 is calculated using the following equation:Based on entomological and demographic information for the study area, and transmission parameters available in the literature, the basic reproductive rate , student , and housewife . The remaining occupations were self-employed (two), cook, maid (two), transportation supervisor, plasterer, timber worker, minor (three), member of the armed forces, driver, machine operator, agricultural producer, teacher, and electronics technician.Thirty-two individuals had lived in the area where the assessments were carried out for less than 5 years. Of these, eleven came from other areas in the same municipality, nine from malaria-free municipalities in the Atlantic Forest, five from other states, and two from other municipalities with malaria in the Atlantic Forest. No information was available for the remaining five. None of those who said they had come from other states were from the Amazon region, and none of the individuals had travelled to the Amazon region during or before the study period.P. vivax.Three individuals reported having had malaria before, one in 2002 and two in 2004. The diagnosis was based on the result of a thick blood smear test, and the parasites were morphologically consistent with Thirteen individuals reported having had fever episodes in the previous 2 years. Of these, two said that the fever had lasted more than 3 days . The patient who reported a 7-day fever was the only individual to report having had a fever on two separate occasions, the second lasting 3 days. None of these individuals were investigated for malaria by the local health services when they had fever.Ten individuals reported having had fever episodes between the first and second assessments; of these, three said that the fever had lasted more than 4 days . A thick blood smear for the individual who had had a fever for 14 days was negative when examined by local health staff. The number of individuals who reported having had a fever between subsequent assessments was two, three, four, six, three, and zero for each of the assessments, respectively. Malaria was not diagnosed at any of the assessments, and the febrile episodes improved spontaneously. Three individuals reported prolonged febrile episodes, one between the fourth and fifth assessments, lasting 21 days, one between the fifth and sixth assessments, lasting 15 days, and one between the sixth and seventh assessments, lasting 30 days.P. vivax, one for P. malariae and one for P. vivax and P. malariae, corresponding to a prevalence of 3.4% in the population sample (2.3% for each species). During follow-up, four new PCR-positive cases  were recorded, corresponding to an incidence of 2.5 infections per 100 person-years or 1.25 infections per 100 person-years for each species in the population sample analysed . A prevalence of this magnitude is similar to that reported by Cerutti et al. [In this follow-up of a cohort of individuals living in an extra-Amazonian area of Brazil endemic for malaria, the initial prevalence of asymptomatic i et al.  in 1527 i et al. –65.Plasmodium carriers remained positive in more than three blood collections, and all those positive for Plasmodium turned negative spontaneously, with the exception of a single carrier who initially tested positive for P. vivax/P. malariae, but in subsequent collections only tested positive for P. malariae. All four individuals who were positive only after the first collection subsequently tested negative for Plasmodium DNA.Throughout the follow-up, only three asymptomatic P. malariae DNA between the first collection and the end of the study period, with the exception of two collections in which the PCR was negative. The parasites that cause quartan malaria, such as P. malariae, appear to be the best adapted to their hosts, who can have a chronic infection for decades without symptoms [P. vivax [Individual no. 33 remained positive for symptoms . These iP. vivax .P. vivax. One of these, individual no. 84, who reported a previous episode of malaria in 2004 and was treated in accordance with Ministry of Health recommendations, was negative in the first assessment in this study, and positive for P. malariae DNA in subsequent assessments.Three of the individuals enrolled in the study reported having had malaria more than 5 years previously, when it was diagnosed based on the result of a thick blood smear that showed parasites morphologically consistent with P. vivax, the probability of remaining infected would have decreased as time passed, along with his capacity of remaining infective. Even if his infection had persisted for 6 months, the mathematical model would have still been applicable and the prediction would have remained valid.Although not probable, it is possible that the infection could have persisted in individual no. 84, producing gametocytes that could help to maintain the transmission cycle. Alves et al.  cited unP. malariae instead of P. vivax when diagnosed by the local health system in the first place. As infections by P. malariae can persist much longer than those by P. vivax [P. malariae are much less frequent than those by P. vivax in the Atlantic Forest system of Espírito Santo, being less important from an epidemiological point of view. Even such an unlikely scenario would not have interfered with the mathematical prediction of the present study, which, incidentally, was constructed taking P. vivax into consideration.There is also the possibility that individual no. 84 was in fact infected by P. vivax , the DNAP. vivax , infectiOf the thirteen individuals who presented with fever during the study period, none had splenomegaly, and only one individual who had a prolonged episode of fever was tested using a blood smear when he was seen by a local physician, with a negative result. In all the followed-up patients who had fever at some time, the condition resolved itself spontaneously without the use of antimalarial medication.Plasmodium DNA and the small sample size, that the study area is indeed endemic for malaria, albeit at a very low level.Since none of the individuals migrated to areas outside the Atlantic Forest, particularly the Amazon region, at any time before or during the study, it is clear, despite the small number of individuals with samples positive for 0 (0.337) and the fact that, in theory, R0 < 1 indicates that the number of infected individuals is decreasing in the study population, it is reasonable to suggest that the persistence of endemic malaria in the study area cannot be explained only by the presence of asymptomatic infected individuals but would require an additional reservoir, such as non-human primates. This explanation would currently appear to be plausible as a monkey found in the wild in the same region was positive for P. vivax DNA [In light of the value of Rivax DNA .P. vivax DNA was amplified in samples of thoraces of anopheline specimens of the subgenus Nyssorhynchus  from the study area, raising the possibility that this subgenus might be involved in malaria transmission in the Atlantic Forest. However, the authors conclude that the low endemicity of malaria is in itself evidence that these species do not participate in the transmission of the disease, as the low parasitaemia would make infection of anophelines with limited vector capacity and competence improbable. The absence of more competent vectors, such as Anopheles darlingi, in the study area leads to the conclusion that if the subgenus Nyssorhynchus is involved in the transmission cycle, its role is a secondary one, i.e. that of an incidental vector. Since it is possible that monkeys could be acting as reservoirs, a mathematical model that can take this into account is required. Development of an accurate model would in turn require further studies of the monkeys in the region.In a study by Rezende et al.  using PCPlasmodium knowlesi malaria has been shown to be a zoonosis. Initially believed to cause only simian malaria, P. knowlesi has been responsible for continued endemic human malaria in regions where there has been a clear decrease in infections by P. falciparum and P. vivax, and has come to represent an obstacle to malaria control efforts in regions of Southeast Asia, particularly Malaysia [Plasmodium simium can occur in humans in Brazil, although only incidentally, and that An. cruzii is abundant in the Atlantic Forest region of the state of Espírito Santo [An. cruzii is considered the probable vector in this region and prefers to remain in the tree canopy feeding on non-human primates, malaria is probably being transmitted as a zoonosis [Plasmodium reservoirs in areas with residual malaria was also suggested by Duarte et al. in a study in which they investigated the prevalence of antibodies against circumsporozoite protein and asexual forms of P. vivax, P. malariae and P. falciparum in monkeys in the Atlantic Forest in the state of São Paulo [An. cruzii females in Juquitiba in the state of São Paulo, argued against transmission by zoonosis. Specifically, they found only human blood in 26 engorged female mosquitoes and failed to find blood from monkeys, other mammals or animals of any other species in any of the specimens [An. cruzii specimens were collected only from the peridomestic environment, rather than also from the forest environment, the habit of mosquitoes to feed on the closest host has to be taken into account. Collections at the forest edge, the true habitat of the monkeys, might have shown that An. cruzii also feeds on these non-human primates. In a recent study in a Yanomami community in the Venezuelan Amazon, 12 of 33 samples PCR-positive for P. malariae had 18S gene sequences identical to those in a Plasmodium brasilianum strain isolated from an infected monkey in French Guiana. As P. brasilianum is morphologically similar and, apart from a handful of mutations, almost genetically identical to P. malariae, the authors speculate that they are the same species. They note the ease with which plasmodia can be exchanged between humans and monkeys, and warn that there is a lack of host specificity for quartan malaria caused by P. brasilianum, which they consider a true zoonosis [P. simium is a variant of P. vivax, with minimal differences in a few molecular markers [P. vivax and P. simium, as well as P. malariae and P. brasilianum, are known to be derived from each other, questions remain as to the direction in which the transfer took place, whether from humans to monkeys or vice versa [Malaysia . In thisMalaysia . Deane (to Santo . The diszoonosis , 37. Theão Paulo . Their fão Paulo , 46. Kirpecimens . The moszoonosis . Similar markers , 46, 71.ce versa .P. vivax, the technique used is able to detect as few as 0.12 parasites/µl [The main limitation of the present study are the characteristics of the sample, which, although large enough to allow new cases to be identified, was too small to allow the detection of possible factors associated with the risk of getting infected. As the sampling of the participants was a random process, despite being triggered by the occurrence of a symptomatic case, some infections could have gone unnoticed. However, the similarity between the prevalence found in this study and that evidenced by a much larger sample in the study of Cerutti et al.  indicatesites/µl , ensurinPlasmodium DNA at the first collection and positive at one or more subsequent collections were counted only once. Furthermore, the infection was considered to be the same when an individual had a sample that was positive for the same species of Plasmodium in a subsequent assessment. In these circumstances, the fact that genotyping was not performed could be considered a limitation, as the possibility of a new infection cannot be eliminated.To calculate the incidence, individuals who were negative for In spite of these limitations, the results presented here clearly indicate a need to improve our understanding of the transmission cycle in these low-endemicity areas. Specifically, genotypic studies with both human and simian hosts, as well as human and simian vectors have to be undertaken in order to fully elucidate the role each plays in the transmission cycle. Of particular note here is the finding based on our mathematical model, namely that the local transmission cycle cannot be maintained by human-mosquito-human interactions alone.Plasmodium spp. in an area endemic for malaria in the Atlantic Forest in Brazil. These results complement previous observations of a low frequency of symptomatic infections [The present study detected a low frequency of asymptomatic carriers of fections , and lowfections , 37. TheAdditional file 1. Additional material.


Plasmodium falciparum infection among school-aged children and to undertake a model-based spatial prediction of P. falciparum infection risk using environmental predictors.In Côte d’Ivoire, malaria remains a major public health issue, and thus a priority to be tackled. The aim of this study was to identify spatially explicit indicators of Plasmodium species-specific infection and parasitaemia using Giemsa-stained thick and thin blood films. Household socioeconomic status was assessed through asset ownership and household characteristics. Children were interviewed for preventive measures against malaria. Environmental data were gathered from satellite images and digitized maps. A Bayesian geostatistical stochastic search variable selection procedure was employed to identify factors related to P. falciparum infection risk. Bayesian geostatistical logistic regression models were used to map the spatial distribution of P. falciparum infection and to predict the infection prevalence at non-sampled locations via Bayesian kriging.A cross-sectional survey was conducted, including parasitological examinations and interviews with more than 5,000 children from 93 schools across Côte d’Ivoire. A finger-prick blood sample was obtained from each child to determine P. falciparum was the predominant species (94.5 %). The Bayesian geostatistical variable selection procedure identified land cover and socioeconomic status as important predictors for infection risk with P. falciparum. Model-based prediction identified high P. falciparum infection risk in the north, central-east, south-east, west and south-west of Côte d’Ivoire. Low-risk areas were found in the south-eastern area close to Abidjan and the south-central and west-central part of the country.Complete data sets were available from 5,322 children aged 5–16 years across Côte d’Ivoire. P. falciparum infection risk and related uncertainty estimates for school-aged children in Côte d’Ivoire represent the most up-to-date malaria risk maps. These tools can be used for spatial targeting of malaria control interventions.The The online version of this article (doi:10.1186/s13071-016-1775-z) contains supplementary material, which is available to authorized users. Malaria is a vector-borne disease that is widespread in sub-Saharan Africa. In 2015, an estimated 188 million malaria cases and 395,000 deaths occurred in Africa .For the implementation of best-practice control strategies and intervention planning, in-depth knowledge of spatial characteristics and factors that influence malaria is needed. Predictive risk mapping has proven to be an important tool for malaria control , 8. In pPlasmodium falciparum prevalence in excess of 60 % [In Côte d’Ivoire, a country highly endemic for malaria, the national malaria prevention and control policy is based fundamentally on the use of long lasting insecticidal nets (LLINs), intermittent preventive treatment with sulfadoxine-pyrimethamine (IPT-SP) and environmental sanitation. The control strategy also includes prompt diagnosis and treatment with artemisinin-based combination therapy (ACT) . No spec of 60 % , 31.P. falciparum prevalence data obtained from the first national school-based survey on parasitic infections in Côte d’Ivoire [P. falciparum infection risk. Environmental factors that govern the spatial distribution of P. falciparum were used to produce a model-based high spatial resolution P. falciparum risk map for Côte d’Ivoire.The purpose of this study was to spatially analyse d’Ivoire –32. A BaCôte d’Ivoire has a warm and humid climate. The average temperature ranges between 25 °C and 32 °C. There are three main seasons; warm and dry from November to March, hot and dry from March to May and hot and wet from June to October. A dense tropical moist forest covers the south-western part of the country; in the middle part of Côte d’Ivoire, a Guinean forest-savannah mosaic belt extends from east to west and a Sudanian savannah covers the northern part.A country-wide cross-sectional survey using the lattice plus close pairs sampling approach was designed, as described elsewhere –33. In sP. falciparum infection status, two drops of blood from finger prick samples were collected from enrolled children and thick and thin blood films were prepared on microscope slides. The slides were air-dried and transferred to nearby laboratories where they were stained with Giemsa and examined under a microscope by experienced laboratory technicians for Plasmodium species identification and parasitaemia. The number of parasitized blood cells were counted by assuming a standard white blood cell count of 8,000 per 1 μl of blood. Ten percent of the slides were randomly selected for quality control.To determine A pre-tested questionnaire was administered to all children participating in the survey. The questionnaire included information on household asset ownership , clinical symptoms  and recent history of diseases  . ChildreData were double-entered and cross-checked in EpiInfo version 6 .Environmental data were obtained from satellite imagery for the year 2011. The sources and the properties of these data are summarised in Table Children were grouped into two age categories: (i) 5–10 years and (ii) 11–16 years. Distance from school to the nearest health facility was obtained from the “Programme National de Santé Scolaire et Universitaire” (PNSSU) and was summarised into three categories, i.e. (i) < 1 km, (ii) 1–5 km, and (iii) > 5 km. The first category included schools located in villages or towns with a health facility. For assessment of socioeconomic status, an asset-based approach was used to stratify children into five socioeconomic groups .P. falciparum infection [Bayesian geostatistical stochastic search variable selection (SSVS) was performed to explore all possible models within a geostatistical framework and select the most important predictors for nfection , 36. In nfection ). DetailP. falciparum infection risk across Côte d’Ivoire (variable selection 1). In a second step, variables at individual level about prevention, treatment, distance from school to the nearest health facility and socioeconomic status, which were not available at 1×1 km spatial resolution, were also explored for selection in order to assess their effect on P. falciparum infection risk (variable selection 2). The possible nonlinear relationships of variables were taken into account by considering the inclusion of categorical predictors in the model. We built both selection procedures to enable the model to choose the best functional form of each predictor, i.e. categorical or linear. With the exception of age, and distance from school to the health facilities that were categorised, predictors were introduced in both functional forms in the model for the variables, which could be expressed either as categorical or linear. The variables and their functional form included in the final models were the ones with posterior probability of inclusion over 50 % (median probability model).A first variable selection that included demographic (age and sex) and environmental variables was performed to build a predictive model of the P. falciparum via MCMC simulation algorithms for estimation of model parameters. Let Yij be the P. falciparum infection status for child j  in school i . We assumed that Yij arises from a Bernoulli distribution with probability pij such as, Yij ~ Be (pij). We modelled covariates Xij and school-specific spatial random effects φi on the logit scale, i.e. logit (pij) = Xijβ + φi, where β represents the vector of regression coefficients, including a constant. Spatial random effects were assumed to follow a multivariate normal prior distribution, φ ~ MVN . The variance-covariance matrix Σ introduced spatial dependency through an isotropic exponential correlation function of distance between locations as follows: Σkl = σ2 exp (−ρdkl), where dkl is the Euclidian distance between a pair of schools k and l, the variance σ2 measures the spatial geographic variability and ρ is a parameter that controls the rate of correlation decay. The range, defined as the minimum distance at which spatial correlation between locations is below 5 %, is calculated as 3/ρ. To complete model specification, prior distributions were assigned to model parameters. For the regression coefficients, non-informative normal prior distributions were chosen such as β ~ N  where β = T. For the variance and the correlation decay, inverse gamma and gamma distributions were respectively assumed, i.e. σ2 ~ IG  and ρ ~ G . Model prediction was done using Bayesian kriging [Geostatistical logistic regression models within a Bayesian framework of inference were performed to analyse the risk of infection with  kriging . We asseP. falciparum infection risk and the observed prevalence. Model uncertainty was assessed by the sum of the standard deviations (SDs) of the predictive distributions.To validate our model, a geostatistical logistic regression model was fitted on a sub-dataset of 73 randomly selected schools . We then predicted the risk at the remaining schools and compared our predictions with the observed prevalence data. Model predictive ability was assessed by calculating the mean absolute error (MAE), which is the mean of the absolute differences between the median of the predicted Geostatistical variable selection and model fit were implemented in OpenBUGS v.3.2.2 . Fortran 95  was used for prediction. STATA version 10  was used for data management and preliminary analysis. ArcGIS version 10.0  was used to display study results on maps.P. malariae and P. ovale were very low; 3.7 % and 0.3 %, respectively. P. falciparum was the predominant species with an overall observed prevalence of 69.2 %. All subsequent analyses focussed on P. falciparum only. The spatial distribution of the observed P. falciparum infection prevalence is shown on map A in Fig. Complete data records were available from 5,322 children aged 5–16 years in 93 schools. Of note, one of the sampled schools refused to participate. The prevalences of r < 0.9). Hence, all of them were considered for selection as potential predictors of malaria risk. Median probability models with their posterior probability, as well as posterior inclusion probability of each predictor for both variable selection procedures are shown in Table We assessed potential correlation between predictors in a preliminary analysis, but none were considered as highly correlated, since the absolute value of the Pearson’s correlation coefficient never exceeded 0.9 : 1.95, 95 % Bayesian credible interval (BCI): 1.23–3.03); forest/savannah OR: 2.30, 95 % BCI: 1.43–3.81). The spatial range was 285 km (95 % BCI: 139–477 km), indicating important residual spatial correlation. Model validation showed that the model predicts a random sample of 20 % of the data with a MAE of 0.11 and a sum of SD of the posterior predictive distribution of 1.81.Estimates of model parameters and model validation results for the Bayesian geostatistical logistic regression model with land cover as predictor are summarised in Table P. falciparum infection; the wealthier the household, the lower the risk for P. falciparum infection. A substantial residual spatial correlation of 259 km (95 % BCI: 123–457 km) was estimated.Estimates of model parameters for the Bayesian geostatistical logistic regression with socioeconomic status are also presented in Table P. falciparum infection prevalence among school-aged children in Côte d’Ivoire. Lowest prevalences (around 45 %) were estimated for small urban aggregated areas in the south-east of the country, close to Abidjan, central-southern and central-western parts. Prevalences above 70 % were found in the north, central-east, south-east, west and south-west of Côte d’Ivoire. Map C in Fig. P. falciparum infection prevalence. High prediction errors were mostly found from central-west to central-east of the country.Map B in Fig. P. falciparum infection prevalence and (ii) produce a smooth risk map of P. falciparum infection among school-aged children for Côte d’Ivoire. To our knowledge, this spatial analysis is the first at the national level based on P. falciparum data collected within a few weeks during the dry season in late 2011/early 2012. The results obtained from this spatially explicit analysis are useful for current and future malaria control efforts in Côte d’Ivoire.The purpose of this study was to (i) identify sociodemographic, environmental and disease prevention indicators associated with Among the environmental covariates, only land cover was selected by the geostatistical variable selection procedure. Precipitation, temperature and distance to freshwater bodies - factors that have previously been associated to malaria risk in Côte d’Ivoire , 22 - weP. falciparum compared to those from urban areas. On one hand, this result suggests that the endemicity of malaria in Côte d’Ivoire is linked to two main vegetation types, namely the forest in the south and the savannah in the north, which basically do characterise the two main ecozones of the country. In contrast, other studies using land cover showed that the forest area was associated with a decrease of malaria incidence [The spatial model with land cover as covariate indicated that school-aged children living in areas with forest/savannah and croplands are at higher risk of ncidence  or risk ncidence . It is iP. falciparum infection risk. Wealthier households were associated with a low risk of P. falciparum infection and is line with results from other studies [P. falciparum infection and parasitaemia are positively associated with low socioeconomic status.Interestingly, the second geostatistical variable selection that included environmental variables, prevention and distance from school to the nearest health facility suggested that only socioeconomic status, as assessed by the wealth index, explained  studies , 41, 42. studies , 31, 43.P. falciparum (> 70 %) were found in the entire north and the south-west (Taï forest region) of Côte d’Ivoire. This is in contrast with lower prevalences obtained from a previous spatial analysis using historical data focussing on children aged < 16 years [P. falciparum prevalence rates in these areas. However, differences in survey designs and large heterogeneities of historical data must be considered to deepen the understanding of potential changes in P. falciparum prevalence and parasitaemia in space and time [In the present study employing recent epidemiological data, very high prevalences of 16 years . Recent 16 years  and prog16 years , 46, whiand time . The desP. falciparum infection among school-aged children across Côte d’Ivoire. Given the high burden of P. falciparum infections in the school-aged population, there is a need for intervention strategies that also target this age group. Notwithstanding, since the end of 2014, the national malaria control programme in Côte d’Ivoire is making LLINs available to the entire population, including school-aged children, thus an important step is taken to tackle the malaria burden in this specific age group. The produced smooth P. falciparum prediction map, in conjunction with uncertainty estimates, represent useful tools for scaling up current and future malaria control interventions. Future predictive risk profiling should include other factors such as population density and more detailed information on intervention coverage in order to better understand the impact of ongoing malaria interventions.This study provided a comprehensive analysis on the spatial distribution of


Measurements of anti-malarial antibodies are increasingly used as a proxy of transmission intensity. Most serological surveys are based on the use of cross-sectional data that, when age-stratified, approximates historical patterns of transmission within a population. Comparatively few studies leverage longitudinal data to explicitly relate individual infection events with subsequent antibody responses.Plasmodium falciparum asexual stage antigens (MSP-1 and AMA-1) was examined using three annual measurements of 691 individuals from a cohort of individuals in a malaria-endemic area of rural east-central Tanzania. Mixed-effect logistic regression models were employed to determine factors associated with changes in serostatus over time.The occurrence of seroconversion and seroreversion events for two While the expected population-level relationship between seroprevalence and disease incidence was observed, on an individual level the relationship between individual infections and the antibody response was complex. MSP-1 antibody responses were more dynamic in response to the occurrence and resolution of infection events than AMA-1, while the latter was more correlated with consecutive infections. The MSP-1 antibody response to an observed infection seemed to decay faster over time than the corresponding AMA-1 response. Surprisingly, there was no evidence of an age effect on the occurrence of a conversion or reversion event.While the population-level results concur with previously published sero-epidemiological surveys, the individual-level results highlight the more complex relationship between detected infections and antibody dynamics than can be analysed using cross-sectional data. The longitudinal analysis of serological data may provide a powerful tool for teasing apart the complex relationship between infection events and the corresponding immune response, thereby improving the ability to rapidly assess the success or failure of malaria control programmes. Malaria imposes a severe public health burden in tropical and sub-tropical regions, particularly sub-Saharan Africa . QuantifPlasmodium falciparum apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-119) recombinant proteins, both of which have been proposed as malaria vaccine candidates [Anti-malarial antibody responses not only contribute to mediating protection from the disease, but also represent a marker of individual, and population, exposure history. Since antibodies persist longer in the human body than the parasite itself , serological markers may represent more robust and sensitive measurements of transmission intensity than entomological or parasitological markers –6. The undidates .Serology has been used as a tool to measure transmission intensity and monitor changes as far back as the 1950s. Nearly all examples of this approach employ cross-sectional serological surveys coupled with serocatalytic models to estimate the average seroconversion and seroreversion rates in the study population , 14. SerThere have been comparatively few longitudinal assessments of malaria serologic data –21, eachThere is a relative lack of longitudinal data aimed at elucidating medium or long-term trends in antibody response to malaria in a large study population. Such insights are required to improve the use of serological outcomes in monitoring malaria transmission within large-scale control programmes. Current approaches have been validated to infer longitudinal trends in malaria transmission based on cross-sectional data but nuances in transmission dynamics may be missed , 7, 8. TA longitudinal, cluster-randomized study was conducted in 24 randomly selected villages in Mvomero district in rural east-central Tanzania from 2011 to 2013. In a two-stage design, villages were enrolled as clusters and households randomly sampled from within each village; six study villages were assigned to each of four groups . A cohort of 5385 people from 962 households (approximately 40 households per village) were enrolled and sampled in three consecutive years during the long rainy season, for a total of 16,155 possible measurements. The first survey was conducted in March and April 2011, with follow-up conducted in the same months of 2012 and 2013. A detailed description of the study design can be found in Kramer et al. .This analysis was restricted to the sub-set of individuals for whom parasitological and serological outcomes, for either AMA-1 or MSP-1, were available at all three time points; namely, those with complete-case data. There were 681 individuals with complete-case data for AMA-1, and 686 individuals with complete-case data for MSP-1. There were five individuals with complete information for AMA-1 data who were missing at least one MSP-1 measurement, and there were ten individuals with complete MSP-1 data who were missing at least one AMA-1 measurement. As a consequence, there were a total of 691 individuals in the current evaluation. The age ranges, and other relevant characteristics, of these individuals are shown in Table At each annual data collection round, participants provided a finger-prick blood sample from which a dried blood spot was stored and a malaria smear was prepared. Blood spots were stored with desiccant at −20 °C prior to and after shipping to the London School of Hygiene and Tropical Medicine, with laboratory analyses conducted on all samples at the conclusion of the study. Thick and thin blood smears were stained with Giemsa solution and examined with a binocular microscope with an oil immersion lens to quantify the parasitaemia. Parasitaemia was measured by counting the number of asexual parasites against the number of leukocytes in the blood film, based on a count of 8000 leukocytes per microlitre. The number of asexual parasites was counted against 200 leukocytes using a hand tally counter. A slide was considered negative if no malaria parasite was observed in at least 200 oil-immersion fields.P. falciparum apical membrane antigen 1  and merozoite surface protein 1  recombinant proteins, which were produced as described [Antibodies to malaria antigens were detected from serum samples of study participants using indirect enzyme-linked immunosorbent assays (ELISA). The technique is described fully in Stewart et al. . Brieflyescribed , 25. AllTwo different classes of models were fitted: one using the continuous antibody titre as the outcome, and another using observed (binary) seroconversion or seroreversion events as the outcome. Separate models of each class were fitted for AMA-1 and MSP-1 . Observed seroconversion events were defined as when an individual who was seronegative in one survey year became seropositive in the following year; observed seroreversion events were defined as when an individual who was seropositive in one survey year become seronegative in the following year. As such, with data from three consecutive years, it is only possible to identify these events in the final 2 years (2012 and 2013). Serostatus  was determined by designating a titre threshold, above which a sample was deemed to be seropositive, and below which was deemed to be seronegative. For each antigen, this threshold was determined by fitting a two-component normal mixture model to the estimated titer distribution; the threshold was calculated as the mean of the ‘seronegative’ (i.e. lower titer) component plus three standard deviations .The continuous antibody titre models were fitted as normal mixed-effects models  with natc) statistic [c = 0.85, RCC c = 0.84; for MSP-1: spline c = 0.72, RCC c = 0.72). In practice, after implementing the lasso approach for each outcome and for both antibodies, all two-way interactions were dropped from each of the six final models. All analyses were performed using SAS/STAT software, version 9.4 of the SAS System for Windows. Copyright © 2013 SAS Institute Inc., Cary, NC, USA.In both classes of models, lasso variable selection was used to find the most parsimonious model . The foltatistic  comparedThe characteristics of the 691 individuals in the analysis sample are shown by survey year in Table Individuals were classified by observed malaria infection ‘trajectory’-in other words, their malaria infection status across time in each survey year; the characteristics of individuals within each trajectory group are shown in Table Table Individual log-titre values over time within each serostatus trajectory for both antibodies are shown in Fig. The distribution of log-titre by serostatus as a function of age within each survey year is shown in Fig. reversion to the alternate antigen, on the other hand, was associated with significantly reduced odds of seroconversion to the outcome antigen : the odds of seroconverting for AMA-1 was multiplied by 0.25 (95% CI 0.13–0.50) when a seroreversion was observed for MSP-1 compared to those who did not experience a seroreversion. The odds ratio of seroconverting for MSP-1 when a seroreversion for AMA-1 was observed in the same year was 0.50 (95% CI 0.28–0.90). Being seropositive in both years for MSP-1 did not impact the odds of AMA-1 seroconversion  compared to being seronegative in both years for MSP-1. However, for those who were being positive for AMA-1 in both years, the odds of MSP-1 seroconversion was multiplied by 1.97 (95% CI 1.10–3.51), compared to being negative in both years.Individuals who were seronegative in one year and seropositive the following year  were defined as having seroconverted. Those who seroconverted were compared to those who remained seronegative in a mixed-effects logistic regression. Table more likely to MSP-1 seroconvert than individuals with no observed malaria infections in either year . Individuals who did not have a malaria infection in the concurrent year but did in the preceding year were significantly less likely to MSP-1 seroconvert . Having a malaria infection in both years was not associated with any difference in the odds of MSP-1 seroconversion , compared to individuals with no observed infections.In the AMA-1 model, neither concurrent  or preceding year  malaria infection status (at the time of sampling) was a significant predictor of seroconversion, compared to individuals without an infection in either year. However, individuals who had a malaria infection in both years had 2.77 (95% CI 1.11–6.89) times higher odds of seroconverting compared to individuals without an infection in either year. By contrast, in the MSP-1 model, those who did not have an observed malaria infection in the preceding year but did have one in the concurrent year were significantly Using a similar principle as for seroconversion, a seroreversion event was defined as when an individual who was seropositive in a given year (2011 or 2012) was seronegative in the following year (2012 or 2013). Those who seroreverted were compared to those who remained seropositive in a mixed-effects logistic regression. Table Having a malaria infection in the current, previous or both years was not significantly associated with AMA-1 seroreversion compared to individuals without any observed infections. For MSP-1, having an observed malaria infection in the previous year but not the current year was associated with higher odds of seroreverting  compared to individuals without an infection in either year, but the effect was not statistically significant. Neither concurrent malaria infections  nor having a malaria infection in both years  were significantly related to the odds of MSP-1 seroreversion, again compared with individuals with no observed infections.concurrent year were correlated: a one unit increase in MSP-1 titre was associated with a 0.21 (95% CI 0.18–0.25) increase in AMA-1 titre, and a one-unit increase in AMA-1 titre was associated with a 0.53 (95% CI 0.45–0.61) unit increase in MSP-1 titre. There was an inverse relationship between outcome antibody titre and titre for the alternate antibody in the previous year. Log AMA-1 titre decreased by 0.11 (95% CI 0.07–0.14) for each one-log increase in MSP-1 titre in the previous year. A similar relationship is observed for MSP-1 titre, which decreased by 0.34 (95% CI 0.25–0.42) for each one log increase in AMA-1 titre in the previous year. The major difference between the two antibodies is the direction of the effect for observed malaria infections in the preceding year. For AMA-1, an observed malaria infection in the preceding year, but not the concurrent year, was significantly associated with higher log-titre . The opposite effect was observed for MSP-1 titre; malaria infection in the preceding year was associated with lower log-titre in the concurrent year .The continuous titre models were fitted as mixed-effects models with the natural log-transformed titre values for each antibody as the outcome. Table This study describes antibody responses to malaria antigens in samples collected over a 3-year period from a cohort in a malaria-endemic population. The profiles of these responses over time were more dynamic than anticipated: seroreversion events were just as common as seroconversion events . Further, the number of events changed dramatically between subsequent surveys, with a 300% increase in the total number of seroconversion events between 2011 and 12 and 2012 and 13 and a corresponding 70% reduction in the total number of seroreversion events over the same time period, which parallels changes in infection prevalence estimates between the surveys. In contrast, studies using cross-sectional data often assume static rates over time, especially for seroreversion , 15. AltDespite the well-studied relationship between age and population-level seroprevalence that forms the basis of most cross-sectional models, such as the reversible catalytic conversion and superinfection models , 10, 30,As expected, on a population level, the occurrence of seroconversion and seroreversion events tracked with changes in the number of observed infection events, with higher incidence of malaria associated with a higher number of seroconversions for both antibodies , 20, 33.increase in the observed number of malaria infections. This suggests the possibility that there was a change in transmission intensity not directly observable through looking at prevalence alone. The apparent relationship between seroreversion and exposure implies that changes in seroreversion rates  may be a useful monitoring tool for understanding changes in exposure, particularly in the context of a highly effective prevention intervention. Further, a cross-sectional approach using standard serocatalytic models is ill suited to estimating seroreversion rates [The present results differ from some of the common assumptions made by cross-sectional analyses; further analysis of longitudinal serologic data may reveal more complexities that cross-sectional methods are ill suited to fully capture. For example, between 2011 and 2012, the number of observed seroreversion events for both antibodies were markedly higher than the number of seroconversion events, despite a slight on rates , 34, andThe results from the continuous titre models support the inferences from the seroevent-based models. The increased odds of a seroconversion event, and decreased odds of a seroreversion event, between the 2012 and 2013 survey years coincides with both an increase in the average log-titre levels for both antibodies from 2012 to 2013 and a concomitant increase in the number of observed infections. It was also observed that if an individual’s log-titre increases for one antibody, it will, on average, increase for the other during the same time frame. This reflects the same phenomenon whereby if an individual seroconverts for one antibody they are more likely to seroconvert for the other. Similarly, the negative coefficient for alternate antibody in the previous year suggests that if an individual seroreverts for one antibody they are less likely to seroconvert for the other. For both AMA-1 and MSP-1, changes in serostatus were correlated, but maintaining serostatus for one antigen did not affect the odds of seroconversion or seroreversion for the other.previous malaria infections were associated with significantly increased log-titre for AMA-1, but significantly decreased log-titre for MSP-1. Consecutive infection statuses were associated with significantly increased log-titre for AMA-1, but did not impact average log-titre levels for MSP-1. This would seem to imply that, while antibody titre is elevated for both during an infection, the response is preserved over a longer time frame for AMA-1 than for MSP-1, which may begin to clear more rapidly following the resolution of the infection. This would account for the differential effects of previous year infection status and consecutive infection statuses on outcomes between the AMA-1 models and the MSP-1 models. Serological and parasite based analysis may be operating on different temporal scales, something that is reflected in both the time required to mount a response and the persistence of antibodies following infection. More work remains to be done to refine the statistical methods necessary to most efficiently leverage continuous serologic data. However, this study demonstrates that the serostatus-based and titre-based models can produce complementary results in a longitudinal framework.Concurrent malaria infections were associated with increased average log-titre levels for both antibodies. However, The stimulation of these antibodies as a response to known infection events is not well understood, and is further complicated by boosting due to repeated infections . While sOne strength of the present study is that the continuous titre models have the outcome antibody titre in the previous year as a predictor of that antibody titre in the current year. The magnitude of that coefficient gives an estimate of the degree to which an antibody response is maintained on a year-to-year basis, controlling for both infection status at point of sampling and titre for the alternate antibody. This would seem to further support the notion that the MSP-1 response decays at a faster rate than AMA-1, since the degree to which elevated titre in the previous year is associated with current titre is reduced. Although this would contrast with the findings of Akpogheneta et al. , who didThere are several important limitations to this study. The sub-set of individuals included in the analysis  represents a biased sample from the overall Mvomero study population: e.g., there were more children under five and adult women used in this analysis compared to the overall sample. However, that these characteristics were not associated with any of our outcomes mitigates concerns about the influence of selection bias on the interpretation of the results . FurtherThe classification of individuals as seropositive and seronegative makes a number of assumptions regarding the distribution of antibody titre in the population and how it relates to discrete biological states . AlthougFinally, the present study relies on serological and parasitological measurements taken a year apart during the March–April rainy season. It is possible that this does not give a granular enough picture of individual infection or titre trajectories over time to make positive determinations about the way these trajectories are shaped by infection events. Undoubtedly, many infection events went unobserved between the sampling time-points; in addition, submicroscopic infections, which may potentially make up a large proportion of all malaria infections , would nThe potential of serologic data in malaria surveillance in a variety of transmission settings has long been acknowledged . SeveralLongitudinal methods, on the other hand, can model time directly; the indicator variable for survey year in the models used in these analyses may act as a direct measurement of year-to-year fluctuations in exposure across the study population. Longitudinal surveillance of events is likely to be more sensitive than cross-sectional methods, particularly to short-term changes in malaria exposure. The present study demonstrates that longitudinal serostatus and titre changes track with other metrics of transmission , while also offering additional insight not possible through prevalence or cross-sectional methods alone . The simultaneous modelling of multiple antigens with different response dynamics offers a novel method for potentially shedding light on the timing of changes in exposure. Further, this approach highlights the potential of the use of the continuous variable of antibody titres in repeated sampling for determining the efficacy of a programme.


Podoconiosis is a neglected tropical disease characterised by a slowly progressive swelling of the foot and lower leg. It is prevalent among subsistence barefoot farmers who live and work in highland areas of the tropics. This study was conducted in Wayu Tuka ‘woreda’ (district), western Ethiopia to determine the prevalence of podoconiosis and assess factors associated with acute adenolymphangitis (ALA) episodes.A two phase, community-based cross-sectional study was conducted between January and March 2015. First, all households in the district were surveyed to determine the prevalence of podoconiosis. This was followed by a second phase in which 366 people with podoconiosis from four randomly selected ‘kebeles’ (subdistricts) were assessed for clinical features of the disease, shoe-wearing habits, personal hygiene, social stigma and functional impairment. Data entered into Epi DATA were then exported to SPSS. Logistic regression analysis was conducted to identify factors associated with ALA.Prevalence of podoconiosis in the population was 3.05% (1197/39 256) (95% CI 2.9% to 3.2%). The prevalence was significantly higher among women than men (3.67% vs 2.4%). Most (92.2%) people with podoconiosis were in the economically active age group (15–64 years) in the first phase survey. Of participants in the second phase of the study, 43% had stage 2 disease and 38.1% had ‘moss’-like skin changes. On average, people with podoconiosis had 23.3 episodes of ALA/year and each person with podoconiosis lost 149.5 days of activity/year. Never walking barefoot and daily foot washing were both associated with decreased odds of ALA .A relatively high prevalence of podoconiosis, frequent ALA episodes and considerable decreases in daily activities were identified in this district. Footwear use and daily foot hygiene were associated with decreased odds of ALA. We recommend prevention and morbidity management interventions to address this developmental challenge. The study used a complete census of all podoconiosis cases in one district. It provides a profile of people with podoconiosis in the district, which is important for rational deployment of limited resources towards prevention and treatment of the disease.A limitation of the study is case identification—this was not supported by serological tests to exclude lymphatic filariasis.A second limitation was that self-report of acute lymphangioadenitis was not validated in any way.Podoconiosis is a disabling and stigmatising neglected tropical disease (NTD) which affects the lower limb. It is caused by long-standing exposure to red clay soil of volcanic origin.3Podoconiosis can be distinguished from filarial elephantiasis through history and clinical examination: it develops first in the foot, it causes bilateral but asymmetric swelling often confined to the lower leg and groin involvement is rare. In contrast, the swelling of lymphatic filariasis is commonly found above the knee and often involves the groin. Another common differential diagnosis is leprosy lymphoedema, but podoconiosis can be distinguished from this because sensation persists in the toes and forefoot, and trophic ulcers, thickened nerves and hand involvement are not found.4Acute adenolymphangitis (ALA) is a common and disabling complication of podoconiosis lymphoedema, yet has been very little investigated until now. According to studies conducted in northern and western Ethiopia, patients with podoconiosis experienced on average five episodes of ALA per year and up to 90 days per year incapacitated by ALA.7Prevalence of podoconiosis is high in many highland parts of Africa: Cameroon,10Recently, podoconiosis and other NTDs have been receiving attention in Ethiopia.The study was conducted in Wayu Tuka ‘woreda’  in east Wollega Zone, Oromia Region, western Ethiopia. It is located 324 km from the capital Addis Ababa at an altitude of 1700–2200 m above sea level and has an average annual rainfall of 2400 mm. The population of Wayu Tuka ‘woreda’ is estimated at 75 970, living in 15 930 households,10.1136/bmjopen-2016-012308.supp1supplementary fileA two phase cross-sectional community-based study was conducted. In the first phase, all the households in the ‘woreda’ were surveyed to determine the prevalence of podoconiosis and to identify peoples with podoconiosis for the second phase study. In the second phase, 366 people with podoconiosis from four randomly selected ‘kebeles’  were approached to assess clinical features of the disease, habits of shoe-wearing, personal hygiene social stigma due to the disease and functional impairment.All individuals who were willing to participate were included, however, in the second phase; people with podoconiosis with mental health problems precluding them answering questions with substantial recall were excluded.Thirty level 4-holder nurses employed as health extension workers and two Bachelor of Science (BSc) nurse supervisors were recruited in the first phase survey, while 10 health extension workers and two BSc nurse supervisors were recruited for the second phase survey. The health extension workers were responsible for house-to-house enumeration of podoconiosis cases and the interviewing and physical examination of people with podoconiosis. The nurses were responsible for supervising the activities of the health extension workers. In households where there was more than one person with podoconiosis, all of them were invited to the interview and physical examination including measurement of leg circumference. All field staff were trained for 1 day before carrying out data collection. The questionnaire included sections from earlier studies on podoconiosis  age of people with podoconiosis was 43.1±13.3 years. Slightly more than 9 in 10  belonged to the economically active age group. The majority (95%) lived in rural areas and were reliant on subsistence farming .Prevalence of podoconiosis in the population was 3.05% . The prevalence was significantly higher among women than men (3.67% vs2.40%). Prevalence disaggregated by age and sex is shown in A total of 366 people with podoconiosis were approached for the detailed study in four randomly selected ‘kebeles’. These participants consisted of 134 (36.6%) males and 232 (63.4%) females. On average, the respondents had lived in the study area for 38.64±18.24 years. The majority of the respondents (337 (92%)) belonged to the economically active age group. More than 75% had no formal education, and the majority  of the study participants were farmers .People with podoconiosis in the four randomly selected ‘kebeles’ were categorised into five clinical stages according to pre-existing clinical staging criteria.ALA is the major cause of morbidity in people with podoconiosis. The majority  of people with podoconiosis had experienced ALA in the year before the survey. The most recent episode of ALA had occurred an average of 2.97 weeks before the study. On average, people with podoconiosis experienced 23.3±14.4 ALA episodes/year and, on average, were bed bound for 6.42±5.39 days, meaning that, on average, each person with podoconiosis lost 149.5 days of activity per year. Nearly half  of the people with podoconiosis had ALA at the time the interview was conducted. More than one-third (38.4%) of people with podoconiosis reported that ALA episodes were worse during the rainy season. The major trigger of an ALA episode was prolonged walking . Many  had retired to bed without treatment at least once in the past year as a coping mechanism. During episodes of ALA, 40% of people with podoconiosis had sought treatment at a podoconiosis treatment clinic while 9.5% had not sought treatment of any kind .In multivariate analysis, never walking barefoot and daily foot washing were associated with lower odds of ALA. People with podoconiosis who never walked barefoot had one-quarter the odds of ALA as those who walked barefoot at times . People with podoconiosis who washed their feet daily had one-twelfth the odds of ALA as those who did not . Variables such as patient's age, clinical stage of disease, mossy appearance and presence of wound were associated in the bivariate analysis, but these associations did not persist after multiple logistic regressions .Approximately one in three  people with podoconiosis mentioned that they had experienced one or more forms of social stigma at school, church, within marriage, at the market place or at feasts. Stigma took the form of being excluded, shunned, pointed or gestured at and not having products bought at market.The experience and attitudes of respondents towards footwear and personal hygiene were assessed. The majority  of people with podoconiosis had no problem finding enough water and took an average of 22 min to reach a water source. The majority  of people with podoconiosis washed their feet at least once per day (mean 1.4±0.086), and 79 (21.4%) washed their feet with soap daily. Almost all  had washed their feet on the night before the interview was conducted. Foot washing behaviour did not change in 97 (26.2%) people with podoconiosis after their leg started to swell. The mean age at first shoes wearing was 25.94±13.83 (range 4–95) years. During the interview, 162 (43.8%) were wearing closed plastic shoes, while 32 (8.6%) were barefoot. A small but important subset  had never worn shoes.Burden assessment using a household census enables the number of people with podoconiosis within a district to be determined, and provides information on their distribution within the district. Such information is important for planning and implementing podoconiosis interventions in endemic districts. Our study identified that the prevalence of podoconiosis in Wayu Tuka ‘woreda’ in 2015 was 3.05%. The prevalence was higher among females than males and increased with age. The male to female ratio was 1:1.58 and the economically active age group (15–64 years of age) was heavily affected. ALA episodes were a major cause of morbidity, and patients' ability to work was impaired by ALA episodes. Patients also experienced significant stigma in a range of settings. The habits of never walking barefoot and daily foot hygiene were both independently associated with reduced odds of ALA.At 3.05% (95% CI 2.9% to 3.2%), the prevalence of podoconiosis in those aged 15 years and older in Wayu Tuka ‘woreda’ was slightly more than that reported from Gulliso ‘woreda’ in west Wollega, 2.8%,21Male to female sex ratio was 1:1.58, which is consistent with several recent studies conducted in other areas, including the national study,5Episodes of ALA had been experienced in the past year by 88.8% of patients, a slightly lower proportion than that found in Gulliso ‘woreda’ (97%). Some patients linked ALA with ‘chagino’ . ALA was not significantly associated with sex or age at onset of illness, consistent with the study in west Wollega.10One in four patients (25%) experienced stigma in this study. In contrast, a study conducted in northern Ethiopia showed that about 13% of patients had experienced one or more forms of social stigma at school, church or in the market place.24Most patients could reach a water source in an average 22 min walking round trip. Another study in western Ethiopia also claimed that patients could access water with a walk of ‘only’ 20 min;This study used a large scale community-based house-to-house survey in all ‘kebeles’ of Wayu Tuka ‘woreda’, followed by a detailed study of patients with podoconiosis. This provides a complete profile of people with podoconiosis in the district. Nonetheless, the study is not without limitations. A clinical algorithm was used to differentiate podoconiosis from other disease causing leg swelling in such areas. Had a serological test been available, it would have helped exclude lymphatic filariasis, the most common phenocopy in tropical areas. However, given that the study area is at 2200 m above sea level, transmission of filaria is extremely unlikely.We recommend strong prevention and morbidity management interventions to address this developmental challenge. These interventions will require collaboration between local government and non-government stakeholders, and integration with existing programmes addressing water and sanitation, neglected tropical diseases and chronic diseases.


Plasmodium in humans. Misdiagnosis is a major challenge in endemic areas because of other disease complications and technical expertise of the medical laboratory staff. Microscopic method using Giemsa-stained blood film has been the mainstay of diagnosis of malaria. However, since 1993 when rapid diagnostic test (RDT) kits were introduced, they have proved to be effective in the diagnosis of malaria. This study was aimed at comparing the accuracy of microscopy and RDTs in the diagnosis of malaria using nested PCR as the reference standard. Four hundred and twenty (420) venous blood specimens were collected from patients attending different General Hospitals in Ebonyi State with clinical symptoms of malaria. The samples were tested with Giemsa-stained microscopy and three RDTs. Fifty specimens were randomly selected for molecular analysis.Malaria is a debilitating disease with high morbidity and mortality in Africa, commonly caused by different species of the genus Plasmodium falciparum; there was co-infection of P. falciparum with Plasmodium malariae and Plasmodium ovale. The sensitivity and specificity of microscopy was 50.00 and 70.59% while that of the RDTs were (25.00 and 85.29%), (25.00 and 94.12%) and (68.75 and 52.94%) for Carestart, SD Bioline PF and SD Bioline PF/PV, respectively. Cohen’s kappa coefficient was used to measure the level of agreement of the methods with nested PCR. Microscopy showed a moderate measure of agreement (k = 0.491), Carestart showed a good measure of agreement (k = 0.611), SD Bioline PF showed a fair measure of agreement (k = 0.226) while SD Bioline PF/PV showed a poor measure of agreement (k = 0.172).Using different diagnostic methods, the prevalence of malaria among the subjects studied was 25.95% as detected by microscopy, prevalence found among the RDTs was 22.90, 15.20 and 54.80% for Carestart, SD Bioline PF and SD Bioline PF/PV, respectively. Molecular assay yielded a prevalence of 32%. The major specie identified was P. ovale in the study area necessitates an expanded molecular epidemiology of malaria within the study area.This study recommends that the policy of malaria diagnosis be changed such that the routine diagnosis of malaria is done by a combination of both microscopy and a RDT kit of high sensitivity and specificity so as to complement the errors associated with either of the methods. The finding of  Malaria is the most common cause of outpatient visits in all age categories in Nigeria and it is the major cause of morbidity and mortality in all parts of Nigeria . It is tPlasmodium lactate dehydrogenase (pLDH), or aldolase, which are usually produced during the erythrocytic cycle [The diagnosis of malaria requires careful clinical examination and laboratory investigation . The micic cycle . At presic cycle . The sucic cycle .Molecular tools are highly sensitive in detecting low levels of infections and accurately detecting species of malaria parasites . Many ofPlasmodium species is essential for the reduction of malaria and for epidemiological studies. The World Health Organization (WHO) has recommended microscopy as the gold standard for the diagnosis of malaria. However, the detection of malaria parasites using microscopy requires intensive training of medical laboratory scientists because the result is subjective and requires expertise. It has a very low sensitivity when performed by poorly trained personnel in endemic areas, especially in primary and secondary healthcare facilities [The accurate diagnosis of infections by cilities , 12. ThiThis comparative study on malaria diagnostic methods is aimed at providing an evidence-based criteria on diagnostic options in malaria endemic areas, when critical decision concerning treatment is required that will provide a high level of specificity and sensitivity.This study was carried out in Ebonyi State, Southeastern Nigeria. Samples were collected from the general hospitals in Ebonyi State. It is a double-blind clinical diagnostic assay of malaria using microscopy and three RDT kits and comparing their results against that of nested PCR, as the gold standard.Simple random sampling technique was used to select the subjects recruited for the study. The study populations comprised of individuals who had shown clinical signs of malaria and in whom test for malaria parasite have been requested for by clinicians and who assented to participate in the study. The subject population included persons of different age groups such as children, adults and pregnant women.Peripheral blood samples were collected by finger prick and by venepuncture. The finger prick samples were collected with sterile lancet after cleaning the finger with 70% alcohol. Three drops of blood from the finger were used to impregnate dry blood spot (DBS) papers . A total of six DBS spots were made on each DBS paper for each specimen. These specimens were preserved for molecular analysis. 2 ml of venous blood specimens were also collected by venepuncture using sterile syringe and needles. The specimens were dispensed into EDTA containers and immediately used to make the thin and thick films as well as the RDT tests.The subjects were screened for malaria parasites using Giemsa-stained malaria microscopy, three RDT kits, among the specimens collected, a total of fifty (50) were randomly selected for nested polymerase chain reaction.Microscopy was performed to detect malaria parasites and the species were identified based on morphological features. Thick and thin film of the peripheral blood specimens were made immediately after collection on a clean, grease-free microscope slide and allowed to air dry on the laboratory bench. The films were stained with 10% Giemsa solution  for 30 min, allowed to air dry and subsequently examined using oil immersion objective lens and all the fields were examined before declaring a slide negative. For each specimen, the thick films were examined first for detection of malaria parasites, the thin films of each specimen were subsequently examined for speciation only in those specimens in which malaria parasites were identified in the thick film.The slides were examined by two malaria microscopists who are certified by the WHO. Each microscopist examined each specimen independently and results were recorded as being positive when both microscopists recorded a positive result.Three different commercially available RDT kits for malaria parasites having different sensitivity and specificity were used to detect malaria parasites in the blood samples. These are; Carestart™  with Batch Numbers MO2K2, SD Bioline PF  with Batch Numbers 082352 and SD Bioline PF/PV  with Batch Numbers 05AD14038. The tests were performed by strictly adhering to the manufacturers’ instructions.The test device was removed from the foil pouch and placed on a flat, dry surface. 5 µl of whole blood was collected with the capillary pipette provided and transferred to the round sample well. Four drops of the assay diluents was added vertically into the square assay diluent well. Results were read after 15 min over a bright light.A dry blood spot was made from each of the 50 selected blood samples on a DBS paper  for PCR assay. Three drops of the blood from finger prick were used to impregnate 3 mm DBS papers  and allowed to air dry on a paper rack. Each filter paper specimen was stored in a plastic bag at room temperature with a desiccator and transported to the laboratory for molecular assay . MoleculPlasmodium genus, in the second reaction the genes for Plasmodium species were identified using methods described by Snounou et al. [Molecular analysis using nested PCR technique based on amplification of 18s rRNA genes as described by Fuehrer et al.  was usedu et al. .DNA extraction was done using the Saponin-Chelex extraction method. Saponin (0.5%) in phosphate buffered saline (PBS) was prepared by dissolving 1 g of saponin and one tablet of PBS  in 200 ml of sterile distilled water, shaken vigorously and allowed to stand till the saponin and PBS dissolved completely. Each of the dried blood spot was cut out from the DBS papers with sterilized scissors with serrated edges and each of the discs were transferred to pre-labelled Eppendorf tubes. 1 ml of the saponin-PBS solution was added to the specimen in the Eppendorf tubes and allowed to stand for 24 h at 4 °C for complete haemolysis.The specimens were washed three times in PBS with intermittent centrifugation at 13,000 rpm for 5 min after each wash. Subsequently, 1 ml of PBS was added to the sediment, and then it was vortexed for 1 min and incubated for 30 min at 4 °C. It was centrifuged again for 5 min at 13,000 rpm and the supernatant decanted.Fifty microlitres of freshly prepared Chelex solution was added to the tubes followed by 150 μl of distilled water to obtain 5% of the Chelex solution. It was then vortexed and incubated in a block heater set at 99 °C for 20 min, centrifuged at 13,000 rpm for 5 min and the supernatants decanted into newly labelled Eppendorf tubes. The supernatants contain the DNA.DNA yield was measured using a nanodrop spectrophotometer  based on maximum absorbance of DNA at 260 nm. 1μl of the DNA sample was applied on the platform of the nanodrop spectrophotometer and a reading was taken after adjustment of absorbance to zero using water as blank. The yield was measured in ng/μl. The 260/280 nm ratio was obtained to give an analysis of the purity of the sample and the concentration of the extracted DNA was also found.Taq polymerase . A species-specific region of the 18 rDNA of Plasmodium was amplified (All the primers used were synthesized by Inqaba biotec S.A). In the first reaction, 10 μl volume equivalent of extracted DNA was added, using pairs of primers targeting an outer region specific to the Plasmodium genus. One microliter of the first reaction product was then used as a template in a second nested reaction to yield specific Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale products.PCRs were performed according to Snounou et al.  nested-pThe temperature profile for the PCR was: 5 min at 95 °C; 25 cycles of 1 min at 94 °C, 2 min at 58 °C, 2 min at 72 °C; followed by the second reaction where the annealing temperature was modified to 65 °C and the cycles repeated 30 times.P. falciparum, P. malariae and P. ovale was detected. In every set of reactions, negative (PBS) and positive controls for P. falciparum, P. malariae and P. vivax (3D7 Pf) were used. The primary reaction yielded a 500 bp PCR product.The PCR products were visualized under UV light on 2% agarose gel after electrophoresis in 0.5X Tris borate EDTA buffer and ethidium bromide staining. A sample was considered positive if a 205 or 125 or 105 bp products for T test. Cohen’s kappa coefficient was used to compare the measure of agreements between microscopy and the RDTs versus nested PCR results as the reference standard. All statistical analysis were calculated at 95% level of significance. The sensitivity, specificity, positive predictive value and negative predictive value were calculated using the formulae presented below. Data analysis was performed with the aid of statistical programme for social sciences (SPSS) version 18.0Data was analysed using descriptive statistics, Cohen’s kappa coefficient and student’s Key: TP is true positive, FN is false negative, TN is true negative, FN is false negative.P. falciparum. Among the subjects studied, 96 (22.9%), 64 (15.2%) and 230 (54.8%) were positive by Carestart, SD Bioline PF and SD Bioline PF/PV respectively  subjects were enlisted for this study. The subjects comprised of 255 females (60.70%) and 165 males (39.30%), having an age range of 2–65 years with a mean age of 34 years. Malaria parasite was detected in 109 (25.95%) of the subjects by microscopy and all the species detected by microscopy were observed to be Based on the results obtained from microscopy, males had a prevalence of 25.45% (42 of 165) while females had a prevalence of 26.27%  cases while P. ovale was present in one (1 of 16) subject. Both P. malariae and P. ovale were present as co-infection with P. falciparum in all the subjects they were found.Among the specimens that were subjected to molecular analysis, 16 (32.0%) were positive while 34 (68.0%) were negative for Cohen’s kappa coefficient was used to compare the agreement between microscopy and rapid diagnostic tests using PCR results as the gold standard. This study found that when compared between microscopy and PCR, there was moderate agreement (k = 0.491). Among the RDT kits, Carestart showed good agreement with PCR (k = 0.611), SD Bioline PF showed fair agreement (k = 0.226) while SD Bioline PF/PV showed poor agreement (k = 0.172).When the agreements between the RDTs and microscopy were compared, Carestart and microscopy had a fair agreement (k = 0.233), whereas SD Bioline PF and SD Bioline PF/PV had poor agreements with microscopy (k = 0.192) and (k = 0.085), respectively.When the agreements between the RDTs were compared, SD Bioline PF and SD Bioline PF/PV had poor agreement (k = 0.169), Carestart and SD Bioline PF/PV had fair agreement (k = 0.257) while Carestart and SD Bioline PF had a moderate agreement (k = 0.465).Molecular methods have generally been accepted to offer excellent sensitivity and specificity and are considered as reference standard for diagnosis of malaria infection . This stThis study observed generally reduced malaria prevalence in the different methods employed. This finding is consistent with that of other studies –19. It iThe low prevalence of malaria among under five children and pregnant women observed in this study is an indication of the positive results of malaria control and prevention programmes, such as free treatment of malaria for these population groups and administration of anti-malarial drugs to pregnant women in antenatal clinics, the introduction of ACT anti-malarial drugs in the treatment of malaria, as well as the sustenance of intermittent preventive therapy (IPT) during pregnancy. It therefore calls for more intensive effort to be geared towards malaria eradication.Different intervention strategies are currently in place to reduce the burden of malaria. However, in spite the low political commitment to these intervention strategies, the overall widespread mass education/awareness about preventive practices through the mass media have contributed immensely in reducing malaria prevalence among the population.Microscopy yielded a prevalence of 25.95% 109 of 420), with a moderate measure of agreement (k = 0.491) when compared with PCR result. This finding is not in consonance with the good measure of agreement with nested PCR observed by Alemu et al. [, with a The sensitivity and specificity of microscopy when compared with the nested PCR method is in contrast to the study by Alemu et al. , which fP. falciparum in some specimens which had co-infection of P. falciparum with either P. malariae or P. ovale. This occurrence was also observed in other studies by Osman et al. [In this study, malaria microscopy only detected n et al.  and Khann et al.  that alsplasmodium parasites. This allows for the detection of low density infections as well as mixed infections which are routinely missed in microscopy and makes nested PCR an ideal confirmatory test for malaria diagnosis [Nested PCR utilizes genus and species specific markers for the detection of iagnosis . The resiagnosis , 24–26.Plasmodium ovale and this finding has not been reported by other studies which had been conducted among the study population. This might be attributed to the absence of molecular techniques in previous studies which used either microscopy or RDTs.This study found the presence of Although microscopy is still considered the gold standard for the diagnosis of malaria, many countries consider shifting their diagnostic policy for malaria to implementing RDTs as standard diagnosis. This is because in many areas, proper microscopy is not possible because of lack of electricity or equipment . MalariaThe three RDTs studied showed different measures of agreements with PCR. Good measure (k = 0.611), fair measure (k = 0.226) and poor measure (k = 0.172) was observerd for Carestart, SD Bioline PF and SD Bioline PF/PV respectively. Besides HRP2 gene deletion which may be responsible for the varying accuracies observed among RDTs, They are affected by extraneous factors such as storage and handling. Most RDTs are not produced in Nigeria hence the climate may affect the efficacy of these RDTs. Also, in countries like Nigeria, RDTs are sold by traders, most of who know little or nothing about the influence of environmental storage conditions on the accuracy of the results. Care must be taken to ensure that when RDTs are purchased in large quantities, storage conditions prescribed by the manufacturers are strictly followed to ensure that the potency of the test kits are adequately preserved and hence assure the quality of results obtained by their use.RDTs in conjunction with microscopy should improve the diagnosis of malaria. However, RDT use should be considered as more cost—effective in the areas characterized by high or moderate intensity malaria transmission and in situations where health services are deficient or absent such as primary health centers . In deveThis study was carried out in an endemic area where technical expertise are deficient as found in many developing countries therefore a combination of microscopy and RDT will surely enhance the sensitivity and specificity of malaria diagnosis in both clinical and research settings.The finding that RDTs had low sensitivities  for Carestart, SD Bioline PF and SD Bioline PF/PV respectively is also comparable to the study by Kashif et al.  who repoThe low sensitivity and high specificity observed in all the test kits studied is in concordance with that of other studies –31. The This study has found microscopy still being a good method for the diagnosis of malaria and having a good measure of agreement with PCR. Hence it is recommended that the laboratory diagnosis of malaria be performed using both microscopy and RDT of high sensitivity and specificity. The combination of these methods will ensure that laboratory reports on malaria diagnosis are of very high accuracy. Furthermore, the impact of routine training and retraining of medical laboratory staff on malaria microscopy cannot be overemphasized.