Information on the epidemiology of malaria is essential for designing and interpreting results of clinical trials of drugs, vaccines and other interventions. As a background to the establishment of a site for anti-malarial drugs and vaccine trials, the epidemiology of malaria in a rural site in central Ghana was investigated.Anopheline malaria vectors in the study area were also carried out.Active surveillance of clinical malaria was carried out in a cohort of children below five years of age (n = 335) and the prevalence of malaria was estimated in a cohort of subjects of all ages (n = 1484) over a 12-month period. Participants were sampled from clusters drawn around sixteen index houses randomly selected from a total of about 22,000 houses within the study area. The child cohort was visited thrice weekly to screen for any illness and a blood slide was taken if a child had a history of fever or a temperature greater than or equal to 37.5 degree Celsius. The all-age cohort was screened for malaria once every eight weeks over a 12-month period. Estimation of Entomological Inoculation Rate (EIR) and characterization of Plasmodium falciparum dhfr gene were prevalent in this population and 25% of infections had a fourth mutant of pfdhps-A437G. The main vectors were Anopheles funestus and Anopheles gambiae and the EIR was 269 infective bites per person per year.The average parasite prevalence in the all age cohort was 58% . In children below five years of age, the average prevalence was 64% . Geometric mean parasite densities decreased significantly with increasing age. More than 50% of all children less than 10 years of age were anaemic. Children less than 5 years of age had as many as seven malaria attacks per child per year. The attack rates decreased significantly with increasing cut-offs of parasite density. The average Multiplicity of Infection (MOI) was of 6.1. All three pyrimethamine resistance mutant alleles of the The transmission of malaria in the forest-savanna region of central Ghana is high and perennial and this is an appropriate site for conducting clinical trials of anti-malarial drugs and vaccines. Over 500 million episodes of malaria occur yearly, predominantly in sub-Saharan African children under five years of age, resulting in the death of approximately a million of these children -3. SeverKnowledge of the epidemiology of malaria is essential for the design and interpretation of the results of trials of drugs, vaccines and other anti-malarial interventions -10. Info2 and a population of approximately 150,000 living in 97 communities. The mean monthly temperature ranges between 18°C and 38°C and the average rainfall is 1250 mm per annum, occurring mainly between May and October each year.Kintampo North and South districts lie within the forest-savanna, transitional ecological zone in the Brong Ahafo Region of Ghana. The districts have an area of 7,162 kmThree hundred and thirty-five children below five years of age (child cohort) and 1,484 participants of all ages  from the study areas were enrolled. They came from sixteen "index houses" selected randomly from the 22,000 houses within the study area that are registered in a database of an ongoing health and demographic surveillance system. The houses were stratified initially into three micro-ecological areas  and weighted such that the number of houses selected from each of these areas was proportional to the target population in each stratum and that only one "index house" was selected from each village. From each "index house", all children less than 10 years of age and a weighted number of 10-19 years and above 19+ years old adults were selected. If an "index house" did not have the required number of participants per age group, nearby houses were visited in order of proximity to the "index house" until the required number for each age category was obtained. An "index cluster", our unit of sampling, formed the cluster of contiguous houses in which recruitment took place. An average of 87 houses represented a cluster. In total, there were 1113 participants in the age group less than 10 years weighted in each cluster, 167 in the 10-19 years age group and 204 participants in the 19+ years age-group. Study participants were recruited in November and December 2003 and followed up over a 12-month period.The child cohort was visited at home thrice weekly to check the axillary temperature and to enquire about any recent illness. If a child had an axillary temperature ≥ 37.5°C or a history of fever within the past 48 hours as reported by the caregivers, the symptoms and signs of the illness were recorded and a finger-prick blood sample was collected to measure haemoglobin concentration and to prepare a blood smear. Children who had an axillary temperature ≥ 37.5°C or a history suggestive of malaria and malaria parasitaemia were treated with sulphadoxine-pyrimethamine (SP), the second line drug for management of uncomplicated malaria and allowed the clinician's judgement to prevail. Participants were observed at days 7, 14 and 28 post-treatment to assess clearance of malaria parasites as well as resolution of symptoms. Children who had a negative smear, had a repeat blood smear taken on the following day and were treated with SP if positive for malaria. If a repeat smear was negative and symptoms had subsided, the thrice-weekly examination of temperature and history taking was resumed. If symptoms persisted, daily temperature measurements, blood smears and evaluation by the medical staff continued. Symptomatic patients with negative smears were evaluated for other conditions beside malaria, treated appropriately and followed up. A referral system to the regional hospital was put in place for any child whose condition deteriorated.The all-age cohort was surveyed once every eight weeks over the one- year study period. At each survey, axillary temperature and a finger prick blood sample was collected to measure haemoglobin concentration using a calibrated haemocue  and to test for malaria parasites. A medical history was obtained and physical examinations performed; information was also collected on use of a bed net by each participant.Thin and thick blood smears were stained with Giemsa after fixing the thin smear with methanol. At least 100 high power fields were examined before a thick smear was declared negative. The number of parasites per micro-litre approximately equals 40 times the number of parasites counted per 200 leukocytes. 10% random selections of smears were independently read by an experienced second reader to ascertain the quality of microscopy. There was a high level of correlation between the microscopists (kappa = 0.94).Plasmodium falciparum chloroquine resistance transporter (pfcrt) and multi-drug resistance (pfmdr1) genes. Polymorphisms in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes were also determined by nested PCR amplifications, and dot blot hybridization as previously described by Noedl et al [Duraisingh et al [msp2) were used for genotyping the Plasmodium falciparum isolates using a nested PCR.Baseline assessment of chloroquine and SP resistance molecular markers and multiplicity of infection was carried out. Parasite DNA was extracted using the Chelex 100 sodium form (Bio-Rad laboratories) according to the procedure previously described . This DNdl et al  and Duragh et al . PolymorPlasmodium falciparum as described above. Filtermats were collected from children who were treated with SP for clinical malaria episodes and actively followed up to ascertain the treatment outcome. DNA was extracted from filtermats with corresponding microscopically slide positive samples on the day of treatment (Day Zero), negative on Day 14 and positive on Day 28 after treatment and analysed using the methods developed and routinely used at LSHTM [The seasonal distribution of parasites within the all-age cohort was determined in 600 participants microscopically positive for malaria. These randomly selected filter paper samples were taken through DNA extraction and analysis, using the chelex method and MSP2 genotyping of at LSHTM  describeAnopheline vectors were morphologically characterized by species using keys [Anophelines were discarded after recording numbers caught.CDC light traps were used to collect mosquitoes in rooms of randomly selected households. Trapping was done a day before the malaria survey in each cluster. All houses within the study area were given equal chances of participation. They were selected without replacement until all houses within a stratified area had been considered. Traps were set in at least four houses in each cluster each month. Additional traps were set weekly in rooms of study participants included in the child cohort or the all age cohort ensuring that at least one trapping took place in each month in each of the sixteen 16) clusters and throughout the whole one year period of the study. A total of 664 successful light traps were set in 708 houses. Occupants of each room in which a trap was set were provided with an untreated bed net to be used for the night that the trap was set in his/her room. Traps were hung approximately 1.5 m above the floor at the foot of the bed/mat of the index person.  clustersAnopheles gambiae and Anopheles funestus, were checked for the presence of circumsporozoite (CS) antigens of Plasmodium falciparum using the sandwiched Enzyme-Linked Immunosorbent Assay (ELISA) as described by Wirtz et al [Anopheles vectors were used as the negative test controls. All positive tested mosquitoes were retested to confirm positives.Heads and thoraces of the two major vectors of malaria, tz et al . PresencAn. gambiae species were subjected to PCR analysis to identify sibling species within the complex as per the Scott et al protocol [An. gambiae s.s. was performed as per the protocol of Flavia et al [kdr alleles associated with knock-down resistance in West Africa was assessed, as described in the Martínez-Torres et al protocol [Sub-samples of protocol . Enzyme ia et al . The preprotocol . An adjuet al [The sample size calculation was based on our assumed estimates of the age-group specific parasite prevalence rates (p) and standard error (s) among children less than 5 years , 5-10 years, 10-19 years , and more than 19 years . A minimum number of 140 volunteers was estimated in each age group to represent a cluster. This allowed us to estimate the age specific parasite prevalence rates to within 6 -10% of the true values for the different age groups. The expected precision of the estimates were calculated using the formulae of Bennett et al .® Visual FoxPro 6.0. All data management processes were done at the computer laboratory of the Kintampo Health Research Centre. Data analysis was done using StataCorp Stata 9, TX USA. Moving averages and proportions were used to provide descriptions of age and seasonal variation in parasite prevalence, density, infection rates and the proportions of parasite clones that persisted over the period.All data collected in the field or the laboratory were logged for traceability, and then batched for double data entry and processing using MicrosoftThe study protocol and instruments were reviewed and approved by the Ghana Health Service Ethics Review Committee and London School of Hygiene and Tropical Medicine Ethics Committee. In each community, a meeting was held among community members, their opinion leaders and the investigators to seek approval for the study to be conducted in the community before the study started. Written consent was sought from each adult participant while care-givers gave a written consent on behalf of their children.The annual prevalence of malaria over the 12-month period in the all-age cohort was 58%  and it ranged from 67.3%  in May-June to 51.9%  in November-December. The average annual prevalence in children below five years of age was 64%  and it ranged from 74.3%  in May-June to 56.8%  in November-December, a statistically significant difference between the two periods. Parasite prevalence increased with age until about ten years. It was relatively high during the rainy season (May-October) but was high throughout the year  Figure . ThroughThe incidence density of clinical malaria defined as any level of parasite density plus reported fever/axillary temperature > 37.5°C among children below five years of age was 7 attacks per child per year. It was 2.3 attacks per year in children below six months of age and peaked in 12-35 month old children (8.6 per child per year). The incidence of malaria with higher parasite density levels was lower than malaria with any parasite density in all age groups  was high. The MOI peaked in March-April and this trend was seen in children and adults Figure .Culex (51.3%), Aedes (1.0%), Anopheles funestus (35.2%), Anopheles gambiae (10.6%), Anopheles rufipes (1.8%) and Anopheles pharoensis (0.1%). Among the sample of mosquitoes assayed by CS-ELISA, 1.5% of An. funestus  and 4.7% of An. gambiae  were infected with P. falciparum. No An. arabiensis was found. The estimated annual Entomological Inoculation Rate (EIR) for both An. gambiae and An. funestus vector species was 269 infective bites per person per year (ib/p/y). Infective bites per person per night ranged from 0-13.5 in the communities  and became less during the major wet season, May-Oct (67 ib/p/y). Transmission was all-year round, but the two main vectors alternated their peak transmission periods  whiles An. funestus transmission peaked during the rains (May to October). This observation reflected in abundance of the vectors as the abundance of An. gambiae was very high at the end of rains whiles abundance of An. funestus was very high during the torrential rains  were identified as S-molecular forms, two (11.11%) as M-molecular forms and one (5.56%) as a hybrid (S-M) molecular form. The pattern was similar for 33 samples collected in the dry season where 11 (68.75%) were made up of S-molecular forms, two (12.50%) of the M-molecular forms and three (18.75%) were hybrids (S-M)-molecular forms (Table kdr resistant genotypes was 0.60 F(R). The genotypes kdrRR was observed in the M-molecular form in comparison with the kdrR/S genotype which was found in the hybrid (M-S) molecular form. All susceptible genotypes kdrS/S were detected in S-molecular forms (Table Only ms Table . The frems Table .Bed net (treated or untreated) use was low for all age groups. The maximum use of a bed net was found among children less than 5 years of age (25.4%). However, <10% of the community used treated bed nets throughout the year.The prevalence of malaria is now falling in several parts of Africa -25 as thThe prevalence of anaemia was very high and consistent with reports in Ghana (84%)  and otheib/p/y) was also relatively high and transmission was mainly by Anopheles gambiae and Anopheles funestus. An arabiensis had no role in malaria transmission in this area unlike other West-African countries such as Mali [An. gambiae s.s. The S-molecular form was predominant in this area with a short dry season contrary the predominantly M-form found in northern Ghana where the dry season is longer [The average EIR . Since 2005, the first line treatment for malaria has been changed from chloroquine and SP to the more effective drug combination of artesunate plus amodiaquine, although other anti-malarials are still used in the private sector. It is likely that as a result of these interventions the level of malaria transmission has fallen in the study area since the investigations reported in this paper were carried out and therefore a follow-up survey needs to be undertaken. Nevertheless, in comparison to many other parts of Africa, the transmission of malaria remains high in this zone because the background incidence of malaria in children was very high and there is transmission throughout the year. Thus, the Kintampo area is an appropriate site for the conduct of malaria treatment and vaccines trials. Since the completion of this study a clinical trial of artemether-lumefantrine, amodiaquine and artesunate and artesunate + chlorproguanil dapsone  and two Findings from this study have facilitated the design of trials of anti-malarial drugs and vaccines in the study area and led to optimal interpretation of the results from such trials. On the basis of the background epidemiology of malaria described in this paper, the Kintampo Health Research Centre is well-equipped to contribute to the phase 3 trial of the RTS, S vaccine starting in mid-2009 and future treatment and vaccine trials.The authors declare that they have no competing interests.SOA was the lead in writing the proposal with contribution from KPA, DC, JG and BG. SOA, KP, JG and DC were involved in the planning of the study. SOA, KP, MD, SN and DC implemented the study and data collection. MD, DD, DE and AA analysed laboratory specimen. GA, MA and EL contributed to data management. SOA, KPA, MA, GA, EA, DC and BG contributed to data analysis and presentation. SOA, KPA, DC, BG, DE and AA wrote the manuscript. All authors read and approved the final manuscript.


Vitamin A deficiency and malaria are both highly prevalent health problems in Africa. Vitamin A deficiency affects over 30 million children, most of whom are in the age-group (under five years) most affected by malaria. Vitamin A deficiency increases all-cause mortality in this part of the population, and malaria is an important cause of death in children at this age. A low serum retinol concentration (a marker of vitamin A deficiency) is commonly found in children suffering from malaria, but it is not certain whether this represents pre-existing vitamin A deficiency, a contribution of malaria to vitamin A deficiency, or merely an acute effect of malaria on retinol metabolism or binding. In this paper, available evidence in support of a causal relationship in each direction between vitamin A deficiency and malaria is reviewed. If such a relationship exists, and especially if this is bidirectional, interventions against either disease may convey an amplified benefit for health. Malaria commonly afflicts populations that are both impoverished and malnourished, and a large proportion of the burden of malaria falls upon children. There were more than 100 million malaria episodes among young children in sub-Saharan Africa in the year 2000, according to a recent estimate .Vitamin A is an essential nutrient required for maintaining immune function, playing an important role in the regulation of cell-mediated immunity and in humoral antibody responses -6. VitamThe relationship between malaria and VAD deserves careful scrutiny. The available evidence associating VAD with malaria is reviewed and discuss whether this association may be causal and bidirectional.Two main dietary forms of vitamin A exist: pre-formed vitamin A, found in foods such as butter, egg yolks and cod-liver oil, and provitamin A carotenoids, found in foods such as spinach, carrots, mangoes and papayas. Approximately 90% of the vitamin A in the body is stored in the liver, and an adult liver can contain enough vitamin A to last more than 1 year. In developing countries a lower than required dietary intake of vitamin A-rich foods commonly results in deficiency, especially among young children .The most accurate measure of an individual's vitamin A status is the hepatic vitamin A content. To assess this requires an invasive technique that is impractical for population studies. Alternative methods for estimating VAD at population level include measuring breast milk vitamin A, dark adaptometry, pupillary responses, conjunctival impression cytology and serum or plasma retinol concentration . Of thesThe prevalence of VAD  among African pre-school children in the year 2000 was estimated to be 32%, affecting 33 million children . The samPlasmodium falciparum-parasitized erythrocytes. The authors concluded that increased parasite clearance and reduced proinflammatory cytokine responses to infection might partly explain the beneficial effects of supplementation with vitamin A in malaria.The immune response against malaria involves both innate and specific acquired immune mechanisms . It has Approximately two thirds  of the 1et al [Is malaria one of the conditions aggravated by VAD? Binka et al  first inP falciparum infection  or anaemia. A recent randomized controlled trial conducted in Burkina Faso [Different findings were reported in two subsequent randomized controlled trials assessing the impact of vitamin A supplementation on slide confirmed malaria. In a study conducted in Papua New Guinea  480 chilina Faso  assessedTwo major methodological challenges complicate any attempt to assign to malaria a causal role in the development of VAD. These are: (i) the lack of enough evidence from prospective studies demonstrating that infection precedes deficiency; and (ii) the fact that the serum retinol concentration (the widely-used marker for vitamin A status) may be affected as part of the host's acute phase response to infection.Associations have been reported between xerophthalmia  and a variety of preceding acute illnesses, including diarrhoea, pneumonia and measles . In one More data are available for this effect in malaria than in other infections. In a recent large case-control study  in MalawInvestigations of this kind cannot demonstrate causality nor its direction, and none of these studies evaluated vitamin A status previous to the infection. An indirect attempt to determine antecedent VAD in patients with malaria was made in a study in Thailand, in which the mean serum retinol concentration was lower in children with malaria than in controls . In thisThe observation that clinical malaria or even asymptomatic parasitaemia is associated with low serum retinol levels seems consistent across the studies referred to above. However, it remains controversial whether the reduction in retinol levels reflects an actual depletion of vitamin A in the liver. A number of possible mechanisms have been proposed by which malaria could lead to VAD .Sick children tend to eat less, and malaria could precipitate VAD in a child who is already malnourished and has precarious hepatic reserves of vitamin A. This effect may be enhanced by the fact that the malaria season is commonly a time when food supplies are scarce. There is evidence that serum carotenes are depressed in malaria patients independently of serum retinol concentrations, suggesting preceding reduced oral intake of carotene-containing compounds .There is an association between helminth infections and malaria  and it hUrinary retinol losses during severe infection can be substantial. A study showed that adults in intensive care with pneumonia and sepsis lose nearly three times the recommended dietary allowance, whereas healthy adults lose <1% of the recommended dietary allowance per day . ImpaireAs part of the acute phase response  cytokineIs the plasma retinol concentration during an acute infectious illness of any value as an indicator of an individual's vitamin A status? A prospective study  assessinThe available evidence suggests that vitamin A supplementation is a good strategy for children living in malaria-endemic regions, because deficiency is common and because supplementation reduces all-cause mortality. Supplementation also reduces the incidence of vitamin A-specific pathology such as xerophthalmia. Although vitamin A supplementation reduces the incidence of uncomplicated malaria by about one-third, it does not appear to reduce the rate of deaths that can be specifically attributed to malaria. Evidence is weak for malaria having a causal role in the development of VAD, although on theoretical grounds and from indirect evidence this is a distinct possibility.Current programmes that deliver separate interventions against malaria and VAD may have a synergistic effect, and coordination of these interventions may provide added benefit against both diseases.RBP: Retinol binding protein; RR: Relative risk; VAD: Vitamin A deficiency; VAS: Vitamin A supplementation; WHO: World Health Organization.The authors declare that they have no competing interests.MAS performed the literature search, reviewed all articles cited and drafted the manuscript. MEM confirmed the findings and conclusions, and provided additional commentary and perspective. All authors contributed to and approved the final manuscript.


This article reports on the strengths, limitations of this methodology and its diagnostic performance in cross-sectional surveys on young children and women of child-bearing age.Prompt and correct diagnosis of malaria is crucial for accurate epidemiological assessment and better case management, and while the gold standard of light microscopy is often available, it requires both expertise and time. Portable fluorescent microscopy using the CyScope®) and the CyScope®. Results from the surveys were used to calculate diagnostic performance (sensitivity and specificity) as well as to perform a receiver operating characteristics (ROC) analyses, using light microscopy as the gold-standard.552 adults (99% women of child-bearing age) and 980 children (99% ≤ 5 years of age) from rural and peri-urban regions of Ugandan were examined for malaria using light microscopy (Giemsa-stain), a lateral-flow test  showed reduced specificity (<40%), resulting in higher community prevalence levels than those reported by light microscopy, particularly in adults (+180% in adults and +20% in children). Diagnostic sensitivity was 92.1% in adults and 86.7% in children, with an area under the ROC curve of 0.63. Importantly, optimum performance was achieved for higher parasitaemia (>400 parasites/μL blood): sensitivity of 64.2% and specificity of 86.0%. Overall, the diagnostic performance of the CyScope was found inferior to that of Paracheck-Pf® is certainly a field-applicable and relatively affordable solution for malaria diagnoses especially in areas where electrical supplies may be lacking. While it is unlikely to miss higher parasitaemia, its application in cross-sectional community-based studies leads to many false positives . Without recourse to other technologies, arbitration of these false positives is presently equivocal, which could ultimately lead to over-treatment; something that should be further explored in future investigations if the CyScope® is to be more widely implemented.Fluorescent microscopy using the CyScope With more than one million people dying every year from malaria, prompt and correct diagnosis leading to accurate epidemiological assessments and better case management is crucial. This is particularly important as fears of emergence of parasite drug resistance to the recently introduced artemisinin-based combination therapy (ACT) are increasing . The bal®  [® is also thought able to quantify infection parasitaemia, by counting the number of malaria parasites per white blood cells, a feature missed by all lateral-flow tests, as well as, being used for direct morphological inspection of red blood cells. Understanding parasitaemia levels pre- and post-treatment is crucial particularly for in-patient case management in health centres, clinics or hospital wards. Recently, a pioneering cross-sectional facility-based analytical study of the diagnostic performance of the CyScope® was conducted in Sudan with very promising results: sensitivity of 98.2% and specificity of 98.3% (gold standard: light microscopy) was obtained [The expansion of parasitological diagnosis leading to better case management even in the most remote areas endemic for malaria will soon rely predominantly on rapid diagnostic tests (RDTs) , and untGermany) , which aGermany) ). Albeit®, and the fact that reagents do not require cold storage, make the method a potentially attractive alternative for malaria diagnosis in the rural setting.The affordable pricing, portability and compact design of the CyScope®, as well as the lateral-flow Paracheck-Pf® test , as RDTs for malaria in children under five years of age and women of child-bearing age during cross-sectional epidemiological surveys in rural and semi-urban environments in Uganda.This article reports on the diagnostic performance of the CyScope®, constituted the 6-month follow-up of the SIMI cohort (baseline surveys took place in April-June 2009). Recruitment at baseline involved a community-wide sensitization (door-to-door); those willing to participate were instructed report to a specific location within the village  the following day. SIMI's recruitment criterion at baseline was to include any mother aged 15 to 55 years of age accompanied by at least one child. Exclusion from the cohort study would occur if mother and child(ren) failed to provide faecal samples for schistosomiasis diagnosis. Individuals from Kasangati town were investigated solely for malaria . The total study population included 552 adults  and their 980 children . All adults gave written informed consent on their behalf, and on behalf of their children. Malaria treatment - ACT (20 mg arthemether/120 mg lumefantrine) for all except pregnant women  - was offered on the basis of a positive Giemsa blood film and/or positive Paracheck-Pf® test in an outpatient setting.The study was conducted between October and November 2009 in six rural villages of Uganda where malaria is particularly problematic and responsible for considerable infant mortality: Bugoigo , Walukuba  and Piida  located along the shoreline of Lake Albert in Buliisa District, Bugoto , Bukoba  and Lwanika  located along the shoreline of Lake Victoria in Mayuge District. To make an assessment in a more urban setting, the town of Kasangati  was chosen . Individuals from Buliisa and Mayuge districts were inspected for malaria as part of a large-scale cohort study investigating maternal and child health in rural Uganda (the schistosomiasis in mothers and infants - SIMI project funded by the Wellcome Trust). This survey, using the CyScopeAll individuals (552 adults and 980 children) were finger-pricked for preparation of thick and thin blood smears, and the latter was fixed in absolute methanol for 30 seconds. The slide was then immersed in a freshly prepared 10% pre-filtered Giemsa stain solution for 5-10 minutes, washed with water and left to dry. Asexual parasites and gametocytes were counted against 200-500 leucocytes and converted to number of parasites per volume assuming 8,000 leucocytes/μL of blood. Although gametocytes are not normally included when estimating an individual's parasitaemia, they were included for this methodology to allow comparison between the numbers of parasitic forms identified by both light and fluorescent microscopy. Slides were considered negative when no parasites were detected after viewing 100 microscopic fields. A successful stain was achieved in 99% of slides (548 adults and 968 children). Reading took place under 1000 × magnifications using a light compound microscope in the field under oil-immersion and using a portable generator as local power supply. As a quality control cross-check, all slides were transported to Kampala and re-read at the Vector Control Division  to ascertain field accuracy (3 slides from adults and 12 slides from children were broken during transportation). All light microscopy was performed by experienced Vector Control Division technicians.®Malaria, Partec, Münster, Germany) and with a lateral-flow test .Rapid testing for malaria was performed by fluorescent microscopy  with the same finger prick blood sample as the thick/thin smears and fluorescent microscopy. Test kits were stored as directed by the manufacturer and quality of package desiccant was checked before use. The fresh blood sample was transferred directly to the sample pad by the provided sample applicator. All tests were labelled with patient ID numbers and village and results were recorded 15 minutes after adding 6 drops (300 μL) of clearing buffer. Presence of both the control and test lines indicated a positive result for 2 test [Diagnostic performance of both fluorescent microscopy and the lateral-flow test as diagnostic tests for malaria was established using Giemsa-stained light microscopy (double reads) as gold-standard. Sensitivity (SS), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) were calculated . The dia2 test .The correlation between light microscopy slide reads performed in the field and back in Kampala was calculated as well as between light microscopy slide reads performed in the field and fluorescent microscopy slide reads. Additionally, and to ascertain not only the correlation but also the agreement between light and fluorescent microscopy, a Bland-Altman plot was performed. The Bland-Altman plot is a tool for comparing two different methods of measuring the same value, when the true value being measured is not known. The purpose of a Bland-Altman plot is to try and determine whether fluorescent microscopy is better than light microscopy, using a hypothesis testing approach . In thesReceiver operating characteristics (ROC) analyses were also performed -15, plot® v 2.8.1. For prevalence values and the various point estimates of diagnostic precision, 95% confidence intervals (CI95) were estimated using the binomial exact method [Data were collected from each individual using pro-forma data sheets, which were then entered using Microsoft Excel™. The data thus collated were analysed using R statistical packaget method . Prevalet method .Ethical permission for the study was granted by the London School of Hygiene and Tropical Medicine (LSHTM 5538.09) and the National Council of Science and Technology, Kampala, Uganda.95 21.1 - 28.5%) in adults and 71.7% (CI95 68.7 - 74.5%) in children. Three infections were identified as non-P. falciparum malaria, likely Plasmodium malariae. The mean parasite count in all positive slides was 1464 per μL of blood  for adults and 9928 per μL of blood  for children. Fluorescent microscopy identified malaria prevalence levels reaching 68.0% (CI95 63.6 - 72.2%) in adults and 86.5% (CI95 84.0 - 88.7%) in children. The mean parasite count in all positive slides was 1149  in adults and 5611  in children. The lateral-flow rapid diagnostic test identified malaria prevalence levels reaching 24.1% (CI95 20.6 - 27.9%) in adults and 68.0% (CI95 63.6 - 72.2%) in children.The number of individuals included in the study was 552 adults  and 980 children . Fever, defined as under-arm temperature higher then 37.5°C, was diagnosed in 2.6% of adults and 6.6% of children, and only five children reported with symptoms of complicated malaria detected visually . Light microscopy identified malaria prevalence levels reaching 24.6% (CIP < 0.0001). This was not observed in adults using any diagnostic test, or in children, using fluorescent microscopy. See Additional File 95) and mean parasitaemia in adults and children detailed by location.Malaria prevalence levels according to both light microscopy and the lateral-flow RDT were significantly lower in children from the Kasangati town than in children living in shoreline villages from Buliisa and Mayuge districts P < 0.000. This wa2 = 0.94) between light microscopy reads performed in the field and those performed in Kampala  between reads by light and fluorescent microscopy  was found to be sensitive (92.1% in adults and 86.7% in children) for diagnosing malaria infections; however, with low specificity, particularly in children (38.8% in adults and 28.6% in children). The lateral-flow RDT (Paracheck-Pf®) significantly out-performed fluorescent microscopy in mothers  and children , with high sensitivity (91.9% in adults and 98.4% in children) and specificity (98.1% in adults and 96.2% in children) at diagnosing malaria infections. See Table Fluorescent microscopy , with a reported sensitivity of 89.6% and specificity of 37.2%, where 65.5% of diagnoses correctly classified. Interestingly, the ROC analysis demonstrated that the parasitaemia threshold for optimum performance by fluorescent microscopy is 400 parasites per μL of blood: sensitivity of 64.2% and specificity of 86.0%, with 74.2% of diagnoses correctly classified.Using fluorescent microscopy as a quantitative diagnostic test (parasitaemia counts) using data from 620 individuals , the resulting area under the ROC curve was 0.8059  and Victoria (Mayuge district) was targeted at women of child-bearing age and preschool-aged children (≤5 years of age) where malaria is known to be problematic and co-infection with intestinal schistosomiasis commonly occurs. Although most mothers were younger than 45 years of age, some older guardians were recruited in an attempted to maximise numbers, which explains ages reaching 54 years. The survey in Kasangati town was staged in the local health centre, and people were recruited from the attending line . Here, mothers with their young children were recruited; however, some fathers also wished to participate, and to increase sample sizes were included in the study. This explains why 1% of our sample of adults was male, and why 1% of our children were older than 5 years of age (maximum age recorded was 8 years).Anopheles gambiae s.s. and Anopheles funestus) [Levels of malaria in young children according to light microscopy were high, particularly in both rural settings (76.1% in Buliisa district and 72.9% in Mayuge district); nevertheless, children from Kasangati town, a far more urbanized settlement, were still highly affected by malaria, with prevalence reaching 47.5%. Mothers were far less affected by malaria, with levels varying only slightly from 20.9% (Kasangati town) to 26.4% (Buliisa district). 21% of the pregnant mothers were infected with malaria. These levels of malaria are of particular concern as many of the children and mothers positive at the time of this survey (Buliisa and Mayuge districts) had been diagnosed and treated (20 mg arthemether/120 mg lumefantrine - children and non-pregnant mothers) in May-June (baseline survey) and September 2009 (3-month follow-up), indicating very high re-infection rates in these rural environments. In fact, previous observations on entomological inoculation rates suggest that individuals in rural Uganda can receive >1,500 infective bites per year (unestus) .P. falciparum, according to light microscopy. In corroboration, additional data from real-time polymerase chain reaction analysis from blood-spots collected at the baseline survey (cohort establishment six months prior to these surveys) reveals prevalence levels of non-P. falciparum malaria varying from 3.0% in Bugoigo village, Bullisa District  to 17% in Bukoba village, Mayuge district  (unpublished). Prevalence levels and proportion of the different Plasmodium spp. reported in this article are within previously reported ranges [The great majority of infections were d ranges -23.® performed well in field conditions . These values also fall within previously reported ranges [P.f. infection may occur in the study areas, a vast proportion occur in co-infection with P. falciparum.The lateral-flow rapid diagnostic test Paracheck-Pfd ranges ,6 and fu® is fairly simple, relatively cheap (£818 for the microscope and £0.40 per test) and was found to be easy to apply even in field conditions, and when parasitaemia is above 400 parasites per μL of blood, positive cases are easy to identify , all Plasmodium spp. fluoresce using this methodology . This excessive diagnosis of malaria positives was repeated in the adults, only this time in all locations surveyed . Importantly, even in hyper-endemic rural environments, this methodology was shown to be significantly less reliable than the already established, cheap and rapid lateral-flow test: the Paracheck-Pf®) with only very few parasite-like fluorescent bodies found in the whole slide , whose fragments could easily be mistaken for parasites . Many false positives were diagnosed with extremely low parasitaemia ; however, high rate of false positives was surprising and should be more carefully examined, as this can lead to ineffective treatment streamlining and drug waste. Further studies are needed to determine the cost-effectiveness and longer term performance of the CyScope® in diagnosing malaria, especially if this methodology is to scaled-up in the rural health care setting.Fluorescent microscopy by CyScopeThe authors declare that they have no competing interests.JRS, NBK, MB and JCSF conceived and participated in the design of the study. JRS, MB, DO and JCSF coordinated the study. JCSF performed statistical analysis. DO and MAd performed fluorescent microscopy reads, FB and MAr performed the lateral-flow diagnostic tests, ANam and ANan performed light microscopy both in the field and in Kampala. JCSF, JRS and MB helped to draft manuscript. All authors contributed towards the final manuscript.Supplemental table. Prevalence (and CI95) and mean parasitaemia in adults and children detailed by location.Click here for file


Anopheles. gambiae s.l. and Anopheles. funestus vectors. The immune response status to Plasmodium falciparum was evaluated in children living in two villages where malaria transmission was ensured by dissimilar species of Anopheles vectors (An. funestus vs An. gambiae s.l.).In sub-Saharan areas, malaria transmission was mainly ensured by An. gambiae s.l. and Gankette Balla village which is exposed to several Anopheles species but where An. funestus is the only infected vector found. In each village, a cohort of 150 children aged from one to nine years was followed during one year and IgG response directed to schizont extract was determined by ELISA.A multi-disciplinary study was performed in villages located in Northern Senegal. Two villages were selected: Mboula village where transmission is strictly ensured by P. falciparum infection were observed in both villages. Specific IgG response increased progressively from one-year to 5-year old children and then stayed high in children from five to nine years old. The children with P. falciparum infection had higher specific antibody responses compared to negative infection children, suggesting a strong relationship between production of specific antibodies and malaria transmission, rather than protective immunity. In contrast, higher variation of antibody levels according to malaria transmission periods were found in Mboula compared to Gankette Balla. In Mboula, the peak of malaria transmission was followed by a considerable increase in antibody levels, whereas low and constant anti-malaria IgG response was observed throughout the year in Gankette Balla.Similar results of specific IgG responses according to age and Anopheles species. These results are discussed according to i) the use of immunological tool for the evaluation of malaria transmission and ii) the influence of Anopheles vectors species on the regulation of antibody responses to P. falciparum.This study shows that the development of anti-malaria antibody response was profoundly different according to areas where malaria exposure is dependent with different  Plasmodium falciparum malaria is a major cause of human morbidity and mortality throughout tropical Africa. In sub-Saharan areas, malaria transmission is caused by several anopheles vectors, mostly Anopheles gambiae sensu stricto (s.s.) and Anopheles arabiensis from the Anopheles. gambiae complex, Anopheles funestus and Anopheles pharaoensis [An. funestus breeds prolifically in swampy habitats with much vegetation, whereas freshwater members of the An. gambiae complex do best in small sunlit pools. The anthropophilic sibling species An. arabiensis and/or An. gambiae s.s. usually predominate in areas where the environmental conditions do not provide plentiful breeding sites for An. funestus [An. funestus [An. gambiae sensu lato (s.l.) is the principal malaria vector in many epidemiological settings of the Afro-tropical region, such as Kenya [An. funestus can play a predominant role in malaria transmission. In Savannah areas, An. funestus has been shown to relay An. gambiae s.l., which reaches its peak of abundance in the early dry season [raoensis ,2. Depenfunestus , or wherfunestus . Thus, Aas Kenya , Tanzanias Kenya , Zimbabwas Kenya , Zaire [as Kenya , and Senas Kenya . Neverthy season .An. funestus (which disappeared as a result of the drought in the 1970s) [An. gambiae and An. funestus vectors in this region provided an opportunity to survey this particular situation in which high risk of intense malaria transmission in populations presenting low anti-malaria immunity is commonly seen [In the Northern part of Senegal, malaria transmission is low, unstable and seasonal with an average of two to seven infective bites/person/year ,12. The e 1970s) . The connly seen .Anopheles vectors infected by Plasmodium associated with the degree of infection/morbidity attributed to malaria in human [P. falciparum [In many epidemiological studies, malaria transmission can be estimated by evaluating the density of in human . Serologlciparum . Recent lciparum ,18; theslciparum .P. falciparum blood stages have been screened using antigenic materiel derived from parasite lysates crude schizont [P. falciparum represent a sort of global "picture" of anti-malaria immunity and gives information about malaria transmission.To explore the risk and dynamics of malaria transmission, a longitudinal survey using an immunological marker, was conducted in Northern Senegal. Specific antibodies to schizont  as a widschizont . This suIn the present study, the objectives wereP. falciparum wide antigens in two sites located in Northern Senegal  where different Anopheles species are responsible for malaria transmission 1) To evaluate IgG responses to 2) To analyse the relationships between specific Ab levels and parasitological and entomological dataP. falciparum in both villages according to the period of malaria transmission3) To analyse the one-year dynamics of Ab responses to P. falciparum are known to be low in children less than 15 years of age [The study was conducted in the north of Senegal, in two villages, Mboula and Gankette Balla, located along the Senegal River Basin, nearby Ferlo and the Lake Guiers, respectively. In this area, the prevalence and intensity of s of age . This sis of age ,13.The study population is in majority from the Wolof ethnic group. A longitudinal study was performed during one year, between June 2004 and June 2005 in both villages. Five passages  were undertaken and, for each passage, a cohort of 150 children aged from one to nine years was selected for each village. The later passage included the distribution of permethrin-impregnated bet nets.Plasmodium species and the number of malaria parasites was counted. Parasite density was defined as the number of P. falciparum parasites/μl of blood. In the same way, capillary blood collection was done for each child at each passage for the determination of specific IgG levels by ELISA.For each child, parasitological measurements of malaria were performed at each passage using thick blood smears (TBS) obtained by finger-prick. The smears were Giemsa-stained to identify The present study followed ethical principles according to the Helsinki Declaration, and was approved by the Ethical Committee of the Ministry of Health of Senegal . Informed consent was obtained from the studied population.Anopheles species [Anopheles infection rate was studied by ELISA (Enzyme-Linked ImmunoSorbent Assay) for P. falciparum circumsporozoite antigen (CSP). For all specimens, only 0.04% An. funestus collected in Gankette Balla was positive for P. falciparum CSP with an entomological inoculation rate (EIR) estimated to 3.00 infected bites. In contrast, transmission was not perceptible in Mboula whereas An. gambiae s.l. is the strict potential collected vector (EIR = 0).Adult mosquitoes were collected in June, September, December 2004 and March 2005 in both villages by human-landing collection. In each village, mosquito populations were caught in three selected households in six collection sites half indoor/half outdoor (7:00 p.m to 7:00 a.m) during two consecutive nights. The number of bites per human per night (BHN) was calculated by dividing the number of mosquitoes caught by the total person-night used for the period. Mosquitoes caught were brought to the laboratory, counted and identified morphologically to  species . AnophelP. falciparum schizont lysate obtained from infected erythrocyte and kindly provided by D. Dive from the Institut Pasteur of Lille.ELISA was used to evaluate IgG directed to total extract of schizont. Total schizont antigen is a soluble extract of 2O2) and absorbance (OD) was measured at 405 nm. Individuals results were expressed as ΔDO value calculated according to the formula: ΔDO = ODx-ODn, where ODx is the individual OD value of infected individuals and ODn was the individual OD value for each serum without antigen. The reproducibility of OD-positive values from IgG responders in the study children was verified in three later assays. A negative control  was used for each assay. A subject was considered an immune responder if this ODx was higher than the ODn + (3 × SD) value.Schizont extract (7.5 μg/ml) were coated on flat-bottom microtiter plates  with 100 μL/well for 2 h 30 at 37°C. Plates wells were then blocked for 30 mn at room temperature with 200 μL of blocking buffer, pH 6.6 , 0.5% gelatin  and washed one time with PBS, pH 7.2, 0.1% Tween 20 . Individuals sera were incubated in duplicate at 4°C overnight at a 1/50 dilution (in PBS-Tween-0.1%). This dilution was determined as the optimum after several preliminary experiments. For detecting human IgG, plates were incubated for 90 min at 37°C with 100 μL of mouse biotinylated mAb to human IgG  diluted 1/1000 in PBS-Tween 0.1%, after three times washing with PBS-Tween 0.1%. Plate wells were then washed four times with PBS-Tween and incubated for 30 minutes at room temperature with 100 μL of peroxydase-conjugated streptavidin . After washing six times with PBS-Tween, colorimetric development was carried out using ABTS (2.2'-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium; Sigma, St Louis, MO, USA) in 50 mM citrate buffer  and R software version 2.3.1. After verifying that values in each group did not assume a Gaussian distribution, differences in Ab levels were tested by Mann-Whitney U-test and Kruskal-Wallis test between more than two groups. The non-parametric Friedman-test matched pair test was used to compare paired sera all along the follow up. Spearman's correlation was used to check for correlations between parameters. All differences were considered significant when P < 0.05.All data were analysed with Graph Pad PrismP. falciparum prevalence in children and the intensity of exposure to both major Anopheles species bites (BHN) along the year follow-up, are presented on Table The percentage of anti-malaria IgG responders, the An. gambiae s.l. was collected by using the human-landing method in Mboula village (only two An. pharoensis mosquitoes collected during the one year follow-up) suggesting that children were practically exposed only to bites by An. gambiae species. The exposure to An. gambiae bites was maximum in September but stayed low (BHN = 3). Whereas no infected An. gambiae was detected, moderate prevalence of P. falciparum was observed during the seasonal transmission (16 to 22%). Indeed, prevalence increased in September to reach a peak in December and March. It could be thus considered that An. gambiae species was the strict vector in Mboula. In contrast, in Gankette Balla, individuals were largely exposed to An. funestus bites as indicated by very high BHN compared to low An. gambiae BHN. In addition, exposure to An. funestus was higher in September and December and this species is the strict vector in this village. A very low prevalence of P. falciparum was observed during the year follow-up.The entomological data indicated that mainly P. falciparum infection was found in Mboula which presented differences according to the passage with a peak between December and March 2005  Figures  and 2B. P. falciparum parasite/μl of blood) and Ab responses in Mboula , whereas in Gankette Balla the correlation  was not significant.In addition, a correlation was observed between the intensity of malaria infection (number of The levels of anti-shizont IgG after the period of transmission (December) according to the age of children was presented in Figure P. falciparum . This result suggests that the age-dependent increase of specific IgG response was not dependent to malaria status. In Mboula, this positive correlation was not significant in uninfected children .In Gankette Balla, specific IgG responses appeared to be correlated with age, even in children not infected by Anopheles species  were presented during one year follow-up  and entomological data evaluating the intensity of exposure to both major p Figure .An. gambiae increased during the period of high malaria transmission (September). This peak of An. gambiae BHN was followed by an increase in levels of specific IgG in December to March . For this purpose, the evolution of anti-schizont IgG levels in relation to the intensity of exposure to Anopheles species was analysed during one year. The IgG response against whole P. falciparum extract antigens was thus assessed in children living on both villages according to intensity of exposure to Anopheles species, age, period of transmission and presence of malaria infection  and could result from a booster effect of the presence of long-lasting presence of the parasite on the specific immunity [P. falciparum IgG responses in both villages need the use of better immunological tools in order to closely evaluate the intensity of transmission. For this purpose, it has been shown that the Ab responses against different sporozoite  or pre-erythrocyte antigens  are positive serological markers of malaria transmission [et al [In spite of the differences of specific IgG responses between villages, positive association of immune response with immunity ,26. The smission ,19,27. Tn [et al  reportedn [et al ,30.P. falciparum [Anopheles species and that the prevalence of P. falciparum infection was very different between villages, did not appear to be a factor of variation of the age-dependent development of anti-malarial IgG responses.It has been also reported that specific IgG responses increased progressively in children aged from one- to five years and then stay high until eight. This influence of age was similar in both villages and appeared to be independent to of malaria status. This intrinsic increase of anti-malaria Ab response with age was commonly reported in children living in malaria areas and in populations highly exposed to infection ,32. Thislciparum . In the An. gambiae s.l. exposure (September) was followed by the high IgG responses in December, which remained stable until March. In contrast, no difference of Ab responses was observed in Gankette Balla according to season, whereas the exposure of An. funestus vector was very high in September-December. This situation of high exposure to An. funestus species accompanied with a very low prevalence of P. falciparum infection was rarely observed in the epidemiological studies which have taken place in Senegal and elsewhere in Africa [Anopheles vectors. Nevertheless, these immune differences could be attributed to the characteristics of malaria transmission in the areas studied. In Gankette Balla, high exposure to larval An. funestus species, resulting in the reconstitution of their breeding sites, could provide optimal conditions to aquatic stages and thus allow the emergence of adults. An. funestus collected in Gankette Balla were young, the most part having never laid and strikingly zoophilic; they seemed to present a very low vectorial capacity . This particular situation could explain the low malaria transmission and low prevalence of P. falciparum observed in this village and could clarify the absence of seasonal variations of anti-malarial Ab response. In contrast, the low, but marked, season-dependent exposure to An. gambiae s.l. vector in Mboula appeared to induce a significant increase of specific IgG response. The bionomics of An. gambiae s.l. is closely related to human populations (anthropophilic and endophilic) and its high susceptibility to human malaria parasite endows this vector with a high vectorial ability [An. gambiae s.l. in Mboula compared to An. funestus (33.18%) in Gankette Balla .In Mboula, the peak of n Africa ,34. Seve ability . This isP. falciparum infection and the level of anti-schizont IgG response. However, it is conceivable that the bite by different Anopheles species vector which caused transmission could influence the anti-malarial Ab responses. Indeed, it is now well known that the saliva of arthropod vectors presents an immunomodulatory effect [P. falciparum protective immunity [An. funestus and An. gambiae species . The identification of the different proteins between both species is actually under investigation. The variation of salivary proteins expression according to mosquito species and their secretion during the bite has been also observed between Aedes species [Anopheles species could show dissimilarity and induces a different immune regulation on the development of anti-malarial immunity. Recently, it has been demonstrated that IgG responses to Anopheles saliva is detected in children living in malaria area and represent a marker of the intensity of An. gambiae exposure [P. falciparum and anti-saliva Ab responses in these villages according to the season is currently under investigation in the same cohort. This project will be one step to validate the hypothesis that the bite by different Anopheles vectors could influence the anti-parasite immune response.The present study shows a close association between the presence of y effect -38. In aimmunity ,40. In t species . It coulexposure . The comP. falciparum IgG response in children was different in two areas where malaria transmission was ensured by different Anopheles species. Previously, it has been demonstrated that in a Leishmania animal model, the immune regulation of Phlebotomus saliva can induce a substantial effect on the development of infection and pathology [The main original results of the present study show that the acquired anti-athology ,44. ThusJBS and FR have equally contributed to the design, acquisition, analysis, interpretation of data and manuscript drafting.ID, LK contributed to conception of study and contributed markedly to the analysis of entomological data.SG, CS, SM, ST, CT, for field activities and microscopic examinations.AMS provided expertise and technical support for evaluation of the immune responses.FS participated in the conception and coordination of the study and helped to draft the manuscript.GR provided the scientific supervision in Saint Louis and revised the manuscript.This study was performed in the framework of the "Pal-Fleuve programme" which is a structure developing multidisciplinary approaches  and complementary researches on malaria exposure/transmission in Northern Senegal. This programme includes four units of research: EPLS (Association Espoir Pour La Santé) – Saint Louis; IRD-UR024 (Epidémiologie et Prévention) – Dakar; UCAD (Université Cheikh Anta Diop) – Dakar and IPD (Institut Pasteur de Dakar).


Anopheles (An.) darlingi saliva in volunteers from the Brazilian Amazon and addresses the association between levels of specific antibodies and clinical presentation of Plasmodium (P.) vivax infection.Despite governmental and private efforts on providing malaria control, this disease continues to be a major health threat. Thus, innovative strategies are needed to reduce disease burden. The malaria vectors, through the injection of saliva into the host skin, play important role on disease transmission and may influence malaria morbidity. This study describes the humoral immune response against P. vivax infection by light microscopy and nested PCR. Non-infected volunteers and individuals with symptomatic or symptomless infection were randomly selected and plasma collected. An. darlingi salivary gland sonicates (SGS) were prepared and used to measure anti-saliva antibody levels. Plasma interleukin (IL)-10 and interferon (IFN)-γ levels were also estimated and correlated to anti-SGS levels.Adult volunteers from communities in the Rondônia State, Brazil, were screened in order to assess the presence of P. vivax presented higher levels of anti-SGS than non-infected individuals and antibody levels could discriminate infection. Furthermore, anti-saliva antibody measurement was also useful to distinguish asymptomatic infection from non-infection, with a high likelihood ratio. Interestingly, individuals with asymptomatic parasitaemia presented higher titers of anti-SGS and lower IFN-γ/IL-10 ratio than symptomatic ones. In P. vivax-infected asymptomatic individuals, the IFN-γ/IL-10 ratio was inversely correlated to anti-SGS titers, although not for while in symptomatic volunteers.Individuals infected with An. darlingi antibody levels can indicate the probable P. vivax infection status and also could serve as a marker of disease severity in this region of Brazilian Amazon.The estimation of anti- Plasmodium (P.) falciparum [Plasmodium (P.) vivax [Plasmodium antibodies [Plasmodium are not well understood.Malaria continues to be one of the most serious public health problems worldwide, exacting a huge impact on human wellbeing, mainly in tropical and subtropical countries. A better understanding of the interactions between the host, the vector and the parasite could be valuable to indicate future strategies. In endemic regions, residents are frequently bitten by both uninfected and infected mosquitoes. There is also a progressive acquisition of immunity, leading to a decreased number of malaria clinical attacks related to increasing age and time residing in the endemic area ,2. Withilciparum  and Plas.) vivax  infectiotibodies . On the tibodies . Such asAnopheles (An.) stephensi increase a pro-inflammatory T helper 1 biased response that limits P. yoelii infection [An. gambiae saliva is immunogenic for travelers transiently exposed to bites in African endemic areas [An. gambiae saliva IgG antibodies were also detected in young children from a seasonal malaria transmission region in Senegal, and antibody levels were higher in patients who developed clinical malaria episodes, suggesting that the estimation of humoral response to Anopheles salivary antigens can serve as potential marker for the risk of malaria [An. dirus salivary protein antibodies occur predominantly in patients with acute P. falciparum or P. vivax malaria, whereas people from non-malarious areas do not carry such antibodies [An. darlingi, is poorly explored. The objective of the present work was to measure the anti-saliva IgG responses against An. darlingi mosquitoes in the Brazilian Amazon and to evaluate the association of antibody levels with different clinical presentations of P. vivax infections.In this scenario, the anopheline vector could play significant role in malaria clinical severity. Mosquito bites can induce immediate, delayed, and systemic hypersensitivity reactions in hosts . Moreovenfection . In humaic areas , with th malaria . Moreovetibodies . Little P. vivax malaria was performed during 2007 (June to August) in Buritis , a recent urbanized municipality, and Demarcação , a riverine community of the Rondônia State, in the south-western part of Brazilian Amazon. In general, Rondônia has a flat topography, with an average elevation of 300 m above sea level. The climate is tropical, with a long rainy season from January till May. It is argued that the environmental changes caused by deforestation have favored the main malaria vector in Brazil An. darlingi [P. vivax accounts for the majority of malaria cases, while P. falciparum infection prevalence is 23.7% [P. malariae achieves 10% in Rondônia [A cross-sectional study investigating determinant factors for asymptomatic darlingi . Within darlingi , with andarlingi . In the is 23.7% . In addiRondônia .P. malariae infection and 16 persons infected with P. falciparum were identified and excluded from the study. Hence, all the volunteers selected were negative for P. falciparum and/or P. malariae infection by both microscopic examination and nested PCR. Other exclusion criteria were chronic alcoholism, severe chronic degenerative disease as well as HIV, HBV and HCV infections. A total of 204 volunteers were used in the study. All the positive cases were followed up for 30 days for the evaluation of malaria symptoms. Individuals who were positive for P. vivax infection and remained without fever (axilary temperature >37.8°C) and/or chills, sweats, strong headaches, myalgia, nausea, vomiting, jaundice, asthenia, and arthralgia for 30 days were considered asymptomatic, while in the presence of any listed symptom they were classified as symptomatic. The volunteers were stratified in three different groups according to the P. vivax malaria diagnosis and the clinical spectrum of the disease. Thus, 80 people were non-infected, 50 had asymptomatic infection and 74 were symptomatic. The baseline characteristics of the volunteers are listed in the Table P. vivax DNA was amplified by nested PCR , responsible for malaria control in the Brazilian Amazon. Active case detection was made by visiting residences in regions pointed by the local health authorities as major areas of disease transmission. The individuals were examined and interviewed by a trained physician, and blood samples were collected for serological experiments. The malaria diagnosis was performed using two methods. First, patients were screened by thick smear examination using field microscopy and the parasitaemia (parasites/μL) was calculated in positive cases. Further, nested PCR was performed in all whole blood samples to confirm the diagnosis (as described below). Two individuals presenting Plasmodium genus-specific primers named PLU5 and PLU6. Positive samples yielded a 1,200-bp fragment, which served as template for the nested reaction. The nested PCR amplification was performed with species-specific primers for 30 cycles at annealing temperatures of 58°C for P. falciparum , and 65°C for P. vivax (Viv1 and Viv2 primers) or P. malariae . The fragments obtained for P. vivax were of 120 bp, whereas for P. falciparum and P. malariae were 205 bp and 144 bp, respectively. The oligonucleotide sequences of each primer used are listed in Table The molecular diagnosis of malaria infection was performed using the nested PCR technique, based on the Snounou protocols, with minimal alterations ,20. The An. darlingi mosquitoes were dissected and transferred to 20 μL of 10 mM HEPES pH 7.0, 0.15 mM NaCl in 1.5-mL polypropylene vials, usually in groups of 20 gland pairs. Salivary glands were kept at -70°C until needed, when they were disrupted by sonication using a Branson Sonifier 450 homogenizer . The homogenates were centrifuged at 10,000 × g for 4 min and the supernatants were used for the experiments. Protein concentrations were measured by the bicinchonic acid method . As the salivary glands used in this study were obtained from field captured mosquitoes, Plasmodium contamination needed to be checked by nested PCR. Briefly, it was performed the DNA extraction of a sample from the same SGS pool used in the serological experiments using the Qiagen Generation Capture Card Kit . Further, the nested PCR was performed as described above, in duplicate samples. There was no amplification of DNA in both duplicates (data not shown).Salivary glands from field captured adult female An. darlingi salivary homogenate (SGS) equivalent to 1.5 μg/mL in carbonate buffer overnight at 4°C, then washed with PBS/0.05% Tween and blocked with PBS/0.1% Tween plus 0.05% BSA. Sera were diluted 1:100 with PBS/0.05% Tween and incubated overnight at 4°C. After further washings, the wells were incubated with alkaline phosphatase-conjugated anti-human IgG  at a 1:5,000 dilution. Following another washing cycle, the color was developed with p-nitrophenylphosphate. The reactions were blocked with NaOH and read at 405 nm using Soft Max-Pro Software v5  ELISA reader. The optical density (OD) values plotted represent the means between each sample duplicate, adjusted for the values from the blank wells.Volunteer's sera were collected and kept at -70°C. Serological tests of all samples were performed in a single experiment, with duplicate samples. ELISA was performed as described elsewhere . Briefly®  according to the manufacturer's protocol.Interleukin (IL)-10 and interferon (IFN)-γ plasma levels were measured using de Cytometric Bead Array – CBA® (GraphPad Software Inc.). For the ordinal variables , differences between groups were calculated using the non parametric Kruskal-Wallis test with Dunn's multiple comparison post test. Chi-square test was used to compare differences regarding categorized variables (Time residing in the area and parasitaemia). Mann-Whitney test was used to compare differences in IgG levels between non-infected individuals and those with symptomatic or asymptomatic P. vivax infection. This test was also used to estimate significance in IFN-γ/IL-10 ratios from volunteers with asymptomatic or symptomatic P. vivax infection. To evaluate the cut off value of IgG anti-SGS predicting malaria infection or asymptomatic infection, we performed Receiver-operator characteristic (ROC) curves, calculated the Area under curve (AUC), and then estimated the likelihood ratio for the discrimination between the conditions analyzed. Fine Lowess curves were plotted to evidence the trend of the data presented in correlation analyzes. Spearman test was used to verify the significance in the correlations between cytokine levels and anti-SGS levels. Differences were considered significant at P < 0.05.Data were analyzed using the GraphPad Prism 5.00P. vivax, anti-SGS values obtained from non-infected individuals were compared to those from either symptomatic or asymptomatic infected volunteers. As seen in Figure An. darlingi salivary antigens than non-infected individuals. The variability probably indicates individual differences in exposure to mosquito bites, even during the period of high malaria transmission, when these data were collected. Previous studies have also demonstrated a high variation in anti-An. dirus saliva antibody titers [In an attempt to check if the measurement of anti-SGS antibody levels could be a suitable method to estimate natural exposure to y titers .P. vivax infection from the non-infected condition. A cut-off point of 0.0855 OD displayed a likelihood ratio to be infected of 2.11 indicating P. vivax infection . LAM, AB and MB-N are senior investigators from CNPq.


Plasmodium falciparum.Intermittent preventive treatment in infants with sulphadoxine-pyrimethamine (IPTi-SP) reduces malaria morbidity by 20% to 33%. Potentially, however, this intervention may compromise the acquisition of immunity, including the tolerance towards multiple infections with Plasmodium falciparum isolates were obtained from children participating in two Ghanaian IPTi-SP trials  at 15 months of age, i.e., six months after they had received the second dose of IPTi-SP or placebo. By typing the polymorphic merozoite surface protein 1 (msp1) and msp2 genes, multiplicity of infection (MOI) was assessed in 389 isolates. A total of additional 133 samples were collected in Tamale at 3, 6, 9, and 12 months of age. Comparisons of MOI between groups were done by non-parametric statistical tests.P. falciparum clones (MOI) ranged between one and six. Mean MOI in Tamale was stable at 2.13 - 2.17 during the first year of life, and increased to 2.57 at age 15 months (P = 0.01). At no age did MOI differ between the IPTi-SP and placebo groups . At 15 months of age, i.e., six months after the second dose, MOI was very similar for children who had received IPTi or placebo  as was the proportion of polyclonal infections . Adjusting for study site, current and prior malaria, parasite density, and season did not change this finding.The number of distinguishable IPTi-SP appears to have no impact on the multiplicity of infection during infancy and thereafter. This suggests that tolerance of multiple infections, a component of protective immunity in highly endemic areas, is not affected by this intervention. Efficient and safe means of controlling falciparum malaria in endemic areas are urgently needed to reduce its intolerable high burden, particularly in young children. Reducing parasite exposure, however, may compromise the acquisition of protective immunity, as has been debated with respect to vector control and was observed following chemoprophylaxis -3.Plasmodium falciparum clones per isolate. Commonly, MOI is assessed by using the polymorphic regions of P. falciparum merozoite surface proteins 1 and 2 genes (msp 1/2) as markers. In that, inter-laboratory differences exist [P. falciparum infections of comparatively short duration in infants preclude tolerance [Multiplicity of infection (MOI) denotes the number of distinguishable es exist , and metes exist . Irrespees exist ,7. The aes exist . Indeed,es exist . In contolerance , and mulolerance -16. The olerance .Intermittent preventive treatment in infants (IPTi) denotes the administration of a curative dose of an anti-malarial, commonly sulphadoxine-pyrimethamine (SP) at the times of routine vaccination, regardless of whether a child is parasitaemic or not. IPTi with SP (IPTi-SP) was initially tested in Tanzania also as an approach to avoid the rebound effect of increasing morbidity following chemoprophylaxis. In that initial study, IPTi-SP reduced the incidence of malaria in infancy by 59% without evidence of a rebound effect . In fiveSo far, only scarce data are available on the effect of anti-malarial interventions on MOI. In Tanzania, MOI was significantly reduced among infants infected despite chemoprophylaxis and this reduction was suggested to be involved in the subsequent rebound in clinical malaria episodes . RTS, S Plasmodium falciparum isolates obtained from children participating in two related, randomized IPTi trials conducted in Tamale, hyperendemic northern Ghana, and in Afigya Sekyere district, holoendemic southern Ghana, were examined. The attempt of identical examinations in the IPTi trial from Lambaréné, Gabon [é, Gabon , was abaé, Gabon ,22. Brieé, Gabon ,22. In té, Gabon . In the é, Gabon ,22. Episé, Gabon ,22.P. falciparum parasitaemia were subjected to assessing MOI. For that, sequences corresponding to the allelic families of P. falciparum msp1 block 2  and of msp2 block 3  were amplified in five separate nested PCR assays [®-agarose gels  and analysed using GeneSnap software . In case of negative or inconclusive PCR results, assays were repeated maximally twice. MOI was calculated as the highest number of fragments for either msp1 or msp2. Of a total of 348 microscopically positive samples collected at 15 months of age, MOI could be assessed in 277 (79.6%). For the Tamale trial, all samples collected at 15 months of age were examined by Plasmodium-specific nested PCR assays [sp1/2 genotyping was extended to additional 142 samples identified to have submicroscopic parasitaemia of which 112 yielded a result for MOI . Also, for the Tamale cohort, a random sample of microscopically positive samples  collected at 3, 6, 9, and 12 months of age was genotyped to illustrate the development of MOI with age . For all samples, processing and DNA extraction were performed in a standardized manner, and all samples were typed at the Institute of Tropical Medicine in Berlin, Germany.On the day of IPTi dose-3, i.e., six months following IPTi dose-2 and at approximately 15 months of age, venous blood samples were collected. These were stabilized by adding an equal volume of DNA-stabilizing buffer , stored at +4°C, and DNA was extracted by commercial kits . All samples with microscopically confirmed R assays . These aplus fever  or fever during the preceding 48 hours reported by mothers without being asked. For incidence estimates, children were regarded as not being at risk for 21 days following an episode of malaria and person-time-at-risk was reduced accordingly. Polyclonal infections had a MOI of >1. Rainy season (> 10 days or >90 mm of precipitation/month) was defined as before [Malaria was defined by the presence of microscopically visible parasitaemia s before .2 tests. In multivariate analysis, known or potential confounders of the proportion of polyclonal infections and MOI were adjusted for by including these in logistic regression models and by applying non-parametric multiple ordinal regression, respectively.Continuous parameters were compared between groups by the non-parametric Mann-Whitney U-test or Kruskal-Wallis test. These tests were also applied to compare MOI between groups but results are primarily presented as arithmetic means since this measure of average more selectively displays (the direction of) differences than medians, which are also shown, nevertheless. Proportions of polyclonal infections were analysed by χThe characteristics of the 1086 children at 15 months of age from Tamale and their 964 peers from Afigya Sekyere are shown in Table vs. 17.4% (176/1013), P = 0.80) and malaria (9.6% (97/1015) vs. 8.9% (90/1013), P = 0.60). Also, GMPDs (95%CI) were alike (1622 (1175-2238) vs. 1791 (1318-2432)/μL, P = 0.66).In Tamale and Afigya Sekyere, IPTi-SP at ages 3 and 9 months had achieved reductions in the incidence of malaria until 15 months of age of 24% (13-33%) and 20% , respectively, largely due to incidence reductions during the first month following each treatment ,22,24. AP = 1.0), and increased to 2.57  at age 15 months (P = 0.01). At no age, did MOI differ between IPTi-SP and placebo groups (P ≥ 0.5). Lacking impact of IPTi-SP on MOI was confirmed when adjusting for parasite density and age by multiple ordinal regression  were similar among former IPTi-SP and placebo recipients. This was true for both microscopically identified and submicroscopic infections  samples of malaria cases. The distribution of MOI in these 277 samples is shown in Figure P. falciparum in the rainy season was observed only in multivariate analysis. Adjusting for these factors, the finding that previous IPTi-SP did not influence the multiplicity of P. falciparum infection was confirmed , each 2 (1-5); P = 0.0006). Submicroscopic infections were arbitrarily set as 1 parasite/μL and included into a separate multivariate analysis as above for the Tamale cohort. Again, no significant impact of IPTI-SP on MOI  or on polyclonal infections  was discernible.In the Tamale cohort, submicroscopic infections showed a lesser multiplicity than those microscopically visible , 2 (1-5) vs. 2 (1-3); P = 0.82).Between months 16 and 24 of follow-up of the Tamale cohort, 12 excess cases of severe malaria were observed in the previous IPTi group . For theP. falciparum, and the rebound of malaria following that intervention has been attributed to this reduction [In holoendemic Tanzania, continuous chemoprophylaxis in infancy reduced the multiplicity of infection with eduction . In the eduction ,22,24,34eduction -17 this The present study analysed multiplicity only, i.e. one actor in the network of diverse immune mechanisms. The importance of this factor is furthermore varying with age and endemicity -17. AlsoP. falciparum infection also affect the assessment of parasite populations, and single assessments may provide only a limited impression of the overall parasite composition in a host [de facto higher incidence of malaria has been missed in Tamale due to the comparatively broader mesh of active follow-up. Also, the use of artesunate for treatment there as compared to long-acting artesunate-amodiaquine in Afigya Sekyere might be involved in a more intense exposure.The present data accord with previous findings from highly endemic areas ,14 in thn a host . In the n a host ,35. The n a host ,40 but cn a host ,42. Consn a host ,39. Unexn a host ,43,44 bun a host , this fin a host ,16,17,43P. falciparum up to the age of 15 months, although it reduced the incidence of malaria in the two trials by 24% and 20%. This effect on malaria could largely be attributed to a prophylactic effect of approximately one month duration following treatment [Vice versa, the reduction of MOI following RTS, S vaccination [IPTI-SP did not influence the multiplicity of reatment ,22,24. Treatment  suggesticination ,30 mightcination ,46, reascination , potenticination , and clucination ,21,22. Acination . The precination  needs toThe authors declare that they have no competing interests.FPM, JM, and MPG designed the study. PZa, KR, SA, RK, HHA, and Pzi were responsible for patient recruitment, and clinical and parasitological examinations. UB did the genotyping, and ID and FPM the statistical analyses. UB, ID and FPM wrote the paper with major contributions of the other authors. All authors read and approved the final manuscript.


Plasmodium falciparum transmission intensities through which communities might expect to transition, as intervention coverage expands.The understanding of the epidemiology of severe malaria in African children remains incomplete across the spectrum of Plasmodium falciparum transmission intensity in these communities were assembled to be spatially and temporally congruent to the clinical admission data. The analysis focused on the relationships between community derived parasite prevalence and the age and clinical presentation of paediatric malaria in children aged 0–9 years admitted to hospital.Paediatric admission data were assembled from 13 hospitals serving 17 communities between 1990 and 2007. Estimates of 2 = 0.73, p < 0.001). Cerebral malaria was reported among 4% and severe malaria anaemia among 17% of all malaria admissions. At higher transmission intensity cerebral malaria was a less common presentation compared to lower transmission sites. There was no obvious relationship between the proportions of children with severe malaria anaemia and transmission intensity.As transmission intensity declined a greater proportion of malaria admissions were in older children. There was a strong linear relationship between increasing transmission intensity and the proportion of paediatric malaria admissions that were infants (RAs the intensity of malaria transmission declines in Africa through the scaling up of insecticide-treated nets and other vector control measures a focus of disease prevention among very young children becomes less appropriate. The understanding of the relationship between parasite exposure and patterns of disease risk should be used to adapt malaria control strategies in different epidemiological settings. Plasmodium falciparum transmission intensities [During the 1980's and 1990's a series of epidemiological observations were reported on the age and clinical patterns of severe malaria in African children across a range of ensities . It appeensities -9.P. falciparum transmission increases, the mean age of severe malaria decreases. Less consistent is the reported relationship between clinical syndromes of severe paediatric malaria and transmission intensity.Subsequent to these earlier studies there has been a renaissance in the clinical epidemiology of severe paediatric malaria across a wide range of different transmission settings, leading to descriptions of severe paediatric malaria from Sudan , MozambiThere are three limitations of many of the reported ecological comparisons. First, they often compare data from very few sites, reducing the contextual ranges of the observations and thus their wider validity. Second, the measures of transmission intensity used in most studies are often imperfect or assumed rather than measured ,17,23,25P. falciparum transmission intensity.To circumvent some of these limitations of cross-site comparisons and increase the power to generalize from observations, data from 17 sites across seven countries are presented. The standardization of metrics used to define transmission intensity in each site have significantly improved, have been temporally matched to the clinical surveillance period and attempts have been made to ensure standardization in surveillance and diagnostic methods between survey observations, to examine the relationship between age patterns of hospitalized paediatric malaria and In this review we have assembled clinical admission data from 17 communities served by 13 hospitals. The surveillance sites were selected on the basis of having established clinical and parasitological surveillance, often to specifically study the pathophysiology, management and epidemiology of severe paediatric malaria.P. falciparum endemic world . A primary diagnosis of malaria was made when a child had a positive blood smear and no other detectable cause for the clinical presentation, after a review of all available clinical and haematological data and where indicated, X-ray and microbiological data. Two clinically important complications of severe malaria in African children are cerebral malaria and severe malarial anaemia ,30. ConsP. falciparum infection prevalence from the communities served by the study hospital sites were assembled from information available as part of The Malaria Atlas Project [PfPR2–10) using algorithms described elsewhere [Cross-sectional estimates of  Project  database Project ,35. Infelsewhere .PfPR2–10 values of 1% in Kilifi North between 2004–07 to as high as 87% in Namawala/Michenga villages in Tanzania in the early 1990s  admission series documented between 1999 and 2001 shows a high proportion of infants while transmission intensity is intermediary between two sites where the infant admissions are much lower.For a number of reasons, that are expanded on in the discussion, the public health and intervention significance of clinical risks in infancy is of programmatic importance. Among the six communities where the PfPR2–10 during each observation period: Kilifi North in the 1990s and 2000s and two closely located communities surveyed between 1999 and 2007 south of Kilifi District Hospital and corresponding to Kilifi South surveyed in 1990's. In the same communities over the two time periods transmission had dropped dramatically. In Kilifi North over ten years PfPR2–10 dropped from 52% to 1% and at Kilifi South (1992–1995) and the nested area of Chonyi (1999–2001) corresponding PfPR2–10 estimates were 77% and 43% respectively. In both areas the age patterns of malaria admissions had shifted toward older children as PfPR2–10 declined but the most dramatic age-shift was observed at Kilifi North with the largest decline in PfPR2–10 over a longer time frame.The observations summarized in Figure PfPR2–10 is shown in Figure PfPR2–10, however, without the very high proportion of cerebral malaria cases reported at Kilimanjaro this relationship is less convincing. Similarly excluding the community of Nyamawala/Michenga in Tanzania where 50% of all admissions had a PCV <15% there appears to be no direct relationship between increasing transmission intensity and increase in proportion of severe malaria anaemia cases  and adoption of new effective therapeutics -41. How PfPR2–10 estimates of transmission contemporary with the clinical observations were used to ensure congruence between exposure and outcome.There are very few serial, long-term clinical observations of severe paediatric malaria in areas where transmission intensity is in transition . Thus toPfPR2–10 is ≥ 40% more than 40% of malaria admissions to paediatric wards were infants, compared to only 10% in areas where PfPR2–10 is < 5%. These observations are consistent with the view that the speed of acquired clinical immunity scales with the frequency of parasite exposure since birth [PfPR2–10 6%) and Bakau (PfPR2–10 2%). These declining risks with age under very low parasite exposure from birth suggest that only a few parasite exposures might confer a degree of clinical immunity [One of the most striking observations was the relationship between increasing transmission intensity and the predominant age of paediatric malaria admissions Figures  and 3b. ce birth . Interesimmunity  or age iimmunity .PfPR2–10  coincidental with vaccine schedules , is likek groups  until onk groups  (Figure –5 years , as thisontinent , suggestPfPR2–10 1% to 33%, however, and may also reflect the difficulties in measuring cerebral malaria in very young children [PfPR2–10 (Figure PfPR2–10 was 87%. The epidemiology of severe malaria anaemia may be more complex than previously thought [In this study series, cerebral malaria appeared to be a more common presentation among children hospitalized with malaria from lower intensity transmission settings compared to areas of high transmission Figure . This obchildren . The pro0 Figure  with the thought ,55 and w thought . A recen thought , thus coPfPR2–10 declines can be made. Nevertheless, it is interesting to note that the one long-term serial study of severe clinical malaria in Africa has investigated the rate of hospitalization during a time of major transmission reduction at Kilifi North [PfPR2–10 of approximately 50% and declined to 1% over ten years, resulted in a ten-fold decline in the risks of hospitalization with malaria in childhood. The focus here has been on better descriptions, across more sites of the age and clinical presentation of hospitalized malaria in childhood likely to be observed with reductions in transmission intensity across Africa as prevention strategies go to scale over the next ten years. Perhaps not surprisingly these results confirm many other observations, using less rigorous inclusion criteria [The incidence of hospitalization has not been examined, largely because the precise calculation of the paediatric populations at risk was not possible across most of the sites studied after the 1990's. Therefore, no specific comments on the overall changing risks of hospitalization as fi North . O'Mearacriteria ,23,25, tThe authors declare that they have no competing interests.EA assembled all the hospital data, restructured the data and wrote the manuscript; AAT, HR, RI and JAB were responsible for the assembly of hospital data from Yemen, Tanzania, Uganda and Kilifi, Kenya respectively and contributed to the final manuscript. RWS was responsible for the project and its overall scientific management, interpretation and preparation of the final manuscript.


Plasmodium Falciparum Malaria is a cause of ICU in coming, appearing like in Sepsis with multi - organ failure, resulting in high mortality 10-50%. Non-immune patient are most susceptible.Malaria is the most important human parasitic disease, affecting about 40% worldwide population, most prevalent in tropical countries. Severe Plasmodium falciparum Malaria and Sepsis.Compare severity between severe Plamodium falciparum Malaria and the first 18 patients admitted with Sepsis in ICU -Sagrada Esperança Clinic during 2007 was compared concerning to age, sex, severity ICU scores (SOFA and APACHE II), organ failure and mortality. We also compared mortality between immune and non-immune patients for Malaria.Severity between the 18 patients admitted with Severe See tables 2plasmodium falciparum malaria and Sepsis, there are similar organ failure rate.There is no significant difference for severity comparing severe Mortality among non-immune patients is high.


A specific retinopathy has been described in African children with cerebral malaria, but in adults this has not been extensively studied. It has great potential as a diagnostic and prognostic tool and pathogenetic marker. Since the structure and function of the retinal vasculature greatly resembles the cerebral vasculature, study of retinal changes can reveal insights into the pathogenesis of cerebral malaria. Obstruction of microcirculatory blood flow is thought to be important in causing both malarial retinopathy and cerebral malaria.A detailed observational study of malarial retinopathy in Bangladeshi adults with P. falciparum malaria was performed using a portable retinal camera. The aims were to establish the prevalence, spectrum and time to resolution of retinal findings in malaria, their effect on visual function and to investigate the role of obstruction of microcirculatory blood flow in its pathogenesis. A second study aimed to establish if assessment of malarial retinopathy in adult malaria using ophthalmoscopy by non-ophthalmologists has clinical and prognostic significance.For the first study, all Plasmodium falciparum smear positive adult patients admitted to Chittagong Medical College Hospital, Chittagong, Bangladesh, during the malaria seasons of 2008-2010 were eligible. Control groups were febrile encephalopathy, sepsis and healthy volunteers and children with malaria. Patients were photographed daily until discharge then weekly until complete resolution of retinal changes. In adults, severity of retinal findings was correlated with markers of microcirculatory blood flow obstruction ) and rheological factors important in microcirculatory obstruction  and red blood cell deformability (LORCA)).The second study recruited unselected patients with severe and uncomplicated falciparum malaria, vivax malaria and healthy controls. Retinal examinations were by two non-ophthalmologists, one using direct and the other indirect ophthalmoscopy.Of 234 adults recruited in the first study, 51/60 (85%) with cerebral, 18/27 (67%) noncerebral and 28/59 (47%) uncomplicated malaria had retinal changes. Of the controls, 9/29 (31%) encephalopathy, 11/28 (39%) sepsis and 4/31 (13%) healthy volunteers had some degree of retinopathy, mostly mild. Moderate-severe retinopathy was found in 67% of those with fatal, 62% of cerebral, 41% noncerebral and 12% uncomplicated malaria, 14% encephalopathy, 0% sepsis and 0% healthy volunteers. Other than papilloedema, moderate-severe retinopathy was highly specific for malaria (98%), and for cerebral malaria in comatose patients (93%). Resolution of signs took median 14 days, and visual function 3-4, days. Severity of retinopathy correlated with severity of malaria, coma recovery time and markers of, and rheological factors important in, microcirculatory obstruction.In 210 adults in the second study moderate-severe retinopathy as assessed by non-ophthalmologists using direct ophthalmoscopy was found to be an independent predictor of mortality in malaria. Although indirect ophthalmoscopy was more sensitive it provided minimal additional prognostic information compared to direct.Malarial retinopathy is highly specific for malaria and was present in the majority of adults with severe disease, particularly cerebral and fatal, but was prominent in very few with uncomplicated disease. It thus has diagnostic and prognostic utility at the bedside, including when assessed by a non-specialist using direct ophthalmoscopy. Retinal findings were similar in adults and children. The strong correlation with markers of and rheological factors important in, microvascular obstruction suggest an important role of microvascular obstruction in the pathogenesis of both malarial retinopathy and cerebral malaria. Although malarial retinopathy lasted around two weeks, impairment of colour vision and acuity were only transient.Malarial retinopathy has potential as a bedside tool to aid diagnosis and prognosis in adults with malaria. Prominent retinal changes are highly suggestive of severe disease, particularly cerebral and fatal malaria. Findings in adults were very similar to those in children, both in the present study and in Africa. The correlation of severity of retinopathy with impaired microcirculatory blood flow suggest this process is central to the pathogenesis of both malarial retinopathy, and cerebral malaria. Studies using fluorescein angiography and magnetic resonance imaging to investigate this further are currently underway.


Conventional blood film microscopy for malaria diagnosis remains a tedious and time-consuming activity requiring examination of 100 to 200 Giemsa stained oil-immersion fields. In expert hands, blood film microscopy is highly sensitive but for the vast majority of laboratories accuracy of diagnosis is little better than 70%, particularly at low levels of parasitaemia. The introduction of lateral flow immunochromatography rapid tests (RDTs) over the past 15 years has gone some way towards addressing this problem but again these tests encounter sensitivity problems particularly when parasite numbers are low. The issue of sensitivity can be overcome by multiplex PCR assay systems but at the present time this technology is restricted to a few major reference laboratories. One sensitive immunological method that has not been applied to malaria diagnosis is direct immunofluorescence (DFA), mainly because the high cost and maintenance of UV microscopes has made their routine use impractical and prohibitively expensive. However the availability of a new generation of low cost, LED fluorescence microscopes  puts this technology in reach of almost any laboratory or field station. One LED adapter allows the conversion of any light microscope into a fluorescent microscope in a matter of minutes. We report here the development of a DFA staining technique for the detection of the five human malaria species. The method is highly sensitive with antibody specificity providing a significant advantage to the operator.P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi) parasites . Stained iRBCs containing parasites can easily be seen under a x40 objective lens with x10 eye piece as shown in Figure Direct fluorescent antibody staining of malaria parasites coupled with the use of low cost LED microscopes should increase the reliability of malaria diagnosis in resource-deprived settings and provide enhanced sensitivity, thus facilitating reliable detection of low density parasitaemias and subsequent early treatment, which may aid in minimizing emergence of drug resistance.