The importance of invasive salmonellosis in African children is well recognized but there is inadequate information on these infections. We conducted a fever surveillance study in a Tanzanian rural hospital to estimate the case fraction of invasive salmonellosis among pediatric admissions, examine associations with common co-morbidities and describe its clinical features. We compared our main findings with those from previous studies among children in sub-Saharan Africa.S. typhi, 45 (29%) were NTS and 97 (62%) were other pathogenic bacteria. Invasive salmonellosis accounted for 59/156 (38%) bacteremic children. Children with typhoid fever were significantly older and presented with a longer duration of fever. NTS infections were significantly associated with prior antimalarial treatment, malarial complications and with a high risk for death.From 1 March 2008 to 28 Feb 2009, 1,502 children were enrolled into the study. We collected clinical information and blood for point of care tests, culture, and diagnosis of malaria and HIV. We analyzed the clinical features on admission and outcome by laboratory-confirmed diagnosis. Pathogenic bacteria were isolated from the blood of 156 (10%) children, of which 14 (9%) were S. typhi and NTS infections. Certain suggestive clinical features  may be used to distinguish invasive salmonellosis from other severe febrile illness.Invasive salmonellosis, particularly NTS infection, is an important cause of febrile disease among hospitalized children in our rural Tanzanian setting. Previous studies showed considerable variation in the case fraction of  Salmonella enterica serotype Typhi (S. typhi) and several non-Typhi serotypes of S. Enterica (NTS) are important causes of childhood bacteremia in African children There is inadequate information on invasive salmonellosis in sub-Saharan Africa. In particular, the importance of NTS sepsis is not widely recognized. An important reason is that most hospitals in the region lack adequate microbiological facilities. Epidemiologic data on invasive Salmonellosis in sub-Saharan African countries is important for assisting clinical management and development of preventive strategies. We conducted fever surveillance in a district hospital in rural Tanzania. We estimated the case fraction of invasive salmonellosis among our pediatric admissions, examined associations with common co-morbidities and described its clinical features in comparison with other febrile illnesses. We compared our main findings with those from previous studies among children in sub-Saharan Africa.Plasmodium falciparum malaria with perennial transmission and two seasonal peaks coinciding with the short and long rains The study was conducted at Teule Hospital, which is the designated district hospital of Muheza in north-eastern Tanzania. The hospital serves a catchment population of about 277,000 of whom 17% are aged less than five years. Child mortality in the area is 165/1000 Haemophilus influenzae type b in March 2009 and hopes to introduce pneumococcal vaccine in 2010 The Tanzanian Expanded Programme of Immunization includes the following: Bacille Calmette-Guérin, live oral polio, diphtheria-whole cell pertussis-tetanus-hepatitis B and monovalent measles vaccines for children, as well as supplemental tetanus toxoid vaccine for women of child-bearing age. Tanzania had just started immunization against Prior to the start and during the course of the study, emergency triage and hospital care guidelines were implemented in the ward P. falciparum malaria: HRP-2 based  and LDH based . From each child, thin and thick blood films were prepared, Giemsa-stained and read by experienced laboratory technicians. At least 100 high power microscopic fields of the thin film were examined to exclude the diagnosis of malaria. Blood for culture was inoculated into a BactALERT™ Pediatric-fan bottle  and incubated in the BacT/ALERT 3D automated microbial detection system. Blood cultures were processed according to standard methods. Colonies with biochemical reactions on API20E suggestive of Salmonellae were confirmed serologically by slide and tube agglutination testing using specific O and H antisera . Sera were tested for the presence of HIV-1 and HIV-2 antibodies according to the National HIV rapid testing algorithm We collected 1 to 10 millilitres of blood (depending on body weight) from each eligible child. Immediate bedside testing included those for hemoglobin concentration  and blood glucose level . We performed two types of rapid diagnostic test (RDT) for Data were double-entered into custom-made data entry programs using MS-Access . Data management programs included error, range and consistency check programs.S. typhi (typhoid fever), NTS, and other  pathogenic bacteria. Malaria was defined as fever with a positive RDT or blood film. HIV infection was defined as a positive Capillus test or SD bioline, confirmed either by a positive Determine HIV-1/2 or a positive Unigold test. Low maternal education was considered as schooling to less than the National Curriculum standard 7. Diarrhea was defined as loose or watery stools ≥3 times per day. A seizure was regarded as abnormal movements with altered consciousness. Desaturation was defined as oxygen saturation less than 90% measured by pulse oximetry. Acute severe malnutrition was defined as the presence of bilateral pedal edema or severe wasting. We also assessed the mid-upper arm circumference (MUAC) of children between 12 to 59 months of age. Signs of shock were temperature gradient in the lower extremities and delayed capillary refill of ≥3 seconds or systolic blood pressure <70 mm Hg. Signs of dehydration were delayed skin pinch >2 seconds or sunken eyes. Prostration was defined as inability to sit unsupported (for children over 9 months of age) or to drink/breastfeed. Coma was defined as Blantyre coma score ≤2 for children less than 2 years of age or a Glasgow coma score ≤10 for older children. Hypoglycemia was defined as blood glucose level of <2.5 mmol/litre. Anemia was defined as hemoglobin of <8 g/dl and severe anemia <5 g/dl.Fever was defined as history of a rise in body temperature as recalled by a care-giver or presence of axillary temperature ≥37.5°C on presentation. Bacteremia was defined as fever with isolation of pathogenic bacteria from blood culture, further classified as those caused by To assess potentially important distinguishing factors, we classified the cases into 5 non-mutually-exclusive groups: typhoid fever, invasive NTS infection, other pathogenic bacteremia, malaria, and those without bacteremia and malaria. Comparisons of categorical data were made using the Chi square or Fishers' Exact test, as appropriate. Comparisons of continuous data were made using student's t-test for data with equal variance or Welch's t-test for those with unequal variance. All analyses were performed using Stata™ v 10.0 .S. typhi and NTS infection from sterile-site specimen  bacterial cultures. The searches were restricted to publications from 1987 to date. For study sites with several publications generated from the same study population, only one citation was included unless the time period varied. We also conducted supplementary searches of the references in retrieved articles. Abstracts were reviewed and if relevant, the article was included.We conducted a literature review to compare our main findings with those from previous studies. Potential articles for inclusion were identified by direct searches of the MEDLINE database through PubMed. We included facility-based studies of children ≤16 years in sub-Saharan Africa that reported case fractions of The fever surveillance was conducted following the principles governing biomedical research involving human subjects. Prior written informed consent was obtained from the parent or guardian of each eligible child. Pre-test counseling was provided before HIV testing in accordance with local guidelines. The study was approved by the National Institute for Medical Research, Tanzania (NIMR/HQ/R.8a/Vol.IX/666) and the International Vaccine Institute - Institutional Review Boards, South Korea (IRB# 2007-017).S. typhi, 45 (29%) had NTS and 97 (62%) had other pathogenic bacteria. Thus, invasive salmonellosis accounted for 59/156 (38%) bacteremic children.From 1 March 2008 to 28 February 2009, 2319 children were admitted to Teule Hospital during study hours . After eS. typhi ranged from 0 to 42% and of NTS from 9 to 84%, depending on the study sampling frame. The number of NTS isolates for every one S. typhi ranged from 0.8 to 166.We compared our findings with those from previous studies among children in sub-Saharan Africa . SixteenS. typhi was the most common isolate among those over 5 to 14 years of age. Other commonly isolated bacterial pathogens included Escherichia coli (27/156 or 17%) and Haemophilus influenzae type B (20/156 or 13%). There were 8 (5%) Streptococcus pneumoniae isolates.Of the 1,502 children enrolled, 806 (54%) were >2 months to 2 years, 520 (34%) were >2 years to 5 years and 176 (12%) were >5 years of age . We rankS. typhi, NTS, and other pathogenic bacteria . Considering those with a positive RDT or blood film as having malaria, children with invasive NTS infection were more likely to also have malaria (33/45 or 73%) compared to those with typhoid fever  and other bacteremia . The proportion of those with a positive RDT and negative blood film was highest among those with invasive NTS infection (20/45 or 44%) compared to those with typhoid fever  and other bacteremia .We present the percentage of malaria and HIV co-infections among children with S. typhi) and malaria were significantly associated with low maternal education. Malaria patients were less likely to present with cough and diarrhea  compared to children without bacteremia and malaria. Children with other pathogenic bacteraemia and malaria were more likely to have seizures and present in coma  compared to children without bacteremia and malaria. Compared to the other groups, children with invasive NTS infection had most frequently received an antimalarial medication prior to admission . Although uncommon, children with invasive NTS were also more likely to be jaundiced . Severe palmar pallor, anemia and hypoglycemia were common among invasive NTS infection, other pathogenic bacteremia, and malaria cases but those with invasive NTS had the lowest mean hemoglobin level (5.7 g/dl) and were most frequently hypoglycemic . Children with invasive NTS died more frequently  compared to all other groups.We compared the admission clinical features and outcome of typhoid fever, invasive NTS infection, other pathogenic bacteremia, and malaria cases with those of children without bacteremia and malaria . Age wasSalmonellae ranked as the most common cause of bacteraemia. Our over-all isolation rate of pathogenic bacteria was similar to that in previous studies that used similar sampling criteria S. typhi and NTS infections S. typhi infections by several-fold. In many sites, pediatric typhoid fever was not detected at all. This is in marked contrast to findings from Asia where a high burden of disease is seen even in the youngest age groups In our study population of children between 2 months to 14 years of age, Malaria has long been suspected to increase the risk and contribute to the seasonality of invasive non-typhi salmonellosis In this study, typhoid fever cases were more common in older children and presented with a longer duration of fever. In contrast, the non-typhoid bacteremia cases, as well as malaria, tended to occur in young children, particularly of poorly-educated women. It is likely that low educational attainment is a marker for low socio-economic status. NTS infection was associated with a considerable increased risk for death.S. typhimurium and S. enteritidis are the predominant serotypes among African children Our study has several limitations. First, it is well known that blood cultures are insensitive for detecting bacteremia. Small blood volumes for culture especially from the younger and sicker patients, as well as the prior use of antibiotics, further decrease the sensitivity of blood cultures. Among our participants, 17% admitted to antibiotic use prior to admission. Thus, it is likely that we have underestimated the case-fractions of hospitalized invasive salmonellosis. This may also have led to an underestimate of the overall number of bacteremic children and contamination of the comparison groups due to false-negative diagnoses. Second, we compared and contrasted features of non-mutually-exclusive groups: typhoid fever, invasive NTS infection, other pathogenic bacteremia, malaria, and those without bacteremia and malaria. It was necessary to use these non-mutually exclusive groups as it is not possible to determine whether malaria or bacteremia was the precipitating event that led to admission. Factors associated with each group could modify the severity of the illness and be responsible for the differences in clinical picture. On the other hand, this comparison yielded characteristics that clinicians could potentially use to help distinguish invasive Salmonellosis cases from other hospitalized patients. Third, we were not able to include children less than 2 months of age in the current fever surveillance since these children were admitted and cared for in a separate area of the hospital. Fourth, serotyping of the NTS isolates was beyond the scope of the study but would have added interesting information. Although NTS cases are a rather heterogeneous group of diseases, previous reports have shown that In summary, we found that in a malaria endemic region in Tanzania, invasive salmonellosis is an important cause of hospitalized febrile diseases among children. Invasive NTS disease is associated with a high risk for death. Certain suggestive clinical features  may be used to distinguish invasive salmonellosis from other severe febrile illness on presentation. We shall continue our fever surveillance in Teule hospital to follow trends in the occurrence and clinical picture of these infections in our community.


Clinical management of malaria is a major health issue in sub-Saharan Africa. New strategies based on intermittent preventive treatment (IPT) can tackle disease burden by simultaneously reducing frequency of infections and life-threatening illness in infants (IPTi) and children (IPTc), while allowing for immunity to build up. However, concerns as to whether immunity develops efficiently in treated individuals, and whether there is a rebound effect after treatment is halted, have made it imperative to define the effects that IPTi and IPTc exert on the clinical malaria scenario.Here, we simulate several schemes of intervention under different transmission settings, while varying immunity build up assumptions. Our model predicts that infection risk and effectiveness of acquisition of clinical immunity under prophylactic effect are associated to intervention impact during treatment and follow-up periods. These effects vary across regions of different endemicity and are highly correlated with the interplay between the timing of interventions in age and the age dependent risk of acquiring an infection. However, even when significant rebound effects are predicted to occur, the overall intervention impact is positive.IPTi is predicted to have minimal impact on the acquisition of clinical immunity, since it does not interfere with the occurrence of mild infections, thus failing to reduce the underlying force of infection. On the contrary, IPTc has a significant potential to reduce transmission, specifically in areas where it is already low to moderate. Malaria exerts a huge morbidity and mortality toll on people in sub-Saharan Africa New strategies designed to deal with such potential drawbacks have been put forward and into practice. Particularly, the administration of anti-malarial drugs to pregnant women through intermittent preventive treatment (IPTp), has been quite successful at protecting them from malaria and placental parasitaemia, and their newborns from malaria-associated low birth weight Because fewer doses are given compared to chemoprophylaxis, concerns about cost and drug resistance are significantly reduced. We will not dwell on how IPT might alter emergence and spread of drug resistance, as it has been studied elsewhere The complexities of malaria epidemiology have huge implications for the unsettled question of whether immunity in treated individuals develops as in untreated ones, and a precise evaluation of IPT interventions efficacy in different endemic settings is required. It is important to define as clearly as possible the conditions under which the current conception of IPTi within the EPI delivery system, and IPTc administered to a specific age range of children, according to a particular schedule will be beneficial. We will explore the possible scenarios by simulating different schemes of intervention and transmission settings, relying on empirical data The establishment of whether (and if so under which conditions) a rebound effect is observed is of major importance to advise control programmes being implemented in the field. So far, reports on outcomes of IPTi interventions have been inconclusive and contradictory to some extent. Whereas all studies show a great decrease (at least 20%) in the number of clinical cases during the treatment period For IPTc, the scarce data that exists refers to studies conducted in seasonal transmission settings, in which reported efficacy reached as much as 90% S) and treated (TS); clinical malaria resulting from infection in a completely susceptible untreated (1I) or treated individual (1TI); recovered with clinical natural immunity (R) or immunity acquired while treated (TR); and mild or asymptomatic infection resulting from exposure of recovered untreated (I2) or treated individuals (IT2). The force of infection is an age-dependent function defined previously in We built a simple model to simulate IPT interventions in a given cohort. Overlaid on a basic topology describing the transmission of malaria in a given population λ0(1−r) (at age zero) converging asymptotically to λ0 as age increases. Parameter k determines how steeply the force of infection increases with age, and r controls the magnitude of that increase.The function is strictly increasing with age, with a minimum A summary measure of transmission is obtained by integrating the force of infection over age as:Λ is proportional to the frequency of infectious individuals, the proportionality constant being the transmission coefficient, Adopting standard assumptions, β a time dependent variable. Adapting a standard sinusoidal function:β0 is the baseline transmission coefficient, and δ is the amplitude, and φ the phase of the variation.The force of infection can be seasonally forced by making a = 0 are The boundary conditions for system (1) at age γ, to the corresponding treated class, represented by subscript T. The dynamics of transmission in the treated compartments mimics that of the basic model, but is governed by different parameters representing the prophylactic effect of anti-malarial drugs, which wanes at a rate γα. Rates of drug clearance and recovery from infection for the treated classes are taken from the literature c to represent the proportion of clinical infections in treated individuals that acquire clinical immunity. We also introduce a parameter σ to account for the reduction in the risk of acquiring a new infection while treated.The transmission parameter values for the non-treated classes were estimated from datasets from 8 regions in sub-Saharan Africa The model dynamics are schematically represented in η) of clinical malaria infections is undergoing first line drug treatment. We consider that 13% of clinical cases are severe To assert the benefits of intervening on a given endemic population, we first simulate our age structured model in equilibrium conditions to obtain the age profile of clinical disease prevalence without any intervention. We use that age profile as the initial condition for the simulation in which IPTi is implemented. We assume EPI coverage of 90% in concordance with the guidelines for the EPI initiative Simulation of IPTc interventions is similar to that of IPTi, except intervention is not continuous in time, but rather for a specific age range. Specifically, interventions occur at predetermined instances over a 1 year period, targeting a given age range. We decided to mimic one study conducted in primary schools in Kenya, in which children aged 5–18 years received 3 doses of anti-malarial drugs at 4 months intervals K) and for the Senegalese site in S) were then defined as:The general time dependent transmission function was adjusted to the two settings simulated here We will present the results either in the form of age profiles retrieved at specific points in time, or time plots, where each point represents the integral of the corresponding age profile. We will convey the malaria scenario in the simulated population as proportions of clinical malaria and parasite prevalence. The former is equal to the proportion of people with clinical malaria in the overall population, in other words, the prevalence of clinical malaria, while the later is the proportion of people infected with a malaria parasite in the population, regardless of symptoms.For the sake of simplicity we have established a nomenclature to define the transmission levels associated to the simulations. The most reliable measure of transmission on a given region is the overall parasite prevalence registered at a given time. We have, then, defined 3 classes of transmission according to parasite prevalence. Henceforth, we will refer to as low transmission settings, those regions where a cross-sectional survey would detect parasites is less than 10% of the individuals. If that value is within the 11–50% range, the region is defined as an intermediate transmission setting, while parasite prevalence above 50% would categorize the region as a high transmission setting.TS class, c, and the reduced risk of acquiring an infection while treated, σ, modulate the age profiles of clinical malaria. We show the age profiles for the parameter combinations that result in the best (green line) and worst (red line) intervention impacts, as well as a parameter combination assuming an empirical estimate for σc (blue line). In c = 1, and the drug does not reduce the risk of having a malaria infection, e line,  . Generalσ and c , albeit at the cost of a rebound effect just after a few months (red line in panel A). The implementation of the same intervention in high transmission regions  (In ove 80%) , indicatThe dynamics of the impact of IPTc on clinical malaria age profiles is shown in the In We also simulated the study conducted in Senegal by Cisse et al In The seasonal model presented here is a valuable tool for the design of intervention schedules tailored to specific regions. A systematic analysis of this aspect is too extensive to be included in the present study.Intermittent preventive treatment (IPT) is a very promising approach to the management of clinical malaria burden in infants and children. However, concerns as to whether immunity is being allowed to build up during treatment, or if disease risk is actually just being postponed to later ages (rebound effect), persist.c, representing the probability of clinical immunity acquisition.Although it is well established that parasites are rapidly cleared from the bloodstream after administration of anti-malarial drugs c = 1), and the risk of infection is not reduced by treatment (σ = 1), the modelled rebound effect is minimised. If immunity is not built up efficiently (c<1), the proportion of individuals still susceptible to clinical malaria after the last dose of prophylactics may be large, thus, causing a rebound effect in later ages. If the risk of infection is reduced by treatment (σ<1), boosting of immunity is less frequent and there is a greater risk of returning to the non-immune compartment. Intervention outcomes in ages following the last drug dose schedule are highly dependent on transmission levels and on how treatment affects infection dynamics, mainly the time for which the drug effect lasts, the drug induced decreased risk of acquiring an infection, and the ability to acquire functional clinical immunity while treated.In the ideal scenario, where immunity is allowed to build up during treatment as efficiently as if the individual goes through a full length of infection , the better the outcome of intervention in terms of effectiveness is, during both treatment and follow-up periods. Even when there is a noticeable rebound effect  and stochasticity. Rather, we focus on the essential mechanisms which characterise malaria transmission on a population of individuals which exhibit identical behaviour. More detailed models which also simulate IPTi interventions The baseline transmission model presented here was fully parameterised using datasets from several Sub-Saharan settings, encompassing a broad range of transmission intensities, and its parameters were subject to an extensive sensitivity analysis in Careful evaluation of the transmission characteristics of the areas under study can be extremely useful in deciding which program will be the most beneficial for each specific region. It has been observed that P. falciparum transmission is decreasing in some settings in sub-Saharan Africa Video S1Dynamics of the impact of IPTc on clinical malaria age profiles(0.25 MB AVI)Click here for additional data file.


Malaria management policies currently recommend that the treatment should only be administered after laboratory confirmation. Where microscopy is not available, rapid diagnostic tests (RDTs) are the usual alternative. Conclusive evidence is still lacking on the safety of a test-based strategy for children. Moreover, no formal attempt has been made to estimate RDTs accuracy on malaria-attributable fever. This study aims at estimating the accuracy of a RDT for the diagnosis of both malaria infection and malaria - attributable fever, in a region of Burkina Faso with a typically seasonal malaria transmission pattern.Cross-sectional study. Subjects: all patients aged > 6 months consulting during the study periods. Gold standard for the diagnosis of malaria infection was microscopy. Gold standard for malaria-attributable fever was the number of fevers attributable to malaria, estimated by comparing parasite densities of febrile versus non-febrile subjects. Exclusion criteria: severe clinical condition needing urgent care.Plasmodium falciparum infection. In the rainy season, this proportion was 841/1,317 (64%) and 623/1,669 (37%), respectively. The attributable fraction of fever to malaria was 11% and 69%, respectively. The RDT was positive in 113/400 (28.3%) fever cases in the dry season, and in 443/650 (68.2%) in the rainy season. In the dry season, the RDT sensitivity and specificity for malaria infection were 86% and 90% respectively. In the rainy season they were 94% and 78% respectively. In the dry season, the RDT sensitivity and specificity for malaria-attributable fever were 94% and 75%, the positive predictive value (PPV) was 9% and the negative predictive value (NPV) was 99.8%. In the rainy season the test sensitivity for malaria-attributable fever was 97% and specificity was 55%. The PPV ranged from 38% for adults to 82% for infants, while the NPV ranged from 84% for infants to over 99% for adults.In the dry season, 186/852 patients with fever (22%) and 213/1,382 patients without fever (15%) had a In the dry season the RDT has a low positive predictive value, but a very high negative predictive value for malaria-attributable fever. In the rainy season the negative test safely excludes malaria in adults but not in children. Plasmodium falciparum strains, prompted a more selective approach. The new policy recommends that malaria treatment should only be administered after laboratory confirmation. This recommendation had initially been limited to older children and adults, but the new edition of WHO guidelines, released on March 9 th, 2010 [The adoption of artemisinin-based combination therapy (ACT) for malaria in most endemic countries, and the availability of new diagnostic tools, such as rapid diagnostic tests (RDTs), have led the World Health Organization (WHO) to recommend a modified approach to malaria management. The previous policy indicated the presumptive treatment for malaria of all patients with fever, unless another obvious cause was found. The increased cost of new treatments, as well as the concern for the potential selection of drug resistant th, 2010 . Moreoveth, 2010 -6.Despite many published papers on RDT performance in different countries -20, impoA recent randomized trial on RDT-based versus presumptive treatment  aiming aThis paper addresses the third and fourth knowledge gaps, aiming at assessing RDT accuracy in an area of Burkina Faso with great seasonal variation in transmission intensity and at estimating, in both seasons, how RDTs perform as predictors of malaria-attributable fever. The two issues are clearly linked, as the proportion of fevers attributable to malaria may vary greatly according to the season.RDTs are designed to detect malaria infection. In an endemic area many individuals are asymptomatic carriers of malaria parasites. A patient with fever and with malaria parasites in blood may be a case of malaria, or an individual with another cause of fever, and with incidental parasitaemia. No malaria test, including RDTs, is designed to discriminate between the two conditions, and indeed, any febrile patient with malaria parasites in blood, detected with whatever method, should be treated for malaria. However, if the parasite density is high, the fever is more likely to be due to malaria. Traditional microscopy provides a quantitative estimate of the parasite density, while RDTs do not, barely indicating the presence or absence of malaria parasites. A false negative RDT result may be of no consequence, if it fails to detect an incidental, low parasitaemia in a patient with another cause of fever. Paradoxically, a positive result might be harmful in the same patient, by confirming a clinical suspicion of malaria. RDTs based on HRP-2 protein have shown a variable accuracy for malaria infection in field studies, with microscopy taken as the gold standard. A recent review reported results ranging from 87.5% to 100% for sensitivity and from 52%% to 99.5% for specificity , the latThis study primarily aimed at estimating how accurately RDTs predict malaria-attributable fever in the low and high transmission seasons. The main steps are outlined below:1. The prevalence of malaria infection was assessed among febrile and non-febrile patients presenting at primary health care centres at the end of the dry and of the rainy season.2. The fraction of fever episodes attributable to malaria infection  was determined in both seasons.3. The accuracy of a rapid diagnostic test on malaria infection was evaluated in both seasons.4. The performance of the RDT as a predictor of malaria-attributable fever was estimated in both seasons.A cross-sectional study was carried out in May and in October, 2006, in 10 primary care health centres of the provinces of Bobo Dioulasso and Banfora, south-west of Burkina Faso, an area with stable malaria and with a seasonal transmission pattern. The lowest transmission takes place at the end of the dry season (April-May), and the highest transmission at the end of the rainy season (October). The study sites were selected with convenience criteria, as described elsewhere .th April to 19th May and 2nd to 20th October, 2006) and giving their (or their guardians') written informed consent were consecutively submitted to a standardized medical examination, and to thick and thin film, by specifically trained research assistants. The research assistants were trained by the study investigators and by professional laboratory staff from an Italian referral centre (see below) for three days preceding each study period: training included the correct execution of malaria smears (thick and thin film) and the execution and reading of the RDT.All (febrile and non-febrile) patients > 6 months consulting one of the study sites for any clinical problem during the study periods . Fever was defined as an axillary temperature ≥ 37.5°C.® Device), in the context of the randomized trial cited above [A random sample of the febrile patients, in both seasons, was also submitted to a malaria RDT  in the conventional way according with WHO criteria. A double blind cross reading of a random sample of 300 slides (thick plus thin films) was carried out in order to check for inter-observer variability, as a double reading of all the > 5,000 slides was not feasible.The reference test was malaria microscopy executed by highly experienced staff from the Centre for Tropical Diseases (CTD) of S. Cuore Hospital of Negrar, Verona, a reference centre in Italy. The thick and thin films were coded locally and transported daily to a central laboratory  for Giemsa staining by local staff, supervised by two senior microscopists from the CTD. Reading was done by the senior microscopists who were masked to the result of the RDT as well as to the clinical status (febrile or non-febrile) of the patients. A number of microscopic fields corresponding to 200 WBC were read in the thick film. The parasite density was calculated , which detects the P. falciparum specific HRP-2 protein. The tests were individually sealed, were transported and stored according to the manufacturer's instructions, and were opened a few minutes before use. They were performed and read by the trained research assistants, coded and stored for future control. For positive results, the time of appearance of the positive band was recorded. RDT reading was checked every evening by the senior microscopists.The RDT used in the study was the test ParacheckData were double-entered at Centre Muraz, Bobo Dioulasso, with Epi Info software . Data analyses were carried out with R 2.8.0 , and Stata 10.1  statistical packages.The primary aim of the analysis was to estimate how RDTs predict malaria-attributable fever in the high and low transmission seasons. The main conceptual steps are summarized below, and detailed in the following paragraphs.1. The prevalence of  malaria infection was assessed in febrile and non-febrile patients.2. The proportion of fevers attributable to malaria was estimated, stratifying by parasite density class and age group. The attributable fraction (AF) was defined as the proportion of fevers, among infected patients, that would not have occurred in the absence of malaria infection. Formulas used for AF calculation are reported below.3. The diagnostic accuracy of RDTs for malaria infection by parasite density and age group was calculated.4. The attributable fractions and the calculated diagnostic accuracy of RDT for malaria infection were combined to obtain an estimate of the sensitivity and specificity of RDT for malaria-attributable fever, accounting for both the expected increasing sensitivity of RDT for infection and the higher likelihood of a fever to be due to malaria at higher parasite densities.All analyses were performed on the rainy and dry season data, separately.P. falciparum asexual forms (any parasite density) among febrile and non-febrile patients. The AF of fever to malaria infection was estimated from the odds-ratios obtained from logistic regression modelling according to methods described for case-control studies [The prevalence of falciparum malaria was estimated as the proportion of patients with a positive slide for  studies  where paRDT sensitivity, specificity, positive and negative predictive value  were estimated for malaria infection on the subset of febrile patients undergoing the RDT, and with microscopy results taken as the gold standard. As for the AF, sensitivity and specificity were calculated for each cross-classification of parasite density and age (20 strata) for the rainy season and for each parasite density stratum for the dry season. In addition, PPV and NPV of the RDT were assessed in febrile patients during the rainy and dry seasons. Confidence intervals were estimated with the Wilson's score method. To asseBased on RDT performances on malaria infection at each level of parasite density and age, its accuracy was subsequently assessed on malaria-attributable fever.The AF-based approach does not allow classifying each individual febrile case with a positive slide as having clinical malaria, or simple malaria infection with another cause of fever. However, through this approach it is possible to estimate the number of malaria-attributable fevers at each stratum of parasite density and age, by multiplying the number of febrile cases in each stratum by the respective AF.The number of true positive RDT results was then calculated in each stratum as the product of the number of malaria-attributable fevers and the probability of a RDT positive test result for febrile patients in the stratum. The total number of RDT true positives was the sum of the RDT true positives in all strata, as in formulas reported in Table As a sensitivity analysis, the assessment of RDT diagnostic accuracy was repeated using logistic regression models for the risk of fever  and for the probability of testing positive at RDT for each individual patient, including malaria infection status (yes/no), log-parasite density, and linear and quadratic terms for age. For the rainy season analyses, age/infection status and age/parasite density interaction terms were also included. The product of the AF and probability for an RDT positive result was added up for all febrile patients to obtain the diagnostic accuracy of RDT for malaria-attributable fever, similar to the stratified analysis.th April 2006). Written informed consent was obtained through the use of an information sheet with detailed explanation of the purpose of the study and the procedures involved. Once the clinical officer had decided that a patient was eligible for inclusion, a research assistant gave the explanation in local language, in the presence of at least one independent witness. In case of agreement, the informed consent form was signed both by the patient (or one of the parents in case of minors) and by the witness. For illiterate people the signature was replaced by the fingerprint.The study protocol was approved by the "Comité National d'Ethique"  of Burkina Faso  were asked their informed consent; 5,236 consenting patients were enrolled in the study  , and conversely, in another case, a previously diagnosed very low P. falciparum parasitemia (< 50/μL) was missed by the second reading . Inter-observer variability in the assessment of parasite density was within acceptable limits: in particular, in three instances only was the class of parasite density (see below) different among observers, and the difference was of one class only. RDT reading by the research assistants presented no problem and was invariably confirmed by the study supervisors on site and by the subsequent control (every evening) by the senior microscopists. Only for seven samples (four in the dry season and three in the rainy season) the result was reported as indeterminate, and in one case (rainy season) it was missing. All eight were then excluded from the analysis of RDT accuracy.The inter-observer variability of microscopy reading was assessed on a randomly taken sample of 300 slides that were blindly re-read by the study microscopists. In one case, a slide previously read negative was subsequently read positive  in the group reporting no fever history in the last three days, and 65/474 (14%) in the group reporting such history. In the rainy season, the proportion of patients with P. falciparum malaria infection was 841/1,317 (64%) and 623/1,669 (37%) in patients with and without fever, respectively; and in particular: 318/938 (34%) in the group with no fever history and 305/731 (42%) in the group with such history. The group with fever history was excluded from the calculation of the AF, in order to avoid misclassification of febrile/non-febrile patients. The RDT was positive in 113/400 febrile patients in the dry season (28%) and in 443/650 febrile patients in the rainy season (68%).In the dry season, 186/852 patients with fever (22%), and 213/1,382 patients without fever (15%) had a The overall population attributable fraction (PAF) of fever to malaria among patients attending health clinics  was 2% in the dry season and 44% in the rainy season. Among those infected, the AF was 11% and 69%, respectively Table  and 4.In the dry season Table , only inWhen results of the rainy season were stratified by age group, it was noted that AF decreased as age increased. For children younger than five years of age a substantial proportion of fever cases was attributable to malaria even at the lowest parasite density. In infants < 1 year old, in particular, even at the lowest parasite density 1-400/μl), the AF was very high (94%), and consistently so at all levels of parasite density. Contrarily to all other age groups, there was no substantial difference in AF across strata of parasite density  fever cases in the dry season, and in 443/650 (68.2%) in the rainy season  for malaria infection in patients with fever was 72% (95% CI: 63-79) in the dry season and 88% (95% CI: 85-91) in the rainy season; the negative predictive value (NPV) was 95% (95% CI: 92-97) and 88% (95% CI: 83-92) in the dry and rainy season, respectively.In the dry season, RDT sensitivity and specificity for malaria-attributable fever were 94% and 75%, PPV was 9%, and NPV was 99.8%. In the rainy season, the test sensitivity for malaria attributable fever was 97% and was consistent across age groups, while specificity ranged between 36% (in children aged 5-14 yrs) and 78% (in adults), and was 55% overall. The PPV ranged from 38% in adults to 82% in infants, while the NPV ranged from 84% in infants to > 99% in adults. The overall PPV and NPV in the rainy season were 63% and 96%, respectively  for malaria infection, as reported in Tables 5 and 6, are within the range found by previous studies ,20,27.et al in Uganda showed that the missed treatment for patients with a false negative malaria microscopy never resulted in severe disease [Although the different test sensitivity in the dry and in the rainy season may be surprising, this difference appears to be caused almost entirely by the different mean parasite density in the two seasons: if the analysis is stratified for parasite density, the sensitivity is very similar in both seasons. The overall sensitivity was lower than 95%, the minimal level recommended by the WHO. However, most false negative results occurred at the lowest parasite density. Over 400 parasites/μl the sensitivity was higher than 95% and approached 100% over 4,000 parasites/μl. Leaving without treatment patients with false negative results at low parasite density might be relatively harmless. Niama-Meya  disease .® may remain positive for weeks after disappearance of trophozoites. A recent study showed that 70% of these tests were still positive 35 days after appropriate treatment [Unfortunately, even at very high parasite density the RDT sensitivity was not 100%. In one of our cases (a 6-year-old child in the rainy season), who had malaria with very high parasite density , the RDT was negative and was confirmed as such by expert reading. This worrisome occurrence, though rare, has been described by others for HRP-based tests , and migreatment . Moreovereatment ,33.In the dry season, after a positive RDT the probability for a fever to be attributable to malaria remains below 10%. It is unquestionable that all patients with a positive RDT should be treated for malaria. A positive RDT, however, should not influence the treatment decision for other, potential causes of fever. In settings/seasons where malaria accounts for a negligible proportion of all fevers, RDTs are claimed to be most useful, and safe ,14. ThisThe picture radically changes in the rainy season. Almost 90% of febrile children below 1 year, and almost 85% of those between 1 and 4 years, had a positive RDT Table . After aNo published study as yet has attempted to assess RDT performances on malarial fever rather than on simple malaria infection. A recent study in Mali used a different approach to estimate RDT accuracy for clinical malaria: the latter was defined with empirical criteria, using as a case definition either the diagnosis by a clinician, or the presence of fever and of ≥ 2000 plasmodia/μl at thick film ,35. The This study provides for the first time a formal estimate of how RDTs predict malaria-attributable fever in a population exposed to a highly variable malaria transmission intensity across seasons. Assessing the RDT accuracy only on infection is of course methodologically easier, but less informative. If RDTs are able to exclude malarial fever when negative, then they can be considered relatively safe even when missing some infections; if they are positive in a high proportion of febrile cases that are not attributable to malaria, then it is crucial to train health care providers not to use the positive test result as a pretext for exclusion of other possible causes of fever.Some limitations must be duly acknowledged. First, the choice of a study setting based on health facilities may be questioned as not being representative of the general population. The use of RDTs in the country is not planned at community level but in health facilities only, and therefore the study population is a sample of the population which is actually targeted for the test-based policy.Alternative methods to the AF approach to the case definition of malarial fever have been suggested . HoweverErrors  are presented in our manuscript only for malaria infection and not for malaria-attributable fever, due to a number of approximations for which the sampling error cannot be determined.® [® almost 90% of test results were positive, with a more sensitive test this percentage would be close to 100%, making it totally useless as a decisional tool.Finally, this study concerned but one out of the many commercially available RDTs that are currently under WHO scrutiny: several tests were more sensitive than Paracheck® . Would aRDTs appear to be most useful during the low transmission season: a negative test safely excludes malaria and would avoid most unnecessary treatments, if prescribers are convinced to rely on the negative result. However, they must also be aware of the low predictive value of a positive test: clinical guidelines should not limit to indicate malaria treatment in this case, but clearly recommend considering other life-threatening diseases, regardless of the test result.In the high transmission season a negative test does not safely exclude malaria in children below 5 years and particularly so in infants. Moreover, most tests are positive in febrile children. Although cost implications are beyond the scope of this paper, it is clear that in such context the cost of the tests would be simply added to that of treatment, with a waste of resources. If these findings were confirmed by other studies, a RDT-based policy should not be recommended in similar contexts for young children. Therefore, in areas with great seasonal variation in malaria prevalence, the optimal policy might not be the same throughout the year. While for adults the indications would not differ, the same is not true for children. From an operational point of view, however, it is unrealistic to suggest a differentiated policy according to the season.The cost-effectiveness of a RDT-based policy, in comparison with presumptive treatment, will be investigated, on the same study population, considering the test performances on malaria-attributable fever and not simply on infection.Despite the study limitations that are duly acknowledged, these data provide enough evidence to suggest extreme caution before moving to a generalized test-based policy in all contexts. In areas similar to this study setting the test-based policy should probably remain restricted to older children and adults, at least until better and conclusive evidence on its safety in young children is produced. General guidelines on malaria diagnosis and treatment can be misleading, should they fail to take into account the local epidemiology.The authors declare that they have no competing interests.ZB conceived the study design, wrote the study protocol, planned the training of research assistant and wrote the training manual (but for the laboratory). Trained research assistants in rainy season. Supervised enrolment and data collection in the field in rainy season. Concurred to data analysis. Wrote the draft and final version of the manuscript. SBS collaborated to the study design and writing of the study protocol. Contributed to draft versions, revised critically the manuscript.JM performed statistical analysis , contributed to draft versions, revised critically the manuscript and approved the final version. CP performed statistical analysis , contributed to draft versions, revised critically the manuscript. AA trained research assistants in dry season. Supervised enrolment and data collection in the field in dry season. Performed bibliographic research. Revised critically the manuscript. FG trained research assistants in rainy season. Supervised enrolment and data collection in the field in rainy season. Performed bibliographic research. Revised critically the manuscript.HT contributed to study design. Supervised enrolment and data collection in the field in both seasons. CL carried out first data analyses. Wrote a Master degree thesis (IMTA - Antwerp) reporting part of the paper findings for RDT accuracy for malaria infection. BN enrolled patients, trained and supervised research assistants, coordinated logistics in the field in both seasons. MG trained and supervised research assistants (laboratory) and local laboratory staff; wrote handbook for the research assistants (laboratory); performed microscopy; designed testing of reproducibility of microscopy reading. JVdE contributed with major inputs to the study design. Critically reviewed all draft versions, extensively collaborating for the final version.All authors read and approved the final version of the manuscript.Supplement Table 1. Calculation of diagnostic accuracy of RDT for malaria - attributable fever during the low transmission seasonClick here for fileSupplement Table 2. Calculation of diagnostic accuracy of RDT for malaria - attributable fever during the high transmission seasonClick here for fileSupplement Table 3. Diagnostic accuracy of RDT for malaria - attributable fever during the low transmission season based on individual level logistic regression modelsClick here for fileSupplement Table 4. Diagnostic accuracy of RDT for malaria - attributable fever during the high transmission season based on individual level logistic regression modelsClick here for file


Plasmodium falciparum diagnosis, especially in settings where quality microscopy is not available. RDTs based on the detection of histidine-rich protein 2 (HRP-2) can remain positive for several weeks after an infection is cured, due to the persistence of HRP-2 antigens. As a result, test specificity may vary between age groups with different prevalence of P. falciparum infection.The use of malaria rapid diagnostic tests (RDTs) has been widely advocated to improve P. falciparum parasites/200 leucocytes). A sample of 598 individuals living in an area of intense malaria transmission had demographic data collected before an RDT was performed. HRP-2 test sensitivity, specificity, positive and negative predictive values were calculated and compared between distinct age groups, using microscopy as "gold standard".A community-based cross-sectional survey, carried out in southern Tanzania in July and August 2004, evaluated the performance of the Paracheck Pf in comparison with microscopy  amongst people less than 25 years of age, but decreased to 81.3% in older adults. The HRP-2 test specificity varied between age groups, ranging from 25% among children of five to nine years of age, to 73% among adults aged 25 or more. The test positive predictive value increased with malaria prevalence, while the negative predictive value was consistently high across age groups.P. falciparum infection. The particularly low specificity among children will lead to the over-estimation of malaria infection prevalence in this group.These results suggest that the performance of HRP-2 tests in areas of intense malaria transmission varies by age and the prevalence of  P. falciparum to chloroquine and sulphadoxine pyrimethamine (SP). As a result, malaria treatment increasingly relies on artemisinin combination therapy (ACT), a drug regimen that is only cost-effective if it is targeted exclusively at patients with a confirmed malaria diagnosis [P. falciparum. HRP-2 persists after cure of active infections, potentially leading to false-positive test results after treatment.Rapid diagnostic tests (RDTs) for malaria were developed in the early 1990's, and welcomed with great enthusiasm as a tool to improve malaria diagnosis, especially in remote areas of Africa where access to microscopy is often limited due to the lack of resources and skilled technicians. In such settings, malaria diagnosis frequently relies on clinical criteria, which have a low specificity, leading to overuse of malaria drugs and potentially exerting increased drug pressure on malaria parasite populations leading to the selection of drug-resistant parasites. The urgent need to deploy affordable and accurate RDTs grew with the emergence of widespread resistance of P. falciparum highly endemic areas as reported in previous studies [According to the World Health Organization's (WHO) recommendations, RDTs should demonstrate a minimum sensitivity of 95% (100% when parasite density is above 100/μL), and a minimum specificity of 90% . HRP-2 b studies -12. With studies ,9,13-15.P. falciparum infection. This is despite the short-comings of microscopy, the performance of which varies according to the way in which the slide is read [et al [A relatively small amount of research has been carried out to investigate how the specificity of HRP-2 based tests, with microscopy as the "gold standard", varies between areas of distinct endemicity and malaria prevalence. Their findings are summarized in Table  is read . Hopkinsd [et al  suggested [et al ,14.The study presented here was carried out in an area of intense and perennial malaria transmission in Tanzania. In such transmission settings, the prevalence of malaria infection varies with age, and so the performance, especially the specificity, of HRP-2 based tests, might vary between distinct age groups. This community-based study aims to estimate and compare the sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) of an HRP-2 based assay between age groups, using microscopic examination of blood smears as the "gold standard".Plasmodium falciparum is the main species responsible for malaria in Tanzania, with Plasmodium malariae and Plasmodium ovale occurring in less than 5% of infections, and usually mixed with P. falciparum. Plasmodium vivax is only occasionally found. The country's national strategy against malaria at the time of the survey relied on early treatment of episodes with sulphadoxine/pyrimethamine (SP) as the first-line drug and amodiaquine as second-line drug, and the promotion of insecticide-treated bed nets to reduce exposure.This study was nested within a household baseline survey carried out in 2004 in the Tandahimba district of Mtwara region southern Tanzania. The study area and survey are described in more detail elsewhere . MalariaEight clusters of 30 households were sampled from sub villages of Litehu and Namikupa divisions of Tandahimba district using a modified EPI-type sampling scheme, such that each household in a division had an equal chance of being included in the sample . A sequeThis cross-sectional survey was carried out during July and August 2004 by two groups of seven experienced field workers accompanied by a supervisor. Villages were visited one day before the survey, and an invitation letter left in each of the selected households. A modular questionnaire was used to collect data on age, sex, location of household, demographic characteristics and various health-related information. All household members were invited for a malaria check by RDT and a small quantity of blood was collected on a filter paper. The teams collected, stained and stored blood slides for subsequent microscopic examination.P. falciparum parasitaemia was the Paracheck rapid diagnostic test device . A Swahili version of the technical sheet was developed to provide the details of the testing procedure for the field workers. A training session was conducted by an experienced laboratory technician before a practice session in the classroom, followed by a supervised pilot of all procedures in the field. Blood films were prepared by experienced laboratory assistants and labelled with a unique sample identification number before transportation to the research laboratory where they were stained with Giemsa and the number of asexual and sexual P. falciparum parasites per 200 leucocytes was counted. All blood slides were read twice by two independent technicians, and the results compared. A third reading was performed in case of positive-negative or species disagreement, or if the ratio of two positive results was greater than 1.5 or less than 0.67 with an absolute difference in parasite density of less than 30 parasites/μl. The definitive parasite count was then calculated assuming a white blood cell count of 8,000/μL.The HRP-2 test used to detect Data were entered into hand held computers at the point of data collection . StandarAfter each linkage, a consistency check was performed for the variables common to the various data sets. Data were analyzed using Stata , according to a pre-defined analytical plan. The study sample was divided into four age groups on the basis of prevalence of parasitaemia: 0 to 4 years, 5 to 9 years, 10 to 24 years, and 25 years and above. Three categories of parasite densities density were created among individuals with a positive blood smear, with cut-off points at 500 and 5,000 parasites/μL.Malaria prevalence was calculated in each category of age, sex and geographical location, and reported with a 95% confidence interval. Using microscopy as "gold standard", each HRP-2 result was categorized as true positive (TP), true negative (TN), false positive (FP) or false negative (FN), so that sensitivity (TP/TP+FN), specificity (TN/TN+FP), positive predictive value (TP/TP+FP) and negative predictive value (TN/TN+FN) could be calculated in each age group, with their 95% confidence interval. The test performance was also evaluated for categories of parasite density. In accordance with the study design, all measurement of malaria prevalence and HRP-2 test performance were performed using the "svy" command in STATA, in order to account for the sampling strategy. Geometric means of parasite densities of positive blood slides between age groups were computed on a logarithmic scale, back-transformed and compared using a one-way analysis of variance (ANOVA).Proportions were compared using Fisher's exact test, and tests for trend were performed using linear regression. A logistic regression model was used to assess the variation of test sensitivity between age groups, adjusting for parasite density. A P-value < 0.05 was considered statistically significant.An information sheet about the study was drawn up in Swahili, and read out to each household head before written informed consent was sought. In the event of a positive HRP-2 test result, study participants were offered treatment, according to the national guidelines. This study was approved by the ethics committees of the Ifakara Health Institute , the Tanzanian National Medical Research Co-ordinating Committee, the London School of Hygiene and Tropical Medicine and the Swiss Tropical& Public Health Institute .P. falciparum infections in 205 of the 598 blood smears. The sample age distribution is presented in Figure Sampled individuals were equally distributed across two divisions (Litehu and Namikupa) of Tandahimba District. Each of those two divisions is divided into three wards, where the sample population was approximately equally distributed Table . Among tPlasmodium falciparum infection prevalence based on microscopy and HRP-2 test, by baseline characteristics, is presented in Table P. falciparum and no significant differences were found between genders or across divisions  and a detailed history of recent drug treatment.The relevance of HRP-2 based assay results depends on the malaria transmission profile, the age of the patient and the aim of testing. HRP-2 test users should be aware of its very low specificity in some population age groups and malaria transmission settings, when compared with microscopy. HRP-2 test performance against PCR should be investigated in different transmission settings to determine the proportion of false positive results due to sub-patent parasitaemia. Comparison against PCR is likely to reduce variation in specificity but strong theoretical grounds remain to expect that the phenomenon of variation in test performance in different age groups and transmission settings will remain.ACT: Artemisinin Combination Therapy; HRP-2: Histidine Rich Protein 2; NPV: Negative Predictive Value; PCR: Polymerase Chain Reaction; PPV: Positive Predictive Value; RDT: Rapid Diagnostic Test; SP: Sulphadoxine-Pyrimethamine; WHO: World Health Organization.The authors declare that they have no competing interests.The study was conceived & designed by DS, JAS, PLA, HM and MT. Substantial contributions to acquisition of the data were made by KS. Initial analysis and interpretation of the data was done by AL, SK, JAS and DS. The manuscript was drafted by AL, JAS and DS. All authors were involved in critical revision for important intellectual content and approved the final version of the manuscript.HRP-2 test performance by age groups and categories of likelihood of malaria infection. (†) Fisher's exact test. (±) Test for a linear trend.Click here for file


Plasmodium falciparum and Plasmodium vivax, are prevalent. In these areas, rapid detection of the malaria parasites and early treatment of infection remain the most important goals of disease management. Therefore, the usefulness of a new rapid diagnostic (RDT), the First Response® Combo Malaria Ag (pLDH/HRP2) card test was assessed for differential diagnosis between P. falciparum with other Plasmodium species in remote villages of Jabalpur district.Malaria presents a diagnostic challenge in tribal belt of central India where two Plasmodium species, A finger prick blood sample was collected to prepare blood smear and for testing with the RDT after taking informed consent. The figures for sensitivity, specificity, accuracy and predictive values were calculated using microscopy as gold standard.P. falciparum, the sensitivity and specificity of the test were 96% and 95% respectively, with a PPV of 85% and a NPV of 99%. The RDT accuracy 95% and J-index was 0.84. For non-falciparum malaria, the sensitivity, specificity and accuracy were 83%, 94% and 92% respectively with a PPV of 69% and a NPV of 97%.Analysis revealed that overall, the RDT was 93% sensitive, 85% specific with a positive predictive value (PPV) of 79%, and a negative predictive value (NPV) of 95%. The accuracy 88% and J-index was 0.74. For The RDTs are easy to use, reliable and simple to interpret. RDTs are more suited to health workers in situations where health services are deficient or absent. Therefore, the test can be used as an epidemiological tool for the rapid screening of malaria. Plasmodium falciparum and Plasmodium vivax are prevalent [P. falciparum parasites [Malaria is a major public health problem in tribal belt of Central India where only two Plasmodium species, i.e. revalent ,2. The erevalent . Furtherarasites . Early dP. falciparum, these RDTs are based on detection of the P. falciparum histidine rich protein 2 (HRP2) or of the Plasmodium specific lactate dehydrogenase (pLDH). Species specific pLDH isoforms have been used to develop a test for P. vivax [® Combo Malaria Ag (pLDH/HRP2) card test was developed in India for differential diagnosis between P. falciparum and the other plasmodium species. To determine the usefulness of new rapid test in low endemic area where both P. falciparum and P. vivax are prevalent, the diagnostic capacity of First Response® Combo Malaria Ag (pLDH/HRP2) card test  was compared with that of expert microscopy, the gold standard. Additionally, the ease of use and accuracy of the test was also assessed.Several rapid diagnostic test (RDTs) kits for malaria exist for situations in which reliable microscopy may not be available ,6. TheseP. vivax . RecentlAnopheles culicifacies and Anopheles fluviatilis breed profusely. Medical facilities are non-existent.Jabalpur district in central India has a mixed rural, urban and tribal population. This work was performed in Bargi PHC located in forest in 25 km radius, from 21 August – 30 September 2007 during peak monsoon season. The terrain of study area is highly undulating and inaccessible. Villages are remote, thinly populated formed of six to 10 hamlets and located in field and forest. The inhabitants are mainly ethnic Gond tribe (60% – 80%) and agriculture is monsoon-dependant. Inhabitants are poor and live in small, dark, mud plastered huts without electricity. During the rains, perennial streams and its tributaries creates small water pool and remain as potential breeding site for several months in which both the malaria vector, ® Combo Malaria Ag (pLDH/HRP2) card test and thick smear before patients received treatment. In all 291 cases were tested by the RDT after taking verbal consent.All fever cases in ten villages were screened for malaria independently by microscopy and RDT to evaluate the performance of the test under field conditions. Make-shift field clinics were established in field where persons of all ages visit for checkup, thus permitting performance of the RDT in all persons suspected to have malaria, whatever their history , clinical status  and other factors that may affect the sensitivity and the specificity of the RDT. A questionnaire was filled for each patient with basic clinical and demographic information after taking verbal consent. The RDT kits were opened only after the patient had been selected and interviewed by the medical staff. Blood was obtained by finger prick for the First Response®Malaria pLDH/HRP2 Combo test contains a membrane strip, which is pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody (test line 2) is pan-specific to lactate dehydrogenase (pLDH) of the Plasmodium species  and the other line (test line 1) consists of a monoclonal antibody specific to histidine-rich protein 2 (HRP2) of the P. falciparum species. The conjugate pad is dispensed with monoclonal antibodies, which are pan-specific to pLDH and P. falciparum specific to HRP2. Blood sample was measured in a calibrated dropper capable of delivering 5 μl sample accurately into sample well followed by two drops of assay buffer (60 μl) into developer well. Test card has one control line to indicate the validity of the test procedure and it's working condition. Control and test lines appeared within 20 minutes in a reading window. Thus, the RDT is designed for the differential diagnosis between P. falciparum and other Plasmodium species. The interpretation of the test is as described below:The First Response P. falciparum .The presence of three bands  or two bands (control and test line 1) indicates a positive result for The presence of two bands (control and test line 2) indicates a positive result for non-falciparum malaria. The pLDH present in the sample reacts with the pan anti-pLDH conjugate and moves through the test strip where the pLDH is captured by pan specific anti-pLDH.The presence of only one band in the control area indicates a negative result. A one-hour workshop, including training in blood collection from finger prick, performance and interpretation of RDT was conducted at National Institute of Malaria Research Field Station Jabalpur (NIMR) under the Indian Council of Medical Research (ICMR) laboratory by one Medical Officer to two Field Laboratory Assistants (FLAs). All specimens were tested on site with the RDT by the FLAs as per manufacturer's instructions. Simultaneously, thick blood smears were also prepared.The blood smears were stained with JSB stain  and examP. falciparum and P. vivax were given standard treatment as per National Vector-Borne Disease Control Programme (NVBDCP). All adult subjects with P. falciparum were administrated the standard oral dose of chloroquine  followed by primaquine (45 mg as a single dose). Non-falciparum cases were given 1,500 mg chloroquine in three days, followed by 15 mg primaquine daily for five days. Infants and children were given proportionally lower doses. Infants were not given primaquine as per National Vector Borne Disease Control Programme.All patients infected with If the results of the RDT testing conflicted with that of the microscopy for any sample, the blood smear was re-examined by a different technician. This microscopist was also blinded to the previous microscopy and RDT results. If this re-examination gave a different result to the first examination, the second result was confirmed by a third examination by another technician.Each RDT was saved as documentation for future reference. An independent staff re-read the saved tests after two months and matched with that original interpretation of results. The RDTs were stored properly (temperature 4 – 30°C) and used within shelf life. Only tests from one batch were used .P. falciparum and non falciparum malaria separately taking microscopy results as gold standard. The figures for specificity, sensitivity, predictive values and efficiency were calculated as suggested by Tjitra et al [The performance of RDT was expressed by calculating the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) for ra et al . Data weP. vivax (14%), 71 with P. falciparum which also include one mixed infection (25%). Table In all 291 patients  with fever were suspected of having malaria (age range < 1–60 years). The mean duration of fever was four days (range 1 – 20 days) and mean temperature was 100.5 ± 1.0 (range 98.6 – 103°F) while in malaria infected persons mean temperature was 100.9 ± 1.1 (range 99 – 103°F). Out of 291, 113 (39%) were found malaria infected, 41 with P. falciparum were 72, of which RDT detected 69 matching positives. The asexual parasitaemias ranged from 80 -111,920 parasites/μl (mean ± sd 8010.5 ± 21595.2). Only three subjects were found as false negatives and 12 as false positives. The sensitivity and specificity of the test for P. falciparum was 96% and 95% respectively. The PPV and NPV were 85% and 99% respectively. The accuracy was 95% and J index 0.84  were tested positive as non-falciparum malaria by RDT.The results of parasite detection by microscopy and RDT were compared in Table 84 Table . Only twP. falciparum (≤ 0.05). However, specificity (94%), accuracy (92%), PPV (69%) and NPV (97%) were not significantly different from the corresponding values for P. falciparum.Out of 41 non-falciparum infections, RDT detected 34 matching positives, seven false negatives and 15 false positives. The asexual parasitaemias ranged from 200 – 14,800 parasites/μl . The sensitivity of the test for non-falciparum malaria 83% which was significantly lower when compared with P. falciparum and non falciparum infections), the sensitivity and specificity were 93% and 85% respectively with a PPV of 79% and a NPV of 95%. A comparison of parasitaemia versus RDT sensitivity showed that with parasitaemia of ≥ 120 parasites/μl, RDT was 98% sensitive for P. falciparum. The only exception was one subject with the parasite count of 840 parasites/μl which was negative by RDT. For non-falciparum infections, the RDT did not identify seven subjects out of 41, some of these, but not all had low parasitaemia (≤ 500 parasites/μl) and one subject with a parasite count of 4,480 parasites/μl.Overall , which is consistent with other studies[P. falciparum and non-falciparum malaria. In field setting, a negative test corresponds in the vast majority of cases to a non-infected individual.Results indicate that sensitivity for non falciparum malaria infections in this study is markedly lower 83%; CI, 69–91) than the corresponding values for r studies,19. It'sr studies. Further9–91 thanP. falciparum at 840 parasites/μl or non falciparum malaria at 4,480 parasites/μl is a serious cause of concern. This can be potentially dangerous, to miss the diagnosis of malaria in a febrile patient which may develop complications in the absence of appropriate treatment. False negative RDT results in samples with higher parasitaemia have been reported in earlier studies but the underlying reasons are not known [P. vivax is no longer considered a mild infection [However, from a clinical perspective, failure to diagnose ot known ,21,22. Pot known . Furtherot known ,25. Amonnfection -28. Analnfection . Clearlynfection . FurtherP. falciparum case shown as non-falciparum infections by RDT could have been due to mixed infections with non-falciparum parasites. Further studies in various epidemiological settings are required to establish accurately performance characteristics of this new test.The two microscopically detected In addition to performance of this RDT, some operational observations were also made. The RDTs were re-read later and recorded that results were not changed as recorded earlier using MAKROmed RDTs in South Africa .In remote and resource poor areas of central India microscopy is not readily available and it can take -four to six weeks before blood smear results are available as materials, supply lines, trained staff are not sufficient. Additionally, daily power cuts for- four to six hours is a major problem. The delay in the diagnosis and treatment of cases contributes to the continuing transmission. To control malaria, programme managers have to depend on EDPT. Given tHowever, despite its advantages over microscopy and clinical diagnosis, the cost of this RDT is high $1.15 per test  and prevents its wide-spread use in malaria endemic areas of developing countries where many patients need a fever screen. Commercial interest in producing RDTs at a cost that many of the tropical countries could afford is a subject of ongoing debate. However, whatever the RDT costs, the cost-effectiveness of the accurate diagnosis of malaria will become apparent as cheap drug CQ may no longer be effective. Furthermore, RDTs in cassette format tend to be simpler to use and this is likely to affect test accuracy and may provide saving through improved diagnosis.In conclusion, the test is reliable and simple to interpret. The test is a potential alternative to microscopy in places where the facilities for microscopy are poor. Therefore, it is reasonable to consider future use of RDTs as an epidemiological tool for the rapid screening of malaria.PKB: Data collection, analysis and interpretation of results. NS: Data collection. PPS: Data collection. MPS: Statistical analysis and interpretation of results. MS: Clinical assessment and treatment of patients. GC: Data collection. APD: Coordination of the study and manuscript preparation. NS*: Study design, preparation of manuscript, and critically reading the manuscript for intellectual content. All authors read and approved the final manuscript.


A group of children aged 6–17 years was recruited and followed up for 12 months to study the impact of schistosome infection on malaria parasite prevalence, density, distribution and anemia. Levels of cytokines, malaria specific antibodies in plasma and parasite growth inhibition capacities were assessed. Baseline results suggested an increased prevalence of malaria parasites in children co-infected with schistosomiasis (31%) compared to children infected with malaria only (25%) (p = 0.064). Moreover, children co-infected with schistosomes and malaria had higher sexual stage geometric mean malaria parasite density (189 gametocytes/µl) than children infected with malaria only (73/µl gametocytes) (p = 0.043). In addition, a larger percentage of co-infected children (57%) had gametocytes as observed by microscopy compared to the malaria only infected children (36%) (p = 0.06). There was no difference between the two groups in terms of the prevalence of anemia, which was approximately 64% in both groups (p = 0.9). Plasma from malaria-infected children exhibited higher malaria antibody activity compared to the controls (p = 0.001) but was not different between malaria and schistosome plus malaria infected groups (p = 0.44) and malaria parasite growth inhibition activity at baseline was higher in the malaria-only infected group of children than in the co-infected group though not reaching statistical significance (p = 0.5). Higher prevalence and higher mean gametocyte density in the peripheral blood may have implications in malaria transmission dynamics during co-infection with helminths. P. falciparum. Previous studies have suggested both exacerbation as well as suppression of symptoms due to one or both pathogens. Thus the possibility of synergistic or antagonistic interactions needs to be taken into account in planning and implementing interventions, so as to adjust control priorities and strategies accordingly. The most notable impact of schistosome co-infection revealed by our studies was at the prevalence of sexual and asexual stage malaria parasites, which may have implications on malaria disease severity and transmission dynamics.Malaria and schistosomiasis are the most prevalent tropical diseases in sub-Saharan Africa and together exert a huge burden of mortality and morbidity. The geographical overlap of these diseases among the individuals and at the population level commonly occurs resulting inevitably in frequent co-infections. It is not clear how helminth infections affect the outcome or the course of malaria caused by  P. falciparum.Malaria and schistosomiasis are the most prevalent tropical diseases in sub-Saharan Africa and together exert a huge burden of mortality and morbidity as well as contributing to underdevelopment of already disadvantaged populationsPolyparasitism appears to be the rule, rather than the exception, both at the population level and among individuals residing in developing countries S. mansoni and malaria had significantly greater rates of hepatosplenomegaly compared to those affected by either disease singly Buck and colleagues reported that helminth infections affect the clinical manifestations of malaria, specifically that subjects infected concurrently with Subsequent investigations revealed a wide range of disparities in findings, fueling further research in the area. Many studies found that helminths increased susceptibility to malaria P. falciparum infection. Passive transfer of immunoglobulin G (IgG) has provided protection against P. falciparum blood stage in South American (Saimiri) monkeys P. falciparum proliferation P. falciparum malaria.There is growing evidence for the protective role of IgG in We have previously described polyparasitism in the study population comprising children in rural areas of Zimbabwe S. haematobium diagnosis and quantitation, a single stool specimen for S. mansoni and other helminth diagnosis, and a further two stool specimens for quantitation. Blood was drawn for malaria and anemia diagnosis. Based on parasitological diagnosis at recruitment, children were grouped as uninfected (N), schistosome-only infected (S), malaria-only infected (M) or schistosome and malaria co-infected (S+M). The same children were subsequently followed up at two further time points of 6 months (T6) and 12 months (T12), respectively for diagnosis of malaria and schistosome infections and collection of blood samples for other study parameters.The study was a prospective 12-month follow-up of a cohort of children from the end of one malaria season to the end of the next malaria season . At eachThe study area is about 40 km east of the city of Mutare and close to the border with Mozambique. The Burma Valley area is located in the eastern province of Manicaland with a longitude of 32° 48′, latitude of 19° 50′ and altitude of 4, 200 m. The area is mesoendemic for malaria with peak transmission during the rainy season (November to May) with lower level transmission throughout the year. Schistosomiasis is also endemic in the area with transmission mainly in the dry July to October period. This is a commercial farming area in which irrigation practices aid the transmission of both malaria and schistosome parasites. The study participants were drawn from children who attended three elementary schools  within a 10-mile radius in proximity to Burma Valley Clinic, which provides health services to the population. These children predominantly belonged to low-income families who were employed as laborers in the estates. Only children aged 6–17 years, attending the three schools and who had been resident in the area for 4 years or more were included in the study.Samples for this study were obtained from children who agreed to participate following the granting of written informed consent by their parents. Samples were collected with ethical clearance from the Medical Research Council of Zimbabwe and the Johns Hopkins School of Public Health Institutional Review Board (USA) (Study number H.26.04.05.20.B2).S. haematobium , if present, are unable to pass through the filter. The filter membranes were stained with Lugol's iodine to enhance the visibility of eggs under the microscope. Children were diagnosed as schistosome infected if S. haematobium eggs were detected in any one or more of their 3 urine samples or schistosome free if none were found.Urine samples were collected from each participant between 10 am and 2 pm, on three consecutive days to account for any diurnal variations in egg shedding by the urine filtration technique S. mansoni infections and other soil transmitted helminths (STH)  infections from participants' samples as described elsewhere S. mansoni eggs were detected in their stool samples or schistosome free if not, and children who were STH infected were also treated but excluded.The formal ether concentration method was used to diagnose P. falciparum burden. P. falciparum parasitemia was estimated by counting the number of malaria parasites in random microscope fields containing 200 white blood cells (WBCs) and then multiplying the result by 40 assuming 8000 WBCs/µl P. falciparum malaria blood stage parasite was conclusive of malaria infection. Gametocyte counts were also recorded. Hemoglobin levels were measured for each subject using the Hemocue digital counter (mg/dl). The children were classified as anemic or non-anemic according to the WHO age/gender cut-off limitsAbout 5 ml of venous blood was collected from each participant into two tubes (EDTA coated and plain tubes). Plasma and serum were stored at −20°C for use in further laboratory tests for antibody and cytokine levels and their ability to inhibit malaria parasite growth. Malaria blood smears were prepared from EDTA blood for diagnosis and determination of Children who were infected with schistosomes were treated with a single dose of praziquantel (40 mg per kilogram of body weight), whilst those who had STHs received a single 400 mg dose of albendazole . Children who tested positive for malaria were treated at the Burma Valley Clinic with a combination of chloroquine and sulfadoxine/pyrimethamine in accordance with Ministry of Health and Child Welfare (Zimbabwe) guidelines (2002).P. falciparum culture (NF54) was grown in vitro and crude antigen  harvested for use in ELISA detection of antibody levels in plasma from the study participants at the different time points. Cultured parasites were harvested by centrifugation  and released from RBCs by treatment with 0.1% saponin (10 min). Parasites were pelleted  and lysed by sonication. The supernatant was collected after centrifugation at 10, 000 g for 20 min and protein concentration measured by the bicinchoninic acid assay (BCA). A 100 µl volume per well of 2 µg/ml of P. falciparum crude antigen was used to coat 96-well plates  overnight at 4°C. Plate was washed 4 times (PBS/0.05%Tween-20) to remove unbound antigen followed by addition of 200 µl of 5% milk/0.1% Tween to block antigen free surfaces of the wells for 1 hr. After washing the plates 4 times, 100-µl plasma samples (at a dilution of 1:200) were added to each well in duplicate. Plates were incubated at room temperature for 2 hrs on a rocking platform followed by 4 washes. Secondary goat anti-human IgG  conjugated to horseradish peroxidase (HRP) at 1:1000 dilution was added (100 µl/well) and the plates incubated at room temperature for 1 hr. Plates were washed and color developed by adding 100 µl of substrate 2, 2′-azino-di- per well and incubated for 30 min in the dark. Absorbance was read at 405 nm using a multi-scan plate reader and results expressed as optical density (OD) values and recorded as ratios compared to a batch of common pooled sera from malaria exposed adults, which served as the positive control to allow for comparability of samples run on different days and different plates. Pooled normal human serum (NHS) obtained from 12 subjects from the USA with no prior exposure to malaria served as negative control serum. The results were expressed as OD ratios by the formula below:42 (FVO) and AMA-1 (FVO) (100 µl per well 1 µg/ml) used to coat plates were provided by Dr S. Singh of the Malaria Vaccine Development Branch, National Institutes of Health and Dr D. Lanar of Walter Reed Army Institute, respectively. For determination of IgG isotypes, the plates were developed with peroxidase conjugated sheep anti-human IgG2 or IgG3  at 1 µg/ml.To quantify IgG isotypes to the merozoite surface protein-1 (MSP-1) and the apical membrane antigen-1 (AMA-1), the same ELISA protocol as above was used. MSP-1in vitroin vitro growth inhibition assays (GIAs). P. falciparum efficiently utilizes acetyl pyridine adenine dinucleotide (APAD) as a co-factor for the formation of pyruvate from lactate P. falciparum parasites  and merozoite surface protein-1 (MSP-1) have been shown to inhibit parasite growth 2, 5% O2 and 90% N2 using an incubator at 37°C for 48 hr before the lactate dehydrogenase assay was carried out.Parasites were synchronized once by 5% D-sorbitol treatment At the end of the 48 hr growth period, a fixed volume of parasites (50 µl) was transferred from each well into a microcentrifuge tube and spun to pellet the RBCs. The supernatant was carefully aspirated without disturbing the pellet and 40 µl of cold PBS was added to each sample and after mixing, the contents transferred to a microtiter plate. Samples were either processed immediately or stored at −20°C pending analysis. Immediately prior to use, LDH buffer  was supplemented with nitroblue tetrazolium (NBT) 0.5 mg/ml, APAD (0.05 mg/ml) and phenazine ethosulfate (PES) (0.02 mg/ml). A 100 µl volume of the complete LDH buffer was added to each well, the plate placed on a rocking platform and the reaction allowed to proceed for 40 min in the dark and absorbance read at 650 nm using a multi-scan plate reader.Statistical analysis of the data was performed using SPSS 8.0 software . The prevalence of anemia was compared according to WHO age/gender based classification and category of infection: not infected (N), schistosome only infected (S), malaria only infected (M) and schistosome and malaria co-infected (S+M) using Chi-square tests. The mean hemoglobin concentration was compared using analysis of variance (ANOVA) and either a t-test (with Bonferroni correction) or the Mann-Whitney test. Statistical significance was designated as P<0.05.S. haematobium and S. mansoni infections among the study subjects at T0 is shown in S. haematobium infection accounted for 51.3% (249/485/), S.mansoni 8.5% (41/485), whereas, a combination of both infected 13.2% (64/485) of the children and 27% (131/485) were schistosome free  and malaria free (median  = 68) children (p = 0.001 Mann-Whitney Test). On the other hand there was no significant difference in S. mansoni egg counts between S. mansoni and malaria infected (median  = 112) and malaria free (median  = 106) children (p = 0.4).We recruited 605 children (299 girls and 306 boys) at the baseline survey. However, a total of 120 children of the 605 were excluded due to presence of other helminth infections or could not be accurately assigned to any diagnostic group due to missing diagnostic data. ome free . There wWherever possible, all available data on children were utilized during analysis, however, in some situations subsets of data were used based on availability of adequate samples. In addition to loss to follow-up, not all samples had adequate blood samples for all diagnostic tests for all children at the subsequent follow-up time points. Baseline diagnostic groups were maintained across follow-up time points for comparison with baseline although the infection status  may have changed at subsequent follow-up time points. P. falciparum malaria infection prevalence was found to be higher among S. haematobium infected subjects compared to their schistosome-free counterparts at T0 (31% versus 25%) (p = 0.064) and T6 (17% versus 7%) (p = 0.053) .Schistosome-infected subjects were treated with praziquantel at each time point, although only a few subjects were re-infected or newly infected. = 0.053) . HoweverA subset of available samples was analyzed for further estimation of prevalence and density of asexual and sexual forms in the M and S+M groups. At T0 only 36% of the malaria-only infected (M) children had gametocytes by microscopy whereas 57% of the co-infected (S+M) (p = 0.06) children had gametocytes . It thusAt recruitment (T0) the baseline mean Hb levels of the four diagnostic groups showed differences . Schistosome infection by itself did not result in a significant drop in mean Hb levels and malaria infection exerted the greatest impact on Hb levels . The difAccording to WHO age and gender-based anemia classification, at T0 38% of the children in group N, 40% in group S, 64% in group M and 63% in group S+M were categorized as anemic. The proportion of anemic children at T0 in the groups M and S+M as compared to group N were significantly higher with p = 0.0001 and p = 0.01, respectively. At T6 and T12 there was no statistically significant differences in anemia levels across diagnostic groups .P. falciparum antigen extract generally increased with time over the one-year study period in all groups. The antibody levels were significantly different among groups at T0  andT6  but subsequently exhibited no difference in antibody activity to malaria antigens among the groups at T12   as well as for the cohort (n = 82) for each diagnostic group at the three different time points. Plasma IgG reactivity to crude = 0.280) . At T0, In order to determine whether there were any differences in plasma levels of antibody reactivity to specific antigens (MSP-1 and AMA-1) at T0, we repeated the ELISA to determine relative antibody levels in different groups with plates coated with the purified recombinant MSP-1 and AMA-1 antigens . The samP. falciparum malaria attacks in vitro growth inhibition .P. falciparum schizonts and S. mansoni egg antigen(SEA) Recent studies have reported cross-reactivity of IgG3 isotype antibodies to There was no statistically significant difference among the diagnostic groups in terms of growth inhibition capacity at T0 (p = 0.406), T6 (p = 0.602) and T12 (p = 0.271) by the Kruskal- Wallis test. An interesting trend was revealed by the evaluation of the subjects' plasma in GIA. At T0  the mean growth inhibition activity of plasma samples from all children included in the analysis was 58% but dropped to 20% at T6  and rose back to more than 60% at the end of the next malaria season at T12 . It is sS. haematobium infection was associated with higher prevalence of malaria. There was neither a significant difference in egg burdens nor malaria prevalence in S. mansoni infected children. Malaria-infected subjects in this population had lower hemoglobin levels than their malaria-free counterparts and schistosome infection did not exacerbate anemia. Specific IgG activity and levels showed a gradual increase over time from T0 to T12 reflecting increase in malaria specific antibody levels as expected in an endemic area. Plasma from malaria-infected subjects showed higher malaria antibody activity in response to malaria antigens. Growth inhibition activity of plasma was higher for malaria-only infected children than that from co-infected children though antibody levels were comparable for the two groups. Mean GIA of plasma samples from all children included in the analysis was higher at the end than at the beginning of malaria seasons. Such fluctuations may need to be taken into account when evaluating vaccine efficacy in areas of seasonal malaria transmission. The most notable impact of schistosome co-infection was revealed by the prevalence of sexual and asexual stage parasites which may have implications on malaria disease severity and transmission dynamics.In conclusion, in the present study it was evident that schistosome co-infection with malaria positively corresponded with higher incidence and density of gametocytes which likely leads to more intense malaria transmission. In children with higher schistosome egg burdens of