Plasmodium vivax, there has been less effort in identifying transmission hotspots. The main aim of this study was to uncover micro-epidemiological mechanisms of clustering of malaria infections at a sub-village level, based on geographical or behavioural features.Malaria elimination needs a concentration of activities towards identification of residual transmission foci and intensification of efforts to eliminate the last few infections, located in so-called ‘malaria hotspots’. Previous work on characterizing malaria transmission hotspots has mainly focused on falciparum malaria and especially on symptomatic cases, while the malaria reservoir is expected to be mainly concentrated in the asymptomatic human population when transmission is low. For A cross-sectional survey was performed in three villages within the highest malaria endemic province of Cambodia. The survey took place in the dry season, when the malaria reservoir is expected to be low and residing in the asymptomatic part of the population. Village and field locations of households were georeferenced, blood samples were taken from as many residents as possible and a short questionnaire probing for individual risk factors was taken. Asymptomatic malaria carriers were detected by PCR, and geographical clustering analysis (SaTScan) as well as risk factor analysis were performed.P. vivax was most prevalent (5.5%) followed by Plasmodium malariae (2.1%) and Plasmodium falciparum (1.6%). Mixed infection occurred in 12 individuals. In two out of three villages geographical clustering of high and low malaria infection risk was clearly present. Cluster location and risk factors associated with the infection differed between the parasite species. Age was an important risk factor for the combined Plasmodium infections, while watching television at evenings was associated with increased odds of P. vivax infections [OR (CI): 1.86 (0.95–3.64)] and bed net use was associated with reduced odds of P. falciparum infections [OR (CI): 0.25 (0.077–0.80)].A total of 1540 out of 1792 (86%) individuals were sampled. Plasmodial DNA was detected in 129 individuals (8.4%). Clusters of malaria carriers were malaria species specific and often located remotely, outside village centres. As such, at micro-epidemiological level, malaria is not a single disease. Further unravelling the micro-epidemiology of malaria can enable programme managers to define the interventions likely to contribute to halt transmission in a particular hotspot location.The online version of this article (10.1186/s12936-017-2169-1) contains supplementary material, which is available to authorized users. Plasmodium falciparum and Plasmodium vivax are the most common. These two species co-exist in many parts of the world including Southeast Asia. Whereas the understanding of the epidemiology of P. falciparum infection and disease is very broad, for P. vivax it is relatively poor . For P. vivax, net or hammock net use was not associated with the odds of infection (p = 0.885). For P. malariae, hammock net use was related to an increased odds of acquiring the infection [OR and 95% CI with No Net Use as reference: 6.866 (1.069–44.108)]. In the univariable analyses no differences in malaria prevalence according to gender, axillary temperature, previous malaria exposure and working activity were found  [In the current study, clusters of high and low malaria risks were identified in two study villages (Chamkar Sann and Phi), whereas in the third study village only clusters of high malaria risk were observed. Interestingly, when looking at different malaria species, the high-risk clusters for ce level . Moreoveus study . In partrelated) , both atAlthough clusters were observed, still a considerable number of infected persons were detected outside the high-risk clusters, and transmission in Tun showed a more homogenous pattern as compared to the other two villages. This can be explained by a truly homogeneous spread of the malaria infections in Tun or due to a limitation of the SaTScan software, which scans only in circular windows  and doesAnopheles mosquitoes extend their flight distance to the limits of their capacity in order to find a blood meal [Radiuses of the geographical clusters demonstrated a consistent pattern. Areas of high and low malaria risk situated in the field (areas of low housing densities) had a relatively large radius, whereas the opposite was true for clusters in the village centre (areas of high housing densities). This might be attributed to the dispersion of malaria vectors . Malariaood meal , 58, andAn. dirus in Cambodia. It was demonstrated that laboratory-reared malaria vectors became mostly infectious when membrane-fed on venous blood of symptomatic parasite carriers, and only very few by feeding on blood of asymptomatic individuals [An. dirus, while several primary and secondary vectors occur in Southeast Asia, an important drawback of the experiment is that it was conducted on the adult (18+) part of the population only. In a separate study in Thailand, it was demonstrated that only non-adult (below 18 years old) individuals became actually infectious to mosquitoes [It is noteworthy that only one symptomatic case of malaria was found by RDT, indicating that the observed clusters are almost exclusively composed of asymptomatic carriers. Bejon et al. showed that groups of homesteads consisting of asymptomatic carriers can act as stable clusters over several years . Therefoividuals . While osquitoes . Immune P. vivax or recrudescences of P. malariae. This means that risk factors can only be interpreted as risk factors for being a P. vivax or P. malariae carrier, regardless of how long ago transmission of infection has occurred.Numerous behavioural, genetic and environmental factors can be the cause of an increased risk of infection. The present study only covered certain behavioural and intrinsic individual aspects to detect a pattern among malaria-infected individuals. It is important that the described outcomes should not be over interpreted, since this study was conducted in an area where malaria prevalence was very low, although participation rate was very high in all three villages (82–91%). Shortly after this survey, a more in-depth social science study was organized in the same study sites to further extend current findings. As in a previous study in this region , in thisAnopheles vector biting times. A more plausible reason for this phenomenon is that the odds of getting infected are reduced when transmission is low, resulting in a shift towards an increased age of getting a first infection [The current study confirms the findings of other studies in low endemic settings , 59, 60,P. malariae prevalence drastically increased among people using a hammock net. The latter result is likely an artefact from confounding with the location and activities associated with using a hammock net.Self-reported use of bed nets was remarkably high (95%) in accordance with a previous study in Cambodia . A reducForest and field activities are known risk factors for malaria, especially in Southeast Asia , 61. In Nevertheless, in the present study most parasite carriers had their main residence in the field houses and close to the border in the village centre, adjacent to forested areas or small streams and rice fields. Other studies have indeed indicated that both proximity to the forest and vector breeding sites are significant risk factors that could elevate the malaria prevalence , 53, 54.Housing structure in the field was quite poor in Chamkar Sann and Tun. Houses were open and inadequately constructed, as compared to the housing facilities in the village centre Fig. . SeveralThe impact of targeting interventions towards high risk areas or malaria hotspots remains elusive. Early results, obtained from a cluster-randomized controlled trial conducted in Kenya were unfortunately not promising . HoweverIn this micro-epidemiology study carried out in three structurally different villages, geographical clusters of high and low malaria infection risk were clearly identified in two villages. The geographical clustering of malaria carriers was observed to be located in remote areas, and was malaria species specific. Risk factors also differed between malaria species. As in macro-epidemiological studies, age is an important factor for vivax malaria in the current micro-epidemiology study. Net use reduced the odds for falciparum malaria. Migration and evening activities most probably are important factors and should be further investigated. As such, at micro-epidemiological level, malaria is not a single disease. In contrast to the macro-epidemiological studies, which are useful in guiding the focus of attention, further detailing the micro-epidemiology of malaria can actually enable programme managers to define the interventions likely to contribute to halt transmission in a particular hotspot location.


P > 0.05). The profound environmental modifications that occurred in the rice zone would have led to changes in vector behavior and consequently to changes in the epidemiological profile of malaria, contrary to the results obtained since the last publications. An entomological study correlated with this study is therefore necessary for effective decision-making for the malaria control in both areas. Future research must now focus on the impact that these profound environmental modifications of rice area are having on malaria control in Burkina Faso.Twenty years after the latest publications performed on the parasitological indices of malaria transmission in northwest of the second city of Burkina Faso, it was important to update the epidemiological profile of malaria in children under the age of 15 years. The objective of this study was to determine and compare the parasitological parameters of malaria transmission by season, area, and age in the two zones (rice and savanna) in the northwest of Bobo-Dioulasso, Burkina Faso. Overall, the results showed that there was no significant difference in the parasitological indices of malaria transmission within children under fifteen years between the rice site and the savannah site and whatever the season ( Febrile illness is the most common and important component of malaria syndrome in sub-Saharan Africa . Childre Anopheles mosquitoes. The landscaped perimeter of “Vallée du Kou” was realised within the framework of cooperation between the Volta (today Burkina Faso) and China in 1969 and construction began in 1970 on 1000 hectares. This perimeter led to health consequences by promoting the proliferation of Anopheles mosquitoes; in particular, the density of Anopheles gambiae, the major vector of malaria transmission in the area, multiplied 20-fold and this was followed by a doubling of the rate of malaria transmission [Furthermore, Burkina Faso, like many other African countries, is also facing major food security challenges. The solution to this challenge has been a policy to promote the development of irrigated crops including rice and is based on water management and the development of irrigation schemes. Although hydroagricultural works can provide enormous benefits, it is also acknowledged that population health can be negatively influenced by them through profound changes in the ecosystem. Indeed, irrigation promotes the proliferation of mosquito breeding sites and affects both the duration and speed of replication ofsmission . The annsmission , 6.However, while past results have been focused on the evaluation of entomological parameters of malaria transmission, few studies have focused on the evaluation of parasitological parameters of malaria transmission in both the rice-growing area and the surrounding savannah.Twenty years have passed since the last publications were published on the parasitological indices of malaria transmission in the area , 8. This An. gambiae s.l. is the major vector of malaria in the study area, with Anopheles gambiae predominating in some places while Anopheles coluzzii is predominant in the rice-growing area; An. funestus and An. arabiensis are present in small proportions and frequencies increase towards the end of the rainy season [The District of Dandé is located in the northern-west of Bobo-Dioulasso . The cliy season , 9.Two localities from this district were retained as our study sites as representative of two ecoepidemiological areas. Study sites in rice and savanna areas are, respectively, the Kou Valley 1 (VK1)  and Samandeni . These two localities are separated by 15 km and are located on the Bobo-Faramana-Mopti road. The populations of the two study sites are composed of about fifteen ethnic groups and the main spoken language is Dioula. Populations are engaged in subsistence agriculture in the savanna area; however in the rice area the main activity is the cultivation of rice. According to the data from Dandé District in 2014, the two localities counted about 4018 and 10555 inhabitants in VK1 and Samandeni, respectively. Children and adolescents (0–14 years) represented 45.1% of the population. The prevalence of malaria in children 0 to 14 years old one year before the start of the survey based on rapid diagnostic tests (RDTs) and reported by the respective health facilities was 29.8% in VK1 and 15% and in Samandeni.We have carried out a random sampling by relying on subjects who were present at the time of our passage in each site until we reached the estimated number of subjects planned for the study. based on a random sampling of 80 to 110 children from 0 to under fifteen per passage, we conducted six passages, three passages in the dry season  and three passages in the rainy season  in each study site. The objective was to reach at least 300 children per season and per site required for the study, considering prevalence of 29.8% in the savanna zone and 15% in the rice zone in rainy season with an accuracy of 10%, standard deviation (1.96) corresponding to the risk of error 5% and 10% of nonrespondents.The study included all children in the village aged between 0 and under 15 years whose parents gave their consent. Those excluded were study children whose parental consent was not obtained and children not residing in the two sites selected. Also, subjects who received antimalarial treatment during the last 14 days are excluded.The study was conducted for one year, from November 2014 to October 2015. P. falciparum parasites in fields containing 200 white blood cells and multiplied that number by 25 to estimate the number of asexual P. falciparum parasites per μL  in phosphate buffer (pH 7.0) for 30 minutes, and examined using oil immersion magnification (100x). Each slide was examined by two microscopists, who estimated the parasite density by counting the number of asexualμL) of blood. Parasite densities obtained by the study site were classified and annotated as follows: 1: less than 1000 parasites/μL; 2: 1,000–5,000 parasites/μL; 3: 5,001–10,000 parasites/μL; and 4: more than 10,000 parasites/μL.The parasite carriage rate or the plasmodic index (PI) was estimated by the proportion of positive numbers from examined thick smears and the gametocytemic index (GI) by the proportion of subjects presenting sexual forms or gametes in the thick smear. Parasite densities of sexual and asexual forms were expressed as the mean number of parasites per microliter (R).Axillary temperatures were measured by skilled health personnel in order to detect any temperature greater than 38°C (the pyrogenic threshold considered in our study). Any child who had fever above 38°C was considered clinically ill if he/she had fever (a core body temperature ≥ 38°C) and malaria parasites identified from the blood smear. Any child that was clinically ill at the survey date was taken to the nearest public health facility for treatment free of charge. The temperature was taken using an electronic thermometer . After its approval, each participant was then informed about the protocol; care was given to all study participants. Oral informed assent was then obtained from each participant. All the study participants considered clinically ill were examined and treated in line with the national malaria guidelines of Ministry of Health, Burkina Faso.Excel and GraphPad Prism 5 software were used, respectively, for entry and analysis data. The malariological indices were determined and compared by site, season, and age range to determine the variations between the two ecoepidemiological areas. The nonparametric Wilcoxon signed-rank test was used to compare longitudinal samples throughout season and the Mann–Whitney test for mean comparison between areas with an alpha of 0.05.P > 0.05).The census of children under fifteen years of selected households in the two study sites counted 1,121 children of both sexes eligible for this study. There were 555 children from VK1 and 566 children from Samandeni. Their distribution according to sex by site ranged from 242 females to 313 males from VK1 and 246 females to 320 males from Samandeni . HoweverP > 0.05).A total of 1121 thick and thin smears were performed during the study period and examined. 558 of them were carriers of malaria parasites and the mean plasmodic index was 49.77% (CI: 34.99–58.01). The plasmodic index was 40.85% (95% CI: 21.89–59.81) and 61.66% (95% CI: 47.55–75.76), respectively, in the rice zone and in the savanna zone. It was higher in the savanna zone and lower in the rice zone, but this difference was not statistically significant (P > 0.05). But it was higher in Samandeni (savanna zone) than in VK1 (rice area) at the beginning of the dry seasons (November 2014: mean PI 80.72% versus 24.69%) and rainy season (June: mean PI 51% versus 17.18%) (P < 0.05). However the evolution of the plasmodic index according to the dry season months showed a higher parasitaemia in January and April in the rice zone compared to the savanna zone (P > 0.05)  .Plasmodium falciparum was the most prevalent in the dry season, as well as in the rainy period, but with a higher proportion in the rainy season whatever the site. Three species of malaria parasites have been observed during the rainy season in the savanna area while two species  were detected only during the dry season in the rice zone. Mixed infections  were observed in positive slides only during the rainy season in the rice zone and in both seasons in the savanna zone. Three cases of mixed infections were found in the rice area compared to 15 cases in the savannah area. No cases of triparasitism were found in either site.The plasmodic index increased with age, as it was very low in toddlers and reached a maximum in children between 5 and 10 years old, but it then decreased in children >10 years old, regardless of the study site. The children between 5 and 10 years old were the most infected and had plasmodic index of 62.33% in Samandeni and 38.15% in VK1 . The leaP > 0.05) and between the two study sites (P = 0.11).For the two biogeographic zones, the plasmodic index showed no significant differences between the different age groups by site . In the savanna zone, gametocytic index was 1.94% (11/566). This difference in the gametocyte rate found between the two sites was statistically significant (P < 0.05). Gametocytes were found in the positive slides, whatever the period and the study site but with a higher proportion in the rainy season in the rice zone (2.70%) compared to the savanna zone (0.5%). The subjects likely to infect vectors became more important in the population during the rainy season in the rice-growing area.Of the 551 thick smears examined for the rice-growing area, twenty-one of these showed gametocytes ofFor all 1,121 specimens examined, the mean gametocytemic index was 2.85%.µL of blood and the highest was 471,889 parasites/µL, all observed in Samandeni during the dry season (November 2014 and January 2015). For a better interpretation of parasite density in order to compare the two study populations, we expressed parasite densities by the geometric mean parasite density (GMPD) in children from 0 to 15 years, taking into consideration the positive and negative slides examined. The difference in geometric mean parasite densities between the rice area  and the savanna  was not significant . However, geometric means have evolved significantly in each study site (P = 0.03). In contrast, they were comparable whatever the season in the two study sites (P < 0.05).The lowest parasite density was 16 parasites/μL of blood were most frequently encountered in children aged 5 to 10 years, 36.75% in Samandeni (208/566) and 22% in VK1 (122/555) at both sites (P = 0.01). There were 4% against 5% of subjects, respectively, in rice and savannah areas who had parasite densities above 10,000 parasites/μL.Parasite densities less than 1000 parasites/ Plasmodium. This difference is not statistically significant (P > 0.05).Of a total of 555 children examined in VK1, 217 were carriers of malaria parasites and 140 were febrile (25.22%). Of the 140 participants with fever, 43.57% were carriers of malaria parasites compared to 56.43% who were not carrying Plasmodium compared to 46.92% with fever but no Plasmodium (P < 0.001).The presence of fever in VK1 was not related to the presence of asexual malaria parasites in the blood (OR < 1). Contrary to VK1, the presence of asexual malaria parasites was strongly related to the presence of fever in Samandeni (OR = 28.61 with 95% CI: 3.60–158.80), with 53.07% of participants having both fever andasmodium . In bothP < 0.01) with a parasite density > 10,000 parasites/µL . However, there was no variation in the different parasite density groups in Samandeni, but the risk of having a febrile attack was higher in the lowest parasite density group (1000 ≤ DP < 5000).The risk of having a febrile attack increased with parasite density group in VK1. This risk became significant  using the criterion of the plasmodic index in children aged 2 to 9 years. The plasmodic indices (PI) obtained confirm in this study area the presence of hyperendemic malaria according to the plasmodic indices observed in children aged 2 to 9 years in the two study sites.The plasmodic index indicates the percentage of subjects carrying P. falciparum malaria in Africa, and there is a very high degree of immune protection in the population. In our study, the predominant plasmodial species in both localities was P. falciparum. This confirms the literature data that report that this species is most common in West Africa [ Plasmodium either alone (95%) or in association with P. malariae (1–4%) or P. ovale (about 1%). These prevalence rates were higher than those of Meetselaar and Van Thiel [ P. vivax were observed, which was congruent with historical epidemiological studies [Stable malaria is referred to when the transmission season is very long and there is little change in incidence during the year and from year to year. In such conditions, climate change is too insignificant to influence anopheline transmission activity, temperature ensures a rapid sporogonic cycle, the vector is highly anthropophilic, and vector survivorship is long. Stable malaria is most often due tot Africa –13. The t Africa , is likean Thiel , in the  studies . In addi studies –17. IndeThis study investigated the epidemiological profile of malaria in ecologically different environments in children under 15 years in Burkina Faso. In Burkina Faso, as in most of the developing countries, food security is crucial and requires water storage and management. The intensification of irrigated crops is a response to this challenge. Our study suggests that hydroagricultural developments in the northwest of the city of Bobo-Dioulasso influences malaria prevalence and morbidity rates in the region. Indeed, the morbidity rate was comparable between rice area and savanna area. We think that the development of irrigated crops, in particular rice cultivation in Sudan zones as in Burkina Faso, has encouraged the proliferation of mosquitoes, some of which are malaria vectors which would lead to increased number of malaria cases. The once stated theory of the “paradox of Vallée du Kou” seems to have changed over time, and now the rice cultivation zone has similar ecoepidemiological characteristics of the nearby savannah zone.


Malaria is one of the most important causes of morbidity and mortality in sub-Saharan Africa. The disease is prevalent in over 75% of the country’s area making it the leading public health problems in the country. Information on the prevalence of malaria and its associated factors is vital to focus and improve malaria interventions.A cross-sectional study was carried out from October to November 2012 in East Shewa zone of Oromia Regional State, Ethiopia. Adults aged 16 or more years with suspected malaria attending five health centers were eligible for the study. Logistic regression models were used to examine the effect of each independent variable on risk of subsequent diagnosis of malaria.Plasmodium species were P. vivax (54%) and P. falciparum (45%), with mixed infection of both species in one patient. A positive microscopic result was significantly associated with being in the age group of 16 to 24 years , 25 to 34 years , and 35 to 44 years  compared to 45 years or older; being treated at Meki health center , being in Shashemene health center , and living in a rural area compared to an urban area .Of 810 suspected adult malaria patients who participated in the study, 204 (25%) had microscopically confirmed malaria parasites. The dominant P. vivax and P. falciparum infection. Thus, appropriate health interventions should be implemented to prevent and control the disease.Malaria is an important public health problem among adults in the study area with a predominance of The online version of this article (doi:10.1186/s12889-017-4577-0) contains supplementary material, which is available to authorized users. P. falciparum and P. vivax are the two predominant malaria parasites in the country, accounting for 60–70% and 30–40% of infections, respectively , those who were in the age group of 25 to 34 years , those who were in the age group of 35 to 44 years were more likely to have positive test result for malaria  as compared to those in the age group of above 44 years. Those who were living in rural areas were more likely to have positive test result for malaria  as compared to those who were living in urban area . As this was a pragmatic study in a real-life rural environment, blood film was available to diagnose malaria infection, rather than rapid diagnostic testing which has a higher sensitivity . On top P. vivax and P. falciparum; and being in the productive age group, living in Meki or Shashemene, and living in rural areas, were risk factors for malaria infection. According to WHO when the slide positivity rate of all febrile patients with suspected malaria is less than 5%, the country could consider transitioning into “pre-elimination” [In conclusion, findings of this study indicate that malaria is an important public health problem among adults in East Shewa with the predominance of ination” . Therefo


Immunity to malaria could be categorized broadly as antiparasite or antidisease immunity. While most vaccine research efforts have focused on antiparasite immunity, the evidence from endemic populations suggest that antidisease immunity is an important component of natural immunity to malaria. The processes that mediate antidisease immunity have, however, attracted little to no attention, and most interests have been directed towards the antibody responses. This review evaluates the evidence for antidisease immunity in endemic areas and discusses the possible mechanisms responsible for it. Given the key role that inflammation plays in the pathogenesis of malaria, regulation of the inflammatory response appears to be a major mechanism for antidisease immunity in naturally exposed individuals. Plasmodium falciparum malaria is partly mediated by direct injuries inflicted on the host by the parasite, as in the case of haemolysis of red blood cells (RBCs) during parasite egress,The clinical manifestation of Humoral responses (antibody responses) have been extensively studied in the context of antiparasite immunity and clinical protection against malaria, given that the understanding of these responses can be harnessed towards the development of vaccines.Antiparasitic immunity, particularly antibody responses, are elicited during cumulative clinical bouts of malaria, but these responses are slowly acquired and/or are not adequately induced at levels that would confer protection in young children. As well as the problem of antigenic diversity and clonal variation on the parasites’ side, the past decade has led to the identification of several ‘faults’ that may explain why antibodies are slowly acquired, and not adequately induced in younger children. A few notable mechanisms include (i) the identification of a subset of memory B cells called ‘atypical’ memory B cells, which are inefficient at secreting antibodiesin vivo. We emphasize the necessity for high antibody concentrations for the generation of significant in‐vitro growth inhibition and in‐vivo protection. Finally, we provide evidence of parasite tolerance at high levels of exposure: a phenomenon that hinges on the control of inflammation.Although eliminating the parasite possibly by antibody‐mediated processes is vital for protection, mechanisms that allow parasite tolerance without manifesting clinical symptoms, as observed in asymptomatic carriers ,Clinical immunity to malaria infection is two‐pronged. This is because during malaria infection, two mutually inclusive processes precede pathology: (i) parasitaemia,α and interferon (IFN)‐γ, than symptomatic individuals.While both antiparasite and antidisease immunities are convoluted, and are both required to curb parasitaemia and disease manifestation, there seems to be a difference in the end‐point targets of both effector processes. Usually, high levels of antibodies correlate with reduced parasitaemias,This suggests that keeping inflammatory mediators low can prevent the manifestation of disease (antidisease immunity). Such a mechanism will complement the role of antibody responses (antiparasite immunity) and other effector processes that mediate parasite killing.Aotus monkeys, where it was demonstrated that serum from chronically infected monkeys could potentiate malaria immunity.The first insight into a role for antibodies in preventing parasitaemia and clinical malaria was seen in in‐vitro growth inhibition or invasion inhibition assay (GIA) results for most of these vaccine candidates demonstrate that high antibody concentrations against these antigens are required for significant growth inhibition. Furthermore, even at such high antibody levels, growth inhibition is rarely absolute. Antibodies against cysteine‐rich protective antigen (CyRPA) (full length) in two‐cycle invasion assays showed 85% inhibition at 10 mg/ml,42,70 and a 40% invasion inhibition was observed for antibodies against Rh5 interacting protein (Ripr) (ectodomain) at 2 mg/ml in a one‐cycle assay.in‐vitro studies showed that high levels are required to attain considerable invasion inhibitions.For merozoites to invade new RBCs, they must sequentially deploy several invasion ligands to engage their cognate receptors on the RBC surface.P. falciparum antigens, including vaccine targets, varied in a Kenyan cohort, with most (96%) glycophophatidylinositol (GPI)‐anchored antigens being detected, whereas rhoptry‐associated proteins, which are mostly conserved antigens, were sparingly (5%) detected.P. falciparum sporozoite‐based malaria vaccine (PfSPZ) might provide a glimmer of hope, but similar to the pattern observed for the RTS,S vaccine, protection was also observed in 64% of vaccine recipients in a controlled human malaria infection and, particularly, antibody levels did not segregate protected from non‐protected individuals.Levels of antibodies against recombinant antigens of vaccine candidates may, or may not, be detected in the plasma of children [by enzyme‐linked immunosorbent assays (ELISAs) or protein microarray] from malaria‐endemic sites in a healthy state (baseline levels), depending on age and history of exposure. For instance, levels of PfRH5 antibodies were detected in < 16% of healthy children from a Malian cohort.per se. For instance, it was observed that Rh5 responders at baseline were associated with a median 34‐day delay to the first clinical bout of malaria compared with non‐responders.Although higher antibody titres, relative to baseline levels, are generally observed during concurrent acute infection in children, several rigorous attempts to determine the specific antibody, or antibody combination, that best serve as correlate(s) of immunity have yielded disappointing results. Usually, high antibody levels are more associated with a delay to first malaria infection, and lower parasitaemias at presentation, than protection in‐vitro findings provided evidence that buttressed the idea of a multicomponent vaccine. Several studies are now being carried out to investigate humoral responses to a large panel of antigens to determine the best combination of antigens to include in a multicomponent blood stage malaria vaccine. Osier et al.Fortunately, promising synergistic inhibitory effects at comparatively lower antibody concentrations are observed when antibodies are used in combination for invasion assays, as seen in PfCyRPA–PfRh5 and PfF2–PfRH5–PfAARP–PfRH2 combinations.α, IL‐1β and IL‐6,α and IFN‐γ,93 may preclude severe disease and, perhaps, the mild clinical manifestations of malaria. Interestingly, there are data from endemic regions of Africa that show higher parasite tolerance  at high exposure levels; and high parasite tolerance appears to be associated with reduced inflammatory responses.The cascade of cytokines released during the erythrocytic stages of infection, particularly pyrogenic cytokines such as TNF‐94 such that in children from areas of low‐to‐moderate transmission intensities, symptomatic malaria is associated with comparatively lower parasitaemiaThe threshold parasitaemia associated with fever (pyrogenic threshold) differs depending on the prevailing transmission intensity,19, MSP‐2 and AMA‐1 declined with increasing altitude (lower exposure levels).This exposure‐dependent disparity of clinical and malariometric features of malaria has led to the investigation of the associated quantitative and qualitative cellular and humoral immune responses. Observations to date show that children who are highly exposed to malaria usually have higher antibody levels compared with children exposed to low‐to‐moderate transmission intensity, although antibody levels generally increase with age in both high‐ and low‐transmission areas.γδ T cells, and CD4+ IFN‐γ responses in naive Dutch subjects compared with observed levels in exposed Tanzanian adults from an endemic region,We and others+ T cells.+ T cells that produce IL‐10 and coproduce IFN‐γ and IL‐10 were observed in children from the high‐transmission area, whereas CD4+ T cells of children from the low‐transmission area predominantly produced IFN‐γ, TNF‐α and/or IL‐2, suggesting increased inflammatory responses in children at low exposure levels.+ T cells from children with ≥ 2 prior episodes of malaria also coproduced IFN‐γ and IL‐10 after stimulation with infected RBCs (iRBCs), whereas CD4+ T cells from children with fewer than two prior malaria episodes produced TNF‐α only, without the co‐production of IL‐10,in vitro, compared to responses from children who have been historically exposed to high transmission but now live in an area of little to no exposure,Additional studies using multiparameter flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) from asymptomatic children with higher exposures revealed a skewness towards an immune‐regulatory effector phenotype of CD4P. falciparum antigens induced proliferative defects in T cells.in vitro with malaria antigens, as opposed to marked proliferation that was observed in PBMCs from uninfected people, and in mitogen stimulated PBMCs from infected individuals.A possible mechanism may involve the direct immunosuppression of immune cells by parasite antigens and/or parasite products, leading to the functional impairment of immune cells. This mechanism is evident from a report where et al.P. falciparum erythrocyte membrane protein (PfEMP)‐1;110,111 however, ‘stunning’ of DCs was later shown to be PfEMP‐1‐independent.in‐vivo studies from endemic areas showed that high numbers of hemozoin containing monocytes/macrophages were associated with decreased cytokine [IFN‐γ and regulated on activation, normal T cell expressed and secreted (RANTES),in‐vitro data of hemozoin‐induced functional impairment of immune cells.Urban As higher parasitaemias,+ T cells were lost after P. chaubadi infection.+ T cells, as ovalbumin‐specific T cells were not depleted in a murine malaria model.P. falciparum‐infected individuals compared with the observed numbers when these individuals were healthy,The loss of T cells is perhaps the commonest observation of malaria infection in murine models and human malaria. One murine study found that 25% of memory T cells were lost in untreated mice 60 days post‐infection, compared with levels in chloroquine treated mice 34 days post‐infection.δ2 subset of γδ T cells was recently observed in the peripheral blood of children repeatedly exposed to malaria infection.in‐vitro stimulation of these subsets of γδ T cells with parasite antigens. In contrast, the same subset of γδ T cells from children with low prior exposure histories proliferated and produced high cytokine levels, but showed lower expression of immunomodulatory genes. Gamma delta T cells have been shown to preferentially expand during malaria infection,δ2 subset secretes high levels of IFN‐γ upon parasite encounter to mediate parasite killing.γ has been associated with immunopathology and severe malaria syndromes,γ‐secreting cells would mean reduced inflammation, as has been observed at high exposure levels. The same group later reported reduced absolute numbers of forkhead box protein 3 (FoxP3)+ T cells in the peripheral blood of children with higher prior exposure to malaria,Increased lymphopenia, particularly of inflammatory cells, may result in a reduced inflammatory response at high exposure levels. A decline in the numbers of VSome ‘atypical’ mechanisms responsible for reduced inflammation might be at play at high exposure levels. Perhaps immune cells have become refractory to stimulation from repeated high exposure levels, and thus require higher antigen doses (i.e. higher parasitaemia) to cause the activation of immune cells. This proposition agrees with the notion of an adaptive cellular interaction, where the activation thresholds of immune cells are being reset to higher levels following each antigen encounter.+ T cells with an ‘exhausted’ phenotype have been observed during acute infections in murine models as well as human models. This exhausted phenotype is mediated by programmed cell death protein (PD‐1) ,A parallel mechanism may involve an increased propensity of immune cells of children who reside in hyperendemic areas to become terminally differentiated, and consequently display exhausted and/or tolerized effector phenotypes.otein PD‐  of malaria.+ T‐cell response, which led to enhanced parasite clearance; and via a mechanism that increased immunomodulation, which was characterized by increased regulatory T cells.As immunoregulation seems to be at the heart of antidisease immunity, some studies have now started exploring the possibility of modulating exaggerated inflammatory responses as a therapeutic choice. For instance, the engagement of immune checkpoint molecules, such as PD‐1 by its ligand PD‐L1 (programmed death‐ligand 1), normally leads to impaired T‐cell function and expansion.γ, a potent inflammatory cytokine that is produced by activated terminally differentiated killer‐cell lectin‐like receptor G1 (KLRG‐1)+ Th1 cells during malaria infection.+ Th1 cells during malaria infection by suppressing the responsiveness of these  cells to IL‐12 and IL‐2 signalling.+ Th1 cells.Another study explored the consequence of restricting the production of IFN‐α, during concurrent malaria infection in treated patients compared with observed levels in placebo controls.In a more clinically relevant context, it was observed in a randomized, double‐blind, placebo‐controlled clinical trial that treatment with Rosiglitazone led to lower levels of pyrexins, such as IL‐6 and TNF‐Considering that the immunopathology from acute inflammatory responses leads to the clinical manifestation of malaria, a scenario where acute inflammation is modulated (by unknown mechanisms including those listed above) may preclude disease manifestation, even when parasitaemia persists. This might be the case in high‐transmission areas, where infections are somewhat chronic in nature, i.e. high parasite exposure in these areas favours repeated infections which mimic one chronic infection. Under this condition, the continuous exposure to parasites by immune cells leads to an increased capacity to tolerate parasite presence. Therefore, higher parasitaemia is required to activate immune cells and cause an inflammatory response that is often mild Fig. b. Mild i150 suggesting a scenario where exposure to blood‐stage parasites was ‘knocked‐down’ among the vaccinated cohort. Considering that vaccinated children were shown to be at higher risk of clinical malaria at the end of the study compared to unvaccinated ‘wild‐type’ children,These observations have important implications for vaccine efficacy studies and future vaccine designs, particularly vaccines that prevent exposure to blood stage parasites altogether. For instance, RTS,S, the most advanced malaria vaccine, was associated with serological evidence of reduced exposure (reduced breadth and magnitude of antibodies),Further work is required to clearly elucidate the mechanisms of parasite tolerance leading to clinical immunity. Ideally, such studies should be longitudinal; for example, using a birth cohort to investigate the evolution of inflammatory responses as well as adaptive immunity from the first infection of the malaria‐naive infant until acquisition of clinical immunity.The paper was written by TWA and GAA. GAA critically reviewed the paper and provided mentorship to TWA.TWA and GAA were supported by funds from a DELTAS Africa grant (DEL‐15‐007: Awandare) and the Royal Society of Tropical Medicine and Hygiene Small Grant (GR000775: Ademolue). TWA was also supported by a Master's fellowship from a World Bank African Centres of Excellence grant (ACE02‐WACCBIP: TWA). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)'s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa's Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (107755/Z/15/Z: TWA) and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government.The authors declare no competing interests.


Dengue fever (DF) and malaria are the two major public health concerns in tropical countries such as Thailand. Early differentiation between dengue and malaria could help clinicians to identify patients who should be closely monitored for signs of dengue hemorrhagic fever or severe malaria. This study aims to build knowledge on diagnostic markers that are used to discriminate between the infections, which frequently occur in malaria-endemic areas, such as the ones in Thailand.A retrospective study was conducted in Phop Phra Hospital, a hospital located in the Thailand-Burma border area, a malaria-endemic area, between 2013 and 2015. In brief, data on 336 patients infected with malaria were compared to data on 347 patients infected with DF.P < 0.0001). In contrast, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were significantly higher in patients with DF as compared to patients with malaria (P < 0.0001). A decision tree model revealed that using neutrophils, lymphocyte, MCHC, and gender was guided to discriminate between dengue and malaria infection.White blood cells, neutrophil, monocyte, eosinophil, neutrophil-lymphocyte ratio, and monocyte-lymphocyte ratio were significantly lower in patients with DF compared to patients with malaria  contains supplementary material, which is available to authorized users. Please see Additional file P. vivax malaria makes up 75% of all malaria cases, and most cases of P. vivax malaria occurred in the WHO South-East Asia Region (74%) [P. vivax being responsible for half of the total cases [Dengue fever (DF) and malaria are the two most common arthropod-borne diseases in tropical countries, especially in Southeast Asian regions, where their endemic areas greatly overlap , 2. The on (74%) . In 2009al cases .Dengue fever is caused by the dengue virus (DENV) in one of the four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4 . Dengue In Thailand, DF and malaria coexist, and are both prevalent. Early differentiation between dengue and malaria could help clinicians to identify patients who should be closely monitored for signs of DHF or severe malaria. Differences in clinical and laboratory features between DF and malaria have been reported, however, published studies vary on information pertaining to the duration of symptoms and demographic of patients.Phop Phra Hospital is located in Tak Province, a territory located in the western part of Thailand, which shares a long border with Kayin State, Myanmar. It is part of the Thailand-Myanmar border area and is the most common destination for people from Myanmar who migrate to Thailand for agricultural work. During malaria endemics, large numbers of patients with fever and other febrile illnesses are admitted weekly to emergency. There is risk of misdiagnosing malaria cases as DF cases in a malaria-endemic area, as the two diseases share similar clinical and laboratory characteristics. In addition, it may be impossible to both examine for dengue and malaria for all patients exhibiting similar signs and symptoms.The objective of this study was to identify clinical and laboratory features that can differentiate between patients infected with dengue and those infected with malaria, as well as to build knowledge about diagnostic markers used to discriminate DF from malaria, which both frequently occur in malaria-endemic areas.A retrospective case-control study designed to differentiate between dengue and malaria was performed between January 2013 and December 2015 at the Medical Technology Laboratory of Phop Phra Hospital, Tak Province, Thailand.In brief, the patients infected only with DF and those infected only with malaria were exhaustively identified based on the date of microbiological diagnosis. The diagnosis of malaria relied on the identification of sporozoa on thick/thin blood film stained with Giemsa and the identification of parasites under a light microscope by medical laboratorists. Thick and thin blood films were prepared by collecting venous blood in an Ethylenediaminetetraacetic acid (EDTA) tube and staining it with diluted Giemsa (1:10). One thousand red blood cells (RBCs) on the thin blood film and 200 white blood cells (WBCs) on the thick smear were examined before classifying a case as negative. EDTA blood were also analysed to determine the complete blood counts (CBCs) using a BC-5200 Hematology Analyzer . The analyzer provided data on RBC count, hemoglobin (Hb), hematocrit (Hct), platelet count, WBC count, neutrophil, monocyte, lymphocyte and eosinophil counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).Dengue can be diagnosed in laboratories using different methods. Nonstructural protein 1 (NS1) antigen detection was performed from day 0 to day 5, and indirect diagnosis based on the detection of specific anti-dengue immunoglobulin M (IgM) or immunoglobulin G (IgG) antibodies in patients’ sera after day 3 . At leas6/μL, Hb < 11 g/dL, and platelets < 100 000/μL were considered lower than cut-off values. Hematocrit > 40%, MCV > 80 fL, MCH > 25 pg/cell, MCHC > 33 g/dL, and red cell distribution width (RDW) >13% were considered higher than cut-off values. Continuous variables were compared using the Mann–Whitney U test. Categorical variables were analysed using the Fisher’s exact test or chi-square test. Statistical significance was set at P-value less than 0.05. Univariate logistic regression model was performed to confirm that associations between variables were independent of age, gender, and nationality. Variables with P-values of less than 0.05 in univariate logistic regression analyses were entered into a multivariate logistic regression model. Conditional multivariate backward stepwise logistic regression was then performed to calculate the adjusted odds ratios (ORs) and 95% confidence intervals (95%CIs). All significant variables in the univariate and multivariate logistic model were then used to determine a diagnostic value of DF versus malaria. The diagnostic accuracy of the hematological parameters was measured by calculating their sensitivity, specificity, predictive values, and ORs and 95%CIs.A normal distribution of continuous data were determined using the Kolmogorov-Smirnov test. Continuous variables were categorized following laboratory or usual cut-off values. For example, WBCs < 4 000/μL, neutrophils < 2 800/μL, lymphocytes < 800/μL, monocytes < 80/μL, eosinophils < 40/μL, basophils < 16/μL, RBCs < 4 × 10A decision tree model  was built to analyse parameters related to dengue and malaria infection. The goal of the analysis was to identify the most important parameters using the demographic data of the study population and hematological parameters.P = 0.299). There were more women (51.6%) in the dengue group and more men (61.3%) in the malaria group. A significant difference relating to gender between the two groups was observed . The large majority of people in both groups were Thai . A significant difference relating to nationality between the two groups was observed  . No significant differences relating to age were observed between the two groups : WBCs, neutrophil, monocyte, eosinophil, NL ratio, and monocyte-lymphocyte ratio (ML ratio). The following parameters were significantly higher patients with DF as compared to patients with malaria (P < 0.0001): RBCs, Hb, Hct, MCV, MCH, and MCHC (P < 0.009). The following parameters were not significantly difference in patients with DF compared to patients with malaria (P > 0.05): lymphocyte, basophil, RDW, and platelet count , neutrophil count less than 2 800 cells/μL , and eosinophil count less than 40 cells/μL   and an eosinophil count less than 40 cells/μL had the highest specificity (94%) to differentiate between patients with DF and those with malaria. Hematological parameters that had both good sensitivity and specificity were: neutrophil count less than 2 800 cells/μL  and a NL ratio of less than 2.8 . Hematological parameters that had good ORs were WBC count less than 4 000 cells/μL (P < 0.05) (see Table P < 0.05)  and dengue infection were not confound P < 0.0 . This was in concordance to another study, which indicated that patients with DF tended to be younger (a quarter of the study participants being below 15 years of age) . It has lciparum . Previoulciparum , 14.Secondly, significantly more male patients were infected with malaria than with dengue in this study, which a previous study also correlated . This maP. vivax (73%), which was similar to a report from the WHO [P. vivax malaria cases are increasing. It is usually assumed that P. falciparum is the most likely Plasmodium species to cause severe malaria, however, there is growing evidence that P. vivax can also lead to severe complications [Third, although over a third of the patients in this study were from Burma, significantly more Thai patients had DF than malaria. This could be explained by the fact that dengue is transmitted in urban areas and not in the forest. Most of the patients with malaria were infected with  the WHO . Nowadayications .P. falciparum strain [In the present study, hematological presentation was more severe and abnormalities were less frequent in patients with DF than in those with malaria. This might be partly due to the fact that patients with dengue presented early after the onset of symptoms. Red blood cells, Hb, Hct, MCV, MCH, and MCHC were significantly lower in patients with malaria. This may due to the parasite’s primary target being RBCs, resulting in an accelerated removal of both parasitized and non-parasitized RBCs by the destruction of RBCs and bone marrow dysfunction , 17. Onem strain .Hemoconcentration and raised Hct are common in patients with DHF . A high White blood cells, neutrophil, monocyte, eosinophil, NL ratio, and ML ratio were significantly lower in patients with DF than in those with malaria. Leukopenia is common in DF patients and a useful diagnostic marker . The totPrevious studies have aimed to establish criteria to differentiate between malaria attacks and DF. A study performed in southern India to compare malaria to other infectious causes of fever found that normal leukocyte counts, moderate to severe thrombocytopenia, renal failure, splenomegaly, and hyperbilirubinemia with elevated serum transaminases were associated with malaria. However, rash, overt bleeding tendency, normal to low leukocyte counts, moderate to severe thrombocytopenia, and significantly elevated hepatic transaminases were associated with DF . A previA low platelet count is a classic feature of both infections. This study confirmed that both patients with DF and malaria had low platelet counts, however, no significant differences between the two groups were observed. In many tropical regions, fever with thrombocytopenia is assumed to be malaria , and fevIn this study, co-infection of dengue and malaria was not found. Previous reports rationalized that the main habitat of malaria vectors is the forest , whereasThis study had certain limitations such as the fact that the amount of days that patients had fever before they were admitted to hospital could not be determined from the hospital’s database. In addition, this study lacks of previous medical histories that may confound the analysis such as Hb diseases, bacteria or virus infection. Further study is needed to validate whether the proposed parameters were likely to be altered during the febrile stage of the illnesses and whether the proposed indicators can differentiate between dengue and malaria.The findings of this study suggest that several clinical and laboratory measures could potentially distinguish between patients with DF and those with malaria. The sensitivity, specificity, positive predictive value (PLV), negative predictive value (NPV), and diagnostic accuracy for all hematological parameters were determined. A tree decision model revealed that using neutrophils, lymphocyte, MCHC, and gender was guided to discriminate of patients with malaria and DF. This model may be used to identify patients with dengue and those with malaria, but it needs to be used in the early stages of the illnesses in order for it to be useful for reducing unnecessary medication and time of hospitalizations.


The burden of sub-patent malaria is difficult to recognize in low endemic areas due to limitation of diagnostic tools, and techniques. Polymerase chain reaction (PCR), a molecular based technique, is one of the key methods for detection of low parasite density infections. The study objective was to assess the additional burden of asymptomatic and sub-patent malaria infection among tribal populations inhabiting three endemic villages in Keshkal sub-district, Chhattisgarh, India. A cross-sectional survey was conducted in March–June 2016, during the low transmission season, to measure and compare prevalence of malaria infection using three diagnostics: rapid diagnostic test, microscopy and nested-PCR.Plasmodium falciparum cases, 77.7% were afebrile and 35.9% had sub-patent infections.Out of 437 individuals enrolled in the study, 103 (23.6%) were malaria positive by PCR and/or microscopy of whom 89.3% were A substantial number of asymptomatic and sub-patent malaria infections were identified in the survey. Hence, strategies for identifying and reducing the hidden burden of asymptomatic and sub-patent infections should focus on forest rural tribal areas using more sensitive molecular diagnostic methods to curtail malaria transmission. The intensification of intervention measures, such as long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS) of insecticides, artemisinin-based combination therapy (ACT) and improved surveillance methods, have resulted in a 37% reduction in malaria incidence globally and a 60% reduction in the malaria mortality rate between 2000 and 2015. Globally a target of a 90% reduction in malaria incidence and mortality has been set for 2030 . All the RDT positives were treated immediately at the point of collection and those detected positive by microscopy/PCR were treated immediately after the results were made available by the village health workers. Complete treatment of all positive cases was ensured by the village health workers and cross-checked by the supervisors.All RDT/slide/PCR positive malaria cases were treated with anti-malarial according to national drug policy  were independently associated with sub-patent malaria infections.Table Keshkal sub-district is a comparatively low malaria endemic area with marked heterogeneity in reported malaria cases across the villages than adjacent sub-districts. Among four primary health centers (PHC’s) in Keshkal sub-district, Dhanora, where this study was undertaken, is the most malaria endemic area. State health department data showed that API varied from 0 to 16 per 1000 population in the past 5 years in the study villages. As part of the wider project in which this study is embedded, active fortnightly surveillance for malaria was carried out since 2015. During the routine surveillance, only one or two febrile malaria positives were detected in the study area every month, but in a cross sectional survey conducted in December 2015 the overall positivity rate ranged from 8.6 to 45.6% in the study sites, which suggests the presence of asymptomatic and sub-patent infections in the community. Hence, the present study was undertaken to estimate the proportion of sub-patent infections, showing that the burden of asymptomatic and sub-patent infections was high compared to what was reported in routine surveillance of fever cases. This could partially be attributed to negligence in health seeking behavior and distance to nearest health facility from the village.Plasmodium parasites without any signs or symptoms of malaria. These inhabitants do not visit the diagnostic facility or seek any anti-malarial treatment due to absence of any symptoms. These individuals constitute a reservoir of parasites and hence a source of infection for transmission. The chances of getting the infection would be higher if they share a common household unit. If a household has a member infected with malaria, it is more likely that there will be other infected persons clustered in that unit [A significant percentage of those infected (77.6%) were harbouring hat unit , 12, 28.The diagnostic efficiency of light microscopy and RDT is compromised in areas where a substantial number of asymptomatic and sub-patent cases exist . This stParasite detection by microscopy is limited for a number of reasons, such as its inability to detect low density of parasite infected red blood cells, and the loss of a number of parasite materials during slide staining especially in presence of scanty parasitaemia . On the In areas with asymptomatic and sub-patent infections, mass drug administration could be a viable alternative to active case detection, since ACT uses an effective drug combination for clearance of sub-patent gametocytes in these reservoirs , 34. SecP. vivax rather than P. falciparum are important risk factors of sub-patent infections. Though P. falciparum infection is more common in the area, P. vivax infection was associated with sub-patent infection. This could be due to this species’ capability to develop gametocytes at very early stage of erythrocytic schizogony, before the onset of any symptoms. Similar observations were reported in a study in Central Vietnam [Risk factor analysis shows that older age (15 years and above) and moderate to severe anaemia (≤9.5 g/dl) and  Vietnam . Sub-pat Vietnam .Asymptomatic malaria can perpetuate unstable transmission conditions and thus frustrate elimination efforts unless these reservoirs are targeted. This study has added to the evidence that in areas of low transmission malaria surveillance programmes need to be strengthened to identify hotspots, diagnose asymptomatic cases and investigate foci of transmission.This study was conducted with small sample size in the selected tribal villages where the annual parasite incidence varied from 0 to 16 per 1000 population; hence generalization of study findings in the wider population should be done with adequate caution. Nonetheless, results are relevant in Indian context which has decided for malaria elimination in near future.A substantial proportion of asymptomatic and sub-patent malaria carriers are present in the population living in the study area with low transmission. The burden of hidden parasite reservoirs is a major challenge for malaria elimination programmes in the country. Hence strategies for locating hotspots of asymptomatic malaria to control and eliminate such foci of reservoirs should be a focus of active case management in forested rural tribal areas.


Faint test bands of Paracheck Pf.® are interpreted as malaria positive according to world health organization (WHO) guideline. However if there are conspicuous number of faint test bands, a performance of Paracheck Pf.® could be influenced depending on whether interpreting faint test bands as malaria positive or negative. Finding out the frequency and accurate interpretation of faint test bands are important to prevent the overdiagnosis and drug resistance.A cross-sectional, descriptive study was conducted to find out the frequency of faint test bands and evaluate the performance of Paracheck Pf.® by sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of diagnosis of Paracheck Pf.® using microscopy as the gold standard. 388 suspected patients with malaria in Malawi were recruited in this study. Malaria rapid diagnostic tests (RDTs) and microscopy were used and patients’ information which includes age, sex, body temperature and signs or symptoms of malaria were recorded.Among all patients involved in the study, 29.1% (113/388) were found malaria positive by RDT. Overall 5.4% (21/388) of all Paracheck Pf.® tests resulted in a “faint test band” and 85.7% (18/21) corresponded with malaria negative by microscopy. Faint test bands which corresponded with malaria positive by microscopy were lower parasite density and there are no patients who showed definitive symptom of malaria, such as fever. When Paracheck Pf.® “faint test bands” were classified as positive, accuracy of diagnosis was 76.5% (95% CI 72%–80.7%) as compared to 80.4% (95% CI 76.1%–84.2%) when Paracheck Pf.® “faint test bands” were classified as negative.This study shows that frequency of faint test bands is 5.4% in all malaria RDTs. The accuracy of diagnosis was improved when faint test bands were interpreted as malaria negative. However information and data obtained in this study may not be enough and more intensive research including a frequency and property of faint test bands is needed for significant interpretation of faint test bands. Malaria is a major public health problem in Malawi with at least six million cases occurring annually . Early aNippon International Cooperation for Community Development (NICCO) is one of the Japanese NGOs which was conducting medical projects including malaria control, schistosomiasis control, maternal and child health activities, mobile clinics and the bicycle ambulance program in Lilongwe district, Malawi. Malaria RDTs were provided in the malaria control and the mobile clinic. While providing RDTs through those activities, it was found that some malaria RDT results were showing faint test bands with lines on the malaria RDT cassette appearing not visible enough and requiring good light to be seen. According to the WHO guideline, faint test bands of the malaria RDT are interpreted as malaria positive . The MinThere are many studies reported about a performance of Paracheck Pf.® or malaria RDTs based on the detection of histidine-rich protein 2(HRP-2). However the topic of the faint test band frequency was referred only in the study by Allen et al. that was conducted in low transmission area of Tanzania . The stuIf there are conspicuous number of faint test bands, accuracy of Paracheck Pf.® could be changed depending on how to interpret faint test bands, whether interpreting them as positive or negative. However the information about faint test bands such as its frequency or its causes are limited. This study was conducted to examine the frequency of faint test bands and the accuracy of Paracheck Pf.® by using microscopy as the gold standard.Malawi is divided into 28 districts and each district is divided into several areas which are called Traditional Authority (T/A) . NICCO’sThis study was conducted during the mobile clinic in T/A Malili targeting the patients who were suspected malaria.This is a cross sectional, descriptive study which was carried out in June to November in 2014 in Lilongwe, Malawi. Among 747 patients who came to NICCO’s mobile clinic during this period, 406 (54%) patients showed and/or complained signs and symptoms of malaria. Among them 388 (96%) patients  were involved in the study. Malaria RDTs using Paracheck Pf. ® were done to all patients who were suspected malaria. The results which include positive, negative and faint test band were recorded and sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy of RDT were evaluated using microscopy as the gold standard. Participants’ information which includes age, sex, body temperature and signs or symptoms of malaria were recorded.Patients who received an anti-malarial treatment within last 14 days and patients who did not agree to participate in the study were not included.Paracheck Pf.®  was used to diagnose malaria. The choice of Paracheck Pf.® was based on its low cost, high sensitivity (98.6%) in its controlled trials, simple to use by health surveillance assistants and was recommended by the Ministry of Health . The tesP. falciparum [The appearance of both a control band and a test band was concluded as a positive for lciparum . The applciparum .In the study site, blood was taken from finger prick and thick and thin smears were made by two skilled lab technologists of Lilongwe District Health Office Laboratory under the Ministry of Health following the guideline of the WHO . Those lParacheck Pf.®b)MicroscopyThe procedure of health surveillance assistants on RDTs was always followed by the protocol and supervised by the lab technologists . Faint tP = 0.0000). There were 17 samples out of 388 that 1st and 2nd readers did not agree. Only those discordant samples were rechecked by the 3rd reader who was the quality control technologist, the tie breaker. The 3rd reader was blinded to the 1st and 2nd readers’ microscopy results and Paracheck Pf.® results.Readings were conducted at CHSU and all procedures were followed by lab technologists as mentioned earlier. Two lab technologists read all samples and recorded the results by the plus system  All lab technologists were blinded each other as well as the results of Paracheck Pf.®. Evaluation of those two readers’ interrater reliability was 95.62% agreement  were children under 5 years old, 70 (18%) were children aged between 6 and 15 years old and 167 (43%) were adult over 16 years old. Fever were seen in 10% of participants and all participants were showed and/or complained other minor signs and symptoms such as cough, feeling feverish, general body pain, headache, abdominal pain, watery diarrhea, and vomit. . Sensitivity of Paracheck Pf.® was measured as the proportion of RDT positive over the total positive determined by microscopy. Specificity of Paracheck Pf.® was measured as proportion of RDT negative over the total negative determined by microscopy. The PPV is the number of RDT true positives divided by the number of all RDT positive test results . The NPV is the number of RDT true negatives divided by the number of all RDT negative test results . The accuracy of diagnosis is a sum of true positives and true negatives divided by true positives, false positives, true negatives and false negatives.Among 388 patients involved in the study, 29.1% (113/388) were RDT positives, 5.4% (21/388) were faint test bands and 65.5% (254/388) were RDT negatives. If faint test bands are interpreted as malaria positive, the percentage of faint test band was 15.7% (21/134) in the malaria positive. Baseline characteristics of patients in RDT positive, negative and faint test band are showing in Table Plasmodium falciparum.From microscopy, 55.8% (63/113) of all RDT positive cases were true positive and 44.2% (50/113) were false positive. Among RDT negative cases, 9.1% (23/254) were false negative and 90.9% (231/254) were true negative. Out of 21 faint test band cases, 14.3% (3/21) were malaria positive and 85.7% (18/21) were malaria negative by microscopy. All microscopic positive cases were identified as Those three faint test band cases showing true positive on microscopy were lower parasite density, one plus (+) or two plus (++), by qualitative reading. Among all 21 faint test band cases, there were no patients with a body temperature over 37.5° which is the major symptom of malaria  of malaria RDT positive cases resulted in false positive. Sensitivity and specificity of the RDT compared with microscopy were 74.2% (CI 63.8% - 82.9%) and 77.3% (CI 72.1% - 81.9%). Positive predictive value (PPV) and negative predictive value (NPV) were 49.3% (CI 40.5% - 58%) and 90.9% (CI 86.7% - 94.2%). The diagnosis of accuracy was 76.5% (CI 72% - 80.7%).  and 83.3% (CI 78.6% - 87.3%) respectively. PPV and NPV were 55.8% (CI 46.1%–65.1%) and 90.5% (CI 86.5%–93.7%). Accuracy of diagnosis is 80.4% (CI 76.1% - 84.2%). When the faint band was interpreted as RDT negative, the performance of Paracheck Pf. ® changed; sensitivity decreased from 74.2% to 70.8%, specificity increased from 77.3% to 83.3%, PPV increased from 49.3% to 55.8%, NPV decreased from 90.9% to 90.5% and the accuracy of diagnosis increased from 76.5% to 80.4%  of them showed faint test bands. If faint test bands are interpreted as malaria positive, the frequency of faint test band was 15.7% (21/134) in RDT positive. This frequency is not very high like the study by Allen et al. which showed the frequency of faint test band as 23.7% among all patients. –48. Howe/388 of tP.falciparum and SD FK60 Malaria Ag P.falciparum/Pan. Faint or weak line intensities occurred in 32.1% (98/305) of true positive results of SD FK50 Malaria Ag P.falciparumIt, and in 29.5% of true positive results of SD FK60 Malaria Ag P.falciparum / Pan. It explained that those faint or weak line intensities were occurred mostly but not exclusively at low parasite densities [Several other studies used different kinds of malaria RDT brands apart from Paracheck Pf. ® were also found. Palen et al. examined the correlation between the line intensity of RDTs and parasite densities using the RDT brands of SD FK50 Malaria Ag ensities . This stAs mentioned previously, totally eight studies recognized the existence of faint test bands and/or reported the faint test band frequency of Paracheck Pf.® or other RDT brands , 49, 50.From our intensive literature search, the sensitivity of Paracheck Pf.® varies from 54% in Nigeria by Flade CO et al.  to 100% When considering the possible factors of faint test band appearance, parasite density could be also one of them. The WHO guideline mentions that the lines can be faint at low parasitemia . Our stuFrom this consequence, it can be said that the patients who show faint test bands should be treated as malaria positive in areas where medical circumstances are limited such as our research area.The information and data obtained in this study were not enough to conclude an accurate interpretation of faint test band since there are limitations of the study such as a small number of faint test band samples, not conducting quantitative reading for blood smears, not using PCR and not observing the patients chronologically. Further studies measuring frequency of faint test band and defining its interpretation are greatly needed. It is because that clinical diagnosis and treatment will be influenced by faint test bands especially in local health facilities of a high malaria transmission area where the malaria RDT is the most reliable and only available examination.If faint test bands are interpreted as malaria positive, the frequency of faint test bands was 15.7% in the RDT positive. The result of sensitivity, specificity, PPV and NPV of Paracheck Pf.® could not make a conclusion of an ideal interpretation of a faint test band. However the accuracy of diagnosis when faint test bands were interpreted as malaria negative is higher than when these were interpreted as positive. Furthermore, according to the result of microscopy as the gold standard, over 85% of faint test bands were malaria negative. From these results, it is possible to interpret faint test bands as malaria negative. This can be applied only if patients do not have risk factors of getting severe malaria such as age below five years, pregnancy, travelers from non-malaria epidemic areas or immune deficiency. Considering the data obtained in this study and the possibility that an inadequate interpretation of faint test band may influence on the patients’ diagnosis and proper treatment, more intensive research focusing on faint test bands, its frequency and evaluating an accurate interpretation of faint test bands using PCR as the gold standard are highly recommended.


Urbanization is a main trend in developing countries and leads to health transition. Although non-communicable diseases are increasing in cities of low-income countries, vector-borne diseases such as malaria, are still present. In the case of malaria, transmission is lower than in rural areas, but is uneven and not well documented. In this study, we wanted to evaluate intra-urban malaria transmission in a West African country (Burkina Faso).Anopheles bites was measured by assessing the level of IgG against the Anopheles gSG6-P1 salivary peptide. Adult mosquitoes were collected using CDC traps and indoor insecticide spraying in some houses of the four neighbourhoods. The Anopheles species and Plasmodium falciparum infection rate were determined using PCR assays.A cross-sectional study on 847 adults (35 to 59 year-old) and 881 children (6 months to 5 year-old) living in 1045 households of four districts  of Bobo-Dioulasso was performed between October and November 2013. The districts were selected according to a geographical approach that took into account the city heterogeneity. Malaria prevalence was evaluated using thick and thin blood smears. Human exposure to Plasmodium falciparum. Malaria transmission occurred in the four districts. Malaria prevalence was higher in children than in adults (19.2 vs 4.4%), and higher in the central districts than in the peripheral ones (P = 0.001). The median IgG level was more elevated in P. falciparum-infected than in non-infected individuals (P < 0.001). Anopheles arabiensis was the main vector identified . Five P. falciparum-infected mosquitoes were caught, and they were all caught in the central district of Tounouma where 28.6% (14/49) of the tested blood-fed mosquito specimens had a human blood meal.In this study, 98.5% of the malaria infections were due to This study showed that urban malaria transmission occurred in Bobo-Dioulasso, in all the four studied areas, but mainly in central districts. Environmental determinants primarily explain this situation, which calls for better urban management. Plasmodium falciparum is responsible for most (99%) of these deaths [Vector-borne diseases continue to be a threat in most low-income countries, particularly malaria . About 3e deaths  in Africe deaths . In Burke deaths . MoreoveAnopheles vectors can adapt to the urban environment. For instance, urban agriculture offers great conditions for the development of anopheline larvae, as observed in small towns, such as Bouaké in Ivory Coast [Anopheles larvae [Urbanization is accelerating in Africa, and demographic projections suggest that this phenomenon will further increase in the future . It is gry Coast , but alsry Coast  and Antary Coast . Human as larvae , 9.Moreover, urbanization is unequal in time and space, and also between continents, countries and within cities . This urPlasmodium species, mainly P. falciparum are transmitted by Anopheles gambiae s.s. [Bobo-Dioulasso is the second largest city in Burkina Faso with an estimated population of 813,810 inhabitants in 2012 . It is lThe study was conducted in November 2013 in four districts: Dogona, Yeguere, Tounouma and Secteur 25  was performed according to the manufacturer’s instructions. People with a positive mRDT were immediately treated for free, as recommended by the National Malaria Control Programme of Burkina Faso.Plasmodium species. Plasmodium falciparum parasites  were counted in 200 leukocytes and parasite density was calculated for a fixed leukocyte count of 8000 cells/μl of blood. All thick and thin blood smears were read independently by two skilled technicians. Comparison of their results indicated that the estimated rates of parasite detection and parasite density did not differ significantly between technicians. Quality control was regularly performed on randomly selected samples, representing 10% of all thick smears. A malaria case was defined as the presence of fever or history of fever during the past 48 h and parasitaemia > 0.Blood smears were fixed and stained with 10% Giemsa and examined at the Institut de Recherche en Sciences de la Santé (IRSS) in Bobo-Dioulasso to identify An. gambiae, was evaluated using enzyme-linked immunosorbent assay (ELISA), as previously described [Anopheles exposed individuals (ΔODneg) was: ΔODneg ± 3 standard deviations = 0.204. An individual was considered to be an immune responder when ∆OD > 0.204.Blood samples for assessing mosquito bite exposure were collected on filter paper . They were stored at 4 °C until use. The IgG response to the gSG6-P1 salivary peptide, which is specific for escribed , 16. Optescribed . The IgGescribed . The aveAn. gambiae complex specimens using the PCR method described by Santolamazza et al. [Mosquito collections were carried out inside and just outside four houses in each of the four districts using CDC traps during two consecutive nights (between 20:00 and 06:00 h). Indoor resting females were caught by spraying four other houses close to the CDC-sampling houses with insecticide aerosols between 06:00 and 09:00 h during the same two consecutive days. Dead or knocked-down mosquitoes were collected on white sheets laid down on the floor of the sprayed houses. Species identification was performed for a et al. . Their ia et al. . The blon = 273) of children and adults randomly selected from the whole sample. Normality distribution was tested using the Kolmogorov-Smirnov and Shapiro-Wilk tests. The non-parametric Mann-Whitney test was used to compare IgG Ab levels between independent groups, and the Kruskal-Wallis test for comparisons of more than two groups. IgG antibody levels between districts were analyzed using analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) post-hoc tests. A P-value < 0.05 was considered as significant. Graph Pad Prism 5.0 Software and Microsoft Excel 2013  were used to produce graphics and perform statistical analyses.The number of households to be surveyed and then the number of participants were calculated in order to observe significant differences between the four districts at the 5% threshold with a precision of 2.6%, based on a prevalence of 5% . Malaria prevalence was calculated as the number of malaria cases reported to the population considered . Malaria prevalence in the four districts as well as in the central and peripheral areas was compared by a Chi-square test. Parasitemia was calculated among those that had a detectable parasitemia. Variance analysis allowed the comparison between districts. The immunological assays were performed on a sub-sample  living in 1045 households (Table P = 0.026). No difference in malaria prevalence distribution among districts was observed in adults. Comparing neighborhoods to two showed that Dogona was significantly different from Secteur 25 and Yeguere, but quite similar to Tounouma (Table P = 0.001). Plasmodium falciparum was identified in 98.5% (205/208) and P. malariae in 1.4% (3/208) of all infected individuals in the four districts. The average parasitaemia was 3872 and 275 P. falciparum asexual parasites/μl of blood in children and adults, respectively (Table P = 0.019).A total of 206 cases of malaria were reported (Table ma Table . Comparin = 1728), a subsample of 273 blood spots was randomly selected to evaluate the exposure to Anopheles bites by assessing the immune response to the salivary gSG6-P1 peptide. The proportion of children and adults tested positive for an IgG response (above the threshold) was 89% (122/137) and 99% (135/137), respectively. No significant difference in the serology analysis based on age was observed among adults  = 1.980, P= 0.1023) and children  = 2.365, P = 0.0570). No significant difference was observed between adults and children,   where the anti-gSG6-P1 IgG responses were comparable   showed that the anti-gSG6 P1 IgG response was significantly higher in the central than in the peripheral districts   and children   indicated that the median anti-gSG6-P1 IgG level was significantly higher in infected than non-infected adults (Mann-Whitney U-test: 01) Fig. .Fig. 5AnCulex spp., 6.8% of Anopheles spp. and 0.2% of Aedes spp. . CDC traps provided 72.3% (201/278) of the Anopheles samples and 86.1% (173/201) of them were collected with indoor CDC traps. Indoor resting females caught by spraying represented 27.7% (77/278) of the Anopheles samples.In total, 4107 mosquitoes were caught in the four districts. The population was composed of 93.1% of An. gambiae (s.l.) females (n = 278) was successful for 273 specimens (four specimens from Tounouma and one form Dogona were not identified) , followed by An. coluzzii  and An. gambiae  . Anopheles arabiensis represented 81.5% (185/227) of catches in Tounouma, whereas An. gambiae was predominant in Dogona  (Table Anopheles coluzzii was only found in Tounouma and Dogona.PCR-based species identification of the d) Table . The mosAnopheles females, P. falciparum was found only in specimens collected in Tounouma, but not in Yeguere, Secteur 25 and Dogona. In addition, in Tounouma, only An. coluzzii  and An. arabiensis  were infected by P. falciparum  showed that in Tounouma, Dogona and Yeguere, ≥ 25% had a human blood meal and ≥ 15% a mixed meal .Analysis of the available blood-fed mosquitoes  [An. funestus specimens was caught after 06:00 h [An. arabiensis was the main vector in Bobo-Dioulasso as previously reported by Dabiré et al. [An. arabiensis could also help understanding the persistence of malaria transmission. Indeed, its exophilic behaviour could reduce the effectiveness of vector control interventions that specifically target endophilic species, thus allowing residual transmission outside houses.The present study was carried out in four districts of Bobo-Dioulasso to evaluate the spatial disparities in malaria transmission to improve disease control in an urban setting. Despite the significant coverage of mosquito nets, the transmission of malaria occurred in the four districts . Prevale545–665) . Further545–665)  in 2012 545–665)  showed t545–665) . On the  06:00 h . Thus, ié et al. , 27. TheAnopheles larvae in polluted water was previously reported in Dakar, Accra, Dar es Salaam, and Lagos [The higher malaria risk in central areas of Bobo-Dioulasso was relatively unexpected. Indeed, in Burkina-Faso, the risk of malaria is generally considered to be more elevated in suburban than central areas because of the persistency of rural features that favour the presence of malaria vectors . The oppnd Lagos , 29–31. nd Lagos –17, 32.An. arabiensis, is an exophilic species against which mosquito nets are less efficient. If sociological factors, such as a good education level, can strongly influence malaria impact within a population, environmental factors may be improved by better urban planning and management, calling for awareness to build sustainable healthy cities.The transmission of malaria was investigated in four districts of Bobo-Dioulasso that were selected to illustrate the great ecological diversity of the urban environment. District disparities in malaria prevalence were observed in children, highlighting that they were particularly exposed to malaria in the central part of the town. Environmental factors, such as urban agricultural activities along the Houet River favor the presence of malaria vectors and then host-vector contact Fig. . Moreove