Plasmodium vivax malaria outbreak occurred among French forces involved in a mission to control illegal gold mining in French Guiana. The findings of epidemiological and entomological investigations conducted after this outbreak are presented here.In December 2010, a Anopheles mosquitoes were sampled at the identified sites using Mosquito Magnet and CDC light traps. Specimens were identified morphologically and confirmed using molecular methods (sequencing of ITS2 gene and/or barcoding). Anopheles infections with Plasmodium falciparum and P. vivax were tested by both enzyme-linked immunosorbent assay and real-time PCR.Data related to malaria cases reported to the French armed forces epidemiological surveillance system were collected during the epidemic period from December 2010 to April 2011. A retrospective cohort study was conducted to identify presumed contamination sites. P. vivax malaria cases were reported , leading to a global attack rate of 26.5 % (72/272). Lack of compliance with vector control measures and doxycycline chemoprophylaxis was reported by patients. Two illegal gold mining sites located in remote areas in the primary forest were identified as places of contamination. In all, 595 Anopheles females were caught and 528 specimens were formally identified: 305 Anopheles darlingi, 145 Anopheles nuneztovari s.l., 63 Anopheles marajoara and 15 Anopheles triannulatus s.l. Three An. darlingi were infected by P. falciparum (infection rate: 1.1 %) and four An. marajoara by P. vivax (infection rate: 6.4 %).Seventy-two Anopheles marajoara was clearly implicated in malaria transmission for the first time in French Guiana. The high infection rates observed confirm that illegal gold mining sites must be considered as high level malaria transmission areas in the territory.The main drivers of the outbreak were the lack of adherence by military personnel to malaria prevention measures and the high level of malaria transmission at illegal gold mining sites. P. vivax malaria and sylvatic Anopheles species.Illegal gold mining activities are challenging the control of malaria in French Guiana. Collaboration with neighbouring countries is necessary to take into account mobile populations such as gold miners. Malaria control strategies in the French armed forces must be adapted to  Plasmodium species) decreased from 4479 to 445  and Repentir [relative risk = 5.35 CI 95 % (3.08–9.30)] . In Repentir, 166 Anopheles mosquitoes were caught: 164 using two MM traps over the entire period and two using two CDC traps for three nights. In Dorlin, 388 Anopheles mosquitoes were caught: 370 using two MM traps over the entire period and 18 using two CDC traps for three nights. Morphological identification was difficult due to the damage caused to specimens by traps and the presence of moisture. It allowed the probable species identification of 398 specimens: 249 Anopheles darlingi, 88 Anopheles nuneztovari s.l., 53 Anopheles albitarsis s.l. and seven Anopheles triannulatus s.l. Morphological identification was inconclusive for 199 specimens (199 An. spp.) , the products of ITS2 PCR were sequenced and compared to available sequences. The sequencing confirmed the molecular identification for 26 specimens. The overall consistency of morphological identification was 91 %. Table An. albitarsis complex caught were molecularly identified using DNA barcoding. At the two sites where they were caught, all specimens were Anopheles marajoara [During the survey in June and July, 595 female arajoara .Table 2DAnopheles females were tested by enzyme-linked immunosorbent assay (ELISA) for P. falciparum andP. vivax strainVK210 and VK247 circumsporozoite protein. According to ELISA, none of those specimens was infected by a human Plasmodium. All the specimens were tested by RT-PCR for P. falciparum and P. vivax. DNA was extracted directly for 243 specimens not tested using ELISA and using the homogenates prepared for ELISA for the 345 others. By RT-PCR, infected anophelines were only found among samples from Dorlin. Three An. darlingi were infected by P. falciparum (infection rate of 1.1 %) and four An. marajoara were infected by P. vivax (infection rate of 6.4 %)  ruts. Some bromeliads were recovered from fallen trees. Anopheles species implicated in malaria transmission.This study reports on one of the largest malaria outbreaks among French forces deployed to French Guiana , 45. ForP. vivax malaria attack rates, ranging from 24.6 to 27.8 %, were observed in the three military units conducting the same mission: to control illegal gold mining in French Guiana. The epidemic curve presents two peaks.High The first peak corresponds to indigenous malaria cases and could be partly attributed to a lack of adherence to doxycycline chemoprophylaxis. Only 40 % of the patients stated proper chemoprophylaxis compliance in the 8 days before onset of symptoms. Comparable results had already been observed in previous malaria outbreaks among French forces , 47. In P. vivax malaria imported cases occurring after terminal doxycycline chemoprophylaxis. These patients did not experience P. vivax malaria or fever during the 4-month mission, and their first clinical episodes were probably hypnozoite-induced relapses. Doxycycline does not affect the liver stage of the parasite, which also means that it does not kill P. vivax hypnozoites and, therefore, does not prevent P. vivax malaria relapses [P. falciparum resistance to malaria drugs, and in order to optimize compliance with a homogeneous prescription, 100 mg of doxycycline once a day is currently the first line chemoprophylaxis in the French armed forces [P. falciparum malaria during deployments to Africa. The involvement of French forces in endemic P. vivax malaria transmission areas is more recent and has led to an increase in P. vivax incidence [P. vivax relapses when they became involved in the United Nations mission in East Timor from 1999 to 2000 [P. vivax, a daily dose of 100 mg of doxycycline during operations and 22.5 mg of primaquine daily for 2 weeks on returning to Australia were administered [P. vivax [P. vivax attacks in order to prevent relapses. Therefore, liver-stage prophylaxis with primaquine for delayed-onset malaria cannot currently be used by French forces.The second peak occurred after leaving French Guiana, with 82 % of relapses , 50, 51.d forces . Malariancidence . The Aus to 2000 , 53. Connistered . Attack P. vivax . HoweverScrupulously observing individual protection measures against mosquito bites is essential in such contexts. Their combined proper use will theoretically reduce exposure to biting mosquitoes, but not enough to significantly reduce the incidence of malaria among non-immune troops , 57. MilThe main entomological investigations were conducted at Repentir and Dorlin. Both sites were identified as presumed contamination locations by the epidemiological investigation. These sites have been subjected to human pressure for many years. After an initial period of legal activity, the mining site of Repentir was rehabilitated before being closed. But the area was then invaded by undocumented gold miners. In December 2010, military intelligence estimated that more than one thousand undocumented people were living and working in this area. The river was diverted for gold mining purposes and ponds and puddles were created. The forest was cleared for gold mining and many excavations were made that were then flooded by the river or filled by rainfall. In some places, the primary forest was destroyed and replaced by savannas, secondary forest or glades. Wells were also dug. The rest of the surrounding primary forest was criss-crossed by footpaths and ATV tracks. In late 2010, French military forces took up positions in this area and undocumented workers left to the nearest illegal gold mining site: Dorlin. Dorlin is an old illegal gold mining site that has been exploited for 20 years. No rehabilitation of the area has ever been carried out. Military intelligence estimated that more than one thousand undocumented people were present here and working at this illegal gold mining site. Many illegal settlements were still present on the site during entomological investigations. Rivers have been diverted; the primary forest has been extensively cleared, replaced by savanna or secondary forest over large areas. Mining excavations have been transformed into lakes and ponds. Backhoes and bulldozers have been used to dig hills in the search for auriferous veins and footpaths and ATV tracks criss-cross the area.Anopheles mosquitoes. Adult Anopheles from four species that could play a role in malaria transmission were collected with traps, in high densities in some places and periods. Larval prospecting was not significant due to the large size of the areas, the high number of potential breeding sites and the limitations of entomological activities due to security measures.Current entomological investigations confirm that these anthropic modifications are highly favourable to the development of Anopheles mosquitoes caught. Species identified were among the most frequently observed Anopheles species in French Guiana and the Amazon Basin [An. marajoara as a P. vivax vector in the French Guiana rainforest. Infected anophelines were found only at the illegal gold mining site of Dorlin. The high infection rates observed confirm that illegal gold mining sites in French Guiana should be considered as high level malaria transmission areas. Thanks to the setting up of a forward operational post and legal mining operations, Repentir site was free of undocumented gold miners for 6 months before the entomological investigation. This could explain the absence of infected anopheles in this area. However, during the 1-week mission spent in Repentir, one P. falciparum malaria case occurred in a legal gold mining operation close to the FOP, suggesting a continuation of low malaria transmission. The use of traps did not enable the calculation of entomological inoculation rates (EIRs) or make it possible to clearly measure the number of infected bites per soldier in Repentir or Dorlin. Nevertheless, the number of Anopheles mosquitoes caught with traps at those sites was very high compared to previous studies in French Guiana. This suggests that anopheles densities were very high at these sites. Furthermore, CDC light traps that seemed to be more attractive to An. marajoara were only used for 3 days, and the abundance of this species was probably underestimated. Considering this, the malaria transmission risk could be considered as very high both at Dorlin and Repentir. These findings are consistent with other studies conducted at illegal gold mining sites including a study in Venezuela where the An. marajoara and An. darlingi association has already been described [Morphological and molecular analysis made it possible to identify 89 % (528/595) of the on Basin . Howeverescribed . The comP. vivax malaria is probably higher, as P. vivax gametocytes are produced within the first cycles of infection [Illegal gold mining has modified the epidemiology of malaria in French Guiana, which was previously limited to the villages located on the main rivers flowing into the territory, in particular the Maroni and Oyapock rivers. Uncontrolled and mobile populations of undocumented gold miners could reintroduce malaria in the malaria-free coastal area of French Guiana, where competent vectors are abundant. The risk of an introduction of nfection . This siP. vivax malaria and sylvatic Anopheles mosquitoes. To that end, the French military health service and Institut Pasteur de la Guyane initiated a large research programme in 2012 to evaluate human, mosquito and parasite aspects of P. vivax malaria transmission at illegal gold mining sites in French Guiana.Despite a decrease in the incidence of malaria in French Guiana, uncontrolled foci of malaria transmission are present in the deep forest. Gold mining is associated with malaria transmission in all the countries of the Guiana Shield. Collaboration with neighbouring countries is necessary to take into account mobile populations such as gold miners. Military personnel are especially exposed during missions at illegal gold mining sites. Malaria control strategies in the French armed forces must be adapted to


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria. FoRH (VSAs for which the frequency of recognition is high)    and VSASmalaria) .Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), an extremely diverse group of multidomain parasite molecules encoded by var genes  SM –, 21, 22,SM –15–, 23–25. var gene sequence data set (var sequence data by classifying and counting the number of expressed sequence tags (ESTs) as described previously  was made and another pool of sera from 100 children with infection (parasite positive) was made. To identify parasite isolates expressing serologically conserved antigens, the reactivity of the IgG antibodies in these 8 pools against 92 parasite isolates from children with severe malaria (n = 47) or nonsevere malaria (n = 45) was tested by immunofluorescence assay (IFA) of live IEs quantified by flow cytometry.To determine the serological conservation of antigens expressed on IEs, we used sera from 800 healthy children aged 1 to 4 years sampled as part of a large cross-sectional survey conducted in 1995  and a seThe following additional assays were performed for comparison: (i) a subset of the parasites was tested for reactivity with these serum sample pools by an agglutination assay, and (ii) parasite isolates were tested for reactivity with a panel of 15 previously described plasma samples from children with acute malaria collected in the study setting between 1994 and 1996 by both IFA and an agglutination assay .To assay the recognition of parasites by serum and plasma antibodies, ring-stage parasite isolates were cultured using standard methods to reach the mature trophozoite stage, when PfEMP1 is expressed on the IEs, and then cryopreserved in liquid nitrogen until the day of assay. To measure the strength of recognition of IEs by antibodies in the serum and plasma samples, IEs were thawed and adjusted to a maximum of 2% parasitemia using blood group O erythrocytes from a single donor. Total IgG binding to the IEs was then measured as described previously , and thevar expression and the strength of recognition of IEs by antibodies in the serum sample pools  was used for all statistical analyses, and GraphPad Prism software (v6) was used for graphical representation of the data. Spearman's rank correlation coefficient was used to assess the relationship between ools see . Unadjusools see  and betwvels see . Each antted see  and d. Dtted see  and var vels see  were usevels see . StatistConsistent with the findings of previous studies, the antibody responses in the serum sample pools measured using IFA were generally higher among parasite-positive children than children in whom the parasite was not detectable , 29 and var gene expression profiles using two approaches: (i) an expressed sequence tag (EST) approach that involved cloning and sequencing of PCR-amplified var sequence tags, which allows assessment of the relative number of group A-like genes expressed by classifying and counting the sequences amplified using a set of universal primers to the DBLα region of PfEMP1   on the basis of a Bonferroni correction for the 104 comparisons performed , we additionally typed the parasites by an agglutination assay, which was generally less sensitive than flow cytometry, and only two pools had detectable responses to >50% of the isolates . Nevertheless, the agglutination score showed positive associations with DC8 var subsets at the conventional level of statistical significance of a P value of <0.05, though these did not meet the cutoff for statistical significance after a Bonferroni adjustment for multiple comparisons was made . The agglutination score did not differ by disease severity whether it was measured in the panel of plasma samples or the serum sample pools .Where enough sample material was available (var genes tend to be expressed mostly in immunologically naive individuals and the PfEMP1 variants expressed by these genes carry epitopes that are conserved over long periods, the variants that actually cause severe malaria may be prevalent over relatively short periods of a few years but then break apart through var gene recombination (A plausible explanation for our observations is that whereas previous studies used a relatively short time interval of up to 3 years between the time of sampling of naturally acquired antibodies and the time of sampling of target IE, this study used a 10- to 12-year time interval. This raises the possibility that, while group A and DC8 bination . This invar gene expression levels, we have found evidence that group A and DC8 PfEMP1 variants, whose bulk expression correlates with the severity of malaria, have restricted antigenic diversity. However, we found no evidence for an association between the serological conservation of IE antigens per se and severe malaria. These data contrast with the working hypothesis that severe malaria is caused by P. falciparum isolates with PfEMP1 variants whose immunodominant epitopes are maintained over long time periods. Further studies are needed to determine the rate of turnover within the local parasite population of PfEMP1 variants associated with severe malaria.In summary, by measuring


Due to limitation of conventional malaria diagnostics, including microscopy, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), alternative accurate diagnostics have been demanded for improvement of sensitivity and specificity.Plasmodium LDH antigens, Plasmodium falciparum-infected human red blood cells (RBC) derived from in vitro culture or patient’s samples were used for evaluation of the performance of fluorescence-linked immunosorbent assay (FLISA). Microscopic examination was used to determine parasite density and the performance of FLISA was compared to ELISA. Finally, sensitivity and specificity of FLISA was determined by human specimens infected with P. falciparum, Plasmodium vivax, Toxoplasma gondii, and amoebae.Serially diluted In vitro-cultured P. falciparum was detected up to 20 parasite number/μL in FLISA but 5120 parasite number/μLin sandwich ELISA. In vitro P. falciparum-infected RBC number was highly correlated with fluorescent intensity (R2 = 0.979), showing that FLISA was reliable for detection of P. falciparum and available for quantification of parasite numbers. Furthermore, eighteen patient samples infected with P. falciparum (n = 9) and P. vivax (n = 9) showed 100% of sensitivity (18/18). FLISA showed 96.3% of specificity (26/27) because one sample of patient blood infected with T. gondii gave a false positive reactivity among healthy donors (n = 9), T. gondii-infected patients (n = 9), and amoeba-infected patients (n = 9).As a result of FLISA, the fluorescent intensity was highly correlated with antigen amount and FLISA was more sensitive than ELISA. FLISA detected at least 0.01 ng/ml of pLDH antigen, which showed 1,000-fold higher sensitivity than ELISA. FLISA has a keen and high performance to detect malaria antigen, suggesting a potential assay as malaria immunodiagnostic. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi, remain a serious health issue and are widespread in developing countries, continuing to cause about 219 million cases and one million deaths annually . In contrast, the eptitope of Plasmodium LDHs shows 76% identities of Emeiria maxima LDH (GenBank accession no. AAN38977.1) and 66% of Toxoplasma gondii LDH (GenBank accession no. XP002368488.1). This epitope has only 26% identities in Homo sapiens LDH (GenBank accession no. CAE11711)  and P. vivax (R2 = 0.967) LDH , between 20 and 5,120 parasites  and P. vivax (n = 9) infected patient’s bloods were applied to FLISA and FLISA showed 100% of sensitivity (18/18). In contrast, non-malaria disease samples among healthy donors (n = 9), T. gondii- (n = 9), and amoeba-infected patients (n = 9) showed one false positive reactivity with T. gondii-infected patient blood. Therefore, specificity of FLISA was 96.3% (26/27)  Figure .Plasmodium blood screening test is advocated to prevent spread by transfusion in blood banks and in ROK, ELISA has been developed to screen and monitor transfusion-transmitted malaria [Plasmodium infection. Therefore, FLISA has a potential as malarial diagnostic to facilitate the accurate diagnosis of Plasmodium infection and it would be an apparent alternative assay to determine Plasmodium parasite density. Because fluorescent intensity is correlated with malaria density with high sensitivity, FLISA may be considered as a feasible solution for high-throughput analysis of malaria infection during blood transfusion in ROK. Further studies should assess the diagnostic performance for other Plasmodium spp. infection and asymptomic parasitaemia patients.Since elimination of malaria infection by WHO, malaria prevalence is getting low but still prevalent worldwide. In Republic of Korea (ROK), after reemergence of malaria near location of demilitarized zone (DMZ) in 1993, the annual incidence has increased rapidly -25. Unti malaria ,28. Ther malaria . The dev malaria . For thiThe authors have declared that they have no competing interests.SJ, DT, JH, JY, WJ, and HJ carried out the assays and analysed the results; SJ, ET and HP wrote the manuscript; SJ, WJ, HJ, ET, and HP conceived and designed the experiments. All authors read and approved the final manuscript.


Plasmodium falciparum malaria and non-typhoid Salmonella (NTS) bacteraemia are both major causes of morbidity and mortality in children in sub-Saharan Africa. Co-infections are expected to occur because of their overlapping geographical distribution, but accumulating evidence indicates that malaria is a risk factor for NTS bacteraemia. A literature review was undertaken to provide an overview of the evidence available for this association, the epidemiology of malaria-NTS co-infection (including the highest risk groups), the underlying mechanisms, and the clinical consequences of this association, in children in sub-Saharan Africa. The burden of malaria-NTS co-infection is highest in young children . Malaria is one of the risk factors for NTS bacteraemia in children, and the risk is higher with severe malaria, especially severe malarial anaemia. There is insufficient evidence to determine whether asymptomatic parasitaemia is a risk factor for NTS bacteraemia. Many mechanisms have been proposed to explain how malaria causes susceptibility to NTS, ranging from macrophage dysfunction to increased gut permeability, but the most consistent evidence is that malarial haemolysis creates conditions which favour bacterial growth, by increasing iron availability and by impairing neutrophil function. Few discriminatory clinical features have been described for those with malaria and NTS co-infection, except for a higher risk of anaemia compared to those with either infection alone. Children with malaria and NTS bacteraemia co-infection have higher case fatality rates compared to those with malaria alone, and similar to those with bacteraemia alone. Antimicrobial resistance is becoming widespread in invasive NTS serotypes, making empirical treatment problematic, and increasing the need for prevention measures. Observational studies indicate that interventions to reduce malaria transmission might also have a substantial impact on decreasing the incidence of NTS bacteraemia. Salmonella enterica subspecies enterica, which can cause disease in humans, and are normally dichotomized into those causing typhoid fever , and non-typhoid salmonella (NTS) serotypes which include Enteritidis and Typhimurium [There are thousands of serotypes of Salmonella, including those grouped as himurium , 2. Infehimurium . Enterichimurium –8. Invashimurium , 10. In Plasmodium parasites that are known to affect humans and include Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, and Plasmodium knowlesi[P. falciparum and is also borne by children in sub-Saharan Africa [Malaria is caused by five species of knowlesi. The gren Africa .Paediatric NTS bacteraemia and malaria overlap in terms of geographical distribution, age groups at greatest risk and seasonality of both diseases in the tropics. The effect of malaria on susceptibility to NTS bacteraemia was suspected in British Guiana in the early part of the last century , but relA literature search was undertaken in the PubMed database using the search terms “Malaria”, “Salmonella” and “Africa”. Articles that reported the occurrence of malaria and NTS in children, were selected. Additional articles were extracted from the references lists of the full publications and from the authors’ personal collections.NTS are one of the leading causes of bacteraemia in sub-Saharan African countries , 24–28. P. falciparum is the most common form in sub-Saharan Africa and is responsible for the vast majority of severe disease. Other Plasmodium species are less common, and their association with NTS has not been systematically studied. Both pathophysiological and/or epidemiological factors may account for the absence of any reported association, although there are a few reports of bacteraemia and P. vivax co-infection outside sub-Saharan Africa [In 2013, there were 104 countries and territories around the world in which malaria was considered endemic . An estin Africa .Although a decrease in malaria burden has been recorded in some African countries in the past decade, there are still a large number of countries where no reduction has been achieved . In manyThere are relatively few published studies on NTS asymptomatic faecal carriage and seasonality in sub-Saharan Africa. A study conducted in The Gambia showed that NTS faecal carriage remained the same throughout the year, as opposed to NTS bacteraemia cases that were seasonal , with thIn most countries in sub-Saharan Africa, the majority of NTS infections are community acquired and believed to be related to contaminated water, food, livestock, and poultry , 46, altGenerally, the risk of NTS bacteraemia is higher in children, especially those under three years of age, compared to adults , 27. In P. falciparum on a blood film together with compatible clinical symptoms, whilst recent malaria has been variably defined based on: the presence of malaria pigment (haemozoin) in blood leucocytes; the presence of gametocytes but not asexual parasites on the blood film; or a positive rapid diagnostic test (RDT) in the presence of a negative blood film. However, many studies have defined malaria exposure in a way that may represent a composite of these groups. Surprisingly few studies have explicitly reported the independent associations of current or recent malaria with NTS bacteraemia.A number of studies have reported malaria and NTS bacteraemia in the same populations of children in sub-Saharan Africa , 62–66, 2 = 9, p < 0.01) [Only two studies that explicitly reported the association of current malaria with NTS bacteraemia in children were identified , 17. A sP. falciparum histidine rich protein 2 (PfHRP2) and negative blood film were about two to four times more likely to have NTS bacteraemia compared to those without any evidence of recent malaria [PfHRP2 and negative blood film were more likely to have NTS bacteraemia compared to those without  [PfHRP2 and negative blood film had a higher risk of NTS bacteraemia compared to those without  [Similarly, four studies that explicitly reported an association with recent malaria were identified , 14–16.  malaria , 16. A h1.0-3.1) . It shou1.0-3.1) , althoug1.0-3.1) , for recDespite some limitations, the aforementioned studies have provided accumulating body of evidence that has been consistent with an association between symptomatic/recent malaria and NTS bacteraemia in sub-Saharan African children. Whilst there is evidence for the association between symptomatic malaria and NTS bacteraemia, few data on the association between asymptomatic malaria infections and NTS bacteraemia, were found. There is some evidence that among children with severe malaria, those with severe malarial anaemia have a higher risk of NTS bacteraemia , suggestSome studies have reported an association between malaria parasitaemia and any Salmonella bacteraemia  , 69, 70.Studying the pathogenesis of NTS bacteraemia in humans is difficult, but findings from animal models have suggested a plausible sequence of events from the arrival of NTS in the intestine to invasion, bacteraemia and dissemination to other organs. Invasive disease begins with invasion of epithelial and M cells of the intestine, facilitated by the proteins of the Salmonella Pathogenicity Island 1, Type Three Secretion System (T3SS1), which induce internalization of bacteria by massive rearrangement of the host cell membrane . BacteriPlasmodium falciparum is particularly distinguished by its propensity for cyto-adherence of infected red blood cells to vascular endothelium, a phenomenon known as sequestration [post-mortem studies of fatal P. falciparum malaria [Numerous mechanisms have been proposed to explain the susceptibility to NTS induced by malaria, including impairments of: mucosal barrier function; macrophage function; neutrophil function; and, antibody production. stration . This is malaria . Impaire malaria , 81. In  malaria .Macrophage dysfunction has been one of the most attractive explanations for the susceptibility to NTS in malaria , becauseRelatively little attention has focused on the role of humoral immunity and the effect of malaria on the natural acquisition of antibodies against NTS. Antibodies play a clear role in protection against NTS bacteraemia, and are usually acquired during the second year of life in African children . MalariaP. falciparum malaria, which correlated with the extent of haemolysis and HO-1 induction, and interestingly persisted for weeks beyond the clearance of acute malaria infection [Despite these many possibilities, one of the most consistent and compelling observations to explain the relationship between malaria and susceptibility to NTS is the causal role of haemolysis. Apart from malaria, several other haemolytic diseases are associated with susceptibility to NTS, namely sickle cell disease and Carrion’s disease (acute Bartonellosis) , 93. Connfection . No studThere is much literature on the clinical presentation of bacteraemia but little specifically on NTS and malaria co-infection, which is the subject of this review. Generally, the signs of NTS bacteraemia are clinically difficult to distinguish from other causes of bacteraemia in children , 101, chBlood culture is the most widely used method for diagnosis of NTS bacteraemia in most studies, although PCR methods have been developed and may prove useful in future studies . The senStreptococcus pneumoniae[Data available on mortality show that the case fatality rate for children with NTS bacteraemia ranges from 12-24% , 101. A eumoniae. Anaemiaeumoniae, 105. PrData on mortality among those with severe malaria and NTS are sparse. If the different severe malaria syndromes are considered in those with co-infection, then those with severe malaria anaemia and those with cerebral malaria have a higher mortality compared to non-bacteraemic and compared to other severe malaria syndromes , 104.The most pragmatic way to prevent malaria and NTS co-infection may be to prevent malaria. There is abundant evidence that conventional strategies to reduce malaria transmission result in reductions in both malarial and non-malarial morbidity and mortality –108. FurTo prevent NTS bacteraemia in children who already have malaria, targeted antibiotic prophylaxis for children at high risk of subsequent NTS infection, for example those with severe anaemia, may represent an effective strategy. Another intriguing possibility is inhibition of haem oxygenase activity with tin protoporphyrin , 109, anPlasmodium fusion proteins, with Salmonella flagellin improving the immunogenicity of the parasite antigen [The most advanced candidate vaccine for malaria, the RTS,S vaccine, may play an important role in reducing malaria episodes, and consequently bolster the effect of conventional control measures . Even bl antigen . Since S antigen , there i antigen , and hav antigen , 115. Thet al. indicates that a quantitative measure of PfHRP2 – which is already the basis of many RDTs – might help to identify those most at risk of having co-incident bacteraemia [Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, Acinetobacter species and Pseudomonas aeruginosa[Prior to the availability of antibiotics, it was reported that some adults with malaria-NTS co-infection were cured with quinine alone, implying that treatment of malaria was sufficient to allow the host response to clear the NTS bacteraemia . Such a teraemia . Childreruginosa.However, antimicrobial resistance among NTS isolates is an increasing problem worldwide, particularly with spread of the ST313 clone of S. Typhimurium. Multidrug resistance to commonly used antimicrobials, such as ampicillin, chloramphenicol and cotrimoxazole, has been reported to be common in a variety of African settings , 118. SeAccumulating epidemiological and preclinical evidence supports the causal association between malaria and NTS bacteraemia. However, the clinical characteristics and consequences of malaria and NTS co-infection are not well defined, although mortality associated with co-infection appears higher than that associated with malaria alone. Future observational studies (case control or cohort) aimed at evaluating the association between malaria  and NTS bacteraemia, should include suitable controls from the communities, in order to confirm this association. At the same time, there is a pressing need for improved point of care diagnostics for severe bacterial infection across sub-Saharan Africa, both for patients without malaria and those whose malaria infection is complicated by severe bacterial infection. Intervention studies , focused on reducing the burden of malaria which include NTS bacteraemia as an endpoint, will help to corroborate the causal relationship. A reduction in the burden of malaria is likely to simultaneously reduce the burden of NTS bacteraemia and should be a priority.


Monitoring and evaluation of malaria programmes may require a combination of approaches to detect any effects of control. This is particularly true at lower transmission levels where detecting both infection and exposure to infection will provide additional evidence of any change. This paper describes use of three transmission metrics to explore the malaria epidemiology in the highlands of western Kenya.Plasmodium falciparum MSP-1 antibodies) and a questionnaire was administered to record household factors and assess use of vector control interventions.A malariometric survey was conducted in June 2009 in two highland districts, Kisii and Rachuonyo South, Nyanza Province, Kenya using a cluster design. Enumeration areas were used to sample 46 clusters from which 12 compounds were randomly sampled. Individuals provided a finger-blood sample to assess malaria infection  and exposure  by rapid diagnostic test (RDT) and 8.5 % (95 % CI 7.0–10.4 %) by PCR and these ranged from 0–13.1 to 0–14.8 % between clusters for RDT and PCR, respectively. Seroprevalence was 36.8 % (95 % CI 33.9–39.8) ranging from 18.6 to 65.8 %. Both RDT and PCR prevalences were highest in children aged 5–10 years but the proportion of infections that were sub-patent was highest in those between 15 and 20 years of age  and those greater than 20 years . Those reporting both indoor residual spraying (IRS) in their home and use of bed nets had lower exposure to malaria compared to those who reported using IRS or bed nets alone.In this highland site in western Kenya malaria transmission was low, but highly heterogeneous. To accurately characterize the true extent of malaria transmission, more sensitive and complementary metrics such as PCR or serology are required in addition to the standard microscopy and/or RDTs that are routinely used. This is likely to be the case in other low endemicity settings.The online version of this article (doi:10.1186/s12936-015-0944-4) contains supplementary material, which is available to authorized users. In recent years, much attention has been given to the debate as to whether malaria elimination is a viable and realistic option for malaria-endemic countries –3, whethMicroscopy has been the historic mainstay of the estimation of infection prevalence. More recently infection prevalence has been assessed by rapid diagnostic tests (RDT). Both approaches have a similar sensitivity with limits of detection in the order of 5–50 parasites/μl . Whilst In low transmission settings, the burden of malaria shifts with infections distributed across age groups. There have been few studies comparing the utility of different diagnostics in these settings , 11, 12.Plasmodium falciparum in this setting; (2) to assess the utility of different diagnostic tools, including RDTs, PCR, and the presence of antibodies to the parasite antigen MSP1-19; and, (3) to identify factors associated with malaria infections/exposures based on the three malaria metrics used.To further understand malaria dynamics in a low transmission setting, a cross-sectional survey was carried out in a highland fringe area of western Kenya . Whilst several studies have reported malaria transmission from either lowland or highland areas of Nyanza Province –20, few Anopheles funestus and Anopheles arabiensis, and P. falciparum is the predominant malaria parasite.Malaria surveys were conducted in the neighbouring highland districts of Kisii Central, Kisii South and Rachuonyo South, Nyanza Province, western Kenya as part of the Malaria Transmission Consortium project . The totP. falciparum infection among different age groups with 95 % confidence, with 80 % power, and assuming a design effect of 2. Based on historical prevalence data for the field area and its vicinity, the prevalence of infection was assumed to be 15 % among children under 5 years of age . Assuming an average household size of six persons and a 15 % non-response rate, a total sample of 1273 people per district was required to in order to estimate age-specific prevalence to a precision of ±3 %.Sample size was calculated to adequately define the prevalence of Survey compounds (which represent a family homestead of one or more houses) were selected using a two-stage sampling design. At the first stage, 46 enumeration areas  were selected randomly from a sampling frame restricted to EAs with a mean altitude of 1400–1600 m  and haemoglobin levels . Blood was also spotted onto filter paper  for subsequent molecular and serological analysis. Individuals with a positive RDT result were treated for malaria at the home according to national guidelines with artemether-lumefantrine  or referred to a health facility if suspected to be pregnant or allergic to Coartem®. Participants found to be anaemic were provided haematinics according to national guidelines at the time of the survey.The survey was conducted in July 2009, at the end of the long rainy season. At each of the households, informed written consent was sought. The head of household was interviewed to assess household wealth indices and structure and individuals were questioned about malaria control behaviours. A finger-prick blood sample was taken from all consenting household members above the age of 6 months to measure parasite prevalence using PfHRP2 RDT . Approval was also provided by the Division of Malaria Control, Ministry of Public Health and Sanitation. Prior to the surveys, meetings were held with all district administrative representatives, chiefs and district health teams to inform them of the study and acquire local approval. Community meetings were held at each of the EAs to explain the methods of study to potential participants. Household heads were approached and the study explained to members of the home. Individual informed consent was sought from all residents of the compound above the age of 6 months by signature or thumbprint accompanied by the signature of an independent witness. Consent for children under the age of 18 was provided by a parent/guardian and children between 14 and 17 years also provided written assent by signature or thumbprint accompanied by the signature of an independent witness. As defined in the Kenya national guidelines, participants below 18 years of age who were pregnant, married, or a parent were considered ‘mature minors’ and consented for themselves .P. falciparum were assessed by ELISA as previously described [P. falciparum MSP-119 was coated on plates at a concentration of 0.5 µg/mL in coating buffer and incubated at 4 °C overnight. After blocking with 1 % (w/v) skimmed milk solution, the plates were washed, and samples were added in duplicate at a final dilution of 1:1000, with a pool of hyper immune serum titrated on each plate. Plates were incubated overnight at 4 °C and 50 µl of HRP-conjugated rabbit anti Human IgG  were added into each well and incubated. After a further series of washes, substrate solution  was added and the reaction was allowed to develop for 15–20 min before addition of stopping solution (2 M H2SO4). Raw optical density (OD) measurements were averaged and normalized against the positive control samples on each plate. The cut-off value of these assays to define seropositivity was determined using a mixture model, which considers those individuals with OD values greater than the cut-off determined by the model as being seropositive. Age adjusted SCR were calculated by fitting a simple reversible catalytic model using the seropositivity data to determine the rate at which individuals convert from seronegative to seropositive by year of age. The seroreversion rate (SRR) was fixed at a reversion rate of 4.057 for both population and cluster specific calculations. The SRR used was obtained according to estimates from previous studies, and was fixed to ensure robust estimates of SCR, particularly with the smaller sample sizes for the cluster-level estimates [Antibody responses to escribed . Brieflystimates , 24.P. falciparum infection was assessed using nested PCR (nPCR). Blood-spotted filter papers were punched with a sterile hole punch and DNA was extracted using a Chelex-saponin method as described previously [The presence of a current eviously . In brieeviously  and a moeviously . Gel eleAll statistical analysis was conducted in STATA  using the survey (svy) command to account for the two-stage sampling design (EA and compound) and stratifying by district. Prevalence and means and the corresponding exact 95 % confidence interval (CI) were calculated assuming a binomial and normal distribution, respectively. Tests of significance were assessed using the Spearman’s and Chi square test as appropriate. Socio-economic status (SES) was generated using a principal component analysis (PCA) using household level wealth indicators collected as part of the questionnaire . IndicatIn total, 3566 people were sampled from 46 EAs with a mean of 78 (range 70–96) people sampled per EA Table . Forty pMalaria infection by RDT was low overall with a mean prevalence of 3.0 % 95 % CI 2.2–4.2 %) but this ranged by EA from 0 to 13.1 %  and PCR   and showed a non-significant negative trend with SCR decreasing by 0.0002 (95 % CI −0.0004 to 0.00001) with every 1 m increase in elevation. SCR was also highly variable by EAs with a similar mean elevation , which provides a measure of transmission intensity [Seroprevalence to the ntensity . The widntensity , 35.In this population, consistent with other findings, age and wealth indicators were both associated with malaria infection by all metrics . ChildreInterestingly, altitude of residence of survey participants was not associated with either measure of current infection in the adjusted analysis and only with malaria exposure, determined by serology, at the highest altitudes. This lack of association may be due to the low RDT and PCR prevalence in this population or due to the pre-selected narrow altitude range. However, this suggests that the association with altitude and current infection may be more useful at a larger spatial scale  or that Regression analyses suggested that those who had received IRS in the past 12 months had an increased risk of being malaria positive at the time of the survey. Although this is at first counter-intuitive, this finding is likely confounded by the fact that areas with higher perceived risk tend to be the ones more like to be targeted and receptive to the use of mosquito control . In 2009It was observed that those who reported using both ITN and IRS resided in a cluster with a significantly lower SCR than those using just a single intervention. This suggests that combining interventions may be more efficient at reducing exposure and driving transmission to even lower levels, as demonstrated in modelling studies –48. Data19 with a comparatively long antibody half-life [One limitation of the serology component of this study was the use of only one antigen  to assess anti-malarial antibody responses. Increasing the number of antigens would most likely lead to marginal increases in the overall seroprevalence estimates due to heterogeneous immunity in the population. However, whilst this means that the serological outcomes presented here can be assumed to be underestimates, responses to non-variant antigens such as MSP-1alf-life  should balf-life . Furtheralf-life .In this study, EAs were sampled from two districts: Kisii is classified as having low and epidemic-prone malaria transmission whereas Rachuonyo has low and stable transmission. However, analysis of malaria burden determined by all metrics at the EA level showed no clear differences, with those with the highest burden being equally in both districts. The cluster with the highest SCR was in fact located in Kisii, the district with lower overall reported levels of malaria burden . This suggests that allocating resources at the district level may not be ideal; pockets of high burden within the overall low transmission area would be missed and may seed transmission to neighbouring areas . AlthougUltimately, in the highlands in western Kenya, malaria transmission is generally low, but highly heterogeneous and difficult to measure accurately using routine diagnostic tools. This study found a large range in malaria burden at the EA level and a large population of sub-patent infections. This suggests that for malaria surveillance and monitoring, more sensitive metrics such as molecular or serological methods may complement current diagnostic tools in order to accurately assess malaria burden in the population, determine reservoirs of infection, identify risk factors and, therefore, better target appropriate interventions.


Methods. Sera samples (803) were collected from healthy volunteers from the three main districts  of Hormozgan Province. A questionnaire was used to record the sociodemographic features of the participants during sample collecting. An in-house ELISA test, using crude antigens obtained from cell culture of Plasmodium falciparum, was adapted and used to detect anti-malaria antibodies in the sera. Results. The overall seroprevalence of malaria was 8.7% (70 out of 803 samples). A significant correlation was found between seropositivity and place of residence, where the highest rate of seropositivity was seen in Bandar Lengeh (west of the province). The highest seroprevalence of malaria (13.2%) was seen in the age group of 11–20 years and also in low educated individuals. Correlation between seropositivity and gender, age, and educational levels of the participants was statistically significant (P < 0.05). Conclusion. Findings of this study indicate that the rate of seropositivity to malaria in this area is not high and this might be linked to the success of malaria control programs during the last decades in the region.The seroepidemiological condition of malaria in three main districts of Hormozgan Province, a low transmission area in southeast of Iran, was investigated.  Anopheles mosquito and is caused by Plasmodium protozoan parasites. The disease is common in many tropical and subtropical areas of the world and is considered as an important life-threatening factor for people living in these regions. A total of 109 countries are considered as malaria-endemic regions and an annual count of 250 million cases of malaria occurs, in which close to 1 million cases are fatal [Malaria is an infectious disease transmitted by theMalaria has been considered as a threat in Iran with a high morbidity rate in the last decades –4. Nowad P. vivax and the main vector for the parasite is Anopheles stephensi [The predominant species of parasite in southern part of Iran istephensi .The use of thin and thick smears for the diagnosis of malaria was conducted in 1900 and even nowadays it is considered as the gold standard in the diagnosis of malaria. Studies have shown that serological methods, for the diagnosis of malaria, can be considered as an effective tool in epidemiological studies and prevention programs of malaria, since these methods are able to detect previous exposure or contact with the disease.Measuring the prevalence of anti-malaria antibodies  is a valuable approach for defining the status of malaria transmission in a given area and also a useful method for malaria surveillance . MoreoveAs ELISA method consists of simplicity and low cost, compared with other serological methods, and is able to evaluate multiple samples simultaneously; therefore, in this study, ELISA method was used for the seroprevalence evaluation of malaria in a low transmission area of malaria in Iran.The current study was conducted in three main districts  of Hormozgan Province, located in the southeast of Iran. Hormozgan is one of the 31 provinces in Iran and its provincial capital is Bandar Abbas. In 2011, Hormozgan Province had a population of 1,500,000 people. Hormozgan Province maintains a long warm (9 months) season and a short (three months) cool season. The province is one of the warm and dry regions of Iran, with warm and humid coastlines in summer, where the temperature climbs up to 52°C. The average annual temperature of the region is about 27°C. The province is considered as a malarious province in Iran.After getting approval from the Ethics Committee of Shiraz University of Medical Sciences, blood samples were taken from 803 healthy volunteers from three main districts of Hormozgan Province including Bandar Lengeh (171 samples), Bandar Abbas (457 samples), and Bandar Jask (153 samples). The samples were taken from the tip of the left second finger of each subject with the help of a disposable lancet. Three drops of blood were collected from each participant onto a filter paper (Whatman 3 MM), dried at room temperature, sealed in a plastic bag, and stored at −20°C until use. Cluster sampling, based on the population of each district, was used for sample collecting. These three districts are in National Malaria Control Program areas in Iran and names of the head of households were available in local health centers. Cases were randomly selected from these files and reached either in their house or in the health centers. Informed consent was obtained from the subjects before sampling. P. falciparum with the help of two media, Albumax complete medium and alternative malaria complete medium (MCM) [ P. falciparum was kindly provided by Professor S. Zakeri . Malaria antigen was prepared from in vitro culture of P. falciparum. Briefly, HEPES was added to the contents of malaria culture flask, containing RPMI medium, and the content was centrifuged at 1800 g for 10 minutes. The pellet was diluted, 10 times, with the same medium and 2 mL of sample was added to 2.5 mL of cold Percoll (60%), in a 15 mL Falcon tube on ice. After centrifugation at 4°C for 15 minutes, the interphase layer containing parasites schizont was isolated. Then, 5% saponin in PBS was added to the sample and mixed thoroughly to eliminate the red blood cells. Sample was immediately centrifuged, 3200 g for 10 minutes, to remove the saponin. The obtained pellet was washed twice with PBS and kept at −20 until use.Trager and Jensen (1976) method was used for continuous culturing ofum (MCM) . Strain μL of PBS-T to each well, corresponding to 1/50 of serum dilution). The plate was placed on a shaker for at least 12 hours at 4°C and centrifuged with a plate centrifuge at 2200 g for 20 minutes. The supernatant from each well was removed and used for ELISA.The blood spot on filter was punched with a paper punch and each punch was placed in a well of a U shaped ELISA microplate. Extraction of sera from filter papers was performed as previously described with some modification . Brieflyμg/mL of crude malaria antigen in coating buffer . The plate was washed with PBS containing 0.05 Tween three times. Unbound sites were blocked, using skimmed milk (5%), and washed twice after 2 hours. Then, 100 μL of serum containing solution, extracted from Whatman paper, was added to each well and incubated in room temperature for 1.5 hours followed by a wash-up as before. Horseradish peroxidase (HRPO) conjugated anti-human antibodies (Sigma) at 1/1000 dilution in PBST were applied to the plate and incubated at room temperature for 1 hour. Following washing, 100 μL/well of chromogen/substrate  was added to the plates and the absorbance values of the wells, at 490 nm, were determined by a microplate reader, after 30 minutes. Negative and positive samples were included in each series. Negative controls sera were collected from people from nonmalarious area and those who had no history of malaria. Positive sera were selected from samples of our previous study [ELISA microplate was sensitized with 5 us study . Cut-offχ2 test was used to assess the univariate correlation of demographic and malaria seropositivity.Standard The mean age of participant was 27.4 years (range 1–74). Males constituted 58.3% of the subjects and 41.6 of them were females. The overall seroprevalence of malaria was 8.7% (70 out of 803 samples). The number of positive cases in Bandar Abbas was 15, in Bandar Lengeh it was 51 samples, and in Bandar Jask it was 2 cases. A significant correlation was found between seropositivity and place of residence. The highest seroprevalence of malaria (13.2%) was seen in the age group of 11–20 years. Seropositivity was higher in low educated individuals. Correlation between seropositivity and gender, age, and educational levels of the participants was significant. Despite the significant advances that have been made over the years to control malaria, the disease is still a major health problem in the world and about one hundred of the countries or regions of the world are among the malaria-endemic areas , 3. In ISeveral studies pointed out that serological markers are valuable tools for evaluating and monitoring of malaria transmission , 9, 13. Moreover, in this study filter paper has been used for sample collecting which has several advantages. Perhaps the most important advantage is the elimination of blood sampling from the vessels, which often in infants and children have been the subject of debate. In epidemiological studies, obtaining blood samples from the vessels due to the required equipment is more expensive than obtaining blood samples from the capillaries. Another important advantage of this method is the ease of transfer and storage of samples before experiment. The method is less invasive and also reduces the risk of disease transmission.Plasmodium antibodies. In a seroprevalence study conducted in Sistan and Baluchestan Province, anti-P. vivax and P. falciparum were detected in 31.6 and 5% of blood donors, respectively, by IFA [ P. falciparum was used in ELISA. This antigen contains pan malaria antigens which react with P. vivax and P. falciparum antigens. Therefore, antibodies against both species of malaria have been detected in the participants in the current study.In the present study, 8.7% of the participants had anti-, by IFA . It is wThe seroprevalence of malaria in this study is lower compared with those studies conducted in other regions of the world. This might be related to the success of malaria control programs in the region and implies that eradication of the disease in the province in the coming years is feasible .A very low level of seropositivity to malaria (about 1%) was reported in a previous study in another district (Bashagard) of the province. The main explanation of this low seropositivity is that the study has measured anti-malaria antibodies  by ELISA, using either PvMSP-119 or PfMSP-119 antigens, instead of using a pan-malaria antigens . Antibod Anopheles biting and the link between malaria susceptibility and gender has not been documented.In this study, seropositivity and gender were significantly associated. In total, women had more antibodies compared to men. The reason for that is not clear since both males and females are equally exposed toIn the current study, the highest seropositive cases were from the age group of 11–20 years and the lowest in the age group of 1–10. The lower seroprevalence in children below ten years indicates less exposure of individuals and probably lack of local transmission of malaria. Higher rate of seropositivity in postsecondary educational level, in comparison with primary and secondary levels, may also be related to the age of the subjects rather than educational level.Some of the seropositive cases in this study may have parasite, though at very low level, and may act as malaria reservoir, contributing to the persistence of malaria transmission. These seropositive asymptomatic cases may be a big challenge for malaria elimination program in any endemic area. P. vivax and P. falciparum in 17.9% and 1.7% of the cases, respectively. The serum samples have been seropositive with P. vivax and P. falciparum antigens in 54.2% and 32.1% of the samples, respectively [The study of Nateghpour et al. on 408 patients with suspected malaria symptoms microscopically detectedectively . The higectively .Taken together, the findings of this study demonstrated a relatively low rate of seropositivity of malaria in Hormozgan Province, southeastern Iran.


Diagnosis is “the act of identifying a disease, illness, or problem by examining someone or something.” When an individual with acute fever presents for clinical attention, accurate diagnosis leading to specific, prompt treatment often saves lives. As applied to malaria, not only individual patient diagnosis is important but also assessing population-level malaria prevalence using appropriate diagnostic methods is essential for public health purposes. Similarly, identifying (diagnosing) fake antimalarial medications prevents the use of counterfeit drugs that can have disastrous effects. Therefore, accurate diagnosis in broad areas related to malaria is fundamental to improving health-care delivery, informing funding agencies of current malaria situations, and aiding in the prioritization of regional and national control efforts. The International Centers of Excellence for Malaria Research (ICEMR), supported by the U.S. National Institute of Allergy and Infectious Diseases, has collaborated on global efforts to improve malaria diagnostics by working to harmonize and systematize procedures across different regions where endemicity and financial resources vary. In this article, the different diagnostic methods used across each ICEMR are reviewed and challenges are discussed. Combating the global malaria burden begins with accurate diagnosis, which guides specific treatment and public health reporting. Reliable malaria diagnosis improves health-care delivery and informs funding agencies of current malaria situations, which is key for prioritization of regional- and national-level control efforts. Adequate malaria diagnostics contribute toward the long-term goal of malaria elimination, but accuracy in both testing and reporting depends on resource availability, health worker expertise, and formal reporting policies that vary among regions. Microscopy and rapid diagnostic tests (RDTs), which detect parasite antigen, are most commonly used to diagnose malaria; each method has its own strengths and weaknesses.Plasmodium parasitemic individuals do not come to clinical attention and cannot be detected by these techniques because of limited sensitivity.6Since 2010, WHO has recommended either RDT or microscopy confirmation of suspected malaria cases before treatment.During the past decade, highly sensitive and specific nucleic acid amplification techniques have been developed to detect malaria parasites including polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and reverse transcriptase PCR (RT-PCR)  hypnozoite carriers, yet developing methods to do so is important because the biology of P. vivax makes elimination of this parasite more difficult.P. ovale or Plasmodium malariae into account, particularly in Africa, or the zoonotic Plasmodium knowlesi in Asia. It is important to identify infections due to both single and more than one Plasmodium species to ensure proper antimalarial drug treatment.P. vivax and P. ovale form dormant hypnozoites that should be treated with an 8-aminoquinoline , despite challenges related to using these medications on population scales because of the potential for adverse events due to the presence of even small percentages of people having some form of glucose-6-phosphate dehydrogenase deficiency.Accurate diagnosis is essential for treating suspected malaria cases, but in many malaria-endemic regions fever is commonly presumed to be malaria without confirmation.Currently, there are many molecular diagnostics platforms being used in the ICEMR network . Even thPlasmodium species causing malaria toward a more complete understanding of the complex interactions among human hosts, malaria parasites, and mosquito vectors in diverse ecological niches taking into account expected and unexpected changes occur over space and time. All 10 ICEMRs implemented light microscopy–based diagnosis as the primary quantification metric. Nine study sites used microscopy plus RDTs; Amazonia ICEMR did not use RDTs for several reasons: microscopy is generally available and accurate; the dominance of P. vivax compared with P. falciparum malaria, combined with the lactate dehydrogenase (LDH)–based RDTs that were not considered sufficiently sensitive; and national policy requires microscopy-based diagnosis. In Peru, one country in which the Amazonia ICEMR is based, pfhrp2- and/or pfhrp3-lacking P. falciparum were first reported, which made the histidine-rich protein 2/3 (HRP2/3)–based RDT inadequate for use as described later in the section, HRP2/3 deletion among the circulating parasites. Among ICEMRs, the choice of different RDTs has been largely dependent on availability and national policy–based recommendations –specific RT-PCR  protocols are well established to assure reliable and comparable results. For light microscopy, double reading of the slides with a third reader for discrepancies is standard among ICEMRs. WHO recommends that parasite quantification be performed against 200 white blood cells (WBCs), and if the parasite count is less than 100 after counting 200 WBCs, the microscopist should continue to 500 WBCs.WHO collaborates with the Foundation for Innovative New Diagnostics to evaluate and standardize the use of RDTs.5All 10 ICEMRs currently use at least one of the molecular diagnostic methods such as conventional PCR, qPCR, and LAMP . SuitablDried blood spot (DBS) sampling is commonly used to preserve whole blood samples because it is an easy, durable method with an efficient use of space and optimal transport conditions. In research setting, DBSs have been used as starting material for PCR, RT-PCR, and serology.P. falciparum asexual stages and young gametocyte stages. Hence HRP2 has been used in RDTs to detect P. falciparum.Plasmodium LDH (pLDH), from the glycolytic pathway, is found in all malaria species and is the second most commonly used RDT antigen.P. falciparum.HRP2 is a protein found only in P. vivax parasitemia is characteristically low), and the recent discovery of some region-specific field isolates lacking the HRP2 protein. In 2010, P. falciparum lacking pfhrp2 and/or pfhrp3 genes were first isolated from infected human subjects in the Amazon region of Peru.pfhrp2 and/or pfhrp3 gene deletions.pfhrp2/3 deletion in P. falciparum strains has serious implications for diagnosis especially for countries and regions where the antimalarial treatment is primarily based on RDT diagnosis. Therefore, it is important to monitor the prevalence of pfhrp2 and/or pfhrp3 gene deletions in regions where RDTs based on PfHRP2 are the primary mode of malaria diagnosis, given the potential need to base procurement decisions on specific RDT detection proteins.RDTs are particularly useful where microscopy is not available, whether because of financial constraints or lack of equipment or sufficiently trained personnel. RDT performance in the field is influenced by many factors such as manufacturing quality, handling and storage conditions, user interpretation, parasite density and limits of detection  compared to pfhrp2 (41%), and 22% of isolates had both genes deleted.pfhrp2 and/or pfhrp3.Plasmodium falciparum with the double deletion of pfhrp2 and pfhrp3 was found in a patient who returned to Europe from Brazil and misdiagnosed using an RDT based on aldolase and PfHRP2; this patient was treated as having P. vivax.P. falciparum isolates from clinically infected subjects; however, it was not possible to amplify exon 2 for both pfhrp2 and pfhrp3.P. falciparum PfHRP2-positive individuals even though they were diagnosed positive by light microscopy and pLDH . Thus far, Guyana is the only country in South America where all P. falciparum isolates have been found to carry pfhrp2; therefore, to date, the performance of PfHRP2-based RDTs here has not yet been affected.71Peru was the first country where revalent % compareBecause they are relatively expensive and widely used, ACTs have become a target of counterfeiters.Given that workers in malaria-endemic populations are very familiar with using RDTs for malaria diagnosis, the southeast Asia ICEMR chose to develop a lateral flow dipstick as the point-of-care tests for qualitative and semiquantitative detection of artemisinins in antimalarial drugs. Specific antibodies are needed for such dipstick assays, hence new monoclonal antibodies have been made for such efforts, which include the differentiation of commonly used artemisinin derivatives including artemether, artesunate, and dihydroartemisinin. The first approach was to immunize mice with artesunate conjugated to bovine serum albumin at the succinate group, which yielded a monoclonal antibody (mAb) that reacted with artemisinin, artesunate, and dihydroartemisinin, but with limited reactivity to artemether,All aspects of malaria control and elimination must have robust quality management and control standards. The concept of quality management for malaria diagnostics covers the entire process including establishment of standard operating procedures for each method and technique used in diagnosis, especially sample tracking and handling, to achieve accurate and reliable test results and reporting. QA aims at improving and standardizing each component of this complicated process to minimize or avoid unreliable results. QC emphasizes test accuracy and precision.ICEMR sites have established several quality control strategies, most of which aim to comply with WHO guidelines .42,93 InTo maintain microscopy quality, refresher training programs for microscopists using reference slides as recommended by WHO were reported.P. falciparum DNA,P. falciparum DNA,98For molecular diagnosis, the inclusion of well-characterized negative and positive controls  was reported from all ICEMRs. Protocols to freshly extract positive and negative controls to assure efficiency and lack of contamination in processed samples remain to be standardized. WHO provides international standards for Although WHO has published standardized protocols for microscopy and RDTs, no such protocols exist for nucleic acid amplification–based diagnostic tests such as PCR. Each ICEMR site has established conventional PCR; variation in details of sample preparation  remains a challenge for effective comparisons of diagnostic and treatment outcomes data. Other molecular diagnostic tools, such as qPCR, LAMP, and other sensitive techniques, are either not yet available in all the ICEMR regions or were not selected as part of the research design.International standardization of diagnostic platforms in settings and research networks such as the ICEMR will be tedious. However, this process will include routine technical training for technicians, monitoring of diagnostic accuracy by external QC procedures, outreach training, reference laboratory support, and regular proficiency testing to ensure comparable results among laboratories. An external QA plan is important to assure valid diagnostic results and accurate reporting. Harmonization of PCR procedures more recently has taken on higher priority given the increasing importance of molecular diagnostics in the ICEMR network and elsewhere in infectious diseases epidemiology, clinical care and research. Access to reference laboratories to assure accurate and available QA/QC in both malaria microscopy and PCR should be readily available, as a matter of public health policy within malaria-endemic regions.Although this article focused on different diagnostic platforms and protocol standardization, being able to harmonize data across platforms is essential, in the case of the ICEMRs, enabling comparable case definitions across the ICEMRs for which diagnostics lead to interventions, whether for disease or surveillance.


The escalating level of mosquito resistance to pyrethroid insecticides threatens the effectiveness of insecticide-treated nets (ITNs) for malaria control in Malawi. An evaluation of the effectiveness of ITNs for preventing malaria in children aged 6–59 months old, after 1 year of mass distribution of LLINs was conducted in Machinga District, Malawi, an area of moderate pyrethroid resistance.A facility-based, case–control study among children 6–59 months was conducted in an area of pyrethroid resistance between March and September 2013 in Machinga District. Cases and controls were children with fever who sought care from the same hospital and tested positive and negative, respectively, for malaria parasites by microscopy.A high proportion of both cases (354 of 404 or 87.6 %) and controls (660 of 778 or 84.8 %) slept under an ITN the night before the survey. In univariable logistic regression, older age , sleeping on the floor versus a mattress (p < 0.001), and open versus closed house eaves (p = 0.001) were associated with increased odds of malaria, whilst secondary education of the caretaker, having windows on multiple walls, and being in the least poor wealth quintile (p < 0.001 for each) reduced the odds of malaria; ITN use was not associated with malaria (p = 0.181). In multivariable analysis, older age (p < 0.001) and secondary education of the caregiver (p = 0.011) were the only factors significantly associated with malaria.This study did not find a significant personal protective effect of ITNs. However, high use of ITNs in the community and recent findings of lower malaria incidence in ITN users compared to bed net non-users from a cohort study in the same area suggest that ITNs provide community protection to both users and non-users alike in this area. Anopheles vectors [Insecticide-treated bed nets (ITNs) remain a cornerstone of global malaria control. Their excito-repellency effect deters mosquitoes from entering houses and causes premature exit from houses where ITNs are in use . As a re vectors , there i vectors  led to tIn this study, results are presented from a prospective clinic-based, case–control study conducted to evaluate the effectiveness of ITNs in preventing malaria amongst children aged 6–59 months in an area of moderate insecticide resistance to pyrethroids in Malawi.Plasmodium falciparum is the dominant parasite species although Plasmodium malariae and Plasmodium ovale have been recorded [Anopheles funestus is the main vector for malaria transmission in the area. Recent data on mortality of An. funestus s.s. exposed to pyrethroids show high levels  of pyrethroid resistance which is way below the 90 % cut-off point recommended by the WHO, in all districts where entomologic resistance monitoring has been carried out using the standard WHO test kits and procedures, including the study area [® Net  ITNs had been completed in the district in July 2012, during which one ITN for every two people was distributed.This study was conducted in the under-five outpatient clinic of Machinga District Hospital in southern Malawi , see Fig. recorded . Anophel® device, Orchid Biomedical Systems, India) were performed on all children and patients testing positive were treated by health facility staff. Data were collected using personal digital assistant  with programmed skip patterns and logic checks.This clinic-based, case–control study recruited cases and controls between March and September 2013. To be eligible to participate, children had to be between 6 and 59 months of age, living within 15 km of the hospital and not participating in another ongoing malaria study. Age-eligible children presenting at the Machinga District Hospital under-five outpatient department with current fever (measured axillary temperature ≥37.5 °C) or history of fever in the previous 48 h (as per caretaker) were invited to participate. After written informed consent was obtained from a caregiver, children were enrolled in the study. A questionnaire about illness history, ITN use, socio-economic status (SES) and other malaria risk factors was administered, and a thick and thin blood smear was collected from each participant. Rapid diagnostic tests for malaria  or by a factor of 10 or more for low-density parasitaemia slide (<400 parasites/μL). Any discrepant readings were resolved by a third microscopist.P. falciparum asexual stage parasites, once blood smears were read later on at the study laboratory. Controls were enrolled children with current or recent fever and a negative blood smear for P. falciparum. Data were analysed using STATA version 12 . A SES index was calculated using principal component analysis taking into account 14 household factors including: electricity, caregiver’s and spouse’s occupation, ownership of various assets, source of water, type of toilet, and material of the floor and roof [Cases were defined as enrolled children with current measured temperature or recent reported fever (within 48 h) and a positive blood smear result for and roof . The firP value <0.05 was considered significant.Univariable and multivariable logistic regression analyses were conducted to examine the relationship between potential risk factors and malaria. Only factors that were significant in univariable logistic regression analysis were included in a multivariable analysis using logistic regression. Odds ratios (ORs), adjusted odds ratios (AOR), and 95 % confidence intervals (CIs) were calculated in the analyses. For all statistical tests, a Written informed consent was obtained from parents or caregivers of each study participant prior to enrolment in the study. The study protocol was reviewed and approved by the institutional review boards of the University of Malawi, College of Medicine, Blantyre, Malawi and the Centers for Disease Control and Prevention, Atlanta, GA, USA.A total of 403 cases and 778 controls had complete data and a household visit and were included in the final analysis. For 34 children (2.6 %), caregivers refused the home visit, and for 71 (5.5 %), homes were unable to be located after the clinic visit. There were no differences by sex between cases and controls. Median household size (IQR) was 4 (3–5) for both cases and controls. A high percentage of both cases (87.6 %) and controls (84.8 %) slept under an ITN the night prior to enrolment, and nearly as many were reported to have consistent net use, defined as sleeping under an ITN all 14 nights in the 2 weeks before illness onset   nor consistent net use immediately prior tothe illness , was associated with a reduction in the odds of malaria , age 36–59 months  and having a caregiver with at least secondary education  remained statistically significantly associated with malaria  and controls (84.8 %), which may be attributable to a national-wide mass ITN distribution campaign approximately 1 year before the study. The ITN distribution campaign was part of a universal coverage campaign that Malawi had in 2012, during which one ITN was distributed per two residents, with an extra net for households with an odd number of inhabitants. Thus, nearly all households in the study area owned at least one ITN during the study period. This lack of an individual-level protective effect could therefore be due to community-wide protective effects of ITNs in the study area. Research has shown that ITNs confer protection on community members who may not sleep under a net, even with modest ITN coverage of 35–65 % use among all ages, especially if the nets are of good quality . The comtive ITN  and redutive ITN . Even intive ITN . ImproveAn. funestus in this area [The finding of widespread resistance to his area  and elsehis area  raises this area . In addihis area . These chis area . Abdullahis area  showed this area  and it cIn addition, the lack of protective  efficacyThis study indicated that caregiver’s education was associated with lower risk of malaria, as was living in a higher SES household and having windows on at least two different walls, although the latter two were only marginally statistically significant when adjusted for other factors. This is consistent with findings from a recent meta-analysis which showed that the odds of malaria were higher in the poorest compared to the least poor children  and receThe study did not find a significant personal protective effect of ITNs in an area of high ITN use and pyrethroid insecticide resistance. The lack of an individual protective effect could be due to overriding community-wide protection from ITNs in this area or limitations in the case–control study design. The use of ITNs should still be continued in the area because of their physical barrier that they provide and their community-wide effect by killing older more epidemiologically important vectors.


Seasonal malaria chemoprevention (SMC) has been shown to be highly efficacious against clinical malaria in areas where transmission is acutely seasonal. SMC targeting depends on a complex interplay of climate, malaria transmission and population distribution. In this study a spatial decision support framework was developed to identify health districts suitable for the targeting of SMC across seven Sahelian countries and northern states of Nigeria that are members of the Nouakchott Initiative.A spatially explicit decision support framework that links information on seasonality, age-structured population, urbanization, malaria endemicity and the length of transmission season was developed to inform SMC targeting in health districts. Thresholds of seasonality, population and receptive risks were defined to delineate SMC suitable health districts and define the age range of children for targeting. Numbers of children were then computed for the period 2015–2020 in SMC districts. For 2015, this was combined with maps of length of malaria transmission seasons and WHO recommended treatment regimen to quantify the number of tablets required across the SMC health districts.A total of 597 Sahelian health districts were mapped, out of which 478 (80.1%) were considered suitable for SMC based on seasonality and endemicity thresholds. These districts had an estimated 119.8 million (85%) of the total population in 2015. In the six years from 2015–2020, it is estimated that a total of 158 million children 3m to <5 years, 121 million of whom were in rural areas, will need SMC to achieve universal coverage in the Sahel. If the upper age limit of SMC targeted children was increased to <10 years in low transmission districts, a total 177 million overall, of whom 135 million were rural children, will require chemoprevention in 2015–2020. In 2015 alone, an estimated 49–72 million SP tablets and 148–217 million AQ tablets will be needed to cover all or rural children respectively under the different scenarios of upper age limits.Our proposed framework provides a standardised approach to support targeting and scale up of SMC by the countries of the Nouakchott Initiative. Our analysis suggests that the vast majority of the population in this region are likely to benefit from SMC and substantial resources will be required to reach universal coverage each year. Seasonally targeted intermittent preventive treatment of malaria in children, also known as seasonal malaria chemoprevention (SMC), has been shown, under trial conditions, to prevent approximately 75% of clinical malaria episodes, including severe malaria, in areas where transmission is concentrated within a few months of the year ,2. In FeP. falciparum transmission [SMC targeting depends on a complex interplay of seasonality, the length of the transmission seasons, endemicity, population distribution and urbanisation. Where routine health systems remain weak, the most reliable sources of malaria risk are those that predict the intensity of smission . Howeversmission . The useP. falciparum infection changes, with older children at greater risk [Where transmission potential in such areas is high due to the continued presence of efficient vectors and large numbers of asymptomatic human hosts, SMC is critical to sustain low disease incidence and maps of receptive risks are better suited to defining endemicity thresholds for SMC. Conversely, as transmission declines to very low levels, the age pattern and the clinical burden posed by ter risk ,8. IncreIn this study, we develop a spatial decision support framework that captures the interplay of seasonality, age-structured population, urbanization, endemicity and the length of transmission season to inform SMC targeting in Burkina Faso, Chad, Gambia, Mali, Mauritania, Niger, Senegal and nine northern states of Nigeria . Layers of information are assembled and analysed to empirically quantify each element of the SMC spatial decision support framework. SMC health districts are defined and the numbers of target children by age range are estimated for the period 2015–2020, coinciding with the baseline and the first milestone years of the new WHO Global Technical Strategy for malaria . For illP. falciparum malaria risk map [P. falciparum malaria risk map [The spatial decision support framework for SMC targeting that we propose in this study  is differisk map , an apprrisk map  is used Detailed descriptions of the assembly of spatial layers of information are provided in the footnotes for Population density mapping techniques ,13 were An approach developed by Cairns and colleagues  was adopP. falciparum malaria transmission [Within health districts that were identified as seasonal, the median number of transmission months was extracted from a map obtained from the International Research Institute for Climate and Society website ,17. ThisPlasmodium falciparum parasite rate (PfPR) data standardised to the age range 2 to just below 10 years (PfPR2-10) at 1 × 1 km spatial resolution for the year 2000 and 2010 was used to define receptive and current risks respectively [PfPR2-10 surfaces were used together with the population distribution surfaces for the same year at matching 1 × 1 km spatial resolutions to compute population adjusted PfPR2-10 (PAPfPR2-10) by health district for both 2000 and 2010. To compute PAPfPR2-10, the mean proportion of the posterior PfPR2-10 for a given year was multiplied with the pixel level population surface to estimate the numbers of people who were likely to be infected per pixel for that year, which was then summed for each health district. The estimated population that was infected was divided by the total population of the health districts for that year to generate the mean PAPfPR2-10, (Section F in Recently published continuous maps predicted from community ectively . The conPfPR2-10 (receptive risk) of ≥5%. Within these SMC health districts, children 3 months to below 10 years were targeted in those where transmission was 5% to 10% PAPfPR2-10 in 2010. In districts where transmission PAPfPR2-10 in 2010 as >10%, SMC was targeted at children 3 months to below 5 years. Projected population of SMC targeted children by age class were extracted for each year from 2015 to 2020 for each health district. Using the year 2015 for illustration, the estimated number of SMC targeted children was multiplied by the median number of malaria transmission months per health district and the SP and AQ tablets required per child per month  and had 2000 PAP. falciparum transmission intensity, as measured by PAPfPR2-10 in 2000  had the defined receptive risk threshold suitable for SMC of ≥5% PA in 2000 . A total in 2000 . 478 , Mali and Burkina Faso account for over 60% of all children living in areas suitable for SMC. From a Nouakchott Initiative regional perspective a bigger impact may be achieved by prioritising these countries. However, the potential impact of SMC is not determined by the size of the population at risk alone but also the strength of the health system tasked to deliver the intervention, the availability of domestic resources in addition to external funding to sustain coverage and other factors such as conflict, general good governance and conflict. In addition, where regional initiatives exist, prioritising one country over another, without the requisite political goodwill, may lead to tension. Decisions on prioritising high population at risk and potentially high return countries must therefore be done on the basis of regional agreements and the existing operational environment to ensure maximum impact. Overall, the primary objective should be that resources are made available to all those countries that are likely to benefit from SMC.The reliability of the various inputs into the spatial decision support framework drive the overall accuracy of the definition of the SMC target health districts and the quantification of the size of the target population of children and the required tablets. Wide variations exist in the number of transmission months within the SMC suitable areas of Sahel and sub-Sahel , key facPfPR2-10 indicate reasonable accuracy based on the prediction to a carefully selected holdout dataset [PfPR2-10 and the size of SMC population, are also based on modelled redistribution of census data, the accuracy of which is largely determined by the spatial resolution and the currency of this data [The model-based geostatistical methods use to predict  dataset . Howeverhis data . All thehis data . DespiteP. falciparum, by far the most pathogenic of the four malaria parasites found in Africa. However, the Sahel sub-region does support P. vivax transmission and significant numbers of clinical cases of P. vivax have been documented in Mauritania, and increasingly in and around Nouakchott [P. vivax in the Sahel and investigate the clinical significance of this parasite and its responsiveness to SMC using SP+AQ.Finally, we have considered uakchott , 32; Maluakchott , 34, Buruakchott  and Nigeuakchott . FurtherP. vivax and the role of SMC to reduce its burden in the Sahel. In addition, there are likely to be other sub Saharan African countries outside of the Sahel that meet both the disease burden and seasonality criteria for SMC suitability [Our spatial decision support framework allows for a common platform for decision making for SMC for the countries of the Nouakchott Initiative. From the estimated numbers of children to be targeted with SMC for the period 2015–2020 and tablets required for 2015 alone, the region clearly needs to invest substantial resources to reach universal coverage of SMC in areas that are likely to benefit the most. Where the financial and opportunity costs of various delivery mechanisms are known , our emptability  but whertability . ExperimS1 File(DOCX)Click here for additional data file.