Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes.Antibody immunity is thought to be essential to prevent severe P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of “virulent” PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children.A 1000 feature P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.Children with uncomplicated and cerebral malaria have similar breadth and magnitude of The online version of this article (10.1186/s12936-018-2323-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria is central to the prevention and clearance of symptomatic disease [P. falciparum infection [Antibody immunity to  disease , but is  disease . Immunitnfection . It is sP. falciparum proteome-wide array encompassed approximately a quarter of the inferred proteome (2320 proteins encoded by 1204 genes) and identified 491 immunoreactive proteins recognized by sera from a cohort in Mali [Although not all studies agree , prior s in Mali .P. falciparum erythrocyte membrane protein 1 (PfEMP1), a parasite variant surface antigen (VSA) encoded by ~ 60 var genes, is critical for cerebral sequestration [In CM, stration , 14. Sinstration , childrestration , 16. Thestration . Studiesstration –25. Breastration , 27, andstration .P. falciparum antibody responses, and magnitude of PfEMP1 specific antibody responses between children with uncomplicated malaria (UM) and children with stringently defined CM  in acute infection and at 30 days of convalescence. A new ~ 1000 feature partial proteome array based upon the 3D7 P. falciparum reference genome that prioritized antigens with relevance to diagnostics and human anti-malarial immunity was used. The antigens represented on this array include vaccine candidates as well as proteins that have been associated with either exposure to P. falciparum or protection from clinical disease. The study results indicate that children with UM and CM have broad seroreactivity to the panel of ~ 1000 P. falciparum antigens and have lowest seroreactivity during acute disease to EPCR-binding “virulent” PfEMP1 antigens represented on the array [Here, partial proteome microarrays were used to identify and characterize differences in global antibody level, breadth and magnitude of he array , 25. DesP. falciparum parasitaemia, and no overt signs of chronic disease, malnutrition, or progression to severe malaria; all were treated as outpatients. Children with CM were admitted to the Paediatric Research Ward at QECH with clinical, WHO-defined cerebral malaria  and were 6 months–12 years in age. Only children with CM with malarial retinal abnormalities (Ret + CM) were included. HIV + children were excluded. Cases reported in this study are a subset of those previously reported [var qRT-PCR typing from acute infection, and (4) sufficient acute and convalescent plasma for the study. Comparable numbers of cases from each group were compared. Age and sex distribution were comparable to the larger group [During the 2015–2016 malaria seasons, children with malaria at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi were recruited to the study  samples using human isotype-specific radial immunodiffusion kits (The Binding Site Ltd.). Plasma was heat-killed prior to use, and all samples were assayed in duplicate.P. falciparum protein features  was developed at Antigen Discovery, Inc. . Proteins were selected based on internal R&D using P. falciparum full proteome microarrays probed with samples collected from subjects during naturally and experimentally acquired immunity (unpublished). Briefly, proteins were selected based on a prioritization scheme as follows: (1) a curated list of P. falciparum proteins prominently featured in the scientific literature and established targets of P. falciparum candidate vaccines and diagnostics, plus the full repertoire of 3D7 PfEMP1, rifin and stevor proteins, (2) top hits from previous studies with protein arrays that identify correlates of protection or exposure, (3) a ranking of proteins based on ADI internal full proteome microarray data. The criteria for ranking proteins with empirical full proteome data employed a scoring system that prioritized: seroprevalence in either naturally immune populations (> 60%) or pre-erythrocytic experimental immunity (> 20%); predicted transmembrane domains; predicted signal peptides; gene ontology (GO) terms for Function, Process and Component that were broadly related to parasite and host cell surfaces, host-parasite interactions and host immune system. Proteins were selected according to the prioritization scheme until a total of 1000 targets were selected, accounting for fragmented proteins. The 1000 full-length or partial P. falciparum proteins represent 762 genes from P. falciparum reference strain 3D7, including 61 PfEMP1s, vaccine candidate proteins, and 176 conserved Plasmodium proteins of unknown function. Protein microarrays were fabricated as described in Additional file A partial proteome microarray with 1000 roteins,  top hits2 scale) was determined relative to signal of expression system controls . Following dilution, the antibody concentration of the hyperimmune serum was 100 μg/ml.Hyperimmune IgG and naïve sera were probed on the array as controls. Hyperimmune IgG was pooled from 834 HIV and HBsAg seronegative Malawian adults [P. falciparum antibodies was performed in R version 3.1.1 [Statistical analysis was performed using Stata 12.1 or Prism 7.0 for Macintosh. Statistical differences were determined by Wilcoxon rank sum , Wilcoxon signed rank sum  or Chi squared test . Analysis of on 3.1.1  and Grapon 3.1.1 .A total of 25 children with Ret + CM and 23 with UM were included in the study Table . Both grAs reported previously , the PfERet + CM cases had significantly reduced levels of total IgG relative to children with UM during acute infection P < 0.001), which normalized in convalescence  portion of each PfEMP1 protein is highly conserved. Therefore, all analyses examining antibody reactivity to PfEMP1 proteins were performed with and without the ATS.By case severity, no significant differences in the breadth and magnitude of IgG and IgM responses to PfEMP1 specific antigens were observed in acute infection Figs. b, c. Aftvs. Ret + CM) were observed, all cases were pooled for subsequent PfEMP1 specific analyses. PfEMP1 antigens with the highest reactivity in acute infection were highly conserved ATS regions or domains associated with CD36 binding  specific IgM responses decline significantly in convalescence for all cases Fig. a. While When the PfEMP1 antigens were classified on the basis of their known or predicted binding phenotype , 43, 5 Evar transcripts in the peripheral blood of each case (both UM and Ret + CM) as previously described [Exposure to PfEMP1 domains during acute infection was determined using relative qRT-PCR profiling of escribed , 32, 44.P. falciparum infection progress to severe disease. Despite evidence of general suppression of antibody response in acute Ret + CM  [Seroreactivity to the newly identified e array)  was foune array) . Here, tNeither a consistent response to the corresponding antigen in children exposed to a specific PfEMP1 variant in acute infection or a general significant increase in PfEMP1 IgG antibody at 30 days convalescence were detected. Prior studies employing in vitro functional assays demonstrated that children seroconvert against the VSA of the isolate seen in acute infection following disease resolution  and subsP. falciparum-specific antibodies recognize conformational epitopes. The cell free in vitro expression system and direct printing protocol used in production of the microarray employed here involves minimal manipulation of expressed proteins and no denaturation steps, but correct conformation of recombinant protein is not readily verifiable.The array was developed using the 3D7 proteome, which lacks some EPCR-binding proteins. 3D7 PfEMP1 proteins may lack sufficient homology to Malawian PfEMP1 to be recognized by specific antibody. The PfEMP1 family is hypervariable with less than 50% sequence identity found in homologous domains of homologous genes , 16. StrP. falciparum antigens than to other PfEMP1 antigens. The median age of our subjects was 3–4 years, whereas peak EPCR-binding domain antibody prevalence was detected in children aged 8–10 years [Plasmodium, studies that have examined acquisition of “protective” antibody responses to VSA or Plasmodium antigens, significant protection begins to appear in children 8–10 years of age [Seroreactivity in both acute infection and convalescence to antigens from all PfEMP1 binding phenotype groups was identified in UM and CM cases. Seroreactivity to EPCR domains is acquired relatively early in life  and may 10 years . Althougs of age , 52. Chis of age   between groups.Additional file 2. Supplemental methods for array design, probing, and analysis.Additional file 3. Annotations of PfEMP1/var genes and domains encoded.Additional file 4. Assessment of P. falciparum and total Ig levels by age and sex. (a) Spearman correlations comparing age vs. acute total IgG level, age vs. 30-day convalescent total IgG level, and total IgG levels across timepoints show significant linear trends. (b) Volcano plots comparing the effect of age (> 5 years/≤ 5 years) on IgG, IgM seroreactivity to P. falciparum and PfEMP1 antigens indicates that for IgG in acute infection, age affects the magnitude of seroreactivity to a few P. falciparum antigens and a general non-significant trend for higher antibodies to all proteins in older children during acute infection . (c) Volcano plots of inverse unadjusted P-values (y-axis) and linear regression effect estimates (x-axis) for comparing the effect of sex  on IgG, IgM seroreactivity to P. falciparum and PfEMP1 antigens indicate that sex does not have a significant effect on seroreactivity in our patient population in acute infection or convalescence, although non-significant trends for higher overall P. falciparum-specific IgG and IgM in males during acute infection and lower PfEMP1-specific IgG in males for both time points could be observed . Dashed lines represent unadjusted P-value of 0.05. Points are highlighted in bold, red if significant after adjustment for the false discovery rate.Additional file 5. Reactivity to markers of prior malaria exposure and antigens of interest in control populations.Additional file 6. Seroreactivity to PfEMP1 antigens in pooled control serum vs. paediatric malaria cases.Additional file 7. CIDR IgG responders at time of acute P. falciparum infection.Additional file 8. Reactivity to head structure/CIDR or ICAM-binding domain in unexposed cases.Additional file 9. Additional exposure model covariates: the effect of cerebral malaria, age, and sex on PfEMP1 antibody level. Volcano plots of linear regression effect estimates (x-axis) for the independent variables  included in the exposure models generated for PfEMP1 expressed domains as determined by qRT-PCR of infected blood samples obtained during acute disease. .


Plasmodium genus. India is set for malaria elimination by 2030. Since cases of mixed Plasmodium species infections remain usually undetected but cause huge disease burden, in order to understand the distributional prevalence of both monospecies infections and mixed species infections in India, we collated published data on the differential infection incidences of the five different malaria parasites based on PCR diagnostic assay. About 11% of total cases were due to mixed species infection. Among several interesting observations on both single and mixed parasitic infections, incidences of Plasmodium falciparum monoinfection were found to be significantly higher than P. vivax monoinfection. Also, P. malariae seems to be emerging as a potential malaria threat in India. Putting all the facts together, it appears that the dream of achieving malaria elimination in India will not be completely successful without dealing with mixed species infection.India is highly endemic to malaria with prevalence of all five species of human malaria parasites of Humans simultaneously infected by different parasites are quite natural and frequent . Around To this extent, malaria is an age-old disease of the human kind, mainly restricted to the tropical and subtropical countries of the globe . Several Plasmodium and abundant distribution of nine species of Anopheles that are vectors of malaria parasites, have resulted in bringing out complex malaria in India [Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi). Therefore, (i) in order to determine distributional abundance of mono-malaria parasite infections as well as mixed malaria parasite infections in all of India in a single communication, (ii) to generate debate on the probable cause of differential interactions of two or more different species of Plasmodium infecting a single individual at a time, and (iii) to synthesize if the current knowledge would be useful for the targeted malaria elimination program in India, we have collated published data (published between 2005 and 2017) on mono-malaria infections and mixed malaria infections based on PCR diagnosis in India [India is highly endemic to malaria and one of the few countries contributing significantly to global deaths and morbidity due to malaria infection . In 2015in India , as a fiin India  and pres P. falciparum (64%), followed by P. vivax (33%). The other three species have meagre contribution  to malaria infection in India  followed by Goa (79%) and lowest in Tamil Nadu (20%) ( P. vivax was evident in Delhi (66%) followed by Uttar Pradesh (62%) and the lowest one was in Chhattisgarh (0.33%) with no cases in Jharkhand  followed by Tamil Nadu (5.3%), Andhra Pradesh (3.3%), Madhya Pradesh (1%), and Asom (0.7%). However, monoinfections of P. ovale were found only in two individuals from Chhattisgarh, and P. knowlesi was restricted to Andaman and Nicobar only in three cases  . Similarharkhand . Interesee cases . It is t 50 : 50 , but at ha drugs . Furtherof India , 37 and of India , 39. The Plasmodium are often found to be in coinfection in a single individual. When data on mixed infections (in any combination among five different species) in a single individual are segregated based on different states, the incidence of mixed infection was found to be highest in Karnataka (30%), followed by Jharkhand (27%), Odisha (23%), Andhra Pradesh (18.5%), Andaman and Nicobar Islands (17.7%), Tamil Nadu (17%), North-eastern states (16.4%), Maharashtra (14%), Chhattisgarh (11.8%), Gujarat and Rajasthan (9%), Uttar Pradesh (5.6%), Madhya Pradesh (4.3%), Delhi (2.9%) and Goa (2.6%), with no cases of mixed infection in Bihar  had the highest cases of mixed infection due to P. vivax and P. falciparum. Similarly, in Jharkhand where not a single case of P. vivax monoinfection was detected, 27% of mixed infections are due to P. vivax and P. falciparum coinfection. Apart from the mixed infection of P. falciparum and P. vivax, coinfections among other species of Plasmodium in India are quite infrequent with an exception of mixed infection involving P. ovale with either P. vivax or P. knowlesi in India. Among all mixed infection cases, one such case  needs significant attention. Mixed infection due to these two species was found to be distributed across several Indian locations, especially in the eastern and western parts  but hasn Odisha  has now  Figures . To be nf Odisha –44. Furt P. vivax monoinfections was statistically significantly positively correlated  to P. falciparum across 18 Indian states. This indicates that Indian populations are susceptible for infection by both the parasite species and therefore explains the unique burden of both P. vivax and P. falciparum in India. This situation is opposite to Vanuatu where negative correlation in occurrence of these two species had been reported [ P. falciparum and P. vivax have different origins and they are placed far in phylogenetic tree as compared to the other human malaria parasites [P. falciparum monoinfection versus P. falciparum + P. vivax, r = 0.6646, P < 0.004; P. vivax monoinfection versus P. vivax + P. falciparum, r = 0.6722, P < 0.003). This means that mixed species infections are prevalent in both P. vivax endemic and P. falciparum endemic localities in India, thereby justifying that both of these parasite species have evolved greatly to coinfect Indians. Incidentally, both monoinfections and mixed infections by P. malariae were statistically strongly positively correlated with different combinations of species . This indicates the adaptive affinity of P. malariae to coinfect with every other species of Plasmodium in a single individual in India. Interestingly, very few cases of coinfection involving P. ovale and P. knowlesi with other species could be found indicating their poor adaptive capacity in a coinfection state in a single human host in India.In order to understand if any defined trend of association exists in the occurrence of different monoinfections and mixed infections, it was interesting to note thatreported . This siarasites . Interes Plasmodium parasites have evolved to create a favourable environment for the parasites that provides abundant opportunity for transmission   , and thae of RBC , cause luggested . Competiuggested . Accordiulocytes , which iulocytes  and suchman RBCs . Conside P. ovale and P. vivax have similar breadth and sequence type of the pir (Plasmodium interspersed repeats) gene repertoires [ Plasmodium genomes reported thus far [ P. malariae contains only a restricted subset of the pir gene family [ pir genes in P. malariae are pseudogenes, host adaptation in P. malariae is less restricted than in P. falciparum [ P. malariae is mostly found as mixed infection with other species of Plasmodium in India  is 84.8% and 82.7% similar to P. malariae and P. ovale, respectively [ P. knowlesi shows its prevalence more as a coinfection with P. falciparum than infecting as a single parasite. Since the three species  are phylogenetically close to each other [ Plasmodium are in the learning phase and therefore seek help of an evolved parasite  that can invade any kind of RBC  mostly occur as coinfections mainly with P. falciparum and to certain extent with P. vivax.Adaptation by Darwinian natural selection at both genetic and phenotypic level is evidenced in many species including malaria parasites in India . Cases onfection ) therefolciparum ; China-MP. ovale ; Switzermalariae ; and Chilciparum ) on the lciparum . Accordilciparum . The epi P. falciparum monoinfections, and mixed Plasmodium species infections in a single individual. However, conclusions made out of the results presented in this communications suffer from several limitations. For example, (i) the PCR diagnostic assay has been performed in many different laboratories without following a uniform molecular biological protocol, (ii) the results of mixed infection came as a by-product of some other research work, and therefore sampling might have been biased, (iii) malaria samples have not been collected in defined period of year, and (iv) data from each and every malaria endemic locations have been included in the present analysis. Even then, the present dataset, prepared out of collation of published literature on mixed malaria infection, provides enough insight on the trends of increasing incidences of P. falciparum monoinfection and occurrence of mixed infection of five different malaria parasites in variable proportions.Clearly, India is heavily burdened with appreciable number of malaria cases with increasing number of P. falciparum monoinfection as well as mixed species infection and entails considerable risks that might come in the way of ambitious malaria elimination program in India. Considering (i) the fact that there is no defined diagnostic method for detection of mixed species infection at the local health centre, (ii) high species diversity of anopheline mosquitoes that are vectors to Plasmodium parasites, and (iii) unavailability of a defined treatment program for mixed species infection and increasing trends of drug-resistant parasites [ P. falciparum malaria and expanding range of P. malariae, (ii) type of infection (monospecies/mixed species), and (iii) distribution of drug and insecticide resistance malaria parasites and vectors, respectively [Over several years, many agendas  for either eradication or elimination of malaria have been prepared and implemented. The Global Malaria Eradication Program (GMEP), which ran from 1955 to 1969, had helped in reduction of malaria cases globally to a significant extent. However, technical challenges and resurgence of malaria in many countries including India led to a shift away from malaria elimination for many decades. Since 2000, there has been an increasing political drive to eliminate malaria , and redarasites , goals sectively , would p


Prophylaxis for high-risk populations, such as forest workers, could be one component for malaria elimination in the Greater Mekong Sub-region. A study was conducted to assess the malaria incidence in forest rangers and the feasibility of malaria prophylaxis for rangers sleeping in forest camps.Plasmodium infections were cleared using presumptive treatment, irrespective of malaria status, with a 3-day course dihydroartemisinin/piperaquine (DP) and a 14-day course of primaquine. Before returning to the forest, study participants were randomly allocated to a 3-day course of DP or placebo. Fifteen days after returning from their forest deployment the participants were tested for Plasmodium infections using uPCR.Forest rangers deployed in the Bu Gia Map National Park, Vietnam were invited to participate in the study. Plasmodium. Seventeen days (median) after enrolment the rangers were randomized to DP or placebo 2 days before returning to forest camps where they stayed between 2 and 20 days (median 9.5 days). One ranger in the DP-prophylaxis arm and one in the placebo arm were found to be infected with Plasmodium falciparum 15 days (median) after returning from the forest. The evaluable P. falciparum isolates had molecular markers indicating resistance to artemisinins (K13-C580Y) and piperaquine (plasmepsin), but none had multiple copies of pfmdr1 associated with mefloquine resistance.Prior to treatment, 30 of 150 study participants (20%) were found to be infected with Anti-malarial prophylaxis in forest rangers is feasible. The findings of the study highlight the threat of multidrug-resistant malaria.Trial registration NCT02788864The online version of this article (10.1186/s12936-017-2091-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum resistance to artemisinins and partner drugs . The study did not detect any impact of the prophylaxis; one infection was detected in both the treatment and in the placebo arm. This finding could be related to the limited sample size and number of infections. Based on the malaria incidence estimate from this study, 892 participants would have to spend 14 nights each in the forest to have sufficient power (90%) for demonstrating significant protection is afforded by DP prophylaxis , followed by An. maculatus (14%), and An. barbirostris (2%). These findings are consistent with earlier entomological studies in Binh Phuoc province. Ngo and coworkers found 17 Anopheles species in the province amongst which An. dirus was most prevalent [An. dirus was most prevalent in southern Vietnam [An. dirus was preferentially anthropophilic, biting outdoors before 22:00. The biting rhythm and resting behaviour of An. dirus reduces the impact of the two most commonly employed control measures, insecticide-treated bed nets and indoor residual spraying. Imaginative interventions such as supplying forest workers with insecticide treated hammocks does not address the biting rhythm and resting behaviour and had a disappointing uptake in field studies [The entomological component of the study detected three Anopheles species. The large majority were revalent . Similar Vietnam . They al studies , 37. In P. falciparum isolates evaluated at baseline carried genetic markers associated with artemisinin and resistance to the partner drug piperaquine. The long terminal half-life of piperaquine affords protection against infection with susceptible P. falciparum isolates for up to 4 weeks [P. falciparum infections after the directly observed administration of a course of DP in the first and second round is cause for concern. Three of the P. falciparum isolates carried markers for artemisinin and piperaquine resistance. The remaining 10 P. falciparum infections detected at baseline with K-13 and plasmepsin markers associated with artemisinin and piperaquine resistance had cleared after a course of DP due to the remaining therapeutic efficacy of DP or immunity. None of the P. falciparum isolates had multiple copy numbers of pfmdr1 associated with mefloquine resistance. This finding supports the notion of an inverse susceptibility between piperaquine and mefloquine and could suggest that the therapeutic efficacy of DP can be boosted by a triple therapy adding mefloquine to DP. Such a triple regimen is currently in clinical trials in the region [The study highlights the challenges of multidrug-resistant malaria to treatment and prophylaxis in the region. Treatment failures with ACT are an increasing challenge for healthcare providers treating falciparum malaria in Vietnam , 5. All  4 weeks . The pree region . Adaptin2 infections acquired during 1000 nights spent in the forest.25 infections would be needed to have sufficient power to show a 75% protection against infection during a forest visit. .12,500 forest nights should suffice to detect 25 infections acquired during stay in the forest.892 participants would have to spent 14 nights each in the forest to demonstrate significant protection is afforded DP prophylaxis.An. dirus and the impact of anti-malarial prophylaxis with DP is increasingly limited by the spread of anti-malarial resistance. Two populations of forest rangers, permanent and short time overlap temporarily in the park and sustain the transmission of Plasmodium infections. Breaking this cycle will require the targeting of all rangers in the forest at the same time. A mass anti-malarial administration campaign targeting all rangers in the National Park could provide a permanent solution. DP is probably no longer appropriate for such a campaign; new drug combinations will be needed. After the interruption of malaria transmission in the National Park preventing reintroduction of Plasmodium infections will remain a constant challenge until malaria is eliminated from Vietnam.The findings of this study illustrate the challenges in preventing malaria in forest rangers in Bu Gia Map National Park. The impact of vector control approaches is limited by the biting behaviour of the dominant vector


This study was initiated from the observation that prevalence of malaria obtained with rapid diagnostic test (RDT)  was higher than in microscopy in a malaria low transmission area of Senegal. PCR was then performed to evaluate the performance of the RDT compared to microscopy in clinical settings.The study included 215 patients suspected of malaria in two peri-urban area of Dakar. Finger-pick blood samples were tested using RDT . Venous blood samples were collected for light microscopy and PCR (gold standard). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated as performance characteristics.Considering PCR as the gold standard, CareStart™RDT showed high sensitivity (97.3%) and specificity (94.1%) with PPV and NPV of 97.3 and 94.1%, respectively, while microscopy had a sensitivity and specificity of 93.2 and 100%, respectively, and PPV and NPV of 100 and 87.2%, respectively.Malaria CareStart™RDT test demonstrated a superior sensitivity compared to microscopy, which is the gold standard for malaria diagnosis. CareStart™RDT could be a useful tool in individuals suspected of malaria even in areas where prevalence is low. Light microscopy detecting human malarial parasites in Giemsa-stained thick and thin blood films still remains the gold standard for malaria diagnosis . AlthougMalaria rapid diagnostic tests (RDTs), based on immunochromatographic parasite antigen detection, are of great value in endemic regions. Since 2010, WHO has recommended either RDT or microscopy confirmation of suspected malaria cases before treatment . HoweverThe objective of this study was to compare CareStart™ RDT to microscopy performed by a WHO-certified level 1 microscopist and determine the test characteristics using PCR as gold standard.The study was carried out in two sub-urban sites of Dakar (Pikine and Rufisque) from October to November 2016. Malaria transmission in Dakar is low, with parasitaemia prevalence estimate of 1.3% and a typical urban malaria pattern . Pikine The study patients were enrolled if they presented with clinically suspected malaria on the basis of fever or history of fever in the previous 48 h to the health clinics in the study areas. Blood samples were collected for RDT and microscopy, and a filter paper collected for PCR. Inclusion criterion for this study was individuals with fever  and without clinical sign of severe malaria.All parasitological examinations were carried out in Aristide Le Dantec Hospital Laboratory, the national reference laboratory of Senegalese National Malaria Control Programme. The laboratory hosts the external competency assessment (ECA) centre for African Francophone countries. Thick and thin blood smears were prepared from collected venous blood samples. Both thick and thin films were made in the same slide. The thin films were fixed in methanol. The slides were stained in 10% Giemsa solution for 15 min. Stained slides were read by two WHO-certified level 1 microscopists in the laboratory. The number of parasites was counted against 200 leucocytes when more than 100 parasites were counted, and 500 leucocytes were required when fewer than 100 parasites were counted. Quantification of parasite density was estimated by assuming 8000 leucocytes/μL of blood. The result was considered negative if no parasite was detected after examining 200 microscopic fields at 1000× magnification. The technician was blinded to the results of the RDT. In case of discordant results between the two readers, a third expert reader was used.Plasmodium falciparum and the other detecting presence of pan malaria-specific antigen (pLDH) of all malarial parasites species. Five microlitre of blood was drawn using a loop provided with the device. The test preparation and interpretation was done following manufacturer’s instructions. Positive results indicate the presence of two or three bands (including control test line). For negative results, only the control line appears. Results were observed and number of visible line was recorded.The same patients were tested for malaria parasites using CareStart™Malaria HRP2/pLDH Combo Test in the fields (Lot Number: MR15A06). CareStart™RDT is an immunochromatographic test coated with monoclonal antibodies in two separate bands, one recognizing the specific histidine-rich protein-2 (HRP-2) associated with the presence of Plasmodium PET-PCR reaction was performed as previously described [From the collected venous blood, two drops of blood were spotted onto filter paper, individually stored in a plastic bag and sent to the Parasitology Laboratory of Le Dantec University Hospital for PET-PCR assay. DNA was extracted using the QIAamp blood kit (QIAGEN™) according to the manufacturer’s instructions and stored at 4 °C until processed. The escribed , 11. SamData were processed in Excel (version 15.27) and analysed using STATA. Diagnostic performance was determined by calculating the test sensitivity (Se), specificity (Sp), predictive values (PPV and NPV), with 95% CI. PET-PCR was used as the reference method. McNemar Chi square analysis was used to determine the significance differences between Se, Sp, NPV and PPV. The p values less than 0.05 were considered statistical significance.Oral informed consent was obtained from the selected patients. Ethical approval with the reference number was obtained from Senegalese National Ethic Committee of Ministry of Health.P. falciparum. The parasite density ranged from 16 to 341,090 parasites/μL of blood with a mean of 36,823 parasites/μL by microscopy.A total of 215 blood samples collected from malaria suspected patients were analysed. The sex ratio was 1.56:1. The mean age was 23 years (range 4–77 years). Among the 215 samples tested, the PPV was 137 (63.7%), 146 (67.9%) and 144 (68.4%) using microscopy, CareStart™RDT and PCR, respectively. Overall, CareStart™RDT detected more positive samples than microscopy and PCR. Species of malarial parasites identified in all study participants were When compared to PCR, CareStart™RDT test showed better Se (97.3%) than microscopy 93.2%) p = 0.0143) and better NPV, 94.1 versus 87.2% (p = 0.0251). However, better Sp (100%) and PPV (100%) were noted in microscopy versus RDT   , giving an overall LDH positivity rate of 32.0%. This positivity rate was even less (17.4%) when parasite densities were less than 1000 , it was reported that CareStart™RDT gave more positive results than microscopy performed by average microscopists. The aim of this study was to compare CareStart™RDT to microscopy using PCR as gold standard. The results of the study confirmed the observation noted by health-centre microscopists. In fact, CareStart™RDT showed better sensitivity than microscopy although specificity was better with the latter.The CareStart™Malaria HRP2/pLDH (Pf/pan) Combo Test is a three-band RDT that detects HRP2 and pan-pLDH antigens . CareStaDespite CareStart™RDT detecting more true positive samples than microscopy in this study, there were also some false positive records. These false positives may be due to the persistence of the HRP2 antigen after treatment . HoweverPfHRP2 gene sequence of parasites varies depending on the geographic locations [PfHRP2 polymorphism did not affect the performance of HRP2-based RDTs [PfHRP2 gene deletion was observed. However, in Mali, a neighbouring country of Senegal, parasites lacking PfHRP2 have been reported to cause false-negative results [Many other factors can affect the performance of RDTs. False-negative can be observed when parasites fail to express the target antigen (gene deletion) or when the parasite express a variant of the protein (polymorphism) which can affect the antigen–antibody binding. ocations . In Senesed RDTs  and no P results  suggestiPfHRP2 gene deletion.Unlike PfHRP2, the pLDH protein does not persist in the blood after effective treatment with anti-malarial . Thus, tHowever, pLDH-based RDTs have several limitations. pLDH-based tests have decreased sensitivity at low parasitaemia . The posAlthough RDT usefulness in low transmission areas or in the detection of low parasite density infections is being questioned, CareStart™RDT showed good sensitivity comparable to that performed by expert microscopist. However, in the wake of reported HRP2 deletion in other countries, microscopy should always accompany the use of RDT. Combination of RDT and microscopy together with the evaluation of malaria RDTs over time should be a powerful tool for diagnosing malaria in endemic countries.


Plasmodium spp. DNA is maintained in the long term in an extra-Amazonian region of Brazil with low incidence, residual malaria transmission.Regions with residual transmission are potential obstacles to the elimination of malaria. It is, therefore, essential to understand the factors associated with the maintenance of endemic malaria in these areas. The objective was to investigate whether the status of asymptomatic carriers of Plasmodium DNA detected in a survey carried out between 2001 and 2004 were reassessed between 2010 and 2011 using questionnaires, PCR and thick and thin blood smear tests three times at 3-month intervals.Asymptomatic carriers of Plasmodium malariae DNA was detected.Of the 48 carriers detected between 2001 and 2004, 37 were located. Of these, only two had positive PCR results and, as in the first survey, P. malariae, both in the first and in the second survey, may be compatible with a long-term carrier state for this parasite. Since most clinical cases of malaria in the region are a consequence of infection by Plasmodium vivax, the epidemiological impact of such long-term carriage would be limited.The findings suggest that untreated dwellers from this extra-Amazonian region, who initially harbour malaria parasites, may become negative without ever developing apparent symptoms of the disease. Although the possibility of re-infection cannot be ruled out, the finding of two individuals harbouring  Although malaria control efforts have reduced the impact of the disease around the world, there were an estimated 212 million cases worldwide in 2015, leading to 429,000 deaths [Plasmodium vivax was identified in 48 of the 51 symptomatic individuals previously diagnosed as malaria cases by both microscopic examination of blood films and polymerase chain reaction (PCR) who were asked to participate in the survey. Plasmodium malariae DNA was identified by PCR in one sample, and in two samples blood smear tests revealed parasites morphologically similar to P. vivax but the PCR was negative. At the same time, 1527 blood samples were collected from residents of the areas where cases were reported and tested by multiplex PCR and microscopic analysis of blood films (thick and thin smears) to identify asymptomatic carriers. Plasmodium vivax DNA was detected by PCR in 23 of the samples, P. malariae DNA in 15, Plasmodium falciparum DNA in nine, and P. falciparum and P. malariae DNA in one. No parasites were observed in the thick smears of these asymptomatic carriers.Residual malaria in the Atlantic Forest in Brazil has a very low incidence in various states along the coast. The greatest number of reported cases in these states is in Espírito Santo , where tPlasmodium species, Plasmodium brasilianum and Plasmodium simium. The genetic similarity between P. vivax and P. simium and between P. malariae and P. brasilianum [Plasmodium DNA in the bloodstream. Consequently, this reassessment enables the verification of long-term positivity for Plasmodium DNA and clinical evolution to apparent disease, contributing to the understanding of the role of asymptomatic carriers in the sustained transmission of malaria among humans in this endemic area.Understanding the transmission chain associated with endemic malaria maintained in such a vast area with such a low frequency of symptomatic and asymptomatic cases is a considerable challenge. According to one hypothesis, this behaviour may be typical of a zoonosis and the human cases may be a result of incidental transmission of locally occurring simian malaria –7, whichsilianum , 9 reinfPlasmodium DNA in the first survey (between 2001 and 2004), 37 were located in 2010 and included in the study. The remaining 11 consisted of three who had died of causes unrelated to malaria , one who refused to take part in the study and seven who had moved and could not be located. Each of the individuals located was asked to provide a blood sample once every 3 months on three separate occasions to investigate the presence of Plasmodium DNA. Before the first collection the participants signed a voluntary informed-consent form. On each occasion a questionnaire was filled out covering demographic data and any instances since the previous collection of febrile illnesses, thick-smear tests for malaria or trips outside the area where the participant lived. A total of 5 mL of blood was then collected in vacutainer tubes  containing EDTA, thick and thin peripheral blood smears were prepared and the subject’s spleen was palpated by the physicians in charge of the project (FECA and CCJ).Of 48 asymptomatic individuals who were positive for g for 10 min in the Protozoology Laboratory in the Tropical Medicine Unit, Federal University of Espírito Santo, to separate the plasma from the red blood cells. These were then aliquoted and stored at − 20 °C in Eppendorf tubes, which were sent to the Protozoology Laboratory at the São Paulo Institute of Tropical Medicine, where DNA was extracted and PCR was performed.The blood collected was centrifuged at 300Thick and thin blood smears were prepared following the method recommended by the World Health Organization . The smePlasmodium 18S RNA gene sub-unit. The first step was carried out in a final volume of 20 µL containing 0.8 µL of each primer, P1UP and P2 (10 µM), 0.25 µL of dNTP mix , 2 µL of 10 × PCR buffer, 1 µL of MgCl2 (50 mM), 0.16 µL of Platinum Taq DNA polymerase-Invitrogen (5 U/µL) and 5 µL of DNA. The amplification was run in an Applied Biosystems thermocycler with the following programme: 92 °C for 2 min, 35 cycles of 92 °C for 30 s and 60 °C for 90 s and one cycle of 60 °C for 5 min.Genomic DNA was extracted from the samples with the NucleoSpin Tissue purification kit (Macherey–Nagel) following the manufacturer’s instructions. The PCR procedure followed Win et al. . BrieflyP. vivax, F2—P. falciparum or M1—P. malariae).The product from the first step was diluted 1:50 in sterile water and used in the second step with the P1 (genus–specific) primer and one of the three reverse species-specific primers , 0.5 µL of dNTP , 2 µL of 10 × PCR buffer, 0.16 µL of Taq DNA polymerase (5 U/µL) and 2 µL of the diluted final product from the first step. The second amplification was carried out in the same equipment as the first, and the cycling parameters were 92 °C for 2 min followed by 18 cycles of 92 °C for 30 s and 60 °C for 1 min and one cycle of 60 °C for 5 min. The amplified products of the second step correspond to species-specific fragments of about 100 bp.P1UP (F):5′ TCC ATT AAT CAA GAA CGA AAG TTA AG 3′P2 (R):5′ GAA CCC AAA GAC TTT GAT TTC TCA T 3′P1 (F):5′ ACG ATC AGA TAC CGT CGT AAT CTT 3′V1 (R):5′ CAA TCT AAG AAT AAA CTC CGA AGA GAA A 3′F2 (R):5′ CAA TCT AAA AGT CAC CTC GAA AGA TG 3′M1 (R):5′ GGA AGC TAT CTA AAA GAA ACA CTC ATA T 3′.The primers used were:P. vivax malaria patients treated at the São Paulo Department for the Control of Endemic Diseases (SUCEN), from P. falciparum cultures and from blood smears positive for P. malariae provided by the Centers for Disease Control and Prevention (CDC) was used as a control.DNA extracted from peripheral blood of The amplified product was run on 2% agarose gel at 80 V for 40 min. The gel was stained in ethidium bromide, and the bands were visualized under a UV transilluminator.Quantitative variables were expressed as medians and interquartile ranges, and categorical variables were expressed as absolute and relative frequencies. The data were analysed with SPSS version 17.0 .Plasmodium DNA carriers in the first survey, 37 participated in the study and provided blood samples on three different occasions between April 2010 and March 2011. Fifteen (40.5%) of the samples from these 37 asymptomatic carriers had been positive for P. vivax DNA in the 2001–2004 survey, 12 (32.4%) for P. malariae DNA, 9 (24.3%) for P. falciparum DNA, and one (2.7%) for P. falciparum and P. malariae DNA.Of the cohort of 48 individuals found to be Of the individuals assessed, 25 (67.6%) were males. Median age at the time of the 2010/2011 survey was 24 years, the interquartile range was 18–40 years and the minimum and maximum age were 7 and 62 years, respectively. The most common occupation was farmer  followed by student . The other occupations were land surveyor, shopkeeper, housewife, bank intern, driver , bricklayer, and agricultural technician  was seen by the paediatric service at the University Hospital, but the aetiology of the splenomegaly could not be determined. The splenomegaly subsequently regressed and the spleen was no longer palpable.Fourteen individuals reported experiencing fever during the follow-up in at least one of the interviews. In all but one case, in which it subsided after a week, the fever lasted 1–4 days. None of those who presented with fever was tested for malaria by the local health services, and their fevers cleared without any specific treatment. In two individuals the spleen was palpable at the costal margin, in one only at the first collection and in the other at all three collections. Blood smears in both patients were negative for Plasmodium malariae DNA was detected in two individuals. Individual no. 15, who was 18 years old, was positive at the second and third collections, while individual no. 19, who was 24 years old, was positive only at the second collection. Both were male farmers. Plasmodium malariae DNA had been detected in these patients’ samples in the first survey as well, although they did not have any symptoms suggestive of malaria in either survey  of the individuals found to be carriers of this species in the first survey. Additionally, none of them developed symptoms compatible with malaria in the period between the first survey and the reassessment.In this study to investigate the persistence of DNA positivity in a cohort of 37 asymptomatic carriers approximately 6 years after an earlier survey, only two patients with a molecular diagnosis consistent with The role of asymptomatic individuals  in the mIn an area such as that studied here, where malaria is uncommon and the population would not generally consider it a cause of fever, even people who have been given guidance about the disease do not normally go to medical centres to be tested for malaria infection, unlike in regions of the Amazon where there is a higher incidence of the disease.P. vivax [In areas of the Atlantic Forest where there is residual malaria and very low endemicity, thick blood smears normally reveal low parasitaemia in symptomatic patients . Most ofP. vivax . As the P. vivax –25.Plasmodium over long periods, potentially maintaining transmission cycles in some regions [In a study in the Amazon region, Alves et al. found that even though they were asymptomatic, patients who had exhibited very low parasitaemia, that could only be detected by PCR 2 months before they were recruited to the study to assess their capacity to infect mosquitoes, remained infective. However, they had less infective capacity than the symptomatic patients. They cited unpublished data showing that 40% of the asymptomatic patients were negative by PCR after 2 months of follow-up and suggested that the limited capacity of asymptomatic patients to infect mosquitoes may be made up for by carriage of  regions .P. falciparum DNA in 10 asymptomatic individuals in the transmission area during the 2001–2004 survey. To date there have been no cases of malaria caused by this species in the region in question, and P. falciparum is not considered an aetiologic agent of malaria in Atlantic Forest systems. Recently, Maselli et al. [P. falciparum DNA in asymptomatic blood donors who had been in the Atlantic Forest system in the state of São Paulo, a state where there have not been any reports of symptomatic cases of this type of malaria among local residents. It is possible that detection of P. falciparum could be a result of the molecular technique used. In the 2001–2004 study [P. falciparum DNA in this region may represent artifacts of the technique, indicating the presence of a species that is not in fact part of the transmission chain [P. falciparum and P. vivax, the latter in a smaller proportion of the infected mosquitoes. This was confirmed by DNA sequencing, which identified P. falciparum in 76% of the positive specimens, most of which were Anopheles cruzii, suggesting that P. falciparum circulates actively among Anopheles in fragmented areas of the Atlantic Forest in this region of Brazil. In light of these unresolved questions, further studies are needed before the role of P. falciparum in the aetiology of residual malaria in Atlantic Forest systems can be determined.An issue that remains to be elucidated is the detection of i et al.  also det04 study . As the on chain . Neverthon chain  identifiP. malariae in the present survey were not re-infected after the first survey. However, the presence of the same species of parasite in both surveys and the potential for infections of very long duration observed for this species of parasite make it possible that they are the same infections. It should be emphasized that asymptomatic individuals are not treated for malaria in the study area. Consequently, specific treatment does not represent confounding. Information about any symptoms experienced by the participants between blood collections was provided by the participants themselves. At the time of the collections, however, they were all asymptomatic. None of the individuals who reported having had a fever sought medical assistance to be tested for malaria and in all of them the symptoms improved without the use of anti-malarial medication.The limitations of this study should also be taken into consideration, as many years had elapsed since the first study, making it difficult to obtain reliable information about the period during which the individuals in the original survey were not followed up. It is not possible to assert without any doubt that the two individuals infected by P. malariae. Therefore, although it has been suggested that asymptomatic carriers should be treated [P. vivax, the epidemiological impact of a possible long-term carriage of P. malariae is limited.The study’s importance lies in its novelty, as it is the first reassessment of asymptomatic carriers following a prolonged interval of time between surveys. In this cohort, even though asymptomatic carriers were not followed up over a very long period and it was not possible to determine with a reasonable degree of accuracy when DNA tests were no longer likely to be positive, most of the patients who were positive for some of the species in the first survey were no longer positive in this study, apart from two patients who were positive for  treated , treatme


Plasmodium spp. are important sources of infections for malaria vectors and thus contribute to the anchoring of the disease in favourable eco-epidemiological settings. The objective of this study was to assess the asymptomatic malaria case rates in Korhogo and Kaedi, two urban areas in northern Côte d’Ivoire and southern Mauritania, respectively.Since 2000, substantial progress has been made in reducing malaria worldwide. However, some countries in West Africa remain a hotspot for malaria with all age groups at risk. Asymptomatic carriers of Cross-sectional surveys were carried out during the rainy season in 2014 and the dry season in 2015 in both settings. During each season, 728 households were randomly selected and a household-based questionnaire was implemented to collect demographic and epidemiological data, including of malaria preventive methods used in communities. Finger-prick blood samples were obtained for biological examination using microscopy and rapid diagnostic tests (RDTs).Plasmodium spp. infection was confirmed in 12.4% (n = 832) and 0.3% (n = 22) of the assessed individuals in Korhogo and Kaedi, respectively. In Korhogo, the prevalence of asymptomatic malaria was 10.5% (95% CI: 9.7–11.2) as determined by microscopy and 9.3% (95% CI: 8.6–10.0%) when assessed by RDT. In Kaedi, asymptomatic malaria prevalence was 0.2% (95% CI: 0.1–0.4%) according to microscopy, while all RDTs performed were negative (n = 8372). In Korhogo, asymptomatic malaria infection was significantly associated with age and season, with higher risk within the 5–14 years-old, and during the rainy season. In Kaedi, the risk of asymptomatic malaria infection was associated with season only : 6.37, 95% CI: 1.87–21.63). P. falciparum was the predominant species identified in both study sites representing 99.2% (n = 825) in Korhogo and 59.1% (n = 13) in Kaedi. Gametocytes were observed only in Korhogo and only during the rainy season at 1.3% (95% CI: 0.7–2.4%).Overall, 2672 households and 15 858 consenting participants were surveyed. Plasmodium spp. infections in Korhogo and Kaedi.Our findings show a low prevalence of clinical malaria episodes with a significant proportion of asymptomatic carriers in both urban areas. National policies for malaria infections are focused on treatment of symptomatic cases. Malaria control strategies should be designed for monitoring and managing malaria infections in asymptomatic carriers. Additional measures, including indoor residual spraying, effective use of long-lasting insecticidal nets is strongly needed to reduce the number of The online version of this article (10.1186/s40249-018-0412-9) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium falciparum infections are asymptomatic in endemic countries : 0.51, 95% CI: 0.30–0.87), 10–14 years  and the rainy season  were significantly associated with the prevalence of asymptomatic Plasmodium spp. infection.In the univariate analysis, sex, age group and season were significantly associated with the prevalence of asymptomatic Plasmodium spp. infection, this reason did not allow us to perform a multivariate analysis. In univariate analysis, the risk of asymptomatic Plasmodium spp. infection was 6.36 times (crude OR [cOR]: 1.87–21.63) higher during the dry season, compared to the rainy season.In Kaedi, by contrast, only the season was associated with asymptomatic Plasmodium spp. with a focus on the distribution of Plasmodium spp. infection in asymptomatic carriers from urban areas of Korhogo in Côte d’Ivoire and Kaedi in Mauritania.The present study assessed the prevalence of Plasmodium infection observed in Korhogo (12.4%) was generally low compared to other reports in urban settings in Côte d’Ivoire [The prevalence of d’Ivoire , 18, 36.d’Ivoire –39. As tP. falciparum setting [The low prevalence of malaria infection observed in Kaedi  may be partly attributed to the divers Global Fund-supported intervention strategies implemented in this area, such as LLINs, IPT for pregnant women and immediate treatment with ACTs. In a recent study conducted in Rosso, a city located along the Senegal River, localised in the Sahelian zone, Ouldabdallahi and colleagues  reported setting . NeverthA recent study conducted in three strata in Mauritania  showed tSchool-aged children (i.e. 5–14 years of age) in Korhogo and preschool-aged children (< 5 years) in Kaedi presented the highest asymptomatic malaria prevalence. It was previously shown that teenagers sleep under bed nets less frequently than the preschool-aged children. Those asymptomatic teenagers raise many issues because they are unknown carriers of the parasite and these reservoirs contribute in maintaining the parasite lifecycle, as documented in malaria-endemic communities , 43. HowIn the current study, parasitaemia increased with age in children aged 1–5 years, and declined thereafter, suggesting a certain immune protection during the childhood obtained at an early age. This explains why children falling into this age category were found to be asymptomatic most of the times.Plasmodium spp. positive febrile participants was 15.4% and 0, 2% in Korhogo and Kaedi, respectively. The prevalence of Plasmodium spp. in feverish participants in Korhogo was higher than in those with no fever. Indeed, the majority of fever cases in both sites was not due to malaria parasitaemia. The association between malaria parasitaemia and fever has been convincingly documented in endemic areas [The proportion of ic areas –46. In uic areas , 22.n = 1040, 0.9%) in Trarza region in the Sahelian zone [Pf-HRP2 mutations reported in Central and South America and Asia [The prevalence of asymptomatic infection in Korhogo was 10.5% (ranging from 8.3% to 12.3%). This prevalence rate was similar to the prevalence of asymptomatic malaria cases documented in a mountainous district in Ghana (11.9%) . This fiand Asia , 51 havePlasmodium results in partial immunity and favours asymptomatic parasite carriers (APCs) [In areas where malaria transmission is high, regular infection with s (APCs) , 48, 52.s (APCs) –55. Thess (APCs) .Anopheles mosquitoes during this period. The rainfall creates more productive breeding sites to renew vectors populations, and hence, malaria transmission intensifies. Moreover, during the rainy period, higher amounts of gametocyte carriers are generated. The observations made in Kaedi do not fit with this common pattern and are different to a report from a southern area of Mauritania [Plasmodium parasitaemia [In the two sites surveyed, malaria infection follows a seasonal variation. However, while in Korhogo malaria was found at the highest prevalence during the rainy season, the opposite was observed in Kaedi where the prevalence of infection was higher during the dry season. In Korhogo, the high malaria prevalence during the rainy season is likely to be due to the abundance of uritania , where muritania  recordedsitaemia . HoweverPlasmodium species were found  out of the three species previously reported from Côte d’Ivoire [Plasmodium were observed during our study  as previously reported from Mauritania [P. vivax was found. This species is mainly found in Nouakchott because of a high density of Duffy-positive P. vivax-susceptible individuals among the population [P. vivax infection among 1050 children screened [P. vivax identified case may be related to people movement from Nouakchott, P. vivax endemic area in Mauritania. P. falciparum was the dominant species in Korhogo and in Kaedi, which is in line with previous studies in both countries [P. falciparum cases [In Korhogo, two d’Ivoire , 36. In uritania , 24, 58 pulation , 23. A rountries , 26, 36 ountries , 59. Yetum cases . Data geOur study has several limitations that should be taken into consideration when interpreting the results. Firstly, the current level of APCs proportion, especially in Kaedi, may be underestimated. Indeed, in low transmission area, parasite density seemed low; therefore, high sensibility diagnostic methods like PCR and loop-mediated isothermal DNA amplification are needed to identify malaria cases that are not detectable via microscopy , 61. FurPlasmodium spp. reservoirs and thus contributing greatly to the anchoring of malaria in favourable eco-epidemiological zones. Ours results have implications for malaria control programme in Côte d’Ivoire and Mauritania, which, currently, do not include APCs in intervention strategies likewise of school-aged children and adults. Hence, the authors recommend further investigations placing emphasis on APCs in each study country in order to design and implement appropriate surveillance and control measures.The study shows that in the urban areas of Korhogo, in northern Côte d’Ivoire and Kaedi, in southern Mauritania, malaria prevalence is low with non-negligible proportions of APCs. We highlight also the implication of school-aged children and adults as Additional file 1:Multilingual abstracts in the five official working languages of the United Nations. (PDF 736 kb)


Malaria remains an important health problem in India with approximately 1 million cases in 2014. Of these, 7% occurred in the Jharkhand state mainly in the tribal population.This study was conducted in Dumargarhi, a tribal village about 42 km east of Ranchi city, Jharkhand, from May 2014 to September 2016. Four point prevalence surveys were carried out during consecutive high (October–December) and low (June–August) transmission seasons. Malaria cases were recorded from April 2015 to April 2016 through fortnightly visits to the village. Adult mosquito densities were monitored fortnightly by manual catching using suction tube method.Plasmodium falciparum (94 cases) and Plasmodium vivax (5 cases), respectively. During the high transmission season the mean density of P. falciparum parasitaemia per age group increased to a peak level of 23,601 parasites/μl in the 6–10 years age group and gradually declined in the adult population. Malaria attack rates, parasite prevalence and density levels in the study population showed a gradual decrease with increasing age. This finding is consistent with the phenomenon of naturally acquired immunity against malaria. Three vector species were detected: Anopheles fluviatilis, Anopheles annularis, and Anopheles culicifacies. The incoherence or complete out of phase pattern of the vector density peaks together with a high prevalence of parasite positive individuals in the study population explains the year-round malaria transmission in the study region.The study area consists of five hamlets inhabited by 945 individuals living in 164 households as recorded through a house-to-house census survey performed at enrollment. The study population consisted predominantly of the Munda  and Oraon  ethnic groups. Study participants were categorized as per their age 0–5, 6–10, 11–15 and >15 years. There were 99 cases of clinical malaria from April 2015 to April 2016 and all malaria cases confirmed by microscopy were attributed to The collection of clinical data from a well-characterized tribal cohort from Jharkhand, India, has provided evidence for naturally acquired immunity against malaria in this hyperendemic region. The study also suggests that enforcement of existing control programmes can reduce the malaria burden further. Plasmodium species. Of these, Plasmodium falciparum and Plasmodium vivax are the main causes of disease and mortality worldwide . RDT positive cases were examined by thick and thin blood smears from finger prick blood samples. All slides collected from the study area were brought to a laboratory at the National Institute of Malaria Research field unit, Itki, Ranchi. Slides were examined by trained microscopists under compound microscope at 100× magnification after staining with Jaswant Singh Bhattacharjee (JSB) stain . Asexual parasites were counted against 200 leukocytes and parasite density was calculated as number of asexual parasites per micro liter of blood assuming a mean normal leukocyte count of 8000/µL. Slide-positive cases were provided anti-malarial treatment as per the guidelines of the National Vector Borne Disease Control Programme of the Government of India  throughout the year. An. culicifacies breeds mainly in pools formed in streams and riverbeds; the most productive breeding sites of An. fluviatilis are slow-moving streams while An. annularis breeds in margins of ponds, rivers and streams with abundant vegetation. The three vector species An. culicifacies (sibling species B & C), An. fluviatilis (sibling species T) and An. annularis (sibling species A) were identified based on their morphological characteristics and cytogentic analysis of their polytene chromosomes [The district is characterized by a large network of streams and other water bodies which provide innumerable and diverse breeding sites for species-specific malaria vectors [omosomes .Entomological survey was conducted fortnightly from May 2014 to March 2016 by manual catching using suction tube method . Indoor 2-test. P values <0.05 were considered to indicate statistical significance.Statistical analysis was done with the R 3.3.0 and GraphPad Prism 5 software. Age was categorized in four levels  and demographic characteristics were compared between villages by using the χ2 analysis) , Jarwadih (n = 103), New Torang (n = 84), Karam Tungri (n = 123) and Old Torang (n = 449)  and P. vivax (5 cases), respectively. The monthly incidence of P. falciparum and P. vivax is shown in Fig. P. falciparum malaria incidence was generally higher after the end of the monsoon season and lower in the hot dry summer months. The mean density of P. falciparum parasitaemia was calculated for the 0–5, 6–10, 11–15, and >15 year age groups, respectively  in CSS1 and 72 were positive for malaria  in CSS3. The parasite rate during the first and second surveys in the high transmission was 46.6 and 22% respectively. During low transmission (CSS2 and CSS4), 254 and 225 individuals were enrolled, 50% of whom had also participated in CSS1. Of these, 51 individuals were malaria positive by the RDT test in CSS2  and 39 were positive for malaria in CSS4 . The parasite rate during the two surveys in the low transmission was 20.1 and 17.3% respectively. The average parasite rate during the high (CSS1 + CSS3) and low (CSS2 + CSS4) transmission season was 35.3 and 18.8%, respectively. A significant variation in the seasonal malaria prevalence  was observed.During the high transmission season (CSS1 and CSS3), 386 and 327 villagers respectively were examined for P. falciparum from CSS1 and CSS2, respectively. The mean P. falciparum parasite density of these slide positive individuals was 6113 (±1456) parasites/μl and 3480 (±1511) parasites/μl during high and low transmission season, respectively  who did not have fever or any other symptom of malaria were classified as asymptomatic P. falciparum carriers. The number of malaria cases and asymptomatic carriers were 4, 15, 10, and 15 and 3, 7, 5, and 27 in the 0–5, 6–10, 11–15, and >15 years age groups, respectively. The mean parasite density of each age group was plotted for malaria cases and asymptomatic carriers, respectively  and 13 25.5%) of the RDT positive samples were also found to be slide positive for 5.5% of t7.8% and An. fluviatilis as the most prevalent vector constituting 49% of all the vector species. An. annularis and An. culicifacies constituted 34 and 17%, respectively. The cumulative annual average of all the vectors captured in the study area during fortnightly surveys was 21 and 79% for vectors resting in human dwellings and cattle sheds, respectively. Some seasonal fluctuations in the month-wise person-hour density (PHD) of An. culicifacies, An. fluviatilis and An. annularis was observed  a high malarial attack rate, and (iii) higher parasite densities compared to adults are indications of hyper-endemicity in this study area. It is generally observed that individuals living in hyper-endemic areas gradually develop an anti-parasite immunity which protects them from febrile malaria. The age-dependent variation of malaria attack rates, parasite prevalence and density levels in the present study was characterized by a gradual decrease with increasing age. This relationship is typical of NAI against malaria and is similar to findings in highly endemic areas of Africa [P. falciparum infected erythrocytes in healthy adults but not in children below 3 years of age [Malaria is a focal disease in India, influenced by several local ecological and social factors . The stuf Africa –24 and Af Africa . The devf Africa . Similarf Africa . Anothers of age . CollectP. falciparum-positive samples was around 44% with a clinical malaria prevalence of 11.4%. This was reduced to 19.3% for P. falciparum-positive samples and clinical malaria prevalence of 8.6% during the next high transmission season. This reduction in the parasite burden in the study population is likely due to the combination of active case detection and ACT of all identified positive cases.Another important observation of the present study was the apparent decrease in the malaria parasite burden between 2014 and 2016. In the first cross-sectional survey during high transmission season of 2014 the rate of An. fluviatilis and An. culicifacies as the primary vectors and An. annularis as the secondary vector for malaria transmission. The climatic conditions in the forest ecotype are more conducive for higher survival of vectors that are associated with a predominance of P. falciparum [An. fluviatilis is widely distributed in India although its role in malaria transmission varies from place to place depending on the local prevalence of different sibling species [An. fluviatilis (species S) is among the most efficient vectors of malaria particularly in hills and foothills of India and it has previously been incriminated as a vector in the tribal districts of Koraput, Orissa [An. fluviatilis (species T), though prevalent in high densities in the foothills of Shiwalik range was not playing role in malaria transmission [An. fluviatilis (species T) has been found to be susceptible to P. vivax [P. falciparum infections  and has been incriminated as an efficient vector of malaria for transmission in the mountainous areas of the Hormozgan province, south Iran [An. fluviatilis (species T) as the primary vector in malaria transmission in this study has been established by way of its high prevalence period coinciding with the peak transmission period, more preference for human biting, high human blood index and susceptibility to plasmodial infections . The other primary vector, An. culicifacies (species C), is responsible for 60–70% of all malaria cases in India despite being predominantly a zoophagic species and being prone to environmental factors with epidemiological implications in different ecotypes [An. culicifacies may be regarded as playing a complementary role in maintaining perennial transmission during pre-monsoon and monsoon months where transmission is low. The secondary vector An. annularis though primarily zoophagic, exophilic and exophagic, is associated with low human biting rate and sporozoite rate, and has a marginal role in malaria transmission in this study area. While An. annularis plays a limited role for malaria transmission in the present study area, it is responsible for year-round transmission as observed in inlands of Odisha [The entomological studies revealed that the study area is under the influence of three vector species lciparum . An. flu species . An. flu, Orissa  and Bast, Orissa . In contsmission . HoweverP. vivax  and P. futh Iran . The rolecotypes , 34. An.f Odisha  and foref Odisha . The preBesides technical inadequacies, the problem posed by malaria in tribal areas of India is multifaceted. Operational constraints such as inaccessible remote areas, poor surveillance, inadequate vector control and general lack of motivation and health awareness all facilitate persistent transmission. Therefore, focus should be on strengthening of health infrastructure at the periphery to ensure early case detection and prompt treatment. This should be supported by quality vector control measures, creating health awareness in the community and ensuring economic growth of the tribal areas. These efforts along with the development of an efficacious malaria vaccine will lead to a sustained reduction in malaria morbidity and mortality.In conclusion, the collection of clinical data from a well-characterized tribal cohort from the state of Jharkhand in Eastern India has provided evidence for the presence of NAI against malaria in this hyperendemic region.


Plasmodium falciparum malaria in febrile patients. Matched urine and finger-prick blood samples from participants ≥2 years of age with fever (axillary temperature of ≥37.5°C) or with a history of fever in the preceding 48 h were tested with UMT and microscopy (as the gold standard). BinaxNOW (Pf and Pan versions) blood RDTs were done to assess relative performance. Urinalysis and rheumatoid factor (RF) tests were conducted to evaluate possible interference. Diagnostic performance characteristics were computed at 95% confidence intervals (CIs). Of 1,800 participants screened, 1,691 were enrolled; of these 566 (34%) were febrile, and 1,125 (66%) were afebrile. Among enrolled participants, 341 (20%) tested positive by microscopy, 419 (25%) were positive by UMT, 676 (40%) were positive by BinaxNOW Pf, and 368 (22%) were positive by BinaxNow Pan. UMT sensitivity among febrile patients (for whom the test was indicated) was 85%, and specificity was 84%. Among febrile children ≤5 years of age, UMT sensitivity was 93%, and specificity was 83%. The area under the receiver-operator characteristic curve (AUC) of UMT (0.84) was not significantly different from that of BinaxNOW Pf (0.86) or of BinaxNOW Pan (0.87), indicating that the tests do not differ in overall performance. Gender, seasons, and RF did not impact UMT performance. Leukocytes, hematuria, and urobilinogen concentrations in urine were associated with lower UMT specificities. UMT performance was comparable to that of the BinaxNOW Pf/Pan tests, making UMT a promising tool to expand malaria testing in public and private health care settings where there are challenges to blood-based malaria diagnosis testing.The need to expand malaria diagnosis capabilities alongside policy requirements for mandatory testing before treatment motivates exploration of noninvasive rapid diagnostic tests (RDTs). We report the outcome of the first cross-sectional, single-blind clinical performance evaluation of a urine malaria test (UMT) for diagnosis of  The current WHO recommendation for parasitological confirmation of suspected malaria cases prior to treatment is a paradigm shift from entrenched presumptive diagnostic practices. Annually, there are 214 million estimated cases of malaria globally  were enrolled per eligibility criteria, with a mean age (± standard deviation [SD]) of 18.6 years , of whom 970 were female (57%) and 721 were male (43%). Among these 1,691 enrollees, 566 (34%) participants were febrile (≥37.5°C), and 1,125 (66%) were afebrile but reported a history of fever in the previous 48 h . HeadachPlasmodium falciparum (BinaxNOW Pf) and aldolase, a pan-malaria antigen found in all Plasmodium species (BinaxNOW Pan), detected malaria in 676 (40%) and 368 (22%) of participants, respectively. The highest prevalence was among febrile participants. Among 566 febrile participants, 204 (36%) were positive by microscopy, 231 (41%) were positive by UMT, 224 (40%) were positive by BinaxNOW Pan, and 317 (56%) were positive by BinaxNOW Pf. Similarly, febrile participants had higher parasite densities (PDs) than afebrile participants  of 1,691 enrollees were microscopy-positive for malaria . The UMTicipants  (2). Thr0% of 1,6P < 0.001). The sensitivity and specificity of BinaxNOW Pan did not differ significantly from those of UMT, while PPVs and NPVs did not differ significantly between UMT and either of the BinaxNOW tests. The negative likelihood ratio (NLR) for UMT differed from that of BinaxNOW Pf and was closer to that of BinaxNow Pan, while the positive likelihood ratio (PLR) for UMT was also closer to that of BinaxNow Pan than to that of BinaxNow Pf , and specificity was 84%  . Among faxNow Pf .P = 1.00), sensitivity was significantly lower than that of BinaxNOW Pf (97%) (P = 0.001) (P < 0.001). The likelihood ratios also varied among the tests.Of 1,125 afebrile participants, UMT sensitivity was 72%, and while it did not significantly differ from that of BinaxNOW Pan (72%) (= 0.001) . The speP < 0.001) (P < 0.001). The PPV and NPV were ≤75% and ≥94%, respectively, for all tests and did not differ significantly from each other. The BinaxNOW Pf also differed from UMT and BinaxNOW Pan with respect to NLR and PLR values in the general study population  were 79%  and 89% , respectively. UMT sensitivity did not differ from that of BinaxNOW Pan (80%) but was significantly lower than that of BinaxNow Pf  (< 0.001) . Specifipulation .P = 0.212), BinaxNOW Pf (P = 0.054), and BinaxNOW Pan (P = 0.358) did not differ between febrile and afebrile participants   curves (AUCs) for UMT ( > 0.05) . A poole > 0.05)  showed tP < 0.001). UMT, BinaxNOW Pf, and BinaxNOW Pan sensitivities increased with higher PDs among all patients, as well as with stratified subpopulations of febrile and afebrile patients (Graded PDs by microscopy compared with UMT performance showed that increased PD was associated with increased probability of fever (patients . While BP < 0.001). The PPVs of UMT did not differ between age categories, but while NPVs of children and adolescents were significantly lower than those of adults (P < 0.001), all values were ≥90% in each category. Upon stratification by fever, the majority of the performance characteristics of the three tests did not differ significantly between the oldest and youngest participants.The sensitivities of all tests decreased with increasing age and increased with higher PDs. In contrast, specificities of all tests were significantly higher in adults than in younger participants in the overall study population , while hematuria was associated with significantly lower specificity (P = 0.001). UMT specificity was lower among participants with urobilinogen concentrations of ≥1 mg/dl than in those with levels of <1 mg/dl (P < 0.001). Specific gravity, which indicates urine concentration, was not associated with UMT performance. All of the 15 rheumatoid factor-positive (RF+) participants tested negative for malaria and were negative by UMT.Gender and season (dry and rainy) did not affect UMT performance. Urinalysis revealed that a higher number of leukocytes (>15) was associated with a significantly higher specificity (Plasmodium parasites: 362 (99%) were infected with P. falciparum, and 3 (1%) were infected with P. malariae. A further analysis of these samples by PCR showed that seven study samples originally found to harbor P. falciparum by microscopy were negative by PCR. Additionally, 58 microscopy-negative samples were positive by PCR; of these 56 were infected with P. falciparum, and 2 were infected with P. malariae. The PCR results of one participant sample indicated infection with Plasmodium ovale, but the source of the infection was originally identified by microscopy as P. falciparum.The original microscopy results showed that 365 individuals were infected with P. falciparum parasites as determined by PCR were removed from the data set. There were no statistically significant differences between original and PCR-corrected microscopy results of discordant samples by UMT and BinaxNOW Pf/Pan, likely reflecting high-quality microscopy methods and well-trained microscopists , but this was expected as a mathematical relationship between prevalence and PPV value is known, whereby increasing prevalence always produces an increased PPV. Likelihood ratios also did not demonstrate clinically meaningful changes.The original microscopy results indicated that 361 study participants were infected with In contrast to microscopy results, those for sensitivity, specificity, and negative predictive values changed by less than 3% regardless of which participant population, total, febrile, or afebrile, was assessed. The largest changes occurred among the positive predictive values (PPVs), with differences between the original and PCR-corrected microscopy results ranging from 3% to 12%. However, this was expected as a mathematical relationship between prevalence and the PPV value is known, whereby increasing prevalence always produces an increased PPV. Likelihood ratios also did not demonstrate clinically meaningful changes.The UMT is the first noninvasive malaria test clinically evaluated on a large scale at the population and community levels. Given its sensitivity and specificity, a disease prevalence of 20% in the study population, and a PLR of 7.2, the posterior probability of disease in a patient who tests positive by the UMT is 59%. Given the NLR of 0.23, the posterior probability of disease in a patient who tests negative by UMT is 4%. This indicates that the UMT could aid in the clinical management of suspected malaria cases. For example, upon receiving a negative UMT result for a suspected malaria case, the clinician now knows that this patient's probability of having malaria parasites detectable by microscopy is unlikely, i.e., only 4%. In contrast, a positive UMT result would indicate that the probability of detecting malaria parasites in this patient by microscopy is 59%.The performance of UMT was higher among febrile participants (who are the intended use population) than among afebrile individuals. High rates of false positives and lower specificities reported among HRP-2-based blood RDTs corroborate our findings –20 and a–21–P. falciparum parasitemia and antigenemia do not correlate directly due to sequestration, while there are no data on the correlation between parasitemia and antigenuria. Therefore, UMT performance metrics must be established de novo and not on the basis of antigenuria reported by RDTs.It should be noted that The availability of the blood-based RDTs has greatly increased malaria testing particularly in the public sector, where about 53% of patients with malaria-like symptoms are tested. In the formal and informal private sector health care settings, only 36% and 6%, respectively, of patients are tested . The UMTViral hemorrhagic fevers  are major global health concerns where the UMT could be invaluable. The Ebola viral proteins connected with infection are present in blood and other body fluids at the time of fever and onset of symptoms , 28 but Programmatically, the UMT could expand access to prompt parasitological confirmation of malaria in public and private health care settings, particularly in areas or settings where invasive blood testing is impractical or restricted.The study was conducted in six health care facilities (HFs) across Ikorodu  and Somolu (urban) local government areas (LGAs) of Lagos State, southwest Nigeria. Malaria is hypo-mesoendemic in Lagos State, with peak transmission during the rainy season (April to September). Ikorodu LGA and Somolu LGA have estimated populations of 535,619 and 402,763, respectively.The HFs in Ikorodu LGA were the following: Imota Primary Health Center (PHC), Imota; Agura PHC, Gberigbe; Ijede Health Center, Ijede; Bayekun PHC, Bayeku; Annex of St. Kizito Clinic/PHC, Oreta; and General Hospital, Somolu, Somolu LGA, Lagos State, Nigeria.+) individuals were enrolled and tested to determine potential cross-reactivity with UMT, as RF is known to elicit proteinuria and cross-reactivity with HRP-2-based RDTs  who presented with fever  or history of fever in the past 48 h. Fifteen blood smear-negative rheumatoid factor positive , assuming that 40% of subjects would have positive blood smears, based on the “Nigeria Malaria Indicator Survey 2010” . RF testing was done on a cohort of microscopy-negative patients.P. falciparum proteins, in urine , and aldolase, a pan-malaria antigen found in all Plasmodium species (BinaxNOW Pan). The test was performed per the manufacturer's instructions and was used as a comparator RDT. Multianalyte urinalysis reagent strips  were used to measure glucose, bilirubin, specific gravity, ketones, blood, pH, protein, urobilinogen, nitrites, serum creatinine, and leukocytes. A direct slide test was used to detect RF . All tests were performed per the manufacturer's instruction.The BinaxNOW (Pf/Pan) blood-based malaria RDT  is a qualitative immunochromatographic assay that detects HRP-2 antigen found in Thick and thin malaria blood films (MBFs) were prepared on the same slide in HFs and transported to the ANDI Center of Excellence for Malaria Diagnosis at the University of Lagos for microscopy. Two WHO-accredited malaria microscopists blinded to UMT and blood RDT (BinaxNOW Pf/Pan) results read the MBFs independently. Asexual parasites were counted relative to 200 to 1,000 leukocytes in thick films. Computed parasite density (PD) was reported as the number of parasites/microliter of blood using the mean PD of the two microscopists if discordance was ≤20%. Discordance in PDs of >20% between the two microscopists required reading by a third WHO level 1 microscopist, and the PD was recomputed incorporating this microscopist ‘s findings.P. falciparum detection and the pan-Plasmodium 18S rRNA gene , using the packages epicalc, pROC, and ROCR.Microscopy was the gold standard (reference test) for sensitivity and specificity calculations. Parasite density (number of parasites per microliter of blood) was computed for all positive MBFs. Exact binomial confidence intervals were calculated for sensitivity, specificity, and positive and negative predictive values . McNemar's test was used to compare sensitivities and specificities between diagnostic tests. The weighted generalized score method was used to compare predictive values . The chi