Plasmodium vivax infection has been gaining attention because of its re-emergence in several parts of the world. Southeastern Turkey is one of the places in which persistent focal malaria caused exclusively by P. vivax parasites occurs. Although control and elimination studies have been underway for many years, no detailed study has been conducted to understand the mechanisms underlying the ineffective control of malaria in this region. Here, for the first time, using serologic markers we try to extract as much information as possible in this region to get a glimpse of P. vivax transmission. We conducted a sero-immunological study, evaluating antibody responses of individuals living in Sanliurfa to four different P. vivax antigens; three blood-stage antigens  and one pre-erythrocytic stage antigen (PvCSP). The results suggest that a prior history of malaria infection and age can be determining factors for the levels and sustainability of naturally acquired antibodies. Significantly higher antibody responses to all the studied antigens were observed in blood smear-negative individuals with a prior history of malaria infection. Moreover, these individuals were significantly older than blood smear-negative individuals with no prior history of infection. These data from an area of sole P. vivax-endemic region may have important implications for the global malaria control/elimination programs and vaccine design. Plasmodium vivax parasites are the main cause of widespread human malaria infections, with at least 35–80 million cases reported each year P. vivax is very high, resulting in renewed attention P. vivax is age-dependent and develops more quickly than immunity to P. falciparumP. vivax parasites have distinct biological features, such as persistence in the host due to hypnozoites/relapses, effective gametocyte production, and rapid and efficient adaptation to mosquitoes P. vivax-specific IgG and IgM, cytokines and chemokines)? What are the host age groups? What are the kinetics of acquired immunity against P. vivax? P. vivax infection has been hampered by the fact that the majority of studies analyzing serological parameters and disease severity have been performed in patients co-infected with P. falciparumP. vivax is the only Plasmodium species present P. vivax in Sanliurfa province 19 antigen (one of the leading candidate vaccine antigens) in serum samples from infected individuals pvmsp1 polymorphisms in the parasite population in Turkey P. vivax antigens in this region. Such an understanding will facilitate the design of effective vaccination strategies against P. vivaxSoutheastern Turkey is one of the few regions in which P. vivax antigens that may be potential vaccine candidates. Total IgM and IgG antibody responses (including IgG subclasses) to three blood-stage antigens, PvMSP119, PvAMA1-ecto and PvSERA4, and one pre-erythrocytic stage antigen, PvCSP, were examined in individuals living in Sanliurfa. Any association between antibody responses to each of the P. vivax antigens and the degree of parasitemia in the patients was investigated. Baseline data provides important information for the elimination programs targeting vivax malaria by drugs or vaccines.As a first step, in the present study, we analyzed naturally acquired antibody responses to four P. vivax transmission was reported in seven Turkish provinces in 2006, and 84% of cases occurred in the southeastern cities of Diyarbakir and Sanliurfa Although the vivax malaria elimination program was launched in 1925 P. vivax candidate vaccine antigens  were examined and total IgM and IgG antibody responses (including IgG subclasses) were measured. In the total study population (n = 195), 79.1% individuals were seropositive for either IgG or IgM against at least one of the four antigens studied, while 62.1% were seropositive for IgG alone, and 65.6% were seropositive for IgM alone , PvAMA1-ecto (21%) and PvSERA4 (16.4%). However, IgM antibody responses to PvSERA4 and PvCSP were observed in the majority of individuals   . The IgGctively) . Taken tP. vivax cases decreased by about 86% from 2002 to 2008 and by 42% from 2004 to 2008. Based on this information that the infection rate had decreased dramatically over the last decade, we examined whether there may be any difference in the antibody responses between younger (< 6 years old) and older individuals. The study population was grouped according to their history of malaria infection . Importantly, the mean age of PN individuals with a history of malaria infection was significantly higher than that of individuals with no prior history of infection . Thus, these observations suggest that in this region, it appears that repeated exposure to malaria correlates with significantly higher percentage of PN individuals.Although 2000–6000 cases of malaria were recorded annually in Sanliurfa up until 2003, the incidence declined rapidly after the government's efforts to control the disease using chloroquine and primaquine nfection . The resP. vivax infections evoked high IgG responses to all four antigens, both in terms of frequency and serum concentration in the PP group with no history of malaria infection   . IgG3 ren = 80; data not shown and n = 115; #P<0.05 and ##P<0.01; χ2 test). However, despite the fact that IgG antibody responses were lower when compared with those in the whole study population or those in PP individuals  in the PN population. This may indicate that one or two infections may induce naturally acquired immunity. Supporting this notion, IgG responses against P. vivax and P. falciparum infection, IgG responses to PvMSP119 increased rapidly during the early ages of infection and were sustained over a long period of time 19. However, in an earlier pilot study, we observed higher sero-reactivity (53.6% IgG responses to PvMSP119) to the C-terminal region of the PvMSP1 antigen (produced in Saccharomyces cerevisiae) in the PP population, which was negatively correlated with parasite levels 19 and parasite number, suggesting the possibility of allelic polymorphisms in the antigen/parasites. In fact, in a recent study, we did find eight substitutions in the PvMSP1 gene that were unique to the Turkish P. vivax population and one of them  was previously unidentified 19 IgG levels and parasite numbers in individuals infected with E haplotype substitutions, but not D haplotype substitutions . It is also noteworthy that other studies from Brazil and Papua New Guinea reported that different individuals show naturally acquired IgG responses to different regions of PvMSP1 In agreement with previously published studies of 19 responses, antibody levels to chimeric PvCSP antigen immediately increased in younger individuals, but the response was weaker than that for PvMSP119, even during patent infection . Therefore, it seems unlikely that more than 4.5% of malaria history negative PN group (presumably naïve) individuals have undetectable level of parasitemia. We cannot exclude, however, another possibility of P. vivax infections causing relapses due to the presence of hypnozoites, even in the absence of mosquito bites, because of ineffective drug treatment P. vivax infection and this certainly limits meaningful evaluations to specific populations as re-infection probabilities may exceed 20% per year Similar to anti-PvMSP1nfection . Howevernfection . One posWe observed antibody responses to PvAMA1-ecto antigens in this region, although low in frequency and accompanied by low serum antibody levels. Children infected at a younger age (<6 years old) developed rapid antibody responses to AMA1-ecto antigen, but these responses were not seen after 6 years of age. However, the current study provides important evidence that IgG antibody responses to PvAMA1-ecto domain are significantly increased in non-infected individuals with a prior history of malaria infection. In addition, the IgG levels to PvAMA1-ecto were negatively correlated with parasite levels, which collectively may suggest that antibodies to PvAMA1-ecto are important and maybe closely related to protection P. vivax SERA4 responses. The serine repeat antigen (SERA) is an abundant asexual blood-stage antigen primarily expressed by Plasmodium parasites during late trophozoite and schizont stages P. falciparum, upon schizont rupture theabundant Pf SERA gene family member, SERA5, is processed to yield a 47 kDa N-terminal, a 50 kDa central, an 18 kDa C terminal, and a 6 kDa domain P. falciparum serine repeat antigen 5 (PfSERA5) has already been exploited as a potential candidate vaccine P. vivax, the highest transcription of PvSERA4 in all field isolates 19 gene polymorphisms observed in this region unpublished observations). However, the polymorphic nature of the recombinant PvSERA4 antigen used in the present study (and the other antigens to a similar extent) may also be one reason that we detected low IgG antibody responses in this region. This, too, requires further investigation.This is the first report analyzing anti-P. vivax endemic region. However, we are far from the conclusion that naturally acquired antibodies to these relatively small numbers of vivax proteins could establish malaria immunity and/or protection. Apparently, new tools such as protein microarrays which represent at least 20% of the parasite proteome are needed to evaluate naturally acquired antibodies which may be associated with naturally acquired vivax malaria immunity in this setting as similar to recently described study for falciparum malaria This study constitutes a first seroepidemiological analysis/survey of antibodies to a variety of blood and pre-erythrocytic stage malarial antigens and provides valuable information in their relation to the age of the individuals living in a sole Nevertheless, our study offers valuable insights to those designing vaccines/drugs especially in the settings where elimination programs have been launched All samples were collected after written informed consent was obtained from the patients , prior to anti-malarial treatment when appropriate. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Authorization was obtained from the Turkish Ministry of Health, Sanliurfa Bureau, and ethical approval was obtained from the Research Institute for Microbial Diseases, Osaka University.P. vivax malaria (as diagnosed by microscopy) P. vivax-infected using thick blood smears after Giemsa staining. The level of parasitemia  was determined as previously described n = 69 from 2004 and n = 11 from 2008) (data not shown); and hence, the two sets of samples were grouped together as “microscopically positive for P. vivax parasites” (n = 80)  in southeastern Turkey during 2004 and 2008. The samples were collected at the peak of the malaria season (July to November) and kept at −20°C until use. All 69 serum samples collected in 2004 were found to be infected with patent  80) see .P. vivax (P. vivax-positive (PP) according to blood smear results; n = 80) and individuals living in the malaria-endemic area but not infected with P. vivax at the time of sampling (P. vivax negative (PN) according to blood smear results; n = 115). The mean (mean ± SD [range]) age of the two groups was 21.1±16 [0–65] years and 20.4±16.5 [0–77] years, respectively, and 56.2% and 41.7% of the respective groups were male. There was a significant difference in body temperature between the PP and PN groups . The mean parasite density was 5502±6386 [160–25560] parasites/µL. The majority of the patients (46.3%) had parasite densities between 1001 and 5000 parasites/µL, whereas only 32.5% of the patients had heavy parasitemia (> 5000 parasites/µL) and 21.3% had very low parasitemia (< 1000 parasites/µL). Mean hemoglobin, hematocrit concentrations, and mean white blood cell counts did not vary significantly between the PP and PN groups (The mean age of the sample population was 20.5±16.2 years [0–77 years] (mean ± SD [range]), and 47.7% of the subjects were male. The samples were divided into four age groups: 0–6 years old (19.5%), 7–15 years old (26.7%), 16–30 years old (33.3%), and >31 years old (20.5%). N groups .Disclosure of malaria infection is compulsory in Turkey and intervention is controlled by the Turkish Ministry of Health, alongside local national malaria control centers, in accordance with the World Health Organization criteria for malaria diagnosis and treatment. Questionnaires filled out by the subjects or their parents ascertained whether they were previously diagnosed and treated for malaria at any of the local malaria control centers. The answers were cross-checked against the records from the malaria control centers. The results showed that those individuals with a history of previous malaria infection had only a single exposure (> 95%).19, PvAMA1-ecto, and PvCSP were expressed using a wheat germ cell-free protein translation system  E. coli as a histidine tagged fusion protein. The PvCSP was designed as recombinant chimeric protein that presumably cover vivax parasite population globally as previously described in detail 77 to Gln484 based on the SalI sequence, PVX_ 092275). Both gene fragments were cloned into pEU-E01-His-TEV-N2 plasmid for the wheat germ cell-free system (CellFree Sciences).PvMSP119 to Lys352 was amplified from gDNA of SalI after comparing sequences with NICA and Chesson. The N-terminal position and prediction of peptide cleavage site was determined using SignalP 3.0 E. coli Rosetta-gami B (DE3) pLysS . Protein expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM. The recombinant protein was extracted into soluble fraction with BugBuster Master Mix (Novagen) and purified using a His GraviTrap . All antigens were stored at −80°C until assayed.PvSERA4 gene sequence corresponding to amino acid regions Val19, PvCSP, PvAMA1-ecto and PvSERA4 were measured by ELISA as previously described 450 measured using sera from 20 Japanese blood donors with no history of malaria exposure. After subtracting the cut-off values, any ROU measurements higher than 0.01 were considered positive. The cut-off values were as follows: anti-PvMSP119-IgG, IgM, IgG1, IgG2, IgG3 and IgG4: 0.094, 0.345, 0.052, 0.324, 0.022 and 0.367, respectively; anti-PvAMA1-ecto-IgG, IgM, IgG1, IgG2, IgG3 and IgG4: 0.145, 0.542, 0.204, 0.32, 0.064 and 0.059, respectively; anti-PvSERA4-IgG, IgM, IgG1, IgG2, IgG3 and IgG4: 0.03, 0.405, 0.019, 0.062, 0.0418 and 0.01, respectively; and anti-PvCSP-IgG, IgM, IgG1, IgG2, IgG3 and IgG4: 0.141, 0.775, 0.14, 0.41, 0.05 and 0.1, respectively.The levels of human IgG, IgM, IgG1, IgG2, IgG3, and IgG4 antibodies against PvMSP1-positive and blood smear-negative for P. vivax. The antibody levels in all age groups, and in the blood smear-positive and -negative groups, were compared using the X2 test. Differences in antibody levels (expressed as ROU) between the groups were analyzed using multiple comparison tests in conjunction with ANOVA. Spearman's rank correlation was used to evaluate correlations between the variables. A P value < 0.05 was considered significant. All statistical analysis was performed using the SPSS statistical package .Data from 2004 and 2008 were pooled and/or divided into two groups: blood smearTable S1Demographic characteristics of the study population. * Significant values were determined between microscopically confirmed parasite positive vs. parasite negative populations by Student's t-test and X2- tests with a level of significance set at P<0.05. a Statistically significant by Student's t-test.(DOC)Click here for additional data file.


Plasmodium falciparum  merozoite ligands that mediate invasion of the erythrocytes through their sialated receptor: Glycophorin A (GPA). The effect of the EBA 175 dimorphic alleles (F and C) on the severity of the disease is not yet fully understood. Therefore this study was designed to assess the distribution of the divergent dimorphic alleles of P. falciparum EBA-175 (F and C) in three different geographical areas in Sudan and the possible association of this dimorphism with the severity of the disease.The Erythrocyte Binding Antigen (EBA) 175 has been considered as one of the most important P. falciparum obtained from patients in three different geographical areas in Sudan were screened for the dimorphic alleles  of the EBA-175 using nested PCR.A sum of 339 field isolates of p = 0.00). There was no significant association between malaria clinical manifestation and P. falciparum EBA-175 F and C alleles frequencies.The percentage of F, C, and mixed F/C alleles were; 41%, 51%, and 8% respectively. F and C alleles showed significantly different distributions in the various geographic areas (This study showed a significant differential distribution of F and C alleles in different geographical malaria endemic areas. No significant association was observed between F and C alleles and different malaria phenotypes. P. falciparum merozoites invade the erythrocytes through multiple ligand-receptor interactions[P. falciparum merozoites.In the pathogenesis of the malaria parasite, ractions-4 providractions. This ilP. falciparum strains mainly use ligands that bind to sialated receptors of the erythrocyte, other strains use ligands that bind to receptors independently of sialic acid. However, switching from sialic acid dependent to independent invasion is reversible and depends on the parasite ligand used[P. falciparum merozoites ligands is the (EBA-175), which utilizes the sialated erythrocytes receptor, GPA, the major glycophorin found on human erythrocytes, in order to invade the red blood cell[P. falciparum "referred to as the F loop" and the second one in CAMP strains "referred to as the C loop"[P. falciparum examined up to date and since merozoites are haploid; each parasite has a C or F segment, but not both or neither[P. falciparum merozoites use the erythrocytic GPA receptor in the invasion process[Some and used,5. Disruand used. This meand used,8. One o the EBA-75, whichood cell,10. Thisood cell. EBA-175ood cell,11. It i C loop",13. F an neither. In spit neither,15. Furt process. Studies process.P. falciparum EBA-175 (F and C) in three different malaria endemic areas in Central and Eastern Sudan and secondly, the possible association of this dimorphism with the outcome of the disease.Therefore, this study was designed to study firstly, the distribution of the divergent dimorphic alleles of This study is a cross-sectional carried out in November 2007 and December 2009. It was conducted in three regions; in the Eastern region  and the Central region of the Sudan (Wad Madani and Sennar). Um-Salala is a small village inhabited by a single tribe of ~1,200 people by 1997, and characterized by stable malaria transmission. Wad Madani and Sennar are two big cities located in central Sudan with a population size of 452,628 and 1.2 million respectively. Both are inhabited by multiple tribes and characterized by unstable malaria transmission[P. falciparum infected malaria patients from the three different malaria endemic areas; Um-Salala 57 samples, Sennar 166 samples and Wad Madani 116 samples. Depending on the World Health Organization (WHO) criteria of severe malaria, a report revised in the year 2000[P. falciparum is the only species under study. The BinaxNOW® Malaria ICT test targets the histidine-rich protein II (HRPII) antigen specific to P. falciparum and a pan-malarial antigen (PMA), common to all four malaria species capable of infecting humans - P. falciparum, P. vivax, P. ovale, and P. malariae.The study was approved by the Ethical committee of the institute of endemic diseases, University of Khartoum. Informed consents were obtained from adults or parents of children before they were enrolled in the study. A questionnaire form includes information about the family and clinical picture for each participant was completed. Peripheral blood samples were collected from a total of 339 microscopically confirmed g for 2 minutes. The supernatants, which contained the DNA, were carefully removed and transferred into new tubes[Chelex extraction method was used for DNA extraction from whole blood spotted and dried on filter papers. Each filter paper punch was incubated overnight at 4°C in 1 mL of 0.5% saponin in phosphate-buffered saline (PBS). The punches were washed for 30 minutes in 1 mL PBS at 4°C, transferred into new tubes containing 25 μL of 20% Chelex-100 and 75 μL of distilled water, and vortexed for 30 seconds. The tubes were heated at 99°C for 15 minutes to elute DNA, vortexed, and centrifuged at 10,000 × ew tubes.P. falciparum, nested PCR that was used as described previously[P. falciparum bands of 205 bp in 2% agarose were selected for a further nested PCR to genotype the EBA-175 dimorphism using specific primers described previously[2, 2.5 μl of 10X PCR buffer, 10 mM dNTPs, 10 mM from each primer, 2 μl DNA, and 5 Unit Taq polymerase, 1 μl bovine serum albumin and 14.8 μl of dH2O. The second PCR end products were separated in agarose gel electrophoresis at 1.5% concentration and visualized using a gel documentation system  for the presence of 714 bp or 795 bp bands representing the C and F segments respectively. The presence of both bands indicates a co-infection with at least two different P. falciparum clones.In order to confirm the infection with eviously using 10eviously as folloStatistical analysis was carried out using the SPSS software version 9. F and C Alleles frequencies among populations were calculated, firstly collectively in all populations, secondly in each of the three different study areas separately. In order to determine the possible association between allele distributions and severity of the disease, comparison between genotypes and phenotypes were carried out using the Chi-squared test. The same test was used to detect the significance of the distribution of different alleles among the overall population. The t-test was used in order to compare between the median-values.The total number of participants recruited in this study was 339 of which 57 from Um-Salala, 166 from Sennar and 116 from Wad Madani. Patients were with different malaria phenotype were included: 276 mild, 37 Asymptomatic and 35 severe. The group of severe malaria includes: 20 individuals less than five years of age with severe anemia, hyperpyrexia, convulsion and hyperparasitemia; the other 15 individuals were more than 10 years of age with hyperpyrexia, convulsion, anemia and hypoglycemia. The age of patients from all three study areas ranged from 1 year to 85 years, where 80% of the patients were less than 20 years old. The age average was 11.86 ± 11.02. Sex ratio was 1:1. The haemoglobin levels ranged from 4.4 g/dl– 18.0 g/dl with an average of 10.1 ± 1.8, and glucose levels ranged from 23–205 mg/dl, with an average of 105.6 ± 33.9 mg/dl.P. falciparum positive blood samples from three different geographical areas have been examined for the dimorphic alleles F and C , 95 (57.23%), and 16 (9.64%) respectively. In Wad Madani, out of 116 samples, 43 (37.07%) were genotyped as F, 66 (56.90%) as C, and 7 (6.03%) as mixed F/C segments. The total number of samples from Um-Salala village were 57 samples out of which, 41(71.9%) showed the F allele, 12 (21.1%) were genotyped as C, and 4 (7.0%) carries a mixed alleles, F/C. The difference of the F and C alleles distribution among the different study areas collectively was significant . No significant differences in the distribution of alleles in central Sudan-Sennar and Wad Madani .The distribution of C and F alleles was assessed in four different age groups; less than 5 years, 5–10 years, 11–20 years and more than 20 years. No significant difference of C and F alleles frequencies within the different age groups .Malaria phenotypes of all patients were categorized according to thecWHO criteria into three main groups: asymptomatic, mild, and severe malaria. All malEleven asymptomatic malaria patients from Wad Madani showed positive result for sickle cell test. The prevalence of F, C and mixed F/C segments among sicklers were 36.4%, 63.6%, and 0% respectively.In Um-Salala village the 57 malaira patients were presented by mild malaria. The genotypes distribution was (72%) for the F allele, (21%) for the C allele and (7%) carried both alleles F/C.Using unpaired (independent) t- test, the parasitemia of 132 samples from central Sudan were related to F and C genotypes, the average parasitemia among C and F alleles carriers was 38317.9/μl, and 35485.7/μl respectively with no significant difference .P. falciparum positive blood samples from three different geographical areas showed that both fragment types, C and F, had nearly the same distribution within the population of Wad Madani and Sennar, being 56.9% and 37.1%, respectively in Wad Madani and 57.23% and 33.13%, respectively in Sennar area. The percentage of the co-infection with both alleles C/F was 6% and 9.64% for Wad Madani and Sennar respectively. The parasites population in Wad Madani and Sennar, can be considered as a single gene pool because of their geographical proximity which may lead to an admixture. The two cities are close to each other so there is frequent population movement between these two big cities.Assessment of C and F distribution in 339 The distribution of the F allele in the third region , was markedly higher than that of the C allele. This could be due to location of Um-Salala village in remote area which is inaccessible during rainy season and could be described as semi isolated area. In addition to that this village is inhabited by one ethnic group of the Massalit tribe and in comparison with Sennar and Wad Madani their big cities subjected to population movement and this will lead to populations admixture and parasite diversity. Moreover there is only one single transmission peak symphonized with Anopheles arabiensis after the short rainy season in Um-Salala village. This reWhile the malaria pattern in Um-Salala is characterized by asymptomatic and mild forms, in Wad Madani the asymptomatic cases are relatively few and almost non for Sennar, where most of the cases were mild but some with severe symptoms.P. falciparum were genotyped for F and C fragments, we observed that the C allele was found to be more frequent than F allele, 7: 4 C to F respectively. This finding contradicts Cramer’s study[In order to detect the effect of the dimorphic allele’s distribution on the out come of the disease, we compared the distribution to the phenotypes of the disease, which was categorized into three groups: Asymptomatic, mild, and severe malaria. There were no significant differences in the distribution of F and C among asymptomatic, mild, and severe clinical forms of malaria among whole populations from the three study areas collectively, p = 0.297. Our finding is similar to Heidari and his colleagues as they ’s study and sugg’s study. The Cli’s study,32. Sick’s study. The higet al.[Furthermore, to find out the relationship between this dimorphism and severity of the disease, F and C distribution was correlated to the parasitemia level of subset of infected individuals. There was no significant difference in average of parasitemia in F and C allele’s carriers. This result is concurrence with Heidari et al. who didnThis study showed a significant differential distribution of F and C fragments in different geographical areas endemic with malaria. It could explain the different patterns of malaria in Um-Salala village and cities in central Sudan. More studies on larger populations are needed to understand the distributional pattern of EBA-175 alleles according to the clinical outcomes of the disease.The authors declare that they have no competing interests.HSM conceived the study, substantial contributions to conception and design, field work, interpretation of data and drafting the manuscript. NHA participated in coordination of the study, recruitment of cases and helped in revising the manuscript. AAMA, involved in the collection of the samples, all field laboratory work, DNA extraction, genotyping and acquisition of data. DAH and WHO are involved in the recruitment of cases and sample collection. AAA contributes in all field laboratory work, sample collection and genotyping. BYN contributes in field station setting and sample collection. AZJ contributes in analysis of the data. MEI involved in coordination of the study and revising the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/469/prepub


Increased travel leads to a heightened risk of imported infectious diseases. Patterns of immigration to countries like Canada have changed such that countries of malaria endemicity are frequented in larger numbers. In keeping with the changes in travel patterns and immigration, the major metropolitan city of Calgary has seen a dramatic rise in malaria incidence over the last decade. Fuelling this rise in Calgary has been the apparent complacence with prophylaxis in individuals visiting friends and relatives and potentially inadequate public health intervention in areas of the city with increased immigration and lower socioeconomic status. In the mid 20The geographical distribution pattern of malaria is highly confined, but it is not limited to tropics and sub-tropics. Annually, 80 to 90 million people from malaria non-endemic countries visit developing countries with high malaria transmission rates Calgary is becoming one the fastest growing metropolitan cities in Canada, exhibiting steady growth in immigrant Ethics approval to conduct this study was obtained from the Conjoint Health Research Ethics Board at the University of Calgary (CHREB Ethics ID 24835). Patient consent was waived per CHREB as this was a retrospective study with aggregate data containing no patient identifiers. This study included patients who tested positive for malaria in Calgary between January 2000 and December 2011. Every suspected malaria case within Calgary was exclusively reported to Calgary Laboratory Services (CLS), a centralized regional medical diagnostic laboratory, for clinical testing Laboratory's diagnostic workflow involved rapid diagnostic test  as a screen and microscopy using thick and thin Giemsa-stained peripheral blood smears for confirmation as previously described Plasmodium species were counted as a single case and were classified as a mixed infection, 3) in case of discordant results between the types of Plasmodium species that patients were infected with, real-time PCR assay results were always prioritized over results that were identified by other detection methods (i.e. microscopy and rapid diagnostic test), 4) discordant results that could not be distinguished by the real-time PCR  were counted as a single case, and were classified as Plasmodium spp.When reporting a case, the following considerations were made: 1) Relapsing infections that were less than 2 months apart were counted as a single case, 2) infections associated with more than one type of A standardized clinical history form was requested for all patients with samples submitted for malaria testing from 2007–2011. Demographic variables reviewed in this study were: gender, age and location of residence. Physicians were required to complete CLS malaria history forms upon requesting an order for specimen malaria testing. For this study, Malaria case history forms (n = 4832) were manually entered into a Microsoft access database. containing information on patient's travel and clinical history, which was further divided into different criteria including the following: pre-travel advice (yes or no), reason for travel , countries with malaria visited, types of prophylaxis taken, and treatment . Each section showed different completion rate, 100%, 81.7%, 93.7%, 100% (82.09% of the people who answered yes listed the name of the treatment), respectively. N/A refers to the sections with insufficient information.When multiple history forms were completed by a single patient regarding their travel and clinical history, the original form was used and complemented with additional information made available from subsequent forms. Patients' postal codes were collected in order to determine the incidence rate of malaria within different city wards for year 2011. Calgary is divided into 14 different wards, and the incidence rate for the each ward is reported as the number of cases per 100,000 people.The database was created using Microsoft Access in order to manage and update the information obtained from the patients' malaria history forms. Data held in Microsoft Access was then exported to Microsoft Excel for the facilitated data analysis. The ANOVA test was used for comparing the means of more than two groups. A p-value≤0.05 was used as the cut-off value for statistical significance between the means. The malaria incidence rate was determined based on the census data provided by the City of the Calgary and Alberta Health Services (AHS) P. falciparum infections (p = 0.05375). P. falciparum and P. vivax infections represented greater than 85% of the reported cases. Of the 295 cases, there were 171(58.0%) P. falciparum infections and 85 (28.8%) P. vivax infections. The remainder of the cases consisted of P. ovale , P. malariae , Plasmodia spp.  where speciation was not possible, and mixed infection . Patients classified as having a mixed infection were simultaneously infected with P. falciparum and P. ovale.Between 2000 and 2011, there were 295 laboratory confirmed malaria cases . On averP.vivax infections were predominant in males (p = 0.01), but for the other etiologic agents, the gender difference in the disease prevalence was insignificant. For all Plasmodium species, the mean age of onset of malaria was 32.03±1.20 years, with a wide age range of 0.8–87 years. One hundred and twenty four (73.8%) cases were observed in individuals between the age of 20 and 59, with the highest proportion in the 25–29 (15.5%) age group. Thirty seven cases (22.0%) were observed in infants and children <1–19, and 54.1% of the patients in this age group were infected with P. falciparum. Seven cases (4.2%) were observed in elderly individuals between the ages of 60–70+, and 71.4% of the patients within this age group were infected with P. vivax.The occurrence of malaria was significantly more frequent in males than in females  . P.vivaxA minority of patients (23.8%) had received advice from a clinic/physician prior to their travel, but the percentage was slightly lower in infants and children (20.0%). Sixty percent of the individuals who had received pre-travel advice took prophylaxis, but the percentage of prophylaxis usage was reduced to 13.5% among the individuals who had not received pre-travel advice. The most common reason for travel was VFR (44%), followed by immigration to Canada (22.2%), tourism (11.1%), business (7.90%), and visiting Canada (1.60%) . The meaP. falciparum, 75.0% of P. ovale, 100.0% of P. malariae, and 100.0% of mixed infections were contracted in Africa. By contrast, the highest proportions of P. vivax (55.6%) and Plasmodia spp. (50.0%) infections were acquired in Asia. P. vivax was the only species contracted in the Americas . Overall, the highest numbers of cases originated from Sudan (n = 20), followed by Nigeria (n = 19), Uganda (n = 15), India (n = 14), Ghana (n = 13), and Cameroon (n = 9).All the reported malaria cases in Calgary were acquired outside Canada . During Patients most commonly took doxycycline (n = 7) prophylaxis, followed by chloroquine (n = 5) . Other pAt the time of the specimen submission, 53.2% of patients were on malaria treatment. Different types of treatments regimens prescribed included the following drugs sometimes used in combination for severe cases: Doxycycline (n = 19), Quinine (n = 24), Atovaquone-Proguanil (n = 23), Chloroquine (n = 9), Artesunate (n = 4), Primaquine (n = 3), Clindamycin (n = 2), Mefloquine (n = 2). Fourteen patients indicated that they have received the treatment but did not list the name of the treatment, 2 of these patients reasoned that this was because they were not able to recall the name of the treatment received. Twelve patients indicated they have received the treatment but failed to indicate the name of the treatmentP. falciparum malaria (parasitaemia level of >2%) as outlined in the WHO Guidelines for the Treatment of Malaria, there were 19 severe P. falciparum patients Based on one definition of severe The malaria incidence rates between the 14 wards defined by the City of the Calgary varied greatly . The higIn order to determine the relationship between malaria incidence rates and the socioeconomic status, social districts which essentially overlap with wards  were evaHuman mobility is essential to the survival and dissemination of the malaria parasite P. falciparum cases which were fortunately not associated with mortality.Taken together, the data point to a troubling correlation where individuals in areas of the city with increased immigration, lower levels of education, and lower average income do not access the health care system for prophylaxis recommendations and thus acquire more malaria. For example, the North East sector of our city has the highest total number of immigrants based on census data and also the lowest average income. In contrast, the Northwest of our city has a significant number of immigrants but higher levels of income and we assume education levels. Census data conducted by health authorities in this province of Canada, also point to increased immigration from South Asia and Africa in the north east of city where the highest cases of malaria occur. This observation has been made before but not quantified in terms of incident rates and correlated to socioeconomic data in Canada P. falciparum malaria is attributed to travel to West Africa and a higher geometric mean parasitemia, whereas P. vivax is linked to travel to South Asia with lower parasitemia. Surprisingly, the 25–29 age group were the single largest contributing age group for positive malaria cases, as opposed to infant or the elderly, and may be associated with patterns of non-adherence to prophylaxis regimens. Limitations of this study include the fact we do not have denominator data for rates of travel to regions to provide risk information. We also cannot provide data on rates of travel by districts in the city but assume higher rates of immigration correlate with increased travel given the fact that imported malaria is linked to VFR in our population. Given the localized nature of increased imported malaria incidence, a case can be made for more concerted public health intervention in these wards of the city through social media, physician offices, and cultural centers targeting both West African and South Asian immigrants.While the reasons for traveling without prophylaxis are likely multifactorial, we speculate that cultural attitudes toward malaria play a significant role. Gender differences may also play a part as significantly more men acquire malaria then women, again perhaps due to adherence rates with prophylaxis as shown by others


Malaria in pregnancy remains a major health problem. Placental malaria infection may cause pathophysiological changes in pregnancy and result in morphological changes to placental villi. Quantitative histomorphological image analysis of placental biopsies was performed to compare placental villous architecture between active or treated placental malaria cases and controls.Plasmodium falciparum and Plasmodium vivax infections. Image analysis of histological placental sections was performed using ImageJ software to measure the number and size (area) of terminal villi, perimeter measurement per villus and total perimeter per unit area, and number of capillaries per villus (vascularity). Histological features of placental malaria were scored and these results were correlated with malaria status and clinical outcomes.A total of 67 placentas were studied from three clinical groups: control patients who did not have malaria (n = 27), active (n = 14) and treated (n=26) malaria cases, including both P. falciparum or P. vivax.Villous size correlated with vascularity (p <0.0001) but was inversely correlated with observed villi per unit area, (p = 0.0001). Significantly greater villous area and vascularity was observed in UK controls. Indices of histological malaria infection were significantly greater in active versus treated malaria cases. Active placental malaria cases showed significantly smaller villous area (p <0.0084), vascularity (p <0.0139) and perimeter (p <0.0006) than treated malaria cases or controls, but significantly more villi per unit area (p <0.0001). Villous size in treated malaria cases was significantly larger than active placental malaria cases (p <0.001) and similar to controls. There was a significant relationship between villous number and anaemia at the time of infection (p <0.0034), but not placental weight, birth weight or gestational age at delivery. No differences were found between histology or villous morphology comparing infections with These results imply that villous size, perimeter and vascularity are acutely decreased during active placental malaria, decreasing the surface area available for gas exchange per villus. However the increased number of villi per unit area offsets this change and persists after treatment. Histopathological and villous architectural changes may be reversed by early detection and appropriate anti-malarial treatment. Malaria infection during pregnancy is a major public health problem in tropical and subtropical regions throughout the world, with substantial risks for the mother, her foetus and subsequently the neonate . MalariaPlasmodium falciparum and Plasmodium vivax, or were ameliorated by effective anti-malarial treatment before delivery.Placental malaria infection causes pathological changes such as inflammation, intervillous fibrin deposition and infarction, that can persist until delivery and affect the function of the materno-placental unit, contributing to adverse birth outcomes and increased perinatal mortality and morbidity . HoweverEthical permission for collection of placental biopsies in the specific studies was granted with each separate study -21. Use In this study a total of 67 placental biopsies were examined, from cases that were divided into three main clinical groups – non-malaria controls (n = 27), active placental malaria cases (n = 14) and treated malaria cases (n = 26).P. falciparum infection, n = 5 with mono-infection by P. vivax and n = 9 with consecutive or mixed P. falciparum/P.vivax infections. Of this latter group, n = 7 had multiple consecutive infections, including at least one episode each with either P. falciparum and P. vivax, and two of whom had proven mixed infections with both organisms simultaneously. Details of the malaria status of the cases are shown in Figure The active or treated malaria-infected patients (n = 40) included n = 26 who had only P. falciparum and P. vivax[Placental biopsies were collected between 2004–2011, from the Department of Tropical Pathology, Faculty of Tropical Medicine and three previously published studies investigating malaria in pregnancy conducted at Shoklo Malaria Research Unit (SMRU) field research station at Mae Sot, Thailand. These were studies of the histopathology of placental malaria, used to identify cases with acute infection at delivery with both  P. vivax, an obse P. vivax,19 and t P. vivax. Women iThe cases included:(i) Control normal placentas from UK women delivering at The John Radcliffe Hospital, Oxford, UK (n = 9), with no fever complicating pregnancy or delivery.(ii) Control placentas from Karen women delivering at SMRU, who had no evidence of malaria infection by active screening by microscopy throughout pregnancy and negative histology at delivery .(iii) Active placental malaria cases (n = 14), where malaria was diagnosed by microscopy and subsequent blood films in the mother, and placenta biopsies confirmed acute placental malaria infection at the time of delivery (from histopathology and UPS studies).(iv) Cases with known single or multiple malaria infections treated during pregnancy at various time points prior to delivery .(v) Single-treated malaria infection in early, middle or late pregnancy (from PCA) but not at delivery (n = 17), selected randomly from patients where mono-infection was proven and subsequent screening microscopy until delivery was negative . ‘Early’Clinical outcome data on the patients included the gestational age at delivery (EGA), the placental weight at delivery (in grams), the birth weight of the baby (in grams), and the presence of maternal anaemia at the time of infection. This was measured using standard haematocrit (HCT) measurement, and stratified according to the criteria used at SMRU for defining maternal anaemia 1: HCT > 30% - normal, 2: HCT 25-30% - mild anaemia and HCT < 25% - severe anaemia. EGA was measured for all Mae Sot patients by dating ultrasound prior to 24 weeks, Dubowitz estimation of gestational age and the last menstrual period. Because malaria in this study group was diagnosed actively and prospectively, and treated when found regardless of symptoms, the possibility of asymptomatic placental infection at delivery was minimal. Each case was defined at delivery as being malaria negative on the basis of smear microscopy on cord blood, maternal blood and placental smears. Summary details of the clinical groups are shown in Table Placental biopsy samples were taken by trained personnel according to established protocols, taking a full thickness biopsy including maternal and fetal surfaces, half way between the placental edge and the cord insertion site. Specimens were fixed in 10% neutral buffered formalin  or Streck’s fixative (PCA study), then subsequently embedded in wax and processed using standard histological techniques to produce paraffin blocks. All processing and histological staining was performed in batches at the same pathology laboratory. Four μm histological sections were cut on a rotary microtome, and stained with both haematoxylin and eosin and Giemsa for histological examination.The morphological study examined terminal, as opposed to stem or intermediate villi, because previous studies on the effects of disease on villous morphometry, and a pilot study in these cases, indicated that the predominant changes caused by disease are seen in these villi. Terminal villi were identified using a series of criteria including thinned trophoblastic lining, lack of muscularized arterioles, and more than 50% of the cross sectional area being occupied by vessels .A total of 67 placenta specimens were used for image analysis by assessing four main parameters: the cross sectional surface area (size) of terminal villi; the number of capillaries per terminal villus; the number of terminal villi per high powered field (hpf); and the perimeter measurement of terminal villi, both the mean perimeter of individual villi and the total perimeter measurement in ten hpf.2), number of capillaries per terminal villous and perimeter of terminal villi (μm).Tissue sections were examined and digital images were captured from ten randomly selected ×400 hpf per slide using an Olympus DP-20 colour video camera interfaced with an Olympus BX41 microscope. The images were scanned at a resolution of 1,600 × 1,200 pixels using Olympus DP-20 BSW software (Version 2.2) and stored. Only the intermediate zone of the placenta was used and areas where villi could not be distinguished, with placental infarction, intervillous fibrin deposition, or arterial vessels forming the primary stem and intermediate villi were avoided, as these can distort the observed villous size ,13. Only2 from the area of red pixels. The mean area of terminal villi for a case was calculated by the ratio between the total area and number of terminal villi in the field across all ten hpf.The images were first subjected to a preprocessing step Figure  which inThe villous vascularity was measured using the cell counter tool in ImageJ to count the number of open capillary spaces, lined by endothelium, in each villous (regardless of whether this contained erythrocytes or not). The number of capillaries per villous was calculated by the total number of villous vessels divided by the number of terminal villi across ten separate hpf.The number of terminal villi in ten hpf was counted by eye with light microscopy in each case. The mean number of terminal villi per hpf in each patient was calculated by the ratio between the total number in ten hpf/ten. Results for individual cases were then combined for comparison between clinical groups.The perimeter of terminal villi (in μm) was measured in two ways. The mean perimeter measurement of individual villous cross sections  was calculated automatically in a similar manner to the villous area by ImageJ, directly from the outlined edge of terminal villi Figure D. The toet al. [The scoring system of Muehlenbachs et al.  was usedThe specimens were prepared and analysed blind to the clinical group. Analyses were performed using the statistical program SPSS version 18  for Windows and STATA v12.0 . Data were described using means (standard deviation-SD) and frequencies (%). Due to the relatively small sample size and unequal variances across groups, non-parametric tests were used for comparisons. Correlations between villous number, size and vascularity and between clinical groups, across all individuals and between clinical outcome measures, were assessed using Kendall’s rank correlation coefficient (tau). The relationship between histoscore parameters and measures of villous architecture between clinical groups, subgroups of the PCA cases, and with clinical outcomes such as anaemia, was performed using the Fisher’s exact test and the Kruskall-Wallis test. Differences were considered statistically significant for p-values less than 0.05. However, as this was an exploratory study, numerous comparisons were made and exact p-values  are reported so that the reader may apply Bonferroni corrections, if preferred .P. vivax as well as P. falciparum infections, and also mixed infection with both organisms. In addition the patients included in this study were frequently tested for malaria and treated when this was detected, so active placental malaria at delivery is rare and the duration of undiagnosed malaria infections during gestation is minimal [Maternal malaria in southeast Asia differs from the African setting in that the disease is not endemic, there is a substantial proportion of  minimal . In malaThere was a significant inverse correlation between villous area and villous numbers observed per hpf  either examining all cases or individual clinical groups  but significantly lower villous vascularity (p <0.0002). In addition, the UK control group had a significantly greater villous area (p <0.0004) and villous vascularity (p <0.0087) than Thai/Karen control cases indicating a difference in observed villous area on a geographical basis  with all treated malaria cases  and active placental malaria cases .There was a significant decrease in villous area in active malaria-infected cases compared to both controls and treated malaria cases (p = 0.0084) Figure . The numThe number of villi seen per hpf was significantly increased in both in active and treated malaria cases compared to non-malaria controls (p <0.0001) Figure . The perVillous size and vascularity was reduced in acute placental malaria cases. The number of villi observed per hpf increased significantly in malaria cases even after treatment, and this effect lasted until delivery. However other changes such as the acute decrease in villous size and changes in vascularity, and the histological features of placental malaria, resolved given early detection and appropriate treatment. In addition, although these smaller villi in malaria cases showed a lower perimeter measurement, the total perimeter available for gas exchange per unit area was increased, possibly a compensatory mechanism.et al. [These results are different to those reported by Crocker and colleagues , who shoet al. . In the et al. [The pathological changes associated with placental malaria were scored according to the system of Muehlenbachs et al. . The preThere were no significant differences between the demographic details of the different clinical groups Table  other thMalaria is the single greatest risk factor for anaemia in pregnant Karen patients and reversal is slow even when malaria and anaemia are treated ,25. BecaP. falciparum (n = 26) or P. vivax (n = 5), and where patients had both species either on separate occasions during gestation or mixed infection with both (n = 9). No significant differences were seen between any of the placental architectural parameters and either mono-infection with either P. falciparum or P. vivax, or in pregnancies where both species had been found, either as a mixed infection or on separate occasions ; ‘middle’  and ‘late’ infection . Comparison showed no significant differences between timing of infection before delivery and any of the parameters of the histoscore or villous architecture  because these processes affect the maternal blood space. Changes in villous vascularity and size in other diseases examined using morphometry developed over a longer period due to poor oxygenation and hypertension, leading to defective stem villous development and hence vascularization of terminal villi ,29,30. AVillous size was interdependent with villous vascularity. If there was a generalized decrease in villous vessel numbers in malaria infection this may cause an acute shrinkage of villous area, which seems more likely than a sudden loss of established villous blood vessels. Placental malaria infection causes acute changes to both maternal and foetal haemodynamics, which reverses rapidly, within three days, after effective treatment ,33. FoetThe pathophysiological effects of malaria infection in the placenta include inflammatory cell localization, cytokine release and direct effects on villous function by parasitized erythrocyte binding -38. CytoP. falciparum, P. vivax, or consecutive infections by both during the course of pregnancy. These results imply that villous architecture changes acutely during malaria infection, causing shrinkage of the terminal villous and compensatory increase in the number of villi which persists following treatment and eradication of the infection. It cannot be excluded that the reversal of placental morphological changes may be related to improvement in maternal anaemia, caused by malarial treatment. However other architectural and pathological changes induced by malaria infection are reversible given early detection and appropriate anti-malarial treatment.Digital image analysis was used to examine pathological changes to villous architecture in patients with active and treated malaria and controls. Villous area was significantly correlated with vascularity and inversely correlated with density of villi per unit area. UK controls showed significantly greater villous area and vascularity than Karen groups. There was a significant relationship between villous number and anaemia at the time of infection, but not placental weight and gestational age at delivery. Active malaria-infected cases showed significantly smaller villous area, vascularity, density, and perimeter measurement than controls. However increased villous numbers in malaria cases caused a compensatory increase in total available villous perimeter. Histology score and villous architecture showed no significant differences between infections with either The authors declare that they have no competing interests.SC, AM, EP, and GDT performed the experiments, analysed data and wrote the paper. UC, EP, PV, RM, MJR, and FN collected samples, helped analyse data and wrote the paper. SC and SJL performed statistical analysis. GDT, UC, EP, RM, MJR, and FN conceived of the study and designed experiments. All authors read and approved the final manuscript.


The impact of insecticide treated nets (ITNs) on reducing malaria incidence is shown mainly through data collection from health facilities. Routine evaluation of long-term epidemiological and entomological dynamics is currently unavailable. In Kenya, new policies supporting the provision of free ITNs were implemented nationwide in June 2006. To evaluate the impacts of ITNs on malaria transmission, we conducted monthly surveys in three sentinel sites with different transmission intensities in western Kenya from 2002 to 2010.Anopheles funestus and An. gambiae, increased about 5–10 folds in all study sites after 2007. However, the resurgence of vectors was highly variable between sites and species. By 2010, asymptomatic parasite prevalence in Kombewa had resurged to levels recorded in 2004/2005, but the resurgence was smaller in magnitude in the other sites. Household ITN ownership was at 50–70% in 2009, but the functional and effective bed net coverage in the population was estimated at 40.3%, 49.4% and 28.2% in 2010 in Iguhu, Kombewa, and Marani, respectively.Longitudinal samplings of malaria parasite prevalence in asymptomatic school children and vector abundance in randomly selected houses were undertaken monthly from February 2002. ITN ownership and usage surveys were conducted annually from 2004 to 2010. Asymptomatic malaria parasite prevalence and vector abundances gradually decreased in all three sites from 2002 to 2006, and parasite prevalence reached its lowest level from late 2006 to early 2007. The abundance of the major malaria vectors, The resurgence in parasite prevalence and malaria vectors has been observed in two out of three sentinel sites in western Kenya despite a high ownership of ITNs. The likely factors contributing to malaria resurgence include reduced efficacy of ITNs, insecticide resistance in mosquitoes and lack of proper use of ITNs. These factors should be targeted to avoid further resurgence of malaria transmission. Malaria is the leading cause of child mortality and morbidity in Africa. There is growing evidence documenting a substantial decline in clinical malaria morbidity and mortality in a number of African countries, including Kenya. This can be attributed to malaria control measures, predominantly to the use of insecticide treated nets (ITNs), indoor residual spray (IRS) and artemisinin-based combination therapies (ACT), which have been implemented on a wide scale ITN coverage and reported malaria incidence are not spatially congruent, and remarkable success has been reported in reducing malaria prevalence and mortality in different settings in Kenya ITNs have been used in rural Kenya since the early 1980s, but the coverage has been low. In 2002, the Ministry of Health (MoH) of Kenya developed an intensive ITN delivery programme targeting pregnant women and children under the age of 5 years. The goal was to achieve a rapid scale-up in net coverage and to reach a target of 60% coverage of populations at risk by 2005 through the commercial retail sector. However, due to persistent low net coverage Here, we report on a nine-year longitudinal survey of both monthly malaria parasite prevalence in school children and vector abundances in one endemic mid-altitude site and two epidemic highland sites in western Kenya, to assess the impact of ITN/LLIN coverage on malaria parasite transmission and the malaria vector population. The limitations of using ITNs as a primary prevention measure to combat malaria are also discussed.Plasmodium falciparum is the primary malaria parasite species Anopheles gambiae s.s., An. arabiensis and An. funestusThe study was conducted in three sentinel sites in western Kenya: two highland sites, one in Iguhu  (mesoendemic) in the Kakamega district and one in Marani  (hypoendemic) in the Kisii district, and one mid-altitude site in Kombewa  (holoendemic) in the Kisumu district. The catchment populations range from 19,000 in Marani, to 23,000 in Kombewa and 24,000 in Iguhu. The topography of the two highland sites is characterized by valleys and depressions surrounded by densely populated hills. The mid-altitude site has a rolling terrain bisected by small streams. The climate in western Kenya generally consists of a bimodal pattern of rainfall, with the long rainy season from April to June, which triggers the peak malaria transmission period, and the short rainy season from October through November. The dry season in Kenya, when the overall rainfall is significantly less than in the wet seasons, occurs from July to September and it is also the coolest season. January and February are the hottest months. Monthly parasitological surveys were initiated in June 2002, and in February and June 2003 in Iguhu, Marani, and Kombewa, respectively. The parasite surveys were conducted using randomly selected school children aged 6–13 years in all study sites. At least 100 volunteer school children from 5–6 primary schools were sampled at each site and each month for parasite prevalence determination since 2002. During high-transmission seasons (May and June each year), we increased the sample size to up to 300 volunteers to increase the statistical power for parasite prevalence determination. The sample size was calculated based on the size of study population and parasite prevalence from the previous study An. funestus or An. gambiae s.l. Longitudinal mosquito adult surveys were started in February 2002, and in February and June 2003 in Iguhu, Marani, and Kombewa, respectively. In each site, 30–40 houses were randomly selected for alternate monthly adult mosquito sampling to eliminate any post spray effect. Indoor resting female mosquitoes were collected using the pyrethrum spray catch (PSC) method An. gambiae complex were further analyzed by three PCR methods. Mosquitoes collected from 2002–2005 were analyzed by the PCR method described in a previous study Adults of the Cross-sectional ITN coverage surveys on randomly selected households were conducted from late 2003 to early 2004, and from January 2006 through October 2010 in the study sites. Information obtained from a household questionnaire survey included number of bed nets owned, number and ages of persons sleeping under bed net, age and condition of bed net (torn or not), types of bed net (regular ITN or LLIN), and retreatment of regular ITNs . The number of LLINs owned was recorded in surveys from 2008 onwards.Ownership of ITNs did not necessarily directly translate to regular usage by either children or adults. An additional questionnaire on actual usage of bed nets was carried out during surveys in early 2009 and 2010 focusing mainly on who (sex and age) and how often  the residents of the household used the ITNs.Bed nets of different ages were randomly collected in 2009 and 2010 from households that owned bed nets. We replaced these nets with the new ITNs being distributed by the Kenya MoH through government hospitals. Thirty nets were tested at each site.An. gambiae s.s. (susceptible Kisumu strain). The Kisumu strain of An. gambiae s.s. is a standard susceptible reference strain originally colonized in western Kenya used in previous studies The insecticidal efficacy of nets was evaluated by a slightly modified WHO Mean monthly weather records from 2002 to 2010 were obtained from the meteorological stations in Kakamega (for Iguhu), Kisii (for Marani) and Kisumu (for Kombewa). These meteorological stations were within 20 km from the study sites. The parameters used in this study include monthly average of daily maximum, mean, and minimum temperature, and monthly cumulative rainfall.Scientific and ethical clearance was given by the Kenya Medical Research Institute and University of California at Irvine institutional review boards. Volunteers were enrolled from the primary schools in the study sites through the primary school administrators with the permission of the division office of the Ministry of Health. Written assent for children (<18 years of age) were obtained by the participants and their parents or guardians. Inclusion criteria included: provision of informed consent, age >6 months at recruit, and no reported chronic or acute illness except malaria. Exclusion criteria include: those who were unwilling to participate in the study, and those who planned to move out of the study area. According to the standard malaria treatment guidelines of the Ministry of Health of Kenya, asymptomatic infections were not treated with antimalarials, but symptomatic volunteers were referred to the local government hospitals or clinics for diagnosis and treatment free of charge.Plasmodium parasite prevalence was expressed as the ratio of positive samples over the total number of samples tested. The abundances of anopheline mosquitoes in each site were computed monthly as the number of females per house per night (f/h/n). Bed net ownership rate was calculated as the ratio of the number of households with at least one bed net over the total number of households surveyed. Population coverage with bed nets was computed as the ratio of the total number of individuals reporting sleeping under a bed net, regardless of the condition and type of the net, over the total number of individuals surveyed. Effective population coverage by bed net is defined as the proportion of the population who slept under LLINs in good conditions, or ITNs in good conditions and routinely being re-treated by insecticides. Ineffective population coverage by bed net is defined as the proportion of the population who slept under torn nets, or slept under regular ITNs but those that were not routinely retreated by insecticides 2-test. Bed net ownership was compared between different survey periods using the χ2-test.y is parasite prevalence, t is time, A, B, C and t0 are parameters. The lowest prevalence month, t0, was in July 2007.To find the trends in the parasite prevalence or vector density dynamics, we performed time-series analyses in the following three steps. First, due to seasonal fluctuations in prevalence and vector density, we used the 12-month moving average to minimize the effect of seasonality. Second, we examined the existence of time-varying trend using the randomness and stationarity tests of differenced series. The randomness of a series was tested using autocorrelation function (ACF) and partial correlation function (PCF), and stationarity was tested by examining whether the mean and variance were constant in time t is time, A, B, C and D are parameters. All parameters, except t0, were estimated using the least square method of nonlinear estimation.For the 12-month smoothed parasite prevalence data in Iguhu and Marani and the 12-month smoothed vector density series in all three study sites, the following model was used:For association between climatic factors and vector density and parasite prevalence, time-lagged cross-correlations were computed based on monthly observations, and a maximum of 3-month time-lag has been used. Software package STATISTICA 8.0  was used to perform all analyses.Plasmodium falciparum was the predominant parasite species, constituting 97.3% to 98.3% of total positive blood samples, and P. malariae comprised 2.3–5.1% and P. ovale less than 1%. There were a few cases of mixed infections of two parasite species in all study sites but the rates of mixed infections were generally low. Due to the low prevalence of other parasite species, only P. falciparum prevalence dynamics was analyzed in the following sections.Over the nine year period, we read a total of 33,640 blood smears, and malaria parasite prevalence rates were 47.1% , 28.4% , and 6.2%  in Kombewa, Iguhu, and Marani, respectively. An. gambiae s.l. and An. funestus, with An. gambiae s.l. accounting for 37.4%, 86.9%, and 67.8% in Kombewa, Iguhu, and Marani, respectively. Thus, An. funestus was the predominant malaria vector in Kombewa, the mid-altitude area, but An. gambiae s.l. dominated in the highlands.Malaria vectors in the three sites comprised Similar trends were observed in all three sites, i.e., declining parasite prevalence from 2002/2003 to 2006/2007, and a rebounding trend in prevalence from 2007 to present, with current parasite prevalence comparable to that five years ago .In Kombewa, parasite prevalence decreased slightly from 2003 to June 2006 , then declined sharply  during the last half of 2006 . Thereafter it gradually increased throughout 2007 (average 30.0% in 2007) and well into 2008, reaching a monthly rate of 49.6% in 2008, about the same prevalence observed before 2006 and significantly higher than 2007  . ParasitP. falciparum prevalence was observed from 2002 with a notable decline in 2005; however, a sharp declining trend occurred after June 2006  and 1.3 f/h/n to 0.24 f/h/n and 0.23 f/h/n after July 2006 in Iguhu and Kombewa, respectively . Meanwhile, An. funestus densities declined from 0.3 f/h/n and 4.4 f/h/n to 0.03 and 0.4 f/h/n in Iguhu and Kombewa, respectively during the same period . Similar to malaria parasite prevalence, malaria vector densities have also resurged since 2007. Compared with 2007, indoor resting abundances of An. gambiae s.l. and An. funestus increased by 8–10 folds in Iguhu in 2009 and 5–7 folds in Kombewa. The mean monthly density of An. gambiae s.l. from January to March 2010 was 1.8 f/h/n in Kombewa, which was the highest since 2003 and was significantly higher than the second highest density of 0.7 f/h/n in 2004. In Marani, the indoor resting vector densities were all below 0.5 f/h/n except in June 2003 when it was 1.0 f/h/n , but decreased significantly to 11.5% in 2010 . In contrast, species composition change in Iguhu was characterized by a significant increase in the proportion of An. arabiensis from <1% in 2003 to 18.8% in 2006 (Fisher exact test P<0.001), then by a gradual declining trend from 2006 to 2010   . Due to R2 = 0.96  fits all vector density data well with a prior setup of D = 0 (EQ 2)  . The res2 = 98.9, df  = 1, P<0.0001), but it has remained almost unchanged since then . In Kombewa, bed net ownership was relatively high (35.9%) in 2004 and increased to 80.7% in 2010 . Population bed net coverage, i.e., the population observed using the bed nets, had reached about 50.3% and 52.3% in Iguhu and Marani, respectively, and 77.9% in Kombewa in 2010. Even though bed net ownership and population bed net coverage were high, a substantial proportion of bed nets was torn or not-retreated ITNs. For example, we found that 20.1% (82/407), 36.6% (106/290), and 46.0% (88/191) of the bed nets being used during 2009 survey in Iguhu, Kombewa and Marani, respectively, were torn (with holes) or they were ITNs in good condition but were not retreated with insecticide. Furthermore, the 2010 survey found that 4.7% and 15.5% of the bed nets in Iguhu and Kombewa, respectively, were not used at all. Therefore, the functional effective bed net coverage in the population was estimated 40.3%, 49.4% and 28.2% in 2010 in Iguhu, Kombewa, and Marani, respectively  and Kombewa . Due to the small number of conventional ITNs tested, the comparison between efficacies of different ages of nets was not performed.The result of contact bioassays of insecticidal test, indicated by mortality rate after 24 hours post exposure in the standard WHO paper cup test, showed that the overall efficacy LLINs varied among the study sites with 92.6% (ranged from 51–100%) and 81.1% (ranged from 24–100%) killing efficacy in Iguhu and Kombewa, respectively. Although older LLINs showed slightly lower efficacy than newer LLINs, ANOVA tests revealed no significant differences in efficacy across the age (in year) of LLINs in both Iguhu . In Kombewa, approximately one quarter of the sampled bed nets caused 100% mosquito mortality and 75% of the tested LLINs showed a mortality rate above 80%, whereas 20% of the tested LLINs exhibited <50% killing efficacy, the minimum efficacy threshold set by WHOPES.Although there were variations in monthly cumulative precipitation and monthly mean maximum and minimum temperatures over the 9 years, the changing patterns in climatic parameters were not associated with changes in malaria parasite prevalence and vector abundances , becauseEncouraged by the progress made in the last decade P. falciparum prevalence was associated with a decline in vector abundance after nationwide implementation of free ITN/LLINs and increased use of ACT treatment for uncomplicated malaria.Malaria prevalence has exhibited a large change over the nine year study period in the three sentinel sites in western Kenya, as indicated by the initial decline of vector abundances and malaria prevalence resurgence since 2007. Without the long-term longitudinal monitoring, it is impossible to detect these changes, especially the rebounding of parasite prevalence and vector density. For example, point data did not show a significant difference in parasite prevalence in Kombewa between 2004 (53.9%) and 2009 (51.7%), leaving one to wonder if the bed net intervention policy in 2006 had any impact on malaria transmission at all. The reasons for the changing pattern of malaria epidemiology are most likely multi-factorial. The decreasing trend in An. gambiae s.s. relative to An. arabiensis in Kombewa and Iguhu in 2010, which was distinct to the sites in the same area in early years in western Kenya Although large numbers of insecticide treated bed nets is being distributed in malaria endemic sub-Saharan Africa, population bed net coverage is still low and did not meet the targeted goal of 80% coverage at our study sites Anopheles mosquitoes might compromise the long-term effectiveness of ITNs and LLINs in killing mosquitoes. The pyrethroid resistant alleles, kdr alleles, have not been detected in either An. gambiae s.s. or An. arabiensis populations in the study areas in 2005 An. gambiae s.s. in Kombewa and Iguhu are resistant to both permethrin and deltamethrin . A change in the vector biting behavior was observed in the study areas, with mosquitoes tending to bite earlier, before bed time  The gradual resurgence of parasite prevalence since 2007 may be explained by several factors. First, good conditions and proper use of bed nets are crucial for preventing parasite infections; the high percentage of torn bed nets and the erratic use of nets may have contributed to the resurgence observed, similar to previous findings P. falciparum, increase among older children as transmission intensity declines because ITN coverage was significantly lower in school-aged children compare to both pre-school children and adults Pf PR  among children aged 2–10 years. Our study provides information on parasite prevalence in slightly older children. Furthermore, the technical capacity to conduct school malaria surveys already exists in most sub-Saharan Africa countries Malaria control in Africa has focused on young children (<5 years age group) and pregnant women over the last decade, but as transmission intensity declines, school aged children will become an important clinical risk group. Brooker et al Current integrated malaria management, including ITNs, IRS, ACT and RDTs, has and will continue to have a substantial impact on malaria morbidity and mortality in Kenya. There was a clear association between the steep decrease in vector abundance and the mass distribution of ITNs. However, the impact was short lived, and it seems that there are major gaps in ITN ownership and actual use. World Health Organization has issued the guidelines on proper use and sustaining high coverage and replacement of old LLINs (3–5 y) in 2005


Copeptin has recently been identified to be a stable surrogate marker for the unstable hormone arginine vasopressin (AVP). Copeptin has been shown to correlate with disease severity in leptospirosis and bacterial sepsis. Hyponatraemia is common in severe imported malaria and dysregulation of AVP release has been hypothesized as an underlying pathophysiological mechanism. The aim of the present study was to evaluate the performance of copeptin as a predictor of disease severity in imported malaria.Copeptin was measured in stored serum samples of 204 patients with imported malaria that were admitted to our Institute for Tropical Diseases in Rotterdam in the period 1999-2010. The occurrence of WHO defined severe malaria was the primary end-point. The diagnostic performance of copeptin was compared to that of previously evaluated biomarkers C-reactive protein, procalcitonin, lactate and sodium.Plasmodium falciparum, 63 non-falciparum infection), 25 had severe malaria. The Area Under the ROC curve of copeptin for severe disease  was comparable to that of lactate, sodium and procalcitonin. C-reactive protein (0.84 [95% CI 0.79-0.89]) had a significantly better performance as a biomarker for severe malaria than the other biomarkers.Of the 204 patients (141 C-reactive protein but not copeptin was found to be an accurate predictor for disease severity in imported malaria. The applicability of copeptin as a marker for severe malaria in clinical practice is limited to exclusion of severe malaria. Arginine vasopressin (AVP), also known as the antidiuretic hormone, is a key hormone in maintaining fluid balance and vascular tone. Despite its clinical relevance, measurement of mature AVP is difficult and subject to (pre)analytical errors. Recently, copeptin, a 39-amino acid glycopeptide that comprises the C-terminal part of the AVP precursor was found to be a stable and sensitive surrogate marker for AVP release ,2. In reThe present study, therefore, aimed to evaluate copeptin as a pathophysiologic predictor of disease severity in patients with imported malaria. The diagnostic performance of copeptin was compared to that of several previously validated biomarkers to provide the clinician with the most accurate tool for clinical decision making in the acute care setting. A reliable test, easily discriminating severe from non-severe disease in malaria, would be clinically useful, especially in the setting of imported malaria, given the observation that most clinicians in non-endemic areas have little expertise with identifying severe malaria but these patients require a rapid triage for parenteral anti-malarials and intensive monitoring.P. falciparum malaria. In the present study, the previously validated biomarkers plasma lactate [The Harbour Hospital is a 161-bed general hospital located in Rotterdam, The Netherlands. It also harbours the Institute for Tropical Diseases, which serves as a national reference centre. In the period 1999-2010 more than 500 cases of imported malaria were diagnosed. The characteristics of the Rotterdam Malaria Cohort in the period 1999-2008 were previously described [ lactate , procalc lactate ,13, sodi® Malaria Test, Binax Inc. Maine, USA) and by conventional microscopy of stained thick and thin blood smears to identify the causative Plasmodium species. In case of Plasmodium falciparum infections, parasite density was determined. When the parasitaemia was less than 0.5% infected erythrocytes, parasites were counted per 100 leucocytes in thick smears. When the parasitaemia was equal or higher than 0.5% infected erythrocytes, infected erythrocytes were counted in thin smear and expressed as a percentage of the total erythrocytes. The number of parasites per microliter was subsequently calculated from these data. Patients were classified as having severe malaria if they met one or more of the WHO criteria for severe malaria, as published [On admission, blood samples were taken for analysis of the red blood cell count, haematocrit, white blood cell count, platelet count, CRP, serum electrolytes, total bilirubin, serum creatinin, liver enzymes, and blood glucose. A separate blood tube was drawn on admission for determination of plasma lactate, which was immediately analysed after isolation of plasma. Malaria was diagnosed by QBC (Quantitative Buffy Coat) analysis, by a rapid diagnostic antigen test for malaria . Normal serum values for procalcitonin are < 0.1 ng/ml. Copeptin was measured with a commercial sandwich immunoluminometric assay . Normal values for serum copeptin are 1.70-11.25 pmol/L [P. falciparum malaria and non-severe non-falciparum malaria were analysed with the Kruskall-Wallis test followed by Dunn's post-hoc tests. Two group comparisons were done by Student's unpaired t-test, Student's unpaired t-test with Welch correction or with the non-parametric Mann Whitney test depending on the distribution of the data. The diagnostic performance of each biomarker was reported as sensitivity, specificity, positive and negative predictive value for severe malaria and their corresponding 95% confidence intervals. Youden's index J (J = sensitivity + specificity-1) was used to identify the most appropriate cut-off point for each biomarker. Of each test a Receiver Operating Characteristics (ROC) curve, a graphical plot of sensitivity (true positive rate) versus 1-specificity , was constructed as a summary statistic and the area under the ROC curve (AUROC) and its corresponding 95% confidence intervals were calculated. The AUROC of each of the biomarkers was compared to that of copeptin in a pair-wise comparison with the method of DeLong et al. [Differences between patients with severe malaria, non-severe g et al. .Plasmodium vivax, 15 with Plasmodium ovale, three with Plasmodium malariae) and 141 patients were diagnosed with P. falciparum infection. Of the patients with P. falciparum infections, 25 (17.7%) patients had severe malaria. There were no cases of severe non-falciparum malaria. The infection was most frequently acquired in Africa (n = 156), followed by Asia (n = 27) and South America (n = 14). Thirty (14.7%) patients reported adequate use of malaria chemoprophylaxis, 24 (11.8%) used it inadequately and 143 (70.1%) patients did not use any chemoprophylaxis (data on prophylaxis use were not available in seven patients). The general characteristics of all patients are shown in Table In total, 204 travellers with imported malaria were included in this study; 63 patients were diagnosed with a non-falciparum infection , South-East Asia (n = 1) and South-America (n = 1). Twenty-four patients with severe malaria did not use any form of malaria chemoprophylaxis and one patient was using it inadequately. At admission to the intensive care unit (ICU), the 25 patients with severe malaria all fulfilled one or more of the severity criteria . The first arterial blood gas analysis on the ICU showed a median bicarbonate level of 21 mmol/L (range 9-25 mmol/L) and a median base excess of -2 mmol/L (range -18-3). The median Glasgow Coma Scale score was 15 (range 11-15). Two patients required mechanical ventilation. Two patients were referred to another hospital for renal replacement therapy because of persistent acute oliguric renal insufficiency. Thirteen patients received exchange transfusion as an adjunct therapy to parenteral anti-malarial treatment. One case fatality was observed. The laboratory results of travellers with severe malaria were further characterized by significantly lower platelet counts and haemoglobin levels and by significantly higher plasma lactate, bilirubin and CRP levels compared to patients with non-severe P. falciparum malaria (Table Twenty-one of 25 patients (84.0%) with severe malaria acquired a Asia n =  and SoutP. falciparum would have been falsely diagnosed as having severe malaria. In contrast, the diagnosis severe malaria would not have been considered in 5 of 24 (20.8%) evaluable patients with severe disease. Addition of either lactate, sodium or copeptin as a secondary selection criterion to the prediction rule did not result in a proper identification of all patients with severe disease on admission. Only after adding procalcitonin (at a cut-off of ≥ 0.9 ng/mL) as a secondary selection criterion to the prediction rule, all evaluable patients with severe malaria would have been correctly identified on admission . See Additional file When focusing on the 25 patients with severe malaria, 11 patients fulfilled a single criterion for severe malaria on admission, 11 patients fulfilled two malaria severity criteria, 2 patients fulfilled 3 malaria severity criteria and one patient fulfilled 4 malaria severity criteria on admission to the ICU, respectively. As a consequence of this distribution a proper stratification by numbers of malaria severity criteria present on admission was not feasible. When the severe malaria patients fulfilling a single malaria severity criterion on admission were compared to the patients with more than one criterion for severe malaria on admission, only for the biomarker plasma lactate a statistically significant difference was found , bacteraemia (n = 18) and urinary tract infections (n = 12). Interestingly, these episodes were related to both community-acquired (n = 30) as well as nosocomial infections (n = 66) . In our These abovementioned observations underline the limited value of these biomarkers for a rapid diagnosis of severe disease in the acute care setting in non-endemic industrialized countries; as for now, a biomarker-based decision rule can therefore certainly not replace the current clinical evaluation. It is noteworthy that all currently studied biomarkers were characterized by high negative predictive values, which may be helpful for exclusion of severe malaria on admission. The combined use of biomarkers may look promising since they allow correct identification of all patients with severe malaria but their use in the current clinical practice is hampered by their poor positive predictive values and lack of prospective validation.In conclusion, copeptin was not found to be a good biomarker for severe malaria in imported malaria. Instead, CRP appeared to be a more accurate predictor for disease severity than the other investigated biomarkers. The main clinical applicability of the current biomarkers or combination of biomarkers is probably limited to a rapid exclusion of severe disease given their high negative predictive values and low positive predictive values.The authors declare that they have no competing interests.MEvW contributed to the data acquisition and analysis and writing of the manuscript. DAH, EJH and JvH participated in the data analysis and revising of the manuscript. YdeR carried out the copeptin measurements and contributed to the data analysis. RK is responsible for collection of patient materials and database management. PJvG participated in the data acquisition and analysis and in writing and revising the manuscript. All authors have read and approved the final version.Figure S1 A two-step decision rule including the combined use of C-reactive protein and Procalcitonin to identify all patients with severe malaria on admission. Legend: SF = severe P. falciparum malaria; NSF = non-severe P. falciparum malaria; NSNF = non-severe, non-falciparum malaria; CRP = C-reactive protein; PCT = Procalcitonin.Click here for fileTable S1 Impact of the number of severity criteria on the level of the biomarker on admission in malaria patients with severe disease.Click here for file