Hemoglobin variants C and S protect against severe malaria but their influence on parameters not directly linked to disease severity such as gametocyte carriage and infection chronicity is less well understood. To assess whether these infection-related phenotypes depend on the host hemoglobin genotype, we followed 500 Malian individuals over 1–2 years and determined their parasitological status during monthly visits and incidental clinical episodes. While adults heterozygous for hemoglobin S mutation were less often parasitemic compared to AA adults , schoolchildren (but not toddlers or adults) with AC genotype carried parasites, including gametocytes, more often than their AA counterparts . AC children were also likelier to be parasite-positive during the dry season, suggesting longer infections, and were more infectious in mosquito skin feeding assays than AA children. Notably, AC school-aged children, who comprise ~5% of the population, harbor a third of infections with patent gametocytes between May and August, when transmission transitions from very low to intense. These findings indicate that schoolchildren with hemoglobin C mutation might contribute disproportionately to the seasonal malaria resurgence in parts of West Africa where the HbC variant is common. Plasmodium falciparum infections can have diverse clinical presentations, even within the same person over time1, and this might impact the potential for onward transmission of the parasite. For example, individuals with asymptomatic infection can carry chronic parasitemia without seeking treatment and might remain infectious to mosquitoes for long periods of time. Consequently, identifying and addressing factors involved in frequent or long-lasting parasite carriage can be important to malaria elimination and control strategies that aim to reduce human-to-mosquito transmission. Here, we assessed whether two hemoglobinopathies, hemoglobin (Hb) S and HbC traits , that are frequent in sub-Saharan Africa3 can influence parasite carriage and transmission.4, and mechanistic hypotheses proposed to explain this protection include impaired blood stage parasite development7, reduced cytoadherence of infected red blood cells9, which is linked to the redox imbalance of hemoglobinopathies and its effects on the export of parasite-encoded proteins11, tolerance to malaria infection related to heme catabolism by heme oxygenase-112, or accelerated acquisition of immunity15. Life-threatening episodes, however, represent only a small proportion of falciparum infections16, including in susceptible individuals1, and these same mechanisms might regulate other epidemiologically important parameters in non-severe infections, such as duration of parasite carriage and infectiousness. To determine how these variants shape the reservoir of malaria parasites in human populations, we analyzed a longitudinal study with frequent follow-up in an area with seasonal transmission in Mali.These two hemoglobin variants have been associated with protection against severe malaria syndromes in numerous epidemiological studiesIn June 2011, 250 individuals  were enrolled, without knowledge of their genetic backgrounds, in an epidemiological study in the village of Bancoumana, southwestern Mali , and 44 in the second year (July 2012–June 2013). In each experiment, 60 female Anopheles gambiae mosquitoes, distributed in two cups, were allowed to blood-feed directly on the skin of volunteers. Fed mosquitoes were kept for one week before dissection and oocyst detection by microscopy. Only assays with at least 10 dissected mosquitoes were included in this analysis.Malaria parasite transmission potential of study participants was assessed by mosquito skin feeding experiments, the most sensitive method to quantify infectivityHemoglobin variants were ascertained using high-performance liquid chromatography : 380 (76.3%), 54 (10.8%), 59 (11.8%), 2 (0.4%) and 3 (0.6%) participants were AA, AS, AC, CC and SC, respectively. Genotypes were equally distributed among individuals from different age groups (P = 0.57) and enrolled in different years (P = 0.66).To assess the association between hemoglobin variants and parasite and gametocyte carriage, mixed effects logistic models with two random effects (study participant and household) were used. Mixed effects Poisson models for the outcomes of infection and gametocytemia incidences during scheduled visits with one random effect (household) were also fit and similar results were obtained (see Supplementary Information). Multivariate estimates were adjusted for transmission season and age. In analyses excluding infants, who might be more resistant to malaria compared to older children, we obtained similar results for all-age models (data not shown). To quantify the importance of infections in individuals with hemoglobin variants to local malaria epidemiology, we used demographic and parasitological data to calculate the proportions of all patent infections that occur in children and adults with different hemoglobin types (Supplementary Information).The datasets generated during the current study are available from the corresponding author on reasonable request.P. falciparum asexual parasites and gametocytes were detected by microscopy at 776/5,457 (14.2%) and 270/5,457 (4.9%) routine visits, respectively. During the transmission seasons (July–December) of 2011 and 2012, 47.7 and 67.9% of the study population, respectively, carried patent asexual parasites during at least one scheduled visit. The number of parasite carriers during dry seasons (January–June) was lower: approximately one sixth and one third of participants had at least one routine visit with patent parasites during the dry seasons of 2012 and 2013, respectively. In parallel, 17.4 and 26.0% of individuals had microscopically-detectable gametocytes during 2011 and 2012 wet seasons, respectively; during dry season months, only half as many participants developed gametocytemia. In addition to routine visits, there were 859 unscheduled visits: asexual parasites were observed at 293/810 (36.2%) visits and gametocytes at 15/808 (1.9%). 609 clinical malaria episodes were diagnosed and treated, mostly (547/609) during wet seasons; 2 were considered severe according to WHO criteria.In Table 18. Individuals with AC genotype also had higher asexual stage parasite counts during asymptomatic scheduled visits , which could indicate tolerance to higher parasite densities for longer periods. However, during clinical malaria episodes, when asexual stage parasite counts were on average much higher , hemoglobin C mutation was not significantly associated with higher asexual stage parasites levels  than AA children with similar age. At the population level, this group was also a major reservoir of malaria infection  host transmission potential and how malaria transmission processes can vary in communities with different frequencies of these alleles would be particularly valuable. In this longitudinal study, we observed associations between hemoglobin variants and frequencies of patent parasite and gametocyte carriages, possibly linked to differences in infection duration. Our findings provide insights into how these protective genetic factors may affect natural infections and could have practical implications for malaria control and elimination programs in areas where these mutations are common, as treating individuals who are more likely to be infected throughout dry seasons is an effective strategy to reduce seasonal peaks of transmission.Since Allison’s observation that individuals with sickle cell trait were protected against microscopically-detectable infection after parasite exposure or inoculation4) have shown that both hemoglobin variants C and S protect against severe malaria. Although it is possible that the mechanisms of protection afforded by these variants target pathogenic events that are unique to severe clinical cases, epidemiological evidence that these variants also influence non-severe clinical malaria risk suggests that heterozygosis for hemoglobin C or S influences pathogenic processes common to all falciparum malaria infections. Experimental work also indicates that these mutations affect malaria parasites in several different ways29, however we still do not understand the importance of these different mechanisms in natural infections and how within-host parasite dynamics in AA individuals and in AS or AC heterozygotes differ, especially in terms of duration of parasite circulation and production of transmission stages.Several studies  of parasite, including gametocyte, carriage. Mosquito feeding assays that closely mimic in natura transmission indicate that these frequent microscopically-detectable gametocyte levels have a direct impact on infectiousness. Although they comprise less than 5% of the population, AC school-aged children contributed about a third of the sexual stage parasite reservoir from May through August, when transmission transitions from low to high intensity. As costs of molecular assays are gradually decreasing, identification of individuals that persistently carry parasites for long duration after infection might accelerate malaria elimination efforts. This may be especially important in areas where programs have effectively controlled malaria, but a persistent reservoir of infection presents a final obstacle to elimination.In summary, this study provides evidence that hemoglobin variants that were, and continue to beSupplementary Information


Plasmodium falciparum and Plasmodium vivax are both endemic. By combining passive case detection data with entomological data, a transmission scenario on the northwestern Thai–Myanmar border where P. falciparum is likely driven by importation was described, whereas P. vivax is also locally transmitted. This study highlights the differences in the level of control required to eliminate P. falciparum and P. vivax from the same region.Cross-border malaria transmission is an important problem for national malaria control programmes. The epidemiology of cross-border malaria is further complicated in areas where Malaria case data were collected from malaria clinics in Suan Oi village, Tak Province, Thailand between 2011 and 2014. Infections were diagnosed by light microscopy. Demographic data, including migrant status, were correlated with concomitantly collected entomology data from 1330 mosquito trap nights using logistic regression. Malaria infection in the captured mosquitoes was detected by ELISA.P. falciparum compared with Thai patients  and cases were significantly associated with seasonal migration. However, P. falciparum infection was not associated with the Anopheles mosquito capture rates, suggesting predominantly imported infections. In contrast, recent migrants were equally likely to present with P. vivax as mid-term migrants. Both migrant groups were twice as likely to be infected with P. vivax in comparison to the resident Thai population . Plasmodium vivax cases were strongly correlated with age and local capture rates of two major vector species Anopheles minimus and Anopheles maculatus , suggesting that a high level of local transmission might be causing these infections.Recent migrants were almost four times more likely to be infected with P. falciparum infections occur mostly in the recent migrant population with a seasonality reflecting that of agricultural activity, rather than that of the local mosquito population. This suggests that P. falciparum was mostly imported. In contrast, P. vivax cases were significantly associated with mosquito capture rates and less with migrant status, indicating local transmission. This highlights the different timelines and requirements for P. falciparum and P. vivax elimination in the same region and underlines the importance of multinational, cross-border malaria control.On the Thai–Myanmar border, The online version of this article (doi:10.1186/s12936-017-1900-2) contains supplementary material, which is available to authorized users. Malaria epidemiology in the Greater Mekong Sub-region (GMS) is complex, with all five parasite species present and a large variety of mosquito vectors , 2. MalaApproximately three-quarters of reported cases in the GMS occur in Myanmar while the incidence rate in Thailand is low and further decreasing . Tak Province represents a malaria hotspot in Thailand with an estimated 6.3 cases per 1000 population in 2015 . The borPlasmodium vivax in this area [Plasmodium falciparum and P. vivax, may exhibit very different dynamics in cross-border transmission scenarios. Plasmodium falciparum is much more dependent on a sustained local mosquito population, whereas P. vivax can endure lower mosquito numbers and highly seasonal mosquito population dynamics [P. falciparum is eliminated first, but local P. vivax transmission is sustained for an extended period of time [Anopheles minimus and Anopheles maculatus are the main malaria vectors constituting >85% of the captured vector population over the course of a year (2012). However, a variety of minor vectors, including Anopheles dirus, Anopheles annularis and Anopheles barbirostris are also present and have been shown to contribute to malaria transmission [Previous studies have rarely integrated entomological with epidemiological surveillance –20. Entohis area . Importahis area . Furtherdynamics . It is,  of time . Vector smission , 26. TheThe study was carried out in Suan Oi village located in Tha Song Yang district in northwestern Thailand Fig. . This ThP. falciparum and P. vivax [Adult mosquitoes were collected using CDC light traps . Traps were placed in or near randomly selected households to approximately cover the extent of the village area. Traps were placed indoors and outdoors from 18.00 to 06.00 h for five consecutive nights per month in a total of 45 locations. There were a total of 266 trap placements (1330 trap nights) from August 2011 to April 2013 with the majority of trap placements in 2012. During some months, no traps were set up due to flooding and political unrest. Captured mosquitoes were transported to and sorted in the laboratory of the Medical Entomology Department, Faculty of Tropical Medicine, Mahidol University, Bangkok. Mosquito species were identified based on morphological characteristics . The heaMalaria patient data were available between August 2011 and December 2014 from the Suan Oi malaria clinic. However, in the present study only the data collected in the 12 months of concomitant mosquito collection were utilized. Malaria was diagnosed by light microscopic examination of Giemsa-stained thick and thin blood smears. If slides were positive, parasite densities were determined by counting a minimum of 1000 and a maximum of 2000 leucocytes. Apart from clinical symptoms, basic demographic information such as age, gender, occupation, nationality, and migration status was also collected. Patients were divided into three groups: Thai nationality (M0), migrants residing in Thailand for 6 months or longer (M1), and migrants who stated that they had been living in Thailand for fewer than 6 months (M2). Since the ethnic background in this region does not allow for determination of nationality, the grouping was done according to The Bureau of Vector Borne Diseases (BVBD) in Thailand. Most M1 migrants are registered with the Ministry of Labour (MoL), which grants permission to stay in Thailand for a period of typically 1–2 years. People in the M2 group are often highly mobile, and are less likely to have registered with the MoL. The provincial government gives permits at the border crossings for entering that district of Thailand for 1–7 days, which can be extended by returning to the border crossing for re-authorization.The study was approved by the Ethics Committee of the Department of Disease Control Ministry of Public Health, Thailand and the Human Research Ethics Committees of Mahidol University and Pennsylvania State University.P. falciparum and P. vivax positivity (as the dependent variable), with the predictors such as age, gender, migrant status, and the monthly An. minimus and An. maculatus capture rates. The mosquito capture rate is defined as the number of mosquitoes trapped per trap and per night. The proportion of malaria-positive patients in each migrant group was plotted over the proportion of all malaria-positive patients to obtain a measure of the contribution of each migrant group to the overall malaria cases. Linear regression was used to correlate overall patient proportions positive for either P. falciparum or P. vivax with the mosquito capture rates. Statistical analyses were conducted in Stata 12 .Logistic regression was used to test for an association of P. falciparum and 354 P. vivax infections. In the same time, a total of 512 An. minimus and 286 An. maculatus were trapped in the 266 trap placements (1330 trap nights), constituting ~85% of the trapped anopheline population. Average number of mosquitoes monthly captured per trap was shown in Figure S1  and P. vivax (Panel c) for the different migration strata  during 2011–2013, number of P. vivax and P. falciparum cases per month were shown in Figure S2  throughout the study period. Most mosquito traps were placed in 2012. Altogether four infected mosquitoes were captured . However, these low numbers precluded the use of sporozoite rates in the present statistical analyses.There were 4225 patient visits to the malaria clinic in the 12 months when mosquito trapping was conducted. The overall patient characteristics are shown in Table P. falciparum infections and were almost four times more likely to present with P. falciparum (odds ratio (OR) 3.84, 95% CI 2.76–5.33, p < 0.001) in comparison with the Thai patients (M0)  (OR 2.49 95% CI 1.33–4.72). This suggests that P. falciparum infections were mostly imported. Long-term migrants were only slightly more likely to present with P. falciparum in comparison with Thai patients  in Fig. Plasmodium falciparum infections were also higher in the rainy season (August) , further indicating that the majority of P. falciparum infections might be imported. Age was not significantly correlated with P. falciparum positivity . Males were significantly more likely to present with detectable P. falciparum  contributed the highest proportion of  contribuP. vivax cases was relatively constant throughout the years. Both, M1 and M2 foreigners were equally more likely to present with P. vivax infection when compared with the Thai population group . However, Thai patients presented a stable contribution to the caseload throughout the year 2011–2013, whereas the contribution of the recent migrants fluctuated and normally peaked in the dry and hot seasons of January and April 2012, coinciding with seasons of increased agricultural activity. Yet, the seasonal peaks in the migrant populations were less pronounced in year 2013 .The overall proportion of An. minimus and An. maculatus, in mosquitoes per trap-year) and the proportion of malaria-positive cases was determined for both parasites. Consistent with local P. vivax transmission, P. vivax slide positivity was more likely in months with high An. minimus and An. maculatus capture rates , there was no association between the proportion of P. falciparum-positive patients and the mosquito capture rate , compared to the Thai population (M0). This may partially be contributed to importation. However, there was no association of P. vivax infection with either the ‘recent’ (M2) or ‘longer-term’ (M1) migrant status. Thus, the difference in vivax infection rate between the migrant and the Thai populations is likely due to the differences in their living conditions, access to healthcare, use of malaria prevention measures and/or socio-economic factors. Migrants predominantly live in semi-permanent dwellings on the edge of the village, closer to potential breeding sites. In addition, the households are poorer, and ITN coverage and other precautions are also lower in this population [P. vivax infection, suggesting of the acquisition of immunity in older populations.This study shows that pulation , 19. A sP. falciparum infections were not associated with the dynamics of the major vectors An. minimus and An. maculatus, but they were ~ four times more likely in recent migrants compared to the resident population. Also, there was no significant age trend in the probability to present with a P. falciparum infection as shown in previous studies in Southeast Asia [P. falciparum infection. Plasmodium falciparum case numbers fluctuated with agricultural seasonality, and more recent migrant (M2) patients presenting with infections were predominantly in the months of harvest season (November–December), which are characterized by an influx of labourers from Myanmar [P. falciparum cases. It has been shown in similar settings on the Thai–Cambodian border that seasonal labourers are at a higher risk of being infected with vivax malaria [In contrast, ast Asia , 34. Fur Myanmar . This su malaria , 35, 36. malaria , 37.A significant limitation of this study is that it was focused on only passively detected cases. However, the findings are in agreement with the results of a recent active case detection survey on a cohort of approximately 500 individuals in the same region, where ‘no citizenship’ (likely to be migrants) was identified as the most significant risk factor for asymptomatic malaria infections . In thisAn. minimus mosquitoes were found positive for P. vivax and one An. maculatus mosquito was found positive for P. falciparum. Upscaled trapping studies may allow for the inclusion of sporozoite rates in the different mosquito populations into statistical models. Even though in this study An. minimus and An. maculatus constituted 85% of the captured vector population, other vectors, especially the deep forest vector An. dirus, should not be ignored as it has been shown to contribute significantly to transmission [An. dirus represented <0.5% of the captured vector population as trapping was conducted inside the village. It is noteworthy that An. minimus and An. maculatus exhibit both anthropophilic and zoophilic biting behaviours. Therefore, fluctuation of cattle populations with the cattle trade may also affect the dynamics of these mosquito vectors [The present study emphasizes the importance of mosquito surveillance. Several smission . However vectors . The mobP. falciparum and P. vivax transmission on the Thai–Myanmar border. This study shows that P. falciparum infections were more concentrated in the recent migrant population and that the seasonality of P. falciparum cases mirrors that of agricultural activity. It can therefore be speculated that P. falciparum may be mostly imported. In contrast, P. vivax cases were significantly associated with the dynamics of the local mosquito population and less with migrant status, strongly suggesting local transmission. These findings suggest that P. vivax elimination may require considerably greater efforts than P. falciparum elimination and that sustained, well coordinated, border-transcending malaria control will be required to attain elimination status in this area and contain the expansion of drug resistance.Entomological surveillance and its association with passive case detection data provided important insights into Additional file 1: Figure S1. Average number of mosquitoes monthly captured per trap.Additional file 2: Figure S2. Number of P. vivax (A) and P. falciparum (B) cases per month reported by Suan Oi malaria clinic.Additional file 3: Table S1. Patient characteristics for the M2 migrant population*. Table S2. Patient characteristics for the M1 migrant population*. Table S3. Patient characteristics for the Thai national population. Table S4. Occupation distribution among the different population strata.


Plasmodium falciparum, were included. The four genotypes were detected by real-time PCR using the MTHFR 677C>T, MTHFR 1298A>C, MTR 2756A>G, and MTRR 66A>G detection kit (Sacace Biotechnologies REF: T01002-96-S). The results revealed frequencies of 90% 677CC, 10% 677CT, and 00% 677TT for MTHFR C677T; 78.6% 1298AA, 19% 1298AC, and 2.4% 1298CC for MTHFR A1298C; 61.9% 2756AA, 33.3% 2756AG, and 4.8% 2756GG for MTR A2756G; and 50% of 66AA, 45% of 66AG, and 5% of 66GG for MTRR A66G. Correlations were found between A2756G MTR genotypes and parasitaemia (P = 0.02), MTRR A66G and hemoglobin genotypes (P = 0.009), and MTHFR A1298C and sex (P = 0.01). This study demonstrated for the first time an association between the A2756G MTR alleles and Plasmodium falciparum malaria in Burkina Faso and gave an overview of the genotypic distribution of the major SNPs influencing the metabolism of homocysteine.This study analyzed the four main polymorphisms of the genes in homocysteine metabolism in malaria patients. Forty-two randomly selected subjects, diagnosed positive for Homocysteine is an intermediate amino acid in the metabolism of methionine. The plasma level in homocysteine depends on many factors, including the folate, B12, and B6 vitamins status. In addition, the genetic polymorphisms of Methylene Tetrahydrofolate Reductase (MTHFR) play a crucial role. Hyperhomocysteinaemia is a risk factor for several pathologies such as cardiovascular disease , renal iPlasmodium falciparum, the parasite responsible for the lethal cases of malaria, uses the pathway of polyamines essential to its proliferation and differentiation, thus imposing an oxidative stress on the host cell due to the use of glutathione [tathione , 9. This Plasmodium in human is therefore influenced by the polymorphisms of these genes which intervene in the remethylation of homocysteine into methionine. Indeed, when the route of remethylation is favored, homocysteinaemia is low causing a low production of glutathione and thus reducing the development of Plasmodium in the human body. On the other hand, if this pathway is blocked, hyperhomocysteinaemia leads to an increase in the transsulfuration pathway and an accumulation of glutathione in the organism necessary for Plasmodium development.Depending on the polymorphisms of the genes responsible for the synthesis of these enzymes, the route of the remethylation can be promoted over that of the transsulfuration and vice versa. The development ofStudies on the possible association of the polymorphisms of genes involved in homocysteine metabolism and malaria in Burkina Faso are missing. However, Chillemi et al. (2005) suggested that the determination of plasma homocysteine levels could be used as a measure of the severity of malaria infection . The genThe present study was a prospective study conducted from September to November 2014 in Koubri. This is a rural municipality located in the southern Ouagadougou at approximately 25 km, with the following geographical coordinates: 12°10′ north and 1°24′ west. Koubri has an average annual temperature of 28.2°C and it falls on average 780 mm of rain per year. However, the locality has numerous reservoirs of water that make the presence of mosquitoes vectors persistent throughout the year, so explaining the endemicity of malaria in the area. Plasmodium falciparum HPRII antigen (histidine II-rich protein) and Plasmodium lactic dehydrogenase (pLDH) common to Plasmodium species in human whole blood. Tick smears were made for each patient for the confirmation of plasmodial species and the parasite count. Finally, the 42 patients with the highest parasite density (density superior to 3000 parasite/μL of blood) were selected for the G-6-PD deficiency study. The age and the antimalarial drugs intakes were directly recorded prior to the blood sampling.The study initially included 182 patients (1 to 79 years) who underwent malaria rapid diagnosis test, SD BIOLINE Malaria Ag P.f/Pan, which enables the detection of theFrom the 42 selected patients, 5 mL of venous blood was collected in two EDTA impregnated tubes. The first tube was used for the hematological analysis; the second was centrifuged to discard plasma and the pellets were stored at −80°C until use for nucleic acid extractions.μL of 1% saponin and the sample spots were deposited on the CELLOGEL with capillary tubes. The migration was made with 150 V in 60 min.Platelet count and hemoglobin were determined with total venous blood using the ABX micro 60 automate. Hemoglobin electrophoresis was performed at alkaline pH (tris-glycine buffer pH 9.5) on cellulose acetate strip (CELLOGEL 5.7 × 14 cm). In brief, whole blood was washed three times with 0.9% NaCl by successive centrifugations at 3000 rpm for 5 min. Finally, the red cell pellet was lysed with 500 μLITE  was used for DNA extracts purity and final concentration assessment.The extraction of DNA from the blood pellet was carried out using the salting out method , 16. TheThe samples genotyping with real time PCR was carried out using “Real Time PCR kit for detection of MTHFR 677C>T, MTHFR 1298A>C, MTR 2756A>G, and MTRR 66A>G” . SaCycler-96  was used for amplification. Thermocycling was performed at 80°C for 2 min and 94°C for 3 min and then 40 cycles at 94°C for 15 sec and 64°C  for 40 sec.P value < 0.05 was considered statistically significant.The data was processed using Excel 2010 (Microsoft) and SPSS® software version 20 . The Pearson Chi test was used for comparisons and any The study was approved by the Ethics Committee for Health Research in Burkina Faso (Deliberation number 2014-9-128). Adults have given their free and informed consent for their participation in the study while the parents or guardians of the minors have given their approval for the participation of the minor.μL while 11.90% had a parasitaemia between 3,000 and 10,000 trophozoite/μL. However, the parasitaemia was below 3,000 trophozoites/μL for 33.34% patients.The study involved 69.05% (29/42) men and 30.95% (13/42) women, the majority of whom were children with 14.28% (6/42) over 20 years of age . The mea−1), while 35.72% (15/42) had moderate anemia (10 < Hb < 12 g L−1) and 23.80% (10/42) had anemia (Hb < 10 g L−1). It should also be noted that 33.33% (14/32) of patients had thrombocytopenia compared to 66.67% (28/42) with normal platelet count. The frequency of the HbAA genotype was 57.14% (27/42) with one of the patients (0.02%) presenting major sickle cell syndrome (SC).With regard to the anemia, 40.48% patients were not anemic (Hb > 12 g LP = 0.009) (P = 0.02). Individuals with 2756AA genotype predominantly (19/26) had high parasitaemia . The analysis of these polymorphisms according to the genus revealed a statistically significant difference for the MTHFR A1298C gene (P = 0.01) with more male subjects carrying the MTHFR 1298AA genotype (26/29 or 89.65%). Finally the data recorded in the present study were compared with previous data recorded in five other countries, namely, China, Pakistan, Brazil, South Korea, and Sweden (= 0.009) . An assod Sweden . The freThis study described the MTHFR C677T, MTHFR A1298C, MTR A2756G, and MTRR A66G mutations. The genotypic frequencies found for the MTHFR C677T mutation are similar to those already reported in Burkina Faso ; 28.6% 6Genotypic frequencies for this gene  in our study are also similar to those found in 20–45 years of age  in Burkina Faso [Our results, however, differ from the genotypic frequencies  reported in Chinese Han adults ; 67.8% AThe results of our study for the mutation MTR A2756G  are also different from the genotypic frequencies  reported in Pakistan  and in JμL of blood) could be more susceptible to malaria than others. An association between this mutation and malaria has been reported in the Northeast region of India [A correlation between MTRR A66G and hemoglobin genotypes was observed in this study. A2756G MTR genotype and parasitaemia showed a statistically significant association; MTR 2756AA individuals with predominantly high parasitaemia  for the genes involved in homocysteine metabolism correlated with simple malaria. Several associations such as MTRR A66G and hemoglobin genotypes; MTR A2756G and Plasmodium falciparum; and MTHFR A1298C infection and sex were observed and suggest a large enough sample for further analysis.Burkina Faso is a malaria endemic country with a high frequency of host resistance polymorphisms to


Plasmodium falciparum AMA-1, MSP-119 and CSP were measured with the aim to describe transmission patterns in meso-endemic settings in Lake Victoria. Two cross-sectional surveys were conducted in Lake Victoria in January and August 2012. The study area comprised of three settings: mainland (Ungoye), large island (Mfangano) and small islands . Individuals provided a finger-blood sample to assess malaria infection by microscopy and PCR. Antibody response to P. falciparum was determined in 4,112 individuals by ELISA using eluted dried blood from filter paper. The overall seroprevalence was 64.0% for AMA-1, 39.5% for MSP-119, and 12.9% for CSP. Between settings, seroprevalences for merozoite antigens were similar between Ungoye and Mfangano, but higher when compared to the small islands. For AMA-1, the seroconversion rates (SCRs) ranged from 0.121 (Ngodhe) to 0.202 (Ungoye), and were strongly correlated to parasite prevalence. We observed heterogeneity in serological indices across study sites in Lake Victoria. These data suggest that AMA-1 and MSP-119 sero-epidemiological analysis may provide further evidence in assessing variation in malaria exposure and evaluating malaria control efforts in high endemic area.As markers of exposure anti-malaria antibody responses can help characterise heterogeneity in malaria transmission. In the present study antibody responses to  Nevertheless, with scaling up of malaria prevention, diagnosis and treatment, the prevalence of Plasmodium falciparum infection in many parts of sub-Saharan Africa declined by 50%, and the incidence of clinical disease fell by 40% between 2000 and 20152.In sub-Saharan Africa, malaria remains one of the leading causes of morbidity and mortality, with 191 million cases and over 390 thousand deaths reported in 2016P. falciparum parasite rate for the population aged 2–10 years (PfPR2–10) is below 1%3. Despite significant increases in coverage of long-lasting insecticide-treated nets (LLINs) through free-mass distribution campaigns4 and the change in first-line treatment to artemisinin-based combination therapy (ACT)5, 10.6% (4.3 million) of the population still live in areas with PfPR2–10 ≥ 40%, mainly counties adjacent to Lake Victoria in western Kenya6. In addition, increased paediatric malaria hospital admissions in 20097 and high prevalence of malaria infection among school children in 20148 highlight the challenge of effective malaria control in this highly endemic region of Kenya.In Kenya, 65% (26 million) of the population live in areas where 10. As a proxy measure of malaria transmission, serological responses to P. falciparum antigens have shown a robust and consistent correlation with estimates of entomological inoculation rate (EIR)10, and thus have increasingly been incorporated in cross-sectional and longitudinal studies to monitor changes in transmission15 and identify hotspots in transmission17.Understanding the intensity of malaria transmission in a community is fundamental to the design and evaluation of malaria control and elimination programmes. The most widely used metric of malaria transmission intensity is parasite prevalence, measured through cross-sectional surveys. In recent years, antibody responses to one or more malaria parasite-specific antigens have been explored as alternative means to estimate malaria transmission intensity20, no such study has been carried out in the adjacent Lake Victoria basin where prevalence is moderate to high with significant local heterogeneity21. In the present study, antibody responses to P. falciparum blood-stages antigens apical membrane antigen 1 (AMA-1), merozoite surface antigen-119 (MSP-119) and circumsporozoite antigen (CSP) were measured to assess malaria exposure and transmission on islands in Lake Victoria. Results from this study provide baseline data to evaluate the planned malaria elimination programme in the study area.Whilst several sero-epidemiological studies have been conducted in the low-transmission western highlands of KenyaA total of 5044 participants were enrolled from five different settings  in January and August 2012. Population coverage varied among settings: 10.5% in Mfangano, 35.7% in Ungoye and 48–90.6% in the small islands. Gender and age distributions were similar across the five settings. The majority of participants were children and adolescents ≤15 years old  and came from the islands . At enrolment, 5.9% (95% CI: 5.2–6.5) of the population were febrile (axillary temperature >37.5 °C), and 20.8% (95% CI: 19.7–22.0) were anaemic (haemoglobin [Hb] level < 11 g/dL). Of all children 12 years and below (n = 3045), 1261  were found to have an enlarged spleen. The prevalence of febrile illness, anaemia, and enlarged spleen varied significantly by study sites (P < 0.001). Further details on the study population are shown in Table P. falciparum infection by microscopy and PCR in the study sites is shown in Fig. P. falciparum parasite prevalence ranged between 4.1 and 32.1% by microscopy, and between 11.2 and 56.2% by PCR. Parasite prevalence was significantly higher in Ungoye than other sites, regardless of detection method . Parasite prevalence by PCR generally peaked in the 11–15 years group and declined thereafter in all study sites  for Ungoye, Mfangano, Takawiri, Kibuogi, and Ngodhe, respectively  and PCR   between Ungoye (43.4%) and Mfangano (45.4%), and significantly lower on the three small islands . Seroprevalence between genders showed a higher prevalence in females in Ungoye (P = 0.023) and Mfangano (P = 0.024), but the differences were comparable in the small islands. Antibody level and seroprevalence increased significantly with age (P < 0.001) in all study areas    . The different serological outcomes for CSP all produced similar trends; similar between the study areas, albeit the differences in antibody responses were statistically lower than AMA-1 and MSP-1ns) Fig. , and thens) Fig. . SCRs we86) Fig. .P. falciparum infected individuals. Antibody responses did not differ significantly between the febrile and the non-febrile groups in all study sites, but the proportions of AMA-1 seropositive individuals were significantly higher (P < 0.05) in the non-febrile groups in Ungoye and Mfangano  among children with enlarged spleen than those with non-palpable spleen in Ungoye and Mfangano, although the differences were not consistently observed on the small islands  infections , Kibuogi (P = 0.008), and Ngodhe (P = 0.037) (Table 19 (P < 0.001) and CSP (P = 0.002) on Ngodhe. Age was significantly (P < 0.001) associated with AMA-1 seropositivity in all five study sites, MSP-119 seropositivity in Ungoye (P < 0.001), and CSP seropositivity on Ngodhe (P = 0.024). Significant association between anaemia and MSP-119 seropositivity was observed in Ungoye (P = 0.012) and Mfangano (P = 0.03) only  associated with seropositivity to AMA-1, MSP-1e P = 0.07  were similar between Ungoye and Mfangano, but significantly higher when compared to the three small islands , SCR based on serology can provide a more robust picture of the malaria transmission dynamics in an endemic area. In this setting, in the area with lack evidence of infections by microscopy, serological and molecular tools enabled a more complete understanding of the ongoing transmission and allowed for an examination of risk factors. Furthermore, as a metric of malaria transmission intensity, EIR is notoriously difficult to measure and the heterogeneous distribution of mosquito bites among individuals within a population makes it even more difficult to standardize32. Previously developed log-log calibration curves that relate SCRs to EIR values can provide a straightforward low-cost measure of malaria transmission10. These serology-based EIR estimates, especially those based on MSP-119 SCR, have shown a robust correlation with measured EIR10, and thus allow for clear differentiation of low-, moderate-, and high-transmission areas.Despite differences in immunogenicity, both 36, making it a suitable marker for monitoring historical changes in transmission intensity14. AMA-1 and MSP-119 are erythrocytic-stage antigens; they are produced continuously and in relative abundance because the erythrocytic-stage parasites go through several cycles of multiplication. In contrast, CSP is a pre-erythrocytic-stage antigen and is available to the immune system when a small number of sporozoites shed this antigen into circulation37. In consequence, the availability of CSP to immune system is of much shorter duration, which may explain its inability to detect subtle differences in exposure and transmission among settings in this study.Antibody responses to CSP did not follow the same pattern as those for merozoite antigens; neither seroprevalence nor SCR showed significant difference among settings. CSP has been previously reported to give reliable estimates of malaria endemicity in hyper-endemic areas and reflect the seasonal dynamics of transmissionP. falciparum antibody prevalence increased with age. When analysed as a whole population, only antibody responses to AMA-1 were negatively associated with anaemia in Mfangano, Takawiri and Ngodhe. In contrast, when analysis was restricted in children ≤12 years, antibody responses to MSP-119 were significantly associated with anaemia in Ungoye and Mfangano, whilst all the antigens tested were independently associated with enlarged spleen in all areas. Associations between measured antibody responses to P. falciparum antigens and the risk of malaria have shown to be inconsistent in multiple studies38. Potential reasons for these inconsistencies include differences in the intensity and stability of transmission, allelic variation of specific antigens and IgG subclass switching39, which may explain some of the differences in findings in our study areas. Nevertheless, antibodies to AMA-1 have been associated with protection from clinical malaria in some studies41, but not others43. Some studies have also documented independent association between antibodies to MSP-119 and protection from clinical malaria44, whilst others have not45. Similar to the responses to blood-stage antigens, antibodies to CSP was associated with a higher odds of having an enlarged spleen, and age-adjusted antibody levels showed apparent high responses in children with enlarged spleen in each study area  were disproportionally represented . The population in the study area consists mainly of people of the Luo ethnic group with Dholuo is primarily spoken, as well as the national language of Kiswahili. Population estimates were 18,600 for Mfangano, 1000 for Takawiri, 700 each for Kibuogi and Ngodhe , and 3,471 for Ungoye (2009 Kenya Population and Housing Census). The area is predominantly rural with most residents depend on fishing and traditional small-scale farming as the main occupations21. Most houses are typically made of mud walls with thatched or corrugated iron roofs.The present study was conducted on four islands in Lake Victoria  and one mainland lakeshore area (Ungoye) in Homa Bay County, western Kenya Fig. . Homa Ba55. Malaria in the study area is meso-endemic but geographically heterogeneous, with prevalence estimates ranging from 14.6% on islands to 44.2% in the coastal mainland21. The major vector responsible for malaria transmission in Lake Victoria is Anopheles gambiae s.l.56, and estimated annual EIR from serological markers were reported as high as 50 infectious bites per person per year14. P. falciparum is the predominant malaria parasite, while Plasmodium malariae and Plasmodium ovale are less common, and no Plasmodium vivax infections are observed21.The climate in Lake Victoria is tropical, with temperatures ranging from a mean annual minimum of 17.7 °C to a mean annual maximum of 34.8 °C, and humidity is relatively high. This region generally experiences a bimodal pattern of rainfall, with the longer rainy season starting in March and ending in July and the shorter rainy season from November to December. In Lake Victoria basin, peak malaria transmission occurs 1–2 months after the rainy season and the mean monthly anopheline vector abundance has been reported to increase by 6- to 8-fold in the rainy season compared to the dry seasonIn this study, seven catchment areas on Mfangano (i.e. large island) were selected to represent the different local environments found on the island. Gulwe and Kagungu are situated in the central highland. On the other hand, Ramba, Wakinga, Mrongo, Ugina and Wakula are situated along the coastal lowlands of the island. Across the island, there are six government health facilities including one health centre and five dispensaries. Meanwhile, on each small island, population is mainly distributed between two main settlements: Kamarach and Kongata on Takawiri Island, Kibuogi A and Kibuogi B on Kibuogi Island, and Bonde and Luanda on Ngodhe Island. Takawiri and Kibuogi are close to Mfangano while Ngodhe is situated to the north of Rusinga Island. Takawiri and Ngodhe are each served by a dispensary, but no public health facility is available on Kibuogi. Ungoye, a small village on the mainland was included for comparative purposes due to its similarity to the islands in environmental characteristics, infrastructure, and access to the health facilities. It is served by a government dispensary. The village is connected by an unpaved road to the nearby towns of Sindo and Mbita, each with a sub-district hospital.This study was carried out in full accordance with all international, Kenyan and Swedish accepted guidelines as written informed consent was obtained from all participants. The Ethics Committee reviewed and approved all the consent procedures. This study was approved by the Kenyatta National Hospital/University of Nairobi-Ethics and Research Committee in Kenya (No.P7/1/2012) and the Committee on the Ethics of Human Research of Karolinska Institutet in Sweden (Dnr 201271239–31/4).Two cross-sectional surveys were performed in January and August 2012, after the short and long rainy seasons, respectively. Both surveys were conducted approximately 2 months after the rainy seasons to coincide with the periods of heaviest malaria burden. In our effort to ensure a high coverage of sampled population, each survey was conducted on different parts of the five study settings. The convenience sampling strategy was used in this study, whereby residents were asked to come to selected survey points such as community-based beach management unit (BMU) halls or school for study participation. Island and village leaders were sensitised to study by trained field workers and together provided information to community members at community meetings. Adult community members willing to participate were asked to read and provide signature on an informed consent form. In case of illiteracy, consent by thumb print was obtained in the presence of an independent literate adult witness. For children ≤12 years of age, consent was obtained from parents or guardians.21. On each small island, surveys were conducted separately between the two main settlements: Kamarach, Kibuogi A and Bonde in January and Kongata, Kibuogi B and Luanda in August. All surveys on small islands were conducted at BMU halls, which are typically connected to marketplaces.Different catchment areas were targeted in each survey Fig. . On MfanTo ensure a balanced representation of all age groups, five age categories were defined per study area: one to five years, six to ten years, 11–15 years, 16–30 years, and >30 years. Age and gender of each participant were recorded. Axillary body temperature was determined using a digital thermometer , and those with temperature exceeding 37.5 °C were considered febrile. Haemoglobin level was measured with the HemoCue Hb 201 Analyzer . A measurement below 11 g/dL was classified as anaemic. Children aged 12 years and below were examined for enlarged spleen by AK according to Hackett’s method, regardless of fever or malaria status.P. falciparum parasite in 100 high-power fields by experienced microscopists as described previously21. Parasite density was determined by counting the number of asexual parasites against 200 leukocytes and assuming that there are 8,000 leukocytes per µl of blood. DNA was extracted from a quartered blood spot (17.5 µl) using the QIAamp Blood Mini Kit  according to the manufacturer’s instructions. The presence of P. falciparum DNA was assessed using a nested PCR protocol (nPCR) as described previously57.A blood sample was obtained by finger prick (approximately 10 µl) for thick and thin blood smears, and two spots of blood (70 µl each) were collected on Whatman ET31 Chr filter paper . Blood spots on filter paper were air-dried and stored in plastic bags at 4 °C short term and at −20 °C for longer term. Blood smears were stained with Giemsa solution and examined for the presence of 58. The reconstituted blood spot solution, equivalent to a 1/200 dilution of serum, was stored at 4 °C until used for antibody test.A 3 mm disk was punched from each dried blood spot and serum was eluted in reconstitution buffer in 0.5 ml deep well plates  as described previouslyP. falciparum malaria antigens namely apical membrane antigen-1  and merozoite surface protein-1 , as well as to the P. falciparum sporozoite antigen NANP5 repeat peptide circumsporozoite protein (CSP) . Briefly, antigens were coated on NUNC-Immuno plates (Sigma) at concentrations of 0.5 µg/ml and 1.0 µg/ml in coating buffer (50 µl per well) for recombinant blood-stage antigens (AMA-1 and MSP-119) and CSP, respectively. The plates were washed in PBS with 0.05% Tween 20 (PBS/T) and blocked using 1% (w/v) skimmed milk solution  in PBS/T for three hours. After washing, 50 µl of reconstituted blood spot solution were added in duplicate at final dilutions of 1:1000 for both MSP-119 and CSP, and 1:2000 for AMA-1 and incubated overnight at 4 °C. A positive control consisting of a pool of hyper-immune sera was included in each plate. The plates were washed and 50 µl of horse-radish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody  were added to all wells at a dilution 1:15,000 in PBS/T and incubated for three hours. After further series of washes, 100 µl of the substrate solution 3,3′,5,5′-tetramethylbenzidine (TMB)  were added. Reactions were stopped after 15 minutes with 50 µl per well of 0.2 M H2SO4. The optical density (OD) at 450 nm was read using Multiskan Go ELISA reader .All sera were tested for IgG antibodies by indirect quantitative enzyme-linked immunosorbent assay (ELISA) to two recombinant blood-stage 58. Seropositivity was determined by fitting a mixture model to normalised OD values assuming two Gaussian distributions, one for sero-negative individuals and another for sero-positive individuals25. The mean OD plus three standard deviations associated with the sero-negative group was used as the cut-off value for seropositivity. A separate cut-off was generated for each antigen. Differences in parasite prevalence and seroprevalence estimates between study areas and age categories were performed using a two-sided test for proportions and the corresponding exact binomial 95% confidence intervals (95% CIs). The titre of antibody responses was estimated using the equation dilution/[maximum OD/(OD test serum – minimum OD) – 1]; the median titre and interquartile range (IQR) are given. Differences in antibody responses were assessed using age-adjusted linear regression. Seroprevalence was stratified into yearly age groups and then analysed using a reverse catalytic modelling approach under a binomial sampling assumption, as described elsewhere13. This method provides estimates of the mean annual rates of conversion to seropositive  and reversion to seronegative  status, averaged over the age of the population. The common SRR was estimated from the model using maximum likelihood as described previously9. Infant under 1 year of age were excluded to remove any influence of maternally derived antibodies9. The serologically-derived annual EIR was estimated using AMA-1 and MSP-119 seroconversion rates and a calibration curve derived from determined values10. Factors associated with P. falciparum seropositivity were determined for each site separately using generalised estimating equations adjusting for correlation between observations from the same variables. The following factors were included: age in years, gender, fever, anaemia and enlarged spleen (children ≤12 years old). Variables that were significant at P < 0.10 in the univariate analyses were added to the multivariate model and retained in the final multivariate model if their association with immune responses was statistically significant at P < 0.05.Statistical analysis was performed using STATA/SE version 13.1  and GraphPad Prism Software version 5.03 . Duplicate ELISA OD values were averaged and normalised against values from blank wells as previously described to adjust for background reactivityThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Figure S1


Plasmodium falciparum and Plasmodium vivax are highly prevalent. The objective of this study was to describe treatment-seeking behaviour and household costs prior to a change to a unified treatment policy of dihydroartemisinin-piperaquine in Mimika district, Papua province in 2006.Malaria remains a significant public health issue in Eastern Indonesia, where multidrug resistant P. vivax compared with those diagnosed as P. falciparum.In 2005 a randomized cross-sectional household survey was conducted to collect data on demographics, socio-economic status (SES), treatment-seeking, case management, and household costs. Information on the cost of illness was also collected from patients exiting health facilities, in order to compare the cost of episodes diagnosed as P. falciparum or mixed infection received the first-line treatment of chloroquine with SP, and 38% (61/159) of those with a diagnosis of P. vivax reportedly received the first-line treatment of chloroquine and primaquine. Overall, public facilities were more likely to prescribe the correct prevailing first-line drug combinations than private providers . The mean cost to the household of an episode of P. vivax was similar to the cost of P. falciparum [US$44.50 (SD: 46.23) vs US$48.58 (SD: 64.65)].825 households were included in the survey. Of the 764 individuals who sought treatment for fever outside the home in the last month, 46% (349/764) went to a public health facility. Of the 894 reported visits to healthcare providers, 48% (433) resulted in a blood test, of which 78% (337) were reportedly positive. Only 10% (17/177) of individuals who reported testing positive for P. falciparum and P. vivax infections was substantial. Engagement with the private sector is needed to ensure that patients have access to affordable good quality, effective diagnostics and anti-malarials for both P. falciparum and P. vivax.Private providers were a popular source of treatment for malaria, but adherence to the national guidelines was low and the economic burden of malaria for both The online version of this article (doi:10.1186/s12936-016-1588-8) contains supplementary material, which is available to authorized users. Great progress has been made in reducing the burden of malaria in the Asia Pacific region, however the ultimate goal of eliminating malaria has been undermined by the emergence and spread of drug resistance. The World Health Organization recommends that once anti-malarial efficacy falls below 90%, treatment policy should be changed with a combination therapy with greater than 95% efficacy .The impact of any anti-malarial drug policy will depend on how it is implemented, and whether it ensures that individuals with malaria can easily access and receive the right treatment and diagnostics at an affordable cost. Early diagnosis of individuals with malaria and administration of effective treatment is critical to both to individual patients in ensuring rapid resolution of symptoms and preventing progression to severe disease, but also the community, by reducing further transmission and spread of anti-malarial drug resistance.In many malaria-endemic countries, the private sector is an important source of care for individuals seeking treatment for malaria and most of the spending is out-of-pocket. The private sector comprises a diverse range of providers including private hospitals, registered pharmacies and unlicensed drug shops. Having an understanding of the role of different types of health care providers and engaging with them appropriately is, therefore, very important if changes in policy are to result in actual change in how patients with malaria are treated. Treatment-seeking behaviour of patients with symptomatic malaria is influenced by socio-economic factors, knowledge and beliefs, and access to healthcare provision. Malaria disproportionately affects rural poor households for whom one episode of illness can have catastrophic results, not only in terms of morbidity and mortality but also in terms of financial burden.The literature on treatment-seeking behaviour for malaria, has shown that individuals often begin by taking treatment at home or from traditional healers and then move on to private drug sellers with few accessing public facilities directly , 3. In sPlasmodium falciparum and Plasmodium vivax are the dominant species , p = 0.007), although they were less likely to have a detectable parasitaemia .Out of the 894 visits to health care providers, a blood test for malaria was reportedly undertaken in 433 (48%) of these visits  was reportedly prescribed during 61% (547/894) of visits: 86% (471/547) of which were prescribed as tablets, 5% (30/547) as an injection and 8% (46/547) as both an injection and tablets. Overall, public facilities were significantly more likely to prescribe anti-malarial treatment than private providers ; however, the majority  of anti-malarial injections were administered by private clinics or doctors.In relation to parasitological testing prior to anti-malarial prescription, 61% (336/547) of the anti-malarial prescriptions followed a blood test. The corresponding figures were 87% (215/247) at public facilities and 40% (121/300) at private providers (p < 0.001).P. falciparum or mixed infection reported receiving the prevailing first-line treatment of chloroquine with SP, with the majority receiving primaquine , chloroquine  and/or quinine . Those reporting a diagnosis of P. vivax were more likely to receive the correct first line treatment of chloroquine and primaquine [38% (61/159); Table Only 10% (17/177) of individuals who reported testing positive for ine 103; 8% and/orP. vivax infection , 63% (69/109) reported taking less than 7 days of treatment  of respondents who reported a positive blood test for malaria, reported being prescribed primaquine. Of those who reported taking primaquine for 32/337 ofP. falciparum as compared with P. vivax infection. The total direct costs per person according to age group are presented in Additional file The mean total direct cost to the household per visit at a private provider was US$12.46 SD = 30.25) compared to US$3.52 (SD = 14.33) at the public sector (p < 0.001). Table 0.25 compOverall, 77% 641/834) of those who reported taking treatment stated that at least one person had reduced his or her usual activities to care for them and 66% (553/834) of respondents reported having at least one companion with them when seeking treatment. Eight percent (66/834) of respondents reported the need for at least one substitute labourer to perform their usual activities while sick and this was significantly more likely in non-Papuans compared to Papuans . Of those who reported a daily wage, the mean was US$10.92 (SD = 8.22). The days lost by the individual and his/her companions, caretakers and substitute labourers are shown in Additional File 1/834 of P. falciparum in 55% (196), P. vivax in 38% (136), mixed infection in 4% (12), P. malariae in 4% (13) and P. ovale in 0.3% (1). The direct and indirect costs related to visits at public providers for P. falciparum and P. vivax are presented in Table P. falciparum infection reportedly spent more money than those with P. vivax for the healthcare visit (p = 0.013), personal transportation to the healthcare facility (p < 0.001) and lost wages for the patient (p = 0.001). Individuals with P. vivax infection spent more money on companion costs (p = 0.034). When the analysis was restricted to children or adults, the only cost that remained statistically significant was for the healthcare visit.A total of 358 patients were surveyed after exiting six public health care facilities . The reported diagnosis was in 55% 19, P. vivaP. vivax malaria with P. falciparum malaria, as well as self-reported adherence to 14 day primaquine.This study from Papua, Indonesia, was conducted in an area with one of the greatest levels of malaria burden in the region and at a time of high levels of anti-malarial drug resistance to prevailing first-line therapies. It is the first study from eastern Indonesia reporting household treatment-seeking behaviour and is one of the few studies comparing household costs of There were several significant findings of relevance to malaria control efforts. Firstly in relation to treatment seeking, almost a third of individuals reporting a fever in the last month did not seek treatment outside of the home. This figure is higher than previous reports from central Java where 88% of households reported seeking treatment or advice outside of the home for their most recent malaria illness . Over haP. vivax infections who reportedly received primaquine, over half of participants reported taking less than three days of the recommended 14 day regimen, a duration ineffective in preventing relapse [Secondly in relation to anti-malarial treatment, of those patients who reported positive blood tests for malaria, few were prescribed the recommended drug regimen for the species that they reported; and for patients reporting  relapse .P. falciparum, but the majority of patients reported receiving it. With such a large proportion of P. falciparum patients reporting primaquine use, it seems unlikely that this is entirely due to patient recall bias in terms of the species diagnosed or the drug prescribed. Such discrepancies in anti-malarial policy and actual practice have been described previously in other settings [Furthermore, primaquine was not recommended for settings , 33. TheP. vivax malaria was US$44.50, compared to US$48.58 for P. falciparum. This likely represents an underestimate of household costs as patients were at public facilities and not at the end of their malaria episode.Thirdly in relation to household costs, the household financial burden of fevers in this region was substantial and did not differ substantially by the species of malaria. The mean cost per fever episode reported in the household survey was (US$53.54) and included the direct costs of the whole illness episode, including subsequent visits to treatment providers, as well as any further indirect costs of time incurred after visits to a healthcare facility. The mean cost of visiting a public provider for P. vivax episodes on the most vulnerable communities.Over 60% of households reported a monthly income less than $500, indicating that a fever episode represented at least 11% of the household’s monthly income. This also represents nearly 5 h of work using the mean reported hourly wage of US$10.92. Individuals in poorer households were at greater risk of fevers, with additional indirect costs likely to accumulate in those who do not take effective treatment. These findings emphasize the enormous economic burden of recurrent Another strength of this study, is the use of Discriminant Analysis of Principal Components to create meaningful SES categories. In this setting, use of the traditional asset index would have resulted in misclassification of households into categories which would have not reflected a useful categorization of household’s actual status. Re-settled Papuans had been provided with house and land by the local government resulting in many of the poorer households having high levels of house and land ownership but little other disposable income. Lower SES status was a risk factor for both recent febrile illness and parasite positivity at the time of the survey. Those with lower SES status were more likely to ever seek treatment at a public facility, a likely reflection of the lower costs incurred. This, along with the higher likelihood of reporting fever in the past month by those with lower SES, highlights the heterogeneity of the population in relation to risk of fever and treatment-seeking behaviour.This study has a number of limitations. Firstly, many of the older male household members were not present at the time of the survey and therefore not included in the analysis. In this region it is often the adult males who are at higher risk of malaria and so it is possible that we underestimated the true burden of fever episodes. Furthermore, males were more likely than females to seek treatment at private providers, which would have impacted the overall results with regards to treatment-seeking behaviour and costs . AnotherA common issue with all surveys aimed at describing treatment-seeking behaviour and case management for malaria, is the unknown malaria status of the individuals who reported having a fever in the past month. Including all fevers in the previous month, enables the potential capture of entire fever episodes; however, they may also result in significant recall bias due to the length of time between the episode and the survey. In order to triangulate these results, healthcare facility exit surveys were undertaken in which patients were interviewed immediately after their visit, thus minimizing the potential for bias and enabling an examination of costs specific to the type of malaria diagnosed. Any treatment or indirect costs occurring other than this healthcare visit, however, would not be captured by this survey and thus potentially impact its cost estimates.This study was undertaken at a time of very high treatment failure to the recommended first-line anti-malarials . The pooP. vivax were similar to P. falciparum, and in view of the relapsing and recurrent nature of malaria in this location, the financial burden of both infections is likely to be considerable. Anti-malarial programmes implementing policy change of first-line treatment will need to engage both public and private sectors if early diagnosis and highly effective treatment is to impact on the whole community.In summary these findings highlight a high level of reported febrile illness, which is most apparent in young children and those from poor households. Despite the provision of free treatment in public health facilities, treatment-seeking in the private sector was higher than expected, with considerably higher costs. The household costs of


Malaria worldwide annual reported cases range between 250 and 500 million and nearly half a million deaths are reported every year. Colombia has a vast expanse of territory with environmental and social conditions conducive to malaria transmission, which is the reason why it has second place in Latin America for the number of cases of malaria.This is a retrospective, paired, case–control study that compares patients with severe malaria and malaria patients without mention of complication. Medical records of patients with confirmed malaria diagnosis were reviewed. The objective of this study was to identify factors associated with complicated malaria in three municipalities of Colombia during the period 2009–2013.A total sample of 180 medical records was collected, 60 corresponding to cases and 120 to controls following a 1:2 ratio. From the total sample, 88.33 % (159) of subjects were originally from Tumaco, 7.78 %, most of the patients of the study (104) were diagnosed and treated in 2013. Among the laboratory findings, the platelet count was found to have statistically significant differences between cases and controls; this was also true for total bilirubin value and transaminases. The most prevalent severity finding was clinical jaundice, followed by elevated bilirubin (25 %) and elevated transaminases (44.4 %). Thrombocytopenia was found in 11/44 cases (20 %) and only five of 60 patients (8.3 %) presented severe anaemia. The multivariate analysis showed that a platelet count <100,000 and clinical jaundice not associated with organ failure, were the two variables associated with severe malaria in the patients described.Most of the cases studied, despite meeting criteria of severity, were shown not to be as morbid or severe as in other countries. Clinical jaundice and thrombocytopaenia are associated with severe malaria, and they can be used by general practitioners or primary care physicians to promptly identify patients who may have severe malaria. Plasmodium falciparum infections. Annual statistics of malaria cases worldwide range between 250 and 500 million, and nearly half a million deaths are reported every year. Severe cases and deaths from malaria occur mainly in the poorest regions of countries affected with social exclusion [Although other Plasmodium species can cause severe disease and death, most of these outcomes are reported in xclusion .Colombia has a vast expanse of territory with environmental and social conditions conducive to malaria transmission, which is the reason why it has earned second place in the number of cases of malaria in Latin America. In 2010 an estimate of more than one million cases of malaria and 1100 deaths from malaria was made in the South and Central American region. Brazil reported the highest number of cases (54.4 %), followed by Colombia (13.1 %) and Venezuela (9.3 %). The largest number of deaths from malaria reportedly occurred in Brazil, Colombia and Dominican Republic [The major complications of malaria described in literature are cerebral malaria, pulmonary oedema, acute renal failure, severe anaemia, bleeding, acidosis and hypoglycaemia . CerebraThis is a retrospective, matched, case–control study that compares patients with severe malaria and malaria patients without mention of complication. The data used in this study were collected from medical records of patients with confirmed malaria diagnosis, hospitalized between 2009 and 2013, in four different hospitals located in endemic areas of the Colombian states of Nariño and Valle del Cauca, covering the municipalities of Tumaco, Buenaventura and Cali. The total data collection time was 14 months, starting in 2012.Considering the findings of the study performed by Tobon et al. , the proPlasmodium species. Matching by year of presentation and municipality was done to assure that the patient base population of cases and controls remained constant. The area where the study was conducted has a population that is constantly changing and moving in between regions due to their economical activities, such as mining, and the effect of the armed conflict that affects the area. The biggest proportion of cases of severe malaria is reported in the young adult population. Given the fact that malaria presentations change importantly with age, a decision was made to not include the age in the matching process so further analysis could be made from the outcomes of the study related with the age. A bivariate analysis was conducted to determine if the variable age was confounding factor or a factor associated with severity. Due to the lack of information regarding previous malaria episodes in the medical records reviewed, matching by this variable was not possible.Matching of cases and controls was performed considering year of malaria diagnosis, municipality of origin and A detailed review of the National System for Public Health Surveillance (SIVIGILA) databases from 2009 to 2012 was performed to identify the medical records of cases and controls available to review in each municipality. Once the data were obtained, a formal request was sent to each institution to obtain access to the medical records. In order to obtain enough medical records of malaria patients, information from malaria public health notification forms located in the Public Health and Statistic Departments of the hospitals were also used.® tool and was filled using mobile devices with internet access to assure real-time accessibility and quality control of the data being collected.An on-line instrument in the form of a questionnaire was designed as well as a guideline with definitions, inclusion and exclusion criteria, to be used by trained doctors to collect the data from the medical records in the designated areas. The instrument included: personal identification, past medical history, history of present illness, clinical characteristics, physical examination, diagnosis, paraclinical and parasitological results on admission, treatment, and case outcome. The instrument was accessible via email, using the SurveyMonkeyTo ensure the instrument was filled out correctly, three independent reviewers evaluated the quality of the information filled out for each medical record, using a constructed validation mesh interdisciplinary with the statistical unit, and provided feedback to the doctors when necessary to correct discrepancies.Plasmodium vivax or P. falciparum, made by thick or thin smears and treated in the municipalities were included in the study.Patients with signs or symptoms of malaria, with confirmed diagnosis of malaria infection with either Cases of sickle cell disease, viral or bacterial infections and/or other diseases that could explain the clinical presentation, as well as cases that originated from a municipality not included in the area of analysis, or cases that were transferred from another municipality to receive medical care were excluded.P. vivax or P. falciparum via thick blood smear slides, and with one or more of the clinical or paraclinical criteria mentioned in the Colombian National Guidelines for severe malaria, were defined as cases, as shown in Table Subjects hospitalized between 2009 and 2013, with diagnostic confirmation of The Colombian National Guidelines differ from the World Health Organization (WHO) guidelines, for severe malaria they define hepatic dysfunction as transaminase values >40 IU. According to medical literature and WHO, malarial hepatic dysfunction is defined as an elevation of three times the upper limit of normal transaminase values. An intermediate value of >80 IU was utilized in this study in order to analyse the possibility of proposing a new cut-off point for the Colombian guidelines, given the low prevalence of hepatic failure and hepatic dysfunction in Colombia .P. falciparum or P. vivax, that did not have any of the severity criteria mentioned for case definition and were proven to be infected and treated in the municipalities, were included in the study.Patients with clinical malaria diagnosis confirmed by thick or thin blood smear slides, proven to be infected with In order to identify inconsistencies and extreme values in the dataset, an exploration of the variables included in the study was performed. A univariate descriptive analysis, including the demographic, clinical and laboratory data was performed using frequency tables and central tendency measures according to the type of variable.Plasmodium species, a Student’s t test was performed to analyse differences between case and control in the numeric variables, and McNemar’s test for categorical variables. Conditional logistic regression was used to calculate the odds ratio and 95 % confidence intervals to find association between independent variables and severe malaria. In order to find association between clinical factors and severe malaria, a multiple conditional logistic regression was performed using the variables that were found to be statistically significant (P < 0.05) or relevant in the bivariate analysis, controlling confounding factors and analysing possible interactions between variables. Verification that the statistical models complied with all the assumptions of the logistic regression was also performed.Given the design of the study, paired case control study 1:2 ratio, paired by year of diagnosis, municipality and ® version 12.Variables considered in the statistic bivariate analysis and statistical model were: gender, age, place of origin, duration of symptoms before admission, symptomatology on admission, presence of co-morbidities, days hospitalized, vital signs on admission, thick and thin blood smear slide results, physical examination on admission, medical treatment received, initial laboratory values, pregnancy status, previous history of malaria, and prior medical attendance before admission. Statistical analysis was processed using STATAEthical approval was granted by the ethics committee that permitted access to patient medical history and data collection for analysis and conversion to a scientific manuscript.The total sample collected for cases and controls was 180, 60 medical records corresponding to cases and 120 to controls, following a 1:2 ratio. From the total sample 88.33 % (159) of the subjects were originally from Tumaco from which 53 were cases and 106 were controls. 5.00 % (nine) from Cali, three were cases and six controls, and 6.66 % (12) from Buenaventura four were cases and eight controls. Regarding the year of medical consultation, six of the subjects were seen in the emergency room in 2009, 17 in 2010, 12 in 2011, 41 in 2012, and 104 in 2013.Half of the malaria cases with confirmed severity criteria were women, and the other 50 % were men. For the control group, 46.67 % (56) were women and 53.33 % were men. As seen in Table Regarding the general system of social security affiliation status, more than 90 % of the cases and controls were affiliated at the time of their medical attention, meaning that medical attention should be provided to them by law in any public health institution.The average number of days hospitalized for the cases was of 4.1 days, (SD ± 1.77), the average days since onset of symptoms until first medical attention according to the information contained in the medical records was 5.72 days for cases (SD ± 4.45 days) and 5.60 for controls (SD ± 4.97 days). From the total sample, 71 % (43) of the cases and 45 % (54) of the controls were referred from other institutions where they received their first medical attention. Hospitals providing basic medical care, first-level institutions were found to be the main medical institutions 90.7 %.P. falciparum (73 %) and 16 P. vivax (26 %). For controls, 87 were confirmed P. falciparum (72 %) and 33 P. vivax (27 %).Forty-four cases were confirmed to be In the medical records, evidence was found that 27 % of the cases and 19 % of the controls took medication without prescription before admission to hospital. The most common medications found to be used by the patients were home-made remedies (12 %) and analgesics (87 %). From the total sample, 29 % of cases and 16 % of controls had a known medical condition at admission (co-morbidities), the most common being gastritis (10), high blood pressure (5), asthma (4), urinary tract infection (4), and diabetes (3).As shown in Table Table The most prevalent severity finding in the cases group was clinical jaundice as described above, followed by elevated bilirubin 15/15 cases (25 %) and elevated transaminases 12/27 (44 %). Thrombocytopaenia was found in 11/44 cases (20 %) and only five of 60 patients (8 %) presented severe anaemia.Table Regarding the neurological signs and symptoms, only one patient presented with loss of consciousness, and one patient presented with multiple seizures; no data was available regarding the presence of signs of prostration or inability to feed oneself. Few patients presented with respiratory complications: three patients showed signs of respiratory distress syndrome and one had evidence of pulmonary oedema through radiography.There was no data available to determine the presence of metabolic acidosis, or hyperlactataemia. Further information about severity findings at the time of admission is presented in Table Malaria mortality and severity in Colombia, especially in the Pacific region, has maintained a decreasing trend given the improvement in knowledge of the population and from the health providers regarding early symptoms and malaria prevention, as well as more coverage and access to early diagnosis and treatment .During the last decade the Pacific area has received help from the Global Fund for AIDS, the US National Institute of Allergy and Infectious Diseases, the Colombian Ministry of Social Protection and National Institute of Health, which has significantly contributed to a reduction in malaria transmission and mortality, given education and training of health personnel and communities , 9. HoweIn this study, the adult population was found to be affected in greater proportion than other age groups: only 11 % of the sampled patients were under 5 years of age, as has previously been described in the literature , 9. The Even though the patients analysed in this study had severity criteria that classified them as having severe or complicated malaria, the severity of the signs and symptoms was not as high as described in areas of Africa or Asia. The literature proposes clinical immunity to malaria in low-transmission settings to be one of the reasons for this phenomenon . The ColIn Colombia, the diagnosis of severe malaria before 2010 was made using the criteria established by the WHO; however in this year the Colombian Ministry of Health created a document based on the literature to adapt the criteria for the Colombian population, which included more conservative criteria, such as anaemia (haemoglobin <7 g/dL in adults and children), renal dysfunction (serum creatinine >1.5 mg/dL), severe thrombocytopaenia , and hyperparasitaemia , and this is currently been used by healthcare professionals in the country to diagnose severe malaria . However3 [3 were found to be a factor associated with severe malaria, and were described in 30 cases, with an odds ratio (OR) of 9.41 IC 95 % .As described in the literature, thrombocytopaenia is common in severe malaria cases in Colombia; in most cases a mild to moderate thrombocytopaenia  is seen and generally does not represent a risk of spontaneous bleeding . In the 3 , which wJaundice was another factor found to be associated with severe malaria in the present study, and it is important to point out that cases found to have jaundice had no organ damage or dysfunction, which suggests that jaundice is a relevant clinical sign in patients where severe malaria is suspected , 11.Severe anaemia significantly contributes to malaria mortality. The literature reports that mild to moderate anaemia in the majority of the population is related to malnutrition or intestinal parasites and other conditions related to poverty. In this study, 85 patients presented with haemoglobin levels <12 g/dL, considered as anaemia for both men and women, but two patients presented with haemoglobin levels less than 7 g/dL, i.e., severe anaemia and a criterion of severe malaria [Cerebral malaria was not observed in this study, and its incidence has been reported to be low in Colombia .Limited data are available regarding the association between malaria severity and co-morbidities, as opposed to infectious co-morbidities. In this study, no association was found between them: OR 1.93 CI . In the population studied, ten cases and 17 controls were found to have self-medicated with home-made remedies and over-the-counter analgesics before seeking medical attention. This needs to be further analysed in other studies, given its possible relation to the time of consultation of patients.Differences between species regarding the prevalence of thrombocytopaenia, cerebral malaria and other severity criteria have been described in the literature, however no conclusion or analysis between species was made in this study given that a match by species was made as part of the design of the study.Plasmodium species, characterization of the affected population, or the treatment options available. Most studies available are retrospective and developed in the Pacific area. More studies that describe the current situation and contribute to characterization of complicated malaria in patients in Colombia are needed [The literature regarding the characteristics of complicated malaria in Colombia is limited, there is a broad spectrum of clinical manifestations, and there is no information regarding multiple infections and the development of immunity, association of severity signs with different e needed , 9.It is of utmost importance for health personnel specially physicians to identify the red flags for severe malaria, described in the Colombian literature as danger signs. Changes in mental status, seizures, jaundice, bleeding among others have been described as danger signs and should be detected early in order to prevent complications and mortality associated with malaria. In this study, jaundice was found to be the main sign associated with severity in the population studied, and it is well-recognized as a danger sign in the Colombian literature.Even though platelet count severity criteria for the Colombian Guidelines and WHO are 50.000 and 20.000/µL, respectively, in this study having platelet counts less than 100.000/µL was found to be a significant danger sign .The research was rigorous, following the methodology described and as mentioned there were three reviewers of the data, however one of the weaknesses is that it was a retrospective study, there were difficulties in collecting data, there was a lack of quality of medical records by omission of relevant clinical data in patients attending consultation for malaria. The data collection was first programmed to be done in specific municipalities selected in Colombia, based in the information of areas of higher transmission but due to administrative issues this was not possible. The data collection was limited to the municipality of Tumaco where medical records were available. Buenaventura Hospital was closed down during the research process which made it impossible to access medical records. In Cali, most of the patients were found to have non-complicated malaria, and there were few medical records that had severity criteria and were considered cases to pair with the controls.The data collected form the medical records reviewed showed that most cases of malaria that occur in the population, despite meeting criteria of severity, have been shown to be not as morbid or severe as in other countries. The study showed that jaundice and thrombocytopaenia are associated with severe malaria, and they can be used with other signs and symptoms by general practitioners or primary care physicians to promptly identify patients to may progress towards clinical complications.


To the Editor—We thank Rossi and colleagues for sharing their findings from Cambodia [Plasmodium falciparum in subjects with suspected malaria. Sensitivity was low at 72% (compared to 52% in our study). Low-density infection, defined as <100 parasites/μL, explained 75% of false-negative results (compared to 76% in our study). With the large sample size of 4382 patients, sampling of all RDT negatives (vs selective sampling employed in our study), and use of quantitative PCR, the study is a useful addition to the few published studies on performance of RDT to assess symptomatic malaria in low-transmission settings [Cambodia , which cCambodia . Using pPlasmodium falciparum strains from Cambodia have been associated with a lower pyrogenic threshold than some African and American strains [As malaria transmission declines, the proportion of low-density infection among symptomatic as well as asymptomatic individuals increases . It is g strains . Early aMissed low-density infections represent missed opportunities to prevent further transmission. They also represent missed opportunities for transmission reduction activities in the community, as passively identified cases may trigger targeted interventions such as active case detection and vector control. On the flip side, overdiagnosis is also a problem. We would like to note that the false-positivity rates, or the percentage of healthy individuals who incorrectly receive a positive test result, were incorrectly reported in both studies. The correct false-positive rates were low at 5.9% in Swaziland and 0.3% in Cambodia . However, due to the low prevalence of malaria, positive predictive values (PPVs) were compromised. Rossi et al report a higher PPV than our study , but a PPV of 89% still equates to overtreatment in roughly one-tenth of patients, and potential “overintervention” in the communities where activities were triggered by passively detected cases. A new RDT with reported sensitivity 10 times higher than current RDTs has recently been launched. While its use has potential to reduce transmission [