Plasmodium falciparum malaria and soil borne helminth attending Kampala International University Teaching Hospital (KIU).Malaria and helminths share the same geographical distribution in tropical Africa. Studies of the interaction of helminth and malaria co-infection in humans have been few and are mainly epidemiological, with little information on cellular immune responses. This study aimed to determine Cytokine profiles among patients co-infected with Plasmodium falciparum malaria were 55 (22.9%) and those with soil-borne helminths were 63 (26.3%). The controls were 89 (37.1%), while those co-infected with Plasmodium falciparum malaria and soil-borne helminths were 33 (13.8%). Cases were defined as having a positive blood smear for P. falciparum malaria, those with helminths or co-infections of the two. Negative controls were those with a negative blood smear for P. falciparum malaria and those with no stool parasitic infections. Patients presenting with signs and symptoms of malaria or those suspected of having helminths were recruited for the study. A panel of five cytokines  were assayed from plasma samples in patients with and without Plasmodium falciparum malaria, patients with and without helminth, and then those co-infected with the two diseases diagnosis was done using thick blood smears stained with 10% Giemsa and stool examination was done following the Kato Katz technique following standard procedures.A case control study of 240 patients were recruited at KIU teaching hospital. Patients with Plasmodium falciparum malaria by sex was 28 (11.7%) and 27 (11.3%) in male and female respectively. The overall prevalence of soil borne helminth was 26.3%, and among those harbouring helminths, 13.8% were co-infected with Plasmodium falciparum. Cytokine levels significantly differed across Plasmodium falciparum malaria, soil borne helminth infected patients and health controls for IFN-γ (P = 0.023), IL-10 (P = 0.008) and TGF-β (P = 0.0001). Cytokine levels significantly differed across Plasmodium falciparum malaria, soil borne helminth infected patients and patients co-infected with Plasmodium falciparum malaria and soil borne helminth for IL-10 (P = 0.004), IL-6 (P = 0.011) and TGF-β (P = 0.003).The prevalence of Plasmodium falciparum malaria and an up-regulation of IL-10 and TGF-β in soil borne helminth infections was demonstrated. We demonstrate that co-infections of Plasmodium falciparum and soil borne helminth lead to an up-regulation of IL-10 and IL-6 and a down-regulation of TGF-β.An up-regulation of IFN-γ during Trial registration No17/10-16 Malaria remains a global burden with approximately 584,000 deaths among an estimated 198 million cases annually . This scHowever, in other related studies no apparent effect of helminths co-infections on malaria risk or disease severity were observed –19. On tP. falciparum and soil borne helminth infection separately [Plasmodium falciparum malaria and soil borne helminth co-infections among patients attending Kampala International University Teaching Hospital in Uganda.Malaria infections are generally characterized by a T helper 1 (Th1) response predominated by a number of pro-inflammatory cytokines with a gradual shift to Th2 response as the disease progresses , 26. Proparately , few stuP. falciparum malaria, those with helminths or those co-infected with both diseases. Negative controls were those with a negative blood smear for P. falciparum malaria and those with no stool parasitic infections. Patients presenting with signs and symptoms of malaria or those suspected of having helminths were recruited for the study. From each patient and control, 5 ml of venous blood was collected into EDTA vacutainers. Malaria diagnosis and parasite quantification was done using Giemsa stained thin and thick blood smears [A case–control study was conducted at Kampala International University Teaching Hospital Out Patients Department (OPD) in Bushenyi district. Cases were defined as having a positive blood smear for d smears . Stool ed smears  and wormd smears . For staUpon recruitment into the study, a detailed clinical history was sought from each patient. Physical examination of patients was performed by a medical office. Data recorded on the clinical form included the patient’s demographic characteristics, clinical presentation, and perceived onset of illness. Children with other stool parasitic infections, those on anti-helminthic and malarial treatment were excluded from the study.g using an Eppendorf centrifuge. Plasma was subsequently aliquoted and stored at − 20 °C in the hospital laboratory refrigerator until further analysis. Samples were later transported in an ice cold box to immunology and molecular biology laboratory at the College of Veterinary Medicine, Animal Resources and Biosecurity (COVAB), Makerere University for cytokine assay analysis.For immunological assays, 5 ml blood were collected from each patient in EDTA vacutainers and centrifuged for 10 min at 3000P. falciparum diagnosis was done using thin and thick blood smears stained with 10% Giemsa. Parasite evaluation was done by microscopic examination of 200 fields at 100× magnification and parasite density expressed as number of parasites/µl [Upon recruitment, sites/µl .Panels of both pro-inflammatory and counter inflammatory cytokines  were assayed from plasma samples using solid phase sandwich enzyme-linked immunosorbent assay  assay following manufacturer’s instructions as previously described . All assStool samples were collected in dry leaky proof plastic bottles and approximately 3 g were used. Stool examination was done following the Kato Katz technique  and wormAll data were anonymized prior to analysis using Graph Pad Prism version 6.0. All numerical variables was summarized using mean and standard error of the mean (SEM). Before analysis of cytokine data, deviation from normality was tested using D’Agostino-Pearson normality test. Because none of the cytokines presented a normal distribution, data were presented as medians. Comparisons between groups were done using Kruskal–Wallis non parametric test followed by Dunn’s post-test at a significant level (P < 0.05).Plasmodium falciparum malaria were 55 (22.9%) and those with soil-borne helminths were 63 (26.3%). The controls were 89 (37.1%), while those co-infected with Plasmodium falciparum malaria and soil-borne helminths were 33 (13.8%) as shown in Table Plasmodium falciparum malaria by sex was 28 (11.7%) and 27 (11.3%) in male and female respectively. Severe Plasmodium falciparum was detected in only 4 (4.5%) patients as compared to those with moderate 8 (9.1%) and low parasite intensity 76 (86.4%). Plasmodium falciparum intensity according to sex was 42 (47.7%), 3 (3.4%) and 4 (4.5%) for low, moderate and severe respectively in males and 34 (38.6%), 5 (5.7%) and 0 (0.0%) for low, moderate and severe respectively in females.In this study a total of 240 patients were recruited. The mean age of patients was 7.6 ± 3.0 years with a sex ratio of 1.1 . Patients with Plasmodium falciparum. The most prevalent soil-borne helminths observed was Ascaris lumbricoides, 72 (75.0%) followed by hookworms, 20 (20.8%). Mixed infection were only of Ascaris lumbricoides and hookworms with a prevalence 4 (4.2%). No Trichuris trichiura infections were observed in the study population. Among the helminths co-infections, Ascaris lumbricoides had the highest prevalence 26 (78.8%) as compared to hookworms 7 (21.2%). No significant differences were observed in soil-borne helminths across sex . Among those harbouring helminths, 33 13.8%) were co-infected with 3.8% werePlasmodium falciparum malaria or soil borne helminth infections, cytokines from each group of patients were compared with health controls. The results showed that cytokine levels significantly differed across groups for IFN-γ  levels of IFN-γ (47.7 pg/ml) as compared to health controls (8.8 pg/ml) and soil borne helminths infected individuals  and healthy individuals  and healthy controls  and soil borne helminths infected individuals  and soil borne helminths infected individuals  and co-infected individuals  higher levels of IL-10 (304 pg/ml) as compared to alaria 77.7 pg/ml alaria 77.7 pg/ml Ascaris lumbricoides, and the hookworm species are common infections in Bushenyi district among the soil borne helminths [Ascaris lumbricoides had the highest prevalence (75.0%) followed by hook worms (20.8%). In this study no Trichuris trichiura infections were detected, similarly to what was observed by Agwu et al. [Plasmodium falciparum malaria prevalence was (22.9%) lower than that reported in previous studies in Uganda 27% [Ascaris lumbricoides had the highest prevalence (78.8%) and results from this study are in range with other studies done elsewhere in Madagascar which reported that among the helminths co-infections, Ascaris lumbricoides had the highest prevalence of 77% [, Plasmodium falciparum malaria infected individual expressed significantly higher levels of IFN-γ (47.7 pg/ml) as compared to health controls (8.8 pg/ml) and soil borne helminths infected individuals (22.8 pg/ml). This is in agreement with a study done in south East Asia and elsewhere which reported that Plasmodium falciparum malaria expressed higher IFN-γ levels  and healthy individuals (26.09 pg/ml). This is probably because IL-10 is a key anti-inflammatory cytokine protective against helminth infections that cause inflammation [Plasmodium falciparum malaria infected individuals (772 pg/ml) and healthy controls (424.6 pg/ml). This is probably because TGF-β has been attributed to wound healing of the intestinal mucosa during tissue repair caused by helminths in the gut [Plasmodium falciparum malaria and soil borne helminths infected individuals [Plasmodium falciparum malaria infected individuals and soil borne helminths infected individuals. Co-infected individuals expressed significantly higher levels of IL-10 (304 pg/ml) as compared to Plasmodium falciparum malaria infected individuals (11.57 pg/ml) and soil borne helminths infected individuals (77.01 pg/ml). Results from this study are in agreement with a previous study by Hartgers who reported high expression of IL-10 in co-infected individuals [Plasmodium falciparum malaria infected individuals (63.7 pg/ml) and soil borne helminths infected individuals (45.37 pg/ml). This is because IL-6 has been shown to possess both pro and anti-inflammatory features [Plasmodium falciparum malaria (772.7 pg/ml) and co-infected individuals (939.2 pg/ml).This down regulation of TGF-β in co-infections is indicative that co-infected individuals lose the protective role of TGF-β against soil borne helminths during infection. IFN-γ and TNF-α levels showed no significance across groups. This is probably because the higher levels of IL-10 in co-infections and soil borne helminths down regulates IFN-γ by supressing T cells from producing IFN-γ hence the decrease [The over prevalence of soil borne helminth was 26.3%). This is in range with what has been reported previously in Uganda , Tanzanielminths . This isu et al.  in Bushe6.3%. Thianda 27%  that repanda 27%  who repoanda 27% , 41 and e of 77% . In our n et al. ). These  malaria . This ef malaria , 44. Alsammation . Also so the gut , 46. TNFof IFN-γ .7 pg/ml ividuals  and probividuals , 49. Lacividuals . Up reguividuals , 52. Co-features , 54. Soiontrols 4.6 pg/ml. malaria .64 pg/mlPlasmodium falciparum malaria and an up-regulation of IL-10 and TGF-β during soil borne helminth infections. We further show an up-regulation of IL-10 and IL-6 during co-infections of Plasmodium falciparum and soil borne helminth infections and a down-regulation of TGF-β during co-infections of Plasmodium falciparum and soil borne helminth infections. However, how these cytokines may influence the clinical outcome of the diseases involved calls for further investigations.In conclusion, our study demonstrates an up-regulation of IFN-γ during


Plasmodium falciparum malaria. However, its impact on the three other main species of human malaria and lymphatic filariasis (LF), which share the same vectors in many co-endemic areas, is not as well characterized. The recent development of multiplex antibody detection provides the opportunity for simultaneous evaluation of the impact of control measures on the burden of multiple diseases.Universal coverage with long-lasting insecticidal nets (LLINs) is a primary control strategy against P. falciparum rapid diagnostic tests (RDTs). Dried blood spots were collected and analyzed for presence of antibodies to three P. falciparum antigens, P. vivax MSP-119, P. ovale MSP-119, P. malariae MSP-119, and three LF antigens. Seroconversion rates were calculated and the association between LLIN use and post-campaign seropositivity was estimated using multivariate regression. The campaign covered 68% (95% CI: 58–77) of the population in Nacala-a-Velha and 46% (37–56) in Mecubúri. There was no statistically significant change in P. falciparum RDT positivity between the two surveys. Population seropositivity at baseline ranged from 31–81% for the P. falciparum antigens, 3–4% for P. vivax MSP-119, 41–43% for P. ovale MSP-119, 46–56% for P. malariae MSP-119, and 37–76% for the LF antigens. The seroconversion rate to the LF Bm33 antigen decreased significantly in both districts. The seroconversion rate to P. malariae MSP-119 and the LF Wb123 and Bm14 antigens each decreased significantly in one of the two districts. Community LLIN use was associated with a decreased risk of P. falciparum RDT positivity, P. falciparum LSA-1 seropositivity, and P. malariae MSP-119 seropositivity, but not LF antigen seropositivity.Two cross-sectional household surveys at baseline and one year after a LLIN distribution campaign were implemented in Mecubúri and Nacala-a-Velha Districts in Nampula Province, Mozambique. Both districts were known to be endemic for LF; both received mass drug administration (MDA) with antifilarial drugs during the evaluation period. Access to and use of LLINs was recorded, and household members were tested with P. falciparum RDT positivity, the most common measure of P. falciparum burden. However, the significant decreases in the seroconversion rate to the P. malariae antigen, coupled with an association between community LLIN use and individual-level decreases in seropositivity to P. falciparum and P. malariae antigens show evidence of impact of the LLIN campaign and highlight the utility of using multiantigenic serological approaches for measuring intervention impact.The study area noted significant declines in LF seropositivity, but these were not associated with LLIN use. The MDA could have masked any impact of the LLINs on population LF seropositivity. The LLIN campaign did not reach adequately high coverage to decrease  Plasmodium falciparum malaria is the principal cause of illness and death in Mozambique. However, the same mosquitoes that transmit P. falciparum parasites also transmit three other species of malaria  and the worm that causes lymphatic filariasis. To date, we do not know how much transmission of the three other species of malaria occurs. We also do not know if control interventions such as the distribution of bed nets reduce the transmission of lymphatic filariasis and non-falciparum malaria. To address this question, we sampled community members immediately following and one year after a bed net distribution campaign in Mozambique. We analyzed their blood for the presence of antibodies to four species of malaria and lymphatic filariasis. We found that a substantial proportion of individuals had antibodies to P. falciparum, P. malariae, P. ovale, and the worms causing lymphatic filariasis. We found much lower rates of seropositivity to P. vivax. Individuals reporting using bed nets had a lower risk of testing positive for P. falciparum and P. malariae antibodies. The proportion of the population with access to and using bed nets was too low to cause a population-wide decrease in malaria transmission. There was a significant decline in lymphatic filariasis seropositivity between the two surveys, but we could not attribute it to the bed net distribution campaign. Measuring antibody levels for multiple diseases simultaneously has utility in assessing intervention impact. Plasmodium falciparum transmission and disease burden in the world [P. falciparum malaria parasites is among the principal causes of outpatient visits, hospitalizations, and deaths. Malaria transmission occurs year-round and P. falciparum prevalence in children under 5 was measured to reach up to 65% in the central and northern provinces in 2015 [Northern Mozambique has one of the highest rates of he world . As in tP. falciparum parasites are the most deadly and common agent transmitted by Anopheles vectors in Mozambique, anopheline mosquitoes are also responsible for transmission of three other species of human malaria–P. malariae, P. ovale, and P. vivax–as well as the lymphatic filariasis (LF) parasite Wuchereria bancrofti. In contrast to P. falciparum, the distribution and burden of non-falciparum malaria and LF in Mozambique, as in most of sub-Saharan Africa, has not been well characterized. Reasons for this include poor diagnostic capability, less severe manifestations of disease, and less attention and funding from ministries of health and international donors. In general, P. vivax has historically been thought to be largely absent from sub-Saharan Africa due to the lack of the Duffy coat receptor in populations originating in West Africa [P. ovale and P. malariae are thought to be in circulation in Mozambique, estimating their incidence of infection has been difficult, particularly since they are typically present at low parasite densities and are difficult to detect through slide microscopy, especially in the presence of a concomitant P. falciparum infection. Lymphatic filariasis has been mapped to be most prevalent in northern and central Mozambique [Although t Africa . Althougzambique , but theP. falciparum infection focus on vector control [Anopheles mosquitoes are targeted by indoor residual spraying with insecticides and the distribution and use of long-lasting insecticidal nets (LLINs), which, in addition to protecting the user with a physical barrier, in practice also function as human-baited insecticidal mosquito traps and can significantly reduce mosquito populations [P. falciparum infections, LLINs are known to reduce transmission of P. vivax [P. ovale and P. malariae [Currently, the most effective strategies for reducing the burden of  control . In Mozaulations . The MozP. vivax  and havemalariae  and the malariae . Althougmalariae , 9, netsmalariae .As part of its monitoring and evaluation program, the Mozambican NMCP periodically evaluates LLIN distribution campaigns. The objectives are to both monitor the operational performance of the campaigns, as assessed through coverage and usage indicators, as well as to measure the impact of the campaigns on malaria prevalence and estimates of transmission. The latter is particularly important amid the rise of insecticide resistance and the potential for diminishing effectiveness of LLINs in controlling malaria transmission. In 2013, the Mozambican NMCP chose to evaluate a LLIN distribution campaign in the northern province of Nampula. Besides measuring coverage and falciparum malaria prevalence, additional components were added to the evaluation to measure the prevalences and assess the impact of the campaign on non-falciparum Plasmodium species. A further component was included to assess the additional impact of the LLIN campaign on LF; this was complex as both districts received MDAs for LF during the evaluation period. These additional objectives were made possible by the recent development of multiplex serology methods that allow detection of antibodies to multiple antigens simultaneously . It was Two consecutive cross-sectional household surveys were carried out one year apart, with the first, baseline survey occurring two weeks following the mass LLIN distribution campaign in 2013. Achieved coverage with LLINs was assessed during the first survey, LLIN usage was assessed during the follow-up survey one year later in 2014, and impact of the campaign was assessed by comparison of biological markers of infection from the first and second surveys.A LLIN distribution campaign encompassing six districts of the northern province of Nampula was implemented in the second half of 2013. Of the six districts, two were purposefully chosen to be included in the survey: the coastal district of Nacala-a-Velha, and Mecubúri District in the interior . Both diP. falciparum parasites, assuming a baseline prevalence of 43% and a design effect of 3.For the survey, twenty enumeration areas (survey clusters) in each district were chosen randomly with probability proportional to size from the full list of census enumeration areas for each district. Four clusters in Mecubúri were inaccessible and were substituted with four randomly chosen replacement clusters. Immediately prior to the start of the survey, trained enumerators visited each selected cluster and compiled a full list of households, recording the name of head of household and the latitude/longitude coordinates of the household. For each cluster, the list of households was randomly sorted, and in the first survey in 2013, survey teams visited households according to the order of the list, continuing until 16 households were visited per cluster. In the second survey in 2014, survey teams revisited households from 2013, matching households based on the name of head of household and coordinates, and no replacement of households was allowed. In each household, all household members present were invited to participate in the survey. The target sample size was 1,320 individuals and 367 households per district, designed to provide 80% power to detect a 10% change between the two surveys in the proportion of the population testing positive for P. falciparum HRP2-specific rapid diagnostic test (RDT) , and had up to six 10 mcL spots of capillary blood collected on filter paper . Individuals testing positive for malaria were treated by survey teams in accordance with national treatment guidelines [Trained teams, each composed of a national-level supervisor and a district surveyor, visited the selected households and, after obtaining consent, administered a household questionnaire. Surveyors collected socioeconomic data, including occupation and education of the head of household and household ownership of goods; generated a roster of household members; enumerated all sleeping spaces and recorded who slept in which sleeping space; visually inspected each sleeping space and recorded the presence and location (hanging or stored) of the bed net designated for that sleeping space, denoting whether or not the bed net bore the marking specific to the distribution campaign; and asked the interviewee about how often on average each bed net was used during the wet and dry seasons. In both years, each household member, regardless of age, present during the visit and providing written consent was administered a idelines . In 2014The surveys were carried out in September 2013 and October 2014 in Nacala-a-Velha and December 2013 and November 2014 in Mecubúri, with data collection lasting three weeks for each survey in each district.After collection in the field, the filter paper was dried overnight, placed into individual plastic bags with desiccant sachets, and then refrigerated prior to shipment to central laboratories in Maputo and later to CDC laboratories in Atlanta. Blood spots were eluted overnight at 4°C at a serum dilution of 1:40 (assuming 50% hematocrit) and further diluted in casein-containing dilution buffer as previously described for a final dilution of 1:400 of serum , 14.Strongyloides stercoralis) (19) from each of the four main human malaria species was cloned and expressed as recombinant Schistosoma japonicum glutathione-S-transferase (GST) fusion proteins [P. vivax MSP-119 expression clone that included the carboxy-terminal hydrophobic tail sequence was used [5 peptide corresponding to the carboxy-terminus of the P. falciparum circumsporozoite protein (CSP) was cross-linked to GST and then coupled to a SeroMap bead as previously described [P. falciparum Liver Stage Antigen 1 (LSA-1) [Strongyloides stercoralis NIE antigen-GST fusion protein, GST fusion partner with no inserted sequence, and the Brugia malayi Bm14- and Bm33-GST fusion proteins were cloned and expressed as previously described [W. bancrofti Wb123 antigen expressed as a GST fusion protein was a gift of T. Nutman . Although only W. bancrofti occurs in Africa, the serological test for the B. malayi antigens cross-reacts with W. bancrofti.A multiplex bead platform  was usedcoralis) . The 19-proteins –16. A P.escribed . The Pl1 (LSA-1)  was syntescribed –21. W. bP. vivax GST/MSP-119, all other antigens were coupled to SeroMap beads  using the buffers and protein amounts previously described [P. vivax antigen was coupled to a BioPlex COOH bead  using the protein amount and buffer previously specified [minus the buffer-only blank value (MFI-bg). Samples (N = 8) that had discordant result between the two runs (coefficient of variation >15%) for >4 positive antigen responses were repeated.With the exception of the escribed . The P. pecified . Total Ipecified . Assays plus 3 standard deviations of these negative control values were considered positive. For the S. stercoralis NIE assay, a cutoff value determined by a receiver-operator characteristic curve analysis using a different lot of coupled beads was translated to the current study bead set using a 2-fold serial dilution curve of a strong positive control sample as an inter-assay standard.For malaria and LF antigens, cutoff values were determined using a panel of 81 presumed negative sera from adult US citizens who had no history of foreign travel. Values greater than the mean A subset of samples (20%) did not have full blood spots available; for these, partly filled blood spots were analyzed. Comparison of average MFI-bg values from full and partial blood spots revealed an average difference of <10%; thus, all samples were included in the final statistical analysis.P. falciparum infection by RDT was calculated for each district and year, stratifying by age. Estimation of coverage indicators and malaria positivity was adjusted taking into account the complex sample design using the R survey package [Demographic characteristics for the heads of household were tabulated, and a socioeconomic status (SES) index was constructed and calculated for each household using a previously described methodology . Househo package . Data weStrongyloides NIE antigen was included as a control antigen to aid in discriminating between the effects of the LLIN and MDA campaigns, as Strongyloides transmission was presumed to be unaffected by the LLIN campaigns but sensitive to the MDA campaigns.For each of the antigens the mean seropositivity, defined as the percent of individuals with MFI-bg above the predetermined threshold, was calculated for the total population and also stratifying by ten age categories. A reversible catalytic model was fit to the seropositivity by age data for each antigen, and the estimates for the serological conversion rate (SCR) and serological reversion rate (SRR) per year were directly calculated from the likelihood model . The SRRPoisson regression, which allows direct estimation of the relative risk,  was usedAll statistical analyses were performed in R version 3.3.2 .The study was approved by the National Bioethics Committee in Mozambique. Adult participants provided written consent prior to enrollment in the study, and also provided written consent on behalf of child participants. CDC investigators provided technical assistance and were not considered to be engaged in the research.A total of 282 households in Nacala-a-Velha and 300 households in Mecubúri were visited in the first survey in 2013 . The totThe coverage attained by the LLIN distribution campaign was low . The camP. falciparum RDT positivity from 2013 in 2014 in either district with overlapping 95% confidence intervals, although the point estimates for RDT positivity were higher in 2014 versus 2013  .For both to 0.35) . The onlPlasmodium species were detected in both young and old individuals in both districts  of the population in 2013 in Nacala-a-Velha and 56% (52–60) in Mecubúri seropositive for antibodies against P. malariae MSP-119, with a corresponding SCR of 0.068 (0.054–0.081) in Nacala-a-Velha and 0.095 (0.077–0.11) in Mecubúri. Similar levels of exposure were observed for P. ovale, with the proportion of the population with antibodies to P. ovale MSP-119 in 2013 ranging from 41% (37–45) in Nacala-a-Velha to 43% (39–47) in Mecubúri, and an SCR estimated to be 0.12 (0.087–0.15) in Nacala-a-Velha and 0.13 (0.093–0.16) in Mecubúri. Much lower levels of antibody positivity to P. vivax were observed, with only 2.7% (1.5–4.5) of the population in Nacala-a-Velha and 3.9% (2.5–5.9) in Mecubúri with detectable antibodies to P. vivax MSP-119. There was a statistically significant difference in SCR for P. malariae between the two surveys in Nacala-a-Velha, with the estimate for SCR for 2014 24% lower than in 2013 . There was a similar reduction in the SCR for P. ovale in Nacala-a-Velha, with a 22% reduction, but this was not statistically significant . The SCRs for P. malariae and P. ovale in Mecubúri did not show a similar reduction, falling by only 1% and 3%, respectively, with neither antigen showing a statistically significant difference in SCR between the two surveys .Robust antibody responses to the three other istricts , S3 Fig,The absolute levels of antibody response and seropositivity by age curves for the three LF antigens were consistent with the geographic distraction of LF , S4 Fig.Strongyloides NIE antigen in 2013. In Nacala-a-Velha, there was no statistically significant difference in SCR  or seropositivity  to the NIE antigen between the two surveys, whereas Mecubúri witnessed statistically significant declines of 35% for the NIE SCR  and 17% for NIE seropositivity  from 2013 to 2014.A substantial proportion of the population in Nacala-a-Velha  and Mecubúri  had antibodies against the control P. falciparum RDT positivity from 2013 to 2014, both individual- and cluster-level LLIN use was associated with lower risk for P. falciparum RDT positivity in the second year after adjusting for age, sex, and SES . Although there was no detected change in overall  and SES . The relP. falciparum RDT positivity observed in these surveys, coupled with the high rates and age distribution of antibody levels to P. falciparum antigens, confirm holoendemic transmission of P. falciparum in the survey area. The levels of RDT positivity are particularly striking given that the surveys were conducted at the end of the dry season, when transmission would be expected to be lowest. Areas of large malaria burden would benefit most from a mass LLIN campaign. However, the coverage indicators provide evidence that the LLIN campaign evaluated here was far from reaching its intended target of universal coverage. Reported usage, ranging from 17% to 40%, was far removed from the 65% threshold postulated to be necessary in providing a demonstrable community reduction in malaria incidence [P. falciparum infection by RDT, mirroring results from previous studies of LLIN effectiveness in Mozambique [The high rates of ncidence . Neverthzambique  and confP. falciparum transmission intensity. The results show that not all antigens are consistently informative in this setting. For example, the P. falciparum MSP-119 antigen, which to date has been one of the standard antigens used to assess population-level P. falciparum exposure [P. falciparum intensity in a setting of such high transmission as northern Mozambique. The transmission intensity is such that virtually all sampled individuals regardless of age had antibodies to MSP-119, evidence that the first P. falciparum infection likely occurs in early infancy. However, the two other P. falciparum antigens included in the assay, CSP and LSA-1, generated seroprevalence curves with increasing likelihood of transitioning to seropositive with increasing age. Both antigens are thought to be less immunogenic than MSP-119, and the data presented here suggest that repeated P. falciparum infections throughout life are needed to generate a consistently high antibody level to each antigen. Due to the slower acquisition of antibodies to these two antigens, the seroprevalence by age data were informative, allowing differentiation between the higher transmission in Mecubúri versus Nacala-a-Velha, an observation also seen in the RDT positivity data. Additionally, P. falciparum LSA-1 seropositivity was lower in LLIN users, further evidence of the protective effect of LLINs against P. falciparum infection.This study highlights the added benefit of simultaneously measuring seropositivity to multiple antigens to estimate exposure , 29 provP. falciparum transmission, the populations in both districts showed substantial serological responses to P. ovale and P. malariae antigens. A small but non-zero proportion of the population showed evidence of exposure to P. vivax, consistent with the results of a 2015 household survey which showed a national 0.2% P. vivax prevalence in children under 5 years of age [19 antigen competition studies have not indicated antibody cross-reactivity in most individuals [19 antibody responses were common even among patients who had high responses to multiple malaria MSP-119 antigens. Although the MSP-119 antigens from P. vivax and P. falciparum share 51% identity at the amino acid level, some of the conserved residues are cysteines and other hydrophobic amino acids that are unlikely to be exposed to the immune response [19 antigens in ELISA studies of sera from a population living in a region endemic for both parasites and did not observe any correlation between the P. vivax and P. falciparum antibody responses [19 antigen from only one species [P. malariae and P. ovale antigens can be solely attributed to assay cross-reactivity.In addition to the two districts being a setting of very high ividuals , and speresponse . Bousemaesponses . In a se species . Thus, iP. ovale MSP-119 and P. malariae MSP-119 in these two districts in 2013 suggest an annual incidence of P. ovale infection of 125/1000 in Nacala-a-Velha and 133/1000 in Mecubúri, and an annual incidence of P. malariae infection of 70/1000 in Nacala-a-Velha and 100/1000 in Mecubúri. This magnitude of incidence would elevate P. ovale and P. malariae as major contributors to malaria burden in northern Mozambique.Although subject to many limitations, the SCR at its most basic definition is a measure of incidence, the annual rate at which individuals acquire antibodies to a certain antigen . Taken aP. falciparum, there was a statistically significant population-level decrease in P. malariae seropositivity and a borderline significant decrease in P. ovale seropositivity from 2013 to 2014 in one of the districts. Since LLIN use was associated with lower post-campaign risk of testing positive for P. malariae antibodies, there is evidence that the LLIN campaign, at least in Nacala-a-Velha where coverage was higher, might have had an impact on decreased P. malariae transmission. The fact that there were detectable changes in seroprevalence and distribution of P. ovale and P. malariae markers and no change in P. falciparum might be due to the differences in magnitude of transmission intensity. One hypothesis is that with such high levels of P. falciparum transmission, there would need to be a much larger decrease in vectorial capacity to result in a detectable change in incidence, whereas non-falciparum transmission might be low enough to be sensitive to smaller changes in vectorial capacity.In contrast to Strongyloides antigen, which should be influenced by the MDAs but not by the LLIN campaign, suggest that there was high enough coverage from the antiparasitic MDAs to reduce LF transmission. In Nacala-a-Velha, however, there was no significant difference in NIE Strongyloides SCR and seropositivity between the two surveys, and thus the population-level declines in SCR and seropositivity to LF antigens could be due to the LLIN distribution campaign. Overall, there was no association between individual or community LLIN use and seropositivity to LF antigens. Given the confounding due to the concurrent use of MDAs in the survey districts, this result cannot be interpreted as evidence of no effect, as the effect might have been masked by the antifilarial MDAs.In both districts there was evidence of significant declines in LF transmission between 2013 and 2014, as seen by decreases in both the SCR and overall proportion of the population seropositive for the LF antigens. The results are robust as they hold across all three different LF antigens included in the assay. However, attributing this change to the LLIN distribution campaign is hampered by the concurrent MDAs of antiparasitic drugs in both districts. Ivermectin is effective against LF microfilariae, in addition to a postulated killing effect on mosquitos feeding on individuals treated with ivermectin, and albendazole kills adult worms. Together, the two-drug combination could be expected to influence antibody levels through its effect on transmission and worm load. In Mecubúri, the substantial declines in SCR and proportion of the population seropositive for the NIE P. falciparum transmission on the backdrop of low LLIN coverage argue for follow-up campaigns in these two districts, both of which will take part in the nationwide universal coverage campaign in Mozambique launched in 2016. Similar evaluations are recommended to evaluate coverage and usage of future campaigns. Finally, the results presented here provide evidence for the enhanced utility of the multi-antigenic and multi-disease assay for quantifying baseline exposure to the non-falciparum malarias and LF, and evaluating the impact of vector control intervention campaigns on these diseases.Several aspects of the study’s design prevent direct inference of a causal relationship between the LLIN distribution campaign and the observed changes in malaria positivity and serological outcomes for malaria and LF. The lack of a control group and the MDA campaigns in the intervening year hamper direct estimation of the impact of the LLIN campaign. In addition, the low coverage and usage resulting from the LLIN campaign and the lower-than-expected sample size limited the ability of the study to assess the impact of LLINs on malaria and LF transmission. Additionally, as IgG against some antigens is known to persist for years following infection , more elS1 Fig(PDF)Click here for additional data file.S2 FigRed line indicates cut-off used to determine seropositivity. MFI: Median Fluorescent Intensity.(PDF)Click here for additional data file.S3 FigRed line indicates cut-off used to determine seropositivity. MFI: Median Fluorescent Intensity.(PDF)Click here for additional data file.S4 FigRed line indicates cut-off used to determine seropositivity. MFI: Median Fluorescent Intensity.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Background. Changes in lipid profile are commonly reported in adult patients with malaria. However, a few studies evaluated lipid abnormalities in children continuously exposed to P. vivax. Objective. To evaluate lipid abnormalities in children with P. vivax infection and to assess if parasite count or the history of malaria correlates with lipid levels at admission. Methods. A total of 75 children were included in the study, from which 43 were slide confirmed infection by P. vivax. Serial blood samples were collected at admission and, on days 7 and 14, evaluated for the colorimetric measurements of triglycerides, very low-density lipoprotein (VLDL), total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL). Results. The levels of total cholesterol, LDL, and HDL were significantly lower in malaria cases. The levels of VLDL and triglycerides were significantly higher in children with malaria. Such changes were transient and were not associated with parasite counting as well as with the history of malaria of patients. Conclusion. There are significant lipid abnormalities in children with low level of P. vivax infection and mild signs and symptoms of the disease, which are not associated with parasitaemia and previous episodes of disease. Plasmodium vivax is the most prevalent species and accounts for 85% of the cases [Malaria is a major public health issue in several tropical countries . In Brazhe cases , 2. Mosthe cases , 7. Plasmodium as well as in complicated or noncomplicated cases [Changes in serum lipid profile have been associated with genetics and with infectious and inflammatory diseases as well as in chronic or acute conditions , 9. In med cases –13. P. falciparum [ P. vivax is the most prevalent species and children have a high incidence of the disease. There is a paucity of data describing lipid abnormalities in children with vivax malaria. Such changes can increase the risk of chronic diseases, as atherosclerosis and related diseases, which is relevant for children continuously exposed to malaria [ P. vivax infection and to assess if parasite count and history of malaria correlate with lipid levels at admission.Most of the studies designated to evaluate lipid abnormalities were performed in adult patients withlciparum , 14, 15. malaria , 17. Add malaria . Thus, tThe study was submitted to Plataforma Brasil under protocol CAAE 2 07199612.0.0000.0018 and approved under the number 261.593/2013.2 and 27,540 inhabitants, from which 7,347 are children aged 2–10. The economy is based on agriculture practices, timber extraction, and fishing. The municipality accounts for 17.4% of malaria cases in the state of Pará and has a constant annual incidence of cases above 50/1,000 inhabitants. Most cases (86.3%) are caused by P. vivax. The municipality has seven health public facilities, including an ambulatory for the diagnosis and treatment of malaria.The study was carried out in the municipality of Anajas in the Marajo Island (00° 59′ 21′′S and 49° 56′ 24′′W). Anajas has an area of 6,912 Km P. vivax and (2) children with negative diagnoses of malaria after microscopic examination of three blood smears. Exclusion criteria included patients with mixed malaria or signs and symptoms of severe malaria , chronic and other parasitic diseases, and obesity and underweight assessed by the body mass index and those who have reported a history of malaria infection within the previous two months.Participants were recruited by spontaneous demand among those who referred for attendance at health facilities of the municipality from January to December, 2014. Two groups of children were randomly included in the study: (1) children with slide confirmed infection by P. vivax were treated with chloroquine and primaquine according to the recommendation of the World Health Organization [Children withnization . Doses oSerial blood samples were collected at baseline (D0) in both groups of children recruited for the study and on days 7 and 14 after the commencement of the treatment in children with vivax malaria. Samples were collected after a fasting of 12 h. Triglycerides, total cholesterol, HDL, and LDL were measured by spectrometer methods using Merck- diagnosis kits, following the good laboratory practices. VLDL was measured by enzyme-linked immunosorbent assay (LSBio Inc.). Samples were analyzed in triplicate. The normal ranges of each lipid followed the recommendations of the Brazilian Cardiology Society .µL [Parasite counts were performed every 24 hours in Giemsa-stained thick films until clearance. Blood films were examined microscopically using 100x (oil immersion) objectives. Parasite density was expressed as the number of parasites per microliter of blood. This was derived from the number of parasites per 200 white blood cells in a thick film, considering a total white blood cell count of 8,000. The limit of detection of parasites was 40/U test was used to compare the lipids levels between groups at admission on the study. A univariate analysis was used to compare lipid levels amongst days of blood sampling in children with malaria as well as to compare lipid levels amongst patients classified according to the number of previous episodes of malaria. Spearman rank correlation was used to estimate the correlations between parasitaemia at admission with triglycerides, total cholesterol, and fractions. Data were analyzed using SPSS software, Release 21 . The significance level accepted was 5%.Data are presented as a mean and standard deviation and, if necessary, as median and range. Chi-square test was used to compare proportions of altered lipid levels between groups of study. Mann–Whitney  P. vivax corresponded to 57.3% (n = 43) of the participants. The baseline characteristics of participants are shown in A total of 75 children met the criteria for their inclusion in the study. Children with positive slide confirmed infection byU = 403.5; p = 0.0011). A similar result was found for VLDL fraction , with the higher values in malaria cases (15.5–68.7 mg/dl), compared to healthy children (15–39.2 mg/dl). The total cholesterol was significantly lower  in malaria cases (82–208 mg/dl) compared to healthy children (ranges: 97–220 mg/dl). As expected, HDL levels were significantly lower  in malaria cases (ranges: 2–49 mg/dl) compared to healthy children (ranges: 32–61 mg/dl). The values of LDL (p < 0.001) were also lower  in children with malaria (9–136 mg/dl), compared to healthy children (14–138 mg/dl). The lipid levels comparison between case and control subjects is shown in At admission, triglycerides levels were higher in patients with malaria (ranges: 71 to 196 mg/dl) when compared to healthy children (68–243 mg/dl) . For total cholesterol, there are no values below the normal ranges in both groups. Additionally, the proportion of malaria cases with values of HDL below normal ranges (92.8%) was significantly higher than the control group (40.62%) . Only children with malaria (97.6%) showed LDL levels below normal range. In the control group, most children (93.7%) showed LDL levels within the normal range and there were no values below normal range.The proportion of patients with triglycerides above normal ranges was also higher in malaria cases (55.8%) when compared to control group (25%)  and VLDL  decreased significantly between D0 and D14. On the other hand, total cholesterol , HDL , and LDL  increased significantly between D0 and D14. The evolution of lipid concentrations versus time is shown in Significant changes in serum lipid profiles were seen in the follow-up, as follows: triglycerides , total cholesterol , HDL , LDL , and VLDL .The number of previous episodes of malaria was not associated with triglycerides , total cholesterol , HDL , LDL , and VLDL .Finally, there were no significant correlations between the parasitaemia at admission with triglycerides ( Plasmodium, human host, or both [ Plasmodium in both phases of its life cycle in the human host [Abnormalities in lipid and lipoprotein levels are commonly reported in physiological and nonphysiological conditions , 16. The or both , 13, 21.man host . These oman host –25. Addiman host , 24, 26. Plasmodium for their optimal development in hepatocytes, since there is no biological pathway for sterol production, which is obtained from the exogenous or endogenous pathways of the human host [In the study, all participants have total cholesterol levels within normal range values. However, such levels were significantly lower in children with malaria compared to the healthy control. Reduction of total cholesterol level is a common finding in patients with malaria and has been associated with metabolic requirements ofman host , 23, 27. Plasmodium [Cholesterol rich lipoprotein levels, such as LDL and HDL, have also decreased significantly in children with malaria at admission, compared to healthy children. These changes agree with most studies designated to evaluate lipid abnormalities in patients with malaria and other infectious diseases , 16, 28.asmodium , 23, 26.On the other hand, triglycerides and VLDL, a lipoprotein rich in triglycerides, increased significantly in children with malaria, when compared to the control. A plausible explanation is an increase in the hepatic VLDL production and secretion in serum accomplished by a decrease in the clearance of this lipoprotein. Additionally, the role of cholesterol ester transfer protein in mediating the exchange of triglycerides from triglyceride rich lipoproteins to other lipoproteins is likely , 26.The severity of infection was associated with abnormalities in lipid levels in different studies, but the results were inconclusive , 31. Som Anopheles bites in this endemic setting and most participants of both groups have a history of malaria. Thus, it may be worth assessing if the history of malaria may influence lipid levels. In the study, the number of previous episodes of malaria was not associated with the lipid levels. A plausible explanation for such finding is the temporality of changes in lipid profiles. Studies evaluating temporal changes in lipid profiles of patients with malaria have demonstrated inconclusive results, ranging from a few days to one year [There is a continuous exposure to theone year , 14, 32.one year , 10, 23.Further studies should investigate the impact of low levels of cholesterol rich protein on the development, cognition, and nutritional status of children continuously exposed to malaria as well as the atherosclerosis risk and related diseases in such patients. P. vivax and mild signs and symptoms. Lipid abnormalities were characterized by decreased levels of total cholesterol, LDL, and HDL and by the increased levels of VLDL and triglycerides. These changes were transient and were not associated with parasite count, as well as with the history of malaria of patients.Changes in the lipid profile are significant in children with low level of parasitaemia by


Plasmodium vivax and P. ovale infections, the follow up period was extended to 12 months to establish treatment failures and relapses. Fifty nine uncomplicated and 15 severe imported malaria cases were reported in Sri Lanka during the study period. Most of these infections originated in either Sub-Saharan Africa or South and Southeast Asia. Having a P. vivax infection and low parasitic counts were significantly associated with relative diagnostic delay. One of the 14 uncomplicated P. falciparum patients and two of the 12 severe P. falciparum malaria patients who were followed up till day 28 had a late clinical failure. The others responded adequately to treatment both clinically and parasitologically. There was no treatment failure reported amongst any other species. This study, which is the first to assess the therapeutic response of imported malaria in Sri Lanka after elimination, demonstrates that the current antimalarial treatment policies and strategies in Sri Lanka have been effective against infections acquired overseas up until the end of year 2016.After eliminating local malaria transmission and being certified as a malaria-free country, Sri Lanka is facing the challenge of imported malaria. At the same time, the country has the unique opportunity to be a case study for other countries in a similar situation by approaching this issue systematically, guided by evidence. This study demonstrates the importance of developing a mechanism to detect imported malaria and adopting an evidence-based approach to study the resistance of imported malaria to anti-malarial medicines. This is a prospective study of patients diagnosed with imported malaria in Sri Lanka and treated according to the national treatment guidelines, over 24 months (2015/2016). The clinical features, time to diagnosis, origin of the infection, infecting species, parasite density and the treatment given were recorded. All patients were followed up for 28 days, and in the case of  Sri Lanka was declared a malaria-free country by the World Health Organization in September 2016 using quPlasmodium falciparum and P. vivax[To date, on a global scale, resistance to currently used antimalarials, including partner drugs used in artemisinin combinations has been documented in two of the five malaria species known to affect humans in nature:  P. vivax. The curAnopheles culicifacies is abundant in the environment favouring onward transmission from an index case[Prior to the commencement of the study, in 2013–2014, 144 imported malaria cases were reported in Sri Lanka, and 89 (62%) of these had been acquired from South and Southeast Asian countries . The second largest source of imported malaria to Sri Lanka was the continent of Africa. The current study on therapeutic efficacy of Sri Lanka’s first-line antimalarial medicines was deemed necessary to inform the most appropriate treatment policies for the country. The receptivity to malaria is high in Sri Lanka as the main vector ndex case. ContinuP.vivax infections[All main countries from which imported malaria cases had entered Sri Lanka , have endemic CQ resistant nfections–11. ThisP.vivax malaria [P.vivax and P.ovale malaria cases.Primaquine is being used as the hypnozoitocidal agent in Sri Lanka and the AMC recommends 0.25 mg of primaquine base/kg/day daily for 14 days for radical cure of  malaria , 12. Cur malaria . TherefoP. falciparum malaria in Sri Lanka and in most other countries where the disease is endemic. Resistance of P. falciparum to artemisinin and its partner medicines[P.falciparum and P.vivax globally, it is necessary that Sri Lanka monitors the therapeutic efficacy of its first line antimalarials to imported malaria for the purposes of informing drug policy and thereby preventing the re-introduction of malaria to the country.Artemisinin combination therapy (ACT) is the first line treatment for medicines is prevamedicines, 16. A rmedicines fell shomedicines. Yet, itEthical approval for the study was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo (EC 14–165) and the Ethics Review Committee of the Sri Lanka Medical Association (ERC/13-053).This study reports cases of imported malaria from January 2015 to December 2016 (24 months) in Sri Lanka and gives a brief description of epidemiological characteristics and the response to anti malarial medicines in patients with a confirmed diagnosis of malaria during this period .P. vivax and P.ovale malaria patients were followed up for a further 12 months with monthly blood smears to monitor breakthrough relapses (due to primaquine failure).Parasitologically confirmed malaria patients reported during the 24- month study period were included in the study. Informed written consent was obtained from all enrolled patients (and from the parent or guardian if the subject was a minor). Every patient diagnosed with malaria was managed as per national guidelines with quality assured drugs . FollowiOnce a patient was confirmed as having malaria  by microscopic examination at AMC headquarters or the Regional Malaria Office by Giemsa staining and examination under the light microscope, another blood smear was taken for the purpose of further scrutiny and quantification of the parasitaemia according to the protocol recommended by the World Health Organization . An entiP.vivax and P.ovale patients were tested for G6PD status using rapid diagnostic test kits .Demographic and clinical data  were collected by an interviewer filled questionnaire. Clinical data were collected on admission, before the commencement of treatment and thereafter daily during the hospital stay. Body temperature before the commencement of treatment was taken as the D0 temperature. Temperature measured at 24, 48 and 72 hours after the commencement of treatment was taken as the D1, D2 and D3 temperature respectively. Haemoglobin level was measured prior to starting therapy and repeated if deemed necessary. All P. falciparum cases were treated with 6 doses of artemether-lumefantrine (AL) scaled by age/weight as outlined in the monogram of the product. AL was given (after a glass of milk) as a tablet of 20 mg of artemether and 120 mg of lumefantrine . If there was evidence of severe malaria, intravenous artesunate was given at a dose of 2.4 mg/kg until the person could tolerate oral treatment (subject to a minimum duration of intravenous therapy of 24 hours), at which point treatment was switched to oral AL at the above dosage and continued to completion of the full 3 days of treatment. All P. falciparum patients were treated with primaquine 0.75 mg/kg as a single dose on the 3rd day at the time of discharge from hospital. If the patient first presented with uncomplicated falciparum malaria and then transitioned to severe disease, they were first treated with oral artemether-lumefantine and subsequently with intravenous artesunate.Uncomplicated P. vivax, P. ovale and P. malariae infection were treated with 25mg/kg of chloroquine for a period of 3 days at the dose of 10mg/kg on days one and two and 5 mg/kg on day 3. Primaquine 0.25 mg/kg daily for 14 days was issued on the 3rd day at the time of discharge from hospital to patients diagnosed with P.vivax and P.ovale after excluding G6PD deficiency. P.knowlesi infection was treated with AL [0 C were given paracetamol 1g 6 hourly. Antimalarial medicines were given under direct observation of health staff. Compliance with primaquine was monitored by AMC staff during the follow up visits. The participants were observed for 30 minutes after giving the drug dose to ensure that there was no vomiting. If vomiting occurred, the full treatment dose was repeated.Patients diagnosed with  with AL . PatientThe primary outcomes measured were; a) Early treatment failure (ETF), b) Late clinical failure (LCF), c) Late parasitological failure (LPF) and d) Adequate clinical and parasitological response (ACPR) as defined according to the World Health Organization guidelines . The folEthical clearance for the study was obtained from the ethics review committee of the Sri Lanka Medical Council and the ethics review committee of the Faculty of Medicine, University of Colombo.According to the estimates of AMC, approximately 2.2 million screening tests for malaria had been performed during the study period of 24 months in Sri Lanka. These included 1,142,466 blood smears in 2015[There were 59 patients with uncomplicated malaria and 15 patients with severe malaria included in the study. Males comprised a majority (93.2%) of the study population . Fifty tFour individuals with severe malaria (26.7%) had acquired the infection from Mozambique. One to two infections each were acquired in other African countries such as Madagascar, Uganda, Tanzania, Nigeria, Ivory Coast and Sudan. Of the two foreigners who had severe malaria, one was a tourist and the other a seaman. Neither were citizens of an African country.P.vivax accounted for a greater proportion of uncomplicated malaria infections (n = 32), followed by P.falciparum, P.ovale infections (n = 18 and 7 respectively). There was one case each of P.malariae and P.knowlesi infections. All except one P. falciparum infections were acquired in an African country, while all but one P.vivax infections were acquired in an Asian country. Severe malaria was solely due to P.falciparum and acquired in the African Continent.Species wise, P.falciparum malaria and 10.1 days (range 1–53 days) for P.vivax malaria. The severe malaria patients who developed symptoms following their return to Sri Lanka were diagnosed within an average of 4.3 days  since arrival in Sri Lanka. The average time taken since the onset of symptoms to diagnosis in all patients with severe malaria was 6.6 days (SD±3.6). A Kaplan Meier survival analysis showed that time to diagnosis from the onset of illness or from arrival in Sri Lanka did not differ significantly between severe and non-severe malaria cases (data not shown). In the uncomplicated malaria cases, the parasitaemia at diagnosis in P.falciparum ranged from 32–53,040/ μl (median: 1510/μl) and in P.vivax, from 160 to 49,655/µl (median: 2725/µl). In severe malaria cases the parastiaemia at diagnosis ranged from 18,985–668,000/ μl   for 23.4/μl) .P.falciparum patients who were afebrile, the rest of the 56 patients presented with fever with or without chills and rigors. Most of the severe malaria patients had an impaired level of consciousness  and followed up for three days. There was no loss to follow up at the end of D3 after which they received a single dose of primaquine. All these patients except four (who left the country) were followed up until D28. Apart from a single patient who had late clinical failure, all responded to a full course of AL treatment  and this time responded well to treatment.All patients had normal G6PD enzyme levels and were treated according to the standard anti-malarial regimens recommended by WHO and AMC of Sri Lanka , 13. Uncreatment . This paP. vivax malaria who left the country within the first 72 hours, the rest completed their chloroquine treatment under direct observation by hospital or AMC staff. Another seven P.vivax patients left the country before completing the 14 day course of primaquine. Two P.ovale patients were lost to follow up by day 14. All other patients with P. vivax (n = 22) and P. ovale (n = 5) infections completed their 14 day primaquine course. One other P.vivax patient was lost to follow up by D28. The treatment efficacy at D28 for the non-falciparum infections was 100%. Eleven  P.vivax malaria patients and four  P.ovale patients completed the 12 month follow up, and none developed a relapse. Two P.vivax patients are still being followed up and the remainder  were lost to follow up as they left the country.With the exception of three patients diagnosed with P.malariae patient who had asexual parasitaemia till D7, other two patients who were positive for P.falciparum showed zero parasitaemia on D4.Regarding the secondary outcomes in the 56 patients with uncomplicated malaria, who were febrile at presentation, the fever clearance rate was 92.8% (52/56) by D3. All cases were afebrile by D4. The parasite clearance rate was 94.9% (56/59) by D3. Except for the Plasmodium species or the initial parasite count . However time to diagnosis (since the onset of symptoms) was significantly longer when the patients had P.vivax malaria    or when malaria) .P.falciparum malaria.For patients with severe malaria, treatment was commenced with IV artesunate in seven individuals and in the other eight, treatment was initiated with oral artesunate but soon replaced with IV artesunate. Early treatment failure was not observed in any of the patients with severe malaria. A majority  had a fever and parasite clearance time of less than 72 hours and all were both clinically and parasitologically cleared by D7. Two of the patients (both Sri Lankans) had late clinical failure with a recurrence of symptoms and parasitaemia at D15 and D19 respectively after an initial “apparent” cure. The presumed source of infection was Congo and Madagascar respectively. In both these patients the initial infection was treated with 3 doses of IV artesunate at a dose of 2.4 mg/kg at which point they were able to tolerate oral medication and thereafter a full 3 day course of AL was administered. The recurrent infections in both patients were re-treated with another three doses of intravenous artesunate followed by a full course of oral AL and both patients responded to treatment clinically and parasitologically. The two foreign patients with severe malaria left the country between D3-D14 after attaining early treatment success so their outcome with regard to late clinical failure is unknown, as with one Sri Lankan. Thus three patients with severe malaria were lost to follow up by day 28. Overall, adequate parasitological and clinical response to this case series was  by D28 . There wP.falciparum, P.malariae and P.knowlesi infections) or one year post treatment .This study assessed the clinical and parasitological response to recommended treatment schedules of imported malaria in Sri Lanka over a 24 month period. Due to the low number of malaria cases being reported in Sri Lanka , every patient diagnosed with uncomplicated malaria by microscopy  was included in this study irrespective of the parasite count at diagnosis. All attempts were made to follow up patients who remained in Sri Lanka until 28 days post-treatment  of patients with tuations , 24, thiP. vivax malaria patients in one of whom the diagnostic delay was nearly two months. Diagnostic delay with P.vivax malaria had been observed to be significant  in a previous study in Thailand and this was attributed to the less severe symptomatology of the infection[P. falciparum may also be explained by a) almost all of them coming from African destinations and b) a significant proportion of them being defense force members on official duty who receive appropriate health education and systematic screening for parasitaemia upon return. It is also possible that people returning from African destinations are more conscious of the risk of malaria due to high transmission intensities in Sub-Saharan Africa and promptly seek medical advice in event of suggestive symptoms. Delay in diagnosis was also associated with lower than average parasitaemia, this may have been due to the resulting lower intensity of symptoms or even atypical symptoms which would not therefore raise a clinical suspicion of malaria. Low parasite densities may have also given rise to false negative RDT or microscopy readings in the early days of the illness leading to a delay in diagnosis.The response rate to treatment in uncomplicated malaria patients was excellent with only one late clinical failure being reported out of fifty nine patients. The clinical and parasitological response rates were similar in the group of severe malaria as well . Delays in diagnosis as measured by the time from onset of illness to diagnosis was a concern, more so for infection. The fasP. falciparum infections (mostly acquired in Africa) and P. vivax infections (mostly acquired in South and Southeast Asia) were highly responsive to the first line chemotherapeutic agents namely; artemether + lumefantrine or choloroquine + primaquine respectively. The only late clinical failure was reported for a P.falciparum infection acquired from Mozambique. The response to AL for P.falciparum infections in Mozambique as measured from sentinel site data is close to 100% however a few cases of artemisinin resistance have been reported [All uncomplicated reported . As illuSri Lanka was declared malaria free in September 2016 but all of its South Asian neighbours except the Republic of Maldives are endeP. falciparum. These observations suggest that routine follow up of patients with P. falciparum malaria is essential at least until 28 days since beginning of treatment. Regarding P.vivax malaria, there were no relapses reported in any of the treated subjects suggesting the adequacy and efficiency of the current radical cure strategies. Both P. ovale and P. malariae infections identified during this study responded to the medicines used. Neither species were endemic in Sri Lanka prior to malaria elimination.Our results indicate that current anti malarial treatment regimens offered in Sri Lanka are effective against all species of malaria among travellers to the country at present. The only treatment failure observed in uncomplicated disease was in a Sri Lankan returning from Mozambique infected with P.vivax malaria where only 11 patients completed their 12 month follow up. Hence the efficacy of primaquine in attaining radical cure cannot be established with certainty. It was not possible to improve these numbers as all foreigners and most of the locals, who defaulted, had left Sri Lanka.The attrition rate during follow up was high for several subgroups. This was especially evident in the case of Anopheles sp. found in abundance, it is important to diagnose early and effectively cure all imported malaria cases to prevent re-establishment of local transmission. This has to be guided by regular monitoring of the species profile of infections, the country of their origin, the time taken to diagnosis, response to medicines and relapse patterns during follow up. The current study which examined these parameters in patients during 2015 and 2016, suggests that a) the prevailing treatment schedules and strategies have been effective against infections imported during the study period; b) there are unacceptable delays in diagnosis of malaria particularly in the case of vivax malaria, and c) P. falciparum infections, almost all of which originated in sub-Saharan Africa, need to be followed up (at least up to 28 days since initiation of treatment) to detect late clinical / parasitological failures. International non-immune travelers acquiring malaria overseas is a useful group to monitor treatment efficacy to guide treatment policies after elimination of malaria. They also serve as a sensitive sampling pool to detect changes in parasite response to medicines in endemic countries where they got infected from.After cessation of local transmission of malaria, Sri Lanka faces the challenge of tackling imported malaria. Since the receptivity to malaria is high in the country with the vector


Plasmodium falciparum malaria susceptibility. However, information on host genetic factors and their relationships with malaria in the vaccine trial site of Iganga, Uganda is limited. The main objective of this study was to determine the prevalence of selected host genetic markers and their relationship to malaria incidence in the vaccine trial site of Iganga, Uganda. In a 1-year longitudinal cohort study, 423 children aged below 9 years were recruited and their malaria episodes were investigated. Host genetic polymorphisms were assessed by PCR–RFLP, haemoglobin electrophoresis and DNA sequencing. Using a multivariate negative binomial regression model, estimates of the impact of human genetic polymorphisms on malaria incidence were performed. In all statistical tests, a P value of <0.05 was considered as significant.Host genetics play an important role in The prevalences of sickle cell haemoglobin trait, G6PD c.202 G>A (rs 1050828) and NOS2 −954 G>C (rs 1800482) variants were 26.6, 22.7 and 17.3%, respectively. Inducible nitric oxide synthase 2  heterozygosity was associated with lower incidence of malaria in all age groups {Adjusted incident rates ratio (aIRR) 0.59; 95% CI [0.386–0.887]; P = 0.012)}. About 4% of study subjects had co-existence of sickle cell Hb trait and G6PD deficiency. Sickle cell Hb heterozygotes (Hb AS) aged less than 1 year experienced significantly more malaria episodes annually than children with normal haemoglobin (Hb AA) {aIRR = 1.98; 95% CI [1.240–3.175]; P = 0.004}. There was no significant influence of the sickle cell trait on malaria incidence among older children of 1–9 years.Mutation  of nitric oxide synthase 2 gene promoter was associated with a lower incidence of acute malaria. The normal haemoglobin  was associated with reduced malaria incidence rates during the first year of life. More understanding of the interplay between host genetics and malaria susceptibility is required. Plasmodium falciparum malaria has been extensively studied and a number of host genes have been suggested to confer specific protection. However, the sickle cell trait and glucose-6-phosphate dehydrogenase (G6PD) deficiency stand out among other host genetic markers reported in genome wide association and multicentre studies . About 88.2% of the participants’ guardians reported owning and using an insecticide treated bed net (ITN) within their households, while 95.3% reported having ever administered an anti-malarial drug to the enrolled child. Throughout the 1-year of longitudinal follow up in this study, malaria episodes were not registered among 217 out of 423 children (51.3%). Among those who experienced malaria episodes  during the 1 year of follow up, the range of annual episodes per child was 1 to 9 .Genotyping of G6PD c.202 G>A (rs 1050828), HBB c.20 A>T (rs 334) and NOS2 −954 G>C (rs 1800482) was performed by PCR for all 423 children who were successfully followed up. DNA amplification by PCR was successful in 97% of the analysed samples. The distribution of G6PD genotypes was as follows: 16.3% (69 of 423) were G6PD c.202 G>A heterozygous females; 1% (4 of 423) were homozygous females and 5.4% (23 of 423) were G6PD c.202 G>A hemizygous males. The prevalence of Hb AS was 26.6% and only one child was homozygous for sickle haemoglobin (Hb SS). Interestingly, 3.9% (16 of 414) of the children had both the sickle cell trait and G6PD c.202 G>A variant. The only variant allele at the NOS2 locus that was examined in this study population was NOS2 −954 G>C (rs 1800482) and it was found to occur at a frequency of 9%. The genotype and allele frequencies of G6PD c.202 G>A (rs 1050828), HBB c.20 A>T (rs 334) and NOS2 −954 G>C (rs 1800482) variants are shown in Table P. falciparum infection, were included in the final multivariate negative binomial regression model to determine the extent to which they were affected by ABO blood group, HBB c.20 A>T (rs 334), G6PD c.202 G>A (rs 1050828) and NOS2 −954 G>C (rs 1800482) gene variants. None of the ABO bloodgroups showed any significant influence on malaria incidence rates in this cohort. In a previous study in the same cohort of children, age, prior malaria history (reported by guardian) and ITN use were identified as independent predictors of malaria incidence [During the 1 year of longitudinal follow-up, a total of 414 new episodes of malaria were recorded. As defined above, these episodes comprised of a child being unwell, having any level of parasitaemia and having a fever either at the time of visit to the study clinic or within the previous 24 h. The overall incidence of these episodes was 0.98 per child/year. Peak incidence (1.25 episodes/child/year) occurred between the age range of 1–3 years, which was about 1.8 times the incidence for older children in the age range of 5–9 years (0.70 episodes/child/year). These episodes of ongoing or recent (past 24 h) febrile illness, that were accompanied by ncidence .HBB sickle cell gene, we could not draw any conclusions about effect of sickle cell homozygosity (Hb SS) on malaria incidence.Overall . Only G6PD c.202 G>A hemizygous males showed slightly higher incidence of uncomplicated malaria than the wild-type individuals . With only four female homozygotes, it was not possible to obtain a confident conclusion of the effect of G6PD c.202 G>A homozygosity on the incidence of uncomplicated malaria . Another multivariate regression analysis was then performed but considering a combination of the female homozygotes and male hemizygotes. G6PD c.202 G>A homo/hemizygous children had slightly higher incidence of uncomplicated malaria reaching 1.37 annual episodes per child compared to 0.97 for G6PD c.202 G>A wild-type . Only few children (16 of 416) showed co-existence of sickle cell trait and the G6PD c.202 G>A variant, thus it was not possible to evaluate the effect of G6PD deficiency on sickle trait protection against malaria in this childrens’ cohort.The only variant that showed significant influence on malaria incidence was the NOS2 −954 G>C (rs 1800482) promoter variant. Crude annual malaria incidence rates were 1.05 episodes per child for the wildtypes and 0.62 per child for heterozygotes. On analysis, NOS2 −954 G>C heterozygotes had significantly lower malaria incidence rates as compared to those without the mutation . Only five individuals carried the homozygous mutation and, therefore, the significance of difference between the malaria incidence for homozygotes and wild-type individuals could not be determined accurately .An age stratified analysis was carried out, adjusting for all the other predictors of malaria incidence including malaria history as reported by guardian, bloodgroup, ITN use and prior antimalarial drug use. As shown in Fig. G6PD c.202 G>A homo/hemizygous children older than 1–3 years experienced higher malaria incidence than their wildtype counterparts . Observable differences in malaria incidence were not seen among the G6PD c.202 G>A genotypes in the other age categories , HBB c.20A>T (rs 334) and NOS2 −954 G>C (rs 1800482) gene polymorphisms and their impact on incidence of uncomplicated in the clinical trials site of Iganga in Eastern Uganda was determined. This is an extension of an earlier longitudinal study [Susceptibility or resistance to infection by  studies , 2 and m studies . Howeveral study  and beneal study . Adjustm20A>T rs 34 and NOal study , 22.P. falciparum infection [Independent of age, the most significant effect observed in this study was the heterozygous NOS2 −954 G>C mutation, which showed a lower incidence of uncomplicated malaria. This single nucleotide polymorphism has been shown to modify NOS2 transcription and increase nitric oxide activity that may be important in parasite clearance , 23 and nfection –26. An enfection . Also, infection .For sickle cell trait and G6PD genotypes, no significant difference in malaria incidence could be detected. However, with age stratification some differences were noted. Sickle trait carriers less than 1 year experienced higher malaria incidence than those with normal haemoglobin. This observation is in contrast with several other investigations in which the sickle cell trait was associated with protection against both severe and uncomplicated malaria . The prec.202 G>A homo/hemizygous children of 1–3 years showed higher malaria incidence than their wildtype counterparts  bearing HbAS plus G6PD c.202 G>A mutation, we could not accurately assess the extent to which G6PD deficiency affects the influence of sickle cell trait on malaria incidence. Studies with larger sample size are needed to test this relationship.Some children with the sickle cell trait also had the G6PD P. falciparum parasitaemia plus present fever or reported history (within past 24 h) of fever. Unfortunately, other causes of fever  such as viral infections were not adequately investigated and thus could not be ruled out. The relatively large proportion (51.3%) of children without any reported symptom of malaria throughout the follow up period of 1 year is interesting, as this finding could partly be due to acquired immunity, other unknown host factors, parasite variance or environmental factors. At any rate, some of these apparently resistant children (51.3%) could as well have experienced one or more attacks of asymptomatic P. falciparum infection that were not included among annual malaria episodes as defined in this study.There were some limitations in this study. The primary outcomes for malaria as defined in this study were a combination of being unwell or ill, any level of In addition, except for ABO blood groups, this study did not investigate other red blood cell (RBC) variants that could potentially modulate malaria incidence such as thalassaemias and other haemoglobin variants .This study showed that 26.6, 22.7 and 17.3% of the population in Iganga clinical trial site carry the sickle cell trait, G6PD c.202 G>A and NOS2 −954 G>C mutations, respectively. The most significant age-independent effect was the heterozygous NOS2 −954 G>C mutation, which showed reduction in incidence of acute malaria infections. Given the high frequency of sickle Hb, G6PD and NOS2 gene mutations in the study site, screening of at least sickle Hb and G6PD in regional endemic areas should be considered so as to ensure proper management of patients including those with acute hemolytic anemia.