Plasmodium falciparum infection might play a role in determining the clinical outcome of malaria infection and could be a fair reflection of the disease transmission rate. This study investigated the genetic diversity of P. falciparum and multiplicity of infection in relation to the severity of malaria and age of patients in Gezira State, Sudan.Multiplicity and genetic diversity of P. falciparum malaria patients diagnosed with microscopy and confirmed using nested PCR were recruited and classified into uncomplicated malaria and severe malaria states according to the standard WHO criteria. DNA was extracted and MSP1 and MSP2 allelic families were determined using nested PCR.A cross-sectional health facilities-based survey was conducted in Gezira State, Sudan in January 2012. A total of 140 P < 0.05). The predominant MSP1 allelic families were MAD20 for uncomplicated malaria and RO33 for severe malaria. The distribution of both FC27 and IC1/3D7 MSP2 allelic families were approximately the same across disease severity. One hundred and eleven P. falciparum isolates (81 %) consisted of multiple genotypes; 71/90 (78.9 %) in uncomplicated malaria and 40/50 (85.1 %) in severe malaria patient groups. Neither MSP1 nor MSP2 allelic families showed association with malaria severity. No statistically significant differences in multiplicity of infection were observed between different age groups.The overall multiplicity of infection (MOI) was 2.25 and 2.30 and 2.15 for uncomplicated and severe malaria respectively. There were no statistically significant differences between uncomplicated and severe malaria (SM) patient groups in MOI with regard to MSP1, MSP2 and overall MOI (Mann-Whitney U-test; all P. falciparum isolates from uncomplicated and severe malaria patients consisted of multiple genotypes. Further molecular epidemiological studies delineate the link between P. falciparum genotype with the malaria phenotype in different regions are encouraged.In this study the majority of  Plasmoduim falciparum is a major public health problem in sub-Saharan countries. In 2015 theglobal burden of the disease reached 214 million cases with 97 countries and 3.2 billion people at risk [Malaria caused by . Plasmodium falciparum is the most virulent malaria parasite species. It exhibits a complex genetic polymorphism which may explain its ability to present with different clinical manifestations of the disease spectrum [P. falciparum genotypes can be linked with more virulent infections [Malaria exhibits a wide range of clinical manifestations ranging from asymptomatic parasitaemia, uncomplicated (mild) to complicated (severe) disease . Moleculspectrum . Moreovefections , 7, and fections .P. falciparum are major blood stage malaria surface antigens [P. falciparum [Molecular approaches helps to increase our understanding of malaria epidemiology as well as clinical manifestation ; these iantigens . They plantigens ; therefoantigens . Also thlciparum .MSP1 gene is located on chromosome 9 and contains 17 blocks of sequences, of which 7 are variable, flanked by conserved regions ; block 2P. falciparum populations in different regions of the country as well as worldwide and linking parasite genotypes to the disease phenotypes are crucially important.Several studies have linked MOI to the severity of malaria especially in areas with high transmission rates , 18. In P. falciparum and multiplicity of infection and their relationship to the disease severity and patient age.In this health care service-based study we aimed to investigate the genetic diversity of Sudan. Gezira state lies on the west bank of the Blue Nile. The geographical coordinates of the state are 14°30'0"N, 33°30'0"E. Plasmodium falciparum is the predominant species , and the Anopheles arabiensis is the main mosquito vector [The study was carried out in Wad-MadaniPediatric Hospital (WPH), Wad-Madani teaching hospital (WTH) and neighboring health facilities in Gezira State, located in the center of o vector . These hP. falciparum malaria by microscopy and nested PCR. All age groups (from age 1 to 80 years) were targeted. Malaria patients were classified into uncomplicated and severe malaria states according to the WHO criteria [P. falciparum and presence of fever (≥ 37.5 °C). Severe malaria patients were having additional symptoms  [A total of 140 patients from those screened for malaria during a cross-sectional study conducted in January 2012 were confirmed positive for Plasmodium species and for parasite count. Parasite density was determined by counting the number of asexual parasites per 200 white blood cells, and calculated per microliter (μl) using the following formula: number of parasites × 8,000/200, assuming a white blood cell count of 8,000 cells per μl [Microscopic visualization of both thick and thin blood films was prepared using 10 % Giemsa stain. The stained slides were examined under a light microscope for detection and identification of s per μl .Genomic DNA was extracted from 100 μl blood samples or from stored blood spotted on Whatman 3 filter paper using 0.5 % Saponin  to free parasites from red blood cells followed by Chelex® 100  method as described by Plowe et al. . DNA wasPlasmodium spp. were identified by 18S rRNA gene-based nested PCR using genus- and species-specific primers as described by Snounou et al. [Plasmodium falciparurm were further analyzed by two highly polymorphic regions of MSP1 (block 2) and MSP2 (block 3) and their allelic types  and (FC27and IC1/3D7), respectively, using nested PCR [2, 0.2 mMdNTPs, 1 μl of each primer and 1 unit of Taq Polymerase . Cycling conditions for the outer PCR were as follows:initial denaturation at 94 °C for 3 min, followed by 37 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 1 min; and primer extension at 72 °C for 2 min. The final cycle had a prolonged extension at 72 °C for 10 min. PCR reactions were incubated in a thermal cycler . Allelic specific positive control and DNA negative control were included in each set of reactions [u et al. . Plasmodsted PCR . In brieeactions .The amplified PCR products were analyzed by electrophoresis on 2 % molecular grade agarose gel  and visualized by UV trans-illuminator , following ethidium bromide staining. The number and size of DNA fragments was estimated using 100 bp DNA ladder .Multiplicity of infection (MOI) was determined by calculating the number of different alleles at each locus; single infections were those with only one allele per locus at all of the genotyped loci. Multiclonal infections were defined as those having more than one allele in at least one locus out of the loci genotyped.P-value ≤ 0.05 at 95 % confidence interval.Data were entered and analyzed using the software Statistical Package for Social Sciences version 20 . The MSP1 and MSP2 allelic frequency was calculated. The mean multiplicity of infection (MOI) was calculated for MSP1, MSP2 and overall MOI. Continuous variables were compared using Spearman’s rank correlation coefficient. Dichotomous variables were analyzed using Chi-square test. Mann-Whitney U-test was used to compare the mean MOI according to severity (disease phenotype). Different age groups’ MOIs were calculated and the statistical significance was tested using Kruskal-Wallis H-test. Statistical significance level was defined as Plasmodium falciparum DNA was detected in all malaria patients by nested PCR  and 130/140 (92.86 %) samples for MSP1   was 2.25. The overall MOI with regard to uncomplicated and severe malaria cases were 2.30 (95 % CI: 2.12–2.48) and 2.15 (95 % CI: 1.94–2.36), respectively. However the differences in MSP1, MSP2 and overall MOI were statistically non-significant . In uncomplicated malaria patients, a total of 14 different-sized alleles for MSP1 were detected compared to 21 different-sized alleles for MSP2  and MSP2 (FC27 and ICI/3D7) allelic families showed the polymorphic nature of the Sudanese P. falciparum isolates (81 %) consisted of multiple genotypes; 71/90 (78.9 %) in uncomplicated malaria and 40/50 (85.1 %) in severe malaria patients. Neither MSP1 nor MSP2 allelic families showed association with malaria severity. The frequency of the identified MSP1 and MSP2 allelic families across the uncomplicated and severe malaria patients is shown in Table In severe malaria patients, a total number of 12 different-sized alleles for MSP1 were detected compared to 20 different-sized alleles for MSP2. The predominant MSP1 allelic families were MAD20 for the uncomplicated malaria and RO33 for the severe malaria. The distribution of both FC27 and IC1/3D7 MSP2 allelic families was approximately the same across disease severity. One hundred and eleven P.falciparum infections (Table P = 0.658). There was a statistically significant positive correlation between age and parasite density . The oldest age group (> 40 year-old) had the highest mean parasite density  .Approximately more than three-quarters of malaria patients in all age groups were found to have a multiclonal ns Table . Multiplns Table : howeverP. falciaprum. To our knowledge, this is the first study to provide data on the genetic diversity and multiplicity of P. falciparum isolates in relation to disease phenotype based on MSP antigen in Gezira State, Central Sudan. In our study there was no association between MOI and malaria outcome (severe versus uncomplicated) which is consistent with other reports from Sudan [In Sudan, very few studies investigated the genetic diversity and multiplicity of infection of om Sudan , Ghana [om Sudan , Gabon [om Sudan , Gambia om Sudan  and Madaom Sudan , but ourom Sudan  and Indiom Sudan  and thosom Sudan  and Nigeom Sudan . HoweverOur reported overall MOI (2.25) was higher than that observed in Gadarif state, Sudan  and lowevs 78.9 %). This observation is in agreement with the study of Shigidi et al. [P. falciparum specific allele families and clinical outcome and several studies reached the same conclusion [In this study we found a remarkably high frequency of multiclonal isolates (81 %) comparable to that (82.3 %) reported in Mauritania . The frei et al.  which rei et al. , who obsi et al. . In thisnclusion , 28, 30.P. falciparum in this study area compared to previous studies in Sudan could be due to some of the following reasons: (i) the followed treatment guidelines in Sudan using ACTas a treatment regimen since 2004 [The observed increase in multiplicity of infection and frequency of multiclonal isolates of nce 2004  which doWith regard to MSP1, inuncomplicated malaria patients the predominant allelic family was MAD20, whearas in severe malaria patients the predominant allelic family was RO33. These results are in contrast to what was reported by Bouyou-Akotet et al. in Gabon . On the The current study found no association between multiplicity of infection and patients’ age group. This is in agreement with previous studies conducted in Muritania , SenegalP. falciparum were higher than previous studies in Sudan. Neither MSP1 nor MSP2 allelic families exhibited significant association with malaria severity. There was no statistically significant difference between different age groups with regard to the multiplicity of infection. The observed high multiclonal isolates may facilitate the spread of resistant strains with time. Further molecular epidemiological studies that would help delineate the link between P. falciparum genotypes with the malaria phenotypes in different regions are encouraged.In this study the multiplicity of infection was comparable between uncomplicated and severe malaria patients. The multiplicity of infection and the distribution of multiclonal isolates of MOI, multiplicity of Infection; MSP1, merozoite surface protein 1; MSP2, Merozoite surface protein 2; PCR, polymerase chain reaction


P. falciparum antigens, and factors associated with these indicators, in three different epidemiological settings in Uganda.Understanding the current epidemiology of malaria and the relationship between intervention coverage, transmission intensity, and burden of disease is important to guide control activities. We aimed to determine the prevalence of anemia, parasitemia, and serological responses to P. falciparum apical membrane antigen 1 (AMA-1) and merozoite surface protein 1, 19 kilodalton fragment (MSP-119). Analysis of laboratory results was restricted to 1949 (87.5%) participants aged ≤ 40 years. Prevalence of anemia (hemoglobin < 11.0 g/dL) was significantly higher in Walukuba (18.9%) and Nagongera (17.4%) than in Kihihi (13.1%), and was strongly associated with decreasing age for those ≤ 5 years at all sites. Parasite prevalence was significantly higher in Nagongera (48.3%) than in Walukuba (12.2%) and Kihihi (12.8%), and significantly increased with age to 11 years, and then significantly decreased at all sites. Seropositivity to AMA-1 was 53.3% in Walukuba, 63.0% in Kihihi, and 83.7% in Nagongera and was associated with increasing age at all sites. AMA-1 seroconversion rates strongly correlated with transmission intensity, while serological responses to MSP-119 did not.In 2012, cross-sectional surveys were conducted in 200 randomly selected households from each of three sites: Walukuba, Jinja district (peri-urban); Kihihi, Kanungu district ; and Nagongera, Tororo district  with corresponding estimates of annual entomologic inoculation rates (aEIR) of 3.8, 26.6, and 125.0, respectively. Of 2737 participants, laboratory testing was done in 2227 (81.4%), including measurement of hemoglobin, parasitemia using microscopy, and serological responses to Anemia was predominant in young children and parasitemia peaked by 11 years across 3 sites with varied transmission intensity. Serological responses to AMA-1 appeared to best reflect transmission intensity, and may be a more accurate indicator for malaria surveillance than anemia or parasitemia. Malaria remains an important public health problem in sub-Saharan Africa, and is responsible for over 10% of the overall disease burden –7. HowevIn 2012, Uganda was ranked fourth highest in number of malaria cases reported globally, and sixth in number of malaria-associated deaths . Uganda P. falciparum exposure and antibody responses to show that it is possible to estimate transmission intensity in a community by calculating seroconversion rates using age specific prevalence of specific antimalarial antibodies, including those to apical membrane antigen 1 (AMA-1) and merozoite surface protein 1, 19 kilodalton fragment (MSP-119) [Monitoring and evaluation of malaria control activities relies heavily on large, nationally representative cross-sectional surveys. In Uganda, these include Demographic Health Surveys (DHS) which have been conducted every 5 years since 2001 and a single Malaria Indicator Survey (MIS) conducted in 2009. Although all these surveys include estimates of coverage of malaria control interventions, indicators of malaria burden are limited to anemia testing and, only in the 2009 MIS, estimates of parasitemia. The annual entomological inoculation rate (aEIR) is generally considered the ‘gold-standard’ measure of malaria endemicity and transmission intensity, but is highly labour-intensive and rarely measured . In areaMSP-119) –27.19 were conducted in three sub-counties with varying levels of transmission intensity based on concurrently measured estimates of aEIR.To better understand the current epidemiology of malaria in Uganda, cross-sectional surveys to estimate coverage levels of control interventions and key malaria indicators, including anemia, parasitemia, and serological responses to AMA-1 and MSP-1This was a cross-sectional community survey conducted in three different epidemiological settings in Uganda. A total of 200 households were recruited in each site, using a list of households randomly generated from a census database. The surveys consisted of a household questionnaire and biomarker survey. Finger-prick blood samples, obtained from all children under fifteen years of age and from a random selection of age stratified adults, were collected for measurement of hemoglobin, thick and thin blood smear, rapid diagnostic test (RDT) for malaria, and to save on filter paper. The protocol for this trial is available as supporting information .Ethical approval was obtained from the Makerere University School of Medicine Research and Ethics Committee (REC REF 2011–203), the Uganda National Council for Science and Technology (HS 1074), the London School of Hygiene & Tropical Medicine Ethics Committee (Reference 6012), and the University of California, San Francisco Committee on Human Research (Reference 027911). For all households fulfilling the selection criteria, written consent to participate in the study was sought. Written informed consent was obtained from the next of kin, caretakers, or guardians on behalf of the children enrolled in the study. Furthermore, written assent was obtained from children aged 8 years and above to participate in the study.Cross-sectional surveys were conducted between January and June 2012 in three sub-counties purposively chosen to represent varied malaria transmission settings in Uganda: Walukuba, Jinja district; Kihihi, Kanungu district; and Nagongera, Tororo district . In eachIn 2011, all households and other key features in the study areas were enumerated and mapped to generate a sampling frame for the surveys. A household was defined as any single permanent or semi-permanent dwelling structure acting as the primary residence for a person or group of people that generally cook and eat together. Household locations were mapped using hand-held eTrex global positioning system (GPS) receivers  and readings were taken from the door of the household, if possible, or from a point that was most representative of the household.We enrolled 200 households from each site using a list randomly generated from the household enumeration database using computer-generated random numbers. Study personnel conducted door-to-door recruitment to identify those households with at least one adult household resident in sequential order according to the recruitment list. Residents not home during the initial contact were re-visited on at least three other occasions before eliminating them from the sample selection process.When a household was identified, study personnel briefly described the purpose of the study to the head of the household or any adult household member present at home in the appropriate language. Households were included if: 1) an adult aged 18 years or older was the head of the household, and 2) the head of the household agreed to provide informed consent. Households were excluded if: 1) no adult resident was at home on more than 3 visits, and 2) the household was vacant. For all households fulfilling the selection criteria, written consent to participate in the study was sought.Upon enrolment, a household questionnaire, based on the Malaria Indicator Survey questionnaire developed by the Roll Back Malaria Monitoring and Evaluation Reference Group , was admul. A smear was considered negative after reviewing 100 high-powered fields. All positive thick blood smears had corresponding thin smears reviewed for species identification. For quality control, all slides were read by a second microscopist and a third reviewer was used to settle any discrepant readings. Hemoglobin concentration was assessed using a battery-operated portable hemoglobinometer  and estimated to an accuracy of 1 g/dL.Thick and thin blood smears were stained with 2% Giemsa for 30 minutes. Thick smears were evaluated for the presence of parasitemia  and gametocytes. Parasite densities were calculated by counting the number of asexual parasites per 200 leukocytes , assuming a leukocyte count of 8,000/P. falciparum AMA-1 (3D7) and MSP-119 (Wellcome genotype) were used as antigens in indirect ELISA as previously described [oC overnight. The plates were washed in PBST, and blocked with 1% (w/v) skimmed milk solution for three hours. After washing, samples were added in duplicate at a final dilution of 1:1,000 to each plate together with a pool of hyper immune serum and the plates were incubated overnight at 4oC. The plates were washed and 50 μl of HRP-conjugated rabbit anti human IgG  were added into each well and incubated for three hours. After a further series of washes, substrate solution  was added and the reaction was allowed to develop for 15–20 min before addition of stopping solution (2M H2SO4). The optical density was read using an ELISA reader at 450 nm.Blood spots were collected onto filter papers , allowed to dry overnight and stored at -20°C with dessicant for serology studies. The recombinant proteins escribed . Briefly19 antibody seropositivity. Raw optical density (OD) measurements were averaged and normalized against the positive control samples on each plate. The cut-off for seropositivity was calculated, as previously described with a separate cut-off value generated for each antigen [Data were collected using hand-held computers which were programmed to include range checks, structure checks and internal consistency checks. All statistical analyses were carried out using Stata version 12 . Malaria indicators of interest were; 1) anemia defined as hemoglobin less than 11 g/dl, 2) parasitemia defined on the basis of expert microscopy results as presence of any asexual parasites on thick blood smear, 3) AMA-1 antibody seropositivity and, 4) MSP-1 antigen . Serocon antigen . Analyse antigen . GraphicAt total of 200 households were surveyed from each sub-county, including 2737 study participants “Mean hemoglobin levels were generally high (> 12.0 g/dL) and severe anemia (hemoglobin < 8g/dL) was uncommon at all sites “The prevalence of asexual parasitemia was significantly higher in Nagongera than the other two sites (p<0.001 for both comparisons), but was comparable in Walukuba and Kihihi, despite the difference in aEIR in the two sites “The antibody response to AMA-1, whether evaluated as seroprevalence, titer, or seroconversion rate, increased with increasing transmission intensity “19, median MSP-119 antibody titers, and seroconversion rates were lower than with AMA-1 and demonstrated different patterns across the three sites “19 was highest in Kihihi, and was significantly lower in Walukuba (31.8%), than in Kihihi  and Nagongera . MSP-119 seroconversion rates were 0.036, 0.089, and 0.064 for Walukuba, Kihihi, and Nagongera, respectively, which corresponded to calculated aEIRs of 2 in Walukuba and 11 in Kihihi, but only 6 in Nagongera, much lower than the aEIR actually observed (125) [19 up to 3 years of age across the 3 sites “19 and no significant relationship from 21–40 years of age across the 3 sites. Only in Walukuba was parasitemia significantly associated with seropositivity to MSP-119 “Seroprevalences of antibodies to MSP-1ed (125) . In the 19 did not follow a consistent pattern with age, and the seroconversion rate was unexpectedly low in Nagongera.In this study, we evaluated the prevalence of key malaria indicators and associated factors in three different epidemiological settings in Uganda. The prevalence of anemia was predominant in the youngest children at all sites and did not correlate well with the level of transmission intensity. Severe anemia was uncommon at all sites. The prevalence of parasitemia was highest in Nagongera, the highest transmission site, but results were similar in Walukuba and Kihihi, despite an almost ten-fold difference in aEIR. The relationship between age and parasite prevalence was similar at all three sites, increasing with age until peaking by 11 years and then decreasing with age. Seroprevalence and seroconversion rates of antibodies to AMA-1 appeared to best reflect differences in transmission intensity between the three sites. In contrast, seropositivity of antibodies to MSP-1Despite the recent increase in donor financing and focus on scaling-up of available malaria control interventions, including ITNs, IRS, IPTp, and treatment with ACTs, coverage levels remain far below targets in Uganda. Although ownership and usage of ITNs has increased substantially in Uganda in the past decade, and ITN distribution has been extended beyond the traditional target groups of children under-five and pregnant women with the recent shift in policy to universal coverage, we found that ≤ 35% of households in the 3 study sites own enough ITNs to cover the household population (one ITN per two residents). Fortunately, Uganda is currently undertaking a national ITN distribution campaign aiming to achieve universal net coverage for the whole country. The same is true for IRS, which was reinstated as a malaria control measure in Uganda in 2006, after a hiatus of about 40 years. Supported by the US President’s Malaria Initiative and other partners, Uganda’s IRS strategy has emphasized implementation in epidemic-prone areas, high-risk situations, such as camps for internally displaced persons or refugees, and more recently, high-transmission areas . But resP. falciparum malaria is commonly associated with anemia in children living in holoendemic malaria areas [Although the etiology of anemia in tropical areas is multifactorial, ia areas ,33–35. Wia areas . Howeveria areas ,38. Paraia areas ,40. HoweParasite prevalence, particularly in children aged 2–10 years, is a commonly used measure of malaria transmission intensity and disease burden across a range of endemicities ,23. Howe19 did not follow the same pattern; antibody prevalence decreased in the first 5 years of life and the seroconversion rate was surprisingly low for the high transmission site of Nagongera. The former may be due to a combination of short-term fluctuations in transmission and high density parasite infections in some young children. Antibody responses to MSP-119 have recently been shown to be more affected by seasonal changes in transmission [19 in Nagongera is consistent with observations in other hyperendemic settings in Uganda and elsewhere [Serological markers are useful adjunct measures of transmission as antibodies can persist for months or years after infection, thereby smoothing out the effects of seasonal or unstable malaria transmission  and reprsmission . The unelsewhere . The reaThis study was conducted in areas with varying malaria transmission, and the findings may apply in areas of similar transmission patterns. The concurrent measurement of aEIR, parasite prevalence and serology enabled us to compare malariometric indices across different endemicity settings. However, studies were conducted at only 3 sites and we could not assess the trends nor draw firm conclusions on causality because of the cross sectional design. Nonetheless, such surveys are important for tracking temporal trends of malariometric indices and could be used to measure the impact of control interventions.In summary, we found that coverage of malaria control interventions remains below targets in these 3 sites in Uganda. Although anemia and parasite prevalence are widely used indicators of the burden of malaria, we found that they did not directly correlate with transmission intensity in our study sites. Seroprevalence of AMA-1 was a better marker of infection risk and disease burden and may be a better indicator than anemia or parasitemia for monitoring and evaluation of trends in malaria burden and the impact of malaria control activities.S1 Protocol(DOC)Click here for additional data file.


Enterobius vermicularis and Strongyloides stercoralis infections. We aimed to determine the extent of enterobiasis, strongyloidiasis, and other helminth infections and their association with asymptomatic Plasmodium parasitaemia, anaemia, nutritional status, and blood cell counts in infants, preschool-aged (PSAC), and school-aged children (SAC) from rural coastal Tanzania.There is a paucity of data pertaining to the epidemiology and public health impact of Plasmodium parasitaemia testing and full blood cell count examination. Weight, length/height, and/or mid-upper arm circumference were measured and the nutritional status determined in accordance with age.A total of 1,033 children were included in a cross-sectional study implemented in the Bagamoyo district in 2011/2012. Faecal samples were examined for intestinal helminth infections using a broad set of quality controlled methods. Finger-prick blood samples were subjected to filariasis and E. vermicularis infections were found in 4.2% of infants, 16.7%, of PSAC, and 26.3% of SAC. S. stercoralis infections were detected in 5.8%, 7.5%, and 7.1% of infants, PSAC, and SAC, respectively. Multivariable regression analyses revealed higher odds of enterobiasis in children of all age-groups with a reported anthelminthic treatment history over the past six months (odds ratio (OR): 2.15; 95% confidence interval (CI): 1.22 - 3.79) and in SAC with a higher temperature . Strongyloidiasis was associated with eosinophilia  and with Trichuris trichiura infections  in children of all age-groups, and with asymptomatic Plasmodium parasitaemia  in infants. None of the investigated helminthiases impacted significantly on the nutritional status and anaemia, but moderate asymptomatic Plasmodium parasitaemia was a strong predictor for anaemia in children aged older than two years .E. vermicularis and S. stercoralis infections were moderately prevalent in children from rural coastal Tanzania. Our data can contribute to inform yet missing global burden of disease and prevalence estimates for strongyloidiasis and enterobiasis. The association between S stercoralis and asymptomatic Plasmodium parasitaemia found here warrants further comprehensive investigations.The online version of this article (doi:10.1186/s12879-014-0644-7) contains supplementary material, which is available to authorized users. Ascaris lumbricoides), whipworms (Trichuris trichiura), and hookworms . Infections are acquired via the ingestion of the parasites’ eggs, for example with contaminated food, or in case of hookworms via larvae penetrating bare skin . The. TheAscaare skin -6]..Ascaris Strongyloides stercoralis and the pinworm Enterobius vermicularis that are mostly neglected in prevalence reports and global burden of disease estimates due to their rather unpleasant and cumbersome diagnosis with the Baermann and/or Koga agar plate methods or adhesive tape test, respectively, and due to difficulties to assess associated morbidity ,7]-,[7]-Stro-,[7]-Strorbidity .S. stercoralis has an intra- and extra-human lifecycle during which the parasite can reproduce asexually and sexually, respectively. Larvae that hatched from eggs within the intestines are able to penetrate the intestinal wall and can perpetuate the lifecycle within the human host for decades after initial exposure . In. InS. stexposure . Skin leexposure ,13]-. It. ItS. stast Asia . Global ast Asia ,19],. Ec. EcE. venal skin . Enterobnal skin . It is enal skin , but recThe neglect of strongyloidiasis and enterobiasis in most studies pertaining to soil-transmitted helminth infections results in a knowledge gap about their potential impact on co-infections and the nutritional status of the host. While it is widely acknowledged that helminth infections modulate immune responses and might influence the acquisition and outcomes of diseases such as malaria, tuberculosis (TB), and human immunodeficiency virus-acquired immune deficiency syndrome (HIV-AIDS), the exact mechanisms are not known -37]. Re. Re37]. http://ec.europa.eu/research/health/infectious-diseases/neglected-diseases/projects/014_en.html). Of course, to obtain reliable results, not only the presence of major helminth species, but also infections with S. stercoralis and E. vermicularis need to be taken into account. Here, we present, to our knowledge for the first time, the extend of strongyloidiasis and enterobiasis, besides other helminthiases, in infants, preschool-aged children (PSAC), and school-aged children (SAC) from rural coastal Tanzania that participated in a cross-sectional study implemented to recruit participants for the IDEA-malaria study arm in the Bagamoyo district, United Republic of Tanzania. We also show anthropometric and haematological characteristics of the study group and associations found between the individual helminth species infections, between helminth infections and asymptomatic Plasmodium infections and between helminth infections and anthropometric status, anaemia, and additional haematological parameters in different age-groups. With this report we aim to shed more light on the distribution and public health importance of two highly neglected soil-transmitted helminth species and therefore to contribute to inform yet missing global burden and prevalence estimates.The IDEA research program is designed to intensively study the immunological interplay between helminth infections and malaria, TB, and HIV-AIDS  in Bagamoyo, United Republic of Tanzania, was approved by the institutional research commissions of the Swiss Tropical and Public Health Institute  and the IHI. Ethical approval for the study was obtained from the Ethikkomission beider Basel  and the National Institution for Medical Research of Tanzania . The study was monitored by the IHI internal monitoring team in collaboration with the Pan African Collaboration for the Evaluation of Antituberculosis Antibiotics (PanACEA) and Swiss TPH.Local district, community, school, and health authorities were informed about the purpose and procedures of the study and their acceptance for the implementation was sought. Individuals eligible to be screened and to potentially participate in one of the study arms of IDEA were informed in detail about the aims of the study, the risks and benefits of study participation, and the extend of time involvement in case of their participation . It was S. stercoralis and E. vermicularis, or praziquantel (40 mg/kg) against Schistosoma infections. Participants with asymptomatic Plasmodium parasitaemia or clinical malaria were treated with Artemether Lumefantrine and children with severe malaria received Quinine.Patients with helminthiases, malaria, TB, HIV-AIDS, and/or other medical conditions received treatment according to the national treatment guidelines of the United Republic of Tanzania. Accordingly, participants of our cross-sectional survey that were infected with helminths were treated with albendazole  against common soil-transmitted helminth infections, All participants included in the present analysis resided in one among 12 villages or small towns in the Bagamoyo district, belonging to the Pwani region in the United Republic of Tanzania. These settlements are located in a rural environment approximately 20-60 kilometres from Bagamoyo, which is a historical town located directly at the coast along the Indian Ocean, 75 kilometres north of Dar es Salaam. The population of the Bagamoyo district was estimated at 311,740 inhabitants in the 2012 census . Mo. Mo49]. Anaemia thresholds were applied according to WHO recommendations as follows: haemoglobin values >10.9 mg/l non-anaemia, 10.0-10.9 mg/l mild anaemia, 7.0-9.9 mg/l moderate anaemia, and <7.0 mg/l severe anaemia for infants and PSAC, and haemoglobin values >11.5 mg/l non-anaemia, 11.0-11.4 mg/l mild anaemia, 8.0-10.9 mg/l moderate anaemia, and <8.0 mg/l severe anaemia for SAC . ReferenA. lumbricoides, 1,000 and 10,000 EPG for T. trichiura, 2,000 and 4,000 EPG for hookworm and 99 and 399 EPG for S. mansoni, respectively. Microhaematuria was classified according to the manufacturer’s suggestion into negative, trace, +, ++, or +++ and S. haematobium egg counts into light (1–49 eggs/10 ml of urine) and heavy (≥50 eggs/10 ml of urine). Asymptomatic Plasmodium parasitaemia was defined by a positive malaria rapid diagnostic test result and/or by Plasmodium parasites detected microscopically plus the absence of unspecific symptoms of malaria at the day of enrolment or over the past seven days . Counts of below 10 parasites per 200 white blood cells (i.e. less than 400 parasites per μl blood) were defined as low grade parasitaemia, and counts of 100 and more parasites per 200 white blood cells (i.e. 400 or more parasites per μl blood) as moderate parasitaemia.A patient was considered to be infected with a helminth species, if the infection was detected with one or several diagnostic methods. For each individual, helminth infection intensity was determined according to Kato-Katz thick smear results as suggested by the WHO . For thiPlasmodium parasitaemia co-infections we calculated observed and expected prevalences for co-infections ,,,58], wh, wh58], S. stercoralis infections were associated with eosinophilia in our study population, a condition that is generally attributed to strongyloidiasis . Ou. OuS. stfections . Novel iS. stercoralis do not mention malaria, and publications summarizing the epidemiology and immunological interplay of helminth and malaria co-infection ignore S. stercoralis, respectively. The only study we found assessing S. stercoralis infections and malaria was on pregnant women from Uganda, and revealed no association ,,. Ot. OtE. vefections ,62],..E. vermiS. stercoralis, E. vermicularis, hookworm, nor any other investigated helminth infection was associated with wasting, underweight, thinness, stunting, or anaemia in our study population. While wasting, underweight, and thinness were rarely seen in the surveyed children and therefore no association could be determined, stunting occurred in 18.5% of SAC and anaemia affected more than half of the examined children. Previous studies conducted in the Kilombero district in Tanzania in the early 1980s had also revealed no association between wasting or stunting and intestinal helminth infection or Plasmodium parasitaemia . Th. ThS. stsitaemia . This misitaemia , with desitaemia , and witsitaemia . The occPlasmodium parasitaemia but not with helminthiases in infants, PSAC, and SAC in our study. It is widely acknowledged that Plasmodium infections, also if low-grade and asymptomatic as a result of semi-immunity, contribute to the development of anaemia -, th, thS. st malaria ,83]. El. ElS. st disease . Moreoveo values  and ratho values . A limitE. vermicularis and S. stercoralis were moderately prevalent in young children from rural coastal Tanzania. A considerable number of infants were infected and prevalences increased with children’s age. Our data can contribute to inform yet missing global burden of disease and prevalence estimates for strongyloidiasis and enterobiasis. The association between S. stercoralis and asymptomatic Plasmodium parasitaemia found in infants of our study population warrants further investigations.The results of our cross-sectional study showed that


Subclinical infections in endemic areas of Southeast Asia sustain malaria transmission. These asymptomatic infections might sustain immunity against clinical malaria and have been considered benign for the host, but if they are associated with chronic low-grade inflammation this could be harmful. We conducted a case-control study to explore the association between subclinical malaria and C-reactive protein (CRP), an established biomarker of inflammation.Plasmodium species. Plasma CRP concentration was measured in 328 individuals with parasitaemia (cases) and compared with: i) the same individual’s value at the first time point when they had no detectable parasites (n = 282); and ii) age- sex- and village-matched controls (n = 328) free of Plasmodium infection. Plasma CRP concentrations were compared against thresholds of 3mg/L and 10mg/L. Subgroup analysis was carried out for cases with P vivax and P falciparum mono-infections.Blood samples from asymptomatic villagers in Pailin, Western Cambodia were tested for malaria by high-volume ultra-sensitive polymerase chain reaction (uPCR) to determine the P.vivax mono-infections had the highest plasma CRP concentrations with the greatest association with parasitaemia.Median plasma CRP level for all samples was 0.59mg/L (interquartile range: 0.24–1.64mg/L). CRP concentrations were higher in parasitaemic individuals compared with same-person-controls (p = 0.050); and matched-controls (p = 0.025). 4.9% of samples had CRP concentrations above 10mg/L and 14.6% were above 3mg/L. Cases were more likely to have plasma CRP concentrations above these thresholds than age/sex matched controls, odds ratio 3.5 (95%CI 1.5–9.8) and 1.8 (95%CI 1.1–2.9), respectively. Amongst cases, parasite density and CRP were positively correlated (p<0.001), an association that remained significant when controlling for age and fever. Individuals with P.vivax mono-infections. As well as interrupting malaria transmission within the community, treatment of asymptomatic malaria infections, in particular radical cure of vivax malaria, may benefit the health of infected individuals.In this setting persistent malaria infections in asymptomatic individuals were associated with moderately elevated plasma CRP concentrations; chiefly evident in cases with  A subclinical reservoir of malaria plays an important role in sustaining transmission in areas of seasonal malaria and presents an obstacle to malaria elimination . The sizThe effects of submicroscopic parasitaemia on the host beyond the risk of developing clinical malaria and contributing to splenomegaly and anaemia are not well-understood, as these infections are below the level of detection by most standard methods. A recent review of the potential burden of subclinical infections beyond clinical cases, implicated these ‘chronic infections’ with higher risk of susceptibility to bacteraemias, anaemia, and all-cause mortality, but the underlying biology and causal pathways are not clear . WhetherPlasmodium species, as this could inform considerations into either radical cure of P.vivax infections, and or wide-spread treatment of otherwise healthy individuals with ACTs to help achieve P.falciparum elimination.If subclinical malaria infection is associated with an inflammatory response this could contribute to similar morbidities, particularly if these persist for prolonged periods. It is also important to better understand if such associations are more or less evident in different C-reactive protein (CRP) is a cytokine-induced acute phase protein and biomarker of inflammation and infection. CRP plays a role in host defence by activating the complement pathway and as a pro-inflammatory mediator. The median Plasma CRP concentrations in the population is 1.5mg/L with an interquartile range of 0.3–3.5mg/L, a range that is stable across sex and ethnic groups. CRP concentrations increase markedly during acute inflammatory events , 12 notaP falciparum infection [P. vivax cases to determine whether there were differences between Plasmodium species.A number of studies have reported a relationship between clinical malaria and CRP –18. Antinfection . There wA better understanding of plasma CRP concentrations in individuals in malaria endemic settings is needed to determine the potential use of CRP as a guide for antibiotics in febrile patients with negative malaria tests. In a recent study CRP was found to be a useful biomarker for distinguishing between viral and bacterial infections in febrile patients in the Mekong . TreatmeP.vivax as compared with P.falciparum subclinical infections?This study therefore addresses the following questions: i) what is the distribution of plasma CRP concentrations in apparently healthy individuals in rural Southeast Asian settings? ii) are plasma CRP concentrations raised in asymptomatic parasitaemic individuals, and are plasma CRP concentrations correlated with parasite loads? iii) is there evidence of a difference in CRP concentrations in individuals with P.falciparum and P.vivax mono-infections were identified in 12% and 35% of these 328 cases, respectively. The remaining cases had either a Plasmodium infection with indeterminate species (51.5%) or a mixed infection (1.5%). All 328 cases were assigned a matched control, surveyed in the same month. In 282 cases a sample without detectable parasitaemia and with a CRP result was available to act as a same-person control.Of 2151 individual villagers for whom uPCR results were available, 328 (15.2%) were found to be positive for malaria parasitaemia in at least one instance with a corresponding plasma sample available for CRP testing . P.falciThe median plasma CRP level for all samples was 0.59mg/L with an interquartile range of 0.24–1.64mg/L and a full range of 0.02-263mg/L; the data are shown in With the age/sex matched controls this difference remained significant when excluding individuals who reported an episode of illness in the previous 48 hours (p = 0.022) but not when excluding individuals with a fever at the time of the survey (while self-reporting as being asymptomatic) (p = 0.103). In the same-person control comparison the differences were not significant once febrile individuals or those with a reported episode of illness were excluded from the analysis.P.vivax infections, with a median of 0.9mg/L, also significantly higher than their matched and same person controls (p = 0.011 and 0.004 respectively). The differences remained significant also with the exclusion of cases with a reported illness in the previous 48 hours and in those with a fever at the time of the survey . There was no significant difference in plasma CRP concentrations between cases with a mono P.falciparum infection and those of either set of controls.Plasma CRP concentrations were highest in cases with In aggregate, 14.6% of asymptomatic villagers had plasma CRP concentrations above a threshold of 3mg/L, and 4.9% had plasma CRP concentrations above a threshold of 10mg/L. There was a significant difference between cases and controls , with 17A similar difference was found with the higher threshold of 10mg/L , with 7.P.vivax cases were more likely to have plasma CRP concentrations above the threshold of 3 mg/L as compared with the age-sex matched controls ). Likewise the OR for plasma CRP concentrations being above a threshold of 10mg/L was 9 in the vivax cases as compared with their age-sex matched controls (95% CI 2.2–80). No differences were found in the odds of having elevated CRP levels above the thresholds in cases with falciparum mono-infections as compared with either set of controls (both p>0.3).In the subgroups of patients with mono-infections, The geometric mean parasitaemia (95% CI) in cases was 2178/mL (1444–3285), P. vivax, where the differences were most pronounced, and due to the persistent nature of the infection.Subclinical malaria infections could have a significant health burden beyond that posed by clinical cases . This stP.falciparum infections and over 85% of P.vivax infections [The uPCR method used in this study has a lower limit of detection of 22 parasites/mL which detects nearly 75% of Individuals with acute illness were excluded by design from the malaria survey but some participants were found to have a fever and/or reported an episode of illness in the last 48 hours, and some of the differences in plasma CRP concentrations between cases and both groups of controls were no longer significant once these individuals were excluded from the analysis. A degree of interdependence between these inflammatory responses to parasitaemia is expected. The frequent presence of fever and/or elevated CRP in individuals with detectable parasites in a community setting supports the notion that what are often referred to as submicroscopic, ‘asymptomatic infections’ could in fact have a detrimental health impact, even if they do not result in health seeking behaviour.There was a higher prevalence of elevated plasma CRP and a higher proportion of febrile individuals in same-person controls as compared with the age/sex matched controls. This might be explained by persisting infections that were not detected by uPCR. There are, however, other potential factors that could explain these differences that were not accounted for in the matching, such as forest goers that could be at greater risk of other non-malarial fevers, as well as other genetic or behavioural predispositions for elevated CRP.Regarding the use of CRP to guide the use of antibiotics in febrile patients with a negative malaria test, this study confirms that plasma CRP concentrations in this rural, Southeast Asian population were above a conservative threshold of 10mg/L in only 4.6% of individuals. This threshold is in fact probably lower than is likely to be used in this context; a recent study identified 20mg/L as a potential threshold , in whicWhile uPCR is the most sensitive test available for these surveys, some infections are still undetectable, and some of the controls, particularly in the same-person control group could in fact be infected. While this might result in smaller differences in plasma CRP concentrations between the groups this would only strengthen the association between parasitaemia and elevated CRP. Second, there is a possibility of confounders and reverse causality, whereby for instance other causes of fever or elevated CRP might trigger relapse of malaria parasites. Larger studies with repeated CRP measurement before and after infection would be needed to explore this. Other possible risk factors and confounders that could be implicated in elevated CRP and/or parasitaemia would need to be included in the matching process. Third, the thresholds of 3mg/L and 10mg/L used to classify clinically significant elevated CRP concentrations are not well defined, although the overall finding of higher risk of elevated CRP in cases versus controls is a consistent one irrespective of the specific chosen threshold. Fourth, and potentially the most important limitation, was the inability to determine the species of 52% of all plasmodium infections, which may introduce bias into the analysis. Species determination was chiefly unavailable from low-density parasitaemias. As parasitaemia was associated with species the unavailability of results may be non-differential and a potential cause of bias.P.vivax infections. Prolonged inflammation and elevated plasma CRP concentrations have been implicated with cardiovascular disease and other common causes of morbidity. If these findings are verified in future studies, this would lend further weight to the treatment of subclinical infected individuals for their own health benefit, as well as for the community benefits associated with interrupting malaria transmission. The findings also reinforce the importance of radical cure of vivax malaria to avoid recurrent inflammation during repeated blood stages, even if these do not always manifest in clinical episodes.This study finds that subclinical submicroscopic malaria infection is associated with elevated plasma CRP concentrations, particularly in Ethical approval for the survey was obtained from the Cambodian National Ethics Committee for Health Research (0029 NECHR), the Oxford Tropical Research Ethics Committee , and the study was registered on clinicaltrials.gov (NCT01872702). The informed consent form allowed for use of the stored samples for further investigations to characterise malaria infections. We obtained written informed consent from all individuals or from the parent or guardian of children.Pailin is an agricultural province on the Thai-Cambodian border to the north of the Cardamom Mountains. As elsewhere in Cambodia, Pailin has seen a marked reduction in the incidence of malaria over the past decade . SeveralThe three villages with the highest malaria incidence from the Pailin provincial health department’s 2012 records were selected for the study. A study malaria team was formed in each village. Meetings were held to mobilize the community to participate and inform the villagers about the study. Before enrolment individual informed consent was obtained from all participants or their guardian. The study population were self-reportedly healthy individuals; people who were acutely sick were not asked to provide blood during surveys.de facto population, including migrants and visitors was invited to participate in the survey and coverage averaged 80%. Those with malaria parasitaemia in the June 2013 survey were thereafter tested monthly for malaria. During surveys tympanic temperature, height, weight, and health status and health history data were collected. A 3ml blood sample was collected by venepuncture from all participants aged 5 years and above and a 0.5ml venous sample from those aged 6 months up to 5 years. Samples were stored in EDTA tubes on ice and transported daily to a hospital laboratory for processing.The survey has been reported previously . In briePlasmodium species [Plasmodium species (P. species). All laboratory tests were blinded to participant malaria and health status.An ultra-sensitive quantitative polymerase chain reaction (uPCR) technique was used to detect malaria parasites. Validation and performance characteristics have been reported elsewhere . Parasit species . Sampleshttp://www.prolab.co.th/en/quality-assurance) using a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) with conjugated detection antibody. CRP tests were performed independently by laboratory staff blinded to all participant data and their case-control status.20ul plasma samples were thawed, split, and tested for CRP by an independent reference laboratory  sex- village- matched control (uPCR negative for parasites) surveyed in the same month.Cases were defined as villagers who were positive for The study used a cross-over design where each malaria patient served as their own control at the next time seen without malaria, as well as having age and sex matched controls from the same village. The associations between parasitaemia and plasma CRP concentrations were compared between cases and controls using the Wilcoxon matched-pairs signed rank test, for each of the control groups separately. The analysis was repeated with the exclusion of cases and controls who reported illness in the previous 48 hours and with the exclusion of febrile individuals at the time of the survey (in whom plasma CRP concentrations might be elevated for other reasons). We then compared the proportions of cases and controls with CRP concentrations that exceeded thresholds of 3mg/L and 10mg/L as predictors of cardiovascular disease  and infeThe parasite counts and plasma CRP concentrations were log transformed and the correlations between them were estimated (Pearson’s test for correlation); this estimation was repeated omitting cases with fever. A linear regression model was used to graph this association, as well as the association between the age/sex matched controls and their respective plasma CRP concentrations, controlling for the presence of fever.P falciparum and P vivax cases in isolation, excluding mixed infections and those where a species could not be identified.These analyses were then repeated for Data were entered into Open Data Kit (ODK) on smartphones before being exported into Open Clinica. Analysis was done in STATA 14.0.


Papua New Guinea exhibits a complex malaria epidemiology due to diversity in malaria parasites, mosquito vectors, human hosts, and their natural environment. Heterogeneities in transmission and burden of malaria at various scales are likely to affect the success of malaria control interventions, and vice-versa. This manuscript assesses changes in malaria prevalence, incidence and transmission in sentinel sites following the first national distribution of long-lasting insecticidal nets (LLINs).Before and after the distribution of LLINs, data collection in six purposively selected sentinel sites included clinical surveillance in the local health facility, household surveys and entomological surveys. Not all activities were carried out in all sites. Mosquitoes were collected by human landing catches. Diagnosis of malaria infection in humans was done by rapid diagnostic test, light microscopy and PCR for species confirmation.P < 0.001). The average population prevalence of malaria decreased from 15.7 % pre-LLIN to 4.8 % post-LLIN . In general, reductions in incidence and prevalence were more pronounced in infections with P. falciparum than with P. vivax. Additional morbidity indicators  showed a decreasing trend in most sites. Mean Anopheles man biting rates decreased from 83 bites/person/night pre-LLIN to 31 post-LLIN (P = 0.008). Anopheles species composition differed between sites but everywhere diversity was lower post-LLIN. In two sites, post-LLIN P. vivax infections in anophelines had decreased but P. falciparum infections had increased despite the opposite observation in humans.Following the roll-out of LLINs, the average monthly malaria incidence rate dropped from 13/1,000 population to 2/1,000  contains supplementary material, which is available to authorized users. Plasmodium species  are endemic and more than ten Anopheles species filling different ecological niches have been incriminated in the parasite’s transmission [P. falciparum malaria, across most parts of PNG [Papua New Guinea (PNG) exhibits a diverse and complex malaria epidemiology . Four Plsmission . Historismission , 4. The smission , 6 led ts of PNG .In 2004, the Government of Papua New Guinea re-intensified its malaria control efforts with the financial support of a round 3 grant from the Global Fund to Fight AIDS, Tuberculosis and Malaria. The central component of this program was the first country-wide free distribution of long-lasting insecticidal mosquito nets (LLIN) . InsectiThis manuscript assesses malaria morbidity and transmission indicators in sentinel sites before and after the LLIN distribution. The study was part of the evaluation of the Global Fund round 3 malaria grant.Sentinel site locations were selected purposively in 2008 in places which were yet to be covered with the LLIN campaign. The sites were located in the Momase and Highlands regions of the main island of PNG Fig.  and consData were collected prior to the LLIN campaign (10/2008–08/2009) and one year later after LLINs had been distributed (10/2009–08/2010). Data collection included clinical surveillance in the local health centre in three sites  over a period of approximately two months pre- and post-LLIN, coinciding roughly with the main malaria transmission season. A household survey in two to three randomly selected villages in the health centre’s catchment area was carried out in five sites  and entomological surveys were conducted in five sites . For operational reasons not all activities could be carried out in all sites . From the same blood sample, thick and thin blood films were prepared on one slide and haemoglobin (Hb) levels were measured using a portable analyser . Anaemia and severe anaemia were defined in accordance with World Health Organization definitions applying age group-specific cut-offs and altitude adjustment . A dry bFor the household survey, three (in the case of Yapsie four) villages were randomly selected from the sentinel health facility’s catchment area. In each village, 30 to 35 households were randomly sampled from a list of all households. A structured questionnaire about the coverage with malaria control interventions was administered to the heads of sampled households. A finger-prick blood sample was collected from household members above five months of age for preparing a microscopy slide, a dry blood spot on filter paper, and for measuring Hb levels as described above. Symptomatic individuals were diagnosed on the spot using a malaria RDT and positive cases were treated according to standard treatment guidelines . AxillarP. falciparum, P. vivax 210 and P. vivax 247 circumsporozoite proteins by enzyme-linked immunosorbent assay [An. punctulatus group were confirmed to species by polymerase chain reaction (PCR) restriction length polymorphism of the ITS2 region [Entomological surveys were conducted in two villages per site. Mosquitoes were collected by outdoor human landing catch from 6 households per village prior to the LLIN distribution and again 12 months later. The number of person-nights per collection ranged from 16 to 48. Collectors worked in pairs with one member collecting from 18:00 to 24:00 h and the second collecting from 24:00 to 06:00 h. Mosquitoes were stored per collection hour and identified to morphological species before storage on silica gel. Lysates from whole mosquitoes were screened for n = 127; household survey n = 87). DNA was extracted from filter papers using the DNeasy Blood and Tissue Kit  or the Favorgen 96-Well Genomic DNA Kit  following the manufacturer’s protocols. The molecular assay was a semi-quantitative post-PCR, ligase detection reaction/fluorescent microsphere assay described in more detail elsewhere [Thin blood smears were first fixed with methanol and thick and thin smears stained with Giemsa and read by light microscopy independently by two PNGIMR microscopists. Discordant reads were confirmed by a senior microscopist. The number of parasites was counted until reaching 200 white blood cells and a slide was declared negative only after reading a minimum of 200 thick film fields. Molecular diagnosis was used to complement missing second reads and to disambiguate discordant species read results  software. Differences in binary and categorical variables between study sites were assessed by Chi-square test and logistic regression, differences in continuous variables by P = 0.02). Sample details including LLIN coverage by site are provided in Additional file n = 5,036) of the pre-LLIN collection in this site and 43 %  of the post-LLIN collection were screened for Plasmodium spp. infection.Clinical surveillance in three sites included 1,325 fever patients pre-LLIN and 680 post-LLIN. The household surveys in five sites included blood samples from 1,967 individuals pre-LLIN and 1,986 individuals post-LLIN. Pre-LLIN and post-LLIN samples had comparable age distributions in all sites with the exception of the Mumeng clinical surveillance sample (P < 0.001) , the average proportion of fever patients with a positive RDT decreased from 57.3 % (95 % CI: 54.6–60.0) pre-LLIN to 17.9 % (95 % CI: 15.1–21.0) post-LLIN resulting in a 87 % drop in the pooled crude monthly incidence rate from 13/1,000 population to 2/1,000  than with P. vivax . Reductions in P. vivax infections were statistically significant only in children below 5 years of age in two sites  but not in older age groups . In Dreikikir, there was no significant reduction in P. vivax in any age group . The decrease in infections with P. malariae was substantial but due to low numbers of cases not statistically significant .Based on PCR-corrected light microscopy, the reduction was more pronounced in the proportion of fever cases infected with P. falciparum over P. vivax  to equal proportions of the two species . Pre-LLIN, P. falciparum infection prevalence peaked in fever patients aged 5–9 years, and P. vivax in 1–4 year olds. Post-LLIN, the P. falciparum peak had shifted to later age groups, while for P. vivax infections a clear peak could not be identified due to the low number of positive patients. Changes in test-positivity and species composition by site are shown in Fig. The parasite species composition in the febrile patient sample changed in Dreikikir (but not in the other sites) from a clear dominance of P = 0.015), and in splenomegaly  in both sites . Reductions in anaemia in Sausi and of severe anaemia in both sites were not statistically significant , anaemia and splenomegaly were assessed in the fever patients. A statistically significant reduction was found in anaemia in Mumeng . The largest reduction was observed in Yapsie, the site with the highest pre-LLIN prevalence of both P. falciparum (18.9 %) and P. vivax (11.8 %). Across all sites, the reductions were more pronounced in infections with P. falciparum than with P. vivax   only in Tabibuga .Across five sentinel sites, the average prevalence of malaria by light microscopy in the general population of the health facility catchment areas decreased from 15.7 % pre-LLIN to 4.8 % post-LLIN . Of those infected with malaria parasites, 12.9 % (95 % CI: 7.7–19.8) had an acute fever pre-LLIN and 8.1 % (95 % CI: 3.0–16.8) post-LLIN . Self-reported recent febrile illness (previous 2 days) was reported more frequently, by 14.3 % (95 % CI: 12.5–16.3) pre-LLIN and 7.9 % (95 % CI: 6.6–9.3) post-LLIN . A reduction was also noted in anaemia from 68.3 % (95 % CI: 65.6–70.9) to 50.1 % (95 % CI: 47.6–52.6) , and in severe anaemia from 4.9 % (95 % CI: 3.8–6.2) to 2.4 % (95 % CI: 1.7–3.3) . Not all reductions reached statistical significance in all sites, partly due to small sample sizes . Acute fever, defined as axillary temperature of > 37.5 °C, was measured in 2.3 % (95 % CI: 1.6–3.2) of individuals pre-LLIN and 1.9 % (95 % CI: 1.3–2.7) post-LLIN  across five sites decreased from 83 (95 % CI: 48– 117) bites/person/night pre-LLIN to 31 (95 % CI: 15–47) bites/person/night post-LLIN (t(141) = 2.69, P = 0.008). The highest mean pre-LLIN MBR (361/person/night) was found in Sausi and dominated by An. farauti 4 , An. punctulatus (-93.3 %) and An. farauti (s.s.) , while An. farauti 4 (-48.1 %), present in large numbers in Sausi, and An. longirostris (-6.3 %) appeared least affected. An. hinesorum and An. farauti 5 were mainly found post-LLIN. Diversity in species composition was lower after the LLIN distribution and in all sites except Yapsie, the species dominant pre-LLIN remained dominant post-LLIN  P. falciparum infections were dominant. Sporozoite prevalence was lowest in Sausi, where the MBR was highest. Post-LLIN, no infected mosquitoes were found in Dreikikir, Mumeng and Yapsie. In the remaining sites, pre-LLIN infection of mosquitoes and monthly entomological inoculation rate (EIR) were higher for P. vivax than for P. falciparum. Post-LLIN, P. vivax infections had decreased, but P. falciparum infections had increased in both sites  carried es Table .Table 3APlasmodium spp. infection in the general population dropped significantly over the same period mainly due to a 76 % reduction in infections with P. falciparum . The prevalence of blood-stage P. vivax parasitaemia decreased less prominently (-60 %) reaching statistical significance in two of five study sites (Finschhafen and Yapsie). A significant reduction was also observed in the incidence of clinical malaria cases in three sites, primarily as a result of an 81 % drop in the proportion of P. falciparum-infected febrile patients . The proportion of cases infected with P. vivax decreased less across all age groups (-46 %), and statistically significantly only in children under 5 years of age. In the Dreikikir site, no significant decrease in the proportion of P. vivax cases was detected in any age group. These findings confirm results from a cohort study in young children conducted near Dreikikir which found that LLIN use was associated with a reduction in both P. falciparum infections and clinical episodes, while it only reduced P. vivax infections but not clinical episodes [The roll-out of LLINs in 2009 was followed by substantial reductions in key indicators of malaria morbidity and transmission in six sites in PNG within less than one year. Average LLIN use in these sites had increased from 6 to 55 %, and 89 % of households in the sites owned at least one LLIN after the distribution  and therepisodes .P. vivax over P. falciparum following implementation of malaria control measures confirms previous observations both from PNG [A relative increase in from PNG , 20, 25 from PNG , 27. Thefrom PNG  and potefrom PNG .P. vivax to relapse from long-lasting liver stages and the higher probability of gametocytaemia are most likely key factors. A recent study confirmed that relapses from P. vivax hypnozoites contribute 80 % of the burden of malaria infection and clinical episodes in PNG [P. vivax blood-stage infections detected post-LLIN roll-out are not only due to new infections from mosquito bites but also due to relapses from hypnozoites established both pre- and post-LLIN [P. falciparum and P. vivax gametocytes, respectively [P. vivax transmission would therefore require a stronger reduction of P. vivax parasitaemia. Also, P. vivax gametocytes appear early in an infection, when asexual densities are still very low [P. vivax infections are thus thought to be gametocyte positive [Anopheles mosquitoes being more likely infected with P. vivax than P. falciparum [P. vivax transmission before people retire to bed. This is further aggravated by the shorter extrinsic incubation period for P. vivax. As LLINs reduce the biting density as well as the lifespan of mosquitoes, an equal reduction in lifespan is more likely to interrupt P. falciparum transmission [P. vivax than P. falciparum gametocytaemia and hence on transmission. However, once the pre-LLIN hypnozoite reservoir is exhausted, P. vivax prevalence can be expected to drop in line with reductions in the P. vivax EIR.The ability of s in PNG . In trops in PNG . Althougost-LLIN . Gametocectively . Reducinvery low  and almopositive  hence inlciparum , 36, thusmission . As a coAn. farauti four in both rounds), only the P. vivax EIR was found to decrease after LLIN distribution, while the P. falciparum EIR increased. The same was observed in Finschhafen, yet with a small number of mosquitoes post-LLIN (n = 67). In the other sites the mosquito sample size post-LLIN was too limited to draw strong conclusion. However, more comprehensive entomological data published by Reimer et al. [Anopheles infections from P. vivax pre-LLIN to P. falciparum one to two years post-LLIN in Madang province and Dreikikir. The different baseline species composition reported by the two studies for Dreikikir is likely related to the low number of infected mosquitoes  caught pre-LLIN in this study. At the same time, a comparison of results from remote Yapsie(i) area, where a 1986 survey in three villages had found 0.4–1.5 % of anophelines (mostly An. koliensis) to be infected with P. falciparum and 0–1.7 % with P. vivax [. Interestingly in that site, neither in 1986 nor in 2009 infected were mosquitoes collected in the central Yapsie station.The entomological results indicate a significant impact of LLINs on transmission but a varying effect by species and study site. In the site with the largest number of mosquitoes pre- and post-LLIN  suggesting generally positive health developments. Splenomegaly, a syndrome closely associated with chronic malaria in PNG , 42, was.  for examhttp://www.ncdc.noaa.gov/), but rainfall data from Madang airport presented by Reimer et al. [This study was not without limitations. Due to financial and operational constraints not all data collection components  could be implemented in all study sites, thus limiting the extent to which trends in these indicators can be compared. Household survey and entomology survey were cross sectional in nature and the clinical surveillance limited to approximately three months each round. While this approach effectively disregards seasonality, pre-LLIN and post-LLIN assessments were implemented during the same time of the year in an effort to maximize comparability. Differences in weather patterns potentially impacting on entomological indicators could not be reliably investigated due to a lack of detailed weather records  and Madang provinces (Madang North coast area). Gathering data from a larger number of locations over multiple years therefore appears essential to improve our understanding of the malaria epidemiology in PNG particularly as some of the observed effect, such as the shift to P. vivax as the most prevalent Plasmodium spp. infection, may be transient.The distribution of LLIN had distinct effects on AOR, adjusted odds ratio; CI, confidence interval; DNA, Deoxyribonucleic acid; EIR, entomological inoculation rate; Hb, haemoglobin; IR, Incidence rate; IRR, incidence rate ratio; LLIN, long-lasting insecticidal net; MBR, Man biting rate; PCR, Polymerase chain reaction; PNG, Papua New Guinea; PNGIMR, Papua New Guinea Institute of Medical Research; RDT, rapid diagnostic test.


Malaria is considered as one of the major public health problems and among the diseases of poverty. In areas of stable and relatively high transmission, pregnant women and their newborn babies are among the higher risk groups. A multicentre trial on the safety and efficacy of several formulations of artemisinin-based combination therapy (ACT) during pregnancy is currently on-going in four African countries, including Zambia, whose study site is in Nchelenge district. As the study outcomes may be influenced by the local malaria endemicity, this needs to be characterized. A cross-sectional survey to determine the prevalence and intensity of infection among <10 years old was carried out in March-April 2012 in Nchelenge district.The sampling unit was the household where all children < 10 years of age were included in the survey using simple random household selection on a GPS coded list. A blood sample for determining haemoglobin concentration and identifying malaria infection was collected from each recruited child.Plasmodium falciparum and the remaining ones  Plasmodium malariae. Anaemia, defined as haemoglobin concentration <11 g/dl, was detected in 51.2% (398/782) children.Six hundred thirty households were selected and 782 children tested for malaria and anaemia. Prevalence of malaria infection was 30.2% (236/782), the large majority  being In Zambia, despite the reported decline in malaria burden, pockets of high malaria endemicity, such as Nchelenge district, still remain. This is a border area and significant progress can be achieved only by concerted efforts aimed at increasing coverage of current control interventions across the border. Malaria remains a major public health problem and a disease of poverty. In areas of stable and relatively high transmission, pregnant women and their newborn babies bear a high burden of malaria morbidity and mortality .In Zambia, sulphadoxine-pyrimethamine (SP) is used for intermittent preventive treatment in pregnancy (SP-IPTp) during the second and third trimester. Until recently, malaria in pregnancy was treated with quinine, which has now been replaced by artemether-lumefantrine (AL) for women in the 2nd and 3rd trimester in pregnancy; quinine is used for the management of malaria during the 1st trimester and for severe cases  <11 g/dl) 55.9%  . ConsideThe rationale of this study was that it gives a more recent data on the prevalence of malaria in a high endemic region. In places like Zambia, where overall figures indicate that malaria is declining, there are still pockets of high endemicity, which are complicated by perfect breeding grounds for anopheles mosquito and cross-border human movements. This could affect national and global malaria control strategies. The primary objective was to estimate the prevalence and risk factors for malaria in Nchelenge, Luapula Province, Zambia.Nchelenge district, Luapula province, is located on the swampy shores of Lake Mweru, and has a population of 178,000 inhabitants, mostly peasant farmers and/or fishermen. The district has one first level hospital, ten rural health centres and two health posts. The survey was done in the catchment areas  of the two health centres, Kashikishi and Nchelenge, where the PREGACT trial is currently implemented.The household was taken as the sampling unit. The households in Nchelenge and Kashikishi health centre catchment areas were previously numbered using the Global Positioning System (GPS). Then the households were selected according to a pre-defined, computer-generated list of random numbers . In seleThe sample size was determined by assuming that malaria prevalence was 22% ; using RAll children <10 years of age in the selected households were asked to be included in the survey. Each study, subject was assigned a unique identifier for identification of biological samples and questionnaire data and to guarantee confidentiality. Information on bed net use, which was by observation, including long-lasting insecticidal nets (LLIN) or previous indoor residual spraying (IRS) was collected onto the structured questionnaires. A blood sample for a malaria rapid diagnostic test (RDT), , thick blood film and for measuring haemoglobin (Hb)  was collected by finger prick. Children with fever (axillary temperature ≥37.5°C) and a positive RDT or only a positive RDT were treated with AL, the first-line treatment in Zambia. Children found to be sick or anaemic were referred to the health care centres for further management.Thick blood films were stained with 10% Giemsa for 10 minutes. Two hundred high power fields were read before declaring the slide negative. The parasite density was determined by counting the number of parasites against 200 white blood cells (WBC) and assuming 8,000 WBC per μl. All stained slides were read by two independent microscopists unaware of the RDT results. In case of discrepant results between the two independent microscopists, a third microscopist read the slide until reaching a satisfactory agreement on positivity/negativity or the parasite density.2 test or Fisher’s exact test. To account for uncertainty in the prevalence estimates, the 95% confidence intervals were computed. Anaemia was defined as Hb < 11 g/dl and created a dichotomous variable where Hb ≥ 11 g/dl was defined as non-anaemic. The risk of anaemia and malaria was estimated by fitting a multivariable logistic regression model and adjusted for possible risk factors to compute odds ratios (OR) and 95% confidence intervals (95% CIs). The variables included in the multivariable logistic regression model were selected a priori via extensive search of peer-reviewed literature. A two-sided p-value of ≤0.05 was used as threshold for statistical significance. The ‘survey logistic’ procedure in SAS 9.2  with the option ‘clustering’ to account for the design effect of clustering within households.Data were double entered, cleaned and analysed using Epi Info™ . Further multivariable logistic regression analyses were performed using SAS version 9.2 . Mean and standard deviation were computed to describe the distribution of continuous variables, while the distribution of categorical variables was described using counts and proportions. Geometric mean and median were computed for continuous variables that did not follow a normal distribution: parasite densities and Hb concentrations. Continuous variables were compared between age groups using t-tests and categorical variable proportions by χBefore the survey, the study protocol was approved by the scientific and technical committee (STC) of Tropical Diseases Research Centre (TDRC), the TDRC Ethical committee and the Ministry of Health. Participation in the survey was voluntary and participants were free to withdraw any time during the capture.669 households were screened, 39 households were not responsive (21 were not available while 18 refused to participate). There was a 630/669 (94.2%) response. At this rate, the results presented are less biased. Out of the households that were responsive majority reported tubed well or borehole as main source of drinking water . 90.0%  households had open pits or pit latrines without slabs as toilet facilities. 96.0%  households reported charcoal as the main source of household fuel. Most houses were made of finished brick walls  and grass thatched roofs or sticks and mud roofs . In terms of owning durable goods, 83.5%  households owned a landline telephone or a cell phone, while a majority  also owned a bicycle  were more represented than boys 40.5%, 317/782) (p < 0.005). The median age was five years , with the 7–10 years age group as the largest age group . Only about a third of children sampled  came from a household with a bed net. Among those coming from a household with bed net 78.3%  reported to have used it the previous night. Coverage of IRS was even lower, only 11.1%  households had been sprayed by the local district health team  and 31.8% (7–10 years old) in the other age groups , the large majority 98.0%, 231/236) /236 PlasP. falciparum was 1,250 parasites/μl  and varied with age, with the highest value in the 1–2 years old group, 1,770 parasites/μl , and the lowest, 590 parasites/μl  in the 5–7 years old group (p = 0.7) , ranging between 1.2 g/dl and 15.4 g/dl. About half (51.2% 398/782), 95% CI, 47.6-54.7%) of the children were anaemic (Hb < 11 g/dl) while 7.4% (58/782),  of them were severely anaemic (Hb < 8 g/dl). Mean Hb and anaemia did not vary with age (p = 0.7). Malaria infection was significantly associated with anaemia  and such risk tended to be higher in children with the highest parasite densities   nor in households sprayed the previous year . The age specific odds show that children 3- < 5 years and those from 5- > 7 years who did not use bed nets the previous night and are in homes not sprayed are more likely to have a malaria infection than those who use bed nets and have their homes sprayed   survey tools, provides a recent estimation of the malaria burden in Nchelenge district. The prevalence of both malaria infection and anaemia was similar to those found in national surveys for Luapula province , a resulIt is important to notice that Nchelenge borders the Democratic Republic of Congo (DRC), with substantial population movements across the borders, and is difficult to reach due to poor terrain, possibly explaining the low coverage of control interventions. Indeed, only a third of the children included in the survey stated to have a bed net and even less used it the previous night. As IRS, only a small proportion of the sampled houses had received this intervention so that any impact on the local vector population is unlikely. It is unknown what the coverage of control interventions may be on the other side of the border but probably not higher than that in Nchelenge, more likely lower. In these conditions, the prevalence of malaria and anaemia found by this survey are not surprising as they reflect the extremely low coverage of ITN and IRS. However, even in case of a better coverage, prevalence may still have been high as the important population movements between Zambia and DRC would maintain the local human reservoir and hence malaria transmission. A significant reduction in the malaria burden in this region may be achieved only through a close collaboration between the national control programmes of both countries. Other countries sharing borders like South Africa, Mozambique and Swaziland in a programme called the Lubombo Spatial Development Initiative (LSDI) have achieved spectacular successes in reducing cross-border malaria burden .The mean parasite density in the children included in this survey, most of them asymptomatic, was relatively high, particularly in the 1–2 years old age group, confirming Nchelenge as an area of intense malaria transmission. Prevalence of malaria would probably have been higher if molecular methods to detect infection had been used. Following continuous exposure to malaria infection, children develop partial immunity and are able to tolerate relatively high parasite densities. Access to treatment seems high as more than 60% of children’s parents stated they had taken an antimalarial treatment the month prior to the survey. However, information on the quality and the source of treatment was not collected during the survey so that it is impossible to know if this was of adequate quality and dosage. The apparently frequent administration of antimalarial treatment is just an indicator of the importance of malaria in this setting.P falciparum gametocytes by microscopy can miss most of the gametocyte carriers [The prevalence of gametocytes was relatively low though probably a higher number of children with gametocytaemia could have been identified by using molecular methods. Detecting gametocytes in thick blood smears, particularly when present at low densities, is a challenge even for experienced microscopists . Indeed,carriers -19 as thcarriers -22.This was the weakness of this study in which gametocytes were examined on thick smears.Coverage by bed nets and IRS was low and this may explain the higher prevalence of malaria infection. However, recent data seem to indicate a problem of emerging resistance to pyrethroids and DDT among the vector population in Nchelenge . NeighboThe logistic multivariable analyses found that children did not use bed nets and are in households not sprayed have a higher risk of malaria infection in the age 2 to 7 years. This is the age group that is just weaned from the mother and would not be under a bed net nor cover themselves from mosquito bites. This study also confirms that malaria affects haemoglobin levels in communities where children bear the brunt of the malaria burden ,26 and hThe limitations of this work is that it was carried out in relatively smaller or narrow geographical focus. This could over- or underestimate the bed net or IRS coverage and indeed malaria prevalence. Since malaria presents with some seasonality, the study was done at the end of the rainy season but there could be differences in the dry season. Sociodemographic data were collected via self-reporting and, therefore, self-report, recall bias, and social desirability bias cannot be ruled out. Cognisant of this potential bias, the study interviewers made efforts to enhance truthful reporting. The nature of the cross-sectional survey does not permit causal inference of the observed association. In conclusion, even though overall figures indicate that malaria is declining in Zambia, there are still pockets of high endemicity such as the Nchelenge district. This district is on the shores of Lake Mweru and neighbouring DR Congo. Malaria will be controlled only by concerted efforts across borders aiming at increasing coverage of preventive intervention and prompt access to diagnosis and antimalarial treatment.The authors declare that they have no competing interests.All authors contributed to the design of the study and assisted with data interpretation. MN and PM coordinated the study and supervised the enrolment, data collection and entry. PM did the laboratory analysis and study participants enrolment. JPVG, MM and UDA participated in analysis of data. All authors participated in the preparation of the manuscript and approved the final version. All read and approved the final manuscript.