Background and Objectives. Malaria infection, caused by Plasmodium falciparum, is the most lethal and frequently culminates in severe clinical complications. Interleukin-22 (IL-22) has been implicated in several diseases including malaria. The objective of this study was to investigate the role of IL-22 gene polymorphisms in P. falciparum infection. Material and Methods. Ten single-nucleotide polymorphisms (SNPs), rs976748, rs1179246, rs2046068, rs1182844, rs2227508, rs2227513, rs2227478, rs2227481, rs2227491, and rs2227483, of IL-22 gene were genotyped through PCR-based assays of 250 P. falciparum infection. IL-22 gene promoter activity. P. P. falciparum infection. P. P. P. P.  We found that the rs2227481 TT genotype (odds ratio 0.254, confidence interval = 0.097-0.663, P. falciparum infection. IL-22 gene promoter activity.  The study suggests that IL-22 polymorphisms in rs2227481 and rs2227483 could contribute to protection against  Plasmodium falciparum is associated with significant morbidity and mortality in humans [Malaria caused by n humans . It affen humans . Malarian humans .Eliminating Countries for 2020 (E-2020)” initiative of the WHO to achieve the goal of a malaria-free country by 2020 [Plasmodium species causing the majority of the malaria cases is P. falciparum (97%) followed by P. vivax (2%).Saudi Arabia is one of the largest countries in the Middle East with a population of over 32 million. The implementation of malaria control measures in Saudi Arabia over the past 50 years, in collaboration with the World Health Organization (WHO), has effectively reduced the magnitude of risk throughout the nation –6. In 20P. falciparum infection. Also known is the fact that malaria parasites have a robust selective pressure on human genetics, particularly in regions where malaria is endemic [P. falciparumare influenced by host genetic factors and assist in establishing the pathological mechanisms underlying the vulnerability to the infection [α) gene are associated with an increase in SMA in P. falciparum-infected patients [α gene have been shown to increase the susceptibility to severe malaria [It is known that the host genetic polymorphisms play an important role in the variation of malaria severity associated with  endemic , 8. Varinfection . For exapatients . SNPs in malaria .IL-22 gene is positioned at chromosome 12q15. Both innate and adaptive arms of the immune system produce IL-22, including αβ T, γδ T, NKT, and innate lymphoid cells (ILCs). Certain nonhematopoietic and myeloid cells can also release IL-22 [Interleukin-22 (IL-22) has been classified as a cytokine of the IL-20 subfamily. The se IL-22 , psoriasse IL-22 , inflammse IL-22 , Guillaise IL-22 .IL-22 gene are associated with several diseases such as ulcerative colitis (UC) [P. chabaudi malaria infection in mice, a higher production of IL-22 was observed in the liver, which is important as it suggests that this cytokine is an essential factor in the protection against the lethal infection caused by P. chabaudi [P. falciparum-associated malaria and pathogenesis. Therefore, this report investigates the plausible role of variations at the IL-22 gene in the complications associated with P. falciparum infection in a Saudi Arabian population.Polymorphisms in the tis (UC) , psoriastis (UC) , bladdertis (UC) , and systis (UC) . It has tis (UC)  and an Ichabaudi . Such a P. falciparum-infected patients admitted to the Malaria Center in the Jazan region, located in the southwest of Saudi Arabia. The study also included 200 randomly selected uninfected healthy individuals as a control group. A thick blood film from each infected patient was used to determine the parasite density according to the “plus system” scale, described in the WHO manual [Venous blood was collected from 250 O manual . Based oThis study was approved by the Research Ethics Committee at King Fahad Hospital (KFCH), Jazan (Registry number 041) and conducted in accordance with the Helsinki Declaration of 1975. Informed consent were obtained from all participants prior to their participation in the study; consent for children were given by their legal guardians. To ensure confidentiality, patient data and biological samples were anonymized prior to processing by laboratory staff. Basic demographic and clinical data required to accomplish the objectives of this study were collected and securely retained for all study participants.IL-22 gene region were selected for genotyping. The IL-22 SNPs included were rs976748, rs1179246, rs2046068, rs1182844, rs2227508, rs2227513, rs2227478, rs2227481, rs2227491, and rs2227483. Candidate SNPs were selected based on the following criteria: (i) a minor allele frequency (MAF) ≥ 5%, as reported by the 1000 genome for the combined populations and (ii) a linkage disequilibrium (LD) threshold (r2) ≤ 0.8 or (iii) existence of evidence for an association with malaria. Marker information of the SNPs selected is shown in Supplementary A total of ten SNPs within the http://bioinfo.ut.ee/primer3-0.4.0/). All primers were tailed with an M13 sequence to facilitate the sequencing of the PCR products. Sequences of the primers and PCR conditions used for each SNP are shown in Supplementary DNA was extracted using the DNeasy Blood and Tissue kit  according to the manufacturer's protocol and stored at -20°C for further experimentation. The PCR was performed using the GoTaq Green Master Mix PCR . Primers flanking SNPs of interest were designed using Primer3 Input (version 0.4.0)  following the manufacturer's instructions. Extracted plasmids with cloned fragments were transfected into HEK293 cells (Human Embryonic Kidney 293 cells), which were harvested and tested for luciferase activity after 48 hours.Gene fragments of the promoter region containing IL-22 rs2227513 A/G variants were amplified and cloned in the pGL3 Basic Vector . Primers were tailed at the 5′ end with specifying endonuclease recognition sites for the restriction enzymes SmaI and BglII . In addition, a few overhang sequences (CGCCTA) were added to facilitate restriction enzyme binding. Primers used for the amplification of the promoter region, PCR conditions, and chromosomal locations are shown in Supplementary http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) was used to conduct a genotypic association test for all the selected variants as the primary test of association. We also performed the allelic, dominant, and recessive model for each variant to explore the possibility of other models of inheritance. The results were expressed as odds ratios (OR) with 95% confidence intervals (95% CI). Testing of deviation from the Hardy-Weinberg equilibrium (HWE) was performed; rs2227513 was excluded from further analyses due to the significant deviation from HWE . The test of association was considered significant if the P value, obtained in a two-tailed test, was <0.05.The SNPs were analyzed and measures of LD, haplotype frequencies, and MAFs were calculated using the Haploview software (version 4.0) . The de Finetti program  . We also found that the T allele was associated with a lower risk of P. falciparum malaria in a recessive model . For IL-22 variant rs2227483, we identified that the heterozygous AT genotype frequency was significantly higher in the control group in comparison to the patient group .The genotype distribution of the SNPs in the IL-22 gene between the = 0.002) . In addiχ2 = 6.13, and P = 0.013) . The IL-22 SNPs were examined in cohort I compared to cohort II+III+IV and cohort I+II compared to cohorts III+IV; however, none of the SNPs showed any statistically significant distribution between the groups  . In addiχ2 = 4.14, and P = 0.044). Also, the rs2046068 A allele was significant in a dominant model   .χ2 = 5.14, and P = 0.023) . The rs2227478 CT genotype was substantially elevated in the 10 years and older age group malaria compared to the younger age group  , with the T allele of rs2227483 associated with the 10 years and older age group .Comparing the malaria group in a 1-9 years age group with a 10 years and older age group, the frequency of the rs976748 CT genotype was significantly higher in the 1-9 years age group compared to the other group  . Under a= 0.030) . SimilarP. falciparum malaria comparing the malaria group in the 10-20 years age group with the 21 years and older group . No dominant or recessive statistically significant relationship was observed between the two groups. In contrast, the IL-22 rs2227483 T allele was associated with a high susceptibility to develop P. falciparum malaria in the 41 years and older age group with a higher frequency of the rs2227483 T allele . For both SNPs, no dominant or recessive relationship was detected.No relationship was observed between the IL-22 SNPs and the risk of ge group . We founP. falciparum-infected group and the control group and two blocks were generated. Block 1 comprised the IL-22 polymorphisms rs1179246, rs1182844, and rs976748, with block 2 consisting of IL-22 polymorphisms rs2046068 and rs2227491 (P = 0.034). In block 2, one haplotype A-G of rs2046068 and rs2227491 was statistically significant and prevalent in the control group but not in the malaria group .A haplotype analysis was done between the s2227491 . One hapP = 0.036).A haplotype analysis was performed between cohort I+II and cohort III+IV of varied parasite density . The disP = 0.056). However, the haplotype C-A of rs2046068 and rs2227491 was significantly high in the 6 years and older age group .A haplotype distribution analysis was done in the malaria group for two age groups, 1-5 years and 6 years and older. The frequency of two haplotypes was found to be either statistically significant or close to significance . The hapP = 0.041).A haplotype analysis was also performed between patients in the 1-9 years and 10 years and older age groups. One single haplotype was statistically significant . The hapP = 0.045).Finally, we assessed the haplotype distribution between patients in the 1-9 years and 41 years and older age groups. The distribution of two haplotypes was significantly different in these two groups . The hapFor the delineation of the molecular mechanisms driving the IL-22 gene, nested deletions from the IL-22 promoter were generated  and clonP < 0.0001).To test whether rs2227513 contributes to the expression of the IL-22, one nucleotide was changed in clone F3/R1 . The IL-P. falciparum and has an efficacy of 39% [P. falciparum-infected patients. The understanding of genetic differences in populations substantially transformed the development of vaccines in general and assisted in developing the most recent malaria vaccine [Globally, malaria remains one of the major causes of mortality in children younger than 5 years . Recently of 39% . The divy of 39% . Such di vaccine , 27.IL-22 gene in P. falciparum malaria infection. The aim was to determine whether there is an association in terms of vulnerability or protection as indicated by the level of parasite density or the age group distribution. IL-22 is suggested to have a protective role in severe anemia-associated malaria infection, as it has been reported that IL-22 is elevated in children infected with P. falciparum [P. falciparum malaria; one correlated with protection against SM (rs2227491) and the second with susceptibility to CM (rs2227478) [r2 = 0.92) with rs2227481 and is significantly associated with the risk of P. falciparum malaria in our population. There is a dearth of data to study the role of the IL-22 gene with vulnerability or protection, parasite density and age-related risk of P. falciparum malaria infection.In this study, we examined the frequency of nine SNPs  in the lciparum , 22, 29.2227478) . HoweverIL-22 rs2227481 T allele with protection against P. falciparum malaria infection in a recessive mode of inheritance, with a similar association found for individuals with a homozygous TT genotype. It was also found that the C allele of this SNP is associated with an increased risk of malaria infection. Our highest significant SNP (rs2227481) is in high LD with rs2227476, previously linked to a predisposition to childhood CM in an African population, potentially by altering the binding site for several transcription factors as suggested by the in silico analysis [IL-22 in the clinical manifestation of P. falciparum-associated malaria. It has been reported that the IL-22 rs2227481 polymorphism plays a crucial role in effector T cell pathways [P. falciparum parasite density in the Saudi Arabian population. The rs2227481 T allele was associated with a low parasite density in a dominant model. However, no relationship was observed for an age-related risk of the infection. The results indicate that the rs2227481 T and homozygous TT genotype may be important in protecting against P. falciparum malaria infection.In the present study, we strengthen the possible relationship between the pathways . ExploriP. falciparum infection. Zhang et al. demonstrated that the A and T alleles of IL-22 rs2227483 were significantly elevated in controls compared to tuberculosis (TB) cases and suggested that rs2227483 A and T alleles are associated with increased production of IL-22 decreasing vulnerability to TB infection [P. falciparum infection, the malaria group possessed a higher TT genotype compared to the controls. Our findings and literature suggest that the variant rs2227483 TT genotype could have a role in the altered IL-22 expression and increased risk of malaria infection.The IL-22 variant rs2227483 is located within putative binding sites for the aryl-hydrocarbon receptor (AhR-ARNT) complex, and it has been suggested that this complex transcription factor induce IL-22 expression . The AhRnfection . Notablynfection . AlthougP. falciparum malaria [IL-22 gene and could influence the expression levels of the protein; however, the conclusion should be substantiated by experimental evidence.As parasite density is associated with a more severe outcome in  malaria , 34, we IL-22 gene polymorphism rs2046068 was not statistically associated with either protection or risk of malaria infection or the level of parasite density. Similarly, Weger et al. did not find any relationship of the rs2046068 genotype and haplotype analysis with chronic plaque psoriasis [The soriasis . In the soriasis . We founIL-22 gene demonstrated statistically significant frequencies between the control and P. falciparum-infected malaria groups. We detected a high frequency of the rs1179246-rs1182844-rs976748 A-T-T haplotype in the control group compared to the malaria group, suggesting the role of the A-T-T haplotype in protecting against P. falciparum infection. Similarly, an elevated frequency of the rs2046068-rs2227491 A-G haplotype was observed in the control group. Wang et al. did not find any association between the rs2227491 and serum IL-22 levels of the control group in SLE cases in a Chinese population [P. falciparum-associated clinical outcomes and the function of IL-22 need to be experimentally substantiated.The haplotype distribution analysis at the IL-22 gene and it is also part of the promoter region of the IL-22 gene. Studies suggest that introns are imperative modulators of gene functions such as RNA editing [r2 = 0.98) with rs976748, associated with a higher risk of P. falciparum malaria infection.The variant rs2227513 is positioned at the first intron of the  editing , 38. In P. chabaudi-induced liver damage [Several studies have shown that variations at the IL-22 gene could contribute to the outcome of different diseases. Mastelic et al. demonstrated a shielding role of IL-22 cytokine against r damage . Also, Hr damage . It was r damage . The latr damage . Our finIL-22 gene may produce different roles in the vulnerability to infection in populations with diverse ethnic backgrounds. A plausible explanation of this disparity could be environmental or other genetic elements. As we did not investigate all the IL-22 gene variants, future case control studies are necessary to confirm the findings of this study and to explore additional links between IL-22 SNPs and the clinical outcome of P. falciparum-associated malaria. It is essential to reproduce a similar study in other populations with a different genetic background to replicate our findings in a larger sample size. In addition, studies to outline the molecular or cellular mechanisms underlying the role of IL-22 in malaria are recommended.This study suggests that variants in the P. falciparum malaria. The IL-22 promoter polymorphism in rs2227513 G allele appears to be associated with higher expression levels of IL-22, which could play a role in the protection against the disease. These findings will increase the current understanding of the mechanisms involved in the pathogenesis of malaria infection.Our data shows that the IL-22 rs2227481 TT genotype and T allele and the rs2227483 AT genotype and A allele may be associated with protection against


Imported malaria is a major challenge for countries that are in malaria elimination stage such as Zambia. Legitimate cross-border activities add to the risk of transmission, necessitating determination of prevalence, characteristics and risk factors of imported and local malaria.This cross-sectional study was conducted in 103 consented child and adult patients with clinical malaria symptoms, from selected health facilities in north-western Zambia. Patient demographic data and blood samples for malaria microscopy and full blood count were obtained. Chi-square and penalized logistic regression were performed to describe the characteristics and assess the risk factors of imported and local malaria in North-Western Province.Plasmodium falciparum and 6.2% other species. The local cases were 72 (88.9%) while the imported were 9 (11.1%) out of the 81 positive participants. About 98.6% of the local cases were P. falciparum compared to 55.6%  P. falciparum among the imported cases. Among the imported cases, 44% were species other than P. falciparum  while among the local cases only 1.4% were. Gametocytes were present in 44% of the imported malaria cases and only in 2.8% of the local cases . About 48.6% of local participants had severe anaemia compared to 33.3% of participants from the two neighbouring countries who had . In the final model, only country of residence related positively to presence of species other than P. falciparum  and presence of gametocytes .Overall, malaria prevalence was 78.6% with 93.8% P. falciparum as the predominant species although importation of Plasmodium ovale and Plasmodium malariae is happening as well. Country of residence of patients is a major risk factor for malaria species and gametocyte presence. The need for enhanced malaria control with specific focus on border controls to detect and treat, for specific diagnosis and treatment according to species obtaining, for further research in the role of species and gametocytaemia in imported malaria, cannot be overemphasized.Malaria prevalence in North-Western Province is high, with  Malaria continues to be among the most prevalent parasitic diseases experienced worldwide . GloballThe malaria burden in Zambia is greatly influenced by climatic factors, such as rainfall and temperature . RainfalFocussing more on North-Western Province, this is a province bordered by two countries, the Democratic Republic of Congo and Angola . This gePlasmodium falciparum accounted for 99.7% of morbidity in the World Health Organization (WHO) African Region in 2018 . Table P. falciparum positive by RDT, only 77 participants were P. falciparum positive by microscopy, rendering 11 from the RDT as false positives. On the other hand, out of the 15 RDT negative participants, four were microscopically positive for other species. Figure From Table Table P. falciparum and 5 (6.2%) for other species. Disaggregating the malaria cases by local and imported, 72 (88.9%) of the 81 positive participants were local cases and 9 (11.1%) were imported.Overall, the 81 out of 103 patients that tested positive for malaria by microscopy translated to a prevalence of 78.6%. The species found in the malaria positive participants were 76 (93.8%) P. falciparum and only one (1.4%) was P. malariae; and five (three from Angola and two from Congo) out of nine imported cases (55.6%) were P. falciparum although one case was a mixed infection of P. falciparum and P. malariae. The remaining four out of the nine imported cases (44.4%) were of species other than P. falciparum broken down as follows: two out of nine P. ovale (22.2%) occurring in participants from Angola and Congo each; one out of nine (11.1%) with a mixed infection of P. ovale and P. malariae, occurring in a participant from Angola and yet another one out of nine (11.1%) with a mixed infection of P. falciparum and P. malariae, occurring in a participant from Congo.Based on the characteristics of both the local and imported malaria cases investigated, namely; species, gametocyte presence and severity of anaemia, this study shows that 71 of the 72 local cases (98.6%) were Plasmodium species picture in the participants resident in Zambia differs significantly from that in participants from the two neighbouring countries. Participants resident in Zambia had P. falciparum (98.6%: 95% CI 95.9–101.3) as the main species, while participants from the two neighbouring countries had a considerable combination of P. falciparum (55.6%: 95% CI 23.1–88), P. ovale (22.2%: 95% CI 4.9–49.4), P. ovale/P. malariae and P. falciparum/P. malariae (11.1%: 95% CI 9.4–11.1) each. Table P. falciparum, three were adult cases (11.5%) and two were children , varying significantly.Figure 2 = 48; p < 0.001) observed in the participants resident in Zambia.With regards to the other two characteristics studied, i.e. presence of gametocytes and severe anaemia, there were four participants (44%) with gametocytes from the two neighbouring countries, which is significantly more than the two cases  and three (33.3%) from the two neighbouring countries and 35 (48.6%) from Zambia  had severe anaemia.In terms of severe anaemia, Table P. falciparum  as well as presence of gametocytes . Thus participants who resided in Angola and Congo were more likely to have species other than P. falciparum as well as gametocytes. Age and gender were not associated with either presence of species other than P. falciparum or gametocytes. Country, age and gender were all not significant in the severe anaemia analysis.Firth Penalized Regression models were run for the significant cases in the bivariate analysis to determine the associated risk factors. Table These results show that: Predicted logit of (species presence) = −3.864 + (3.664)*Country; and Predicted logit of (gametocyte occurrence) = −3.339 + (3.139)*Country.P. falciparum were 39.1 times  of the malaria cases were local, as expected, although a few (11.1%) imported cases were also detected. These were of patients from the two neighbouring countries, Angola and Congo, who accessed the selected health facilities in Zambia during the period of our study. The two countries Angola and Congo have been reported to be among the leading countries in the importation of malaria from Africa to other countries, such as China . With reDiscussing this study conducted in a moderate malaria transmission zone, in perspective these surveys, makes it clear that imported malaria occurs in low , 36, higP. falciparum as well as P. malariae and P. ovale, referred to as “species other than P. falciparum” in this study, were present in patients attending the selected health facilities of north-western Zambia. A prevalence of 93.8% P. falciparum and 6.2% of other Plasmodium species was found in this study. These findings are consistent with the Zambia 2012 MIS study, which demonstrated prevalence of 88.3% P. falciparum species and 11.6% non-falciparum species [Plasmodium species other than the main Plasmodium falciparum were present, especially in areas that have proximity to border areas [Plasmodium falciparum are present in Zambia, in low proportions [Plasmodium falciparum accounts for more than 95%, while P. malariae makes up 3% and P. ovale 2% [This study determined the characteristics of both the local and imported malaria cases based on species, gametocyte presence and severity of anaemia. The two models, show that country of residence is related to both species and gametocyte presence.  species . Like th species  also demer areas . The Zamportions . Accordiovale 2% .P. falciparum, were missed by the initial RDT screening and were only detected by microscopy. This is attributed to the P. falciparum-specific RDTs employed. As a matter of fact, the P. falciparum-specific RDTs are the only kits available in most health facilities, as per recommendation and provision by the Ministry of Health [P. falciparum are present, they will be missed and misdiagnosed [In this study, the species other than f Health . This ref Health . Howeveriagnosed , 45. It falciparum species at the border areas of Zambia with the two neighbouring countries. All conditions permitting, this is a possibility as one study suggests that individuals who enter malaria regions can influence epidemiological dynamics [P. falciparum and their gametocytes, as part of the process for planning and revising of control and elimination efforts.Apart from species, this study also assessed gametocyte presence in the blood smears collected and examined. Gametocytes are a natural phenomenon where malaria infections ultimately transition from asexual to sexual forms when in a human being –48 and adynamics . It is nP. falciparum than among those infected with other species. This does not rule out the fact that severe anaemia still occurs with species other than P. falciparum [P. falciparum is virulent, and the heavy parasite burden associated with it lends to development of anaemia rapidly causing severe red cell haemolysis due to parasitaemia [P. falciparum malaria diagnosis in Zambia over the other species. With regards to other species, studies have shown that P. malariae is characterized by chronic infections, while P. ovale has dormant hypnozoites stages, which may explain the lower haemolysis rate [P. ovale. Severe anaemia cases were observed in both local participants as well as those from the two neighbouring countries, and in infections involving both P. falciparum and other species. It is, therefore, necessary for control programmes to consider studying the extents of anaemia severity that different species may be associated with so as to appropriately manage the infections. Severe anaemia is a recognized proxy for severe malaria and has been used to prioritize management of cases [With regards to severity of anaemia, the proportion of mild or moderate anaemia was higher than that of severe anaemia overall in our study. However, the cases of severe anaemia were higher in patients residing locally in Zambia than those residing in neighbouring countries. The fact that severe anaemia was not significant in the modelling is highlighted, although noting also that the proportions of severe anaemia were higher among patients with lciparum , 28. Thisitaemia . This cosis rate . In spitsis rate  in the cof cases . It is rof cases .Age, gender and country of residence as risk factors for presence of particular malaria species and gametocytes were evaluated. However, only country of residence was significantly related. Although there were more females than males and more children than adults with malaria, these factors were not of significant relationship based on penalized logistic regression modelling. Notwithstanding, it is necessary for a country participating in malaria elimination programmes to consider exploring age and gender, as potential factors at play especially that these have been significant elsewhere . The varIn this study, the country malaria patients resided in, was related to the malaria species they carried. While such information on species per country is available  and has Plasmodium species by RDT was limited to P. falciparum specific tests, which could have been improved with RDTs that can detect all species; its capacity to confirm both species and gametocytes was limited to microscopy, which could have yielded a more accurate picture with more sensitive methods; it focussed only on malaria-infected humans and their travel history to confirm imported cases, when further tests to confirm onset of infection and detection of infection in vectors, could have increased accuracy.This study was limited on the following scores: it was conducted outside the peak malaria transmission season in Zambia, which could have underestimated the cases detected; it excluded patients without signs and symptoms of malaria and thereby possibly missing asymptomatic cases; its screening capacity to detect all P. falciparum as the predominant species. Importation of P. ovale and P. malariae is happening in the province through human travel across borders with the two neighbouring countries. This study demonstrates that geographic residence of patients is a major risk factor for malaria species and gametocyte presence. It is relevant in that it highlights the status of local and imported malaria and risk factors that could be used for targeted response. The need for enhanced malaria control with specific focus on border controls to detect and treat, for specific diagnosis and treatment according to species obtaining, for further research in the role of species and gametocytes in imported malaria cannot be overemphasized.Malaria prevalence in North-Western Province is high, with


Plasmodium infections in community surveys is essential for effective malaria control. Microscopy and rapid diagnostic tests (RDTs) are the major techniques used to identify malaria infections in the field-based surveys. Although microscopy is still considered as the gold standard, RDTs are increasingly becoming versatile due to their rapid and adequate performance characteristics.Precise detection of Plasmodium falciparum (HSPf) tests compared to SD Bioline Pf and microscopy in detecting P. falciparum infections. A total of 397 individuals aged five years and above were tested for P. falciparum infections. The sensitivity, specificity, positive, and negative predictive values (PPV and NPV) of microscopy, Pf RDT and HSPf RDT was determined using PCR as the gold standard method.A malaria prevalence cross-sectional survey was carried out in north-western Tanzania in 2016, aimed at appraising the performance of high sensitivity P. falciparum infections determined by microscopy, SD Bioline Pf, HSPf and PCR was 21.9, 27.7, 33.3 and 43.2%, respectively. The new HSPf RDT had significantly higher sensitivity (98.2%) and specificity (91.6%) compared to the routinely used SD Bioline Pf RDT(P < 0.001). The positive predictive value (PPV) was 81.8% and the negative predictive value (NPV) was 99.2% for the routinely used SD Bioline Pf RDT. Moreover, HSPf RDT had sensitivity of 69% and specificity of 76.8% compared to microscopy. The PPV was 45.5% and the NPV was 89.8% for microscopy. Furthermore, the analytical sensitivity test indicated that the newly developed HSPf RDT had lower detection limits compared to routinely used SD Bioline RDT.The prevalence of HSPf RDT had better performance when compared to both microscopy and the currently used malaria RDTs. The false negativity could be associated with the low parasite density of the samples. False positivity may be related to the limitations of the expertise of microscopists or persistent antigenicity from previous infections in the case of RDTs. Nevertheless, HS PfRDT performed better compared to routinely used Pf RDT, and microscopy in detecting malaria infections. Therefore, HS Pf RDT presents the best alternative to the existing commercial/regularly available RDTs due to its sensitivity and specificity, and reliability in diagnosing malaria infections. Recent reports have showed a significant decline of malaria burden in some endemic countries, whereby the incidences have declined by over 50% in the past two decades . HoweverPlasmodium infections [Plasmodium antigens, the Plasmodium lactate dehydrogenase (pLDH) and P. falciparum histidine-rich protein 2(HRP2) [TM Malaria Pf test , and ParaSight-F test3  [P. falciparum based rapid test, there are RDT kits capable of detecting more than one Plasmodium antigen and species, such the combo test. However, ongoing field evaluation have reported poor limit of detection for most rapid tests. Recently, manufacturers have developed highly sensitive RDTs based on HRP2 antigen detection, including the Alere™ Malaria Ag Pf Ultra-Sensitive rapid diagnostic test  and SD Bioline Malaria Ag Pf High Sensitive. These second generation PfHRP2-based RDT kits have been reported to have a ten-fold sensitivity over their predecessors and make them highly suitable for malaria control and elimination programmes [ Various efforts have been made to reduce the burden of malaria in the world, particularly in sub-Saharan Africa. The last decade has seen a substantial progress made through adoption and scale-up of various malaria interventions –8. Despifections , 22. The 2(HRP2) . The HRP152 USA) –26. Besiogrammes , but theThe limitations shown by current rapid tests for malaria detection, has necessitated further innovations on sensitivity and reliability of the test kits . IncreasThis was a cross-sectional study, which was conducted for two months. The study participants were patients aged ≥ 5 years presenting with clinical features suggestive of malaria at the dispensaries, and the outpatient departments at the health centres and district hospitals.P. falciparum, but Plasmodium malariae and Plasmodium ovale are present either alone or in mixed infections (Plasmodium vivax is uncommon) [The study was conducted in three sites in Tanzania, namely Magu District Council in Mwanza region, and Geita Town Council and Geita District Council in Geita region. Selection of the two regions was based on the Tanzania HIV and Malaria Indicator Survey of 2011/2012  which shncommon) .Taking the prevalence of malaria of 29% in 2015, the desired probability of 5% and the level of confidence of the selected sample size of 95%. As suggested by the manufacturer (Standard Diagnostics Bioline), a minimum of 300 clinical samples was obtained from subjects presenting with symptoms relating to malaria, as described in the inclusion and exclusion criteria. It was thus estimated that, from each of the three study sites a minimum of 100 specimens would be collected for testing the sensitivity of the HSPf.Since this study involved patients with symptoms related to malaria, the following criteria guided the selection of patients;Patients aged 5 years and above were recruited for the study.History of fever or other symptoms suggestive of malaria during the past 24 hours with or without fever at presentation (axillary ≥ 37.5 °C).Informed consent from the participants/parent or guardian of children.Patients who have taken malaria drugs within 4 weeks before commencement of the study.Patients below 5 years of age.Presence of febrile conditions due to diseases other than malaria  or other known underlying chronic or severe diseases .Presence of general danger signs and hospitalization with multiple blood sampling.Thick and thin blood smears were made on the same slide, air dried and transported to the NIMR Mwanza laboratory where they were stored. The slides were later stained with 10% Giemsa for 15 minutes and examined for malaria parasites by two independent technicians (double blind). The counts of the two technicians were accepted and used to calculate the average parasite density.Asexual parasitaemia was quantified against 200 to 500 leucocytes, assuming a white blood cell count of 8,000/µl as recommended by the WHO . A slidewww.standardia.com).The Pf RDT and HSPf RDT tests were performed according to the manufacturer’s instructions , DNA from blood samples of an individual never exposed to malaria, and PCR water were used as positive and negative controls and were included in each set of PCR for quality control. The PCR was carried out in a thermo-cycler , followed by gel electrophoresis to determine whether blood sample was individuals as either parasite positive or negative.Approximately 3 ml of venous blood was collected from participants into EDTA vacutainer tubes. Using a Pasteur pipette, blood was taken from the vacutainer tube and one drop was placed on each malaria RDT cassette and results were read after 30 minutes. Participants with a positive test result on the routinely used RDT were treated, in line with Tanzanian national treatment guidelines. DNA was extracted from remaining whole blood using the QIAamp DNA Mini Kit , following manufacturer’s instructions. A nested PCR was used to amplify species-specific sequences of the small sub-unit ribosomal ribonucleic acid (18S SSU rRNA) genes of escribed . DNA froThe analytical sensitivity test was conducted to determine and compare the detection limit of the HSPf RDT and the routinely used Pf RDT. Two replicates of blood specimens with higher, mid and low parasitaemia were used in the assay. The two-fold serial dilutions were done using a malaria negative whole blood specimen. Prior to their use in the assay, the malaria negative status of the diluents was confirmed using the HSPf RDT, the Pf RDT and blood smear slides.Data were analysed using STATA  software version 12. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) of microscopy, Pf RDT and HSPf RDT, were determined using PCR as the gold standard, using 2 × 2 contingency tables and compared using the McNemar’s test (sensitivity and specificity) for paired data . Exact 9A total of 397 study participants aged 5 years and above were screened for malaria parasites. Among study participants, 57.2% (227/397) were females and the median age of participants was 24 years IQR, 13–40). Participants from Magu district were younger than those from Geita district  were positive for P. falciparum infections. Three blood samples were positive for P. malariae and one blood sample was positive for P. ovale. Moreover, 87 (21.9%) out of 397 blood samples were positive by microscopy  and 132 (33.3%) were positive for 7.7% and P. malariae and P. ovale infections, respectively, detected by PCR. Of the 21 individuals who tested positive for P. malariae, 18 had mixed infection of P. falciparum + P. malariae, whereas 8 individuals who tested positive for P. ovale infections, 7 had mixed infection of P. falciparum + P. ovale. Moreover, three individuals had triple mixed infections of P. falciparum, P. malariae + P. ovale. The parasite density for P. falciparum ranged from 40–1,000,000 parasites/µL.Furthermore, 21 individuals (5.3%) and 8 individuals (2.1%) had Parasite positivity was compared for the different testing methods. The HSPf RDT detected a higher proportion of parasite positivity than the two malaria tests routinely used, i.e. Pf RDT and microscopy. However, PCR detected the highest proportion of malaria parasite positivity than the other methods. PCR also detected more individuals with low parasitaemia (below 100 parasites/µL), but the difference was borderline (p = 0.090).P. falciparum infections  and 199 out of 217 PCR-negative samples , with a PPV of 79.3% and NPV of 68.2%. The routinely used Pf RDT correctly identified 99 out of 171 PCR-positive P. falciparum infections  and 214 out of 225 PCR-negative samples , with a PPV of 90.0% and NPV of 74.8%. Whereas HSPf correctly identified 117 out of 171 PCR-positive P. falciparum infections  and 210 out of 225 PCR-negative samples , with a PPV of 86.6% and NPV of 79.5%  and specificity (91.6%) compared to the routinely used Pf RDT (P < 0.001). The positive predictive value (PPV) was 81.8% and the negative predictive value (NPV) was 99.2% for the routinely used Pf RDT. Similarly, HSPf RDT had significantly higher sensitivity (69%) and specificity (76.8%) compared to microscopy. The PPV was 45.5% and the NPV was 89.8% for microscopy (P < 0.0001).Although the sensitivity of the different tests decreased with increasing age of participants (> 15 years old), the sensitivity, specificity and predictive values of HSPf RDT was the highest among the three tests. The sensitivity of HSPf RDT among the different age groups was 80% 95% CI:64.4–90.9) in five to ten years old, 91.2% (95% CI: 76.3–98.1) in 11 to 15 years-old, and 55.7% (95% CI: 45.2–65.8) in those above 15 years of age (p < 0.0001). No significant change in the specificity of microscopy was observed across the three age groups. Similarly, the sensitivity of routinely used Pf RDT was lower in those above 15 years-old  than in five to 10 years , and  in those aged 11 to 15 years (p < 0.0001) (Table .4–90.9 iThe selected blood specimens with higher parasitaemia had parasite densities of between 874 parasites/200WBC and 1384 parasite/200WBC (Fig. The blood specimens with medium parasitaemia, between 468 and 574 parasites/200WBC, were used to study the sensitivity of the two RDTs. The HSPf RDT had a minimum detection limit after 64 two-fold dilutions, while that of the Pf RDT was 16 two-fold dilutions.The blood specimens with low parasitaemia, between 42 and 64 parasites/200WBC, were further used to study the sensitivity of two RDTs. The HSPf RDT had a minimum detection limit after 16 two-fold dilutions, while that of Pf RDT was 4 two-fold dilutions.Plasmodium infection, requires intense diagnosis to achieve accurate result with specificity and sensitivity. Therefore, it was necessary to use venous blood in order to avoid to perform several pricks on the same individual at the same time as it has been done in Thailand [Detection of the malaria parasite reservoir is central for control given the renewed focus on transmission reduction leading to elimination. This study used venous blood sampling for determination of sensitivity and specificity of the different tests though both venous and DBS are used in the field setting. However, it should be noted that the decision of the choice of sampling methods depends on the choice of DNA extraction method which needs to be individualized depending on the level of laboratory facility, volume of testing, duration of samples storage and cost benefit analysis before it is adopted for use as it has shown in Uganda and Ethiopia , 34 and Thailand , 37, BraThailand and TanzaThailand .P. falciparum infections, respectively. In line with earlier studies, low parasite density might have affected the proportion of positive infections detected by microscopy and RDT [pfhrp-2 gene or by the prozone effect reported by others [In this study, the positivity by HSPf RDT was higher compared to microscopy and Pf RDT, though PCR had the highest positivity rates. However, compared to PCR as the gold standard, microscopy, Pf RDT and HSPf RDT detected only 40.9%, 57.9% and 68.4% of  and RDT . Similar and RDT . Accordi and RDT , 40–42.  and RDT . On the y others –47. Nevey others , 49. Fury others , 49–53. y others –56.The continued use of the current routinely used Pf RDT in detecting malaria in asymptomatic, low-density parasite infections poses a challenge in achieving malaria elimination. The strategy to eliminate malaria by 2030 will require highly sensitive diagnostic tools to detect the reservoir of low-density and submicroscopic parasite infections .P. falciparum infections might improve detection over the current routinely used RDT or microscopy; but microscopy remains a preferable option, when parasite density needs to be determined in the absence of PCR. PCR allows for the detection of low-density infections and, even more importantly, mixed infections which are routinely missed in microscopy, making it an ideal confirmatory test for malaria diagnosis, but unfortunately difficult to implement on a large scale.Given the observed higher sensitivity values of HSPf RDT compared to Pf RDT and microscopy, the data presented here suggest that, the use of HSPf RDT to diagnose


Malaria elimination efforts can be undermined by imported malaria infections. Imported infections are classified based on travel history.A genetic strategy was applied to better understand the contribution of imported infections and to test for local transmission in the very low prevalence region of Richard Toll, Senegal.Genetic relatedness analysis, based upon molecular barcode genotyping data derived from diagnostic material, provided evidence for both imported infections and ongoing local transmission in Richard Toll. Evidence for imported malaria included finding that a large proportion of Richard Toll parasites were genetically related to parasites from Thiès, Senegal, a region of moderate transmission with extensive available genotyping data. Evidence for ongoing local transmission included finding parasites of identical genotype that persisted across multiple transmission seasons as well as enrichment of highly related infections within the households of non-travellers compared to travellers.These data indicate that, while a large number of infections may have been imported, there remains ongoing local malaria transmission in Richard Toll. These proof-of-concept findings underscore the value of genetic data to identify parasite relatedness and patterns of transmission to inform optimal intervention selection and placement. Senegal is characterized by distinct malaria transmission zones, ranging from very low in the North (incidence < 1/1000) to moderately high in the South (incidence > 400/1000). Richard Toll, a northern district, reports one of the lowest incidence levels in Senegal (0.2/1000 persons in 2017) –5. Thus,malariogenic potential [Understanding the risk and pattern of malaria transmission in a given geographical area provides the foundation for intervention selection and targeting to decrease malaria burden, eliminate transmission, and prevent re-establishment of malaria. The risk of malaria transmission is the product of the risk of local spread, rate of importation, and infectivity, and is referred to as the otential . The Worotential . Thus, mPopulation genetic signatures may reflect changes in transmission dynamics  and can To test the hypothesis that genetic relatedness can be used to differentiate imported and local infections, this pilot project applied population genetic analysis to samples collected during malaria elimination efforts in Richard Toll, Senegal. These activities included case investigation of all passively identified malaria infections (index cases) with reactive active case detection targeting the household and neighbouring households of the index case between 2012 and 2015 [®. A standardized questionnaire was filled out for all participants to collect information on basic demographic information including travel history, with a traveller defined as a resident who spent at least one night outside Richard Toll district in the previous 14 days. A total of 759 discarded positive RDT samples were obtained from either index cases (n = 673) or secondary infections found during RACD (n = 86) between 2012 and 2015 in Richard Toll district.Routine case investigation was carried out in Richard Toll, Senegal between September 2012 and December 2015 through a partnership with the Senegal National Malaria Control Programme , PATH’s Malaria Control and Elimination Partnership in Africa (MACEPA), and local health facility and community health workers [Nucleic acid material was extracted from the RDT and pre-amplification  with molGenotypes were called by their base designation for DNA: A, T, G, or C. Missing alleles are indicated by an “X”; and ascertained alleles that are called bi-allelic  are designated by an “N”.Any 24-SNP barcode that was missing 4 or fewer assays was included in the analysis and considered as successfully genotyped. This means that barcodes missing 0, 1, 2, 3, or 4 assays were included in the analysed samples. Any sample missing 5 or more assays of the 24 SNPs in the barcode were regarded as unsuccessfully genotyped.This study included all samples that passed genotyping quality control for calculating the proportion of infections that were monogenomic . The hypothesis that the means were significantly different between household sample sets from travellers and non-travellers was assessed using a two-tailed Fisher Exact Test for small sample sizes, with a significance level of α = 0.05 (Mathematica 10.4.0).The hypothesis that the proportion monogenomic and polygenomic  indicated that they had travelled and spent at least one night outside Richard Toll district in the previous 14 days , was performed on genotyping data from both Richard Toll samples and clinical samples collected during research conducted in Thiès. A total of 426 Richard Toll samples and 1,516 Thiès samples met the criteria of being monogenomic and missing either no or one single nucleotide polymorphism (SNP) assay .To ask whether infections from travellers contribute to a risk of local spread of these imported infections, this study took advantage of the RACD programme, in which all household members received an RDT,  and testRichard Toll represents a region with one of the lowest levels of malaria incidence in Senegal. Most malaria cases in Richard Toll are detected in individuals who had travelled in the previous 2-week period, thus are classified as imported infections  of genotHowever, genetic relatedness among infections from within Richard Toll revealed patterns of ongoing local transmission. Evidence for local transmission includes, first, that multiple genetically identical parasite lineages found across multiple transmission seasons were detected in Richard Toll; and second, that highly related infections were more likely to be found within households that lacked recent travel. While the sample numbers in this study are small, both of these findings are consistent with local and focal transmission. Given that only one household was found both multiple identical genotypes and a travel history suggests that local transmission of an imported malaria case may be a relatively rare event.Successful malaria elimination benefits from identifying the origin and means of transmission for the few remaining cases in regions of very low transmission. It is important to determine the rate of importation and the risk of local spread in these regions approaching elimination . The WHOThis pilot activity demonstrates the feasibility and likely success of genetic approaches for mapping potential transmission patterns related to imported and local infections. Samples from other sites and additional genetic data, such as obtained from sequencing, will improve resolution and understanding of gene flow between geographic regions. In addition to improvements in sequencing parasite genomes directly from clinical samples, development of population genetic strategies, including analysis of identity by descent, will help define the relationships between infections in settings approaching malaria elimination. The use of identity by descent may help delineate relationships between parasite infections through quantification of the proportion of the genome shared between two parasites derived from a common ancestor, as well as the length of these shared genomic tracts, to estimate the number of generations between individual parasite infections. Challenges of how to address polygenomic infections remain, and may reveal important patterns of transmission, particularly in regions of high malaria burden. Incorporation of other data including human mobility with genetics and modelling these data will also improve our ability to evaluate the impact of imported malaria infections to ongoing transmission of malaria . These aAdditional file 1. Schematic of Richard Toll sample analysis. A total of 759 samples were received and genotyping yielded 649 samples that passed genotyping . Of these 649 samples, 473 were monogenomic .Additional file 2. Travel history and genotyping data.Additional file 3. Richard toll parasite genotyping data.Additional file 4. Thiès parasite genotyping data.Additional file 5. Pairwise comparison of genotyping data using identity by state (IBS) analysis.Additional file 6. Persistent parasite lineages.Additional file 7. Household comparisons.Additional file 8. Map of infections. A. Map of infections collected in 2015 and relative location of Thiès. The distance between Richard Toll and Thiès is approximately 150 miles. The sites of households from which samples were collected in 2015 are shown, with red indicating infections in individuals with a travel history and blue indicating infections from individuals with no travel history (see B). No household locations were collected in any other year. B. Inset from panel A is shown enlarged to indicate the relative locations of households from which parasite infections were identified in 2015. Infections identified in individuals who reported a travel history are shown in red, while those from individuals who reported no recent travel are shown in blue. Tear drop markers indicate infections identified through reactive active case detection (RACD), with index case marked red for positive travel history or blue for no travel history. All of these RACD households with a travel history (red teardrops) had parasites that were different from one another; and all these RACD households with no travel history (blue teardrops) shared genotype infections, with one exception being the household in dark blue in the center of Richard Toll (identified with an asterisk). This household had parasites that were genetically distinct but the index case reported no recent travel. C. Location of passive case detection infections that share a parasite genotype. To provide an example of household location where shared infections were identified by passive case detection at four households with the same parasite genotype are shown in blue teardrops, and a different parasite genotype found among two households are shown in red teardrops. The two infections from the red household were both detected on the same date at the clinic. The infections for the blue households occurred on different dates: (1) September 7; (2) September 12; (3) November 8; (4) November 10. Note that household 4 was positive for additional infections and corresponds to the household in panel B as indicated by the red arrow. The geographic distance between household 1 and 2 is 3.3 miles; between household 2 and 3 is 14 miles and between household 3 and 4 is 0.8 miles.


At threshold levels yielding 99.5% or more diagnostic specificity, diagnostic sensitivities against polymerase chain reaction–confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 92.7%, 71.5%, 46.1%, and 83.8%, respectively. P. falciparum culture strains and samples from Peru indicated that HRP2 and Pf LDH combined improves detection of P. falciparum parasites with hrp2 and hrp3 deletions. This array can be used for antigen-based malaria screening and detecting hrp2/3 deletion mutants of P. falciparum.Malaria antigen detection through rapid diagnostic tests (RDTs) is widely used to diagnose malaria and estimate prevalence. To support more sensitive next-generation RDT development and screen asymptomatic malaria, we developed and evaluated the Q-Plex Unfortunately, RDTs recognizing the common epitopes found in HRP2 and HRP3 result in failed diagnosis for clinical P. falciparum infections because of partial or complete deletions of hrp2 and hrp3, and consequently no expression of proteins.4 Characterization of hrp2/3 deletions by polymerase chain reaction (PCR) is time consuming and expensive for larger scale clinical studies.4 Antibodies targeting a conserved epitope in pLDH can detect all human Plasmodium species Pan malaria lactate dehydrogenase (Pan LDH). pLDH can also be used for species-specific detection for P. falciparum and P. vivax infection using antibodies that recognize species-specific epitopes in P. falciparum-specific lactate dehydrogenase (Pf LDH) and P. vivax–specific LDH (Pv LDH).6There are more than 200 million cases of human malaria worldwide, with some countries in the control phase of malaria and others seeking to eliminate malaria.10 Two quantitative multiplex tests, bead-based assay and chemiluminescence-based ELISA, have been described.12 The bead-based assay detects Pan aldolase, Pan LDH, and HRP2, whereas the ELISA detects HRP2, Pv LDH, Pan LDH, and C-reactive protein (CRP). An expanded version of this ELISA including an assay for Pf LDH was developed for research use and is now commercially available as the Q-Plex™ Human Malaria Array . In this study, the diagnostic performance of this new test was assessed.The performance of RDTs is driven by the underlying relationship between antigen concentration and parasite density. This relationship is defined by multiple factors including antigen turnover and decay rates, affinity characteristics of the antigen capture reagents, whether infections are recent or asymptomatic, and probably also by host factors such as previous exposure to malaria.13 A randomly selected subset of samples was used in this study. Seventy-five clinical samples with P. falciparum or P. vivax infections from symptomatic infections in Vietnam, which were also collected under informed consent and IRB approval, were purchased from Discovery Life Sciences . Twenty-six P. falciparum clinical samples were obtained with informed consent and with IRB approval from Universidad Peruana Cayetano Heredia  (UPCH 52707). Plasmodium malariae– and Plasmodium ovale–infected blood samples were obtained from the Foundation for Innovative New Diagnostics  Specimen Bank.Clinical samples from asymptomatic infections in Myanmar and Uganda were collected under informed consent and with institutional review board (IRB) approvals, as described previously.P. falciparum laboratory strains were used in this study, W2, D10, Dd2, and HB3 , and 3BD5 . The wild-type strain W2 is hrp2+/hrp3+, D10 and Dd2 are hrp2−/hrp3+, HB3 is hrp2+/hrp3−, and 3BD5 is hrp2−/hrp3−. All strains were cultured and harvested at the ring stage (> 99%) after synchronization, as described previously.12 The parasite number was determined by microscopy. Parasite-infected red blood cell pellets were used to generate a dilution series derived from a pool of uninfected blood .Five Supplemental Methods). The speciation of all field samples except those from Vietnam was confirmed by quantitative PCR methods.13 Photo-induced electron transfer real-time PCR was used to characterize samples from Vietnam . The samples from Peru were previously characterized for the presence or absence of hrp2 and hrp3 by PCR and confirmed for HRP proteins by conventional ELISA.14Quantification of malaria antigens and CRP was performed using the 5-Plex (Quansys Bioscience) . The detail in data analysis is described in Supplemental Table 1 and Supplemental Table 2). The 5-Plex was challenged with a validation sample set consisting of 462 blood samples from PCR-confirmed positive and negative cases with P. falciparum and P. vivax infection from Uganda, Myanmar, and Vietnam. All four malaria biomarkers, HRP2, Pan LDH, Pf LDH, and Pv LDH, found in significantly higher amounts in the respective malaria-positive samples are compared with the negative control group (P < 0.0001) . Receiver operating characteristic (ROC) analysis was conducted, and the area under the ROC curves (AUCs) with 95% CIs for each biomarker was calculated in relation to the discrimination between malaria cases and controls. The AUCs were 0.979 (95% CI: 0.960–0.998) for HRP2, 0.939 (95% CI: 0.911–0.968) for Pf LDH, 0.926 (95% CI: 0.898–0.954) for Pv LDH, and 0.948 (95% CI: 0.928–0.968) for Pan LDH .The analytical performance of the 5-Plex was evaluated based on antigen quantification as determined from the pixel intensity on the array spot relative to a standard curve  and the HB3 (hrp2+/hrp3−), to as few as 0.8 p/μL and 0.3 p/μL, respectively. In parallel, the Pf LDH assay detected parasites at 66.7 p/μL and 22.2 p/μL, respectively . Screening of hrp mutants with the Pf LDH assay using hrp2 deletion strains D10 and Dd2 and 3BD5, a hrp2/3 double mutant detected parasitemia of 7.4 p/μL, 22.2 p/μL, and 66.7 p/μL, respectively.A dilution series of five hrp2/3 deletions by PCR.14 All 26 samples were accurately identified as P. falciparum by the 5-Plex by detecting either HRP2 or Pf LDH. A subset of nine from 12 (75%) samples with hrp2/3 deletion in this panel was correctly identified as HRP negative with the 5-Plex HRP2 assay  were positive by the Pan LDH assay and only 5/17 (29.4%) were positive by the Pf LDH assay . The P. malariae samples had relatively high parasite densities in the range of 75.2–988.3 p/μL. Of the eight PCR P. ovale–positive samples, 3/8 (37.5%) were positive by Pan LDH assay, with parasite densities in the range of 0.1–8.0 p/μL. None (0/8) was positive by Pf LDH assay. The P. malariae and P. ovale samples were negative with the HRP2 and Pv LDH assays.The 5-Plex was further investigated with 17 P. falciparum infections, including those with hrp2/3 deletions, P. vivax infection, and also detect P. ovale and P. malariae. Similar multiplex assays have been developed on the Luminex bead-based platform but are not available as commercial kits.16The 5-Plex is able to differentiate 12 the performance of the new 5-Plex is significantly improved: the prozone effect in the HRP2 assay is mitigated along with the wider dynamic range ,17 and through the Pf LDH, it can identify samples with suspected hrp2/3 deletions.In contrast to the previously described 4-Plex array,P. falciparum infection using HRP2 was 92.7%. When identifying P. falciparum and P. vivax infections through the Pf LDH and Pv LDH assays, the sensitivity was reduced to 71.5% and 46.1%, respectively. The Pf LDH assay does show cross-reactivity with P. malariae. The sequence similarity of the related proteins from P. malariae and P. falciparum may contribute to cross-reactivity. In addition, elevated levels of CRP, an acute phase inflammatory protein, were consistently observed in febrile malaria-positive individuals, in contrast to asymptomatic malaria-positive individuals , supporting the correlation of the CRP level with complications in malaria.19At a specificity of > 99.5%, the sensitivity for detecting any species of malaria using the Pan LDH assay was 83.8% and for ™ Human Malaria Array can be used on whole blood samples to operate high throughput screening tool for malaria surveillance including the identification of hrp2/3–deleted P. falciparum before resorting to more complex molecular tests for the gene deletions.4 Although HRP2 persists after malaria clearance, the pLDH level rapidly declines on treatment.20 The capability of the array to simultaneously quantify these malaria antigens may be of significant value to the future studies that could provide deep insight into the malaria infection status classification as well as the speciation of suspected malaria.The data presented in this study suggest that the Q-PlexSupplemental materials


Malaria in pregnancy remains a major public health problem in Africa and Ghana and has been associated with a variety of pregnancy-related adverse complications. The development of effective and timely health policies for the prevention and control of malaria and anemia in pregnancy; requires current and consistent data on the prevalence and risk factors. We report the prevalence and risk factors of malaria and anemia from three major hospitals across three regions in Ghana.This multicenter cross-sectional study comprising a total of 628 pregnant women was conducted at the antenatal care units of the Achimota Hospital in the Greater Accra Region (n = 199), St. Michael’s Hospital in the Ashanti Region (n = 221), and Effia Nkwanta Regional Hospital in the Western Region (n = 211). Questionnaires were administered to obtain socio-demographic, obstetrics and clinical data. Venous blood, stool and urine samples were collected for hematological profile and parasite identification using microscopy. Risk factors were evaluated using logistic regression models.P. falciparum malaria was 8.9%. Factors independently associated with malaria were self-reported mosquito exposure  and severe exposure: aOR = 10.46, 95% CI (3.86–28.34)) and non-use mosquito repellents ). Multiparty  and parity ≥3: aOR = 0.11, 95% CI (0.03–0.45)) and age ) reduced the odds of infection. The overall prevalence of anemia was 42.4%. The prevalence of mild, moderate and severe anemia were 35.7%, 6.1% and 0.6%, respectively. The use of water other than purified water  and well water: aOR = 2.45, 95% CI (1.35–4.44)), increasing gestational age  and third trimester: aOR = 7.20, 95% CI (3.06–16.92)) and malaria ) were independent risk factors for anemia.The overall prevalence of Although the prevalence of malaria is relatively low, that of anemia remains high. We recommend increasing efforts to make ITNs more available to strengthen malaria prevention. Public health education programs could help improve uptake and proper use of ITNs. To help reduce anemia in pregnancy, women should be empowered economically and interventions that reduce malnutrition should be encouraged. Women should be educated on early initiation of antenatal care to enhance surveillance, identification and treatment of anemia. Malaria is a parasitic disease, mostly prevalent in sub-Saharan Africa, Asia, and Latin America . Data frMalaria infection during pregnancy is indeed a major public health problem in tropical and subtropical regions with an estimated 24 million pregnant women being infected in sub-Saharan Africa each year . The risAnemia in pregnancy is an important global public health problem. The WHO defines anemia in pregnancy as having haemoglobin (Hb) concentration of less than 11 g/dL . It is eThe detrimental effect of malaria and anemia on the mother and fetus underscore the need for more aggressive and sustainable preventive and therapeutic measures. The WHO recommends sleeping under insecticide-treated nets (ITNs) to reduce contact between mosquitoes and humans and use of intermittent preventive treatment (IPTp) with sulphadoxine-pyrimethamine (SP) at each scheduled antenatal visit after the first trimester . ITNs haThere is thus the need for effort intensification to ensure compliance and reduce malaria and anemia in pregnancy. This would require the availability of reliable statistics on the prevalence and risk factors of malaria and anemia in pregnancy across the country. Although some cross-sectional studies have been conducted in Ghana –26, the P. falciparum. Effia Nkwanta Regional Hospital is located in the Sekondi-Takoradi Metropolis, the capital of the Western Region of Ghana. The Metropolis has a total land area of 192 km2; an average annual growth rate of 3.2% and the prevalence of malaria in pregnancy in the region was 26.1% in 2015 .Blood pressure was measured with an automated blood pressure apparatus  from the right arm after the study participant had been made to sit for at least five minutes. The average of the two readings taken five minutes apart was recorded. Height was measured without wearing shoes using a wall mounted ruler, to the nearest 0.1 m. Waist and hip circumferences were measured to nearest centimeter using a measuring tape. Waist to hip ratio (WHR) = WC (m)/HC (m), waist to height ratio and (WHtR) = WC (m)/height (m) were calculated. Body mass index (BMI) was calculated using the equation; [BMI (kg/m2) = weight/height3 EDTA tubes for full blood count (FBC) and malaria parasite detection. FBC was performed using the Mindray BC-3000Plus Auto Haematology Analyzer. Ten percent Giemsa-stained thick and thin films were prepared on clean grease-free slides (thin films were fixed with methanol) for malaria microscopy. Examination and reporting of both thick and thin films were performed independently by two trained microscopists. Thick films were used for malaria parasite count and thin films were used to identify the specific species of Plasmodium. A film was considered positive when both microscopists recorded a positive result and for the same species. A film was considered negative only after observing at least 200 high-power fields (HPF) without finding parasites on a thick film. In the case of discordant results, a third microscopist was employed to break the tie. The tests were performed immediately after sample collection to minimize storage variabilities. Additionally, to each participant, two well-labelled, wide-mouth and screw-capped containers were given and instructed to bring their early morning stool and urine samples the following day. The stool and urine samples were immediately transported in cold boxes at 4°C to the laboratory department of each hospital for processing and examination. Urine and stool routine examination were done for each urine and stool sample, respectively. For all stool and urine parasitological tests, samples were considered as positive if the eggs/ova/cysts/trophozoites were detected. For quality control purposes, all slides were prepared and examined in duplicates.Five milliliters (5 ml) of venous blood sample was collected from each participant and dispensed in KAnemia was defined as hemoglobin (Hb) level < 11g/dl; mild anemia was defined as 9g/dl ≤ Hb < 11g/dl; moderate anemia was defined as 7g/dl ≤ Hb < 9g/dl and severe anemia was defined as Hb < 7g/dl .Statistical analysis was performed using the R Language for Statistical Computing version 3.6.0 . CategorA total of 628 pregnant women with mean age of 28.44±6.19 years participated in the study. Most of the participants were married (454/72.3%), had secondary education (416/66.2%), were employed (516/82.2%) and a total of 166 (26.4%) self-reported having been severely exposed to mosquito bites within the past month. Less than half of the participants (282/45.2%) used ITN and of this, only 2 (0.3%) treat their ITN. Notwithstanding, a higher proportion of the participants used mosquito repellents (468/74.5%), with mosquito insecticide spray being the most commonly used (324/69.2%) .Schistosoma haematobium infection upon urine microscopy. Stool examinations were negative for both intestinal flagellates and helminths among all participants , had secondary education (38/9.1%), were employed (48/9.3%), did not use ITN (42/12.2%), self-reported severe exposure to mosquito bites (32/19.3%), were in their first trimester of pregnancy (30/11.7%) and were nulliparous (32/16.5%). Univariate analysis revealed statistically significant associations between malaria and age, marital status, source of drinking water, self-reported mosquito exposure, parity, use of ITN and mosquito repellents .Multivariate logistic regression analysis identified age , p = 0.001) and multiparty , p = 0.013 and parity ≥3: aOR = 0.11, 95% CI (0.03–0.45), p = 0.002) to be independently associated with lower odds of malaria. Self-reported mosquito exposure , p = 0.029 and severe exposure: aOR = 10.46, 95% CI (3.86–28.34), p<0.0001) and non-use of mosquito repellents , p<0.0001) were associated with increased odds of malaria infection .Most of the participants who had anemia were 20–30 years old (140/41.9%), married (174/38.3%), had secondary education (172/41.3%), were employed (212/41.1%), used purified drinking water (104/31.3%), were in the second trimester (160/47.6%) and were nulliparous (94/48.5%). There were statistically significant associations between anemia and marital status, source of drinking water, gestational age, parity and malaria based on univariate analysis .In multivariate analysis, use of water other than purified water , p<0.0001 and well water: aOR = 2.45, 95% CI (1.35–4.44), p = 0.003), increasing gestational age , p<0.0001 and third trimester: aOR = 7.20, 95% CI (3.06–16.92), p<0.0001) and malaria , p = 0.007) were independent risk factors for anemia. Being married was associated with lower odds of anemia , p = 0.044) after controlling for the hematuria, proteinuria and use of medication  .This study aimed to evaluate the prevalence and risk factors of malaria and anemia from three major hospitals across three regions in Ghana. The overall prevalence of malaria among the study population was 8.9%, with prevalence in the Ashanti, Greater Accra and Western regions being 5.5%, 10.1% and 11.4%, respectively. Self-reported exposure to mosquitoes and non-use of mosquito repellents were identified as a major risk factor for malaria in pregnancy; whereas increasing maternal age and parity reduced the risk of malaria. On the other hand, the prevalence of anemia was 54.5%, 34.3% and 37.1% in the Ashanti, Greater Accra and Western regions, respectively. Overall, the prevalence of anemia was 42.4%, with 35.7%, 6.1% and 0.6% presenting with mild, moderate and severe anemia, respectively. The use of unpurified/groundwater, malaria and gestational age were identified as major risk factors for anemia in pregnancy. Married pregnant women were less likely to develop anemia in pregnancy.P. falciparum parasitemia among pregnant women seeking antenatal care [Our reported malaria prevalence of 8.9% is different compared to other studies in the country. In the Greater Accra region of Ghana, Stephens et al., reported 5.0% prevalence of tal care . Also intal care . In the tal care  reportedtal care . Clerk etal care  found a tal care  observedtal care . Furthertal care  and PCR tal care . Of notetal care . This coIn assessing the risk factors of malaria in pregnancy, we found age, marital status, source of drinking water, self-reported exposure to mosquitoes, parity, use of ITN and mosquito repellents to be possible contributors. After adjusting for potential confounding factors in multivariate analysis, we observed that, compared to younger pregnant women, older women (20–30 years old) had reduced odds of malaria infection. This finding is in harmony with studies by Clerk et al.  in GhanaAnemia in pregnancy still is a major public health problem and it is associated with maternal death, still births, low birth weights and impairment of fetal development , 17. OurIn this study, we found an association between anemia and marital status, source of drinking water, gestational age and parity. In the multivariate analysis, being married was associated with lower odds of anemia in pregnancy. This finding could be due to the important roles played by spouses during pregnancy. Studies in Ghana and Uganda have shown that, apart from the economic contribution, husbands play key roles in the frequency of a pregnant spouse’s ANC visits, with some men taking up the responsibility of ensuring that their wives attend ANC clinics and conforms to the directives of their physician/nurse. Thus ensuring the overall well-being of their pregnant spouses and thus contributing to reducing anemia in pregnancy , 43. We Interestingly, controlling for confounders, women who drank groundwater/unpurified water presented with over two-fold increased risk of anemia compared to the drinking purified water. Groundwater is used by over 90% of the Ghanaian population for several purposes. Evidence suggests some level of dissolved iron in groundwater which can improved the iron status . AntitheAnother finding of this study was that increasing gestational age was independently associated with increased odds of anemia in pregnancy. This finding is similar to a study in Ghana by Dicko et al.,  who founImportantly, among the pregnant women with anemia, 5.1% of them had malaria. Regression analysis from our study, revealed that malaria increases the risk of anemia by two-folds among the population. Other studies have also reported increased risk of anemia with malaria among pregnant women in Ghana and elsewhere , 53–55 TThe strength of this study lies in establishment of multiregional prevalence of malaria and anemia among pregnant women in the middle and lower belt of Ghana along with presenting reliable data on the risk factors of malaria and anemia among pregnant women in Ghana. However, some limitations need to be acknowledged. First, although the data was obtained from three major hospitals which serves three regions of the country; hence could represent the current state in those regions, data from the northern belt is not provided in this study; thus, the findings is not to be generalized as data for the whole country. Further studies including the northern sector of the country is warranted. Additionally, although microscopy remains the standard for malaria diagnosis in Africa, the test method has been shown to be less sensitive. Thus, it is possible that the prevalence of malaria may have been underestimated due to missed cases. Future studies should consider the use of more sensitive diagnostic methods like polymerase chain reaction. This study was also conducted using a convenient sampling method.Although the prevalence of malaria is low (8.9%), that of anemia remains high (42.4%) among pregnant women in Ghana. The major risk factors of malaria among pregnant women in Ghana are exposure to mosquitoes and non-use of mosquito repellents whereas the risk factors for anemia in pregnancy are use of unpurified/groundwater and gestational age. Increasing maternal age and parity reduces the risk of malaria whiles married pregnant women have lower risk of anemia in pregnancy. Malaria is a significant contributor to anemia in pregnancy; however, other factors are similarly important. We recommend increasing efforts to make ITNs more available to strengthen malaria prevention. Public health education programs could help improve uptake and proper use of ITNs. To help reduce anemia in pregnancy, women should be empowered economically and interventions that reduce malnutrition through micronutrient supplementation and promotion of iron-rich products should be encouraged. Additionally, community-level health education that enhances involvement of men and family members during pregnancy as well as adopting an integrated approach to bridge the rural–urban divide with respect to access to improved drinking water is recommended. We also recommend educating women on early initiation of antenatal care to enhance surveillance, identification and treatment of anemia.S1 File(DOCX)Click here for additional data file.


The effort to reduce the burden of malaria should target transmission in the community by accurate identification of asymptomatic infections. In malaria-endemic areas, asymptomatic malaria infection is still associated with complications. Malaria during pregnancy is characterized by anaemia and placental malaria, leading to low birth weight and perinatal morbidity and mortality. This study aimed to provide reliable data on the burden of asymptomatic malaria among pregnant women in malaria endemic areas of North-Shoa, Ethiopia.Cross-sectional study was carried out to assess the prevalence and predictors of asymptomatic malaria in pregnant women from November 2018 to January 2019. Multistage sampling technique was employed to include 263 study participants. Data were analysed using SPSS version 20.0 statistical software. In all comparisons, p-values ≤ 0.05 was considered as statistically significant.Plasmodium falciparum was a dominant species 9 (3.4%) and Plasmodium vivax accounted for 6 (2.3%) of Plasmodium infections as detected by microscopy. Multivariate analysis showed that ITN usage and haemoglobin level had a statistically significant association with Plasmodium infection after adjusting other possible factors. Compared to those who were using ITN always, the odds of Plasmodium infection was 18.16 times higher (95% CI 1.84–179.07) in pregnant women who were not using ITN, and 5.19 times higher (95% CI 0.55–49.21) in pregnant women who were using ITN sometimes. Asymptomatic malaria infected pregnant women were 3.78 times (95% CI 0.98–14.58) more likely to be anaemic compared to non-infected pregnant women.The prevalence of asymptomatic malaria infection was 5.7% (15/263) and 3.4% (9/263) by using microscopy and RDTs, respectively. The present study showed asymptomatic malaria is prevalent in pregnant women and it has statistically significance association with the haemoglobin level of pregnant women. This indicates pregnant women have to be screened for asymptomatic malaria to avoid health consequences of malaria infection during pregnancy for the mother and fetus. Plasmodium species is common in malaria endemic areas [Anopheles mosquitoes and may progress to symptomatic disease [There is an estimated 219 million malaria cases globally and 435,000 deaths due to malaria each year with the highest mortality reported in Africa [ic areas , 7. The ic areas . Asympto disease , 9. More disease . The eff disease .Plasmodium falciparum and Plasmodium vivax account for 60–70% and 30–40% of cases, respectively [Approximately, 75% of the landmass of Ethiopia is estimated to be “malarious” with about 52 million people living in these areas and about four to five million people affected by malaria annually. ectively –14. In ENorth-Shoa is one of the 10 Zones in the Ethiopian Amhara Regional state. Based on the 2007 Census conducted by the Central Statistical Agency of Ethiopia (CSA), this Zone has a total population of 1,837,490, of whom 928,694 are men and 908,796 women. The zone has an area of 15,936.13 square kilometers. Thirty-nine percent (39%) of the zone is exposed to malaria. Among the malaria endemic areas of the zone Karakore, Ataye and Senbete towns were selected. These towns are among the hot areas in North-Shoa zone and commonly affected by malaria. The towns have governmental and non-governmental health facilities. Considering the governmental health facilities, Ataye town has one district hospital and one health centre, while Karakore and Senbete town has one health centre each. These health institutions give service for the patients in the towns and their surrounding areas.This cross-sectional study was conducted in malaria endemic areas of North-Shoa, Ethiopia from November 2018 to January 2019. The study was conducted among apparently healthy pregnant women. Pregnant women with absence of disease symptom/sign within the past 48 h, axillary temperature ≤ 37.5 °C, permanent residents in the area, and those willing to participate in the study and signed the informed consent were included. Individuals treated with anti-malarial drugs in the previous 6 weeks prior to data collection and those who undergo any kind of long-term medical treatments, and unwilling individuals were excluded.Sample size was calculated using asymptomatic malaria prevalence of 9.1% reported from a study conducted in the rural surroundings of Arbaminch Town . The ranMultistage sampling technique was used to select study subjects. Three malaria endemic areas were selected by simple random sampling using the lottery method from the known five malarious areas of North-Shoa zone. From each selected areas one health institution was selected by simple random sampling using lottery method. The three selected health institutions were Ataye district hospital, Senbete Health Center and Karakore Health Centres. The sample size was then distributed proportionally to the selected Health Centres based on the size of their pregnant women who are following antenatal care. In the selected health centre, the pregnant women were selected by simple random sampling by using the sampling frame Fig. .Fig. 1SaData were collected using microscopy and RDT methods to diagnose asymptomatic malaria in pregnant women. Structured questionnaire was used to collect socio-demographic and other associated factors. To ensure reliable data collection the questionnaire was pre-tested before the actual data collection and training was given to data and sample collectors on data/sample collection procedures.Plasmodium was observed in 200 high-power fields. On the other hand, thick blood films were taken as positive if one or more malaria parasites have been observed; and, negative if no parasites were seen after examining 1000 white blood cells.Thin and thick blood smears were prepared, stained with 3% Giemsa and examined microscopically from each asymptomatic pregnant woman. Thin smears were considered positive for malaria if one or more malarial parasites were seen; and, negative if no asexual form of Malaria diagnosis using CareStart Combo malaria  test was performed. Rapid diagnostic test results were read by the laboratory technicians. The result was read by a second experienced laboratory technologist who was unaware of the result. In case of disagreement it was read by the principal investigators. Positive and negative blood samples were collected and RDTs were stored in appropriate temperature and humidity and it was checked throughout the study for its validity.Plasmodium infection. In the univariate analysis variables significant at P value of 0.25 were entered to multivariate logistic regression analysis model. P-value less than 0.05 were considered statistically significant in all comparisons.Data were entered and analysed using SPSS version 20 statistical package. Frequencies were used to determine the prevalence of asymptomatic malaria in pregnant women. Binary logistic regression was performed to assess the predictors of asymptomatic A total of 263 pregnant women were involved in this study. The age of the study participants were ranged from 16 to 41 years with the mean age of 27.8 years (SD = ± 5.34). Majority of study participants were house wives (39.2%) and 95.4% were married. The mean family size in the present study was 3.28 (SD ± 1.35). About 36.5% the pregnant women were multigravidae, 52.5% of them were in their second trimester of pregnancy and majority (96.6%) of them were following antenatal care  test with a specificity and sensitivity above 90%. Forty-six percent (46%) of pregnant women had a haemoglobin level of < 11 g/dL and 13.0% of them were infected with malaria.The prevalence of asymptomatic malaria infection was 5.7% (15/263) and 3.4% (9/263) by microscopy and RDTs, respectively. Plasmodium infection at P-value of < 0.05 . Among women who used ITNs sometimes the odds of infections were 5.19 times higher (95% CI 0.55–49.21) when compared to women who always used ITNs. There was statistically significant association between anaemia and asymptomatic malaria infection both in univariate and multivariate analysis. Asymptomatic malaria infected pregnant women were almost 4 times (95% CI 1.98–14.58) more likely to be anaemic compared to non-infected pregnant women.Socio-demographic characteristics and other predictors were analyzed using binary logistic regression. In univariate analysis, family size, ITN usage and haemoglobin level showed significant association with 05 Table . In multPlasmodium  more likely to be infected by Plasmodium infection among pregnant women was 5.7% and 3.4%by using Giemsa-stained blood smear microscopy and CareStart™ HRP2/pLDH combo rapid malaria test, respectively. This prevalence was lower than the finding reported from the rural surroundings of Arbaminch town, South Ethiopia, which was 9.1% and 9.7% by microscopy and RDT, respectively among pregnant women [In the present study, the prevalence of asymptomatic nt women . This mint women . While, On the other hand, the prevalence of asymptomatic malaria in the present study was higher than the prevalence of 3.1% by microscopy and 4.8% by RDTs reported from Nigeria teaching hospital . The difPlasmodium infection was higher using microscopy than RDT which disagrees with a report from the rural surroundings of Arbaminch Town, South Ethiopia [The prevalence of asymptomatic Ethiopia . HoweverEthiopia , 17.Plasmodium infection were higher in pregnant women who have no habit of ITN usage than those who used nets always. This finding is in line with the fact that effectiveness of insecticide-treated net usage is considered as one of the most important prevention and control tools [There was statistically significant association between ITN usage habit and plasmodium infection in pregnant women. This was in agreement with the finding reported from the rural surroundings of Arbaminch town . The oddol tools . In contol tools .The present study showed asymptomatic malaria is prevalent in pregnant women and it has statistically significance association with the haemoglobin level of pregnant women. This indicates pregnant women have to be screened for asymptomatic malaria to avoid health consequences of malaria infection during pregnancy for the mother and fetus.


The connection between malaria-associated morbidities and farming activities has not been succinctly reported. This study aimed to address the connectivity between farming activities and malaria transmission.Plasmodium falciparum was based on the presence of ring form or any other blood stages. Individuals who are either critically ill or lived in the community less than 3 months were excluded from the study.The study took place in the agricultural setting of Nigeria Edu local government  between March 2016 and December 2018. A pre-tested structured questionnaire was administered to obtain information on their occupation and malaria infection. Infection status was confirmed with blood film and microscopic diagnosis of P = 0.005), particularly among rice farmers (2991.6 parasites/μL blood) compared to non-farmer participants. The occurrence and parasite density follow the same pattern for sex and age (P < 0.05). Children in the age of 6 to 10 years  and ≥ 11 years  groups were two-and four-fold more likely to be infected with malaria. The analysis revealed that the proximity of bush and stagnant water to the farmer  and non-farmer  habitations influence malaria transmission.Of the 341 volunteers, 58.1% (52.9% in Shigo and 61.4% in Sista) were infected (parasitaemia density of 1243.7 parasites/μL blood). The prevalence and intensity of infection were higher among farmers (71.3%, 1922.9 parasites/μL blood, This study highlights farming activities as a risk factor for malaria infection in agro-communities. Integrated malaria control measures in agricultural communities should therefore include water and environmental management practices. Plasmodium falciparum  accounts for the majority of malaria-associated morbidities and mortalities, particularly in holoendemic regions  of African region, in which 50%, 71% and 65% of cases occurred in South-East Asia, Eastern Mediterranean and Western Pacific, respectively  and dry (October–March) seasons. The majority of the inhabitants are Nupe, Hausa and a few are Yoruba and Igbo. Our study participants are primarily females (62.5%), uneducated (59.5%) with 67.4% peasant farmers growing crops such as rice, beans and sugar cane with little wage . The comPrior to the study, the community leader and medical personnel in Shigo, Makwagi, Sista and Ogo-Oluwa villages in Bacita community were visited and briefed on the concept and benefit of the study. The participation in this study was made voluntary and the volunteers were randomly screened based on the study criteria. All farmers and non-farmers who live in the rural communities in Nigeria for more than three months were recruited. Individuals that had not stayed in the community for more than three months or critically ill that were unable to respond to the interview were excluded. After the participant screening, 341 participants were considered suitable for represent the entire estimated population of 6,210 people living in our study population with confidence interval and percentage of 5.16 and 48%, respectively.P. falciparum-positive if the ring form or any other blood stages is present by identifying using the criteria previously described  was done to obtain the odd ratio for the comparative analysis of risk factors among farmer and non-farmers participants.The prevalence of falciparum malaria was calculated as the proportion of positive samples. The data generated were presented by using descriptive statistics and subjected to Chi-square statistical and one-way analysis of variance (ANOVA) by using Statistical Package for Social Science (SPSS) version 16 to determine the significance in the relationship of infection and intensity rate with age, gender, and other socio-demographic and economic factors. A P. falciparum and the results of microscopic examination identified 198 (58.1%) positive cases, which ranged from 52.9% in Shigo to 61.4% in Sista and the overall average parasitaemia density of 1243.7 parasite/μL of blood. The highest (1376.9 parasite/μL of blood) and lowest (1093.2 parasite/μL) parasitaemia densities were recorded in Sista and Makwagi, respectively (P < 0.05). Analysis of infection for various types of farming activities revealed that rice and yam plantations were highly associated with the prevalence of infection. The highest parasite load was observed among rice farmers (2991.6 parasite/μL of blood).Blood smears of 341 volunteers were screened for the presence of ectively . In thesP = 0.034). Gender wise, infected males harboured more parasites than their female counterparts . The proximity of bush to human habitation influenced the occurrence of malaria infection in farmers  and non-farmers  study populations . Age groups of 6–10 years and ≥ 11 years with odd ratios of 2.168 and 3.750 were two and four times more likely to be infected with ulations .Malaria remains the most devastating health problem in many tropical countries, particularly in Nigeria . The higMeanwhile, the high prevalence and parasite density observed in this study could be attributed to similar environmental conditions prevailing in these localities and period of sampling, rain season that coincides with the peak of infection as previously reported by Babamale and Ugbomoiko . This isReports have shown that water distribution influences malaria vector populations and parasite transmission , 21. TheAnopheles species, a principal vector of malaria parasites. This has also been previously reported among farmers in African regions (Our study further revealed that rice farmers recorded high occurrence of infection when compared to yam and sugar cane farmers. This observation is probably due to the fact that rice, one of the staple crops in Nigeria, relies on flooded paddies, which provide breeding sites for  regions , 27. How regions .The prevalence and intensity of malaria with respect to age and sex among the farmers conflict with the normal trend previously reported in endemic communities of the general population. For instance, many reports in malaria high transmission zones showed that younger age groups are often susceptible to infection , 29–31. Overall, the data analysis of this study showed that farming activities influence the pattern and transmission of malaria infection. Unlike in non-farmer populations, infection is significantly higher among males and adult individuals that directly or indirectly engaged in farming activities. This observation is identical to the reports of Uneke et al.  in SouthLastly, our bivariate analysis also revealed that the proximity of bush and bathroom to human habitation are essential transmission factors for malaria infection. This further suggests that the malaria vector is inextricably tied to waterlog common in the study area as previously reported , 40. MosIn conclusion, farming, a sustainable means of livelihood of many rural dwellers, poses a great risk for the acquisition of malaria infection. Protection of local farmers through provision of efficient healthcare services and insecticide-treated nets should be extended to many malaria-endemic rural communities to afford them the opportunity of integrated control measures to curb the transmission of malaria infection in their various settings. Also, enlightenment on water and environmental management practices in this type of community will no doubt go a long way in reducing the incidence of the disease.