Plasmodium falciparum antigens. The project the age-dependent distribution of alleles or haplotypes of three P. falciparum malaria vaccine candidates, Circumsporozoite Protein (csp), Erythrocyte Binding Antigen 175 (eba-175) and Serine Repeat Antigen 5 (sera5) in a region of intense malaria transmission in Uganda.The development of malaria vaccines is constrained by genetic polymorphisms exhibited by Plasmodium falciparum DNA was extracted by chelex method. Alleles of sera5 and eba-175 were determined by polymerase chain reaction (PCR) amplification followed by resolution of products by agarose gel electrophoresis. Allele calling was done using gel photographs from ethiduim bromide stained gels. Haplotypes of csp were identified by sequencing 63 PCR products using the P. falciparum 7G8 laboratory strain sequence as a reference. The data were analysed using SPSS 16, EQX for windows and Chi-square test was used to calculate associations , Excel was used to generate graphs. The BioEdit and NCBI blast software programs were used to analyse the sequences from which csp haplotypes map was constructed.A cross-sectional study was carried out between August and November 2009 in which 250 study participants were selected from a population of 600. Finger prick blood samples were collected after informed consent from participants below 5 years, 5–10 years, and above 10 years of age. Blood was used for microscopy, RDT and dried blood spots. Eba-175 FCR3 (48/178) and CAMP (16/178) alleles were observed, the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older participants. Sera5 alleles ORI (6/204) and ORII (103/204) were observed in the population, ORII was more prevalent and was significantly associated with age , parasite density  and clinical outcomes . There was marked csp diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar to a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. There was an age-dependent distribution of csp haplotypes with more haplotypes being harbored by children < 5-year of age, (10/16) compared to adults (2/16). Interestingly, the csp haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed.eba-175 FCR3 allele, sera5 ORII allele, and csp haplotypes are targets of host immunity and under immune selection pressure in Apac District. These molecules could provide alternative malaria vaccine candidates as sub-unit vaccines.This data suggest that the  In addition, malaria also causes an unmeasured impact on local economies, human health in general, and longevity [csp), Serine Repeat Antigen 5 (sera5), and Erythrocyte Binding Antigen-175 (eba-175). CSP, the major component of the most advanced malaria vaccine candidate  is found on the surface of sporozoites. This protein contains two major B cell epitopes consisting of tandem repeats . Another vaccine candidate that has been considered is based on the sera5, also known as the P126 antigen. This is the largest parasite protein, it accumulates in the parasitophorous vacuole of trophozoites and schizonts and is processed into three fragments  [sera5 derived BK-SE36 candidate vaccine is not only safe and immunogenic but protects children against malaria attacks over a year of follow up. Field studies have shown that high titers of IgG anti-SE36 antibodies are associated with protection against malaria in Ugandan children, adults and against placental malaria and low birth weights in pregnant women [Eba-175 of P. falciparum is localized in the merozoites and is thought to be one of the key players during the fast cascade of interactions between the parasite and host molecules before the merozoites completely invade the erythrocyte [csp, eba-175, and sera5. This was to obtain baseline data on these antigen alleles in an area of high malaria endemicity. The study was conducted on the hypothesis that there is no significant association between candidate antigen polymorphisms and participant’s age, clinical presentation/outcomes and parasite density.Malaria is one of the most severe public health problems worldwide. It is a leading cause of morbidity and mortality in many developing countries, where young children and pregnant women are most affected. Nearly half the world’s population lives in areas at risk of malaria transmission in 91 countries and territories. In 2016, malaria caused an estimated 216 million clinical episodes, and 445,000 deaths. An estimated 90% of deaths in 2016 were in the World Health Organization (WHO) African Region [ongevity . There aAnopheles funestus [Plasmodium falciparum is the dominant parasite species, Plasmodium malariae being responsible for ~ 3% of the infections and Plasmodium ovale was previously not observed [The study was conducted in Apac District, a rural district in Northern Uganda located between Kwania Lake and the Victoria Nile . Apac District covers an area of 6684 square kilometres and ranges in altitude between 1350 and 1500 m above sea level. The rainfall pattern is bimodal with a dry season from November to February and two short rainy seasons from April to May and from September to October. According to surveys conducted in 2001–2002, this area experiences perennial holoendemic malaria , with pafunestus . Plasmodobserved P. falciparum malaria in holoendemic areas is characterized by high level of parasitaemia and symptomatic infections in early childhood, and immunity to clinical disease is associated with cumulative exposure [The study population comprised of all patients presenting to the health facilities in the selected parishes for clinical management and those who came as a result of mobilization for the study. These were categorized into three age groups of children below 5 years, 5–10, and 10–75 years residing in four selected parishes of Apac district, i.e. Abedi, Atopi, Akere, and Apac parish. Sampling points were health centers for the parishes of Abedi and Akere. For the case of Apac, the main hospital was used as a sampling point. For Atopi, there being no health facility, Atopi primary school was used as a sampling point. Unpublished data (Med Biotech Labs) indicated that each of these target parishes had a unique malaria prevalence among the selected age groups; Apac town residents < 5 years and > 10 years with 68.4% and 16.3%, respectively; Abedi parish < 5 years and > 10 years with 59% and 20.1%, respectively; Akere parish < 5 years and > 10 years with 69% and 20.8%, respectively; whereas Atopi residents < 5 years and > 10 years had 75.9% and 24.1% prevalence, respectively. This data is consistent with findings from other studies which showed that exposure . This foexposure The inclusion criteria were a positive malaria test for the symptomatic and asymptomatic cases. Those that showed symptoms similar to malaria infection but had negative malaria RDT were excluded from the study. The asymptomatic cases are those who did not present with the cardinal clinical signs for malaria such as high body temperature (above 37.5 °C), general body weakness, headaches and vomiting yet had positive malaria RDT. Symptomatic cases are those who presented with the above signs and also had positive malaria RDT.Participants were recruited in October 2009 in the four parishes of Apac District. Sampling was done in Apac District Hospital, two health facilities of Abedi and Akere Health Centers II, and a primary school in the parish of Atopi, known as Atopi Primary School. Before the sampling days, community meetings were organized to explain the purpose of the study and to invite people to attend sampling points. At the health facilities and hospital, all clients attending the facilities for clinical care, antenatal visits, or who came specifically to benefit from the screening offered by this study were selected for enrollment. People were sequentially enrolled for the study at all study sites. Written informed consent or in case of illiteracy, consent by thumb printing was obtained from each participant older than 16 years of age and from parents or guardians of younger participants. Each participant enrolled in the study underwent a clinical examination during which an axillary temperature was taken twice the higher of the two was recorded. A single blood sample was obtained by finger prick (approx. 0.3 mL) for thick and thin blood films, for filter paper blood collection  and Rapid diagnostic tests . This RDT has an estimated detection rate of 97.5% for parasite densities > 2000 parasites/μL and 54.4% for parasite densities of 200/μL.Questionnaires were used for data collection. They were written in English and translated to Luo. Three trained laboratory technologists and nurses were involved in the administration of the individual questionnaires. The questionnaires were first pretested to make sure they elicit the relevant information sought for. Questionnaire target information included demographic data, mosquito net use, type of anti-malarial taken, and when last taken, how long the person had been sick, whether the treatment had been sought and from whom. Interviews were conducted either in English or Luo depending on the subject’s preference.sera5, eba-175, and csp variants.Following questionnaire administration and obtaining consent, finger prick blood was collected from each of the patients. Briefly, the patient’s index finger was cleaned using an alcohol swab, dried with a piece of cotton wool and Vaseline applied onto the cleaned area, this to prevent blood from spreading over the finger. The finger was gently massaged to encourage blood flow to the tip of the finger after which the distal part of the finger was pricked by the use of a sterile lancet. Blood samples were tested using three approaches: (1) blood smears made on microscopic slides for parasite density determination, (2) RDT for rapid malaria diagnosis and, (3) spotting blood onto filter papers for later DNA extraction for molecular characterization of P. falciparum parasites were seen in any of the fields. The thick blood slides were examined later under oil immersion at high magnification (100×) to determine parasite density. Parasite density was calculated from the number of parasites per 200 white blood cells (WBC), assuming an average normal WBC count of 8000/µL.Thick blood films were made from each patient at recruitment. The smears were stained with 3% Giemsa for 30 min. A thick film was considered negative if no parasites were seen in at least 100 high-power fields or if fewer than 500 leucocytes were observed per 100 fields and if reading did not reveal asexual parasites or gametocytes. A slide was considered malaria positive if Rapid diagnostic testing, RDT of malaria infection was carried out to ensure quick treatment administration. The RDT kits were obtained from Orchid Biomedical Systems, Goa, India. The test was carried out following the procedure from the kit given by the manufacturers. Briefly, a finger prick was made on the index finger of the patient and the sample applicator was used to take a loop full of blood. This blood was then applied onto the sample pad, well A and six drops of the lysing buffer was dispensed into well B and the kit was left to stand for 15 min. After which a reading was taken.Plasmodium falciparum DNA was extracted from dried blood filter paper samples by the Chelex-100 method, as previously described [P. falciparum csp, N-terminus (octamer repeat domain—OR domain) of sera5, and eba-175 were analysed by nested/semi-nested PCR amplification of parasite DNA as described previously [P. falciparum HB3, K1 and 3D7 laboratory strains obtained from Malaria Research and Reference Reagent Resource Center (MR4) were used as templates. The details of these procedures are described below. A pair of primers, CSo101 5′-AATCAAGGTAATGGACAAGG-3′ and CSo102 5′-CTAATTAAGGAACAAGAAGG-3′ were used to amplify the full 1193-bp fragment of CSP (GeneDB Accession Number Pf3D7_0304600) in the primary or outer PCR. A second primer pair; CSo101 nucleotides 940–959, 5′-AATCAAGGTAATGGACAAGG-3′ and CSo104 nucleotides 1278–1297 5′-GGAACAAGAAGGATAATACC-3′ was used to amplify the PCR products of the outer PCR in a semi-nested PCR [escribed , 15 Brieeviously –18, For Pf3D7_0207600 was amplified by an outer PCR using primer pairs p126A (5′-AAT GAA GTC ATA TAT TTC CTT G-3′) and p126B (5′-CAA TGT TGT TCT TAA TTC GAT A-3′) followed nested PCR using the primer pairs p126C nucleotides 1–21, (5′-GTG TTA TAT TTA ACA AAA ATG-3′) and p126D, nucleotides 450–471 (5′-CTT ACA GGA TTG CTT GGT TCG-3′) [eba-175 (GeneDB accession number Pf3D7_0731500) was amplified using primers pairs EBA-1 forward (nucleotides 2336–2356). 5-CAAGAAGCAGTTCCTGAGGAA-3′ and EBA-2 reverse (nucleotides 3060–3083) 5′-TCTCAACATTCATATTAACAATTC-3′ in an outer PCR and primer pairs EBA3 forward nucleotides 2296–2319 5′-GAGGAAAACACTGAAATAGCACAC-3′ and EBA4 reverse (nucleotides 3068–3091) 5′-CAATTCCTCCAGACTGTTGAACAT-3′ for the nested PCR [A repetitive gene region encoding 3434 bp of the P126 antigen  . The PCRsted PCR –20. The Plasmodium falciparum multiplicity of infection might play a role in determining the clinical presentation and outcomes of malaria infections and might contribute to disease transmission rate.In order to determine the multiplicity of infection in the study population, the merozoite surface proteins (MSP) were amplified using MSP1 and MSP2 specific primers as described elsewhere . Plasmodcsp sample was treated as an independent entity. The BioEdit and NCBI blast software programs were used to analyse the sequences from which csp haplotype patterns were constructed using 7G8 (MR4) laboratory strain sequence as a reference. Variables were summarized using frequencies and proportions and presented using bar charts. Association between polymorphisms of csp, eba-175, and sera5 on one hand and subject parameters on the other hand were assessed using Pearson's Chi-Square test; P-values of < 0.05 were considered significant.All data were checked for completeness, coded, and entered in an Excel spreadsheet, transferred to SPSS and analysed. The nucleotide sequence of each Approval to conduct this study was granted by Uganda National Council of Science and Technology, Med Biotech Laboratories Institutional Review Board, Makerere University ethics review board and Apac District Administration Board. Information about the study was provided to the participants and informed consent was directly obtained from study subjects above 18 years old. Study subjects considered minors (< 18 years old), informed consent was obtained from the parents or guardians, and assent to the study was directly obtained from the infant. In all cases, the decision of the minor, if possible, took precedence over the consent of the adult parent/guardian. Only consenting adults and assenting minors were recruited into the study.A total of 250 participants based on the RDT and microscopy results were selected for the study. These were residents of Apac, Atopi, Abedi and Akere parishes, age range from 6 months to 75 years. The males comprised of 41% (106/250) and females comprised 58.7% (144/250). Anti-malarial drug use in the last 2 weeks was reported by 31.6% (79/250) while 68.9% (172/250) reported regular use of insecticide-treated nets  in the target population was 41.7% (250/600). Only those with a positive malaria test (41.7%) were included in the study, the rest were excluded. The parasitaemia/parasite densities were divided into two categories; < 5000 parasites/µL and > 5000 parasites/µL. This was done to enable determine the difference or any association between parasite density and patient parameters such as age, allele distribution, and clinical presentation/outcomes in all categories. Parasitaemia across the parishes was fairly comparable, although Abedi had the highest number of participants with a parasitaemia of > 5000 parasites/µL and Atopi had the highest number of participants with a parasitaemia < 5000 parasites/µL  and ORII (199 bp) were identified by nested PCR analysis in the study region  distribution was highest in the 5–10-year-olds. Overall, there was a significant association between sera5 allele distributions and age (P < 0.0001). Individual analyses of the distribution of alleles with age category presented a divergent statistical picture with ORI, ORII, and ORI/ORII showing P values of 0.466, 0.002, and 0.476, respectively. This shows that only ORII was significantly associated with age. ORII allele frequency was significantly higher with a frequency of 11/19 (57.9%) in participants with > 5000 parasites/µL, while the ORI allele was significantly lower with a frequency of 6/169 (2.4%) in the same participants  and FCR3  were identified by nested PCR analysis and the FCR allele predominated with an overall percentage of 63.3% in the study population . With regards to clinical outcomes, the FCR3 allele was more frequent  in the asymptomatic group while the CAMP allele was more frequent in the symptomatic group. However, the association between eba-175 allele distribution and clinical outcomes was not significant (P = 0.854). The eba-175 FCR3 allele was less frequent  in > 5000 parasites/µL was and more frequent  in the < 5000 parasites/µL group. The CAMP allele, on the other hand, was more frequent in the lower parasitaemia group, that is the < 5000 parasites/µL group . There was also no significant association between eba-175 allele distribution on one hand, and bed net use (P = 0.555) and gender (P = 0.33) on the other. The project further analysed gender, bed net use and found that there were significant associations with allele distribution in both groups.Two ion Fig. b. Districsp extracted from P. falciparum field isolates was amplified by PCR  amino acids at region X (amino acids 308–317). Only two sequences had R (arginine) instead of E (glutamic acid) at region 308–317. While 12.5% (2/16) of the haplotypes were found in the adults, 62.5% (10/16) were found in children < 5 years and 25% (4/16) were found in the 5–10-year olds. There was no significant association between age and CSP haplotypes (P = 0.091). Haplotypes frequencies were unequally distributed among the four parishes studied with Akere having the highest number of haplotypes 37.5% (6/16) while Apac and Atopi had the same frequency, 31.3% (5/16). Seventy-five percent (12/16) of the haplotypes were observed in patients with symptomatic malaria whereas 25% (4/16) were observed in those with asymptomatic malaria. Statistically, there was no significant association between clinical presentation and haplotype distribution, (P > 0.5). However, 69.0% (11/16) of haplotypes were in those with > 5000 parasite/μL while 31.0% (5/16) were in those with < 5000 parasite/μL. Very importantly, out of 17 haplotypes observed in the Ugandan population, only two (U20123 and U40028) were similar to one haplotype (E12) observed in Sierra Leone and the rest were unique to the Ugandan study population  in Apac District; (2) to demonstrate presence or absence of associations between polymorphisms, age, parasite density, and clinical presentations/outcomes.This study aimed to (1) investigate the polymorphism, distribution, and frequency of three major malaria vaccine candidate antigen alleles  and bed net use (P = 0.86). The association between ORII fragment with age, parasite density, and clinical outcomes could be an indication that it is a target of immunity. First, the ORII allele was more prevalent in the < 5 years age group  while having a low prevalence in the > 10 years participants (who have developed immunity over time and can clear parasitaemia). Second, the project documented a high prevalence of ORII in participants with a parasitaemia of > 5000 parasites/µL of blood and lower prevalence in subjects with lower parasitaemia. Third, the ORII allele was more prevalent in the symptomatic malaria group by comparison with the asymptomatic group. These observations strongly support the notion that the ORII allele of sera5 is a target of anti-malarial immunity and can provide potential use in sub unit vaccines.P. falciparum is localized in the merozoites. After the initial contact of the merozoite with the erythrocyte, orientation takes place and the apical end makes contact with the erythrocyte surface [Eba-175 gene is divided into several domains including three cysteine-rich regions  [eba-175 sequences have a low level of deduced amino acid polymorphism in its cysteine-rich region II [eba-175 alleles/fragments, CAMP and FCR-3, were defined by nested PCR of 178 field isolates in the three study parishes (isolates from Abedi did not amplify). The FCR3 fragment was highest in Apac parish, with prevalence 33%, the CAMP and mixed alleles were highest in Atopi parish with proportions of 10% and 36%, respectively. Overall, the FCR3 fragment had the highest proportion while the CAMP fragment was the least frequent in this study population. This data is consistent with data from Nigeria, Gambia, Gabon, and South Africa in which the FCR3 fragment dominated [eba-175 fragment type [eba-175 allele distributions and age, with the FRC3 allele being more prevalent among the < 5 years' age group. The high FCR-3 prevalence in high malaria-endemic areas, especially among the < 5 years old subjects and the low proportion in adults, suggests that FCR3 allele is a target of immune selection pressure. By contrast, the CAMP allele was more prevalent in the > 10 years, an age group known to have acquired immunity against malaria. The CAMP allele was more or less equally distributed in the four parishes as opposed to the FCR-3 allele and mixed alleles that had unequal distribution. This difference in observations between the findings and those of other studies could be explained by random shifts in parasite allele frequencies in different geographical regions [Erythrocyte binding antigen-175 (eba-175) of  surface . Eba-175 and F3) . Most ebegion II , 27. Theegion II . The ebaominated , 28 Howeominated . Previouent type . In this regions , as wellP. falciparum sporozoites. It consists of a sequence comprising a central repeat that is flanked by polymorphic N-terminal and C-terminal non-repeat regions [csp antigen is the primary component of the RTS,S malaria vaccine, which is one of the leading malaria vaccine candidates probably going to be the first malaria vaccine to be deployed in immunization programmes.CSP is the most abundant protein on the surface of the  regions , the NAN regions . It has  regions . The cspcsp among wild isolates circulating in Apac District, in northern Uganda. Field isolates were amplified by PCR and 61 products were sequenced. Previous studies from moderate to low malaria endemicity settings showed less diversity within this region [csp (Th2R/Th3R). Twenty-six percent (16/61) of the Ugandan sequences had the P. falciparum 7G8 reference sequence haplotype and  of the haplotypes identified were unique to the Ugandan isolates (haplotypes not observed in other studies). One haplotype, which appeared in two samples  was similar to a haplotype that was previously identified in The Gambia and Sierra Leone [P. falciparum 7G8 haplotype at the amino acid level, when aligned with the 7G8 nucleotide sequence showed identities of 97–99%. This confirms the authenticity of the Ugandan sequences and their uniqueness. In this study, the single haplotype that was observed in Asia (Asia type) was not detected [P. falciparum antigens in different geographical regions and the uniqueness of parasite strains from an endemic region in Uganda.The results here represent the first molecular study on the genetic diversity of T-cell epitope regions in the C-terminal portion of s region . Resultss region , which sra Leone . The remdetected . In all,csp gene, all the Ugandan isolates had a basic amino acid  while the 3D7 laboratory strain had an acidic amino acid . This again underscores the divergence of Ugandan csp sequences. The absence of the prototype 3D7 sequence in the Apac field isolates could have serious implications on the vaccine accuracy of RTS,S malaria vaccine in this region. This, however, needs to be further investigated, considering the sample size and time from this study. Despite the small sample size, haplotype distribution seemed to take an interesting trend. The majority of haplotypes were observed in the age group under 5 years and also among the symptomatic malaria group. This was not statistically significant. This is comparable to previous data [P. falciparum alleles and haplotypes, as was the case for the three selected malaria vaccine candidate antigens, sera5, eba-175, and csp. For sera5, the increased prevalence of ORII allele among individuals with parasite density above 5000 parasites/µL, in symptomatic individuals, and in children < 5 years strongly implicate this allele as a target of immune selection pressure. This observation indicates that the ORII allele stimulates immunity against malaria. This finding further confirms host responses against sera5 as a vaccine candidate. The FCR3 allele of eba-175 is also an important target of host immunity in the study region. The age-dependent distribution of haplotypes with more haplotypes in the < 5-year-olds, as compared to adults, confirms the role of immunity in haplotype selection. The Ugandan csp haplotypes were unique to the population and the 3D7 vaccine type was not observed. The uniqueness of these haplotypes raises concerns about the effectiveness of RTS,S vaccine in this population when deployed, as polymorphisms in the csp gene could be a result of immune selection pressure [Furthermore, the 3D7 haplotype sequence which is used for the development of the RTS,S malaria vaccine was also not observed in the 61 Ugandan isolates sequenced in this study. This, however, does not rule out the existence of this haplotype (3D7) in Ugandan isolates. It was also observed that at position 326–342 (ThR2) of the ous data , in whicP. falciparum infective bites [MOI in the study population was found to be significantly associated with age, parasite density and clinical presentation. These findings are consistent with studies conducted in Sudan which showed that even within the same individual; MOI tends to decrease with age . This cove bites .P. falciparum at csp locus should be carried out to determine the prevalence of the 3D7 vaccine strain haplotype. This might confirm the uniqueness of the Ugandan strains reported in this study and could provide insights on the future of RTS,S vaccine deployment in this region.Country or region-wide genotyping of sera5, eba-175, and other malaria vaccines candidate antigens genes should also be carried out to identify baseline circulating parasite genotypes before deployment of these vaccines. This could provide insight into future development of more subunit vaccines against malaria.Countrywide genotyping of sera5 and eba-175 subunit vaccines.Studies to confirm the immunogenicity of ORII and FCR3 fragments should be undertaken; this will guide development of more effective It is important to clearly define the genotypes of the parasite population circulating in a region before rolling out a new vaccine. In this regard, the authors recommend the following as important future studies.eba-175 FCR3 allele, sera5 ORII allele, and csp haplotypes are targets of host immunity and are under immune selection pressure in Apac District. These molecules could provide alternative malaria vaccine candidates as sub-unit vaccines. This work also raises the need for continued monitoring of parasite population dynamics as a tool for monitoring emergence of drug resistance as well as to provide understanding of how these populations are developing mechanisms survive, which could guide both drug and vaccine development. In addition, further studies on evolution of parasite genotypes can also provide insight on development of future insecticides.These data suggest that the


Plasmodium falciparum can result in detrimental outcomes for both mother and infant, including low infant birth weight, preterm birth, maternal anemia, spontaneous abortion, and maternal and/or infant mortality. Maternal anemia is a particularly complex outcome, as the body must both maintain erythropoiesis and tolerance of the growing fetus, while directing a Th1 response against the parasite. Underlying the pathogenesis of PAM is the expression of variant surface antigens (VSAPAM) on the surface of infected red blood cells (iRBC) that mediate sequestration of the iRBC in the placenta. Naturally acquired antibodies to VSAPAM can block sequestration and activate opsonic phagocytosis, both associated with improved pregnancy outcomes. In this review, we ask whether VSAPAM antibodies can also protect mothers against malarial anemia. Studies were identified where VSAPAM antibody titres and/or function were associated with higher maternal hemoglobin levels, thus supporting additional protective mechanisms for these antibodies against PAM. Yet these associations were not widely observed, and many studies reported no association between protection from maternal anemia and VSAPAM antibodies. We discuss the epidemiological, biological and technical factors that may explain some of the variability among these studies. We appraise the current evidence of these complex interactions between PAM-specific immunity and maternal anemia, propose potential mechanisms, and discuss knowledge gaps.Pregnancy-associated malaria (PAM) caused by  Pregnant women are especially vulnerable to malaria compared to non-pregnant adults in Africa  and 11 mPlasmodium falciparum infection in high transmission settings, primigravid women have an especially high risk of poor pregnancy outcomes, and this risk decreases with gravidity . VSAPAM malaria . In this malaria . In anot malaria . OpsonizPAM antibody levels, and pregnancy outcomes. Studies should be conducted in many diverse settings to reveal context-specific effects on the acquisition of PAM immunity and the prevalence of maternal anemia. Treatment practices and circulation of other pathogens in a particular area can also affect humoral immunity.As the pathways leading to anemia in PAM are complicated and multifactorial, further research is critical to better understand any potential underlying immune mechanisms in response to malaria infection that contribute to or protect women from anemia. Longitudinal studies should be continued in and outside of Africa to increase the body of knowledge available on infection, VSAPAM antibodies are associated with higher hemoglobin levels in mothers, it will be exciting to unravel the underlying mechanisms of protection. We propose a basic model for how these antibodies that target placental iRBCs could block downstream pathogenic effects that lead to anemia, yet the mechanisms that directly contribute to this process need to be delineated. While the data support a role for these antibodies in blocking sequestration in the placenta and promoting opsonic phagocytosis, emerging evidence linking the innate and adaptive immune systems merit exploration. For example, data is emerging on the role of the complement system in uncomplicated malaria. The antibody-mediated activation of the classical complement pathway was observed in samples from children and was an indicator of protective immunity (in vitro (In those studies where VSAimmunity . In addiin vitro . FurtherThis review highlights the complexity of the body’s response to PAM and the large gaps in the current knowledge on immune mechanisms underlying protection. Pregnant women and young children remain at great risk of morbidity and mortality from malaria. Continued research in this area will add to our understanding of PAM and potentially reveal novel therapy and vaccine strategies to combat this disease.MCW conducted the literature review. MCW and SKY jointly wrote the manuscript. All authors contributed to the article and approved the submitted version.MCW holds a CIHR Frederick Banting and Charles Best Canada Graduate Scholarship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor declared a past co-authorship with one of the authors, SY.


Anaemia is a global health problem and women in reproductive age (WRA) are amongst the most affected population. Its consequences include low birth weight and maternal mortality. This study aimed to assess the prevalence of anaemia and to identify its determinants in Sudanese women in reproductive age.A population-based cross-sectional study was conducted in Sudan in 2016. A multi-stage stratified cluster sampling design was executed with consideration of rural population, urban population, and internally displaced persons/refugees camps residents. All women in reproductive age (15–49 years), classified by pregnancy status, in the targeted households were surveyed and personal characteristic data were collected. Their haemoglobin level and malaria infection  were assessed. The World Health Organization (WHO) haemoglobin level cut-off for defining anaemia and severe anaemia in pregnant and non-pregnant women was used. Logistic regression analyses were performed.p = 0.003), NPW , and WRA . Other identified determinants of anaemia in NPW was living in camps  and in WRA was being in the poorest economic class .A total of 4271 women (WRA) of which 421 (9.9%) pregnant women (PW) were included in the study. The overall anaemia prevalence in WRA was 35.6%. It was 36.0 and 35.5% in PW and non-pregnant women (NPW), respectively. The average haemoglobin level was found to be 113.9 g/L (SD 16.3) and 123.2 g/L (SD 15.7) for PW and NPW respectively. Severe anaemia prevalence was 1.2% in each group. In the logistic regression model, anaemia was associated with malaria infection in PW (aOR 4.100, 95%CI 1.523–11.039, Anaemia is a public health problem in Sudan. The study supported the association between malaria infection and anaemia, but not with low and moderate malaria transmission areas. Resources need to be allocated for all anaemic populations with special attention for the populations in most need and interventions need to be implemented based on local variations. Malaria control interventions, specifically case management, may have a major impact in reducing anaemia prevalence in low to moderate malaria transmission areas. Anaemia is a world-wide problem, but affecting more the low and middle-income countries with young children and pregnant women as the most affected population groups , 2. The Causes of anaemia include blood loss, infections, acute and chronic diseases, micronutrient deficiencies, splenomegaly, and haemoglobinopathies , 4. AmonThe magnitude of the anaemia problem is classified based on its prevalence into mild public health problem when the level of anaemia prevalence is less than 20%, moderate public health problem if the prevalence is between 20% and less than 40%, and severe public health problem if it is equal to or more than 40% . The gloThis cross-sectional study was conducted in Sudan in 2016. Sudan is a low-middle income African country with 40.852 million US$ GDP per capita as estimated for the year 2018 . The couA population-based household survey to measure the prevalence of anaemia in WRA (15–49 years), disaggregated by pregnancy status, was combined with a malaria indicator survey and carried out on November 2016 just after the rainy season. A multi-stage stratified cluster sampling technique was used to select the study clusters using the number of households and population in popular administrative units (PAUs) based on the 2008 census projected to 2016. All (8) internally displaced persons (IDP) camps and 4 randomly selected refugees camps were chosen for the study. Out of the national number of clusters calculated (509 clusters), the number of camps clusters has been assigned following the size of the population resident in these camps. Then, remaining clusters have been allocated equally to states. Within states, the allocation was in proportionate to rural/urban population ratios. In each cluster of more than 150 households, segmentation was done. Then, in a one randomly selected segment, 20 households were randomly selected. In each household, all WRA were enrolled in the survey. All households and participants were assured of their right to participate in this study and written consent was obtained from them before enrolment. Ethical clearance for this study was obtained from the Sudan federal ministry of health, ethical and technical review board.Plasmodium falciparum and Plasmodium vivax specific histidine-rich protein II malaria rapid diagnostic test (RDT) .Data from households were collected in Arabic language using a questionnaire designed in a digital personal data assistant (PDA) device by trained health personnel. Locally developed web-based survey application with its data validation functions was used for data collection. A finger-prick capillary blood sample was taken by trained laboratory assistants from study participants for testing of haemoglobin level using field battery-operated device  and for malaria testing using In this study, the WHO standard cut-off points for anaemia and severe anaemia for WRA is used –22. Any Malaria infection is considered in the study when malaria parasite is identified in the study population using malaria rapid diagnostic test. Study participants who were found to be positive for malaria were treated according to the standard treatment guidelines while anaemic individuals were referred to the nearest health facility for medical attention. Using malaria parasite prevalence in children aged 2 to less than 10 years old, states that showed a parasite prevalence of less than 10% are considered as malaria hypo-endemic and those with a prevalence of 10% to less than 50% are considered malaria meso-endemic ones , 24. No The dependent variable in this study is the anaemia as per the definition described above. Independent variables included pregnancy status, age, type of residency, education level, wealth status, sanitation facility, water source, listening to radio , having health insurance, having malaria infection, and the level of malaria endemicity.p < 0.05 was not selected to be entered in the model. Odds Ratios with its 95% confidence intervals (95%CI) and P. values were reported. P. value of less than 0.05 was considered significant.The analysis was done using SPSS 21. Descriptive analyses were done to describe the data and to demonstrate prevalence among PW, NPW and WRA. Mean haemoglobin level and standard deviation (SD) were generated and compared between PW and NPW and within anaemic sub-groups using T-test and ANOVA. Chi squire test was used to assess statistical differences in categorical data and T-test for continuous data. Then, multivariable analysis was done to examine the association between independent variables and anaemia (the outcome) as well as the strength of the association. Only independent variables that demonstrate a statistically significant effect on anaemia and/or on mean haemoglobin level in the bivariate analysis were further taken for the multivariable analysis. The logistic regression model was built based on a backward (likelihood Ratio) stepwise method. Original haemoglobin level variable was used to check for the absence of multicollinearity among independent variables. A variable that showed no statistical significance at p < 0.001).In this survey, a total of 4271 WRA were examined for their haemoglobin level. Of them, 1385 (32.4%) were in urban areas, 2654 (62.1%) were in rural areas and 232 (5.4%) were in camps. The PW involved in the study were 105 in urban areas, 295 in rural areas and 24 in camps giving a total of 421 (9.9%) PW. The mean (SD) of the age among PW was 27.1 (6.6) and it was 28.3 (9.0) years in NPW  haemoglobin level was 113.9 (16.3) g/L and 123.2 (15.7) g/L in PW and NPW, respectively. Table p = 0.004) but not PW (p = 0.252).Based on this study anaemia is classified as severe public health problem in both PW and NPW residents in camps  compared to rural  and urban  residents (Table p = 0.001). The mean of the haemoglobin level in PW was significantly lower in Camps residents (109.2 g/L (SD 23.1); p = 0.018), and in women using unsafe sources of drinking water (113.3 g/L (SD16.7); p = 0.044 for open source), and in women with malaria infection (100.8 g/L (SD18.9) p < 0.001) (Tables p = 0.005).There were no significant statistical differences in the type of place of residence, level of education, economic status, sanitation facility used, source of drinking water, listening to the radio, having health insurance, or variations in the level of malaria endemicity between anaemic PW and non-anaemic PW according to findings of the bivariate analysis. There was a higher prevalence of anaemia (69.6%) with malaria-infected PW compared to non-infected PW p = 0.00. The meap = 0.004), wealth index ; p = 0.008), type of sanitation facility ; p = 0.011), and malaria infection . Compared to this, haemoglobin level in NPW was found to be associated with the type of place of residence (lower levels in camps residents (121.0 g/L (SD18.3)); p = 0.023), and with malaria infection ); p < 0.001) (Tables p = 0.016), type of sanitation facility (p = 0.005), and malaria infection (p < 0.001) (Table P < 0.001).There were no significant statistical differences between anaemic and non-anaemic NPW’s age, level of education, source of drinking water, listening to the radio, having health insurance and level of malaria endemicity as revealed by the bivariate analysis. However, anaemia in NPW was found to be associated with the type of place of residence ; orest 42.%; p = 0.fecation 9.2%; p =p = 0.006), among the poorest , among those who were practising open defecation , and among those with malaria infection  (Tables p = 0.033), wealth index (p = 0.010), and malaria infection (p = 0.003). Pregnancy status was not found to have an association with anaemia in WRA (p = 0.916) (Table p < 0.001) compared to those with non-malaria infection.When anaemia status was combined in PW and NPW, the univariate analysis showed no significant statistical differences between anaemia in WRA and age, level of education, source of drinking water, listening to the radio, having health insurance, and level of malaria endemicity. A higher prevalence of anaemia was observed in WRA in camps residents  affecting their whole life including their health status. This study classified PW and NPW living in camps as affected by a severe public health problem. It is also noticed that NPW in rural areas look like as being less affected by anaemia compared to urban and camp women. Severe anaemia is seen with high prevalence both among PW and NPW in camps compared to the rural and urban population. Overall results of this study stand at giving more priority for PW and NPW at camps in regards to anti anaemia interventions. Many studies documented the high burden of anaemia in IDPs and Refugees camps –37.P. falciparum malaria, and hence be more prone to the pathogenesis of anaemia. Placental parasitaemia may be present in the absence of peripheral parasitaemia and thus contribute to anaemia. In such settings, the effect of P. falciparum infection during pregnancy is affecting first pregnancies at the most [Our findings of the association between malaria and anaemia were reported by several previous studies , 38. Malthe most , 42. In the most . ResultsThis study did not find an association between pregnancy status and anaemia. In a systematic review involved four countries from Africa anaemia, iron deficiency, and iron deficiency anaemia prevalence were found to be lower in PW compared to WRA . The samThe association between anaemia and parasitic infection is well established. Evidence showed good impact in reducing anaemia through interventions against helminths in high-risk communities . This reIn this study, the association between economic status and anaemia was found for WRA. Results show that the poorest are more affected by anaemia than the richest women. This research is supporting other findings . A studyMany studies showed the lack of association between anaemia in pregnancy and mothers’ age and level of education , 29, 32.This study has various strengths and limitations. Study limitations include the low frequency of severe anaemia that limits further analysis to find out its determinants. Another issue is that the level of haemoglobin/anaemia neither adjusted for smoking nor the altitude. More is that pregnancy status in this study was not validated by any test or procedure and was depending on women response. This study did not address parity, previous obstetrical complications, or screening for haemoglobinopathies, enzymopathies, haematological disorders, or chronic diseases. Neither participants’ use of pica nor the use of iron or folic acid supplements were assessed in this study. The cross-sectional design of this study and the nature of the logistic regression model developed with a limited number of variables are the additional limitation of this study. One major strength of this study is that it is the first large-scale study that provided anaemia prevalence estimates for all states of the country. This will set real-life baseline estimates for monitoring progress in the fight against anaemia. Other study strengths include disaggregation of results by pregnancy status, coverage of IDPs/refugees camps population, the focus on determinants of anaemia, and presentation of results by both anaemia prevalence and haemoglobin level.With the level of anaemia prevalence identified in this study, WRA in Sudan are facing a challenging health problem, both at national and sub-national levels. The problem is severely affecting camps residents. The study established the association between malaria infection and anaemia. With the context of Sudan, low and moderate malaria transmission was not found to affect anaemia prevalence. Thus, early diagnosis and effective treatment of malaria cases are of utmost importance in such situation.Concerning anaemia, Sudan health authority needs to consider subnational level variations and give priority to the people most in need when designing interventions and allocating resources. Achieving progress in access to prompt and effective malaria case management is likely to have a high impact on reducing the burden of anaemia. Improving living conditions as well as reducing poverty may contribute to improving the haemoglobin level of affected communities.


Plasmodium falciparum poses a major threat to malaria control and elimination interventions. Characterization of the genetic diversity of P. falciparum strains can be used to assess intensity of parasite transmission and identify potential deficiencies in malaria control programmes, which provides vital information to evaluating malaria elimination efforts. This study investigated the P. falciparum genetic diversity and genotype multiplicity of infection in parasite isolates from cases with uncomplicated P. falciparum malaria in Southwest Ethiopia.Genetic diversity in P. falciparum microscopy and qPCR positive blood samples were collected from study participants aged 6 months to 60 years, who visited the health facilities during study evaluating the efficacy of artemether-lumefantrine from September–December, 2017. Polymorphic regions of the msp-1 and msp-2 were genotyped by nested polymerase chain reactions (nPCR) followed by gel electrophoresis for fragment analysis.A total of 80 msp-1 and msp-2 genes, the efficiency of msp-1 and msp-2 gene amplification reactions with family-specific primers were 95% and 98.8%, respectively. Allelic variation of 90% (72/80) for msp-1 and 86.2% (69/80) for msp-2 were observed. K1 was the predominant msp-1 allelic family detected in 20.8% (15/72) of the samples followed by MAD20 and RO33. Within msp-2, allelic family FC27 showed a higher frequency (26.1%) compared to IC/3D7 (15.9%). Ten different alleles were observed in msp-1 with 6 alleles for K1, 3 alleles for MAD20 and 1 allele for RO33. In msp-2, 19 individual alleles were detected with 10 alleles for FC27 and 9 alleles for 3D7. Eighty percent (80%) of isolates had multiple genotypes and the overall mean multiplicity of infection was 3.2 (95% CI 2.87–3.46). The heterozygosity indices were 0.43 and 0.85 for msp-1 and msp-2, respectively. There was no significant association between multiplicity of infection and age or parasite density.Of 80 qPCR-positive samples analysed for polymorphisms on P. falciparum populations in Chewaka district, Ethiopia, suggesting that both endemicity level and malaria transmission remain high and that strengthened control efforts are needed in Ethiopia.The study revealed high levels of genetic diversity and mixed-strain infections of  The intensification of malaria control interventions has resulted in its global decline, but it remains a significant public health burden across several malaria-endemic countries . The 201P. falciparum strains, primarily due to repeated exposure to mosquitoes infected with multiple parasite strains [Genetically-distinct malaria parasites in natural populations have an extremely high rate of genetic recombination during the sexual stages in a mosquito host, often resulting in multiple strains being transmitted simultaneously . This di strains . This ge strains .P. falciparum infections are essential parasite indices that could determine the potential impact on the selection of drug-resistant parasites. Although many polymorphic antigens have been described in several stages of the parasite life cycle, merozoite surface protein 1 and 2 (msp-1 and msp-2) seem to be the most appropriate to distinguish parasite populations [msp-1 and msp-2 typing are widely used in anti-malarial drug efficacy trials to distinguishing recrudescent parasites from new infections [Genetic diversity and multiplicity of ulations –14. Thisfections –17. Studfections  and Meyefections  showed tP. falciparum [He) and MOI. However, this does not occurred in all settings . Hence, accurate assessment of the parasite’s genetic diversity across malaria endemic regions could help plan or develop new control and elimination strategies. The MOI, which identifies the number of clones within a particular infection, can serve as a measure of the level of malaria transmission as well as identify hotspots [P. falciparum infections for informed interventions to be implemented [P. falciparum population structure in Ethiopia could not be assessed due to the lack of genetic data and systematic genetic surveillance study. Chewaka district in Southwest Ethiopia experiences frequent epidemic outbreaks of malaria. Parasite genetic diversity and multiplicity of infection studies have also been found to be important in the surveillance of strains circulating in a particular transmission area especially in Southwest Ethiopia because there was so limited information available on the genetic structures of P. falciparum [msp-1 and msp-2 genes of P. falciparum isolates from uncomplicated malaria patients in Chewaka district, Southwest Ethiopia.Declining malaria transmission as a result of scaling-up interventions has been shown to affect the parasite population genetics pattern and population structure of lciparum –22. The lciparum , 23 and lciparum , 24–29 tsettings , 21. In settings . Hence, hotspots , 32. Malhotspots , and hashotspots –37. Parahotspots –40. The lemented . The efflciparum –44. Thiskebeles (villages). As in most other areas, malaria transmission in Chewaka follows rainy seasons, with transmission peaking in the months between September and December and between April and May. The main malaria control strategy in the district includes long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS) and malaria case management with ACT [The study was conducted in Ilu-Harar Health Centre, Chewka district, Buno Bedele Zone, Southwest Ethiopia during September–December 2017. Chewaka district is located in Buno Bedele zone, Oromia regional state, Ethiopia about 570 kilometres southwest of Addis Ababa. It is situated in lowland areas of Dhidhesa valley, which lies below 1500 m above sea level. The district has 26 administrative with ACT , 6. In 2with ACT .P. falciparum infected blood spots were collected during a therapeutic efficacy study of artemether-lumefantrine (Coartem®), between September and December 2017. The PCR analysis of msp1 and msp2 gene markers showed that three cases were recrudescence and a single case of re-infections was observed in the study. The observed recrudescent parasitaemia was between 14–21 days after treatment start. However, the recrudescent parasitaemia resolved quickly after initiated re-treatment in all cases with the same regimen (unpublished data). The participants were aged between 6 months and 60 years, were residents within Chewaka area, and had presented to the local health centre. Febrile patients with axillary temperatures ≥ 37.5 °C, positive for asexual P. falciparum mono-infection giving written consent were included in the study. Children aged less than 6 month, pregnant women and individuals suffering from any other diseases were excluded.A total of 80 P. falciparum mono-infection, approximately 50 μl of whole blood was spotted onto filter paper (Whatman® 927 mm) and air dried. The blood spots were individually placed into plastic bags with desiccant and transported to the Jimma University Clinical Trial Unit (JU-CTU) and stored at −20 °C for a maximum of 3 months prior to further analysis.After consent was obtained, the blood samples were obtained by finger prick and malaria infection was diagnosed using microscopy and qPCR. Whenever a participant tested positive for asexual TM Genomic DNA mini Kit  according to the manufacturer’s instructions. DNA was checked for purity and quantity using Nanodrop spectrophotometer (ND 1000), and stored at −20 °C until used for PCR amplification and detection.Genomic DNA was extracted from whole blood using proteinase K-base method  according to the manufacturer’s instructions for qPCR species identification and parasite density determination. For nested PCR, the DNA was extracted from stored dried blood spots collected on enrollment (Day-0) and on any day after day 3 were deemed to have recurrent parasitaemia using Pure LinkPlasmodium spp. genomes was analysed for the in vitro quantification of all Plasmodium spp. genomes by targeting the 18S ribosomal RNA (18S) gene according to the protocol of Primer designTM Ltd [Plasmodium falciparum genome was analysed for in vitro quantification of P. falciparum genomes by targeting the plasmepsin 4 gene according to the protocol of PrimerdesignTM Ltd [Primer design genesig  standard Kit for gnTM Ltd . SpeciesgnTM Ltd . Each reP. falciparum isolates was carried out by Nested PCR amplification of the two highly polymorphic regions of msp-1 (block2) and msp-2 (block3) genes as reported previously [msp-1 and two allelic families of msp-2 (FC27 and 3D7) have been reported elsewhere [msp-1 and msp-2 (IC/3D7 and FC27), with an amplification mixture containing 250 nM of each primer, 2 mM of MgCl2 and 125 µM of each dNTPs and 0.4 units Taq DNA polymerase . The cycling conditions in the thermocycler , for initial msp-1 and msp-2 PCR were as follows: 5 min at 95 °C, followed by 25 cycles for 1 min at 94 °C, 2 min at 58 °C and 2 min at 72 °C and final extension of 5 min at 72 °C. For msp-1 and msp-2 nested PCR, conditions were as follows: 5 min at 95 °C, followed by 30 cycles for 1 min at 95 °C, 2 min at 61 °C and 2 min at 72 °C and final extension of 5 min at 72 °C [msp-1 and msp-2 amplified products were then performed through electrophoresis on 2 and 3% ethidium bromide-stained agarose gel, respectively, and after migration, the DNA fragments were visualized by UV trans- illumination. A standard curve is then drawn by measuring the distances traveled (in cm) from the well, of the bands of the size marker, according to the mathematical function: f (x) = y; where f (x) = the actual distance traveled by the band on the gel; y = log10 (bp). The size of an unknown strip is then determined by plotting the distance travelled on the x-axis, then projection on the coordinate axis to determine the size in base pairs. For individual samples, alleles were identified according to band size  was defined as the minimum number of P. falciparum genotypes per infected subject and estimated by dividing the number of amplified PCR fragments reflecting the parasite genotypes by the number of positive samples in the same marker [P. falciparum msp-1or msp-2 genes. Alleles in each family were considered the same if fragment sizes were within 20 bp interval [The e marker . The sizinterval .He) for each antigenic markers was calculated based on the allele frequencies, using the formula: p value < 0.05 was chosen as threshold significance for the various statistical tests. All statistical analyses were performed using SPSS version 20.0 .Spearman’s rank correlation coefficients were calculated to assess association between multiplicity of infection (MOI) and geometric mean parasite density and age. The heterozygosity index (He), which represents the probability of being infected by two parasites with different alleles at a given locus, was calculated by using the Genetic Analysis in Excel toolkit (GenAIEx) . Brieflyfferent) . StudentP. falciparum samples was analysed for msp-1 and msp-2 genes. The estimated frequency of msp-1 and msp-2 gene amplification reactions with family-specific primers was 90% (72/80) and 86.3% (69/80), respectively.Of the 80 patients enrolled 57 (71.2%) were males, mean (± SD) age of participants was 20.96 (± 13.6) years. Participants had asexual parasitaemia ranging from 3699 to 14, 744 parasites/µL with a geometric mean of 12,513 parasites/µl . The parasite DNA from the 80 P. falciparum isolates within the allelic families of msp-1 and msp-2 with a total of 253 distinct fragments detected. The msp-1 gene analysis showed 63, 50, 31 fragments belonged to K1 , MAD20 (34.72%) and RO33 (21.5%) allelic families, respectively. The msp-2 gene analysis showed 58, 51 fragments belonged to FC27  and IC/3D7 (46.8%) allelic families, respectively.Polymorphism analysis was assessed in 80 The proportion of K1, MAD20 and RO33 types were 20.8, 4.2, and 4.2%, respectively. The remaining 70.8% (51/72) were polyclonal infections. Among polyclonal infections carrying two allelic types, the frequency of samples with K1/MAD20, K1/RO33, and MAD20/RO33 was 31.9, 5.6, and 5.6%, respectively. Infections with all three allelic types were detected in 29.2% of cases . Among msp-1 isolates, six K1 (130–300 bp), three MAD20 (180–220 bp) and one RO33 (150 bp) allelic families were noted.Allele genotyping demonstrated the highly polymorphic nature  of msp-2, a total of 19 different alleles were identified  and 1.6 (95% CI 1.46–1.70), respectively, while 51/72(70.9%) and 40/66(58%) of isolates contained multi-clonal infection at least with 2 clones, respectively. The heterozygosity index, which represents the probability of being infected by two parasites with different alleles at a given locus, was 0.43 for msp-1and 0.85 for msp-2 loci. No significant correlation between multiplicity of infection and parasite density of patients  or multiplicity of infection and age . According to age, and parasite density the MOI was similar between individuals of different age and parasite density with-out significant difference  harboured more than one parasite genotype identified by the presence of two or more alleles of one or both genes. with the overall mean MOI i.e., parasite clones per sample was 3.2 (95% CI 2.87–3.46). When considering P. falciparum parasites impacts malaria transmission and malaria control strategies [P. falciparum may hold the key for effective disease surveillance and control programmes, especially in Southwest Ethiopia as so far there is very limited information available on the genetic structures of P. falciparum. As the country moves towards malaria elimination, understanding the genetic diversity and population structure of the malaria parasite populations in hotspots is crucial to guide monitoring and evaluation of malaria control strategies and anti-malarial interventions. The present study provides a detailed assessment of genetic diversity and multiplicity of infection of P. falciparum parasites from Chewaka district, Southwest Ethiopia.The genetic diversity of rategies . Geneticthe msp-1 and msp-2 loci showed considerably diverse and extensive allelic polymorphisms in P. falciparum populations in the analysed samples. However, the number of alleles may have been underestimated due to the limitations of the technique used. Indeed, the numbers of alleles (bands) detected may be underestimated due to sensitivity of the PCR technique used as minor fragments (< 50 bp) cannot be detected on the agarose gel and also similar sized fragments may be classified as identical leading to a false impression of similarity. Within allele families, alleles of the same size may have different amino acids motifs [P. falciparum. A total of 10 and 15 different alleles for msp-1 and msp-2, respectively, were obtained from the parasite isolates in Chewaka district, Ethiopia. This genetic diversity was consistent with the diversity found in Kolla-Shele area, Southwest Ethiopia  in 2015 [msp-1: 12; msp-2: 22) in 2018 [msp-1: 15; msp-2: 20) in 2018 [msp-1: 26; msp-2: 25) was found in Bioko Island, Equatorial Guinea in 2018, even though this area has comparable malaria endemicity patterns [msp-1 as also demonstrated in previous studies in Africa, including Southwest Ethiopia [In this study, allele-specific PCR typing of s motifs , 52, whi in 2015 , in Nort in 2018 , and Bra in 2018 . In contpatterns . K1 was Ethiopia , BrazzavEthiopia  and GaboEthiopia . HoweverEthiopia , CentralEthiopia  and BiokEthiopia  the MAD-msp-2 showed that FC27 was the predominant allelic family as also demonstrated in previous reports from Benin [In this study, the RO33 family showed no polymorphism with only a single allele 160 bp). This is similar to findings in Congo [0 bp. Thiom Benin  and Centom Benin , but in om Benin  and BrazP. falciparum strains co-infecting a single host, has been shown to be a common feature in most malaria-endemic areas and was reported to vary with age, parasite density, immune status, epidemiological settings and transmission intensity [Multiplicity of infection (MOI), i.e. the number of different ntensity –60. In tntensity –44, Brazntensity  in 2011 ntensity . In contntensity  in 2018 ntensity  in 2018.The results of this study show that age has no association on multiplicity of infection similar to other studies , 44, 50,P. falciparum populations that are genetically diverse. Antigenic marker genotyping carried out in African regions like Burkina Faso, Sao Tome, Malawi, Uganda and Tanzania have identified P. falciparum populations with alleles occurring at a frequency below 10 percent with a very high He level of 0.78 to 0.99 [msp-2 (He = 0.85), than for msp-1 (He = 0.43), suggesting a large genotype diversity within the msp-2 locus, which was higher than previously reported from Northwest Ethiopia (msp-2: He 0.62) in 2018 [High transmission regions like those in many African countries are commonly characterized by  to 0.99 . This st in 2018 . Djibout in 2018 .Despite the lack of entomological data from Chewaka district, the number of clones co-infecting a single host can be used as an indicator of the level of malaria transmission or the level of host acquired immunity , 64, 65.®) in the region. In this study, the association between the dominant allelic families and the manifestation of the disease was not examined because all samples were collected from uncomplicated malaria patients. Thus, the relationship between malaria severity or clinical symptom and genetic diversity could not be addressed in the present study. The collection of samples throughout the year (not just in high transmission season) would potentially give a better understanding of the true diversity in the region. Despite these limitations, the data from the present study has confirmed the high genetic diversity profile and mixed-strain infections of P. falciparum populations in Chewaka district, Ethiopia, potentially reflecting both the endemicity level as well as the fact that malaria transmission remains high in Southwest, Ethiopia.The limitation of the present study is the small number of isolates analysed, which were collected during a therapeutic efficacy study of artemether-lumefantrine (CoartemP. falciparum parasites harbouring multiple genotypes and also infections with high MOI study indicate the extensive genetic diversity and complexity of P. falciparum infection in the region. More effort is needed to control malaria transmission and prevent the emergence of resistance alleles in the study area.The high level of polyclonal infections with Additional file 1: Table S1. Primer sequence used for PCRs to screen and genotype samples collected in study of genotyping diversity of P. falciparum parasites in Chewaka district, Ethiopia.Additional file 2: Table S2. Primer sequence used for PCRs to screen and genotype samples collected in study of genotyping diversity of P. falciparum parasites in Chewaka district, Ethiopia.Additional file 3: Figure S3. Prevalence of Plasmodium falciparum msp-1 alleles in clinical isolates from Chewaka district, Ethiopia.Additional file 4: Figure S4. Prevalence of Plasmodium falciparum msp-2 alleles in clinical isolates from Chewaka district, Ethiopia.


This study aimed at detecting PfHRP2 and pLDH malaria antigens in urine and salivary specimens of suspected malaria patients using RDT kits, and identifying factors influencing the detection of these antigens.Malaria rapid test kit (SD Bioline RDT kit) was used to detect malaria antigens, PfHRP2 and pLDH, in blood, urine and saliva samples received from patients suspected of malaria. Subsequently, malaria parasitaemia was determined. From the same patients, body temperature readings and haemoglobin concentrations were recorded. Also, micro-haematuria and saliva occult blood were determined. Relative to blood, the sensitivities and the performance of urine and saliva as alternative samples were evaluated.A total of 706 suspected malaria patients provided all three specimens. Prevalence of malaria by microscopy and RDT was 44.2% and 53.9%, respectively. Compared to blood, the sensitivities of urine and saliva were 35.2% and 57.0% respectively. Haemoglobin concentration < 9.9 g/dL, body temperature > 38.7 °C and occult blood influenced the detection of malaria antigens in both urine and saliva. Furthermore, the antigens were not detected in urine and saliva when parasitaemia was < 60,000 parasites/µL and < 40,000 parasites/µL, respectively.Saliva, with or without blood contamination, was found to be more efficient that urine samples. Therefore these non-blood specimens have the potential to be used as non-invasive samples for malaria diagnosis. However, this approach is useful in severe to moderate anaemia, hyperthermia, parasitaemia > 60,000 parasites/µL and samples contaminated with blood. Plasmodium falciparum account for 90–98% of all malaria cases [Malaria is one of the highest killer diseases predominant in sub-Saharan Africa (SSA). In 2018, about 212 million new infections and 381,000 deaths were reported in SSA [ia cases .Invasive phlebotomy can cause adverse effects, such as pain or bruising at the site of puncture, fainting, nerve damage and haematoma  in patieDue to the potential demerits of invasive phlebotomy, diagnosis of diseases based on non-invasive procedures have been suggested and evaluated in some studies . Urine aPrevious studies in Ghana and in the Philippines detected PfHRP2 antigens in saliva of malaria patients using enzyme immuno-assays , 13. UsiP. falciparum specific antigens in urine and salivary specimens from the same patients using readily available RDT kits and identify factors influencing the detection of these antigens in urine and saliva of P. falciparum infected individuals.Even though previous studies identified PfHRP2 and pLDH in salivary and urine samples, detection of these proteins were done mostly with sensitive diagnostic techniques (enzyme immuno-assays and fluorescent immuno-assays), which are only available in reference and specialized laboratories. Malaria is mostly endemic in rural and peri-urban areas where there is lack of technical expertise to perform complex analytical assays and cost involved in establishing these assays is always high. Therefore, this study was designed to detect PfHRP2 and pLDH Blood, urine and saliva specimens were collected from clinically suspected malaria patients from Oduman Health Centre, Kotoku Health Centre, Mayera Health Centre and Pokuase Health Centre. These health centres are located in rural communities in the Ga West Municipality in the Greater Accra Region of Ghana. Samples were collected from October 2018 to May 2019.Clinically suspected malaria patients included in this study were at least 5 years old. Patients less than 5 years and those unable to provide urine and saliva were excluded from the study.2p(1 − p)/d2, where n = sample size, p = prevalent of malaria in Ga West Municipality, z = confidence level at 95% , d = margin of error at 5% . The prevalence of malaria in Ga West Municipality was unknown so it was estimated at 50.0%. Therefore, the minimum sample size was calculated to be 384.Sample size was determined using the formula: n = z3EDTA tubes , 30 mL of urine and saliva (approx. 5 mL) were collected from the same patient. Due to difficulties in obtaining early morning urine, samples were collected between 6 a.m. and 10 a.m. Urine and salivary samples were frozen at − 20 °C till they were transported to Ga North Municipal Hospital Laboratory, Ofankor-Accra for analysis. However, whole blood was stored between 2 and 8 °C prior to analysis. Sample storage ranged between 15 and 48 h.Blood samples (approx. 4 mL) were collected by venipuncture into KMalaria parasites were quantified with the aid of Giemsa staining (pH = 6.8). Exactly, 6 µL of whole blood was used to prepare thick smears measuring approx. 2 cm in diameter. Air dried thick films were stained with 10% Giemsa for 10 min and asexual stages of parasites estimated using the WHO protocol for malaria parasites estimation . In brieMalaria parasites antigenaemia (PfHRP2 and pLDH) were detected by SD Bioline rapid diagnostic test kit  following manufacturer’s recommendation. In summary, 5 µL of well-mixed whole blood was dispensed into the sample window of the kit followed by four drops of buffer. Test results were read at 15 min.PfHRP2 and pLDH antigens were detected in saliva and urine samples using SD Bioline rapid diagnostic test kit with some modifications. Optimized sample volumes were 20 µL of saliva and 30 µL of urine specimens. About 5 s after addition of samples into the sample window of the RDT kit, four drops of buffer were added. Test results were read at 15 min.Blood in saliva and urine specimens were detected using the blood determinant portion of Uritest 10E urine reagent strip  following the manufacturer’s procedure. Blood in the specimens were determined based on the pseudoperoxidase action of hemoglobin and erythrocytes which catalyzes the reaction 3,3′,5,5′-tetramethyl-benzidine and buffered organic peroxide. The blood determinant portion of the urine reagent strip was immersed in the sample  for about 10 s. Results were read at 45 s. The resulting colors ranged from yellow-green to dark green depending on the amount of blood present in the sample. The test kit had sensitivity of 1 intact red blood cells/10 mL or 0.3–0.6 mg/L of haemoglobin.Haemoglobin concentrations were determined using Urit 5200  fully automated haematology analyzer. Well-mixed whole blood was aspirated by the analyzer and haemoglobin concentrations were determined automatically using cyanide free colorimetric method. Haemoglobin concentrations in g/dL were generated within a minute.Laboratory data were processed by Microsoft Excel 2016. The sensitivities, specificities, positive and negative predictive values were determined based on the following formula and their respective 95% confidence intervals as well as the Chi-square goodness of fit of each technique were determined by SPSS Version 24 .where PPV and NPV are positive and negative predictive values respectively.Accuracy was calculated based on the formula: Sensitivity × Prevalence + Specificity × .A total of 864 suspected malaria patients were recruited for this study. Of this number 706 81.7%) patients were able to provide all three samples  for analysis. Table .7% patieP. falciparum parasitaemia in Ga West Municipality by microscopy was 44.2% (312/706) while PfHRP2/pLDH antigenaemia was 53.9% (381/706). Highest malaria parasitaemia and antigenaemia was seen in Mayera Health Centre while relatively lower cases were seen in Pokuase Health Centre. Of the total malaria RDTs done on blood samples, 95% (362/381) were PfHRP2 while 5.0% (19/381) were both PfHRP2 and pLDH. Of the total of 706 urine samples tested, malaria antigens were detected in 134 samples (18.9%). Of the 134 positive urine samples, both PfHRP2 and pLDH antigens were detected in 15 (11.2%) samples while the rest contained only PfHRP2 antigens. In saliva samples, 217 out of 706 samples (30.7%) were found to contain malaria antigens. Both PfHRP2 and pLDH were detected in 92.2% (200/706) of saliva specimens while only PfHRP2 was detected in 7.8% (17/706) of saliva specimens. The distribution of the results in each study site is presented in Table The overall prevalence of In all, RDT detected PfHRP2/pLDH antigens in 132 (34.6%) patients with haemoglobin concentration less than 9.9 g/dL whilst in patients with haemoglobin concentration 10.0–12.5 g/dL and > 12.5 g/dL, PfHRP2/pLDH antigens were detected in 170 (44.6%) and 79 (20.7%) blood specimens. Over half of the salivary specimens were found to contain occult blood (52.2%), while only 29.4% of the urine samples were micro-haematuric. The overall mean temperature of the patients was 38.6 ± 0.72 °C. The distribution of the clinical findings among the four study sites are presented in Table Table In urine samples without blood contamination, 22 samples were found to contain malaria antigens as well as their corresponding blood samples. As many as 325 urine and corresponding blood samples were found not to contain any of the malaria antigens. However, in 269 samples, no malaria antigens were detected while in their corresponding blood specimens, malaria antigens were detected. Based on the foregoing, without blood contamination, the sensitivity of urine samples to detect malaria antigens dropped to 7.6% while the sample remain very specific, with an accuracy of 50.2%. In salivary samples, using the figures presented in Table In the 132 patients with haemoglobin concentrations less than 9.9 g/dL, malaria antigenuria was detected in 113 85.6%) samples while 123 (93.1%) salivary samples were found to contain malaria antigens. As haemoglobin concentration increases, the number of urine and salivary samples found to contain malaria antigens drastically reduces. Rate of detection of malaria antigenuria  and salivary malaria antigen  increased with increasing malaria parasitaemia. It is noteworthy that malaria antigenuria was not detected in patients with parasite count less than 60,000 parasites/µL of blood. But in the case of salivary specimens, parasitaemia was detected when parasitaemia was above 40,000 parasites/µL  of them whereas 28 out of 182 salivary samples (15.4%) without occult blood were found to contain malaria antigens. However, malaria antigens were detected urine of all malaria patients exhibiting haematuria while in the 269 samples without haematuria, 22 (8.2%) were found to contain malaria antigens. It was also observed that as body temperature increased the rate of detection of PfHRP2/pLDH antigens in urine and salivary specimens also increased  [P. falciparum HRP2 antigens were detected in saliva of malaria patients using ELISA technique, and even though the sensitivity was low (43%) with comparatively long turnaround time (approx. 2 h and 15 min) [In spite of these limitations, higher detection rate of va (73%) , 25. Agava (73%)  while in 15 min) , this re 15 min) , 27.Plasmodium antigens in the glomeruli [Again, in this study, urine samples of 35.2% of acute malaria patients were found to contain PfHRP2 and pLDH proteins. It has been previously reported that kidney involvement is not uncommon in falciparum and malariae malaria . Haemodylomeruli . Proteinlomeruli  made it lomeruli  whilst tlomeruli , and pLDlomeruli . Thus, tP. falciparum infected patients, over 85% of salivary and urinary PfHRP2 and pLDH were identified in haemoglobin concentration less than 9.9 g/dL, body temperature greater than 38.1 °C, occult blood in saliva and urine and parasitaemia of > 60,000 parasites/µL of blood.In Plasmodium falciparum is an obligatory intraerythrocytic parasite so destruction of red cells containing parasites and uninfected red cells resulting in anaemia is very common [P. malariae infections [y common . In sevey common  in urinefections . Based ofections , so onceIt has been reported that glomerular capillary dilatation, haemorrhage into the interstitium, in small and large renal vessels occur during pathogenesis of fever , also acFinally, even though detection of malaria antigens improved with increased parasitaemia, Gbotosho et al.  found otP. falciparum specific antigens in urine and saliva. Sensitivity of detecting malaria antigens in these specimens improves in severe to moderate anaemia, hyperthermia, parasitaemia > 60,000 parasites/µL of blood and occult blood in urine and saliva. Available RDT kits were validated for blood samples where malaria antigens concentrations are very high. It is recommended that RDT kits be developed for other body fluids where lower amount of malaria antigens could be present and may degrade faster.The overall prevalence of malaria detectable by microscopy and RDT in Ga West Municipality was 44.2% and 53.9% respectively. Of the total patients found to be positive by RDT, 35.2% and 57.0% had malaria antigens detected in their urine and saliva respectively; confirming the possibility of detecting Study design did not exclude individuals with other co-morbidities that could enhance or reduce the detecting of malaria antigens in urine and saliva. Also, determination of PfHRP2 antigen levels in these alternative samples was not done.