Plasmodium infection among children is a serious public health problem. Asymptomatic malaria infection among humans serves as a significant reservoir for transmitting Plasmodium to uninfected Anopheles mosquitoes, fueling malaria endemicity and asymptomatic malaria may progress to clinical malaria. Therefore, prompt and accurate diagnosis of malaria infection is crucial for the management and control of malaria, especially in endemic areas. This study assessed the point prevalence of asymptomatic malaria infection and evaluated the performance of malaria Rapid Diagnostic Tests (RDT), light microscopy and nested PCR (nPCR) for the diagnosis of asymptomatic malaria infection in a paediatric population in the Atwima Nwabiagya North district, Ghana.Plasmodium infection based on histidine rich protein-2 (pfHRP-2)-based malaria RDT, light microscopy and nPCR.This cross-sectional study enrolled 500 asymptomatic children aged ≤ 5 years. After consent was obtained from a parent, blood samples were collected from each participant to assess for P. falciparum infection. Likewise, microscopy presented with an excellent specificity and high accuracy in detecting both P. falciparum  and P. malariae . However, the sensitivity (56.4%) and reliability (56.4%) of microscopy was low for both P. falciparum.The point prevalence of asymptomatic malaria by microscopy, RDT, and nPCR were 116/500 (23.2%), 156/500 (31.2%), and 184/500 (36.8%), respectively. Using nPCR as the reference, RDT presented with a perfect sensitivity (100.0%), specificity (100.0%), accuracy (100.0%), and reliability (100.0%) in detecting asymptomatic Plasmodium infection among children in Atwima Nwabiagya North district, Ghana. In the absence of the more sensitive PCR, pfHRP-2-based malaria RDT provides substantial diagnostic sensitivity, specificity, accuracy and reliability and is superior to microscopy.The findings of this study indicate a high point prevalence of asymptomatic  Malaria is a pervasive parasitic disease in the tropical and subtropical regions which is mostly prevalent in sub-Saharan Africa, Asia, and Latin America . CurrentClinically, the diagnosis of malaria is often based on signs and symptoms alone. However, due to overlapping symptoms between malaria and other infectious conditions, a malaria diagnosis based solely on signs and symptoms may be inaccurate leading to improper use of anti-malarial medication or the delay in proper diagnosis and treatment of an alternative condition . As a reIn many developing countries, microscopic examination of Giemsa-stained blood smears is considered the “gold standard” for malaria diagnosis and a mandatory test prior to antimalarial therapy. Though it is cost-effective, malaria microscopy is limited by several factors including quality control, limited availability of microscopes, time consuming for optimal film preparation, examination, and interpretation, diagnostic biases as a result of its dependence on operator’s experience and low diagnostic sensitivity –14. FurtPlasmodium falciparum Histidine-Rich Protein-2 (pfHRP-2), parasite-specific Lactate Dehydrogenase (pLDH) or Plasmodium aldolase to detect parasitemia  and P. malariae  were identified in this study. There were no mixed infections (Table 2).A total of 500 children with mean age and haemoglobin level of 2.21 years and 10.26 g/dl, respectively, were included in this study. A higher proportion of the participants were male (51.2%), between 1–2 years of age (47.2%), and resided in the community of Barekuma (28.4%). A total of 189 (75.6%) were anaemic and the point prevalence of mild, moderate, and severe anaemia was 55.2%, 16.8%, and 3.6%, respectively. Only Fig 2).The overall point prevalence of asymptomatic malaria by microscopy, RDT and nPCR were 116/500 (23.2%), 156/500 (31.2%), and 184/500 (36.8%), respectively (Table 3).The point prevalence of asymptomatic malaria was 98 (19.6%) for males and 86 (17.2%) for females based on nPCR. Using microscopy and RDT, the point prevalence among males was 66 (13.2%) vs 86 (17.2%), respectively, and 50 (10.0%) vs 70 (14.0%) among females, respectively. Upon stratification by age, the point prevalence of asymptomatic malaria was highest in children between 1–2 years of age based on nPCR (16.4%), microscopy (11.2%), and RDT (15.6%), respectively. A similar observation was made after age groups were stratified by gender. Children residing in the community of Worapong (13.2%) presented with the highest point prevalence of asymptomatic malaria, followed by Abira (8.0%), Barekuma and Adankwame (4.4%), and Barekese (4.0%), with the lowest being Esaaso (2.8%). Children in Abira , Barekese , and Worapong  had significantly higher odds of asymptomatic malaria compared to Barekuma. A similar observation was made when using microscopy and RDT. In addition, children experiencing any anaemia had an increased odds of asymptomatic malaria .Nested PCR detected 156 P. falciparum infection. Microscopy presented with a similar performance with respect to P. malariae infection. However, the sensitivity (56.4%), accuracy (85.6%) and reliability (56.4%) of microscopy was attenuated for detecting P. falciparum (Table 5).RDT presented with a perfect sensitivity (100.0%) specificity (100.0%), accuracy (100.0%), and reliability (100.0%) in detecting Plasmodium infection among a paediatric population in the Atwima Nwabiagya North district of Ghana. Asymptomatic malaria in children under 5 years has been reported in some African countries Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The submission by Dickerson and colleagues describes a comparison between light microscopy, rapid diagnostic tests and nPCR (as reference) to assess malaria asymptomatic infection in children less than 5 years. The methodology globally sounds good and is clearly reproductible. Their results are in line with relative literature.The paper is clearly written and the discussion is thoughtful as they point out limits of their work.This paper should, as the authors emphasize, galvanize far more intensive surveillance for these asymptomatic bearers.My minor comments are detailed below.Line 65: there is no reference between bracketsLines 120 -121: Have you collected any information about LLIN use?Lines 160-161: the sentence ‘Only P. 161 falciparum and P. malariae were identified in this study. There were no mixed infections by nPCR’ should be moved to Results section.One question: The PCR you used cannot diagnose P. vivax. As you consider this PCR as the reference, I think that there is a little bias. Could you explain this choice ?Reviewer #2: General comments;This manuscript addresses an important area which is highly relevant to malaria control in areas which are transitioning from holo/hyper-endemic to hypo-endemic transmission of malaria. However, the manuscript needs minor revision to better present the key questions addressed, the findings, and clearly show the novelty.1. Title: Well written but it can be rephrased to give a clear message. The authors were assessing the prevalence of asymptomatic P. falciparum infection in paediatric population in Ghana, but also compared the diagnostic tools, so they should rephrase these two sentences in order to capture reader's attention just from the title.2. Background: The concept of malaria prevalence in endemic areas and in the country where the study was conducted needs to be thoroughly explained. The authors should clearly indicate the relevance of conducting this study. A number of this kind of studies have been conducted in Ghana, I suggest that, the authors should clearly show that the study was conducted in (Atwima Nwabiagya) Northern district in Ghana, not just Ghana in general.3. Results: The authors need to check their results the way they have reported them. What I know and from several reports, asymptomatic malaria occurs in children above 5 years of age and not less than 5 because of immunity. But their results indicate children of 1-2 years appear to be asymptomatic compared to other age groups. They need to justify this.- Figures are also missing in the document that I review.- Parasite density is also important to be shown- There should be parasite density of the results obtained by microscopy. This will add value and will be a relevant information as well.4. Discussion: this section is poorly written. There are some facts that need to be considered in order to reflect the rationale of the study and the findings. Authors should handle all issues raised and re-write this section accordingly.5. Study strength and limitations: This section is missing. They should have clearly pointed this out.6. Other minor commentsThe authors should check the language throughout the manuscript and correct typos. I have done my comments in the manuscript in track changes.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentManuscript TDickerson Malaria RDT Ghana-Reviewed DS.docxSubmitted filename: Click here for additional data file. 14 Apr 2020The Editor,PLOS ONEDear Sir/ Madam,RESPONSE TO REVIEWERSThe authors appreciate the timely and scrupulous review of our manuscript (PONE-D-20-02713). The authors note seventeen (17) comments in total. Kindly find below the responses to the reviewers’ comments. Tracked changes have been employed to highlight manuscript texts revised per the reviewers’ recommendations. Revised text indicated in this response are in “quotation marks”.Reviewer #1: Line 65: there is no reference between bracketsResponse 4: Reference #4 has been added to the appropriate textLines 120 -121: Have you collected any information about LLIN use?Response: Thank you for your comment. Because objective of the study is to determine the prevalence of asymptomatic malaria infection and evaluate the performance of RDT, microscopy and PCR for malaria diagnosis, we did not collect other data such as use of LLIN. However, we have included it as a limitation of the study (lines 279-282).“This study is however limited the fact that we used only a single brand of malaria RDT; the prevalence may not be the same when other commercially available test kits are used. Also, data on the use of long-lasting insecticidal nets was unavailable. Additionally, the study was conducted in a peri-urban setting and the findings may not be generalizable to other areas. Thus, we recommend that further studies be conducted in the larger population.”Lines 160-161: the sentence ‘Only P. 161 falciparum and P. malariae were identified in this study. There were no mixed infections by nPCR’ should be moved to Results section.Response: Thank you for your comment. The statement has been moved to the results section (lines 178-179)“Only P. falciparum  and P. malariae  were identified in this study. There were no mixed infections”One question: The PCR you used cannot diagnose P. vivax. As you consider this PCR as the reference, I think that there is a little bias. Could you explain this choice?https://www.who.int/malaria/publications/country-profiles/profile_gha_en.pdf?ua=1/ and https://www.severemalaria.org/countries/ghana and http://www.ghanahealthservice.org/downloads/GHS_Antimalaria_drug_policy.pdf ). We did not include P. vivax because reports of its infection in the Ghana is rare.Response: Thank you for your comment. The PCR primers were selected based on the occurrence/prevalence of the different species of Plasmodium in Ghana. It is known that there are currently five major clinically relevant Plasmodium species, namely, P. falciparum, P. ovale, P. malariae, P. vivax and P. knowlesi. However, only P. falciparum, P. ovale, P. malariae have been documented to occur in Ghana .“Prevalence of asymptomatic Plasmodium infection and the comparison of microscopy, rapid diagnostic test and nested PCR for the diagnosis of asymptomatic malaria among children under 5 years in Ghana”2. Background: The concept of malaria prevalence in endemic areas and in the country where the study was conducted needs to be thoroughly explained. The authors should clearly indicate the relevance of conducting this study. A number of this kind of studies have been conducted in Ghana, I suggest that, the authors should clearly show that the study was conducted in (Atwima Nwabiagya) Northern district in Ghana, not just Ghana in general.Response: Thank you for your comment. The relevance of the study has been clarified and (Atwima Nwabiagya) Northern district as the study site has been highlighted (lines 96-101)“Although a number of studies on asymptomatic malaria in older children have been conducted across Ghana [30-32] and children under 5 in neighboring African countries [33-35], there remains a dearth of published data on asymptomatic malaria in children under 5 years in Ghana, particular in the northern sectors of the country where adequate health facilities are wanting. This study assessed the prevalence of asymptomatic malaria infection and evaluated the performance of malaria RDT, light microscopy and nested PCR (nPCR) for the diagnosis of asymptomatic malaria infection in children under 5 years old in Atwima Nwabiagya North district, Ghana.3. Results: The authors need to check their results the way they have reported them. What I know and from several reports, asymptomatic malaria occurs in children above 5 years of age and not less than 5 because of immunity. But their results indicate children of 1-2 years appear to be asymptomatic compared to other age groups. They need to justify this.- Figures are also missing in the document that I review.- Parasite density is also important to be shown- There should be parasite density of the results obtained by microscopy. This will add value and will be a relevant information as well.Response: Thank you very much for your comment. The data has been rechecked and the results remained the same. In this study, simple random sampling procedure was used to recruit asymptomatic children. The higher prevalence of asymptomatic malaria among 1-2 years old children could be due to the greater number/size of 1-2 year aged children obtained in this study. This could also highlight the distribution of asymptomatic malaria cases in children under 5 in the region. The figures were included during the submission. Parasite densities have been included (table 1 and lines 178-179).“Only P. falciparum  and P. malariae  were identified in this study. There were no mixed infections”4. Discussion: this section is poorly written. There are some facts that need to be considered in order to reflect the rationale of the study and the findings. Authors should handle all issues raised and re-write this section accordingly.Response: Thank you for your comment. The discussion section has been revised, taking into consideration all comments attached (line 216-227)“Based on PCR, this study reports a high prevalence (36.8%) of asymptomatic Plasmodium infection among a paediatric population in the Atwima Nwabiagya North district of Ghana. Asymptomatic malaria in children under 5 years has been reported in some African countries [33-35]. In Ghana, Crookston, et al. [46] reported an asymptomatic malaria prevalence of 31.8% based on PCR among children less than five years of age in Kumasi, Ghana which is similar to our study finding. Other studies in Ghana such as those by Dinko et al. [31] in Kumasi and Danquah et al. [47] in northern Ghana reported asymptomatic malaria among older children. The prevalence found in the studies by Dinko et al. (76.6%) and Danquah et al. (89.7%) are higher compared to this study and the discrepancy may be linked to the fact that older children tend to be the major asymptomatic carriers of Plasmodium compared to younger children , possibly as a result of protective immunity acquired over the years.We also found asymptomatic malaria to be more prevalent among male children compared to females, similar to the findings of Golassa et al. in Ethiopia [29]. The higher prevalence of asymptomatic malaria among male children compared to females could be explained by the fact that, in Ghana, males are more exposed to both daytime and nighttime outdoor activities than females and thus, at higher risk of mosquito bites compared to females who are usually indoors …”5. Study strength and limitations: This section is missing. They should have clearly pointed this out.Response: Thank you for your comment. We have included a section for study strength and limitations (line 273-282)“Study strengths and limitationsThe strength of the study is in the reporting of the prevalence of asymptomatic malaria infection among children less than 5 years old in the northern sector of Ghana. We also highlight potential hotspots for malaria screening and treatment during national and regional malaria surveillance exercises. The study also corroborates previous reports on the usefulness of molecular detection methods for asymptomatic malaria diagnosis. We showed that, in the absence of PCR, RDT performs better in the diagnosis of asymptomatic malaria among children compared to microscopy.This study is however limited the fact that we used only a single brand of malaria RDT; the prevalence may not be the same when other commercially available test kits are used. Also, data on the use of long-lasting insecticidal nets was unavailable. Additionally, the study was conducted in a peri-urban setting and the findings may not be generalizable to other areas. Thus, we recommend that further studies be conducted in the larger population.”6. Other minor commentsThe authors should check the language throughout the manuscript and correct typos. I have done my comments in the manuscript in track changes.Response: Thank you very much for your comment. The language and typos have been reviewed and revised and all comments included as tracked changes have been addressed.Miscellaneous Author Responses: • The 2010 Population and Housing Census of Ghana is the latest published data. A newer census is scheduled to take place in 2020.• The authors initially chose to use the term point prevalence for malaria as our study reports the prevalence of malaria cases among individual asymptomatic children at a single point in time. Whereas point prevalence addresses the question of whether a person currently has malaria, period prevalence addresses the question of whether that subject had malaria at any time during the period under investigation. Since the study did not test individual children for malaria at multiple times over a defined period of time, we are unable to report the period prevalence for asymptomatic malaria infection. Thank you once again for the timely review and scrupulous of our manuscript. Looking forward to hear favorably from you.Sincerely,Ty DickersonCorresponding authorAttachmentResponse to Reviewers 28March2020.docxSubmitted filename: Click here for additional data file. 24 Apr 2020Point prevalence of asymptomatic Plasmodium infection and the comparison of microscopy, rapid diagnostic test and nested PCR for the diagnosis of asymptomatic malaria among children under 5 years in GhanaPONE-D-20-02713R1Dear Dr.  DICKERSON,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: We are thankful to the authors and approve this updated version of the manuscript. The authors replied to all of our comments.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: Yes: Dieudonné M. Mvumbi 8 Jul 2020PONE-D-20-02713R1 Point prevalence of asymptomatic Plasmodium infection and the comparison of microscopy, rapid diagnostic test and nested PCR for the diagnosis of asymptomatic malaria among children under 5 years in Ghana Dear Dr. DICKERSON:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! 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In the past decade, national malaria control efforts in Papua New Guinea (PNG) have received renewed support, facilitating nationwide distribution of free long-lasting insecticidal nets (LLINs), as well as improvements in access to parasite-confirmed diagnosis and effective artemisinin-combination therapy in 2011–2012.Plasmodium falciparum and Plasmodium vivax infections and investigate risk factors at the individual and household level, two cross-sectional surveys were conducted in the East Sepik Province of PNG; one in 2005, before the scale-up of national campaigns and one in late 2012-early 2013, after 2 rounds of LLIN distribution (2008 and 2011–2012). Differences between studies were investigated using Chi square (χ2), Fischer’s exact tests and Student’s t-test. Multivariable logistic regression models were built to investigate factors associated with infection at the individual and household level.To study the effects of these intensified control efforts on the epidemiology and transmission of P. falciparum and P. vivax in surveyed communities decreased from 55% (2005) to 9% (2013) and 36% to 6%, respectively. The mean multiplicity of infection (MOI) decreased from 1.8 to 1.6 for P. falciparum (p = 0.08) and from 2.2 to 1.4 for P. vivax (p < 0.001). Alongside these reductions, a shift towards a more uniform distribution of infections and illness across age groups was observed but there was greater heterogeneity across the study area and within the study villages. Microscopy positive infections and clinical cases in the household were associated with high rate infection households (> 50% of household members with Plasmodium infection).The prevalence of P. falciparum and P. vivax infection rates in the studies villages in East Sepik Province. Understanding the extent of local heterogeneity in malaria transmission and the driving factors is critical to identify and implement targeted control strategies to ensure the ongoing success of malaria control in PNG and inform the development of tools required to achieve elimination. In household-based interventions, diagnostics with a sensitivity similar to (expert) microscopy could be used to identify and target high rate households.After the scale-up of malaria control interventions in PNG between 2008 and 2012, there was a substantial reduction in  Papua New Guinea (PNG) has the highest transmission of malaria in the Western Pacific Region, where it accounted for 80% of the reported confirmed cases in 2018 [Anopheles punctulatus group of mosquitoes, of which An. punctulatus, Anopheles koliensis, Anopheles farauti, and Anopheles farauti 4 have been incriminated as major vectors [An. farauti tend to bite mostly during the early hours of the evening, and in the case of An. punctulatus and An. koliensis, peak activity takes place in the early hours of the morning [East Sepik Province (ESP) has historically been an area of PNG with a high malaria burden, with a peak prevalence of 70% (by light microscopy (LM)) in children 5–9 years of age in the early 1990′s , whilst  vectors –14. The  morning .Plasmodium falciparum and Plasmodium vivax are the most abundant species in PNG, though Plasmodium malariae and Plasmodium ovale are also present, usually in combination with the former two species [Plasmodium vivax was the predominant species prior to the introduction of mass spraying and drug administration programmes in the 1950’s, which were intended to eliminate malaria [P. falciparum followed, which remains the dominant species today [ species . Plasmod malaria , 16, 17,es today , 17. Howes today –22. Thises today , 21, 23.Plasmodium species is important to consider in co-endemic regions such as PNG, given the distinct biological differences of the two main Plasmodium species. While declining prevalence and morbidity have been documented on a regional level, the effects of decreasing malaria transmission on a smaller geographical scale remain to be investigated. Heterogeneities in transmission and disease burden have been described at various scales in PNG [P. vivax can be resilient [While the previously reported reduction in nationwide prevalence and incidence of malaria and all-cause mortality rates in young children are significant achievements , 21–24, s in PNG  and can s in PNG , and espesilient .To investigate the impact of the renewed malaria control efforts on the epidemiology of malaria within communities, data from two cross-sectional community surveys conducted in the Maprik and Wosera-Gawi districts of East Sepik Province, PNG; one in April–May 2005, before the implementation of national LLIN distribution campaign and one in October 2012–February 2013, were directly compared. Risk factors for parasite prevalence, and spatial heterogeneity were investigated in addition to gametocytaemia and complexity/multiplicity of infection. Understanding the key factors related to heterogeneity and residual malaria transmission in PNG is critical to support the desired transition from nation-wide malaria control to sub-national elimination strategies.2 with low hills, plains and riverine plains with a wet tropical climate [Two cross-sectional community surveys were conducted in the Maprik and Wosera-Gawi districts in the East Sepik Province of PNG; one in April–May 2005 , before  climate , 9. The All eligible and willing residents of households in the local communities in the study area were invited to participate and following written informed consent, demographic information , history of febrile illness, household or village location by GPS receiver and blood samples were collected. The design of both studies was similar, and details of the 2005 study have been previously described . All detCapillary blood (250–300 μL) was collected into K + EDTA microtainers, thick/thin films were prepared and haemoglobin levels were measured (Hemocue). The collected blood was centrifuged, the plasma removed and stored at − 80 °C, and the red cell pellet stored at -20 °C until DNA extraction. If febrile illness was reported, a rapid malaria diagnostic test (Carestart Pf/Pan) was performed and those positive by RDT treated with artemether-lumefantrine (Coartem). For RNA preservation, 50 μL of whole blood was immediately transferred to a tube containing 250μL of RNAProtect (Qiagen) and stored at -80 °C within 8 h of collection until RNA extraction.Giemsa-stained thick and thin films from the 2005 survey were examined by LM (minimum of 200-high powered fields) by two independent experienced microscopists, with discrepancies adjudicated by a third independent microscopist, as previously described . All sliP. falciparum, P. vivax, P. malariae and P. ovale [The presence of parasite DNA in all blood samples was investigated by molecular methods . In 2005 DNA was extracted from red blood cell pellets using QIAmp 96 DNA Blood kits (Qiagen) and a post-PCR, ligase detection reaction/microsphere assay (LDR-FMA) was used to determine the presence of P. ovale . In the P. ovale . SpeciesP. ovale . The qPCP. ovale . Copy nuP. falciparum (2005 and 2012/13) and/or P. vivax (2012/13 only) gametocytes were recorded separately from asexual stages during microscopic examination of all blood slides. Gametocyte detection by qRT-PCR was performed on samples from 2012/13: RNA was extracted from all P. falciparum and/or P. vivax qPCR-positive samples using the Qiagen RNeasy plus 96 kit, according to the manufacturer’s procedures. A genomic DNA removal by gDNA eliminator columns and DNase (Qiagen) step was included in the procedure. Absence of gDNA was confirmed by qPCR and presence of parasite RNA after extraction was verified by qRT-PCR with the same primers and probe as the QMAL qPCR described above. P. falciparum and P. vivax gametocytes were detected by qRT-PCR of the highly expressed gametocyte markers pfs25 and pvs25 as previously described [pfs25 and pvs25 qRT-PCRs in the laboratory set up in PNG, were 6 copies/transcript per reaction for both assays.The number of observed escribed . The limP. falciparum and P. vivax LDR-FMA/qPCR positive samples, high-resolution Pfmsp2, Pvmsp1f3 and PvMS2 genotyping was performed as previously described [P. falciparum a nested multiplex PCR approach was used to amplify 3D7 and/or FC27 family alleles of Pfmsp2 using family-specific primers labelled with a fluorescent dye, or a multiplex primary PCR was used to amplify the markers Pvmsp1F3 and PvMS2 with fluorescently labelled primers. The PCR products were analysed by 1.5% agarose gel electrophoresis and the number and size of alleles were then determined by capillary electrophoresis using a 23 ABI 3730XLs platform  with the internal size standard GSLIZ500 and data were subsequently analysed using GeneMarker 2.4.0 demo version (SoftGenetics). The multiplicity of infection (MOI) for P. falciparum was defined as the number of Pfmsp2 alleles counted within a sample, and P. vivax MOI was defined by the number of Pvmsp1f3 or PvMS2 alleles, whichever was higher.To genotype escribed , 34. BriP. vivax or P. falciparum asexual parasites by LM (any density). Symptomatic infections were defined as febrile illness (current or previous 48 h) in the presence of parasites as detected by LM and/or MM. Parasite densities were log transformed and the geometric mean per μL whole blood are reported. Four categories were used to describe anaemia: non-anaemia, mild, moderate or severe, which were defined based on haemoglobin concentrations (measured by Hemocue) and stratified by age and gender as per the WHO recommendations .Raw data from the 2005 survey  was rean2) and Fischer’s exact tests for categorical characteristics and Student’s t-test for normally-distributed continuous variables. Tests were two-tailed and the confidence level was set at 95%. Univariable analysis of factors associated with P. falciparum or P. vivax infection (determined by MM and/or LM) and high rate household were conducted using logistic regression. Multivariable logistic regression models were built with variables with p < 0.15 in univariate analysis and variables were selected using likelihood methods for models with minimal Akaike information criterion (AIC). Analyses were performed using Stata 12. Genetic diversity analyses were done using FSTAT software version 2.9.3.2 to define allele frequencies, and the expected heterozygosity (He). Household, village and health facility location data were collected using a handheld GPS receiver (Garmin GPSmap62sc). Maps were constructed with collected GPS coordinates using ARC GIS Pro 10.4Differences between the two studies were investigated using Chi square  were excluded because of missing demographic or LM data, while insufficient finger-prick blood sample for LDR-FMA analysis led to exclusion of an additional 96 (3.5%) individuals. Overall 2527 participants from 659 households with complete demographic and infection status data were available for comparison with 2012/13 data. A total of 2500 participants in 14 villages from the same areas participated in the 2012/13 cross-sectional survey. Of these, 14 (0.6%) were excluded from the analysis because of missing LM results. Overall, 2486 participants from 704 households with complete demographic and infection status data were available.P. falciparum and/or P. vivax) had dropped from 3.6% to 2.3% (p = 0.007). There was a lower prevalence of anaemia in 2012/13, and accordingly, mean haemoglobin levels were significantly higher in 2012/13 (11.1 ± 1.8 g/dL) compared to 2005  , followed by P. vivax . The prevalence of both species dropped with similar proportions; in other words, for both species the prevalence in 2012/2013 was roughly 16% of the prevalence in 2005  in 2012/13 compared to 2005 while the proportion of P. ovale by MM remained similar. The majority of mixed infections in both years consisted of P. falciparum - P. vivax co-infections, followed by P. falciparum with P. malariae and/or P. ovale, as determined with MM. The proportion of P. falciparum infections that were sub-microscopic decreased significantly from 2005 (47.2%) to 2012/13 , but not for P. vivax . The geometric mean density of parasites by microscopy was similar in both years. There was a significant increase in the proportion of malaria infections with any species that were symptomatic, increasing from 7.0% in 2005 to 25.3% in 2012/13 , as there was a higher rate of people reporting febrile illness in 2012–2013   by that method was significantly higher than with LM . Plasmodium vivax gametocytes detected by LM were not recorded in 2005. In 2012/13, the prevalence of P. vivax gametocytes was 0.8% by LM and 0.9% by qRT-PCR , there was no difference in 2012–2013. In 2012–2013, current or recent fever was a predictor of P. falciparum infection . In contrast, participants with recent/current febrile illness were more likely to be P. vivax infected in 2005, but not in 2012/13   compared to P. falciparum . Similarly, P. vivax infections were successfully genotyped by at least one of two markers Pvmsp1f3 and PvMS2 (63.5% in 2005 and 64.7% 2012/13). The total number of different alleles detected by the markers was lower in 2012/13 than in 2005, but the most common alleles were similar between the 2 years  and from 2.2 to 1.4 for P. vivax . However, the proportion of P. falciparum multiple clone infections (MOI > 1) remained constant , while the proportion of P. vivax multiple clone infections decreased from 58.2% in 2005 to 31.1% in 2012/13 . In addition, 54% of P. vivax single clone infections were sub-microscopic in 2012/13, and MOI was significantly associated with sub-microscopic P. vivax infections in both years . Despite the significant decrease in prevalence, the overall genetic diversity (He) was high in both years for both species . As expected, the proportion of households that were infected in 2012–2013 was highest in villages with relatively higher prevalence of infection. In 2005, the majority of households had 2 or more individuals infected , whereas in 2012–2013, the majority of infected households had only 1 individual infected (61.7% of infected households) , while in 2012–2013 only 27.8% of households had an individual with a LM and/or MM-detectable infection , whereas in 2012–2013, this had decreased to 27.6% (p > 0.001) and the proportion of high-infection rate households  was associated with prevalence in the village in both years . A higher proportion of reported bed net use in the household was associated with a reduced risk of the household being a high rate household in 2005  and equally indoors and outdoors, making it highly susceptible to LLINs. A recent study reported an increase in prevalence of An. punctulatus and a decrease of An. koliensis, another late biter, in an adjacent area in East Sepik province, as well as a shift to earlier mean biting times of An. punctulatus in addition to significantly reduced man-biting-rates and annual entomological inoculation rates after the LLIN campaign [Renewed political and financial will to strengthen malaria control at the beginning of the millennium, resulted in the PNG National Department of Health launching a new campaign to quickly achieve high levels of LLIN ownership and usage. Nationwide free LLIN distribution took place between 2004 and 2009 and resulted in a significant increase in ownership of bed nets  . LLINs icampaign . Proximicampaign –51, but Cultural factors, socio-economic status and education level play an additional role in risk of infection and can vary across villages and households, as well as human behavioural and genetic factors , 52–56. P. falciparum and P. vivax appears to have been maintained at relatively high levels. Multiplicity of infection and especially the proportion of multiple clone infections has decreased. The proportion of multiple clone infections correlated well with prevalence and might be a suitable indicator of hotspot areas and areas of high transmission. Further investigation of markers that are not under selective pressure is required for a more detailed analysis of the impact of malaria control on genetic diversity, differentiation and population structure in this area [Despite the decrease in prevalence and significant geographic heterogeneity, the genetic diversity of both his area , 58.Although the prevalence of malaria has decreased across all age groups and there is no marked shift in the peak age of infections , this difference in timing is unlikely to substantially contribute to the observed decrease in prevalence between surveys. The ‘wet’ season is usually from October–April, whereas the so-called ‘dry’ season runs from May–September, however, in most parts of the lowlands of PNG there is perennial malaria transmission, with limited seasonality , 8. PrevP. falciparum infections in that study (47% vs 41%) [P. falciparum and P. vivax infections in the 2012/13 study is potentially influenced by the difference between qPCR and LDR-FMA and thus the data from this earlier survey will appear to have a slightly higher proportion of sub-microscopic infections than when qPCR was used. In addition, 2nd reads of microscopy slides in 2012–2013 were performed based on qPCR results, potentially resulting in a lower proportion of sub-microscopic infection.A limitation of the study was that the MM used to detect infections in the two time-periods was not the same. However, a previous study directly comparing qPCR and LDR-FMA reported substantial agreement between the two methods . While t vs 41%) , this isThis study was not a formal component of the monitoring and evaluation of the national malaria control programme, with the primary aim to delve into the impact that reduced transmission is having on the epidemiology of malaria rather than assess the programme itself. Prevalence reported in this study is much higher than provincial averages from the national reports , 62, whiP. falciparum and P. vivax prevalence than MSAT [Identifying and targeting focal points and hotspots of malaria is highly relevant for malaria control, since these are likely to be the areas where residual malaria transmission will persist, and can become an obstacle in efforts to eliminate malaria , 63, 64.han MSAT  and implAlthough Papua New Guinea’s strengthened malaria control programme successfully reduced malaria prevalence nationwide between 2005 and 2013, with a substantial impact in ESP, areas with high ongoing transmission remain. In this study, LLIN coverage and use, socioeconomic factors, vector density/behaviour and environmental factors are the likely drivers of this heterogeneity and resilience in the face of control. In order to further reduce transmission in these areas, additional surveillance approaches, novel tools and community engagement strategies may need to be layered on top of sustained nationwide LLIN coverage and effective case management.Additional file 1. Definition of anaemia. Definitions of anaemia that were used in the analyses stratified by age and sex. Values represent haemoglobin levels in g/L. Adapted from [ted from .Additional file 2. Detailed demographic and clinical characteristics of study participants. Additional participants characteristics to Table Additional file 3. Allele frequencies of Pfmsp1, Pvmsp1f3 and PvMS2 alleles in both surveys. Allele frequencies A) Pfmsp1 alleles in 2005 (top) vs 2012/13 (bottom) of all alleles (left) and split into 3D7 (centre) and FC27 (right) allele families, and B) Pvmsp1f3 and PvMS2 in 2005 (top) vs 2012/13 (bottom).


Placental malaria (PM) is a major public health problem associated with adverse pregnancy outcomes such as low birth weight (LBW), preterm delivery and maternal anemia. The present study is aimed to determine the prevalence of placental malaria among asymptomatic pregnant women in Wolkite health center, Gurage zone, Southern Ethiopia.Facility-based cross-sectional study was carried out from June 2019 to August 2019. A total of 230 pregnant women were involved in the study where socio-demographic data, medical and obstetric history were collected using pretested structured questionnaires. Blood samples were collected at delivery from maternal capillary, placenta and umbilical cord for the detection of malarial parasite. Maternal hematocrit was determined to screen for anemia.Plasmodium falciparum and Plasmodium vivax were detected by microscopy. All babies with positive umbilical cord blood films were born from a mother with placental malaria. Maternal anemia was recorded in 58.3% of the women. In univariate analysis, placental malaria was significantly associated with LBW (p < 0.001) unlike parity and maternal anemia.In this study, the prevalence of placental malaria, peripheral malaria and umbilical cord malaria was 3.9% (9/230), 15.2% (35/230) and 2.6% (6/230) respectively. Placental malaria among asymptomatic pregnant women is low in Wolkite health centre, Gurage zone in Southern Ethiopia. Moreover, placental malaria was strongly associated with LBW. Thus, further strengthening the existing prevention and control activities and screening of asymptomatic pregnant women as part of routine antenatal care service is very essential. Malaria in pregnancy (MIP) is a major preventable cause of maternal morbidity and poor birth outcomes in Sub-Saharan Africa . GloballP. falciparum erythrocyte membrane protein-1 (PfEMP-1) to bind to host receptors like chondroitin sulfate A (CSA) in the placental intervillous space leading to placental malaria (PM) [Placental malaria infection is characterized by the accumulation of parasitized red blood cells (pRBCs) in placental intervillous spaces that express unique membrane-bound proteins from the ria (PM) . It is aria (PM) .P. falciparum infections. However,parasite accumulation in the placenta contributes to maternal anemia and can lead to infiltration of mononuclear cells in the placental intervillous space, which is a risk factor for LBW [The symptoms and complications of malaria in pregnancy depend on the malaria transmission intensity and the individual’s level of acquired immunity . Pregnan for LBW . On the  for LBW .As major strategies, WHO has recommended the use of insecticide-treated mosquito nets (ITNs), effective case management of malaria and administration of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) in African countries. Ethiopia does not support IPTp-SP due to cost-effectiveness considerations and low malaria transmission in most parts of the country , 10. TheP.vivax accounted for a higher share of 69.7% and P. falciparum accounted for 29.3% [Gurage Zone is a malaria-endemic area that has unstable and seasonal malaria transmission. In a previous study, the prevalence of microscopically confirmed malaria cases among symptomatic patients was 8.6% where or 29.3% . In malaor 29.3%  and suchor 29.3% . We hypoor 29.3% . Hence, This study was conducted in Wolkite health center, which is found in Wolkite town and located at 158 km southwest of Addis Ababa, the capital city of Ethiopia. Wolkite town is the capital of Gurage Zone, has latitude and longitude of 8°17'N37°47'E and an altitude of 1910- 1935 meters above sea level. Based on the 2007 Ethiopian Census, the town has a total population of 28,856, of whom 15,068 were males and 13,788 were females [Wolkite health center provides service to patients coming from various districts of Gurage zone and an average of 980-1080 women give birth every year.P. falciparum were treated with Artemether-Lumefantrine (AL) and chloroquine was used for the treatment of P. vivax. Infants with umbilical cord malaria were treated with artesunate. All treatments were according to the national treatment guideline. Women who were anemic were treated with ferrous sulfate and folic acid.A facility-based cross-sectional study was conducted among asymptomatic pregnant women in Wolkite health center from June 2019 to August 2019. During the study period a total of 230 pregnant women participated in the study. Pregnant women with absence of clinical symptoms mainly fever or history of fever, chills and headache within the last 24 hours, axillary temperatures ≤ 37.5°C and those who were willing to participate and signed the informed consent in the labor ward were included in the study. Participants who took antimalarial drugs, had multiple pregnancies and those with serious delivery complications that led to a referral to other hospitals and those who were unwilling to participate in the study were excluded. Women with After obtaining written informed consent, socio-demographic characteristics of the participants were collected using pre-tested structured questionnaires in the labor ward. An enrollment form containing demographic data, past medical and obstetric history was completed for every participant by trained midwives. A general clinical examination was performed for each woman with reference to antenatal care (ANC) follow up, bed net use, parity, newborn sex. Axillary temperature was measured by an electronic instrument to the nearest 0.1°C at delivery. All newborns were weighted immediately after birth using an electronic scale to the nearest 10 grams and newborn sex was recorded. Newborns were classified as normal birth weight  or LBW (<2500 g) according to the WHO guidelines .At delivery, placental blood films were prepared by the incision of approximately 1.5 cm of the placenta on the maternal side and a drop of blood was placed on a slide. The umbilical cord blood samples were obtained by wiping away excess blood from a clamped cord to avoid contamination with maternal blood. Then, a small cut was made with a lancet and a drop of blood was taken to prepare blood film. Maternal capillary blood samples were collected for blood film and hematocrit determination. All specimens were correctly labeled and sent to the hematology laboratory for hematocrit analysis.Packed cell volume (PCV) of maternal peripheral blood collected after delivery was determined using the standard method. Based on their PCV value women were considered as anemic if PCV is < 33% and were classified as mild (PCV 27–32.9%), moderate (21.0–26.9%) and severe (PCV < 21%) .Even though placental histology is a gold standard method to diagnose placental malaria  due to lBoth thick and thin smears of the maternal peripheral blood, placental blood and umbilical cord blood were prepared. After proper labeling and air-drying, thin films were fixed with absolute methanol for 5 s while both thin and thick films were stained with freshly prepared 3% Giemsa solution diluted with buffered water (PH 7.1–7.2) for 30 min and examined under 100X magnification . Thick bAccording to WHO, parasite density estimation from parasites counted against high power field (HPF) (100× objective and 10× eyepieces) on the thick film was calculated as ;\documeThe standard volume of blood per HPF is assumed to be 0.002. A negative blood film was reported if no parasite were identified after 100 HPFs were observed.Data quality was ensured using standardized data collection materials, pre-tested questionnaires, properly trained data collectors and intensive supervision during data collection. Pre-analytical, analytical and post-analytical stages of quality assurance that are incorporated in standard operating procedures (SOPs) were strictly followed while performing laboratory analysis.p-value <0.05 was considered statistically significant.The collected data were entered and analyzed using the Statistical Package for Social Sciences (SPSS) version 20 software. Univariate analysis was performed to determine the association of placental malaria with pregnancy and neonatal outcomes. Pearson Chi-square test or Fisher’s exact test was used for categorical variables whereas Student’s t-test was used for continuous variables. A Two hundred forty-two women visited Wolkite health center for delivery from June 2019 to August 2019. Of these, 12 were excluded due to prenatal death, twin delivery and referral to a different hospital due to delivery complications. From 230 normal deliveries, 53.9% of the babies were females and 46.1% were males. The age of the participants ranged from 18 to 40 years old with a mean (SD) age of 24.7 (5.1) years. The majority (72.2%) of participants were in the age group of 18–27 years. Sixty-four percent of the participants resided in Wolkite town and more than 90% of the women reported using bed nets during their pregnancy and attended ANC , 15.2% (35/230) and 2.6% (6/230) respectively. 30, 15.2%p < 0.001)  Table .Table 3p = 0.04). Of 230 babies delivered, 7.4% (17) had LBW (< 2.5 kg) and 66.7% (6/9) women with placental malaria had LBW babies. The difference was statistically significant (p < 0.001).The mean birth weight (SD) among women with placental malaria was 2.2 (0.2) as compared to those without malaria 3.4 (0.4) and the difference was statistically significant (p = 0.05) (Table The mean hematocrit (HCT) among women was 31.0 (6.2) and anemia (HCT < 33%) was recorded in 57.8% (133) of the women. Of these, mild anemia was seen in 61% (81) of the women, moderate anemia in 31.5% 42) women and severe anemia was seen in 7.5% (10) of the women. From 9 women with placental malaria, 8 (88.9%) of them were anemic and among 221 women without placental malaria, 125 (56.6%) of them were anemic. The difference was not statistically significant  and abroad Guinea (15.8%) , 25. Theet al. and Fehintola et al. which reported malaria prevalences of 50.8% and 40% in Nigeria [The prevalence of umbilical cord parasitemia in the present study was 2.6% and all positive babies were born from a mother with both peripheral and placental malaria. There was a strong association of placental malaria with umbilical cord parasitemia (p< 0.001). This prevalence was in agreement with the previous study done in unstable area of Ethiopia (1.6%) and abroad Burkina Faso (1.4%) , 33 but  Nigeria , 34. Con Nigeria . Further Nigeria .The prevalence of LBW in the present study was 7.4% which was comparable with studies in other African countries, including Nigeria 6.7%  and TanzIn our finding, placental malaria was strongly associated with LBW (p<0.001). LBW can be caused by either preterm delivery and/or intrauterine growth restriction. It has been explained in the literature that the sequestration of pRBCs, monocytes in the placenta induce altered cytokine profiles and complement activation that cause placental tissue injury that leads to placental insufficiency, fetal growth restriction which results in LBW , 42.The prevalence of anemia was 58.3% in this study with the majority of women having mild anemia. In the present study, there was no statistically significant association of placental malaria with maternal anemia. The prevalence of anemia in this study was higher than studies conducted by Bassey et al. (31.9%)  and CissThe prevalence of placental malaria among asymptomatic pregnant women was low and all babies with positive umbilical cord blood films were born from a mother with placental malaria. Placental malaria was significantly associated with LBW. Thus, further strengthening the existing prevention and control activities and screening of asymptomatic pregnant women as part of routine ANC is essential.Moreover, future studies with more sensitive diagnostic techniques including placental histology and PCR should be conducted to determine the burden and effect of submicroscopic infection in pregnant women in high transmission areas.


Anopheles salivary peptide could be a pertinent and complementary tool to assess the risk of malaria transmission and the effectiveness of vector control strategies. This study aimed to obtain first reliable data on the current state of the Anopheles gSG6-P1 biomarker for assess the level of exposure to Anopheles bites in high malaria endemic areas in Cameroon. Blood smears were collected from people living in the neighborhoods of Youpwe  and Manoka , both areas in the coastal region of Cameroon. Malaria infection was determined using thick blood smear microscopy, whereas the level of specific IgG response to gSG-P1 peptide was assessed by enzyme-linked immunosorbent assay from the dried blood spots. Of 266  malaria endemic residents  randomly included in the study, Plasmodium infection prevalence was significantly higher in Manoka than in Youpwe . The anti-gSG6-P1 IgG response showed a high inter-individual heterogeneity and was significantly higher among individuals from Manoka than those from Youpwe (p = 0.023). Malaria infected individuals presented a higher anti-gSG6-P1 IgG antibody response than non-infected (p = 0.0004). No significant difference in the level of specific IgG response to gSG-P1 was observed according to long lasting insecticidal nets use. Taken together, the data revealed that human IgG antibody response to Anopheles gSG-P1 salivary peptide could be also used to assess human exposure to malaria vectors in Central African region. This finding strengthens the relevance of this candidate biomarker to be used for measuring human exposure to malaria vectors worldwide.The specific immune response to the  Plasmodium falciparum decreased by 18% with the largest reductions recorded in Southeast Asia, Latin America, and Africa following the introduction of artemisinin-based combination therapies (ACTs) [Although the global malaria incidence and mortality due to s (ACTs) , this dis (ACTs) . The vass (ACTs) .Plasmodium-positive samples to accurately estimate the sporozoïte index [Assessment of malaria transmission intensity (MTI) by mosquitoes is an important determinant of the malaria’s burden estimation and evaluation of effectiveness of control and prevention strategies. It is central to efforts to control and eradicate malaria, and currently estimated by the entomological inoculation rate (EIR) usually interpreted as the number of infective bites received by an individual per unit of time . Unfortute index , 8. The te index  remain tte index , 11. Howte index , 13. White index , 15, thete index , 16, 17.An. gambiae Salivary Gland Protein-6 peptide 1) has been validated as a relevant biomarker of Anopheles bites in various settings including West and East Africa [In this context, there was an increased need to develop novel approaches for an efficient assessment of malaria transmission intensity. Previous studies have shown that immunological markers based on the quantification of human antibody responses to malaria vector salivary antigens could be useful for measuring MTI –22. Moret Africa –29, Amert Africa  and Asiat Africa , 32. Mort Africa , 27–29. t Africa , 33, 34.Anopheles bites in a continental suburban area and rural island in the coastal region of Cameroon. It will be suitable to compare the exposition to Anopheles bites and malaria transmission between such distinct areas, because it’s important to know, if all strategies used in the fight against malaria are working as well as in rural area than in sub-urban area. Because, the distinct geographical nature of the two sites  will induce difference in the malaria transmission risk.To our knowledge, there are currently no published studies that have scientifically evaluated the useful of this biomarker to measure malaria transmission intensity in Central African region in the published literature. Therefore, to provide concrete data on the use of human IgG Ab response to the gSG6-P1 salivary peptide as an epidemiological tool for assessing exposure of human population to malaria vectors in Cameroun, we conducted a prospective population based study to estimate the level of exposure to A cross-sectional study was conducted during the small rainy season in May 2017 and was carried out in 2 localities  in the coastal region of Cameroon.The Island of Manoka  is located in the archipelago of Manoka in the rural area of the 6th district of Douala, the most industrial city of Cameroon. This location is populated by around 40, 000 inhabitants. The main activities on this island are: fishing, production and trade of smoking fish products. Habitations are in temporary materials (houses built on stilts) with opened spaces between the walls of the roof. The hydrographic network is made up of many temporary streams: sources, pools and swamps that also constitute potential breeding sites of mosquitoes. The environment is predominantly marshy here.Youpwe is located in the sub-urban area in the continental zone, the 6th district of Douala , the economic capital of Cameroon. An estimated 3,200 people populated this neighborhood. Youpwe serves as a crossroad for trade activities between Douala and the Manoka Island. The main activity here is the general trade, and the selling of products coming from Manoka: fresh and smoked fish, crabs, fresh and dried shrimps. Habitations are mostly built with cement and overall there is no space between the roof and the walls. The environment is made up with many water collections observed: ponds, streams, reserves for household activities and they constitute the mainly breeding site for mosquitoes.The populations of each of the study sites were sensitized a week earlier by community relay agents who invited them to come to the hospital  on a date set in advance. Once at the hospital, after the study had been presented to the populations, all individual who met the eligibility conditions and agreed to participate in the study were recruited after signing the informed consent. To limit the selection and information biases, participants were enrolled consecutively and participation in the study was voluntary.The target population were all local residents who gave consent to attend health district of the locality to attend health district of the locality. Eligibility for inclusion as defined as local resident who had not travelled out of the study site within the last 3 weeks. Pregnant women, children under 6 months and individual with severe clinical signs of malaria as defined by WHO, were excluded.This study was conducted in accordance with ethics directives related to research on humans in Cameroon. The study received an ethical clearance from the National Committee of Ethics for Research for Human Health (N ° 2015 / 08/622 / EC / CNERSH / SP) and was approved by the Cameroonian Ministry of Public Health and, administrative authorization was obtained from all the localities. Before enrollment and the administration of questionnaire, subjects were informed on the purpose and process of the investigation , and a signed informed consent was obtained from all those who agreed to participate in the study in accordance with the Helsinki Declaration. Participation was voluntary, anonymous and without compensation. All patients were free treated in accordance to the treatment guidelines from the Cameroon National Malaria Control Program.They were open-ended (OEIQ) and closed-ended (CEIQ) interview questions, including a single answer, and multiple choices questions. Data collection sheets were used to collect data on socio-demographic characteristics . The second part focused on i) possession and use of LLINs, ii) use of insecticide products .Anopheles gSG6-P1 salivary peptide. The presence of P. falciparum and other species of malaria parasites was detected by RDT testing  and thin blood smears. P. falciparum parasitaemia was determined by microscopic examination of Giemsa-stained thick blood smears. Parasite density was determined on the basis of the number of parasites per 200 leukocytes on a thick film, assuming total leukocyte counts of 8,000 cells/microL of whole blood.Venous blood samples (3 ml) were collected into EDTA vacutainer tubes and 50 microL were spotted on Whatman filter papers (3M) and stored at 4° until use for the quantification of the level of human IgG Ab response to the Anopheles gSG6-P1 salivary peptide.For each blood sample, blood spotted on Whatman (3M) filter papers was collected and analyzed by direct Enzyme-Linked Immunosorbent Assay (ELISA) to estimate total antibody levels against Anopheles-specific salivary peptide gSG6-P1, the recombinant antigen sequence identification was designed using bioinformatics as previously described by Poinsignon in 2008 [For  in 2008 . It was et al [™) at 1/2 000 dilution in PBS/Tween 1% for 1h30min at 37°C. Streptavidine-Peroxydase conjugate  was then added at 1/2000 dilution and incubated for 1h at 37°C with 100 μL/well. Finally, the plates were subsequently incubated with 100 μL/well of 2,2-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium (ABTS) (Thermo scientific) substrate solution  supplemented with 10 μL of 30% of oxygenate water-H2O2) for 2 hours in the dark and at room temperature for the colorimetric development, then optical density (OD) was read at 405 nm.The level of IgG Ab response to gSG6-P1 salivary peptide was measured on whole blood elute from a dry spot as previously described by Dramé et al . BrieflyIndividual results were expressed as the ΔOD value: ΔOD = ODx—ODn, where ODx represents the mean of individual OD in both antigen wells and ODn the individual OD in the well without gSG6-P1 antigen . For each sample, the experiment was validated only if the coefficient of variation between the two Ag+ well (%CV) was ˂20%. Samples with CV ˃20% were re-analysed.Etablissement Française du Sang—Normandie”, coming from France; 609 chemin de la Bretèque—BP. 558–76235 BOIS GUILLAUME Cedex) with no travel history to malaria endemic countries were used as negative controls. Each ELISA experiment was validated by including on each plate three blank wells containing only PBS-Tween 1%in place of blood plasma elute. The cut-off value was defined as a mean OD of negative control plus three times standard deviation (SD).Blood sample (34) from European volunteer blood donors  and Stata software (version 11 SE).Categorical variables were expressed as frequencies, whereas numerical variables were presented as means+/- Standard deviation (SD) or 95% CI  if they were normally distributed. The exact Fisher test was used to compare qualitative variables. After checking the non-Gaussian distribution, the non-parametric Mann-Whitney U-test was used to compare antibody levels between two independent groups. Only p A total of 266 individuals from whom blood samples collected were included in the two areas, 153 (57.5%) selected in suburban area (Youpwe) and 113 (42.5%) in rural area (Manoka-Island) .The mean length of residence of the individuals surveyed in the study area was higher in Manoka Island compared to Youpwe and the mean of this variable was not significantly different (p = 0.210). Overall, no significant difference was observed for mean age between Youpwe’s population and Manoka’s population (p = 0.565). The entire age range was 6 months to 94 years for Youpwe’s population and 6 months to 85 years Manoka’s population. In each locality, the mean age was 22.12±19.2 with 95%CI (19.05–25.19) and 23.71±20.5 with 95%CI (19.88–27.53) in Youpwe and Manoka Island respectively. Youpwe was predominantly female  while, in Manoka Island men accounted for more than half of the population  and this difference was statistically significant (p = 0.032). Overall, no difference was observed for an educational level between the two localities (p = 0.05) in our study population, however this difference was significant when comparing individuals one to another in each locality for primary and secondary school level. .Both populations used numerous malaria prevention strategies, including long lasting insecticidal nets (LLINs), ventilation, coils and spray bombs . It’s imvs 82.3%, p = 0.3750) and, among them 95.7% declared use of LLINs more in Manoka than in Youpwe but this difference was not statistically significant (p = 0.0868). Additionally, those who use treated nets have been reported to use them every night  in most of responders, more by Youpwe’s population than Manoka’s population (p = 0.0342). No significant difference was observed when comparing reports on the frequency of LLINs use for children compared to adults. Men reported more LLINs use than women  in the Manoka Island, whereas in Youpwe women reported more sleep under ITNs than men  but this difference was not statistically significant. The environment was mainly swampy and the houses with spaces between the walls and the roof were more important in Manoka compared to Youpwe and, this difference was statistically significant (p<0.0001).Overall, 84.6% of responders reported to have LLINs with a higher proportion of individuals reporting LLINs ownership in Youpwe population  and the majority of infected people were found in the Island of Manoka, with a prevalence of 63.7% vs 12.4% in Youpwe. , p <0.0001)  were detected by RDT positively infected with <0.0001) .Anopheles bites in Cameroon, analyses were performed based on specific blood antibody levels and positivity for individuals exposed to Anopheles bites and European negative controls. As shown in To determine if IgG antibody response to gSG6-P1 salivary peptide constitutes tool for detecting human exposure to To determine if IgG Ab response to gSG6-P1 peptide is associated to the level of exposure to Anopheles bites, we compared the level of this response in Youpwe and Manoka given that since in a preliminary study reported higher exposure to Anopheles bites (aggressiveness) in Manoka (HBR = 20.29 b/p/n) than in Youpwe (HBR = 14.50b/p/n) . The levPlasmodium infected population (RDT and/or microscopy positives) and, non-infected population (negative to both tests)  . There wh tests) .As shown in Anopheles bites, the level of anti-gSG6-P1 peptide IgG Ab response was significantly lower in individuals using strategies such as insecticidal twist or spray cans  than those who did not in Manoka island  with a significantly difference (p = 0.0021). In Youpwe, the level of IgG Ab response to gSG6-P1 peptide was not significantly different irrespective of additional measures taken to prevent Anopheles bites.To investigate if the level of specific IgG Ab response to gSG6-P1 salivary peptide constitutes tool for evaluate the impact of preventive measures against malaria infection, analyses revealed in overall population no significantly difference observed according to the use of anti-mosquitoes bites strategies. No difference was observed between individuals that reported using LLINs and those that did not in each area. The level of anti-gSG6-P1 peptide IgG Ab response was higher among people who reported using LLINs frequently  than those who did not use LLINs  in both Youpwe and Manoka Island but no significantly difference was observed. Concerning the use of other strategies to prevent Anopheles gSG6-P1 salivary peptide to assess in a preliminary study and for the first time, the level of human exposure to Anopheles bites in malaria endemic sittings in Cameroon, a Central African region. Specific IgG Ab response to gSG6-P1 peptide was detected in almost all individuals (99%) contrary to non-exposed foreigners (European negative control). This data suggest the use of this specific antibody response to gSG6-P1 peptide as a pertinent marker to assess human exposure to Anopheles mosquitoes in Cameroon. This finding is similar to those reported in other regions in Africa [Anopheles bites differ among individuals. Considering that, the similar data previously reported that this biomarker can discriminate the level of exposure to Anopheles between two individuals living in the same endemic area [Anopheles bites regardless of the gender [This study aimed to use a biomarker based on human IgG Ab response to n Africa , 36–40, n Africa  and Asian Africa , 32,whern Africa , 30, 37 n Africa , 32, 40.e gender .Anopheles bites [Anopheles as shown in our other studies conducted in these areas and where we also reported higher densities of Anopheles mosquitoes breeding site in Manoka Island than in Youpwe [Interestingly, the population of Manoka Island had higher levels of specific IgG anti-gSG6-P1 response than Youpwe. Several studies reported that the level of IgG Ab response to gSG6-P1 peptide is significantly associated with the level of exposure to es bites , 30, 36 es bites . This hin Youpwe , 42, 43.n Youpwe , 42. Togn Youpwe .Plasmodium infected individuals developed higher immune response than non-infected [Plasmodium infected individuals than non-infected. This reinforces the idea that, in addition to being a biomarker of human exposure to malaria vectors bites, the gSG6-P1 peptide could also be used as a tool for assessing the risk of malaria transmission and therefore a tool for predicting malaria morbidity as it was also previously observed in Senegal [The level of the IgG Ab response to gSG6-P1 salivary peptide was previously reported to be positively associated with malaria infection status, infected , 40, 41.Regarding the level of antibody response and malaria infection according to studies areas, there is significant difference between infected vs non-infected individual in Youpwe whereas there is no difference in Manoka Island , suggestAnopheles (An.) coluzzii was 13, 96 b/p/n indoor and 34.76 b/p/n outdoor in Manoka, and 4,75 b/p/n indoor and 24,83 b/p/n outdoor in Youpwè and, also that density of An. coluzzii was significantly higher outdoor than indoor in the both localities [Anopheles bites compared to those that do not is consistent with other studies conducted in Senegal and Angola [In contrast, to previous study in Senegal and Angola , 37, 38,calities . HoweverAnopheles salivary peptide gSG6-P1 were recognized by the different blood samples in Cameroon, with the highest reactivity observed among people with infection and living in the area with high endemicity level. Although our two study sites are geographically closed, environmental and ecological differences may result in a difference of exposition to Anopheles bites and therefore to malaria transmission. Thus, further studies including entomological, ecological and environmental data could allow having a better idea of the situation. These observations could be very important in the decision making regardless of the vector control strategies implemented by the NMCP. Moreover, given the complexity of the vectors distribution in Central Africa with secondary vectors , it required to conduct another studies in the localities hosting these vectors and, to assess the potential of this biomarker candidate to also measure exposure to malaria secondary vectors.The present study shows for the first time that a specific response to specific


Plasmodium falciparum infection throughout the year in Brazzaville.In the Republic of Congo, hot temperature and seasons distortions observed may impact the development of malaria parasites. We investigate the variation of malaria cases, parasite density and the multiplicity of From May 2015 to May 2016, suspected patients with uncomplicated malaria were enrolled at the Hôpital de Mfilou, CSI « Maman Mboualé», and the Laboratoire National de Santé Publique. For each patient, thick blood was examined and parasite density was calculated. After DNA isolation, MSP1 and MSP2 genes were genotyped.. However the multiplicity of infection was higher at the Hôpital de Mfilou .A total of 416, 259 and 131 patients with suspected malaria were enrolled at the CSI «Maman Mboualé», Hôpital de Mfilou and the Laboratoire National de Santé Publique respectively. Proportion of malaria cases and geometric mean parasite density were higher at the CSI «Maman Mboualé» compared to over sites P-value <0.01).At the Laboratoire National de Santé Publique, malaria cases and multiplicity of infection were not influenced by different seasons. However, variation of the mean parasite density was statistically significant (Higher proportions of malaria cases were found at the end of main rainy season either the beginning of the main dry season at the Hôpital de Mfilou and the CSI «Maman Mboualé»; while, lowest proportions were observed in September and January and in September and March respectively. Higher mean parasite densities were found at the end of rainy seasons with persistence at the beginning of dry seasons. The lowest mean parasite densities were found during dry seasons, with persistence at the beginning of rainy seasons. Fluctuation of the multiplicity of infection throughout the year was observed without significance between seasons.The current study suggests that malaria transmission is still variable between the north and south parts of Brazzaville. Seasonal fluctuations of malaria cases and mean parasite densities were observed with some extension to different seasons. Thus, both meteorological and entomological studies are needed to update the season’s periods as well as malaria transmission intensity in Brazzaville. Malaria is still one of the major health problems worldwide. The global incidence of the disease in 2017 has been estimated at 219 million of cases with 435,000 deaths [Climate change has been noticed worldwide with impact on rainfall, temperature and humidity; three factors that are known to affect malaria seasonality as well as, transmission intensity –4. SeverIn areas with seasonal and intense malaria transmission, the human parasite reservoir declines through the dry season until the beginning of the wet season at which time vector numbers begin to rise . Thus unEntomological surveys are encouraged for this purpose. However, parasitological data such as parasite density should be able to supplement entomological data for better understanding of local seasonality and heterogeneity of exposure .Plasmodium falciparum infection (MOI), defined as the minimum number of Plasmodium falciparum genotypes per infected subject, is thought to be a useful parasitological indicator of transmission or host acquired immunity level [Plasmodium falciparum infection [To predict the effect of intervention outcomes in seasonal malaria settings, it is also necessary to understand the dynamic of natural acquired immunity or premunition across a seasonal time scale . The multy level , and to ty level . Howeverty level , 15, whinfection , 17.In the Republic of Congo, malaria is still the leading cause of attendance in health facilities with 52, 8% outpatient consultation, 44, 1% hospitalization and 28% of deaths due to malaria; and the most vulnerable are pregnant women and children under 5 years old .A study conducted in 2006 in Republic of Congo jointly by the WHO and the Ministry of Health and Population showed that the transmission dynamic of malaria in the country follows two different patterns: (1) a year-round perennial transmission in forest areas, with an estimated entomological inoculation rate (EIR) of 200–1000 infective bite/person/year, and (2) a seasonal transmission in savanna areas where the high transmission period lasts 7–10 months and is directly correlated with the rainfall and the EIR is estimated to be 80–200 infective bites/ person/year . In thesPlasmodium falciparum malaria cases, parasite density and the MOI in Brazzaville, the Republic of Congo.This study investigated the seasonality of , estimated at 4312715 inhabitants as described elsewhere [The study was conducted in Brazzaville, the political capital hosting 38%  of the total population of the Republic of Congolsewhere , 21. Duelsewhere , 21.Plasmodium falciparum and Anopheles gambiae s.s. is the predominant vector. Two rainy seasons are observed each year with the main one during the months of February to May, and a short one from October to November [Malaria infection is primarily due to November –24. The From May 2015 to May 2016, patients with clinical signs of uncomplicated malaria, presenting at the laboratory at each of the three study sites were invited to participate in this study. Exclusion criteria were pregnancy, severe malaria or other severe illness as judged by the attending physician. The number of representative patients to be included in each site was estimated taking into account the proportion of malaria reported in each health center, 1 year before starting the study as described elsewhere . Thus, 3Genomic DNA was extracted from samples collected on the Watman filter paper using QIAamp DNA mini Kit  according to the manufacturer’s instruction. Extracted DNA was stored at − 20 °C until use.Plasmodium falciparum genotyping was performed using the nested PCRs technique. The MSP1 and MSP2 genes in their highly polymorphic loci, namely MSP1 block 2 and MSP2 central region were used as markers for this genotyping as described previously [eviously , 27. PCRP-values ≤ 0.05 were considered as statistically significant.Data collected were summarized with average and standard deviation for age and compared using Student’s t-test for two independent samples or the Bonferroni method for one-way analysis of variance (ANOVA) for multiple comparisons. Categorical responses were expressed as a percentage, and comparisons were made using Pearson’s χ2 test (or Fisher’s exact test if appropriate). The effect of seasonality on malaria infection was assessed using logistic regression method but no relationship was found, because some subgroups had few patients with malaria. So, the non-parametric Kruskal-Wallis test was used for determining the differences in prevalence of malaria infection between seasons. All analyses were done with SPSS statistical software for Windows, Version 24.0 (Chicago: SPSS Inc.). All tests were two tailed and A total of 416, 259 and 131 patients with suspected malaria were enrolled at the CSI «Maman Mboualé», Hôpital de Mfilou and the LNSP respectively. Sociodemographic as well as clinical characteristics of these patients are summarized in Table .Out of 259 patients enrolled at the Hôpital de Mfilou, gender and age were recorded for 257 of them, while with regards to the CSI « Maman Mboualé», out of the 416 recruited patients, 410 had records on gender. Both proportion of malaria cases and geometric mean parasite density were higher at the CSI «Maman Mboualé» compared to other sites P-value <0.001). With regards to gender, males were more infected than females at the Hôpital de Mfilou .All malaria patients recruited at the LNSP were more than 5 years old. While the proportion of malaria cases was not influenced by age at the Hôpital de Mfilou Table , patientP-value <0.417). With regards to MSP2 gene, a total of 25, 27 and 4 different alleles of FC27 and 3D7 were identified at the Hôpital de Mfilou, the CSI « Maman Mboualé», and the LNSP respectively, with no statistical significant predominance of any specific family between sites  . While the MOI was not influenced by age, patients with less than 5 years had significantly higher parasite densities compare to those with the age greater than 5 years old . However, the difference of the mean parasite density was statistically significant  with the highest peak in November, corresponding to the rainy season.A particular profile has been found at the LNSP with very low malaria cases without significance between seasons . A contrasting profile was observed at the CSI « Maman Mboualé», where the highest proportion of malaria cases were obtained during the main rainy season, in April, May including the beginning of dry season in June. Progressive diminution of malaria cases has been noticed from July to March, reaching the lowest proportion in December and March corresponding to the beginning of dry season and main rainy season respectively . When taking all tree sites together the highest proportions of malaria cases were confirmed during the rainy seasons, while lowest proportions were registered in September, December and March .At the Hôpital de Mfilou, highest proportions of malaria cases have been found at the beginning of the study in May and June 50%), November (40%), February (38.9%) and April (33.3%) corresponding to the months of rainy seasons and the beginning of dry season for June  at the end of the study Fig. . HoweverP-value <0.043). When taking all tree sites together, similar profile with the CSI «Maman Mboualé», has been observed .By considering the CSI «Maman Mboualé», tree high peaks of mean parasite density have been identified in July, December and May; thereafter, persistence decrease has been noticed during the dry season, including the rainy seasons Fig.  correspoP-value = 0,853).Particular profile has also been found at the LNSP regarding the MOI with no infection in some alternative months , and the MOI did not vary significantly over the year  without any clear pattern and devoid of seasonality. From the beginning of the study in May, the MOI increased reaching a peak during the main dry season in July, thereafter persistence of decrease was observed, with the lowest MOI in September corresponding to the end of the main dry season . Persistence of decrease of MOI was observed from June to August and an increase in September during the main dry season, while high peak of MOI was observed in October with a decrease in November during the short rainy season. Inversely, lower peak was observed in December with an increase in January during the short dry season. Thus there was no clear pattern and devoid of seasonality. When taking all tree sites together, significant variation of MOI was also observed over the year  with similar profile with the CSI «Maman Mboualé» from May at the beginning of the study to November, but slightly increase of MOI from December to May at the end of the study.Concerning the CSI «Maman Mboualé», tree different peaks were observed, with highest MOI registered at the beginning of the study in May; follow by those in October and the lowest in December. Significant variation of MOI was observed within the main dry, short rainy and dry seasons, while MOI was slightly stable during the main rainy season from February to May . While, clear impact of dry season has been observed at the Hôpital de Mfilou with lowest proportions in September and January, at the CSI «Maman Mboualé», lowest proportions of malaria cases were found in September and March. Higher mean parasite densities were found meanly at the end of rainy seasons with persistence at the beginning of dry seasons. Inversely to the lowest mean parasite densities founded during dry seasons but with persistence at the beginning of rainy seasons. This could be due to environmental particularities including the humidity relative to the presence of swampy areas around the Hôpital de Mfilou as well as the “Fleuve Congo” river, surrounding the CSI «Maman Mboualé», which may maintain the multiplication of mosquitos until the beginning of dry seasons, while low level of humidity may influence mosquitos multiplication at the beginning of the rainy season. The outcome of this study is in agreement with those in Nigeria –32. AddiFluctuation of the MOI throughout the year was observed without any clear pattern and devoid of seasonality at the Hôpital de Mfilou and the CSI «Maman Mboualé». The findings presented in this study disagree with the results of previous study in Senegal and Ghana , 33. ThePlasmodium falciparum symptomatic population suggests that malaria transmission is still variable between the north and the south of Brazzaville. Seasonal fluctuation of proportion of malaria cases and mean parasite density was observed. However, persistence of high proportions of cases and mean parasite densities at the beginning of dry seasons and their lower values at the beginning of rainy seasons may be due to the season distortions observe in Brazzaville. Thus, both meteorological data and entomological studies are needed to update the season’s periods as well as malaria transmission intensity.With the lack of recent entomological data in Brazzaville, this study conducted throughout the year on


Plasmodium falciparum is the dominant malaria parasite in sub-Saharan Africa. However, Plasmodium vivax is becoming widely spread throughout Africa. The overuse of vector control methods has resulted in a remarkable change in the behaviour of mosquito that feeds on human as well as on vector composition. The aim of this study was to identify Anopheles mosquito species in vivax malaria endemic regions and to investigate their role in P. vivax circumsporozoite protein (Pvcsp) allele diversity.In spite of the global effort to eliminate malaria, it remains the most significant vector-borne disease of humans. Anopheles gambiae complex were confirmed to species level using molecular analysis. A subset of blood-fed anopheline mosquitoes were dissected to determine the presence of natural infection of malaria parasites. In addition, the rest of the samples were investigated for the presence of Pvcsp gene using nested-PCR.Mosquito samples were collected from Central Sudan  and Eastern Sudan  using pyrethrum spray catch (PSC) and CDC light traps. Mosquitoes were identified using appropriate morphological identification keys and Anopheles arabiensis (94.2%), Anopheles funestus (0.5%), and Anopheles pharoensis (5.4%). None of the dissected mosquitoes (N = 108) showed to be infected with malaria parasite. Overall P. vivax infectivity rate was 6.1% (63/1037) by Pvcsp nested PCR. Co-dominance of An. arabiensis and An. pharoensis is reported in Sennar state both being infected with P. vivax.A total of 1037 adult anopheline mosquitoes were collected from New Halfa (N = 467), Rural Khartoum (N = 132), and Sennar (N = 438). Morphological and molecular identification of the collected mosquitoes revealed the presence of P. vivax infection among wild-caught anopheline mosquitoes in Central and Eastern Sudan. While An. arabiensis is the most abundant vector observed in all study areas, An. funestus was recorded for the first time in New Halfa, Eastern Sudan. The documented Anopheles species are implicated in Pvcsp allele diversity. Large-scale surveys are needed to identify the incriminated vectors of P. vivax malaria and determine their contribution in disease transmission dynamics.This study reported  Plasmodium falciparum), predominantly in sub-Saharan Africa [Plasmodium falciparum is considered to be the most important malaria species responsible for more than 99.7% of malaria cases [P. vivax [P. vivax infections and even deaths [Plasmodium vivax stands for about half infections outside Africa [P. falciparum and for the accepted paradigm that Africans are protected from P. vivax infection by genetic factors [The estimated malaria cases in 2019 was 229 million cases occurred worldwide resulting in 409,000 malaria related death, owing the deadliest parasite  to invade the red blood cell [ood cell . Duffy aood cell , so infeood cell , 13. Howood cell , 15.P. falciparum is responsible for 91.2% of malaria cases while P. vivax makes 8.8% of the cases [P. vivax infections is increased throughout the country with an overall prevalence of 26.6% [Anopheles mosquitoes in transmitting malaria parasites depends on several factors including their preference to feed on humans [Plasmodium [Anopheles gambiae complex, the Anopheles funestus group, and the Anopheles nili complex [In Sudan, he cases , 17. Howof 26.6% , and preof 26.6% , 19. Then humans  and theiasmodium , 22. The complex , 24. Met complex , 26, tha complex , 28.Anopheles arabiensis is the main if not the only malaria vector in Sudan [Anopheles species at malaria foci in Sudan, such as An. funestus sensu stricto (s.s.) [Anopheles pharoensis [An. nili sensu lato (s.l.) [The vast majority of studies stated that in Sudan –32. Howeo (s.s.) , Anophelaroensis  and An. o (s.l.) .Plasmodium circumsporozoite protein (CSP) is an abundant surface protein expressed on the surface of sporozoite and oocyst [Plasmodium vivax circumsporozoite (Pvcsp) gene has three distinctive variants  [d oocyst  its exprd oocyst . This prd oocyst . Plasmodax-like) –41.P. vivax malaria is endemic and to investigate their possible role in P. vivax circumsporozoite protein (Pvcsp) gene allele diversity.The present study was conducted to identify the anopheline mosquito fauna in regions where The study was conducted in two regions, Central Sudan  and Eastern Sudan  .An. arabiensis 94.2% (976/1037), An. pharoensis 5.4% (56/1037), and An. funestus s.s. 0.5% (5/1037) , Rural Khartoum (N = 132), and Sennar (N = 438). Out of the total, 644 mosquitoes were blood-fed, 393 were unfed, while 117 were gravid . None was found infected with sporozoites. CSP gene analysis showed that sixty-three samples showed presence of Pvcsp gene, with overall infectivity rate (6.1%). Details of numbers of infected mosquito species among study sites are presented in Table A subset N = 108) of blood-fed mosquitoes were dissected to detect natural infection with malaria parasite  were detected  gene allele diversity.The present study was conducted to identify the species of Updating the existed knowledge about the vector composition and relative density in malaria endemic areas are essential entomological and epidemiological indicators for the disease burden, transmission season, and monitoring the vector control methods –50.P. falciparum is responsible for more than 95% of clinical malaria cases while revealing 3% were due to P. vivax. However, recently there an increasing numbers P. vivax cases reported in many parts of the country [Previous studies in Sudan , 31, 51  country .An. arabiensis was the most abundant Anopheles species, followed by An. pharoensis, and the least was An. funestus, supporting that An. arabiensis is the principal malaria vector in Sudan. For the first time, An. pharoensis was found positive for P. vivax. In addition to Lewis in 1956 [An. pharoensis, which is considered to be a secondary vector. Other studies in Sennar [An. arabiensis to harbouring malaria parasites. In contrast to Himeidan et al. and Lewis [An. pharoensis and Anopheles multicolour in New Halfa, this study showed the presence of An. funestus in the same area but in a small number.Results of this study demonstrated that  in 1956 , no furtn Sennar , and Gezn Sennar  reportednd Lewis , 53, whoPlasmodium parasites during their sexual life cycle such as mature gametes, zygotes, ookinetes, oocysts. Appearance of sporozoites usually requires approximately two weeks from the time of ingesting infected blood meal with malaria parasites by mosquito vectors [In this study, sporozoites were not observed in the dissected blood fed mosquitoes. This result is similar to previous studies conducted in Sennar and Khartoum , 54. One vectors .P. vivax was not detected in any mosquito collected in Rural Khartoum in accordance with a previous study conducted in Khartoum state [P. vivax were identified  and a marked difference in infectivity rates of the identified mosquitoes (An. arabiensis versus An. pharoensis) was demonstrated in the two study sites. This variation in Pvcsp allelic distribution perhaps is due to adaptation of parasites to local mosquito species thus transmit distinct P. vivax variants/haplotypes with different efficiency [Plasmodium vivax is becoming a serious health problem exhibiting a wide range of hosts children adults and even pregnant women [P. vivax malaria infections could be attributed to misdiagnosis of the infection using rapid diagnostic tests (RDTs) [In this study, um state . In the ficiency , 57. A bnt women . It has nt women , 60. Thes (RDTs) , or the s (RDTs) .Assessment of the impact of vector control interventions on malaria transmission requires more data about entomological indicators including the identification of the vector composition, distribution, and density .An. funestus, into Eastern Sudan. The study also confirms the role of Anopheles species in Pvcsp allele diversity in Sudan. These findings suggest changes in malaria epidemiology in Sudan that requires further entomological, parasitological, and epidemiological studies to accurately determine the distribution and density of malaria vectors countrywide, and to investigate their role in the malaria transmission. Additionally, the potential association between vector species and different Plasmodium species they transmit, need to be investigated thoroughly. Furthermore, the susceptibility of the malaria vectors in Sudan to the currently applied vector control tools must be urgently investigated.The findings of this study are very alarming mainly because it showed the expansion of the efficient malaria vector distribution,