In Sub-Saharan Africa (SSA), where malaria transmission is stable, malaria infection in pregnancy adversely affects pregnant women, fetuses, and newborns and is often asymptomatic. So far, a plethora of primary studies have been carried out on asymptomatic malaria infection in pregnant women in SSA. Nevertheless, no meta-analysis estimated the burden of asymptomatic malaria infection in pregnant women in SSA, so this meta-analysis was carried out to bridge this gap.2). Publication bias was assessed by visual inspection of the funnel plot and further quantitatively validated by Egger’s and Begg’s tests. The pooled prevalence and pooled odds ratio (OR) and their corresponding 95% Confidence Interval (CI) were estimated using the random-effects model in Stata 15 software.PubMed, Web of Science, Scopus, Embase, and ProQuest were systematically searched for relevant studies published until 4 August 2020, and also the expansion of the search was performed by October 24, 2020. We assessed heterogeneity among included studies using I-squared statistics . According to species-specific pooled prevalence estimate, Plasmodium falciparum was dominant species , followed by Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, respectively, found to be 3% , 0.8% , and 0.2% . Asymptomatic malaria-infected pregnant women were 2.28 times more likely anemic  than in non-infected pregnant women. Asymptomatic malaria infection was 1.54 times higher  in primigravida women compared to multigravida women.For this meta-analysis, we included 35 eligible studies. The overall prevalence estimate of asymptomatic In SSA, asymptomatic malaria infection in pregnant women is prevalent, and it is associated with an increased likelihood of anemia compared to non-infected pregnant women. Thus, screening of asymptomatic pregnant women for malaria and anemia should be included as part of antenatal care. Plasmodium falciparum is the etiologic agent for the vast majority of malaria cases in 2018 within the World Health Organization (WHO)-African Region [Malaria remains a substantial public health challenge that disproportionately affects SSA: in 2018, 213 million (93%) malaria cases worldwide and 405, 000 of all malaria-related deaths (94%) worldwide occurred in SSA [Plasmodium species in peripheral blood, an axillary temperature <37.5°C, and an absence of malaria-related symptoms, given the lack of standard definition [P. falciparum, while few infections are caused by other species of Plasmodium [Clinical presentation of malaria during pregnancy vary depending on the intricate interplay of malaria transmission intensity in particular geographical settings, parasites, and the level of acquired immunity , 4. In lfinition , 9. In masmodium , 11.In pregnancy, malaria affects not only pregnant women but also the fetus and newborn child , 12. MalIn 2018, the WHO set a global goal for malaria elimination in at least 35 countries with malaria transmission in 2015 –19. FollS1 Checklist).This systematic review and meta-analysis was carried out following Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines . On October 24, 2020, we have expanded the search by including additional databases such as African Journal Online (AJOL), google scholar and open grey. The full description of the literature search approaches can be found in the We applied the PICOS  approach to include eligible studies. (i) P: healthy pregnant women with no symptoms/signs suggestive of malaria usually fever (axillary temperature <37.5°C). (ii) I: none (iii) C: those pregnant women who have been tested negative for asymptomatic malaria infection, or groups  Vs multigravida (at least two previous pregnancy), and first and second trimester Vs third trimester). (iv) O: asymptomatic malaria infection is characterized by the detection of Plasmodium infection in the absence of clinical symptoms of malaria. The detection of Plasmodium infection can be performed using rapid diagnosis methods, microscopy or molecular methods, or in combination. And anemia is characterized as a level of hemoglobin < 11 g/dl  (v) S: oAll studies which were retrieved using databases and manual hand searching, potentially relevant for inclusion, were stored in EndNote X 8 for management. First, duplicate studies were automatically excluded, and then titles and abstracts of the remaining studies were screened. Second, we have removed titles and abstracts which appear irrelevant to inclusion. Third, after deduplication and screening of titles and abstracts, potentially relevant studies have been scrutinized for full-text (where available) and ineligible studies were removed. Fourth, literature in the full-texts were filtered based on pre-specified criteria.After the selection of eligible studies, the following data were extracted using a pre-prepared excel sheet: Author/s and year of publication, country, diagnostic methods, number of pregnant women tested for asymptomatic malaria infection, number of asymptomatic malaria positive study participants. Where the data is available, the following data were also captured; gravidity, gestational age , and hemoglobin status. Two authors (YY and MJA) independently conducted study selection and data extraction. And any inconsistency or disagreement between the two authors was resolved by consensus.S2 File).Two authors (YY and MJA) independently assessed the quality of included studies using a Hoy et al. , risk ofmetan written command in Stata software (version 15). To synthesize the pooled odds ratios (ORs), we used 2 x 2 tables to extract data (S3 File). When there were zero cases, we added 0.5 as a contingency correction. ORs and 95% Confidence Interval (CI) were calculated to examine the relationship between asymptomatic malaria infections and anemia, gravidity, and gestational age. The overall pooled prevalence and pooled ORs estimate were computed using the DerSimonian-Laird method for the random-effects model, based on the inverse variance approach for measuring weight. Inconsistency index (I2 statistics) was used to assess the magnitude of heterogeneity among included studies, with I2 value >25%, 25–75%, <75% were taken as low, moderate, and high heterogeneity, respectively [All statistical analyses were carried out using ectively  and Beggectively . We did Fig 1 shows electronic data sources and studies selection process. Initially, a total of 703 records were identified from PubMed, Web of Science, Scopus, Embase, and ProQuest. After removing 393 duplicate records, 310 remaining studies were screened based on titles and abstracts and 35 records were further excluded because we were unable to find full-texts. Of 35 studies, 32 records titles and abstracts were not related to the objectives of this study, while 3 potentially relevant reorders were published other than English language. The remaining 275 studies were screened by critically reading the full-texts. Of the 275 full-text studies, we excluded 240 studies with reasons: 51 of them were conducted outside of SSA; 147 of them did not report the prevalence of asymptotic malaria in pregnancy despite studies were conducted in SSA; 36 of them were reviews, and 6 of them were case reports. Twenty-six studies were deemed eligible for inclusion and three additional relevant studies were found from the references list of included studies that were not identified by our electronic database search strategies. Also, additional six eligible studies were identified through searching African Journal Online and google scholar. In the end, a total of 35 studies were included in this meta-analysis.S1 Table. All included studies were conducted in fourteens different countries; the largest number of included studies  was conducted in Nigeria, while three in Ethiopia, two in DR Congo, two in Burkina Faso, two in Tanzania, one in Ghana, one in Kenya, one in Malawi, one in Madagascar, one in Liberia, one in Uganda, one in one in Zambia, one in Mozambique, one in Gabon, and one in Cameroon. The largest number of included studies (n = 20) used microscopy to diagnose asymptomatic malaria, while the remaining nine, four, and two used molecular methods, microscopy and rapid diagnostic test, and rapid diagnostic test, respectively. The sample size of eligible studies ranges from 50 to 2459. All included studies were conducted between 2002 and 2020, with 9, 7, and 18 of the included studies were conducted between 2002 and 2010, 2011–2014, and 2015–2020, respectively. The risk of bias assessment of included studies showed that, respectively, 17 and 18 of them had a low and moderate risk of bias.The characteristics of 35 eligible studies included in this systematic review and meta-analysis is presented in S1 Fig). To quantitatively confirm the result of the funnel plot, we performed Egger’s regression test and Begg’s correlation test. Both Egger’s regression test (P = 0.287) and Begg’s correlation test (P = 0.966) showed the absence of publication bias. Additionally, we performed the trim and fill method and there were no filled studies that pinpoint the lack of detectable publication bias. We have not assessed publication bias on pooled ORs estimates because of the limited number of included studies.We evaluated small-studies effects (publication bias) on our pooled prevalence estimates by visual inspection of the funnel plot. According to the qualitative evaluation of funnel plot symmetry, it seems there is no evidence of publication bias  . Of the thirty five included studies, twenty-seven had reported prevalence of species-specific asymptomatic malaria infection. According to species-specific pooled prevalence estimate , P. falciparum was dominant species , followed by P. vivax, P. malariae and P. ovale, respectively, found to be 3% , 0.8% , and 0.2% . Mixed infection pooled prevalence estimate of P. falciparum and P. malariae, P. falciparum and P. vivax, and P. falciparum and P. ovale was , , and , respectively.The overall prevalence of asymptomatic malaria infection was 26.1% (95%CI: 21–31.2%) using a random-effects model, with very high heterogeneity  and microscopy , compared to studies that used microscopy and rapid diagnostic method  and rapid diagnostic method . Subgroup analysis of pooled prevalence estimate stratified by risk bias assessment was  and  for low and moderate risk of bias, respectively. Also, subgroup analysis stratified by region was 19% , 31% , and 22%  in Eastern, Western and Central African countries.Overall pooled prevalence estimate of asymptomatic malaria infection was significantly higher in studies that diagnosed asymptomatic malaria using molecular methods  showed that the odds of anemia in pregnant women infected with asymptomatic malaria infection was 2.28 higher than in pregnant women not infected with asymptomatic malaria . Pooled odds of asymptomatic malaria infection in primigravidae women was 1.55 times higher  compared to multigravida pregnant women . Pooled odds of asymptomatic malaria infection in the first and second trimester was 1.24 times higher than pregnant women in the third trimester, albeit statistically not significant  .We included nine studies to explore the relationship between asymptomatic malaria infection and anemia in pregnant women. The pooled odds ratio estimate . Also, the overall estimate of asymptomatic malaria infection was not impacted by an increase in sample size  .We carried out a meta-regression analysis to explore the trends of asymptomatic malaria infection in pregnant women over the past two decades. The result of the meta-regression analysis pointed out that the overall prevalence of asymptomatic malaria infection is not showed a statistically significant rise between the years 2002 and 2020 .We performed a sensitivity analysis to assess the robustness of our pooled prevalence estimate. The result of sensitivity analysis demonstrated that the overall estimate of asymptomatic malaria infection prevalence was robust and it was not influenced by a single study. The highest and lowest overall prevalence of asymptomatic malaria infection was 26.79% (95%CI: 21.54–32.04%)  and 24.5Asymptomatic malaria infection is a challenge for malaria elimination campaigns in such a way that asymptomatic individuals do not show overt clinical symptoms, they are less likely to seek health service, so they become a silent natural reservoir of infection that facilitates sustainable malaria transmission , 18. ParPlasmodium infections over time which leads to the development of partial anti-disease immunity [This study demonstrated that asymptomatic malaria infection was prevalent in SSA, with an overall pooled prevalence of 26.1%. The overall prevalence of asymptomatic malaria infections in the present study was significantly higher than studies conducted in low malaria transmission areas including Thailand 11.4%)  and Bang%  and Baimmunity , 37. Subimmunity , 39. ThiP. falciparum was the predominant species with an overall prevalence of 22.1% while P. vivax, P. malariae, and P. ovale overall prevalence was 3%, 0.8%, and 0.2%, respectively. This result is in agreement with the WHO 2018 report that indicated P. falciparum accounted for 99.7% of malaria in the WHO African Region [Among twenty-seven included studies that reported species-specific asymptomatic malaria infection, In this study, the result of the pooled odds ratio revealed that asymptomatic malaria-infected pregnant women were 2.28 times more likely to be anemic than pregnant women who were not infected with asymptomatic malaria infection. This result is comparatively similar to primary studies conducted in Ethiopia  and DR CP. falciparum results in sequestration and adherence of infected red blood cells with chondroitin sulfate A (CSA) on the surface of the placenta, which is mediated by parasite-encoded variant surface antigens (VSA) [The finding of this study showed that the prevalence of asymptomatic malaria infection was 1.54 times more likely higher in primigravida women compared to multigravida women. Studies conducted in stable malaria transmission areas have also shown that asymptomatic malaria infection is reduced in increasing gravidity , 44. Thens (VSA) . Anti-adns (VSA) . Since pns (VSA) , 46.In this study, some limitations are noticed. First, we performed a literature search using the English language, and we excluded three studies published other than the English language. This might not significantly impact the result although language bias was difficult to be avoided. Second, studies included in this meta-analysis are not from all African countries. Taking into account the expected variation across countries, the findings of this study might not be representative of the whole African countries. Third, a high level of heterogeneity was observed among studies that could be related to heterogeneity in diagnostic methods and publication years of studies. Given these aforementioned inherent limitations, the result of this study should be interpreted with caution.P. falciparum, P. vivax, P. ovale, and P. malariae were detected from asymptomatic malaria-infected pregnant women: However, P. falciparum was the predominant causative agent of asymptomatic malaria. Again, the prevalence of anemia in pregnant women infected with asymptomatic malaria was higher than in non-infected pregnant women. Also, primigravidae women were more likely infected with asymptomatic malaria infection compared to multigravida pregnant women. Therefore, laboratory diagnosis of asymptomatic pregnant women for malaria and anemia should be included as part of antenatal care follow-up. Also, further large-scale studies using a highly sensitive method should be conducted to better understand the burden of asymptomatic malaria in pregnancy in SSA.The findings of the present study demonstrate that asymptomatic malaria infection is prevalent in pregnant women in SSA. S1 Checklist(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S1 FigAbbreviation: se of t, standard error of t.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.


IFNL4 gene. Carriers of the IFNL4-dG allele but not the IFNL4-TT allele are able to produce the IFN-λ4 protein. Patients with hepatitis C virus that do not produce the IFN-λ4 protein have higher rates of viral clearance suggesting a potential inhibitory role of IFN-λ4 in liver-tropic infections.Interferon (IFN)- λ4, a type III IFN, production is controlled by a dinucleotide frameshift variant (rs368234815-dG/TT) within the first exon of the Plasmodium falciparum, which has a well-characterized liver stage infection, would be more susceptible to clinical malaria relative to their IFNL4-rs368234815 allele. A cohort of 122 children from a malaria holoendemic region of Kenya was analysed. Episodes of clinical malaria and upper respiratory tract infections (URTIs) were determined using information collected from birth to 2 years of age. The dinucleotide frameshift variant IFNL4-rs368234815-dG/TT was genotyped using a TaqMan assay.In this study, it was investigated whether children infected with IFNL4-rs368234815 allele was evaluated. It was found that children that carried the IFNL4-rs368234815-dG allele had an increased number of clinical malaria episodes. In addition, there was a significant association between earlier age of first malaria infection with carriers of the IFNL4-dG allele .In this cohort, 33% of the study participants had the dG/dG genotype, 45% had the dG/TT genotype, and 22% had TT/TT genotype. The number and time to first episode of clinical malaria and URTIs with respect to the P. falciparum malaria in Kenyan children.The results suggest that the ability to produce IFN-λ4 negatively affects host immune protection against The online version contains supplementary material available at 10.1186/s12936-021-03689-z. Plasmodium, with the species Plasmodium falciparum contributing the greatest morbidity and mortality. Transmission occurs through the bite of an infected Anopheles mosquito. Sporozoites injected by the mosquito travel to and infect hepatocytes, where they develop to form thousands of merozoites that then infect erythrocytes, resulting in the clinical presentation of the disease.Malaria remains a major global health burden. In 2019, there were 409,000 deaths due to malaria and an estimated 229 million cases worldwide, placing roughly half of the world’s population at risk of infection [IFNL4 gene controls the production of IFN-λ4 protein. Roughly half of the world population are carriers of the IFNL4-dG allele and are able to produce the IFN-λ4 protein, whereas carriers IFNL4-TT allele cannot [IFNL4-dG, is still retained at high frequencies in the African populations remains unclear.Type III interferons (IFNs) are antiviral cytokines with a broad antiviral activity that induce hundreds of interferon-stimulated genes (ISGs) , 3. Theye cannot . The TT e cannot . This ise cannot . The reaIFNL4-rs368234815 frameshift variation has been studied in individuals infected with hepatitis C virus (HCV) [IFNL4-dG allele (IFN-λ4 is produced) is associated with unfavorable clinical outcomes and the IFNL4-TT/TT genotype (IFN-λ4 null) with higher rates of viral clearance; the mechanism behind this paradoxical finding remains unknown [IFNL4-dG allele is associated with reduced clearance of RNA viruses that cause respiratory infections [IFNL4-dG allele have different disease risks compared to carriers of the IFNL4-TT/TT genotype. Because HCV is a hepatropic virus, it was reasoned that carriers of the IFNL4-dG allele might also have differential risk of malaria infection where infection of hepatocytes occurs during the primary stage of infection. In this study, the association between IFN-λ4 polymorphism and risk of clinical malaria and upper respiratory tract infections (URTIs) was investigated in a prospective birth cohort of Kenyan children.The effect of the  unknown –9. One p unknown . More refections . These sSamples and clinical data collected by the Chulaimbo Antenatal Postnatal (CHAP) study, a prospective cohort study conducted in Kisumu, Kenya between 2011 and 2015 were used. Details of this cohort have been described elsewhere , 13. BriP. falciparum parasites in the blood. Finger-prick blood samples collected in EDTA at ~ 6 months of age were stored at −80 °C prior to shipment of samples to the USA.The clinical data was collected by completing a structured questionnaire each time the child came for a clinic visit; children then underwent a physical evaluation and any medical findings were included in these questionnaires. URTIs were diagnosed by physical exam by the study clinical officer. Only cases where a diagnosis of URTI were included in the analysis. A small number of cases of pneumonia (< 3%) were identified and these were not included in the URTI definition. Clinical malaria episodes were diagnosed by clinical presentation and confirmed by thick and thin blood smears stained with Giemsa and visualized by microscopy to detect IFNL4-rs368234815 by custom TaqMan genotyping assays, using Genotype Master Mix (Qiagen), on BioRad iQ5, with standard conditions as previously described [Genomic DNA was extracted from whole blood using DNeasy Blood & Tissue Kit (Qiagen) and genotyped for G6PD gene were amplified by PCR using the following primers: A- Forward (5′-CAGCCACTTCTAACCACACACCT-3′), A- Reverse (5′-CCGAAGCTGGCCATGCTGGG-3′), A + Forward (5′-CTGTCTGTGTGTCTGTCTGTCC-3′) and A + Reverse (5′-GGCCAGCCTGGCAGGCGGGAAGG-3′). The PCR amplicons were subsequently subjected to restriction enzyme digestion using NlaIII with resulting fragment sizes visualized by horizontal gel electrophoresis. For A- an uncut product was found from the normal locus, whereas two DNA fragments, 218- and 134-bp, were generated in the mutant locus. For A + 2 DNA fragments, 243- and 52-bp, were found to be associated with the normal locus and 3 DNA fragments, 141-, 102-, 52-bp, were generated for the mutant locus.Glucose-6-Phosphate dehydrogenase deficiency (G6PDd) was characterized as previously published . BrieflyHb-A/S trait was characterized as previously published . BrieflyIFNL4 alleles was evaluated. Only malaria episodes or URTIs that were reported on clinic visits forms were counted for. A Negative binomial regression model with an offset for the total number of sick and follow-up visits was used to evaluate the relationship between IFNL4 alleles and the number of infections; malaria episodes and URTIs were modeled separately. Estimates from the negative binomial model were exponentiated and reported as rate ratios. To evaluate the time to first malaria infection, a Cox proportional hazards model was fit using the survival package (v 3.1–8) [G6PDd and sickle cell trait with right-censoring at the end of the 2-year study; the proportional hazards assumption was checked and was not violated. Adjustments for gravidity and maternal malaria exposure did not improve model fit or change conclusions about IFNL4 associations, so they were not included in the final cox proportional hazards model. To evaluate time to first URTI, Kaplan Meier estimators were calculated, and a log rank test was used to test for differences between IFNL4 alleles. Tables were created with the Table 3.6.0) [Using data from questionnaires collected on all clinic visits, the relationship between malaria episodes and upper respiratory tract infections (URTI) with respect to v 3.1–8) , 17 and v 3.1–8)  in R. The 3.6.0) .Table 1SP. falciparum and upper respiratory tract infection frequency, clinical data collected from 122 children that were part of a previously described birth cohort based in Western Kenya where malaria transmission is holoendemic [IFNL4- rs368234815 dG allele  and the IFNL4-rs368234815 TT/TT genotype, the relationship between maternal malaria during pregnancy and IFNL4 was analysed and no significant difference was found between the two groups . Furthermore, for this population, there was not a significant relationship between birthweight and maternal malaria exposure .To evaluate whether genetic variants in IFN-λ4 play a role in oendemic  was analIFNL4-rs368234815 polymorphism using a custom TaqMan genotyping assay ; both of which are associated with resistance to blood stage malaria infection [IFNL4- dG allele frequency in the study population was 78% with a genotype frequency of 33%, 45% and 22% for dG/dG, dG/TT and TT/TT genotypes, respectively. This distribution was consistent with Hardy–Weinberg equilibrium , telling us that the genotype frequencies seen are a simple function of allele frequency and will remain constant from one generation to the next in the absence of evolutionary influence or other disturbing factors. In addition, 23 (19%) study participants had G6PD deficiency  and 24 (20%) were found to be carriers of the sickle cell trait (Hb-A/S)   were grouped together and compared to those that did not have a dG allele (IFNL4-rs368234815 TT/TT genotype- herein IFNL4-TT/TT).To address whether there were effects on malaria or URTI, health records from a total of 1,520 clinic visits occurring between birth to 2 years of age were reviewed for the 122 children. Children that were carriers of the IFNL4-rs368234815 polymorphism and the number of URTIs during the first 2 years of life. It was found that URTI’s rate was 11.80% lower for those that had the IFNL4- TT/TT genotype relative to those that had an IFNL4-dG allele after adjusting for the number of visits   was found   . For this population, there was not a statistically significant relationship between either G6PDd or sickle cell trait and time to first malaria infection, although there was a trend towards reduced hazard of earlier episodes of first malaria (G6PDd (A-) unadjusted hazard ratio: 0.62, 95% CI 0.31, 1.23; p-value: 0.173 and sickle cell trait  unadjusted hazard ratio: 0.77, 95% CI 0.39, 1.52; p-value: 0.307). Because of the known association of G6PDd and sickle cell trait with protection from clinical malaria, a multivariable Cox proportional hazards model to control for the G6PDd and sickle cell trait was performed. There was no difference in the direction, magnitude, or statistical conclusion following adjustment  Fig. a. AlthouIFNL4- dG allele was protective against P. falciparum malaria in an infant cohort in Kisumu, Kenya a malaria-endemic region with a high rate of transmission. It was found that carriage of the IFNL4- dG allele (IFN-λ4 is produced), increased the number of clinical malaria episodes and was associated with an earlier time to first malaria infection during the first 2 years of life as compared to children with the IFNL4- TT/TT genotype. These suggests that the ability to produce IFN-λ4 negatively affects the ability to protect against P. falciparum in Kenyan children.Malaria remains a major burden worldwide but more so in the African population which has the highest malaria associated morbidity and mortality especially in children . A numbeIFNL4- dG allele and HCV infection and found that the ability to produce IFN-λ4  has a negative effect on viral clearance either spontaneously or after treatment [IFNL4- dG allele carriers, IFN-λ4 is the first response mounted against P. falciparum in the liver but this response alone cannot clear infection and other IFN responses are inhibited. It was recently shown in a rodent malaria model using Plasmodium berghei, that liver stage infection is sensed by the host and activates a type I IFN response that is able to control parasite load and mediate host resistance to reinfection [IFNL4- dG allele carriers (produce IFN-λ4) type I IFN response would be suppressed, via the negative regulators induced by IFN-λ4, and the host would not be able to control parasite load, leading to a higher number of malaria episodes.The results presented here are consistent with several studies that have looked at the relationship between carriers of the reatment –9. Why ereatment . One posreatment . Based onfection . This leIFNL4-dG allele [IFNL4- dG allele had more URTIs during the first 2 years of life, although this was not statistically significant. These results are consistent with the study in Rwanda and suggests that the capacity to express IFN-λ4 may increase the risk of URTIs.Type III IFNs are known to be the first line of defense for respiratory RNA viruses . For exaG allele . This st, IFNL4-dG allele, is still conserved in the African population and has not changed to the derived human-specific allele, IFNL4-TT allele, remains unknown. Carriage of the IFNL4-dG allele is clearly not providing protection against HCV [P. falciparum malaria in children. Undoubtedly there are other selection pressures conserving this allele in this population.The reason why the ancestralinst HCV –9, respiinst HCV , gastroiinst HCV , human cinst HCV , and nowPlasmodium species Plasmodium yoelii and infection of mice, it was reported that the absence of IFNλ signaling decreased parasite burden and increased early antibody titers [IFNL4-dG allele) has during malaria infection is due to its effect on blood or liver stage of P. falciparum infection? It is shown here that after adjusting for G6PD deficiency  or sickle cell trait , both associated with resistance to blood stage malaria infection, carriers of the IFNL4 TT/TT genotype had a reduced risk for malaria infection. These results would argue that IFNL4 action is more important for modulating the liver stage of malaria infection as compared to a role in blood stage infection.Interestingly, in a study using the rodent y titers . This suIFNL4 alleles. In addition, the study was not designed to evaluate the cause of URTI’s and, therefore, might have missed disease specific effects. There is also a likelihood that not all children came to the clinic when they were exhibiting symptoms potentially resulting in a failure to capture some episodes of infection. It is important to mention that since these children were part of a cohort study, they had access to regular medical care potentially reducing the frequency of infections and the severity of malaria observed. One of the questions that was not addressed in this 2-year study is if the ability to produce IFN-λ4, which correlates to earlier time to first infection and more clinical malaria episodes during infancy has any long-term detrimental effects on this pediatric study population.A main strength of this study is that it was a prospective cohort study that followed children from birth to 2 years of age and this allowed us to measure malaria episodes over time. A limitation of this study was a small sample size, which in some cases (e.g. URTI’s) hindered the ability to detect a significant difference between carriers of the different IFNL4- dG allele) negatively affects the ability to protect against P. falciparum malaria during infancy in children living in a malaria holoendemic region of East Africa. These results are consistent with a longitudinal study of children in Mali, West Africa [In conclusion, this study suggests production IFN-λ4 , had a detrimental effect on carriers, increasing the frequency of infections and time to first malaria infection during infancy, presenting a risk for the host. This finding is paradoxical from the known role of interferons in inducing an antiviral state in cells suggesting a different mechanism for function of IFN-λ4 in parasitic infections. Production of IFN-λ4 suppresses the type I IFN response, via the induction of negative regulators. In rodent models, the type I IFN response can control Additional file 1: Fig. S1. Representative allelic discrimination plot for genotyping of IFNL4-rs368234815 polymorphism by custom TaqMan genotyping assay. A clear separation of the homozygotes is shown, heterozygotes on the other hand show presence of both alleles, with different expression levels between samples. HapMan controls are shown in duplicate, inside boxes, and five randomly selected study subjects gDNA samples are also included.


Malaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets.Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing.Cross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae, 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus (s.s.). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax, which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium-infected mosquitoes was successively confirmed by sequencing analysis.Overall malaria prevalence in our study population was 55.0% (752/1367) and P. vivax infection in Cameroon.Our study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired  Malaria remains a deadly scourge in many parts of the world, with 409,000 deaths from this disease reported globally in 2019. Over the past 10 years, a declining trend has been observed in the global incidence of cases and deaths due to malaria, 57 and 10%, respectively [Anopheles species present in Cameroon, only 16 are able to transmit the disease to humans [Anopheles gambiae, An. coluzzii, An. arabiensis, An. funestus, An. nili and An. moucheti [In Cameroon, malaria remains a serious public health problem. Transmission is heterogeneous across the country, ranging from perennial transmission in the southern forested regions to seasonal and unstable transmission in the northern Sudano-savannah and Sahelian regions. Of the 52 formally recognized o humans , and themoucheti , 8. ThesPlasmodium species are known at the present time to cause malaria, among which P. falciparum is considered to be the most prevalent and virulent parasite, responsible for 82–100% of all malaria cases depending on the locality [Plasmodium are also present in Cameroon, including P. malariae, P. ovale and P. vivax [. The latter is the most geographically widespread malaria parasite that causes disease in humans outside of Africa and is especially prevalent in Southeast Asia and the Americas [Pv absence in Africa” [P. vivax infections in Duffy-negative individuals across Africa, and in the intervening time different studies have reported P. vivax infections in Mauritania [P. vivax with human infections in both Duffy-positive and -negative individuals has been reported, albeit at low frequencies. However, no study has yet looked at which local malaria vector species is involved in the transmission of P. vivax.Nevertheless, the effectiveness of control measures is threatened by the rapid expansion of insecticide resistance in vector populations –13, chanlocality –26. OtheP. vivax –30. The Americas , 32. IndAmericas , 34. ThiAmericas , and ind Africa”  began touritania , 38, Ethuritania , 40, Equuritania , Mali [4uritania , Democrauritania , Benin [uritania , Nigeriauritania  and Cameuritania –30. In CP. vivax detection as this species was recently reported in other parts of the country, and we conducted entomological surveys to identify its putative vector, which has not been investigated in earlier studies. Our main goal was to examine the potential risk of transmission of P. vivax and to determine which local malaria vector species could be implicated in its transmission in this region of Cameroon.In this study, we aimed to characterize malaria parasites and vectors circulating in an area of seasonal transmission in the Adamawa region. We focused on The study was conducted in the town of Tibati , which is the capital of Djerem department in the Adamawa region of Cameroon. The population of Tibati is estimated to be 108,502 persons. The town is characterized by a predominantly wet tropical climate with two seasons, a dry season extending from November to February and a rainy season from March to October during which the intensity of transmission is high. The annual rainfall ranges from 1192 to 2023 mm, and the average annual temperature is around 23.6 °C. There is a dense hydrographic network of several rivers, such as the Djerem, Meng and Tomi rivers, in the area around Tibati .n = T2 × P(1 − P)/M2, where n = sample size; T =  confidence level at 95% ; P = malaria prevalence in the Adamawa region (34.2%) according to the Programme National de Lutte contre le Paludisme 2019 [M = margin of error at 5% (0.05). Blood from asymptomatic carriers was collected from individuals living in randomly selected houses in the community, from eight neighborhoods within the Tibati city perimeter   with fever or a history of fever in 2015 and from asymptomatic individuals in the community in 2017. Patients with clinical malaria were recruited among patients in three randomly selected health facilities: the Missionary Hospital of Ngaoubela, the District Medical Center and the Integrated Medical Center located at around 7 km, 600 m and 100 m from the city center, respectively. The minimal estimated sample size of 346 participants was determined by the formula sme 2019 ); and M Plasmodium-positive patients were treated with an ACT according to the recommendations of the Ministry of Health of Cameroon .Blood was collected from volunteers of all ages independently of gender and ethnicity. Adults (≥ 18 years old) or legal guardians were asked to provide written informed consent for underaged volunteers (< 18 years old). Blood was obtained by fingerprick in volumes of 5 and 100 µl for thick smears films and dried blood spots on Whatman™ grade 1 and FTA paper , respectively. Thick blood smears were stained with 10% Giemsa for 20 min  and exam d-phenothrin, 0.01%) was sprayed into all the corners of the house. After 15 min, mosquitoes that had fallen onto the sheet were collected, counted and identified at the genus or species level. Anopheles mosquitoes were identified using morphological identification keys [Anopheles specimens were dissected individually, and the carcasses and head-thoraces were stored separately in tubes containing a desiccant, archived and kept at − 20 °C for further molecular analysis.Mosquito collections were carried out in the morning from 6:00 to 10:00 a.m., and written informed consent was obtained from household owners prior to the start of collection. A white sheet was used to cover the floor and all flat surfaces, and the pyrethrum residual  . BrieflyTo optimize the DNA extraction and allow for a better separation of DNA from some nucleases, we used the Qiagen DNeasy® Blood and Tissue Kit  to isolate DNA from the blood samples collected in 2017, in accordance with the manufacturer’s instructions, and resuspended the isolated DNA in 60 µl elution buffer.Genomic DNA from individual mosquito carcasses was isolated using the 2% cetyltrimethyl ammonium bromide (CTAB) method, following the protocol of Collins et al. . DNA froPlasmodium infection status of blood collected in 2015 was determined by a multiplex PCR [P. falciparum, 300 bp for P. vivax, 375 bp for P. ovale and 412 bp for P. malariae.The plex PCR , which tplex PCR . The ampAnopheles gambiae complex by PCR–restriction fragment length polymorphism (RFLP) [Anopheles funestus group were identified according to the protocol of Koekemoer et al. [Genomic DNA from mosquito carcasses was used to identify members of the m (RFLP)  and membr et al. . All PCRPlasmodium-infected mosquitoes, as previously described [Plasmodium species, including the aquaglyceroporin gene  for P. falciparum, the enoyl-acyl carrier protein reductase gene  specific for P. vivax, the P25 ookinete surface protein gene  for P. ovale and the circumsporozoite gene  targeting P. malariae [A simple quantitative PCR (qPCR) was performed to identify escribed . Selectemalariae . Reactio An. gambiae complex and the members of An. funestus group were identified as described above. Detection of Plasmodium in blood spots and mosquito head-thoraces for these samples was achieved by multiplex qPCR in the Lightcycler 96 real-time PCR system, according to the protocol published by Mangold et al. [. The 18S rRNA gene of the P. falciparum 3D7 clone was used as positive control for Plasmodium species differentiation.For the samples from 2017,d et al. , with slPlasmodium-positive mosquitoes were sequenced using the Big Dye Terminator v3.1 Sequencing Kit (Applied Biosystems) and run on an Applied Biosystems 3130xl Sequencer at the GenSeq technical facility of the Institut des Sciences de l’Evolution de Montpellier. Sequences were verified using SeqScape software (Applied Biosystems).The qPCR products of Plasmodium infections among population age groups and mosquito species. The non-parametric Kruskal–Wallis test was used to assess differences in parasite densities between age groups. The significance threshold was set at alpha = 0.05. The relative risk (RR) was computed to estimate the protection associated with LLIN use. R software version 3.5.3 with maptools and ggplot2 packages was used to generate the map and figures, respectively , while melting temperatures of qPCR-positive controls were retrieved by using functional bases (without package).Data were stored in Microsoft Office Excel files  and transferred into GraphPad Prism 7  for statistical analyses. The Mann–Whitney test was used to compare mean parasite densities according to clinical status, for both trophozoites and gametocytes. The Chi-square test was used to compare the prevalence of A total of 1367 participants were enrolled in this study, 418 through the health centers (symptomatics) and 949 at the community level (asymptomatics). The main characteristics of the participants are detailed in Table  vs 38.75 ± 7.85, Mann–Whitney U = 1992; P < 0.0001) and asexual stages . The mean asexual parasite densities significantly decreased with age in blood samples from both symptomatic and asymptomatic participants . The sex ratio  was 0.7 (558/809). Mean parasite densities were significantly higher in symptomatic patients from the health centers than in asymptomatic individuals recruited in the community, for both sexual (6199 ± 2626Plasmodium. The infection rate was 41.8% (175/418) in samples from symptomatic patients and 60.8% (577/949) in samples from asymptomatic individuals, which is significantly different . Plasmodium falciparum was the most prevalent Plasmodium species collected during both collection periods and represented 98.8 and 92.9% of human infections in 2015 and 2017, respectively. Plasmodium malariae and P. ovale each accounted for 0.6% of clinical infections. In comparison, in samples from asymptomatic persons a higher prevalence was recorded for P. malariae (22.9%) while P. ovale was rare (0.9%). Mixed infections were only found in asymptomatic carriers and were mostly represented by P. falciparum–P. malariae . No P. vivax infection was detected in any blood sample.Of the 1367 samples, malaria diagnostic tools revealed 752 (55.01%) infections with χ2 = 48.39 and χ2 = 49.91 respectively; P < 0.001). Gametocyte carriage was significantly higher in asymptomatic individuals than in symptomatic patients . Heterogeneity in malaria infection was observed between neighborhoods, with infection rates varying from 48.2 to 71.2% inSabongari and Ngaoubela, respectively  and An. funestus (s.l.). Molecular identification of specimens of An. gambiae sibling species showed that this fauna was composed of two species: An. gambiae  and An. coluzzii . Anopheles funestus (s.s.) was the only species identified within the An. funestus group and represented 35.1% (208/592) of anopheline mosquitoes. The distribution of the three species according to the two collection periods is shown in Fig. An. coluzzii and An. gambiae mosquitoes was detected.A total of 592 iae 212; 5.8% and Anopheles were subjected to qPCR to detect infected mosquitoes. The results were obtained according to the melting temperature value given by an optical reading of the corresponding melting curve. In total, 26 (4.39%) Anopheles mosquitoes were infected by a Plasmodium sp., of which 24 could be clearly identified according to melting temperature. However, the remaining two samples (YK60 and NG239) gave a bimodal curve and were assigned as putative co-infection. YK60 was suspected to be P. malariae/P. ovale co-infection, with the main peak assigned to P. malariae and the minority peak to P. ovale . Variation in mosquito infection rate was observed between neighborhoods in 2017, with Yoko (5/9) recording the highest number of infected mosquitoes , P. malariae (19.2%) or P. ovale (15.4%). A single sample was found by qPCR to be positive for An. coluzzii with P. vivax.All Plasmodium-positive mosquitoes were processed for sequencing, and the sequence alignment of the P. vivax infection is presented in Fig. All This study was performed to assess epidemiological and entomological parameters of malaria in Tibati, a locality situated in the Adamawa region of Cameroon. At the present time, little data exist on malaria in this area.The incidence of malaria is known to vary according to epidemiological settings, age and clinical status of the study population and season, as well as with treatment guidelines and control interventions. Malaria prevalence in our study area varied from 42% in symptomatic patients to 61% in asymptomatic individuals in the community. This prevalence value is 15-fold higher than that reported by Songue et al.  in MeidoPlasmodium is therefore crucial for providing appropriate treatment regimens to patients. Determining the burden of malaria is also important for applying effective malaria control strategies in endemic settings and for determining their efficacy. In Cameroon, a nation-wide mass distribution campaign of LLINs took place in 2011, 2015 and 2019, and the NMCP reports a LLIN coverage of > 80% [P. falciparum prevalence [Almost half (42%) of the patients who went to the health care centers for consultation/treatment were diagnosed with malaria infection. Malaria remains the primary cause of fever in febrile patients, but other infections, such as Dengue, Chikungunya or Zika, likely also circulate in the area. Indeed, clinical symptoms of malaria are non-specific, and inter-epidemic arboviral infections are regularly reported in central Africa –64. Accuof > 80% . In our evalence . It mustevalence , 68.Asymptomatic infections represent the major reservoir of parasites . In our Plasmodium species in human blood samples, among which P. falciparum was the most predominant vector species, accounting for over 95% of malaria infections. These data are consistent with previous reports in other epidemiological settings, indicating that P. falciparum predominates across Cameroon [Plasmodium malariae and P. ovale were found as mono- and co-infections with P. falciparum, but at low prevalence and these species were rarely found in symptomatic individuals. In the present study, no P. vivax infection was detected either in symptomatic or asymptomatic samples. Circulation of P. vivax in Cameroon has been recorded since 2014 in different epidemiological settings, even though previous reports documented P. vivax infections only in travellers returning from Cameroon [P. vivax infections in Cameroon were mostly identified at low frequencies, representing < 15% (13/269) of malaria infections in Bolifamba, southwest region [P. vivax infections among symptomatic patients seeking medical attention at the New Bell district hospital in Douala, Littoral region [P. vivax in the present study suggests that the parasite circulates at very low frequency; as such, larger samples would be necessary to confirm this species in the area. The prevalence of P. vivax in Africa differs between epidemiological settings, and it has been suggested that P. vivax is more prevalent in areas with lower malaria burden [In this study, we identified three Cameroon , 79, 80.Cameroon , 82. In t region , 6% , and the change in the density of this species is likely due to modifications of environmental cues, such as the presence of permanent breeding sites (e.g. lakes and rivers with emergent vegetation) that are favorable for the proliferation of this mosquito species[An. funestus mosquitoes benefited An. coluzzii, which represented 9.7% of collected anophelines in 2015 and 47.4% in 2017. The presence of An. gambiae was stable over the two collection periods. The study area is irrigated by the Meng river and is characterized by the presence of agricultural and fish farming sites that create temporary sites which are favorable for the development of An. gambiae (s.l.), including An. gambiae and An. coluzzii [Anopheles coluzzii has a known high capacity of adaptation in urban environments [An. coluzzii. Nevertheless, the use of the IRS method, which is a widely used method for sampling endophilic mosquitoes, could have favored the collection of endophilic resting Anopheles over other mosquito species.We performed entomological surveys to identify mosquito vectors responsible for malaria transmission in Tibati. To date, six main vector species, namely Cameroon , 6, 84.  country , 85–89. o species, 90. Thicoluzzii , 92. Anoronments –95, and Plasmodium infection rates did not differ between An. gambiae, An. coluzzii and An. funestus, and thus these three species are considered to transmit malaria at equal efficiency. Sporozoite infection rates fell within the range reported in a previous study conducted in the Adamawa region [ vs 4.39%; X2 = 2.791, P = 0.0948); this reduction, although not significant, could be due to the use of LLINs following the massive country-wide distribution campaign in 2015 [We found that a region  and in oa region , 84. A s in 2015 . The perP. falciparum, P. ovale, P. malariae and P. vivax. A single An. coluzzii mosquito was carrying P. vivax and the fact that only one mosquito was infected by P. vivax confirms the low circulation of this species in our study area. Further longitudinal studies or more in-depth sampling would increase the probability to detect P. vivax infections in both human and mosquito populations, but it remains that this one mosquito represents the first report of Anopheles infection by P. vivax in Cameroon. Transmission of P. vivax by anopheline mosquitoes has already been reported in sub-Saharan Africa, but never by An. coluzzii. Mosquito infections with P. vivax have been reported for An. arabiensis in Mauritania [An. funestus in Madagascar [An. gambiae in Kenya [P. vivax infections in mosquitoes, but identification of the vector species was not recorded [P. vivax infection in mosquitoes (10.99%) as compared to human population (4.55%) [P. vivax-infected mosquito we identified was an An. coluzzii specimen, a species that has shown greater susceptibility to malaria infection in a study from Cameroon [Plasmodium vivax can then be considered to circulate in the study area and may also be underestimated, as a proportion of P. vivax infections in humans are “hidden” since hypnozoites lie dormant in the liver for several months (or years) where they are undetected.All three mosquito species were mono-infected with uritania , Madagasuritania , 99 and uritania , for An.dagascar , 101, Kedagascar  and for in Kenya . Also, ain Kenya  and Equarecorded . Interes 4.55%) . The P. % . The PP. vivax infection in the human population. Regular monitoring of malaria infections will be necessary to assess the true circulation of P. vivax in the area; we cannot exclude the possibility that the P. vivax-positive mosquito got infected while feeding on a non-resident as Tibati is a cross-border city with a high circulation of people.A limitation to this study is that mosquito samples were obtained at a single collection time and, therefore, the results only provide a one-shot picture of malaria transmission in the study site. Longitudinal surveys would be necessary to follow the dynamics of malaria transmission and, particularly, will be crucial to perform parasitological and entomological surveys before and after the implementation of control interventions, such as LLIN distribution. A second limitation is that sample sizes were small, which is possibly the reason we did not detect An. funestus, An. gambiae and An. coluzzii, were responsible for the transmission of the disease, with all three species contributing equally to Plasmodium transmission. Parasitological indices suggest low bednet usage and that the implementation of control interventions in the area is needed to reduce the malaria burden. We identified hotspots of disease transmission, and these sites should be the target of malaria control efforts. In addition, the presence of P. vivax in an An. coluzzii mosquito prompts for regular monitoring as the spread of this species could introduce further complexity into malaria epidemiology and control measures in this area.We have provided a picture of the epidemiological and entomological malaria situation in Tibati, a small town in the Adamawa region of Cameroon. Malaria prevalence varied from 42% in symptomatic patients to 61% in asymptomatic individuals, and this finding highlights the high malaria endemicity in the area. Three major vectors, namely


P. falciparum, the most virulent and predominant malaria parasite primarily targeted in malaria control and elimination program, is paramount to assess intensity of parasite transmission. Analysis of the block 3 region of the msp-2 gene of P. falciparum provides strong molecular evidence to evaluate the real picture of malaria epidemiology to fine-tune the ongoing control and elimination programs in the region. Thus, this study was aimed at examining the status of such polymorphic gene and its implications in Adama and its surroundings.  Despite significant progress achieved globally in reducing malaria burden, still it is one of the major public health and economic problems in Ethiopia. Investigation of the local genetic polymorphism of msp-2 allelic families were genotyped by using nested polymerase chain reaction targeting its 3D7 and FC27 allelic variants followed by gel electrophoresis for fragment analysis.  148 isolates from patients with uncomplicated falciparum malaria were collected in the study from September 2019 to August 2020. Tween® 20 and the Chelex method were employed for parasite DNA extraction.  Seventeen different polymorphic forms of msp-2 allelic fragments were detected in the study area. Moreover, 47 (31.8%) and 41(27.7%) were detected for 3D7 and FC27 allelic families, respectively. Furthermore, the multiclonal allele type accounted for 60 (40.5%). The mean MOI was 1.4, and the heterogeneity index (He) is 0.49 indicating nearly intermediate malaria transmission in the study area. P. falciparum in the study area, demanding further scale up of the ongoing control and elimination efforts. The study revealed nearly intermediate genetic diversity and mean MOI of  P. falciparum and P. vivax, respectively [P. vivax and P. falciparum are the two coendemic species in the country, sharing the overall burden of 30% and 70%, respectively [World Health Organization (WHO) report shows that in 2019, the number of malaria cases was 229 million, out of which 409,000 deaths were recorded. More than 90% of all malaria cases and death were only from sub-Saharan Africa (SSA) [ectively . Malariaectively , which iectively .P. falciparum are another challenge.In Ethiopia, even though the various intervention efforts made so far have significantly reduced the disease burden, this promoted the country to move forward and plan for malaria elimination strategies in selected areas . HoweverP. falciparum infection is characterized by having higher genetic diversity, which is implicated in its evolutionary fitness, and consequently having more survival advantage by overcoming the control efforts [In a malaria endemic region,  efforts . Genetic efforts , 12 but msp-2 gene [msp-2 gene located on chromosome 2. It is composed of five blocks of which the central block (block 3) is the most polymorphic part [msp-2 has been used extensively to describe the parasite populations as a discriminatory and informative marker for strain differentiation [msp-2 gene is dimorphic and existing in two main allelic families: FC27 and 3D7 [P. falciparum and its multiplicity of infection (MOI) describes the level of malaria transmission and the effectiveness of ongoing malaria control and elimination strategy. For successful malaria elimination, information on the genetic profile of the parasite population in different geographical areas and factors that determine gene flow between locations is paramount. Even though genetic diversity of P. falciparum has been extensively studied in different parts of the world, there is limited information in Ethiopia. Therefore, the aim of this study was to examine genetic polymorphism of the block 3 region of the msp-2 gene of P. falciparum and its MOI in Adama town and its surroundings, Oromia Regional State, Central Ethiopia. Attempt was made to analyze the spatial and temporal feature of such polymorphism in the study area and its comparison with different localities showing brunt of falciparum infection and endemicity.The most commonly used technique for molecular characterization of such polymorphism in a malaria endemic area is the use of nested amplification of merozoite surface proteins (MSP) like the block 3 region of the p-2 gene , 14. MSPhic part , 16. Thuntiation . Due to  and 3D7 . TherefoThe study samples were collected from three districts in Central Ethiopia , including Adama town administration from September 2019 to August 2020. Adama is the major town next to the main capital in Ethiopia, located at a distance of about 99 km southeast of Addis Ababa. Adama District includes Wonji located at 8 km south and Awash Melkasa situated at a distance of 15 km southeast of Adama. Other health facilities are located in neighboring districts like Modjo located at 16 km northwest and Olanciti situated at 23 km northeast of Adama. The population projection of the catchment study area estimated to reach 800,000 inhabitants. Adama and its neighboring districts are located in the rift valley area of Central Ethiopia , where mP. falciparum-infected malaria patient in the study area. The age of study participants ranged from one to sixty-six presenting to selected health facilities. The inclusion criteria for this study were uncomplicated malaria cases due to P. falciparum and having history of fever onset since 24 hours of the clinical examination. Health facilities were selected based on a record of higher patient caseloads at their respective location. Through finger prick, P. falciparum-positive blood samples were spotted on Whatman™ 3MM filter paper for DBS preparation, by trained laboratory technologists, and it is temporarily stored together with relevant patient data. The prepared DBS is later transported to the Adama regional laboratory research center and kept at -20°C prior to molecular laboratory work. Malaria microscopy was conducted for symptomatic patients as per the national parasitological diagnosis of malaria at each health facility. The identified falciparum-positive microscopic slides were collected from sample collection sites for further analysis. Two independent WHO-certified laboratory technologists for malaria microscopy proofread the slides for malaria parasite species identification and parasitaemia level, at the Adama malaria diagnostic center. Parasite density was calculated by averaging the two closest counts. The parasite density per microliter (μl) of blood was estimated by counting the number of WBC by the field examined assuming that 8000 WBC were present in 1 μl of blood. The number of parasite density per microliter (μl) of blood was calculated by using the following formula [A total of 148 blood spotted samples (DBS) were collected from September 2019 to August 2020, from  formula , 22:(1)μl blood), L2 (50-499 parasites/μl blood), L3 (500-4999 parasites/μl blood), L4 (5000-49999 parasites/μl blood), and L5  [For comparison with ranked-order variables during data analysis, parasitaemia were categorized into five levels: L1 (<50 parasites/l blood) .msp-2 gene amplification were conducted at malaria research laboratory, Aklilu Lemma Institute of Pathobiology, Addis Ababa University (AAU). The extraction of genomic DNA of P. falciparum DBS was carried out by using 0.5% Tween® 20  to lyse RBC and tracked by treatment with 6% Chelex® 100  in a water bath at 96°C [The parasite genomic DNA extraction and its  at 96°C .P. falciparum msp-2 gene (block 3) was used as a genetic marker for the genotyping of parasite populations. Nested PCR of the msp-2 (block 3) polymorphic region was performed by slight modification of primers and methods from the previously described reports [μl amplification mixture containing 1x FIREPol® Master Mix (4 μl), 0.5 μl of each primer (M2-OF and M2-OR), 11 μl of nuclease-free water aliquot, and 4 μl of DNA template. In the second (N2) amplification reaction, 20 μl amplification mixture containing 1x FIREPol® Master Mix (4 μl), 0.5 μl of each primer FC27 (B1)/3D7 (A1) and FC27 (B2)/3D7 (A2), and 13 μl of nuclease-free water aliquot to 18 μl to which 2 μl of DNA template was added for the amplification of each allelic variants was used.The polymorphic region of the  reports , 24  (USA) and photographed to estimate band size in relation to the 50 bp DNA ladder   is defined as the number of distinct parasite genotypes coexisting within the given infection. In the present study, it is based on genotyping of the C27 gels , 27. Ampe marker , 29, whimsp-2 gene in the isolates. Descriptive statistics were used to calculate the frequency of each msp-2 allelic family in relation to the total number of genes successfully amplified for that locus. Size polymorphism in each allelic family shows that one band represents one amplified PCR fragment derived from a single copy of the P. falciparum msp-2 gene. When alleles in each family were less than 20 bp, they were considered the same. The Pearson chi-square test was conducted for statistical comparison of categorical variables. P < 0.05 was used to test the level of statistical significance to accept or reject the hypothesis. All statistical tests were performed by using SPSS version 20.0 . In addition, geometric mean of parasite density was calculated by using the Microsoft Excel 2016 version.Allele frequency represents the incidence of the allelic variant (3D7 or FC27) in the sample isolates. It could be determined by dividing the number of times the allele of interest (3D7 or FC 27) is observed in a population by the total number of copies of all alleles detected for the P. falciparum with different alleles at a given locus was calculated by the formula:n is the number of the isolates analyzed and p represents the frequency of each different allele at a locus.The heterogeneity index (He) representing the probability of being infected by two different populations of P. falciparum clinical isolates were successfully amplified for the msp-2 gene. Of which, 105 (71%) were males. The age of the study participant ranged from 1 to 66 (mean ± SD) (27.0 ± 13.6∗) years. Parasite density ranged from 64 to 104,320 parasites/μl with a geometric mean of 5654 parasites/μl. Of all study participants, 85 (57%) were from urban inhabitants. Of all the study subjects by occupation, students, daily laborers, and farmers alone accounted for 113 (76%) P. falciparum malaria cases in the study area. Moreover, P. falciparum infection was found to be significantly related (X2 = 0.017) to occupation type of the patients  isolates were from urban inhabitants and 57 (38.5%) of the isolates from Adama  = 29.6, P ≤ 0.001). This shows heterogeneity in its distribution.From the total successfully amplified om Adama . Patientariation , which wmsp-2 gene of P. falciparum in Adama and its surroundings revealed its polymorphic nature. The frequency of 3D7 and FC27 was 31.8% (47/148) and 27.7% (41/148), respectively. Moreover, 40.5% (60/148) carried both 3D7 and FC27 alleles, with mean MOI of 1.4 and the heterogeneity index (He) of 0.49  and ten FC27 (250-700 bp) different size polymorphic alleles were observed.Allele genotyping of the  of 0.49 . In addimsp-2 gene allelic variant among symptomatic patients tends to increase with age groups, although the variation was not statistically significant (X2 = 0.09). Likewise, though not linearly, the mean MOI slightly increased with age groups  = 1.1, P = 0.56).Moreover, analysis of the urban rural distribution of the patterns , showingP. falciparum msp-2 provides comprehensive molecular evidence for effective disease surveillance that could be ultimately translated to core interventions in the control and eventual elimination of malaria.Ethiopia has now moved forward in targeting a nationwide malaria elimination program by 2030. For effective implementation of this strategic target, one of the key intervention strategies is improving malaria surveillance and response . In thisP. falciparum isolates in the study area was higher in male (71%)   . The maj= 0.017)  which ists alone .msp-2 block 3 region of the P. falciparum genetic profile revealed in this study was the first in its kind in the study area. The authors examined seasonal and spatial distribution of msp-2 allelic variants in the study area . In complement with this finding, Funwei and his colleagues [msp-2 allelic variant and its multiple infections vary from urban-rural location depending on transmission intensity and independent of seasonal change. The major factor that might have contributed to such inconsistent report could be differences in local epidemiology of malaria parasite flow between urban and its surrounding rural areas. In the present study, analysis of the variation of msp-2 allelic frequency across different age groups has shown increment in parallel with P. falciparum clinical prevalence during the study period. Moreover, in this study, mean MOI slightly increased with age when compared with younger children . This could be due to differences in local microecological factors, change in parasite vector interaction, and spatial heterogeneity of the study sites under consideration [In the current study, analysis of the ral area  during tlleagues  reportedlleagues  reportedchildren . This fichildren  might beia level . This coudy area  showed aderation , 48.P. falciparum genetic diversity in Central Ethiopia by using one of the most polymorphic msp-2 genes, the study has some technical limitations. Firstly, the association between the observed dominant allelic families and disease severity was not examined, because all samples were collected from uncomplicated malaria patients. Secondly, the number of alleles maybe underestimated due to the limitations of techniques (nested PCR amplification and gel-electrophoresis) used. Therefore, further characterization of such polymorphic region needs to be designed by increasing the sample size and implementation of advanced techniques like analysis of microsatellite markers followed by capillary electrophoresis and DNA sequencing analysis for samples isolated from symptomatic and asymptomatic cases for further study.Even though, this is the first study to analyze P. falciparum clinical isolates among symptomatic malaria patients. Therefore, to sustain such declining phase of malaria transmission in Central Ethiopia, in addition to the classical epidemiological study approach, the ongoing malaria control and elimination program should be accompanied by similar molecular surveillance for targeted intervention. Moreover, the present study revealed the absence of significant variation in allele frequency in both urban and its rural counterpart and similar allele frequency during the major and minor malaria season, reflecting frequent inbreeding among the existing parasite strains in both settings. This indicates strong evidence for the presence of similar malaria epidemiology in the urban and rural location of the study area demanding a similar intervention strategy. Thus, the findings described in this study will serve as baseline molecular evidence for further research on areas having similar malaria epidemiology to make the control and elimination efforts effective.The present study revealed a nearly moderate transmission rate of


Malaria and sexually transmitted/reproductive tract infections (STI/RTI) are leading and preventable causes of low birthweight in sub-Saharan Africa. Reducing their impact on pregnancy outcomes requires efficient interventions that can be easily integrated into the antenatal care package. The paucity of data on malaria and STI/RTI coinfection, however, limits efforts to control these infections. This study aimed to determine the prevalence and associated factors of malaria and STI/RTI coinfection among pregnant women in rural Burkina Faso.A cross-sectional survey was conducted among 402 pregnant women attending antenatal clinics at the Yako health district. Sociodemographic and behavioral data were collected, and pregnant women were tested for peripheral malaria by microscopy. Hemoglobin levels were also measured by spectrophotometry and curable bacterial STI/RTI were tested on cervico-vaginal swabs using rapid diagnostic test for chlamydia and syphilis, and Gram staining for bacterial vaginosis. A multivariate logistic regression model was used to assess the association of malaria and STI/RTI coinfection with the characteristics of included pregnant women.The prevalence of malaria and at least one STI/RTI coinfection was 12.9% , malaria and bacterial vaginosis coinfection was 12.2% (95% CI: [9.3–15.9]), malaria and chlamydial coinfection was 1.6% (95% CI: [0.6–3.8]). No coinfection was reported for malaria and syphilis. The individual prevalence was 17.2%, 7.2%, 0.6%, 67.7% and 73.3%, respectively, for malaria infection, chlamydia, syphilis, bacterial vaginosis and STI/RTI combination. Only 10% of coinfections were symptomatic, and thus, 90% of women with coinfection would have been missed by the symptoms-based diagnostic approach. In the multivariate analysis, the first pregnancy (aOR = 2.4 [95% CI: 1.2–4.7]) was the only factor significantly associated with malaria and STI/RTI coinfection. Clinical symptoms were not associated with malaria and STI/RTI coinfection.The prevalence of malaria and curable STI/RTI coinfection was high among pregnant women. The poor performance of the clinical symptoms to predict coinfection suggests that alternative interventions are needed. In sub-Saharan Africa (SSA), 880,000 stillbirths and 1.2 million neonatal deaths occur each year . IntrautAntenatal care (ANC) is a unique opportunity to screen, treat and prevent health conditions that negatively affect the pregnancy outcomes. The intermittent preventive treatment of pregnant women using sulfadoxine–pyrimethamine (IPTp-SP) is recommended to prevent malaria infection and its associated negative effects on the pregnancy outcome . In the In Burkina Faso, the prevalence of malaria infected pregnant women during the peak transmission varies between 20 and 50% in the rural area depending on the climatic patterns , 16. DesThe study was conducted in the antenatal clinics of three peripheral health centers  of the Yako health district, northern part of Burkina Faso. In this area, malaria transmission is holo-endemic with a peak of transmission during the rainy season (July–November) . The heawww.pactr.org, registration number PACTR201808177464681). At enrollment, pregnant women were screened for malaria and STI/RTI  between August 2019 and September 2020.This was an ancillary study of a main project comparing the efficacy of IPTp-SP versus IPTp-SP plus azithromycin to prevent low birthweight  (Standard Diagnostics Inc. Korea) for syphilis test on blood samples, and the one-step chlamydia antigen rapid Test SD-Bioline Chlamydia (sensitivity 93.1% and specificity of 98.8%) (Standard Diagnostics Inc. Korea) for chlamydia detection on cervical swabs. Hemoglobin level was measured using a portable spectrophotometer . The antenatal clinics provided HIV counselling and testing; however, these data were not included in this analysis as the approved study protocol did not include HIV data collection. Malaria was tested by standard microscopy at the laboratory of the Clinical Research Unit of Nanoro (CRUN) and bacterial vaginosis (BV) tested by Gram staining and the determination of the Nugent score : 21–30). Nearly all women had partners (married or living together) at the time of the survey (95.3%) and 55.6% of them were unemployed (housewives). The overall highest educational level was low as nearly four in ten women (41.5%) had never been at school and only 2.2% reached university level. The body mass index (BMI) was normal (from 18.5 to 25 kg per mNearly one-tenth (9.5%) of women had at least one clinical symptom such as fever (axillary temperature ≥ 37.5 degree centigrade), vaginal burn, pelvic pain, vaginal discharge, or vulvar/perianal itching at the clinical and obstetrical examination. The most common symptom was vaginal itching (20/402), and followed by vaginal discharge (18/402). A high proportion (63.5%) of pregnant women were also anemic .Most of the pregnant women declared to have their first sexual intercourse before their 20th birthday (68.9%) and only a minority of them (5%) confirmed the use of condom during their last sexual intercourse. Alcohol use defined as a daily uptake of more than one “standard drink”  was common (18.2%). Nearly three-quarter (77.6%) of women declared the use of insecticide treated bed net (ITN) the night before their visit.One third of women (30.1%) visited the health center for their first pregnancy, whereas the other two-third (69.9%) visited for their second or subsequent pregnancies. Miscarriage and stillbirth were frequent in the study setting (15.4%).Plasmodium falciparum species : [13.7–21.3]) and all were due to the The prevalence of malaria and at least one STI/RTI coinfection was 12.9% (95% CI: [9.8–16.7]) and that of malaria and bacterial vaginosis coinfection was 12.2% (95% CI: 9.3–15.9]). Cases of syphilis were detected but no coinfection with malaria was reported (Table –15.9). Cp value = 0.01). Anemic women  and unemployed women  were also at increased risk of coinfection but did not reach significance level. In the final multivariate model, malaria and at least one STI/RTI coinfection was significantly associated with the first pregnancy   and anemia  tended to be associated with malaria and STI/RTI coinfections but without reaching statistically significance level .In the univariate analysis, women under 20 years of age, and women at their first pregnancy were at significantly higher risk of coinfection  increased the risk of coinfection. Indeed, previous study conducted in the country reported much lower rates of bacterial vaginoses with 6.4% in 2008 , and 12.We did not report any coinfection of malaria and syphilis although, separately, both infections were reported. The low prevalence of syphilis reported in this study (0.6%) is likely the reason behind this absence of coinfection between the two infections. However, the low prevalence of syphilis and the absence of coinfection in this study should not undermine the systematic screening of these infections as the cost effectiveness of such intervention is well established even in a context, where the rate is lower than 2% , 27. In Chlamydia Trachomatis [We reported a low proportion of pregnant women coinfected with malaria and chlamydial coinfection in the study settings. The use of rapid diagnosis test in this study likely understated the actual prevalence of chlamydial infections and thus its coinfection with malaria. Indeed, PCR-based diagnostic demonstrated higher prevalence in East Africa with 13% of women infected with chomatis , 29. GivWe noted that first pregnancy women were at higher risk of malaria and STI/RTI coinfections. The high susceptibility of primigravid women to malaria , combineAs expected in this study setting, there was no direct link between clinical symptoms and coinfection. Indeed, only 10% of cases were symptomatic, and yet nearly 90% would have been missed and not adequately managed. This confirms that clinical symptoms cannot reliably predict neither malaria infection  nor STI/In this study, we described malaria and curable sexually transmitted/reproductive tract coinfection in pregnant women in a rural health district of Burkina Faso. Some limitations are worth noting. Important infections such as gonorrhea were not tested due to the absence of rapid test or an easy to implement laboratory diagnostic method and this understated the actual magnitude of coinfection in the study population. In addition, the blood smear microscopy-based diagnosis of pregnancy malaria might have understated the prevalence of malaria and STI/RTI coinfection in the current study through its relative limited performances. Gestational age was collected using the mother’s knowledge of last menstrual period or the obstetrical examination in majority of cases due to the absence of ultrasounds, and this process is frequently prone to errors. However, the study is worth emphasizing the burden of malaria and STI/RTI coinfections and suggest that improved approaches are needed.In this study population, the prevalence of malaria and sexually transmitted/reproductive tract coinfections were high and were not predicted by the clinical symptoms. Thus, exploring an alternative approach to systematically prevent malaria and treat STI/RTI would be of public health interest.