Malaria is a major public health concern among pregnant women in sub‐Saharan Africa. Within the region, Nigeria has the highest malaria cases. This study aimed to determine the prevalence and factors associated with malaria parasitaemia among pregnant women at a booking clinic in Ibadan, Nigeria.A cross‐sectional study was conducted between January and April 2021 at the University College Hospital in Ibadan, Nigeria. A sample of 300 pregnant women participated, and anaemia and malaria were diagnosed using packed cell volume and Giemsa‐stained blood smears, respectively. Data analysis was done using SPSS 25.0.p < 0.05.The study found that 26 (8.70%) pregnant women tested positive for malaria parasitaemia. Factors such as age, religion, level of education, and occupation were significantly related to the prevalence of malaria parasitaemia among pregnant women with Our study identified a high prevalence of malaria parasitaemia among pregnant women with demographic factors such as age, religion, level of education, and occupation significantly associated. Targeted malaria control interventions for pregnant women with low levels of education and low‐income occupations are necessary, with more research needed to evaluate their effectiveness. The World Health Organization reports that in 2018, approximately 228 million new malaria cases were reported worldwide, with most of them in Africa.Pregnancy increases the risk of severe disease for women infected with malaria due to immune system suppression.Most infections in pregnancy are asymptomatic, undetected, and untreated, leading to negative outcomes for both the mother and the foetus.Pregnant women in Nigeria are particularly vulnerable to malaria infection due to various factors such as poor access to quality healthcare services, poverty, and demographic characteristics.Studies have found that demographic factors such as age, level of education, and occupation are significantly associated with malaria prevalence among pregnant women in Nigeria.The current prevalence of malaria in pregnancy at the University College Hospital in Ibadan booking clinic is unknown. A prospective review of malaria in pregnancy protocols is necessary to provide updated evidence and information. The results of this study will contribute to developing an evidence‐based protocol for malaria during pregnancy in this clinical setting and similar settings in Nigeria, sub‐Saharan Africa and beyond.22.1The study was carried out at the Department of Obstetrics and Gynaecology Department of University College Hospital (UCH) in Ibadan, Nigeria, during the first quarter of 2021. UCH is a tertiary care medical center linked with the College of Medicine at the University of Ibadan, which was established in 1952 and had 850 available beds. The Obstetrics and Gynaecology department, one of the largest in the hospital, has 24 consultants and 29 resident doctors in training, with 180 total beds, 120 for obstetrics and 60 for gynaecology. The department typically sees 2000–2400 annual prenatal appointments and delivers 2500–3000 births, with 900–1100 spontaneous vaginal deliveries and an average of 1250–1500 C‐sections, with 750–850 as emergency and 500–600 as elective procedures.2.2This study was designed as a cross‐sectional investigation conducted at the antenatal clinics of University College Hospital in Ibadan, Nigeria. The study aimed to determine the prevalence of malaria parasitaemia among pregnant women at booking and identify demographic factors associated with the prevalence of the disease. Ethical clearance was obtained from the Ethics Committee of the University of Ibadan/University College Hospital before the commencement of the study.A simple random sampling method was used to recruit participants for the study. The process involved assigning a unique identifier to each pregnant woman attending the antenatal clinic at the University College Hospital in Ibadan. The identifiers were then entered into a randomization tool, a Microsoft Excel sheet, which generated a list of 300 identifiers. The list of identifiers was used to recruit participants from the antenatal clinic until the desired sample size of 300 was achieved. This method ensured that each pregnant woman had an equal chance of being selected for the study without any biases or pre‐selection based on any characteristics. Using a simple random sampling method, the study aimed to ensure that the sample was representative of the pregnant women attending the antenatal clinic at the University College Hospital in Ibadan.Women were eligible for participation if they attended the antenatal clinic and agreed to participate. However, those who had already received antimalarial treatment or prevention, those who declined to participate, or those with sickle cell anaemia were excluded from the study. The principal investigator verified each participant's eligibility and obtained their informed consent, and the allocation numbers were recorded on the participant's forms for data analysis purposes.Informed consent was obtained from each participant before the beginning of data collection. The process of obtaining informed consent was conducted thoroughly, which involved explaining the study's purpose and procedures, including the risks and benefits of participation. Participants were informed of their right to refuse or withdraw from the study at any time without consequence. During the informed consent process, potential participants received a copy of the informed consent form to read and sign. For participants who could not read or write, the informed consent form was read aloud and explained in detail by the research team. Participants who agreed to participate in the study were asked to sign or thumbprint the informed consent form.Data collection was carried out using a structured questionnaire and laboratory investigations. The questionnaire was designed to collect detailed demographic characteristics such as age, religion, level of education, and occupation. The laboratory investigations utilized the gold‐standard microscopy method to test for malaria parasitaemia, the recognized diagnostic technique for detecting malaria.1.The prevalence of malaria parasitemia among pregnant women at booking in Nigeria is high at the time of booking.2.Sociodemographic factors such as age, religion, level of education, and occupation are associated with malaria parasitemia among pregnant women at booking in Nigeria.The study considered two main hypotheses:2.3The study obtained blood samples from pregnant women visiting the antenatal booking clinics who met eligibility criteria and provided informed consent. The samples were collected by the head researcher and sent to a laboratory for examination. The principal investigator then refined and analysed the information using participant data forms.2.4The sample size of 300 was calculated using the Cochrane formulawhereN = minimum sample sizeZ = standard score corresponding to a given confidence level. At a 95% confidence level or 5% significance level (0.05), Z = 1.96.p = prevalence of malaria parasitemia and prevalence of 38.8% in a previous study in Northcentral Nigeria was usedd = precision limit or proportion of sampling error at 6% (0.06)N = 275.392N = 275Considering an attrition rate of 10% = 25.3Total sample size = 275 + 25 = 300.2.5Statistical analysis was conducted using the Social Sciences Statistical Package (SPSS‐25.0). The resulting data was analysed to generate tables, percentages, and Chi‐square tests.2.6The study was granted ethical approval from the Institutional Review Board of the University of Ibadan/University College Hospital. Participants were thoroughly informed about the study procedures and allowed to participate voluntarily. They were assured that declining to participate would not impact their future medical care. The study team obtained written consent from all individuals who agreed to participate.3This study investigated the prevalence of malaria parasitaemia among pregnant women using a sample of 300 patients drawn at the University College Hospital, Ibadan booking clinic. Table Table Table χ2 = 299.000, p < 0.05; χ2 = 10.942, p < 0.05; χ2 = 249.347, p < 0.05; and χ2 = 141.811, p < 0.05). In contrast, factors such as marital status and tribe were not significantly related to the prevalence of malaria parasitemia.The social‐demographic characteristics of the respondents significantly associated with the prevalence of malaria parasitaemia among pregnant women at the booking stage are presented in Table χ2 = 40.746, p < 0.05; χ2 = 141.811, p < 0.05; and χ2 = 40.746, p < 0.05). Nullipara and multipara were also observed to have a higher number of nonprevalent malaria parasitaemia compared to primipara.Table 4The study aimed to assess the prevalence of malaria parasitaemia among pregnant women during their first prenatal visit to University College Hospital in Ibadan. The study considered the sociodemographic characteristics and pregnancy history. The findings revealed that 8.7% of pregnant women had malaria parasitaemia. This figure is comparable to the rate found in Ethiopia but lower than that in Ghana, Burkina Faso, and Malawi, which reported rates of 10.2%, 20.4%, 18.1%, and 19%, respectively.Our study investigates the social demographic factors that may have a significant relationship with malaria parasitemia in pregnant women. Our findings show that the patients’ education level and occupation were significantly associated with malaria parasitaemia. In particular, higher levels of education can lead to increased health awareness, which may contribute to a lower prevalence of malaria. This finding is consistent with previous research, where women with a high school education or higher were 0.53 times less likely to have malaria than those without education.This study investigated the relationship between parity and malaria prevalence among pregnant women. The results revealed a statistically significant association, with primigravida having a higher malaria prevalence than multigravida. This finding may be explained by the acquired immunity that develops with increasing parity, reducing susceptibility to malaria infection in multigravida. These findings are consistent with a study conducted in Uganda, where primigravid women had an 84% higher risk of microscopic parasitaemia at enrollment and nearly three times the risk of placental malaria compared to multigravida women.The use of insecticide‐treated nets (ITNs) has been demonstrated to be a highly effective means of reducing the burden of malaria among pregnant women. However, in the current study, only 39.7% of participants reported using ITNs. This finding highlights the need to improve access to and utilization of ITNs in the study population. This result is consistent with a study by Njumkeng5This study had some limitations. One potential limitation of this study is that it was conducted in a single hospital in Nigeria and, therefore, may not represent pregnant women in other regions or healthcare settings. Furthermore, the study relied on self‐reported participant data, which may introduce the possibility of recall bias or social desirability bias. Finally, the study did not include a comparison group of nonpregnant women or men, which limits the ability to conclude the prevalence of malaria parasitaemia in the general population. Statistical limitations of the study include the lack of adjustment for potential confounding factors in the analysis of the relationship between occupation and malaria parasitaemia and the lack of multivariable analysis to identify independent risk factors for malaria parasitaemia among pregnant women. Despite these limitations, the study's findings provide valuable information on the prevalence of malaria parasitaemia among pregnant women in Nigeria and its associated risk factors. These findings can guide the development of targeted interventions to reduce the burden of malaria in pregnancy and improve maternal and foetal outcomes.6The findings of our study demonstrate a noteworthy prevalence of malaria parasitaemia among pregnant women within the targeted study population, thus raising concerns regarding the health implications and management strategies associated with this condition. Demographic factors such as age, religion, level of education, and occupation were significantly associated with malaria parasitemia. Specifically, younger pregnant women, those with lower levels of education, and those in low‐income occupations were at a higher risk of malaria infection. To address this issue, targeted malaria control interventions are urgently needed for these high‐risk groups of pregnant women. These interventions can include increasing access to insecticide‐treated nets, providing regular malaria screening and treatment during prenatal visits, and promoting health education programmes focusing on preventing and managing malaria during pregnancy. However, further research is needed to evaluate these interventions’ effectiveness in reducing malaria burden among pregnant women. This could involve conducting randomized controlled trials to assess the impact of interventions on malaria prevalence and assessing the cost‐effectiveness of different interventions to inform policy decisions.Olufiade P. Oyerogba: conceptualization; data curation; formal analysis; writing—original draft; writing—review & editing. Aduragbenro Adedapo: conceptualization; formal analysis; project administration; writing—original draft; writing—review & editing. Titilayo Awokson: methodology; project administration; writing—original draft; writing—review & editing. Akin‐Tunde Odukogbe: conceptualization; methodology; project administration; supervision; writing—review & editing. Nicholas Aderinto: project administration; writing—review & editing.The authors declare no conflicts of interest.The study received ethical clearance from the University of Ibadan/University College Hospital Institutional Review Committee. Participants were informed of the study procedures and allowed to participate voluntarily. Those who chose not to participate would not be affected in their future care. Written consent was obtained from all participants before their inclusion in the study.The lead author Nicholas Aderinto affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


Haemoglobinopathies such as sickle cell disorder and glucose-6-phosphate dehydrogenase (G6PD) deficiency as well as differences in ABO blood groups have been shown to influence the risk of malaria and/or anaemia in malaria-endemic areas. This study assessed the effect of adding MNP containing iron to home-made weaning meals on anaemia and the risk of malaria in Ghanaian pre-school children with haemoglobinopathies and different ABO blood groups.n = 860). Participants were randomly selected into clusters to receive daily semiliquid home-prepared meals mixed with either micronutrient powder without iron (noniron group) or with iron  for 5 months. Malaria infection was detected by microscopy, blood haemoglobin (Hb) levels were measured with a HemoCue Hb analyzer, the reversed ABO blood grouping microtube assay was performed, and genotyping was performed by PCR–RFLP analysis.This study was a double-blind, randomly clustered trial conducted within six months among infants and young children aged 6 to 35 months in rural Ghana . The prevalence rates of sickle cell traits (HbAS and HbSC) and sickle cell disorder (HbSS) were 17.5% and 0.5%, respectively. Blood group O was dominant (41.4%), followed by blood group A (29.6%) and blood group B (23.3%), while blood group AB (5.7%) had the least frequency among the study participants. We observed that children on an iron supplement with HbAS had significantly moderate anaemia at the endline (EL) compared to the baseline level (BL) (p = 0.004). However, subjects with HbAS and HbAC and blood groups A and O in the iron group had a significantly increased number of malaria episodes at EL than at BL (p < 0.05). Furthermore, children in the iron group with HbSS (p < 0.001) and the noniron group with HbCC (p = 0.010) were significantly less likely to develop malaria.The prevalence of G6PD deficiency among the study participants was 11.2%. However, the prevalence of G6PD deficiency in hemizygous males (8.5%) was significantly higher than that in homozygous females (2.7%) (Iron supplementation increased anaemia in children with HbAS genotypes and provided less protection against malaria in children with HbAC and AS and blood groups A and O.NCT01001871. Registered 27/10/2009.clinicaltrials.gov Identifier: https://clinicaltrials.gov/ct2/show/record/NCT01001871.Registration number:  Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) enhances parasite clearance, which reduces the risk of clinical malaria [Haemoglobinopathies and ABO blood types have been shown to influence the risk of severe malaria and anaemia –3. It ha malaria –6. Moreo malaria , 8. Gene malaria –11.Over the past two decades, iron (Fe) intervention studies in many parts of the world, including Ghana, have shown that the use of microencapsulated iron micronutrient powder (MNP) “Sprinkles”  is cheaper, safer because it is cost effective and has less side effects compared to the use of iron droplets or syrups, and more effective in reducing anaemia rates by up to 60% –21. MoreNonetheless, there is limited knowledge about the effects of iron-fortified micronutrient diets on ABO blood groups, haemoglobin and G6PD genotypes and the risk of contracting malaria and anaemia among pre-school children living in malaria endemic areas. Furthermore, the geographical distribution of ABO blood groups, haemoglobin and G6PD genotypes are often associated with anaemia and malaria protection among certain ethnic groups, but their effect among iron-fortified infants and young children is unknown , 16, 23.This retrospective community-based, double-blinded, cluster-randomized controlled trial (RCT) was conducted in 2009 among children aged 6 to 35 months over a 6-month period in a rural setting in the middle belt of Ghana . The chiPlasmodium falciparum [This study was conducted in Wenchi Municipal and Tain District located within the forest-savanna zone in the middle belt of Ghana. The mean temperature in the area ranges between 18 and 38 °C with an average rainfall of 1250 mm per annum, occurring mainly between May and October, as previously reported elsewhere . Malarialciparum . At the lciparum , 25, andlciparum . In 2010lciparum . There alciparum . A compolciparum .Approximately 2220 infants and young children were screened. Those on weaning foods in addition to breastmilk, free from major illness, afebrile and living in the study area for the duration of the trial were enrolled in the study. Parental consent was obtained after the possible risks and benefits had been discussed with the parents or guardians (caregivers of the eligible child) in the appropriate local language and signed. Therefore, in 2013, the nutritional status was defined by WHO as follows; Moderate wasting (MW): -3 ≤ weight-for-height (WLZ/WHZ) < -2: Moderate stunting (MS): -3 < length-for-height (LAZ/HAZ) < -2: if height or length was measured differently according to the age: Moderate underweight (MUW): -3 ≤ weight-for-age (WAZ) < -2 as previously described in details elsewhere , 28. TheA detailed overview of the context of trial recruitment and randomization has previously been published elsewhere . SubjectHealth Organization (WHO), International Nutritional Anemia Consultative Group (INACG), and United Nations Children's Fund (UNICEF) recommended that the ‘Sprinkle’s formulation should contain 12.5 mg of elemental iron (as microencapsulated ferrous fumarate) plus ascorbic.acid (30 mg), vitamin A (400 μg), and zinc (5 mg) , 32. The+ analyzer  was used to measure haemoglobin levels, and children found to be severely anaemic (Hb < 70 g/L) were immediately referred. The Malaria Rapid Diagnostic Test (RDT)  was performed quickly in the field, and a 3-day course of treatment was given to those who tested positive for malaria. They were only enrolled later upon recovery if any other eligibility requirements had been met. The remaining sample was transferred to the haematological, malaria microscopy and immunogenetic testing laboratory.Baseline (BL) and endline (EL) MNP intervention of the child’s health was assessed (including axillary temperature), and a capillary blood sample of 500µL from the finger or heel was taken into a 0.5 mL ethylene diamine tetra-acetic acid (EDTA) tube. A HemoCue Hb 201During the MNP intervention period and at six-month follow-up, children found to be febrile  or have had a fever in the past 48 h were, blood sampled of 100μL in 0.5 mL EDTA for haematological and malaria tests. Anthropometric, socioeconomic, demographic, adherence to MNP intervention, insecticide-treated bed net use and participants' morbidity data were collected during the study as previously described in details elsewhere , 23.. Human genomic DNA was purified from blood samples in EDTA tubes stored between 2 and 8 °C using the QIAamp® DNA Mini Kit  protocol based on published methods [Both thick and thin smears were prepared on the same slide at the laboratory for malaria parasitaemia and speciation. Thin films were fixed with methanol, and both smears were stained with Giemsa. Two independent microscopists read each slide, while a third microscopy was conducted in case of over 50% discrepancy as previously described , 23. Com methods , 35.2  and 10X ProofStart PCR Buffer  containing 15 mM MgCl2. The cycling conditions were as follows: initial denaturation at 95 °C for 15 min, 34 cycles of denaturation at 95 °C for 45 s, annealing at 65 °C for 45 s and extension at 72 °C for 120 s, followed by a final extension at 72 °C for 10 min in a Peltier thermal cycler (PTC) – 200 model . The PCR product was analyzed by electrophoresis  at 90 V for 60 min using 1µL of blue DNA loading dye  on 3% (w/v) agarose  with ethidium bromide  staining, and pictures were taken using the Ultra-Violet Product (UVP) Biospectrum AC Imaging system with Biochemi-camera .Polymerase chain reaction (PCR) for haemoglobin genotyping amplified a 358 base pair region within the β-globin gene in human genomic DNA using primer sequences SC1F: 5’-AGGAGCAGGGAGGGCAGGA-3’ and SC2R: 5’-TCCAAGGGTAGACCACCAGC-3’ based on published methods . PCR ampMnlI and DdeI  were added to the two separate PCR products and incubated at 37 °C for 3 h and run on 3% agarose gel electrophoresis for 45 min to further differentiate into their various genotypes [Thereafter, restriction fragment length polymorphism (RFLP) was used to differentiate between the point mutations (which translate into the various sicklings) within the amplified gene region. The enzymes enotypes .2.Polymerase chain reaction for G6PD genotyping amplified 354 base pair  and 296 base pair  DNA regions within the G6PD gene in human genomic DNA using primer sequences 5’-CAG CCA CTT CTA ACC ACA CAC CT-3′5’-CCG AAG TTG GCC ATG CTG GG-3’ for allele 202 (354 base pairs) and 5’-CTG TCT GTG TGT CTG TCT GTC C-3’’5’-GGC CAG CCT GGC AGG CGG GAA GG-3’ for allele 376 (296 base pairs) manufactured by Sigma-Aldrich Inc. PCR amplification was performed in a 50μL reaction containing 20 – 40 ng genomic DNA, 20 mM of each primer , 25 mM of each of the four deoxynucleotide triphosphates (dNTPs), 5.0 units of ProofStart DNA Polymerase (QIAGEN Ltd. UK and Ireland) and 10X ProofStart PCR Buffer (QIAGEN) containing 15 mM MgClNlaIII and FokI (New England BioLabs Inc.) and incubated at 37 °C for 3 h. Thereafter, 3% agarose gel electrophoresis was run for 1 h. The restriction enzyme NlaIII cleaved allele 202 PCR product (354 base pairs), while the FokI restriction enzyme cleaved allele 376 (296 base pairs) to enable discrimination between the various polymorphisms [The cycling conditions were as follows: initial denaturation at 95 °C for 10 min, 34 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 60 s, followed by a final extension at 72 °C for 5 min in a PTC – 200. The PCR product was analyzed by electrophoresis on 3% (w/v) agarose with ethidium bromide staining, and pictures were taken using UVP. The PCR products were digested with restriction enzymes orphisms .https://clinicaltrials.gov/ct2/show/record/NCT01001871.The study protocol was reviewed and approved by the ethics committees of Kintampo Health Research Centre Institutional Ethics Committee; Approval ID: KHRC/IEC/FEA/2009–2, Ghana Health Service Ethics Committee, Food and Drug Authority of Ghana and Hospital for Sick Children Research Ethics Board (REB), Toronto, and Canada REB File No.: 1000013476 approved the study as previously reported in detail elsewhere , 23. ThiAll methods were performed in accordance with the relevant guidelines and regulations by the Declaration of Helsinki. The trial was overseen by an independent Data and Safety Monitoring Board (DSMB). International and local health policymakers with experience in randomized controlled trials, nutrition, paediatrics, statistics, and social sciences made up the members of the DSMB. At the end of the recruiting phase and halfway through the intervention stage, the DSMB's statistician summarized the accumulated outcome data for any major adverse effects. The primary caregivers gave their consent for their children to participate in this study. For the interim analysis, if the iron group had any more major adverse events  than the noniron group, the trial would be terminated.p-value < 0.05 was considered statistically significant.Clinical and epidemiological data were entered into a data processing system for Visual Fox Pro version 9.0 , imported into STATA version 14.0 (Statcorp Inc. TX. USA) and SigmaPlot version 11.0 (Systat Software Inc. CA. USA) for analysis. World Health Organization anthropometric software was used to transform the age, weight and length of the study participants into growth indices: weight-for-age, weight-for-length and length-for-age z-scores. Infants with z-scores less than -2 standard deviations of the median reference length-for-age (LAZ), weight-for-length (WLZ) and weight-for-age (WAZ) were classified as stunted, wasted and underweight, respectively . An overA total of 2220 children were screened from 22 communities and over 80% were recruited to receive the multi-micronutrient powder Fig. . This stp > 0.05)  Table . Of 409 5) Table .Table 1Bp = 0.005). The sickling traits (HbAS & HbSC) and disorder (HbSS) among participants were 17.5% and 0.5%, respectively  were significantly more likely to develop anaemia than blood group A children. However, ABO blood group were not statistically associated with malaria (Table p > 0.05) were not significantly less likely to develop malaria (Table p < 0.05). Conversely, no significant association were found between G6PD genotypes and anaemia  (Table The children from the iron group with HbAS had significantly higher moderate anaemia at EL than at BL p = 0.00 Table 4p = 0.004p < 0.05). The mean number of malaria episodes among participants with blood groups A and O in the iron group was also significantly increased compared with children in the noniron group (p < 0.05) (Table p = 0.03)  was significantly higher than that among females (2.7%) among the study participants. Similarly, Carter et al.  reported. . This ma. , with va. . The pre. –53. Bloo. , Cameroo. , Brazil .  and Ghan. . The G6P. . Our obs. , Ghana [.  and Nige. .. [. [The iron group HbAS and noniron group HbAC children were less protected against anaemia at the end of the MNP intervention. However, studies have shown that the presence of HbS alleles enhances the removal of parasitized sickle trait red blood cells (RBCs) via the spleen and reduces the cytoadhesion of parasitized erythrocytes, resulting in increased anaemic status among affected haemoglobin genotypes . In agre.  demonstr. . While p. . Further.  and Pate. [. , a study. [. . Albiti . [. .The O blood group has anti-A and B antibodies with no terminal saccharide moiety, making its RBCs more haemolysis-stable than the non-O blood group . However. (2014), who reported the existence of a protective association for the G6PD A- variant against uncomplicated malaria in Burkina Faso [. [This study was performed during the rainy season (March to September), and malaria parasitaemia was high in both groups. Although malaria parasitaemia was higher in the noniron group than in the iron group, there were similarities between the groups. However, there was a higher risk of malaria for children with G6PD A-A- in both groups. These findings were inconsistent with investigations by Ouattara et alina Faso , and a s Faso [. , who est Faso [. . However Faso [. . Moreove Faso [. . The reaMoreover, our study found that the iron group with HbAS and HbAC was less protected against malaria because the administration of iron increased haemolysis of AS red blood cells by promoting auto-oxidation of haemoglobin S (ferroptosis) and the generation of free oxygen radicals (due to failure of glutathione-dependent antioxidant defences), which damaged the red cell membrane and further damaged it by the deposition of ferritin-like and haemosiderin-like iron on the red cell surface and eventually excessive loss of erythrocytes. Our study was consistent with similar research among pregnant Gambian women with HbAS who had a lower anaemia status when offered iron supplements . HoweverFurthermore, blood group A and O participants were less protected against malaria in the Fe group. The possible reason for this could be that malaria parasites easily mimic antigen A, with the erythrocytes of blood group A forming larger and stronger rosettes that selectively allow merozoites to prefer A antigen receptors for RBC invasion. Therefore, the presence of iron acts as a nutrient for the parasites, thereby increasing parasitization and the rapid expression of surface cell membrane proteins to enhance rosette formation and cytoadherence in the endothelia of the microvessels. In addition, iron elevated macrophage-facilitated phagocytes of malaria parasitized RBCs of blood group O, which increased their susceptibility to malaria.Iron supplementation increased anaemia in children with haemoglobin AS genotypes and offered less protection against malaria for children with haemoglobin AC and AS and blood groups A and O. Therefore, this new evidence will inform global and national policy on the risks and benefits of iron interventions and guide the implementation of programs to prevent and treat iron deficiency disorders in malaria endemic regions.


Plasmodium species from studies conducted in Mauritania and published since 2000.Understanding malaria epidemiology is a critical step toward efficient malaria control and elimination. The objective of this meta-analysis was to derive robust estimates of malaria prevalence and 2 index and Cochran’s Q test. Publication bias was assessed with funnel plots and Egger’s regression tests.The present review followed the PRISMA guidelines. Searches were conducted in various electronic databases such as PubMed, Web of Science, and Scopus. To obtain pooled prevalence of malaria, meta-analysis was performed using the DerSimonian-Laird random-effects model. Methodological quality of eligible prevalence studies was assessed using Joanna Briggs Institute tool. Inconsistency and heterogeneity between studies were quantified by the I2 = 99.8%, P < 0.0001) by microscopy, 25.6%  by PCR and 24.3%  by rapid diagnostic test. Using microscopy, the prevalence of asymptomatic malaria was 1.0%  against 21.46%  in symptomatic malaria. The overall prevalence of Plasmodium falciparum and Plasmodium vivax was 51.14% and 37.55%, respectively. Subgroup analysis showed significant variation (P = 0.039) in the prevalence of malaria between asymptomatic and symptomatic cases.A total of 16 studies with a good individual methodological quality were included and analysed in this study. The overall random effects pooled prevalence of malaria infection (symptomatic and asymptomatic) across all included studies was 14.9% (95% confidence interval [95% CI]: 6.64, 25.80, IPlasmodium falciparum and P. vivax are widespread in Mauritania. Results of this meta-analysis implies that distinct intervention measures including accurate parasite-based diagnosis and appropriate treatment of confirmed malaria cases are critical for a successful malaria control and elimination programme in Mauritania.The online version contains supplementary material available at 10.1186/s12936-023-04569-4. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae) has been reported in Mauritania [Plasmodium falciparum and P. vivax are by far the most predominant malaria species in the country [P. falciparum prevails, P. vivax is rare [P. vivax is the major malaria species in northern Saharan arid zone, notably in oases [P. falciparum and P. vivax across the two ecological zones in Mauritania, i.e. Sahelian and Saharan zones, respectively, are still unknown. Anopheles arabiensis is widespread in the country [In Mauritania, malaria is endemic with a seasonal and unstable transmission –4. The turitania –6. Plasm country , 3, 6. Iin oases , 6. Howe country –12. It iP. falciparum-specific histidine-rich protein-2 (HRP2)- and Plasmodium genus-specific plasmodial lactate dehydrogenase (‘Pan’ pLDH)-based rapid diagnostic test (RDT). Studies on the performance of some RDTs for malaria reported contrasting levels of sensitivity and specificity to detect malaria [Malaria diagnosis is generally established in health facilities either by presumptive clinical diagnosis or, more often, by  malaria , 13. Mic malaria , is rarePlasmodium species, mass distribution of long-lasting insecticidal nets (LLIN), intermittent preventive treatment in pregnant women using sulfadoxine-pyrimethamine (IPTp-SP), and more recently, seasonal malaria chemoprevention (SMC) in children under five years of age [The main target of the current malaria control strategy of the Mauritanian government is to eliminate malaria from the country by 2030 . This sts of age , 17. HowPlasmodium species distribution [Plasmodium species distribution is essential for informed decisions on appropriate control strategies.During the past two decades, numerous studies on malaria prevalence have been conducted in Mauritania by different investigators using various approaches in different ecological conditions and epidemiological context , resulting in heterogeneous malaria positivity rates and ribution , 18–27. ribution , 29. The2  and sub-Saharan Africa with a surface area of 1,080,000 kmThe total estimated population was 4.7 million in 2020, of whom approximately one fourth are living in Nouakchott, the capital city . In the https://www.ird.fr/bureau-du-chercheur) for papers published in French. The database search was performed using a combination of terms/keywords and Boolean operators according to each database instructions  guidelines . Publishth January 2023) in either English or French were included in the analysis. The inclusion criteria were original study conducted in Mauritania to assess malaria prevalence with basic information concerning the sample size, diagnostic methods, Plasmodium species, and the main characteristics of the enrolled subject. The exclusion criteria were as follows: studies published before January 1, 2000, reviews, letters to editors, editorials, commentaries, expert opinions, books, book chapters, brief reports, articles in which only the abstract was available, conference abstracts, theses, case reports, and case series. In addition, articles in which malaria prevalence was not determined and studies with reported malaria prevalence based on small  sample size were also excluded.Studies published between January 2000 and January 2023  quality assessment tool for prevalence studies . The too® Statistical Software version 20.115 . Cochrane Q test and Inconsistency index (denoted I2 statistics) were used to evaluate the magnitude of heterogeneity among studies included in meta-analysis. A Cochrane Q value with P < 0.05 indicated significant heterogeneity among studies. I2 values < 25%, 25–75% and > 75% were interpreted as low, moderate, and considerable heterogeneity, respectively.A quantitative pooling of results was done to perform a meta-analysis using MedCalc2 = 99.5%, P < 0.001). As a result, a random-effects model was performed to estimate the Der Simonian and Laird's pooled effect. Subgroup analyses were conducted to explore the potential sources of heterogeneity, including the following variables: size of the study population, eco-epidemiological zone of the study settings, malaria diagnostic method used, status of study participants with respect to malaria (whether symptomatic or asymptomatic). Forest plots were used to display point estimates and confidence intervals.Preliminary analysis showed a high degree of heterogeneity among the included studies  . The majority of the subjects  were enrolled in Nouakchott. Participants in 11 of 16 (68.7%) included studies were febrile patients with suspected malaria recruited at health facilities, while they were asymptomatic in 3 (18.8%) studies, both asymptomatic and febrile subjects in 1 (6.2%) study, and only malaria-positive patients in 1 (6.2%) study  were from the southern Sahelian region and 18,104 (45.1%) from the northern Saharan region (P = 0.0018) between the proportions of males  and females .Enrolled participants belonged to all age groups in 13 of 16 (81%) included studies and children under 14 years in 3 (18.8%) studies. The sex of the participants was reported in 13 of 16 (81.2%) studies. Overall, there was significant difference  in 5 (31.2%) studies, and both RDT and microscopy in 6 (37.5%) other studies. In the remaining 5 (31.2%) studies, malaria diagnosis was based on microscopy alone (n = 3), microscopy and PCR (n = 1), or RDT alone (n = 1).P. falciparum, P. vivax, P. malariae, and P. ovale) were diagnosed among enrolled patients with a majority of cases  being infected by P. falciparum, followed by those infected by P. vivax . Rare cases of P. falciparum-P. vivax mixed infections (n = 4) and P. vivax-P. ovale (n = 2) were also recorded  consisted of 178 P. falciparum-P. vivax mixed infections, 6 P. falciparum-P. malariae mixed infections, and 1 P. falciparum-P. vivax-P. malariae mixed infection  by microscopy , 25.6%  by PCR , and 24.3%  by RDT   observed in asymptomatic individuals in Kaedi situated along the Senegal River , while tPlasmodium falciparum and P. vivax were the two dominant species using microscopy (n = 12 studies) with a pooled prevalence of 51.1%  and 37.5% , respectively  of the global malaria infections and 96.2% (178/185) of the mixed infections.95% CI: 2.5, 77.4 2 > 97% in all subgroups, P < 0.0001), sub-group meta-analysis was performed by eco-epidemiological zones of the study sites (i.e. Sahel vs. Sahara), sample size of enrolled subjects by study , malaria diagnostic method (microscopy vs RDT vs PCR), and type of malaria cases (i.e. symptomatic vs. asymptomatic)  Fig. .Fig. 5SuP = 0.963) in malaria prevalence between studies having sample size > 1,000 study participants  and those having sample size < 1,000 study participants . However, a significant difference (P = 0.039) was observed between malaria prevalence in symptomatic patients  compared to asymptomatic individuals . The estimated prevalence of malaria in the Sahelian zone was 11.7% , while the estimated malaria prevalence in the Saharan zone was 21.8  (P = 0.510). There was no significant difference between malaria prevalence according to the diagnostic method employed (P = 0.441).Accordingly, the meta-analysis showed no significant difference .The presence of publication bias was evaluated subjectively using funnel plots and objectively using the Egger’s test and Begg’s test. The studies’ effect sizes were plotted against their standard errors to generate the corresponding funnel plot Fig. . Visual Plasmodium species using data from 16 eligible prevalence studies involving 40,138 participants from 18 study sites in Mauritania. The overall prevalence of malaria was 14.9%, 24.3%, and 25.6% using microscopy, RDT, and PCR, respectively. Although PCR consistently showed a higher sensitivity in detecting malaria cases than microscopy and RDT, the low performance of routine microscopy observed in this meta-analysis may be due to several factors, including lack of skilled and competent laboratory technicians in many health facilities which negatively impacted the quality of microscopic diagnosis of malaria as previously reported [P. falciparum and P. vivax infections were 51.1% and 37.6%, respectively. The high proportion of P. vivax observed in Mauritania is unique in West Africa, where P. falciparum is widespread and predominant. Elsewhere in Africa, P. vivax is also present at a relatively high prevalence rate in the Horn of Africa and Madagascar [P. vivax malaria, notably in Africa, which should recognize Mauritania as a P. vivax endemic country.The present systematic review and meta-analysis were carried out to determine malaria prevalence and reported , 36. Nevreported , 38, butreported . On the P. falciparum and P. vivax is about 3:2 [Indeed, the epidemiological situation in Mauritania is somewhat comparable to that of the Horn of Africa, particularly in Ethiopia, where the ratio of bout 3:2 . Moreovebout 3:2 , 27.P. vivax infections, which increases the risk of misdiagnosis by microscopy and RDT [According to the subgroup analysis, the pooled prevalence of malaria was found to be very low (1%) among asymptomatic subjects compared to symptomatic subjects 21.6%). The difference in malaria prevalence between the two sub-groups is likely due to the low level of parasite density commonly observed among asymptomatic subjects, making them mostly undetectable by microscopic examination of blood smears and RDT for malaria . In addi and RDT , 6, 20.%. The diAsymptomatic subjects rarely seek medical treatment. Therefore, they serve as an important parasite reservoir by maintaining parasite transmission . AsymptoThere are several limitations in the present study. Although only symptomatic/asymptomatic status of the enrolled participants has shown a significant contribution to the observed heterogeneity among studies, numerous other factors can explain the observed heterogeneity. For instance, differences in methodology, the characteristics of study participants, the existing variation in the environmental conditions, such as rainfall, residents’ lifestyles, and knowledge and practice of the study participants towards malaria, are potentially important factors, but many of these variables were not addressed in the studies included in the present review to allow further investigation. One of the most likely reasons for this variation could be that some of the studies were conducted during the years with unusually high amount of rainfall, such as in 2013 in Nouakchott   and in 2P. falciparum, P. vivax, P. ovale, and P. malariae), PCR only detected three of them . Even among experienced microscopists, parasite misdiagnosis could occur due the morphological similarity between blood stages of P. vivax and P. ovale [P. ovale. However, PCR performed on these samples showed that these cases were actually P. vivax [Plasmodium species , geographic zones (Sahelian versus Saharan), and study population (symptomatic patients versus asymptomatic carriers).In addition, the present study revealed a discrepancy between microscopy and PCR in detecting malaria parasites. While microscopy detected four human malaria species commonly found in Africa (i.e., P. ovale . For insP. ovale , microscP. vivax . This fiP. vivax . DespiteP. falciparum and P. vivax seem to evolve in distinct geographical zones with minimal interaction, as evidenced by low proportions of mixed P. falciparum-P. vivax infections revealed by PCR, with P. vivax in the Saharan zone and P. falciparum in the Sahelian zone. However, this situation has not been taken into consideration, and malaria control strategy in Mauritania has been designed to target only P. falciparum in the entire country [The results of this study have repercussion on Mauritanian malaria elimination programme . First,  country . Secondl country . FurtherP. falciparum and P. vivax are widespread in Mauritania. This epidemiological situation implies that distinct intervention measures, including accurate parasite-based diagnosis and appropriate treatment of confirmed malaria cases, are critical for a successful malaria control and elimination programme in Mauritania [The present meta-analysis revealed that uritania .Additional file 1: Table S1. Summary of search keywords/terms.Additional file 2: Table S2. Excluded studies reporting malaria prevalence in Mauritania and reasons for their exclusion.


COVID-19 has been causing significant mental health problems and other health-related issues. Despite the fact that COVID-19 has a significant impact on chronic disease patients, there is scant research on insomnia, common mental health disorders (CMD), and their associated factors among chronic disease patients.The purpose of this study was to assess the prevalence of insomnia and common mental disorders (CMD) and their associated factors among patients with pre-existing chronic NCDs in Sidama, southern Ethiopia.A multicenter cross-sectional study was undertaken between June 1 and September 1, 2021. The study included 633 participants. CMD and insomnia were assessed using a 20-item Self-Reported Questionnaire (SRQ-20) and a 7—item Insomnia Severity Index (ISI) scale, respectively. To describe the various variables, descriptive statistics were used. We performed multivariable logistic regression analysis to identify independent factors associated with CMD and insomnia. A value of p < 0.05 was considered statistically significant at a 95% confidence interval.The prevalence of insomnia and CMD was found to be 39.3% and 46.8%, respectively. Being merchant , having a diagnosis of diabetes mellitus , comorbid diagnosis , low social support  and moderate ), symptoms of insomnia  and poor quality of life (QOL)  were independent predictors of CMD. We also found out that, having cardiovascular disorders (CVDs) , CMD , and poor QOL  were significantly associated with insomnia symptoms.Our study suggests that substantially high prevalence of CMD and insomnia. Significant association between CMD and occupation, diagnosis, comorbidity, social support, insomnia, and QOL were found. We also revealed that having CVDs, CMD, and poor QOL were significantly associated with insomnia symptoms. Therefore, dealing with the mental health problems of patients with chronic NCDs is an essential component of public health intervention during the COVID-19 pandemic. Noncommunicable diseases (NCDs) are a group of chronic diseases that significantly increase morbidity and mortality in the community, including cardiovascular disease, stroke, cancer, diabetes, and mental health issues. NCDs have been exerting a significant global burden and considered as primary public health agenda for decades . NCDs acCOVID-19 also known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV2), first appeared in the year 2020, causing a global health crisis and the collapse of several healthcare systems . The ongThe COVID-19 pandemic is claimed to be the cause of a significant increase in the global prevalence of mental disorders such as sleep problems, anxiety and depression. In addition, WHO expressed concern regarding the influence of COVID-19 on an individual’s mental health and psychosocial implications . EvidencPeople may exhibit signs of depression, anxiety, and stress such loneliness, denial, insomnia, despair, boredom, and irritability, and they even face a greater chance of committing suicide . IsolatiVs 24%)  .A study was conducted during the initial period of COVID-19 outbreak in China, and it indicated that 8.1% had moderate to severe level of stress, 16.5% had moderate to severe symptoms of depression, and 28.8% had moderate to severe symptoms of anxiety . A natioFurthermore, sleep disturbances were associated with higher levels of mental distress . Sleep iIt has been established that the COVID-19 pandemic significantly affects the development of the illness and survival of NCD patients. As a result, worries regarding the mental health of NCD patients have increased. Comorbid anxiety and depression make it difficult for patients to take their medications, which decreases therapeutic safety and efficacy and raises the risk of disability and death. Comorbid mental problems, however, are frequently ignored and sometimes not adequately treated . DespiteWe conducted a cross-sectional study at four hospitals in Sidama National Regional State, southern Ethiopia, between June 1 and September 1, 2021 . The Hawassa University comprehensive specialized hospital, with about 500 beds in southern Ethiopia, serves a population of over 18 million in the Sidama region and surrounding regions of southern Ethiopia, Oromia and Somalia. It is the only specialized hospital in the region. It was provided inpatient services for COVID-19 patients and isolation center in the region and surroundings. Yirgalem General Hospital is found in Yirgalem town and it is one of the General Hospitals in Sidama region serving about 4 million people in the region and nearby. Adare general hospital is located in Hawassa city and serves for communities from Hawassa and nearby cities. Leku primary hospital is one of the primary hospitals found in Sidama region located around 25 kilo meter away from Hawassa city.α/2)2 × p × (1 − p)/d2, where n is the sample size, z is the standard normal score set at 1.96, d is the desired degree of accuracy and p is the estimated proportion of the target population. By taking p = 50% (because there were no similar study in the area and to get adequate or maximum sample size), Zα/2 = 1.96 and d = 5%, the computed sample size was 384; and by taking 10% non-response rate, the total sample size computed was 422. Using design effect 1.5, our final sample size was estimated to be 633 . Sample size was proportionally allocated to each hospital based on the patient flow of the hospitals.In this study we have included patients with chronic NCDs such as diabetes, hypertension, chronic cardiovascular diseases, respiratory diseases such as asthma, and others who had regular follow-up visit at the selected hospitals. Individuals with pre-existing chronic medical illnesses receiving follow-up care in the outpatient departments (OPDs) of the four hospitals were contacted and requested to take part in the study if they met the following criteria: (I) patients above the age of 18 with confirmed chronic NCDs; (II) patients with stable clinical condition and able to understand the objective of the survey; and (III) patients with no known serious mental or neurocognitive problems. Patients with pre-existing chronic NCDs who were admitted to the emergency or inpatient units for any reason were not included in the study. The required sample size was estimated using single population proportion formula: , insomnia, and quality of life (QOL). The questionnaire was written in English before being translated into Amharic, the local language. The questionnaire was translated from English into Amharic by native speakers of the language who are fluent in English, then back-translated into English by other translators to guarantee consistency. Finally, the Amharic version of the questionnaire was used to collect data.Data were collected by nurses who had received 2 days of training on data collection processes and assessment tools. A pretest was performed on 5% of the sample to discover potential difficulties with the data collection procedures, as well as to assess the consistency of the questionnaires and the competency of the data collectors. The investigators reviewed each questionnaire for completeness on a regular basis during data collection.Social support of patients with pre-existing chronic NCDs was measured using the three-item Oslo social support scale (OSSS). The scale has a score of minimum 3 and maximum 14. It has three categories. A score of 3–8, 9–11, and 12–14 indicates poor, moderate, and strong social support, respectively .The World Health Organization developed the SRQ-20, a 20-item screening tool for common mental disorders . There aThe nature, intensity, and effects of insomnia are evaluated using the ISI, a seven-item self-assessment questionnaire . The folTo analyze the impact of the COVID-19 pandemic on QOL, we used an adapted version of 12 items , from thp-values of <0.05 were considered as statistically significant.For analysis, the data collected were entered into Epi-data version 3.1 and exported to SPSS version 24 for Windows. Various variables were described using descriptive statistics such as frequency, percentage, mean, standard deviation, and median. Binary and multivariable logistic regression were used to identify independent factors of insomnia and CMD. The Hosmer-Lemeshow test was used to assess model fitness. At 95% confidence interval (CI), A total of 633 patients with pre-existing chronic NCDs were involved in this study. Majority were male 357(56.4%), 410(64.8%) participants were married, 352(55.6%) were Protestant by religion and about one-fourth 175(27.6%) were illiterate. Of the total participants, about one-fifth 144(22.7%) were farmer by occupation and majority 353(55.8%) were urban residents. The mean age of the respondents was 46.49 ± 17.71 as described in About half 336(53.1%) of the participants were diagnosed with diabetes mellitus followed by hypertension 111(17.5%) and nearly one-third 200(31.6%) participants had comorbid diagnosis. Majority 364(57.5%) of the participants were ill for ≤5 years, 61(9.6%) and 49(7.7%) participants were using alcohol and khat in the past three months, respectively. Among the total participants, more than one-third 224(35.4%) and about half 332(52.4%) had poor and moderate social support, respectively. Moreover, 293(46.3%) of participants were poor QOL .The prevalence of insomnia and CMD were found to be 39.3% (95% CI = 35.3–43.3%) and 46.8% (95% CI = 42.8–50.6%), respectively as shown in In a multivariable logistic regression model, occupation, diagnosis, comorbid diagnosis, social support, insomnia, and quality of life were all significantly associated with CMD. When compared to government employees, being a merchant is 66%  less likely to develop CMD. Regression analyses revealed that having a diagnosis of diabetes mellitus  and a comorbid diagnosis  increased the likelihood of reporting CMD significantly more than having a diagnosis of hypertension and their counterparts. Poor social support  and moderate social support  were both associated with an increased risk of developing CMD. Individuals with symptoms of insomnia  and poor QoL  were more likely to report CMD as shown in The COVID-19 pandemic appears to be having a severe impact on the mental health of individuals with pre-existing medical conditions. In this study, we examined the prevalence and predictors of COVID-19 pandemic related common mental disorders (CMD) and insomnia in patients with chronic medical conditions in south Ethiopia. CMD and insomnia levels were surprisingly high, according to our research. Being a merchant, having diabetes mellitus, having a longer illness duration, having comorbid diagnoses, having low social support, having comorbid insomnia, and having a poor quality of life (QoL) were significant predictors of CMD. Additionally, a significant association between a high risk of insomnia and a diagnosis of cardio-vascular disorders, concurrent CMD, and poor QoL was found. The prevalence of insomnia and CMD indicates a need for mental health and psychosocial support (MHPSS) during the COVID-19 pandemic.CMDs such as anxiety and depression were found to be more common in pre-existing physical conditions with a higher risk of COVID-19 infection in previous studies , 55. OurHowever, lower prevalence were reported in Ethiopia (22.8%) , and IndThis result of this study revealed that the prevalence of insomnia among chronic medical conditions was found to be 39.3%. Previous investigations and systematic review reports in different situations and populations provided almost approximate results in line with our findings –72. SimiBeing a governmental employee increases the likelihood to have CMD as compared to merchants. This finding is consistent with a recent study . The civSimilarly, those with inadequate (poor and moderate) social support were more likely to have CMD than those with strong social support, which is consistent with previous studies , 88, 89.This study indicated that patients diagnosed with diabetes were more prone to develop CMD as compared to hypertension. Mental health problems such as depression is more likely to occur in people with diabetes –94. SeveIn this study, participants with CMD were more likely to be at risk to develop insomnia and vice versa. In general, there is evidence that mental health problems are associated to sleep disturbances and that insomnia is related to the psychological stress. That is, a bidirectional relationship between insomnia and psychological problems were clearly demonstrated . FurtherOur study also showed that participants who had poor QoL were more likely to develop CMD and insomnia. Similarly, recent studies have discovered that poor QoL is associated with insomnia and poor sleep quality , 108 andThere were certain limitations in this study. First, due to the study’s cross-sectional methodology, it was unable to demonstrate causal links between the variables investigated. Second, because all of the data was self-reported and the participants’ responses were not independently validated, social desirability bias and recall bias could have occurred. Third, because the study’s participants were all people with chronic illnesses, the findings cannot be generalized to the general public.This study highlights the prevalence of CMD and Insomnia among patients with chronic NCDs were found to be considerably high. Occupation, diagnosis, comorbid diagnosis, social support, insomnia, and quality of life were all significantly associated with CMD. We also found out that, having CVDs, CMD, and poor QOL significantly increases the likelihood of having insomnia symptoms. As a result, addressing mental health issues among patients with chronic CMD is an essential component of public health interventions during the COVID-19 pandemic.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Hawassa University, College of Medicine and Health Sciences, Institutional Review Board (IRB) with reference number IRB/076/13. The participants provided their written informed consent to participate in this study.MA, BD, AG, SH, and ET participated in the conception and designed the study and involved in the data collection. MA, BD, and SD do the analysis of the study. MA and SD prepares the manuscript for publication. BD, AG, SH, SD, and ET critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.


Plasmodium species can be difficult. This study evaluated the performance of the MC004 melting curve-based qPCR for the diagnosis of malaria in routine clinical practice in non-endemic setting. High-quality malaria diagnosis is essential for effective treatment and clinical disease management. Microscopy and rapid diagnostic tests are the conventional methods performed as first-line malaria diagnostics in non-endemic countries. However, these methods lack the characteristic to detect very low parasitaemia, and accurate identification of the Plasmodium falciparum samples determined by both microscopy and qPCR showed the potential of the MC004 assay to estimate the parasite load of P. falciparum. Eight Plasmodium infected patients were followed after anti-malarial treatment by the MC004 assay and microscopy. The MC004 assay still detected Plasmodium DNA although no parasites were seen with microscopy in post-treatment samples. The rapid decline in Plasmodium DNA showed the potential for therapy-monitoring.Whole blood samples were collected from 304 patients with clinical suspicion of malaria and analysed by both the MC004 assay and conventional diagnostics. Two discrepancies were found between the MC004 assay and microscopy. Repeated microscopic analysis confirmed the qPCR results. Comparison of the parasitaemia of nineteen Plasmodium species identification, the ability to indicate the Plasmodium parasite load, and can potentially detect submicroscopic Plasmodium infections.Implementation of the MC004 assay in non-endemic clinical setting improved the diagnosis of malaria. The MC004 assay demonstrated superior The online version contains supplementary material available at 10.1186/s12936-023-04617-z. Plasmodium parasites transmitted by infected Anopheles mosquitoes in (sub)tropical areas. The World Health Organization (WHO) considers malaria a leading public health concern due to its potential lethal complications. In 2021, the WHO estimated 247 million malaria cases, leading to 619,000 malaria deaths worldwide [Plasmodium falciparum is responsible for the majority of malaria deaths worldwide [Malaria is a life-threatening infectious disease caused by orldwide .Early and accurate diagnosis of malaria is essential for both effective clinical management and malaria control. High-quality malaria diagnosis is important, as misdiagnosis can result in significant morbidity and mortality , 5. In nPlasmodium species, stage levels, and allows determination of the parasite density (parasitaemia). The major limitations of microscopy are the need for experienced microscopists, and decreased accuracy when the parasite density is low [Plasmodium species can be difficult or even impossible and the limit of detection (LoD) of the Giemsa-stained thick blood smear (5 to 50 parasites/μL of blood) is not always sufficient to detect low-density infections [Microscopic examination of Giemsa-stained thick and thin blood smears is the most widely used method, and remains the gold standard for the diagnosis of malaria. Microscopic examination provides inexpensive and rapid detection and identification of the y is low , 7. Misdfections , 8, 9.P. falciparum and lactate dehydrogenase or aldolase common to all Plasmodium species. RDTs have reasonable sensitivity to detect P. falciparum, but lack sensitivity for the detection of other Plasmodium species [P. falciparum strains [RDTs are immunochromatographic tests that are inexpensive, easy to use and results can be obtained within 5 to 15 min. The most widely used RDTs are based on the detection of histidine-rich protein II (HRP-II) of  species , 11. The species . Another strains .Plasmodium DNA have been proposed as a confirmatory method. Several nucleic acid amplification tests (NAATs) have been developed for the detection of Plasmodium DNA. These include conventional polymerase chain reaction (PCR), qPCR and loop-mediated isothermal amplification tests (LAMP), which mostly detect genus- or species-specific DNA-sequences of the Plasmodium parasite [Plasmodium species [To overcome the limitations of microscopy and RDTs, molecular assays based on the detection of parasite , 15. NAAparasite –21. The  species –21. In a species , 23.Plasmodium species. Especially the nucleotide sequences of Plasmodium vivax, Plasmodium knowlesi and Plasmodium cynomolgi share high similarity, which may cause misidentification and mistreatment [Plasmodium DNA up to several weeks after effective anti-malarial treatment, potentially leading to over-diagnosis of recrudescence [P. falciparum, P. vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, P. knowlesi (including differentiation of three strains) and P. cynomolgi (including differentiation of three strains) [However, a well-known issue with NAATs is the possible occurrence of cross-reactivity between different reatment , 25. Furdescence . The MC0strains) .Plasmodium parasites compared to standard microscopy [Furthermore, a prospective cross-sectional study was performed in malaria endemic setting  to further assess the performance of MC004 for the detection and identification of croscopy . The preP. falciparum infected patients; one P. vivax, one P. malariae and one P. ovale were available and also included. These follow-up samples were used to examine the correlation between the RFU (Relative Fluorescence Units) of the melting curves determined by the MC004 assay and parasite density (parasitaemia) determined by microscopy. The follow-up samples were not collected systematically, but determined based on the clinician’s request for laboratory testing.The performance of the MC004 assay (melting curve-based qPCR) in routine clinical practice was determined by analysing clinical samples received from different laboratories , the Netherlands, Breda , the Netherlands, Regional Laboratory for Medical Microbiology & Public Health—Haarlem, the Netherlands, Noordwest Ziekenhuisgroep—Alkmaar, the Netherlands). In total, 318 ethylenediaminetetraacetic acid (EDTA) whole blood samples obtained from 304 patients with clinical suspicion of malaria were included in the study. All patients had travelled to malaria-endemic areas and presented with signs and symptoms of malaria. A series of post-malaria treatment samples (one to four samples per patient) from five All blood samples were taken as part of routine blood sampling and examined by microscopy (Giemsa-stained thick and thin blood smear) and a rapid diagnostic test (RDT). The MC004 assay was performed blindly (retrospectively) after the initial diagnosis by microscopy. For the follow-up patients, only the first sample had been examined by all available diagnostic tests; RDT, microscopy and the MC004 assay. The results of the MC004 assay were compared with the results by microscopy.P. falciparum by targeting Histidine-Rich Protein II (HRP-II), P. vivax by P. vivax-specific parasite lactate dehydrogenase (Pv-pLDH) and pan-pLDH for Plasmodium species [P. falciparum and pan-aldolase for Plasmodium species, following manufacturer’s instructions [The Palutop + 4 Optima  immunochromatographic test (ICT) was used for rapid diagnostic testing of the clinical samples from Result Laboratory according to the manufacturer’s instructions. The test can detect and differentiate between  species . The Binructions .Plasmodium species and in the case of P. falciparum or P. knowlesi also the parasitaemia was calculated, following the Dutch standard guidelines for thin smear microscopy [The thick and thin blood smears were stained with 3% Giemsa in phosphate-buffer (pH 7.2), followed by microscopic examination (1000 × magnification) by two experienced technicians, according to WHO recommendations . A bloodcroscopy , 31.DNA was extracted from 200 µL of (EDTA) human whole blood using the QIAamp DNA Blood Mini QIAcube Kit  on a QIAcube instrument , following the manufacturer’s specifications. DNA was eluted with 100 µL elution buffer and the processed specimen samples were stored at − 30 °C.Plasmodium species that cause malaria in humans [Plasmodium species and discriminates between 11 Plasmodium species/strains . The MC004 assay involves two main steps. (1) asymmetric target amplification by two different primer sets, primer pair 1 designed to amplify all 11 Plasmodium species/strains and primer pair 2 designed to amplify only P. vivax, P. knowlesi, and P. cynomolgi. (2) detection and differentiation of the amplicons using probe-based melting curve analysis. The three different molecular beacon probes were labelled with either Texas Red, Cy5, or Cy5.5. The PCR reaction was performed in a final volume of 25 µL, including 23 µL MC004 Mastermix  and 2 µL of extracted DNA. Amplification was performed using the CFX96 Touch Real-time PCR Detection System  and the following settings: 95 °C for 3 min, 50 cycles of 95 °C for 15 s, 60 °C for 30 s and 68 °C for 40 s. Followed by a melting curve step: gradual temperature increase from 25 to 69.4 °C (0.4 °C per 5 s).The previously described MC004 assay  is a single tube multiplex qPCR for the detection and identification of n humans . The assP. falciparum DNA, diluted in EDTA whole blood to a concentration of 1 × 10–3 IU/mL) [Plasmodium species come with the MC004 assay as provided by MRC Holland.Positive and negative controls were used to monitor run validity. Each run included two negative controls: elution buffer added as template and a fresh uninfected human whole blood specimen processed as a separate sample. This control should produce an amplification curve, if no amplification curve was observed, this may be a sign of inhibition and makes the result invalid. To guarantee the analytical sensitivity of the assay, every run also included a positive control for limit of detection  . PositivP. falciparum samples were used to examine the association between the Cq-value determined with the MC004 assay and the parasitaemia determined by thin blood smear microscopy. The parasitaemia based on Cq-value was calculated using the previously described calibration curve of the MC004 assay [Nineteen positive 04 assay . For theLinear regression analysis and calculation of R-squared were performed using Microsoft Excel for Mac 2016. Confidence intervals and P-values were calculated using GraphPad Prism 9 for macOS (version 9.5.1), modules ‘Simple linear regression’ and ‘Correlation’.P. falciparum, three P. vivax, two P. malariae, one P. ovale wallikeri and one P. ovale curtisi. No P. knowlesi, P. cynomolgi or mixed infections were detected in the examined patients. The remaining 270 patients tested negative for the presence of Plasmodium DNA. Twenty-seven (100%) of 27 P. falciparum PCR positive samples were identified by microscopy, with parasitaemias ranging from < 0.1% to 35.8%. P. vivax was identified in 2 of 3 cases (67%), P. malariae in 2 of 2 cases (100%) and P. ovale in 2 of 2 cases (100%) of the PCR positive samples . However, after blind re-examination of the blood smear P. vivax trophozoites were seen. Furthermore, a mixed infection of P. falciparum and P. vivax was identified by microscopy, whereas the MC004 assay only detected the presence of P. falciparum DNA. The RDT showed the presence of the HRP-II, Pv-pLDH and pan-pLDH band . Blind re-examination of the blood smear identified the presence of only P. falciparum trophozoites (parasitaemia 1.3%). Therefore, both sensitivity and specificity were 100% for the MC004 assay compared to microscopy.Analysis of the data showed two discrepancies. In one P. falciparum infected patients (parasitaemia ranging from < 0.1 to 3.1%), one P. vivax, one P. malariae and one P. ovale wallikeri infected patient were followed after starting anti-malarial treatment. The follow-up samples (one to four samples per patient) were examined by microscopy and the MC004 assay. In one P. falciparum (parasitaemia < 0.1%), one P. vivax and one P. ovale wallikeri infected patient, the MC004 assay became negative during follow-up (no melting curves detected). In all three cases, no malaria parasites were seen with microscopy although malaria DNA was still detected by the MC004 assay, see Table P. falciparum and after 7 days of treatment for P. vivax and P. ovale wallikeri, shown in Fig. Five P. malariae and four P. falciparum) stopped when no parasites were seen anymore with microscopy , 8 days for P. falciparum (parasitaemia 3.1%) and 3 days for P. malariae, P. falciparum (parasitaemia 0.1%) and P. falciparum (parasitaemia 0.1%).The follow-up of the other five patients , P. vivax (Pv-pLDH), and pan-pLDH band . Although cross-reaction of P. falciparum with the P. vivax Pv-pLDH antigen is a known issue for RDTs, misunderstanding of the RDT bands could have influenced the microscopic examination [P. falciparum parasites were seen.In the case of the misidentified mixed infection by microscopy, the RDT showed the presence of the mination , 35. BliP. knowlesi, P. cynomolgi or mixed infections were detected by the MC004 assay during this study, indicating that these infections are rare in non-endemic setting. The same observation was reported by Gier et al. for the period of 2008–2015 in the Netherlands and Calderaro et al. for the period of 2013–2017 in Italy [No in Italy , 37.P. falciparum, although, in order to ensure correct interpretation, their limitations should be taken into account. Besides the already mentioned limitation, one should be well aware of the fact that P. falciparum variants are circulating that lack HRP-II. Correct identification of these P. falciparum variants by HRP-II-based assays may be compromised [Regarding the performance of the RDT, the results highlight that the use of RDTs is useful especially for the diagnosis of promised .Plasmodium DNA although no parasites were seen anymore with microscopy. The MC004 assay became negative within 7 days after starting anti-malarial treatment for one P. vivax and one P. ovale infected patient, and between day 4 and 20 for a P. falciparum infected patient . A P. falciparum infected patient with a parasitaemia of 3.1% showed melting curves around 1000 RFU at day 8 after starting anti-malarial treatment, whereas the same RFU was observed at day 12 for a P. falciparum infected patient with a parasitaemia of only 0.2%. Other factors that could have influenced the parasite clearance rate are the drug concentration, drug resistance and host malaria-specific immunity [A higher parasite clearance rate was expected for the patients with a low parasitaemia (≤ 0.2%) compared to the patient with a high parasitaemia (3.1%), since less immunity , 40. Accimmunity . NeverthOne additional benefit of the MC004 assay is its high negative predictive value. According to the UK guideline, the microscopic examination should be repeated three times (every 12–24 h) in order to rule out the diagnosis of malaria . Since oP. falciparum samples were used to evaluate the calculated parasitaemia by the MC004 assay with the parasitaemia determined with microscopy. In fourteen of the nineteen samples the parasitaemia levels determined by the MC004 assay were inside the 95% confidence intervals of the parasitaemia levels determined by microscopy. In five samples, including the two highest parasitaemia levels of 4.2% and 3.5%, the parasitaemia levels determined by the MC004 assay fell outside the confidence interval . Further research would be needed to evaluate quantification of parasitaemia in severe or complicated malaria (> 4%).This study showed that the MC004 assay has potential for indication of parasitaemia in uncomplicated The present study had limitations. During the study period, no mixed infection were observed, as mixed infections are rare in non-endemic setting , 37. Thefalciparum malaria. The MC004 assay demonstrated more accurate Plasmodium species identification, the ability to indicate the parasite load of P. falciparum, and can potentially detect submicroscopic Plasmodium infections. In addition, this study indicated the potential of the MC004 assay for therapy-monitoring reflected by the RFU of melting curves.The MC004 melting curve-based qPCR assay showed 100% sensitivity and specificity for the diagnosis of malaria within routine non-endemic setting. Patient health care and clinical disease management were improved with the implementation of the MC004 assay, especially in the case of non-Additional file 1: Fig S1. Melting curves of one P. malariae patient and four P. falciparum (parasitaemia ranging from 0.1-3.1%) patients that were followed after starting anti-malarial treatment. The specific melting curve pattern was used to identify the Plasmodium species. The x-axis shows the temperature (°C). The y-axis shows the negative derivative of fluorescence (RFU) with respect to temperature (T). The Plasmodium species is indicated in the left corner of each figure. The amount of days after the start of anti-malarial treatment is indicated at the right of the melting curves. Red curves correspond to the Texas Red labelled probe, purple curves to the Cy5 labelled probe, and brown curves to the Cy5.5 labelled probe. For the sake of clarity, not all follow-up samples are included in the figures.


Aortic pulse wave velocity (Ao-PWV) predicts cardiovascular and kidney disease in type 2 diabetes (T2D). Klotho is a circulating antiaging hormone (sKlotho) with putative cardiorenal protective effects. The relationship between sKlotho and Ao-PWV in diabetic kidney disease (DKD) is unknown.2+]i) responses and expression of proteins associated with proosteogenic HASMC phenotypes.In a cross-sectional cohort study, the correlation of sKlotho measured by a validated immunoassay, and Ao-PWV measured by applanation tonometry, was investigated in 172 participants with T2D and early stage DKD  on stable renin angiotensin system (RAS) inhibition. In cultured human aortic smooth muscle cells (HASMCs) stimulated with angiotensin II (AngII), the effects of recombinant human sKlotho pretreatment were assessed on intracellular calcium ([Ca2+]i transients and reduced osteogenic collagen (Col1a2) expression.Mean (range) age of the cohort was 61.3 years (40–82) and 65% were male. Mean (±SD) Ao-PWV was 11.4 (±2.3) m/s, eGFR 78.8 (±23.5) and median (interquartile range) sKlotho of 358.5 (194.2–706.3) pg/ml. In multivariable linear regression analyses, we observed a statistically significant inverse relationship between sKlotho and Ao-PWV, which was independent of clinical risk factors for cardiorenal disease. Pretreatment of cultured HASMC with sKlotho significantly attenuated AngII-stimulated [Cain vitro, these findings may explain the enhanced risk of cardiorenal disease in DKD.In individuals with T2D and early DKD, lower levels of sKlotho are associated with increased Ao-PWV. Taken together with the direct effect of sKlotho on mediators of aortic wall stiffness   The study was approved by the institutional research ethics committee and undertaken with adherence to the Declaration of Helsinki (Research study identifier number-10/H0802/86). All participants had T2D as defined by the World Health Organization criteria for diagnosis and classification of diabetes mellitus,Brachial blood pressure was measured in triplicate in the supine position by an automated sphygmomanometer . Mean arterial pressure (MAP) and pulse pressure (PP) were calculated from systolic blood pressure (SBP) and diastolic blood pressure (DBP) values according to the following formulas:MAP = (SBP + 2 × DBP)/3 and PP = SBP − DBP.L= df-dc)) and Ao-PWV was computed as L/ΔT.Ao-PWV was determined from carotid and femoral pressure waveforms obtained noninvasively by applanation tonometry  using the Sphygmocor system  as previously described.,,g  and the supernatants were stored at -80 °C (<24 months) with no freeze–thaw cycles before analysis. This approach has been shown to preserve the sensitivity of the immunoassay used herein.Plasma sKlotho levels were measured in duplicate by enzyme linked immunoassay .−1/100 μg ml−1) in a 5% CO2/95% humidified air incubator at 37 °C as described before.,HASMC  were cultured in Dulbecco's Modified Eagle Medium  containing 10% (v/v) fetal calf serum, 1% (v/v) L-glutamine and penicillin/streptomycin  as described before.2+]i.Intracellular i, whereas the effect of sKlotho pretreatment on protein expression was evaluated by unpaired t-test. Graphical representation was performed with Prism , also used to determine statistical significance at P < 0.05.For studies using HASMC, data denote mean ± SEM of measurements in at least 3 independent HASMC cultures. A 2-way analysis of variance with Tukey multiple comparisons was used to compare the effects of sKlotho and AngII on i mobilization in response to AngII (100 nM) exhibited a typical biphasic pattern of initial rise and subsequent return to baseline with a small but sustained plateau phase  and RAS inhibition are all known to influence arterial aging as well sKlotho levels and Ao-PWV.,,,et al. mentioned previously, 33.9% of the participants had diabetes whereas 85.6% were on treatment with RAS inhibitors. Moreover, the authors looked across all stages of CKD, which was not exclusively caused by diabetes. Although there was statistical adjustment for the presence of DM and the use of RAS inhibitors in their model, the authors do not report the prevalence of DKD in their cohort and whether these patients were on standard of care treatment with RAS inhibition. In contrast, in our study, we excluded people with kidney disease not related to diabetes and chose a cohort with relatively preserved eGFR with eGFR >45 ml/min. Of note, advanced CKD, diabetes, and use of RAS inhibition will all influence both klotho and Ao-PWV or arterial stiffness.There are several important differences between studies reporting no association between sKlotho and aortic stiffness and the present work. Most of previous studies report findings from participants without diabetes who, predominantly, did not receive RAS inhibitors. Furthermore, these studies included people with a wide range of kidney function levels. Diabetes The conflicting data in this area of research is most likely related to the different inclusion criteria in different studies and the populations studied described above. Therefore, conversely to previous studies, we studied patients with T2D and DKD, who were all on RAS inhibition with eGFR above 45 ml/min. Moreover, we utilized Ao-PWV, the gold standard measure of aortic stiffness in contrast to several of the other studies which used brachial ankle or other indices of arterial stiffness.There is no defined consensus or standardization of normal ranges for sKlotho levels with concentrations ranged from 239 to 1266 pg/ml (mean 562 ± standard deviation of 146 pg/ml) reported in healthy adults32Similarly for Ao-PWV there is no agreed or standardizes normal ranges defined with a rise observed with increasing age in healthy people. In a large population based study Ao-PWV values in people without cardiovascular disease, hypertension or diabetes ranged from a mean of 6.2 m/s in those under the age of 30 to 10.9 m/s in those above 70 years of age.In the 2018 European Society of Cardiology/European Society of Hypertension guidelines on the management of arterial hypertension a Ao-PWV > 10 m/s is considered an estimate of significant alterations in aortic function in middle aged hypertensive patients. Both diabetes and CKD accelerated vascular aging and increase Ao-PWV.In our cohort in people with diabetes and early stage CKD the dispersion of sKlotho and Ao-PWV values around a median and a mean of 358.5 pg/ml and 11.4 m/s respectively are expected findings although we acknowledge that especially for sKlotho there are no established and validated reference ranges either in healthy subjects or disease states such as diabetes and CKD.2+]i transients. The culture milieu utilized here was informed by the lack of significant differences in serum phosphate or calcium levels in our clinical cohort (data not shown). By culturing and treating HASMC in standard media, we aimed to recapitulate the early stages of T2D when renal function is preserved and before perturbations in mineral metabolism linked to kidney damage occur. This may explain why alpha-actin expression was not affected by sKlotho pretreatment. sKlotho was previously shown to preserve VSMC contractile function;, however, in the presence of high ambient phosphate levels.,2+]i imbalance,2+]i increases are partly mediated by prolonged activation of AngII type I receptor.2+-permeable channels such as TRPV5 and TRPC6 that regulate intracellular Ca2+ dynamics.,2+]i increases in response to AngII.We have previously demonstrated the protective effects of sKlotho against AngII-mediated oxidative stress, apoptosis, and senescence of HASMC.sKlotho pretreatment before chronic HASMC exposure to AngII also reduced Col1a2 protein levels, a key mediator of VSMC osteochondrocytic transformation. Our results are consistent with findings in cultured mouse aortic smooth muscle cells, where klotho gene deficiency increased autophagy, leading to upregulation of scleraxis, a key transcription factor of collagen synthesis.Our study has several limitations. Because of the cross-sectional study design, a causal relationship between lower sKlotho levels and higher Ao-PWV cannot be inferred. The number of participants studied was relatively small; however, this was in the context of selective inclusion criteria, which allowed us to only study patients with DKD and preserved renal function. We did not observe, in bivariate analyses, a significant association between Ao-PWV and FGF-23 and vitamin D in a subset of our cohort. However, a limitation of our study is that we did not have these measurements for the whole cohort and therefore further studies are needed to determine if these variables can influence the interrelationship between sKlotho and Ao-PWV. Because we excluded people with advanced CKD, which is more often associated with dysregulated FGF-23 levels and because we also excluded people receiving vitamin D or calcium or phosphate supplements it is unlikely these variables may have influenced our results, but further studies are needed to confirm this.We also acknowledge the need for further experiments with HASMC in the presence of hyperglycemia. In T2D, hyperglycemia is one of multiple factors affecting vascular calcification and its impact also needs to be balanced against the effects of hyperinsulinemia.In conclusion, people with T2D and increased Ao-PWV have lower circulating sKlotho levels and this may in part explain their enhanced risk of cardiorenal disease. Our findings in primary HASMC, where pretreatment with klotho negates the detrimental effect of RAS-mediated vascular changes offer a potential mechanism underlying our clinical observations.The present findings establish the platform and scientific rationale for further research to elucidate the role of sKlotho on aortic stiffening and its potential use as a treatment to ameliorate aortic aging (reduce Ao-PWV) in diabetes and early stage renal dysfunction.All authors declared no competing interests.