Asymptomatic malaria infections largely remain undetected and act as a reservoir for continuous transmission. The study assessed the prevalence of submicroscopic asymptomatic malaria infections and anaemia in two rural low (300 m above sea level) and highland (700 m asl) settings of Korogwe District north-eastern Tanzania.Plasmodium.A cross-sectional malariometric survey involving individuals aged 0–19 years was conducted in June 2018 in the two rural villages. Venous blood was collected from eligible study participants for estimation of haemoglobin level, detection of malaria by rapid diagnostic test (RDT), quantification of malaria parasitaemia by microscopy, as well as dried blood spot (DBS) for determining submicroscopic infections by PCR targeting the small subunit of the ribosomal ribonucleic acid (ssrRNA) of human P. falciparum positive by PCR. Of the 211 RDT and 81 microscopy positive, 130 (61.6%) and 33 (40.7%), respectively, were defined as being asymptomatic. Of the 565 individuals, 135 (23.9%) were anaemic (haemoglobin < 11 g/dL) out of which 5.2% were severely anaemic. The risk of being anaemic was significantly higher among individuals with asymptomatic malaria as compared to those without malaria as confirmed by RDT (AOR = 2.06 (95% CI 1.32–3.20) while based on microscopic results there was no significant differences observed . Age and altitude had no effect on the risk of anaemia even after adjusting for asymptomatic malaria.Out of 565 individuals tested, 211 (37.3%) were malaria positive based on RDT, whereas only 81 (14.3%) were positive by microscopy. There was no significant difference in the prevalence between the highland and the lowland village, p = 0.19 and p = 0.78 microscopy and RDT, respectively. Three out of 206 (1.5%) RDT/microscopy negative samples were Asymptomatic malaria is associated with an increased risk of having anaemia in the study communities. The findings highlight the need for targeted interventions focusing on asymptomatic infections which is an important risks factor for anaemia in the community and act as a source of continued transmission of malaria in the study area. Malaria claims lives of more than 400,000 people annually . ReporteIn Tanzania, in addition to ACT use, successful implementation of other malaria preventive strategies including wide use of long-lasting insecticidal nets could most likely explain the observed decline in malaria prevalence in the general population from 18 to 7% between 2008 and 2017 . VariousIn malarial endemic settings, repeated infections cause a partial non-sterilizing immunity to malaria resulting into asymptomatic and often, chronic infections that are rarely detected and may end up not being treated . Many ofDetection of malaria parasites by microscopy (gold standard) or rapid diagnostic test (RDT) are challenged by a number of factors including limited availability of expert microscopists, low detection rates, especially when the parasitaemia is too low and high level false positive rates with RDT due to circulating parasite antigens . The useDespite the realized success in malaria control from various control interventions that have resulted into a significant decline in malaria related morbidity and mortality in north-eastern Tanzania –8, thereP. falciparum malaria parasitaemia in the lowland village has been on decline from 78% to 2003 to 13% in 2008, whereas in the highland village, the prevalence decreased from 25 to 3% in the same period [This cross-sectional study was conducted in two villages of Korogwe District, namely Mkokola  and Kwamasimba  in north-eastern Tanzania. The prevalence of This study collected data from a cross-sectional malariometric survey as part of the main study entitled “Malaria Research Capacity building for field clinical trial in Tanzania” (MaReCa Project-TMA 2015 SF-998) in two study villages. Analysed data included malaria epidemiological data collected in specially designed questionnaires  from individuals aged 0–19 years. Laboratory data were obtained from RDT, haemoglobin measurements by HemoCue machines, and typing for submicroscopic parasitaemia by PCR from parasite DNA extracted from dried blood spots (DBS). The prevalence of submicroscopic infection was determined from DBS that were selected based microscopic negative blood smears and were as well RDT negative. The DBS  were selected from RDT and blood smears negatives to determine the prevalence of submicroscopic infections.TM, Somerset, NJ USA) with both histidine rich protein 2 (HRP-2) and parasite lactate dehydrogenase (pLDH) following the manufacturer’s instructions. Haemoglobin (Hb) measurement was done in the field using HemoCue 301 machine . All participants with uncomplicated malaria based on RDT and other clinical algorithms as per clinician discretions were treated using artemether–lumefantrine (AL) according to the existing Tanzanian malaria treatment guidelines [The cross-sectional malariometric survey was conducted in June 2018 soon after cessation of the long rainy season with concomitant peak malaria transmission. Socio-demographic information including age, gender and place of residence were recorded using standardized questionnaires. Axillary body temperature was measured using digital thermometers . Symptoms related to malaria such as fever, vomiting, convulsion, headache, loss of appetite and general body malaise were also recorded. Also study participants were asked if they have been taken any anti-malarial treatment within 14 days prior to enrollment into the study. Venous blood was collected in ethylenediaminetetraacetic acid (EDTA) vacutainers and blood remaining in the butterfly tube was used to prepare DBS on Whatman filter paper , dried at room temperature and stored in silica gel). Malaria was tested by using thick and thin blood smears microscopy, as well as RDT . Asexual parasites were counted against 200 leukocytes and sexual parasite counts were done against 500 leukocytes. If the asexual parasite counts were less than 10, reading was done per 500 leukocytes. All slides were read twice by two independent, certified microscopists and results from the two readings with a difference of less than 50% were considered definitive. Smears with discordant results were re-examined by a third reader blinded to the first and second readings. Results from the two independent readers that were in agreement were considered final. A slide was declared negative if no parasites were seen after scanning 200 high power fields. All microscopists who read the blood slides participated in proficient microscopic examination and were certified by the National Institute for Communicable Diseases (NICD), South Africa.Plasmodium species was done by Plasmodium genus and species-specific PCR targeting the small subunit of the ribosomal ribonucleic acid (ssrRNA) genes of human Plasmodium using conditions as previously described [Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale. Plasmodium falciparum forward and reverse primers 5′-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT-3′, 5′-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3′, respectively. Plasmodium malariae forward and reverse primers 5′ AAA ATT CCA TGC ATA AAA TTA TAC AAA-3′, 5′-ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA-3′, respectively. Plasmodium ovale forward and reverse primers 5′-ATC TCT TTT GCT ATT TTT TAG TAT TGG AGA-3′, GGA AAA GGA CAT TAA TTG TAT CCT AGTG-3′, respectively.Malaria parasite DNA was extracted from randomly selected DBS that were collected from RDT negative individuals and confirmed to be negative by microscopy. The DNA was extracted using QIAamp DNA Blood mini kit , as per manufacturer’s instructions. After extraction, the eluted DNA was dispensed into 1.5 mL Eppendorf tubes and stored at − 20 °C. One microlitre of parasite genomic DNA was amplified using a thermocycler . Detection submicroscopic infections of escribed . BrieflyP. falciparum 3D7 strain and DNA extracted from DBS collected from microscopy confirmed P. falciparum positive cases whereas for P. malariae and P. ovale parasite DNA was extracted from microscopy confirmed P. malariae and P. ovale cases. Negative controls used was ultrapure water. Ethidium bromide stained 2% agarose gel was used to resolve amplicons under Ultra Violet-BioDoc Analyzer .For each species targeted positive and negative controls were run together with the extracted DNA samples. To ensure quality of the PCR, positive and negative controls were included in each set of PCR run. As positive control for 2-test. Univariate and multivariate logistic regression analyses were used to assess the associations between anaemia with asymptomatic malaria, gender, altitude and age. Effects sizes were presented using odds ratio (OR) with 95% confidence intervals (CI). P-value of < 0.05 was considered statistically significant.Data were entered and validated using a double entry system in Microsoft access (Version 2007). Data analysis was done using STATA statistical software package version 13.1 . Proportions between groups were compared using χThe study enrolled individuals aged 0–19 years with permanent residence in the study area. These individuals (< 19 years) willingly consented to participate or their parents/guardians gave an informed consent for their participation. Individuals with positive RDT/microscopy and temperature above 37.5 °C and as well with signs of malaria, such as vomiting, convulsion, general body malaise, headache and/or loss of appetite, were considered symptomatic. Asymptomatic malaria individuals were defined as those individuals who had axillary body temperature below 37.5 °C, no history of fever for the past 48 h and were malaria positive by RDT, microscopy or PCR . SubmicrA total of 565 study participants were enrolled of whom 280 were from the highland village and the remaining (n = 285) were from the lowland village. The majority of the participants were children aged below 5 years (42.0%), followed by 5–9 years (25.7%) and the least were those aged 17 –19 years (11.5%), Table p < 0.01)  of the RDT negatives samples were found slide positive. Plasmodium falciparum gametocytes were detected in 6.2% (5/81) of the samples by microscopy. The prevalence of submicroscopic infection based on PCR was 1.5% (3/206)  were detected by PCR. The results indicated that the majority of the study participants 10.6% (60/565) who mentioned the use of anti-malarial within the past 2 weeks have taken ALU, while one participant have taken sulfadoxine–pyrimethamine.The overall malaria point prevalence in the two villages was 37.3% 211/565) as determined by RDT, but with almost 2.5-fold drop in prevalence 14.3% (81/565) as revealed by microscopy (Table 11/565 asp = 0.25). Of the anaemic cases, anaemia seemed to vary with age groups but the difference was not statistically significant (p = 0.22)  were anaemic (Hb < 11 g/dL). Among the anaemic cases, 78/135 (57.8%) had mild anaemia, 50/135 (37.0%) had moderate anaemia and 7/135 (5.2%) were severely anaemic. There was no significant difference in the prevalence of anaemia between the low- and highland villages. There was no statistically significant difference in anaemia statuses between males and females (2) Table .The associations between asymptomatic malaria and anaemia (Hb < 11 g/dL), gender, age and altitude are explored in Table The present study revealed a high prevalence of asymptomatic malaria in this rural population of north-eastern Tanzania. Interestingly, children aged above 5 years who are usually forgotten in many malaria control interventions were particularly vulnerable. It is high time now that instead of focusing on underfive and pregnant women, targeted malaria control interventions also consider school aged children who seem to be most vulnerable. Such a high proportion of asymptomatic malaria among those aged above 5 years suggests that school aged children (and above) are a major reservoir of malaria which is in line with other malaria studies from school aged children in Tanzania –20. ThisP. falciparum which is implicated for the more severe form of malaria pathogenesis. However, there were very few infections with P. malariae, and a P. ovale infection was not detected.As previously reported , the majP. falciparum HRP2 gene deletion. The observed percentage of RDT negative which were found slide positive was very low hence chances of having HRP2 gene deleted samples in PCR analyses for submicroscopic was very low as well. Moreover, the current information reported on P. falciparum HRP2 gene deletion in Tanzanian community surveys shows HRP2 gene deletion is uncommon [As expected, the study has noted remarkable differences in the detection of malaria by using microscopy and RDT. Notwithstanding, a better picture would have been possible if all RDT positive and all microscopy negative results could be subjected to species diagnostic PCR in order to rule out chances for false positives due to circulating parasite antigens or inability of detection by microscopy for submicroscopic infections. However, upon subjecting a calculated portion of both RDT and microscopy negative samples to malarial species diagnostic PCR, only 1.5% of the negative samples were confirmed to be sub-microscopic infections that were missed by both RDT and microscopy. Unfortunately, due to limitations of the study we did not have the opportunity to explore all RDT positive/microscopy negative samples by PCR. By PCR the current study analysed a subset of samples that were both negative by RDT and microscopy, this might have an impact on the results for submicroscopic as there were possibility of DBS selected to have false negative results by RDT as results of uncommon .From this study, it is most likely that RDT may have overestimated the actual malaria point prevalence in the study area as previously reported elsewhere , 23, 24.P. falciparum HRP-2 in the blood stream for at least 2 weeks even after successful clearance of infected erythrocytes [It is worth mentioning, at least from an epidemiological viewpoint, despite being false positive and having the loophole for overestimating the actual point prevalence, in a resource limited setting like in Korogwe Tanzania, the use of RDT still provides some important information about current/recent infection and could guide attending clinician on what management to offer while ruling out the possibility of having malaria infection at least for those who are RDT negative. The challenge with the HRP-2-based RDTs is the detection of circulating hrocytes .The current study observed no significant differences in the prevalence of asymptomatic malaria between the high- and lowland villages as revealed by either RDT and/or microscopy results. This finding is in contrary with a previous observation from the same study area in the period of 2003–2008 , howeverThe overall prevalence of anaemia in this study, defined as Hb level < 11 g/dL was found to be in the category of moderate anaemia (20.0–39.9%). According to the WHO criteria on assessment for severity of anaemia , this fiP. falciparum gametocytes were observed by microscopy which is contrary to previous studies that have shown a high prevalence of gametocyte carriage in settings with high burden of asymptomatic infections [In this study, only few fections . Therefofections .pfhrp2 gene deletion.The submicroscopic component of the study analysed DBS samples by PCR that were all RDT negative. This might have an impact on results presented for submicroscopic as there where possibility of false negatives due to In this study, asymptomatic malaria has been shown as a risk factor for having anaemia in the two study communities of north-eastern Tanzania. The findings highlight the need for targeted interventions focusing on asymptomatic infections which sustain the potential for continued transmission of malaria in the study area. Furthermore, the fact that children above 5 years of age were particularly vulnerable to malaria as opposed to under-fives warrants for the introduction and rolling out targeted malaria control interventions focusing on school aged children.


Malaria continues to be a major cause of morbidity and mortality in Cameroon. With all efforts being made to eliminate malaria, it is imperative to describe the epidemiology of the disease in different parts of the country in order to inform control policies. This study aimed to present the differences in the prevalence and intensity of malaria and the anaemic status of children living in different areas of the North West region of Cameroon.This study was carried out from April 2016-July 2017. Blood samples were collected from children via finger pricking. Stained thick and thin blood films were examined through microscopy (x100) to detect the presence of parasites and to estimate the geometric mean parasite density (GMPD). Packed cell volume (PCV) values were determined by micro-centrifugation. Data was analysed using SPSS to determine proportions and test for significance levels between these.Overall prevalence of malaria was 45.3%. Awing and Obang recorded the highest prevalence while Mankon and Nkwen recorded the lowest (p=0.01). The GMPD of infection was highly heterogeneous between the different localities (p=0.03). Age significantly affected the prevalence of malaria (p=0.02). Sex did not affect the prevalence nor the GMPD of malaria infection (p>0.05). Overall mean PCV value was 32.9±3.9. Localities in urban settings recorded the highest mean PCV values compared to those in rural settings (p=0.68). Sex and age did not affect mean PCV values (p>0.05).Plasmodium species in a particular area so as not to waste resources that would only be of limited effectiveness and value to the populations at risk.Malaria still remains a major problem in the North West region of Cameroon. Malaria control interventions should therefore be based on evident spatial and temporal heterogeneity of  Malaria is a disease of great public health concern, especially in tropical and sub-tropical parts of the world, where some 3 billion people in more than 100 countries live at risk . DespiteCameroon is blessed with the greatest biodiversity in Africa. The highland plateau of the West and North West regions constitutes the savanna zone. However, continuous human activities e.g. changing agricultural practices, deforestation, etc., have resulted in changes in landscape ecology and climatological parameters. These changes have not only impacted the biogeography of the region but also favour the transmission of both new and existing infectious diseases. Several studies carried out in different parts of the country show differences in the prevalence of malaria ,5,6. HowAccording to WHO , CamerooAnaemia is a known complication of malaria and other infections. It has a profound effect on the quality of life of people by inducing such symptoms as loss of stamina, rapid heart rate and shortness of breath . AnaemiaThis study was carried out in six localities  in the North West Region of Cameroon, characterised by savanna, with two distinct seasons; the dry season from November to February and the rainy season which is wet and cool which spans from March to October. The region is made up of many towns and villages, which can be categorised as urban, semi-urban or rural depending on characteristics that include land use, household density, types of housing, access to public transport, access to utility services and access to social services.A cross-sectional study was carried out between April 2016 and July 2017. Children, aged 4-16 years, of both sexes, were used as proxy to estimate the parasitological indices and haematocrit values. Children selected were those whom a malaria test and haematocrit tests had been requested for by a medical practitioner. Children were selected because they are a high-risk group for malaria and constitute the majority of hospital consultations in many health facilities throughout the region.Authorisation for the study was obtained from the North West Regional Delegations of Public Health and the chief medical officer from the respective clinics. The chiefs and their council members in each locality were visited and sensitised on the benefits of the study. One clinic or hospital in each locality was used for sample collection. Participation for blood sample collection was voluntary and parents/ legal guardians had to fill and sign an informed consent form, which explained the benefits of the study .et al. [The sample size for this study was calculated based on a prevalence obtained by Mbenda et al.  using thet al.  with for2(0.9)(0.1)/(0.05)2, so n = 138.29 x 6 = 830.where Z = standard number deviate (1.96), n = desired sample size, p = prevalence of malaria, 90%, q =1-q (proportion in the population that does not have the characteristics being measured), e = desired level of precision, n = (1.96)To get the number of participants in the different localities, we multiplied by the 6 since we worked in 6 localities. However, in some localities we could not reach the required sample size because many potential participants were not available due to socio-political unrest or had moved into other towns.Prior to blood sample collection, demographic information such as the age, sex, and place of residence were recorded. Using sterile disposable lancets, finger pricks were performed. Thick and thin blood films were prepared using the method described by Cheesbrough . The codThe slides were read under x100 (oil immersion) objective by an experienced microscopist. A second experienced microscopist, blinded to the first reading, read all thick smears and any discrepancies  were resolved by a third microscopist. Parasite densities were determined from thick blood smears by counting the number of asexual parasites or sexual parasites per 200 white blood cell count (WBC) and converted to number of parasites/μl blood assuming a standard WBC of 8,000/μl. A smear was considered negative if no parasites were seen after review of 100 high-powered fields.Haematocrit values were determined by micro-centrifugation of blood samples in heparinised capillary tubes. PCV values of ≤20% were diagnostic of severe anaemia, values of 21-29.9% of mild anaemia and values ≥30% were considered to be normal values .Data was entered in Excel and analysed using statistical software SPSS version 20. The Chi square test was used to assess significance levels for differences between proportions. Descriptive statistical analyses were performed to compute the mean and standard deviations of parasite counts and haematocrit values in the general study group, and in both males and females. The prevalence of malaria was determined for localities in the different settings. Intensities were obtained by calculating geometric means and expressed as geometric mean parasite density (GMPD) per locality. Simple Chi square analysis was performed to compare different proportions. The Kruskal-Wallis test was used to compare parasite densities in the different localities. All tests were performed at the 5% significance level.Across all six localities, the total number of children sampled was 876. 42.6% (n=373) of these were males and 57.4% (n=503) were females. The proportion of pupils per age group were as follows: 27.3% (n=239) for the age group 4-8 years; 57.3% (n=502) for the age group 9-12 years and 15.4% (n=135) for the age group 13-16 years. The mean age (±SD) of the study population was 9.84 2.45 years. Out of the 876 children sampled, 397 (45.3%) were positive for malaria recording a parasite density of 690.89 ; see Females recorded a higher prevalence 48.7% (n=245) of malaria than males (40.7% (n=152)), although this difference was not significant (P=0.21); see The age group 9-12 years recorded the highest prevalence 56.9% (n=226) of malaria while the age group 13-16 years recorded the lowest prevalence 15.1% (n=60). Differences in prevalence of malaria was significant with respect to age across the different localities P=0.02, . ParasitOverall, a mean PCV value of 32.97±3.94 was recorded (n=872); see Males recorded a higher mean PCV (33.09±3.93) than females (32.81±3.94), although there was no significant difference (P=0.532) in between sexes. The highest mean PCV value (34.44±3.35) was recorded in the age group 4-8 years while lowest mean PCV value (31.37±3.11) was recorded in the age group 9-12 years; no significant difference (P=0.831) was recorded in the mean PCV value with respect to age groups. The missing 0.5% accounts for 4 PCV tubes which got broken in the course of transportation from the field to the laboratory.et al. [The results of this study show that the prevalence of malaria in different localities in the North West region exhibits a highly heterogeneous profile. The overall prevalence of 45.3% recorded in this region suggests that malaria still remains a major problem in this part of Cameroon. This high prevalence of malaria recorded, despite all the efforts made by the government to curb the disease burden, is probably due to the fact that many of the inhabitants in this region do not use the long-lasting insecticide-treated bednets (LLINs) that were distributed to different homes, coupled to other factors which favour the occurrence of malaria such as temperature, rainfall, relative humidity, presence of breeding sites and topography ,13. The et al. , in someAmongst the localities sampled, the prevalence of malaria was higher in the rural areas than in urban and semi-urban areas. Obang and Awing recorded the highest prevalence, which is probably due to the fact that children living in these localities are more exposed to malaria transmission as a result of higher vector density, poor quality housing, poor drainage systems and difficulties in assessing healthcare facilities ,16. AlthThe prevalence of malaria was higher in females than in males, although the difference was not significant. This disagrees with other studies that have reported higher prevalence rates of malaria in males . There iThe mean intensities of malaria infection varied greatly between the different localities and the difference was significant. The great heterogeneity observed in the mean intensities of malaria infection is probably due to the fact that parasite density distribution is not uniform among pupils in the different localities. Generally, younger children (4-8 years) were found to have higher parasite counts than their older school mates, although none presented with clinical signs or symptoms. It is known that immunity to malaria infection builds up with multiple exposures to the infection. Thus, as children get older they have probably had several attacks thereby developing some partial immunity which aids in reducing the parasite load and suppressing clinical manifestations of the disease.et al. [We found out that urban localities recorded higher mean PCV values than rural localities, although the difference was not significant. The higher mean PCV values recorded in children in urban areas coupled with a low prevalence and GMPD/ μl of blood probably reflects the impact of less malaria transmission intensity when compared to the rural areas. The findings of this study differ greatly from that of Sumbele et al. , who fouet al.  and theset al.  there arThis study showed that the prevalence of malaria in localities of different settings in the North West region of Cameroon shows a heterogeneous pattern. Localities in rural areas (Awing and Obang) recorded the highest  prevalence while those in the urban areas  recorded lowest . The highest mean intensity of malaria (839.42 parasites/μl blood) was recorded in Awing whereas Nkwen recorded the lowest (496.14 parasites/μl blood). Malaria prevalence in children aged 9-12 years was higher than in other age cohorts. Generally, younger children (4-8 years) were found to have higher parasite counts than those in other age groups, although none presented with clinical signs or symptoms. Higher mean PCV values were recorded in the urban localities while lower mean values were recorded in the rural localities. Assessing variations in malaria prevalence/ intensity can be useful when allocating (limited) resources to malaria management and control. Governments, through health facilities, should improve sensitisation and education on malaria in order to reduce ignorance and lack of awareness which makes people neglect their health. Also, people should be educated on food substances that can improve their haematocrit values.


Malaria is a significant cause of morbidity and mortality in children aged under 5 years in Mozambique. The World Health Organization recommends seasonal malaria chemoprevention (SMC), the administration of four monthly courses of sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ), to children aged 3-59 months during rainy season. However, as resistance to SP is widespread in East and Southern Africa, SMC has so far only been implemented across the Sahel in West Africa.Plasmodium falciparum to antimalarial medicines in the study districts. In addition, it is important to understand whether SMC is a feasible and acceptable intervention in the context of Nampula Province, Mozambique.This protocol describes the first phase of a pilot project that aims to assess the protective effect of SP and AQ when used for SMC and investigate the levels of molecular markers of resistance of This study will adopt a hybrid effectiveness-implementation design to conduct a mixed methods evaluation with six objectives: a molecular marker study, a nonrandomized controlled trial, an analysis of reported malaria morbidity indicators, a documentation exercise of the contextual SMC adaptation, an acceptability and feasibility assessment, and a coverage and quality assessment.Ethical approval for this study was granted by the Mozambican Ministry of Health National Bioethics Committee on September 15, 2020. Data collection began in October 2020, and data analysis is expected to be completed by August 2021.This research will make a unique contribution to our understanding of whether the combination of SP and AQ, when used for SMC, can confer a protective effect against malaria in children aged 3-59 months in a region where malaria transmission is seasonal and SP resistance is expected to be high. If the project is successful, subsequent phases are expected to provide a more comprehensive assessment of the effectiveness and sustainability of SMCs.DERR1-10.2196/27855 The National Malaria Control Program’s strategic plan for 2017-2022 focuses on burden reduction in highly endemic areas and on sustaining gains in low transmission areas toward elimination [An estimated 409,000 people die from malaria each year worldwide, and children aged under 5 years are particularly vulnerable, comprising 67% of all malaria deaths in 2019 [a burden . Malariaround. The intervention can reduce the incidence of clinical episodes and severe malaria by approximately 75% [Seasonal malaria chemoprevention (SMC) is “the intermittent administration of full treatment courses of an antimalarial medicine during the malaria season to prevent malarial illness, with the objective of maintaining therapeutic antimalarial drug concentrations in the blood throughout the period of greatest malarial risk” . The curtely 75% . In Burktely 75% . SMC coutely 75% . In termtely 75% .Plasmodium falciparum is sensitive to both antimalarial medicines used in SMC. In 2019, 21.7 million children were targeted in 13 countries [SMC has been recommended by the WHO since 2012, for use in areas where more than 60% of annual malaria incidence occurs within 4 consecutive months, where there is a high burden of malaria in children, and where SPAQ retain their antimalarial efficacy . To dateHowever, there is a potential risk of enhancing drug resistance and the potential for impaired development of naturally acquired immunity in children . The impChallenges include the logistical burden of SMC distribution, particularly during the rainy season when access to remote areas may be compromised; however, adopting a decentralized, integrated approach through community-based distributors may support the sustainability of SMC . The potdhfr) and dihydropteroate synthetase (dhps) genes [dhfr and dhps mutations was 5%-6% [The WHO recommends that SMC is suitable in areas where the efficacy of SPAQ combination remains over 90% . Resistas) genes . Accordias 5%-6% , with moas 5%-6% . HoweverClinical responses to SP are seriously compromised in many regions of the world, and SP is no longer recommended for the treatment of malaria episodes. However, it has been difficult to determine whether the efficacy of SP for chemoprevention is also compromised . Extant The Ministry of Health, National Malaria Control Programme Midterm Review of the Malaria Strategic Plan 2017-2022 has recommended SMC as a malaria control strategy to decrease transmission and accelerate impact in the highest burden locations . This isGiven the potential impact of SMC to avert many malaria infections and deaths, it is essential to investigate the role of drug resistance on the protective effect of SMC in Mozambique and to assess the feasibility and acceptability of implementing SMC in this context. In collaboration with the National Malaria Control Programme in Mozambique, Malaria Consortium will pilot SMC from November 2020 to February 2021, to a target population of approximately 72,000 children aged under 5 years in 2 districts of Nampula Province.We will evaluate the SMC pilot with two primary aims: to determine the protective effect of SPAQ when used for SMC in this context and to assess the feasibility and acceptability of implementing SMC in terms of coverage, quality, and stakeholder perceptions. The study objectives are as follows: (1) to determine the baseline prevalence of SPAQ resistance and any increase in resistance prevalence after one annual round of SMC, (2) to determine whether receipt of SPAQ is associated with a reduction in the odds of clinically significant malaria outcomes, (3) to assess the change in reported malaria morbidity indicators through routine data, (4) to document the adaptation of SMC implementation to the Mozambican context, (5) to explore the feasibility and acceptability of SMC among stakeholders, and (6) to evaluate the process of SMC implementation in terms of distribution quality and coverage.The study will be conducted in Malema, Mecuburi, and Lalaua districts in Nampula Province, northeastern Mozambique  and 2. STo identify suitable districts for SMC, an SMC suitability ranking was conducted by the WHO for all provinces. These criteria included a variety of factors such as (1) seasonality eligible for SMC , (2) mortality  data, (3) access to care (highest ranking given to areas where access to care was poor), and (4) treatment-seeking behavior (highest ranking given to areas where treatment-seeking behavior was poor). Using these four main categories, the average will be calculated to estimate the final ranking and identify the top 20 suitable districts to maximize the impact of SMC. From the list of suitable districts, an additional consideration was taken given the importance of implementing the intervention in an area where no other new interventions were taking place so that an evaluation could be implemented aiming at attributing change to the intervention under investigation. Hence, Malema and Mecuburi districts in Nampula Province were selected, with Lalaua as a comparator, as no indoor residual spraying or new long-lasting insecticidal nets were targeted in these areas, ensuring a robust evaluation component for this pilot study.Methods section. The Lalaua district will serve as a control area . Data collection across the study components was conducted between October 2020 and May 2021 .The methods are organized according to the study objective, described in more detail below, with an overview provided in Ethical approval for this study was granted by the Mozambican Ministry of Health National Bioethics Committee on September 15, 2020. Only participants who met the inclusion criteria and provided written informed consent will be included.Plasmodium falciparum dihydrofolate reductase (dhfr), Plasmodium falciparum dihydropteroate synthase (dhps), Plasmodium falciparum chloroquine resistance transporter gene (pfcrt) and/or Plasmodium falciparum multidrug resistance gene 1 (pfmdr1) mutations, compared with children living in a neighboring district with similar epidemiologic characteristics but not receiving SMC. Children enrolled in both areas will be aged 3-59 months and will have similar clinical and parasitological characteristics. Health facility–based cross-sectional surveys will be conducted in October 2020, before the SMC distribution begins (baseline), and in March 2021, after one complete round of SMC (endline), to measure the prevalence of molecular markers associated with resistance to SPAQ in symptomatic children aged less than 5 years with a positive RDT attending selected health facilities in the intervention and control areas. Monitoring the prevalence of alleles associated with drug resistance will be done by collecting blood samples from symptomatic children with evidence of infection. During the surveys, fingerpick blood samples will be collected on Whatman 903 (10534612) filter papers (dried blood spots). Sample collection will be performed in 2 selected first-level health facilities in each district of the intervention area and also in 4 selected health facilities in the comparator district, with a total of 8 health facilities across all 3 districts. The main outcome measure is the prevalence of molecular markers associated with SP  and AQ  resistance in blood samples collected from symptomatic children aged less than 5 years with a positive RDT attending the selected health facilities. The prevalence will be assessed in areas with SMC and with no SMC at baseline and at the end of the project.The purpose of this study is to determine the baseline prevalence of SPAQ resistance markers and any eventual increase after one round (four monthly cycles) of SMC as assessed via molecular markers in the population. Specifically, to detect prevalence over time in the proportion of symptomatic children, using a positive rapid diagnostic test (RDT), residing in 2 districts where SMC will be implemented who carry parasites with dhfr and dhps sextuple mutants is 0% [P=.05) between baseline and endline. This will permit confirmation of a prevalence of 5% or higher in dhfr and dhps sextuple mutants. Assuming a 10% loss of samples or uninterpretable analysis, the number will be rounded up to 300 samples per area (intervention and comparator districts) per survey per country. control area). The sample size will be determined using the WHO protocol for drug efficacy testing . The surts is 0%  in the iPlasmodium falciparum infection among them based on routine data reporting.Samples will be collected from selected health facilities from the list of all health facilities in the control and intervention areas, based on attendance of children with fever or history of fever and prevalence of The study population will include children aged 3-59 months attending the selected health facilities in both the intervention and comparator districts. The number of children to be screened in each health facility will depend on the proportion of malaria RDT-positive cases over the total number of children meeting the eligibility criteria. Children who do not meet the inclusion criteria, for example, those with a negative RDT result, will be referred within the same health facility for further assessment and appropriate treatment.Caregivers of children meeting all the inclusion criteria mentioned earlier will be interviewed using a short paper questionnaire to record personal identifiers of the children such as date of birth, sex, date of interview, and residence location. The study staff will approach them as part of their clinical visits.dhfr, dhps, pfcrt, and pfmdr1 mutations. Other molecular markers of antimalarial resistance will be considered for analysis as comparators. Specimens will be labeled anonymously , dried, stored in individual plastic bags with desiccants, and protected from light, humidity, and extreme temperature until analyzed. All samples will be batched and stored at 4 °Cat the Centro de Investigação em Saúde de Manhiça laboratory . Dhfr, dhps, pfcrt, and pfmdr1 genotypes will be determined using nested mutation-specific polymerase chain reaction, sequencing, and/or polymerase chain reaction-restriction fragment length polymorphism. The prevalence of dhfr mutations at codons 51, 59, and 108 and dhps mutations at codons 431, 437, 540, 581, and 613 and pfcrt codons 72-76 and pfmdr1 codons 86, 184, and 1246 will be calculated among the sampled children, and differences in the proportions of samples with each of these mutations between baseline and endline will be tested using the z test.A blood sample (4 drops) will be collected through finger prick onto a filter paper (Whatman 3MM) to determine the prevalence of leakage of SPAQ across the district boundary.A nonrandomized controlled trial will be conducted to determine whether the receipt of SPAQ is associated with a reduction in the odds of clinically significant malaria outcomes and to estimate the protective effect of SPAQ. There will be two arms: one (control arm) in the comparator district and the other in one of the SMC intervention districts. Communities will be randomly selected in both the intervention and control districts. Eligible children (aged 3-59 months at the first SMC cycle) will be recruited through random selection. Exclusion criteria include the presence of a severe, chronic illness and a history of a significant adverse reaction to SP or AQ. Selected children will be treated appropriately and retained during the trial. In the control district, communities bordering the intervention district will be avoided because of potential unintended The study will be powered to have an 80% chance of detecting a 40% reduction in incidence with statistical significance at the 5% level. It is assumed that study participants, in the absence of SMC, would experience 0.2 clinical episodes per child per high-transmission season (corresponding to the SMC round) of sufficient severity to present to a health facility . The samThe primary outcome of this study will be visits to a health facility for suspected malaria by eligible children and confirmation of malaria diagnosis using an RDT during the 5-month study period, which includes a period of 28 days after the final administration of SPAQ.Households will be randomly sampled from selected communities in both the control and intervention arms and 1 eligible child recruited at random in each household. A short baseline questionnaire will be administered to collect individual data on each child and to confirm their eligibility after the caregivers provided their consent. In the intervention arm, the questionnaire will be administered before the intake of SPAQ for SMC. The primary end point will be recorded through passive surveillance by study clinicians in both the intervention and comparator areas during the 4-month study period. Children recruited into the study presenting at clinics will be identified using information on SMC record cards, on which a child’s information and SMC doses are recorded, and data on health facility visits including suspected malaria cases and results of RDTs will be matched to baseline questionnaire data to build a database for analysis.Failure defined as a visit to a health facility for suspected malaria and/or a confirmed case of malaria and timing of this event based on the original date of health facility attendance during the study period and the estimated protective effect calculated.Data will be analyzed using multivariate logistic regression with odds ratios estimated for the association between allocation to the intervention arm and (suspected and confirmed) malaria diagnosis at any point during the study period, converted to a percentage estimate of protective effect. To compensate for the lack of preintervention random allocation of participants to intervention or control groups, covariate adjustment will be made for potential confounders that may influence respondents’ odds of experiencing a clinically significant case of malaria. Covariates, measured at baseline, will include sex, age, parental education, and long-lasting insecticidal net use and all of these will be operationalized as categorical variables. Analyses will also use a multivariate Cox proportional hazards model with multiple Cox regression. We will investigate the impact of SMC on reported malaria morbidity (as a primary end point) in children aged 3-59 months in the study area. Differences in rates of malaria incidence by age at the health facility level will be investigated using a quasi-experimental design based on regression discontinuity analysis, taking advantage of the discontinuity in eligibility for SMC based on age.Before obtaining data on malaria indicators at the health facility and district levels, a data quality audit will be conducted to assess the quality of the data that will be used for analysis. The quality of data from districts where SMC is implemented will also be compared with that of data from the comparator district. Health management information system data on health facility attendance for children aged 0-13 years (inclusive) will be extracted from health facility registers as full line listings, covering November 2020 to May 2021 in both implementation districts and the control district by photographing the relevant pages of the registers and entering data directly into Excel tables as appropriate. While monthly suspected malaria cases presenting at each health facility will be calculated, other variables including date of health facility attendance, sex, date of birth , whether a malaria test was performed, the type of test performed (RDT or microscopy), test results , malaria mortality, and any other variables collected routinely for all child attendees  will be obtained. Observations from registers will be categorized by children’s ages according to the smallest possible increments. Incidence curves  for suspThe purpose of this component is to systematically capture the process of how the SMC model implemented by the Malaria Consortium and partners in West and Central African countries is adapted to the Mozambican context. Specifically, this will take into consideration the existing government health system, structures, personnel, and service delivery at different levels. In view of the current COVID-19 pandemic, all of the aforementioned activities and tools will be appropriately adapted to this outbreak context, which will constitute an additional aspect to be documented as part of the model adaptation. Previous study protocols, implementation reports, tools, and materials from other countries where the Malaria Consortium has implemented SMC will be reviewed and collated. Relevant materials will be adapted and translated for the Mozambican context. All of these adaptations will be captured using a template process documentation tool.A key component of investigating the feasibility and efficacy of SMC in Mozambique is understanding the views and experiences of those involved in its implementation and use.To explore the feasibility and acceptability of implementing SMC, a qualitative assessment using key informant interviews and focus group discussions (FGDs) will be conducted. This study component will examine how SMC is viewed, experienced, and engaged with by different stakeholder groups, including policy makers, implementers, and beneficiaries.The study population includes health workers and community volunteers who are involved in SMC implementation, caregivers of children aged 3-59 months, community members, community leaders, and key decision makers such as health officials at different levels of the health system. Purposive approaches to sampling will be adopted to include those best able to provide insight into the topic being explored and a range of views and experiences relating to SMC . ParticiWithin each participant group, participants with a range of different experiences will be identified, where possible. For example, for caregivers, efforts will be made to include participants of different genders, ages, living in more rural and urban locations; those considered more marginalized, such as adolescents, older caregivers, and people living remotely; and those with different socioeconomic status and education levels. Although we presume that most caregivers will be female, we will attempt to include male caregivers where possible and relevant. We will also aim to include those with different experiences relating to SMC, that is, those who refused and those who agreed to receive SPAQ for their children. For health workers and community-based volunteers, those of different ages and experience and seniority levels will be identified for recruitment. For community members, those with potentially different perspectives will be included, for example, village leaders, older men, older women, and younger women. For key informants, implementing partners and those from district, provincial, and national levels and with a range of different potential insights, for instance, relating to malaria prevention policy making, program management, and drug supply chain management, will be identified for recruitment. In addition to identifying participants purposively, the end-of-round approaches to sampling will be adopted, with the final sample size determined based on evidence of achieving data saturation, when additional participants do not generate new findings relating to the topic of investigation ,38. We ethick descriptions [Data will be generated through in-depth interviews and FGDs, based on topic guides, to explore views on SMC, areas relating to SMC acceptability, and reflections on the implementation experience. Topic guides will serve as prompts for the interviewer but will be flexible and participant led. In-depth interviews will aim to encourage participants to elaborate and provide in-depth accounts of their views and experiences relating to SMC, or riptions , includiFGDs can facilitate an understanding of social norms, providing access to a range of perspectives , and the. To evaluate the coverage provided in the SMC pilot, an end-of-round survey will be conducted in March 2021. The objective of the end-of-round survey is to retrospectively determine coverage by surveying caregivers of eligible children aged 3-59 months and ineligible children aged 60-119 months, as leakage into the older age group has been anecdotally observed in other SMC countries [The aim of this component is to evaluate the process of SMC implementation in terms of quality, coverage, and adherence to COVID-19 safety guidelinesountries . CaregivThis study aims to achieve a representative sample of children aged 3-119 months in households grouped within clusters identified from health facility catchment areas across the 2 intervention districts of the SMC pilot .For this objective, the design will be a cross-sectional cluster randomized survey to evaluate the coverage level and quality of SMC piloted in the Malema and Mecuburi districts. Households refusing to participate in the study will be replaced with the next eligible household until the estimated sample size is attained.svysampsi command, based on the following assumptions: (1) the assumed intracluster correlation is 0.2; (2) 15 eligible children per cluster (b=cluster size); (3) the (design/cluster effect) = 1 + (b − 1) intracluster correlation = 1 + (15 − 1) 0.2 = 3.8; (4) the coverage rate of SMC in children aged 0-4 years of at least 80% (and in children aged 5-9 years of at most 20%); (5) a margin of error of 5%; (6) finite population adjustment is applied ; (7) nonresponse rate of 5%; and (8) assumed ratio of children aged 5-9 years to those aged 0-4 years of 0.88.The end-of-round survey will use multistage random samples of households in areas covered by the Malaria Consortium’s SMC pilot and will intend to achieve a representative sample of the target population at the district level to estimate the coverage of SMC at the level of individual eligible children. The sampling protocol aims to achieve a self-weighted sample with sampling units selected with a probability proportional to size. Only at the last stage of sampling  will a constant number of eligible children (1 child per household) be selected. The survey will be powered to provide an estimate of SMC coverage for children aged 3-59 months with a margin of error of 5%, while also providing a representative sample of children aged 60-119 months. The main sampling frame for the selection process will be a list of villages. Villages will then be randomly selected using the probability proportional to size. Villages will be the primary unit of sampling through which households and eligible children will be selected randomly. This may be reviewed once the study starts if this approach is not feasible in practice. A primary caregiver in this survey refers to any individual, aged at least 18 years, with the primary responsibility of feeding and daily care of at least one child aged less than 5 years, in a household where he or she has been a resident before the start of the SMC pilot or 1 month before the last cycle of SMC. The sample size calculation was performed using Stata 16 using the The sample size calculation revealed that a sample of 1842 children was required. It was decided that the survey would include 120 clusters, each comprising 15 children (n=1800), with 60 in each of the two districts. A constant number of households (15) will be randomly sampled from these areas, with the assumption that the populations in each sampling unit were of approximately equal size. One eligible child will be sampled from each compound in the absence of a household-level sampling frame, under the assumption that households contain a similar number of eligible children. Sampling at each stage will be conducted without replacement; once a supervision area or cluster has been selected, it should no longer be eligible for further selection.The data were collected using a questionnaire developed by the Malaria Consortium. The survey questionnaire will be translated into Portuguese and Macua languages and uploaded into the SurveyCTO software application that will enable direct, field-based computer-aided personal interview and remote capture of the data and transfer to a netbook computer.Although the coverage surveys will be the main method to determine coverage, two additional data sources will be analyzed and compared with survey results: (1) administrative data based on SMC tally sheets completed by distributors and data compiled via summary forms and end-of-cycle reports, and (2) (coverage=doses delivered/target population) and stock consumption data (coverage=SPAQ coblister packs received before the campaign−SPAQ coblister packs left at the end/target population).Data will be collected by administering questionnaires to the identified respondents in the sampled compounds within the communities. The survey questionnaires will be administered by a trained research team. All surveys will be administered using SurveyCTO, an electronic data collection platform for smartphones, and data will be uploaded to a remote server after each day of data collection. Interviews will be conducted in local languages using the questionnaires provided by the Malaria Consortium, with data collectors translating from the Portuguese questionnaire on the spot and assigning responses to predefined answer categories in SurveyCTO. For the age eligibility indicator, survey respondents may be asked to present a birth certificate or vaccination card to the data collector to verify the child’s date of birth. The duration of data collection is expected to last for 7 days during the first week of March 2021. Data analysis will be carried out using Stata 16. Coverage will be calculated using the proportion command. Population size weights will be applied using the svy command as appropriate for estimates of coverage indicators when it is not possible to achieve a self-weighting sample. All indicators of interest will be calculated as proportions by district and an average across both districts.The associated study protocol and data collection tools will be made available upon request from the corresponding author. Quantitative data sets will be available from the corresponding author upon reasonable request after the completion of primary analyses and dissemination of results. Qualitative study data sets will not be available, as they may include identifiable information that could compromise participant identity.Data collection and analysis from all six objectives will be completed by September 2021.This study will provide a unique contribution to the evidence on the prevalence of SPAQ resistance in Mozambique and more broadly in the region, and to what extent it may challenge the protection that SMC confers. Furthermore, this first evaluation of SMC implementation in this context will generate insights on the enablers, challenges, perceptions, feasibility, acceptability, quality, and coverage of this intervention in situ. This study is vital, as it is the first time that SMC is implemented in East and Southern Africa.Recent evidence from the ACCESS-SMC program showed that the protective effectiveness of each monthly treatment was similar to that observed in randomized controlled trials ,45. In 2Molecular markers of SPAQ resistance occurred at a low prevalence in previous studies in the Sahel, consistent with the effectiveness of SMC observed in several case-control studies. However, there is evidence of selection for resistance to SP in parasites sampled from the same age group in the areas where SMC will be implemented for the first time in Mozambique. Therefore, resistance to both SPAQ needs to be monitored via standardized methods, across all regions in Mozambique where SMC is used, to provide early warning of loss of effectiveness.Leakage of SPAQ to older age groups not targeted by SMC programs raises a concern for the development of drug resistance, as doses administered are unlikely to offer sufficient protection against malaria transmission. This also influences the secondary data analysis in the same way; leakage reduces the apparent effect size in the targeted age group.This research will be a novel contribution to our understanding of whether SPAQ, when used for SMC, can confer a protective effect against malaria in children aged 3-59 months in a region where malaria transmission is seasonal and SP resistance is expected to be high. In addition, the findings from this study will inform future SMC implementation in Mozambique and other countries and potentially enhance the quality of SMC distribution in terms of quality, coverage, and acceptability. Subsequent phases are expected to provide a more comprehensive assessment of the effectiveness and sustainability of SMCs.


Plasmodium spp. diagnostic capabilities, access to testing is limited in some cases by the almost absolute requirement for blood from potentially infected subjects as the only sample source for all conventional methods. A rapid test on non-invasive specimen with comparable performance to microscopy for the screening or diagnosis of all participants is invaluable. This study sought to compare conventional and non-invasive diagnostic tools for detecting Plasmodium falciparum.Malaria remains a significant health challenge in sub-Saharan Africa, with early diagnosis critical to reducing its morbidity and mortality. Despite the increasing χ2 test whereas differences in group means were assessed using analyses of variance.This was a cross-sectional study, carried out between March and August 2019 to evaluate and compare the diagnostic performance of a PfHRP2/pLDH-based malaria rapid diagnostic test (mRDT) on patients’ blood, saliva and urine relative to conventional light microscopy and nested PCR at outpatient clinics in the Buea and Tiko health districts of Southwestern Cameroon. The significance of differences in proportions was explored using the Pearson’s P. falciparum infection as confirmed by PCR. The sensitivity and specificity of presumptive diagnosis, light microscopy and mRDT on blood, saliva and urine were 86.9% and 19.7%, 77.8% and 96.1%, 75.8% and 96.6%, 74.5% and 93.1%, and 70.7% and 81.8%, respectively. The agreement between mRDT on saliva (k = 0.696) and microscopy (k = 0.766) compared to PCR was good.A total of 359 individuals of both sexes, aged 1–92 years, were enrolled into the study. Of the 301 individuals tested by light microscopy and mRDTs on blood, saliva and urine, 84 (27.9%), 81 (26.9%), 87 (28.9%) and 107 (35.5%) respectively were positive. However, only 34.3%, 90.5%, 91.4%, 83.9% and 65.4% febrile, light microscopy and mRDT positives on blood, saliva and urine respectively had The study highlighted the low performance of presumptive diagnosis, reinforcing the need for parasitological tests prior to antimalarial therapy. The higher PfHRP2/pLDH mRDT parasite detection rates and sensitivity in saliva compared to urine suggests that the former is a practical adjunct to or alternative worth optimising for the routine diagnosis of malaria.P. falciparum infection by light microscopy, rapid diagnostic tests and nested PCR.Flow chart for diagnosis of  Malaria remains a significant health problem especially in sub-Saharan Africa despite a steady decrease in cases and deaths over the last decade . The disEarly diagnosis and treatment remains critical to preventing death and reducing the disease and its transmission , 5. AlthRapid diagnostic tests are vastly sensitive and specific compared with microscopy but also compare favourably with the polymerase chain reaction (PCR) , the mosPlasmodium spp. diagnostic capabilities, access to testing is challenged, particularly in community and private healthcare settings, by the almost absolute requirement for blood from potentially infected subjects as the sample source for all methods [Despite increasing  methods , 13. Blo methods . In addi methods  and lack methods  continue methods . A non-iP. falciparum infection [P. falciparum histidine-rich protein 2 (PfHRP2) antigen [Saliva and urine are two common alternatives to blood for the diagnosis of infectious diseases that are readily available and easy to collect. Saliva has been effectively used to detect the 2019 novel coronavirus, SARS-CoV-2 , 22. Thenfection , 24, parnfection  and P. f antigen , 27 in s antigen , 29. ConPlasmodium spp. transmission is perennial in this area, with Anopheles gambiae being the dominant vector species and Plasmodium falciparum accounting for most malaria infections [This study was carried out in the Buea (4° 10′ 0″ N 9° 14′ 0″ E) and Tiko (4° 4′ 30″ N 9° 21′ 36″ E) health districts, located at the foot of mount Cameroon in the Southwestern Region of Cameroon. The area has an equatorial climate characterized by daily temperatures ranging between 20 and 33 °C, average annual rainfall of 2625 mm, relatively high humidity and precipitation and two major seasons; a long rainy season (March–November) and a short dry season (November–March) [fections .P. falciparum detection using PCR.This cross-sectional study was carried out in the Regional Hospital, Buea and the Baptist Hospital, Mutengene from March to August 2019. A sample size of 293 participants was estimated using the general formula for comparing two independent proportions of alternative diagnostic tests, assuming 95% confidence interval and 80% power to detect a difference of 10% from a sensitivity or specificity of at least 70% as described . IndividHaemoglobin (Hb) concentrations were determined using a hemoglobinometer  and anemia was defined as Hb < 11.0 g/dL .Plasmodium spp. in blood, saliva and urine was assessed using the PfHRP2/pLDH malaria rapid diagnostic kit  and according to the manufacturer’s instructions. Briefly, 5 µl of sample from each was placed in the sample window of the RDT cassette and three drops of diluent added. The results were then read after 15 min, with the presence of two (or three), one or no distinct line indicative of a positive, negative or invalid result respectively.The presence of Thin and thick blood smears were prepared and allowed to air dry. The thin films were fixed with methanol and both smears stained with 10% Giemsa for 15 min as described . Subsequg for another 2 min. The supernatant (DNA extract) was then transferred into new Eppendorf tube and kept at 4 °C for use within hours or at − 20 °C for long term storage.DNA was extracted from dried blood on filter paper using the Chelex method as previously described , with soP. falciparum 18S rRNA gene was then amplified by nested PCR as described previously [The eviously  using spTaq master mix, 0.5 µl each of forward and reverse primers and 4.5 µl of nuclease free water and 2 µl of extracted genomic DNA (primary PCR) or 5.5 µl of nuclease free water and 1 µl of primary PCR product (nested reaction). The cycling conditions of both reactions were the same, except that the annealing temperature and the number of cycles were increased from 55 to 61 °C and 25 to 30 during the nested reaction. The nested PCR products were visualized on a UV transilluminator and a molecular imager relative to a 100 bp molecular weight marker, following ethidium bromide stained 2.5% agarose gel electrophoresis in 0.5 × TAE buffer using a power pack  at 100 V for 25 min. A sample was considered positive if a 205 bp band was detected. P. falciparum 3D7 DNA and distilled water served as positive and negative controls, respectively in every set of reactions.All PCR amplifications were carried out on a thermal cycler , in a total volume of 15 µl, consisting of 7.5 µl of 2 × χ2 test whereas the differences in group means were assessed using analyses of variance. The performance of each diagnostic test was assessed by estimating the sensitivity, specificity, positive and negative predictive values using the following formula and their respective 95% confidence intervals as well as the Chi-square goodness of fit of each technique. Kappa test was also used to determine the degree to which RDT compares to microscopy and PCR. A probability (P) value < 0.05 was considered as statistically significant at a 95% confidence interval.Data obtained was analyzed using IBM SPSS Statistics 20 . Patients without results for any diagnostic test or with indeterminate PCR results were excluded from the analysis. Levels of parasitaemia were log-transformed before analysis. The significance of differences in proportions of qualitative variables was explored using the Pearson’s Sensitivity = true positive/ × 100%Specificity = true negative/ × 100%Positive predictive value (PPV) = true positive/ × 100%Negative predictive value (NPV) = true negative/ × 100% .A total of 359 individuals of both sexes, aged 1–92 years, were enrolled in this study. The characteristics of the population are summarized in Table P. falciparum infection, 98 (32.6%) were positive by PCR while 84 (27.9%), 81 (26.9%), 87 (28.9%) and 107 (35.5%) cases were detected by light microscopy and mRDTs on blood, saliva and urine respectively. Majority  of the participants had axillary temperature ≥ 37.5 °C and/or history of fever at enrollment, with 13 (24.5%) and only 85 (34.3%) of the afebrile and febrile individuals respectively having a positive test for P. falciparum by PCR. The detailed result of the trial profile of the five diagnostic methods is shown in Fig. P. falciparum by PCR. Conversely, of the 217 microscopy negative participants, 196, 193 and 167 respectively were negative following mRDT on blood, saliva and urine; and 5, 10 and 6 shown to be P. falciparum infected after PCR  between febrile status and age group, with all children below 9 years of age, having a febrile illness at enrolment. The proportion of malaria parasite infected participants as detected by microscopy (P = 0.332), blood mRDT (P = 0.459), saliva mRDT (P = 0.171), urine mRDT (P = 0.458) and PCR (P = 0.215) was independent of age.Of the 301 participants tested for ion, 98 3.6% were P. falciparum-infected. Thus, among the samples thought to be infected with the malaria parasite on the basis of the participant being febrile or diagnosed positive by light microscopy or via mRDT on blood, saliva or urine, 163 (65.7%), 8 (9.5%), 7 (8.6%), 14 (16.1%) and 37 (34.6%), respectively were confirmed to be P. falciparum negative by PCR. Furthermore, 24.5% (13/53) of the afebrile as well as 10.1% (22/217), 10.9% (24/220), 11.7% (25/214) and 14.4% (28/194) malaria parasite uninfected participants by microscopy, and mRDT negative on blood, saliva and urine respectively had P. falciparum infection. The sensitivity and specificity of all five test methods, with their corresponding positive and negative predictive values are shown in Table PCR is the most sensitive diagnostic technique and therefore the reference standard in this study. As such, participants found to be positive by any other diagnostic method, but not by PCR, were considered “false” positive. PCR confirmed 34.3% (85/248) febrile and 90.5% (76/84) microscopy positive individuals as well as 91.4% (74/81), 83.9% (73/87) and 65.4% (70/107) of mRDT positives on blood, saliva and urine respectively as P < 0.001 each) in the performance of mRDT on both saliva and urine when compared to blood mRDT.Cohen’s kappa coefficient was used to assess the agreement between tests and the most sensitive method, PCR Table . As suchP. falciparum parasitaemia recorded by light microscopy is shown (Table Variation of the sensitivity (based on febrile status and mRDT diagnosis) with wn Table . The higThe requirement of blood for almost all malaria diagnostic tests limits access mainly due to concerns about disease transmission, blood taboos, needle-sticks injuries  and compThe use of a commercial blood PfHRP2/pLDH detection test to effectively identify malaria parasite-infected individuals in saliva and urine confirms limited reports of the utility of mRDT as non-invasive, non-microscopic yet rapid tools for the detection of the parasite , 29. TheAlthough a higher sensitivity has been recorded in urine samples from India , detectiP. falciparum [This study also assessed the performance of different methods in detecting malaria parasite infection among febrile participants, with PCR outperforming blood smear microscopy and HRP2-based malaria RDT as reported previously , 46, detlciparum  suggestsOverall, the proportion of confirmed malaria parasite infected individuals was low (32.6%) in spite of the high proportion (82.4%) of participants with axillary temperature ≥ 37.5 °C and/or history of fever at enrollment. This finding is consistent with previous reports  and is sAlthough the sensitivity of presumptive diagnosis was quite high, the extremely low specificity recorded in this study vastly overdiagnoses the disease , 53. ThiAlthough light microscopy agreed pretty well with PCR as reported previously , a 9.4% . The use of HRP2-based mRDT (SD Bioline) in this study was informed by the recommendation of HRP2-based assays in areas where P. falciparum is predominant [RDTs in combination with microscopy would improve malaria diagnosisdominant  and repodominant  and elsedominant . Howeverdominant  perhaps dominant , since fdominant . Patientdominant .However, the mRDT could not detect the target antigen in eight matching saliva/blood samples with microscopy and PCR positive results. These false negatives may be due to the low parasitaemia (310–3500 parasites/µl blood) recorded in this study, consistent with low multiplicity of infection and/or malaria parasitaemia in the area , 34. TheWhen compared with PCR, the sensitivity of mRDT though consistent with previous reports in areas of low transmission , 51 was P. falciparum but not other species such as P. malariae and P. ovale that have been reported in the area [The study had a number of limitations. Firstly, the mRDT and nested PCR diagnostic tools could only detect the area , 31, posThis study highlighted the low performance of presumptive diagnosis and the need for parasitological tests prior to antimalarial therapy. A commercial blood PfHRP2/pLDH detection test was effectively used to identify malaria parasite-infected individuals using saliva and urine samples. Although still below optimum as recommended by WHO, saliva had higher detection rates and sensitivity compared to urine, making it a practical adjunct to or alternative worth optimising for the routine diagnosis of malaria. Overall, the proportion of confirmed malaria parasite infected cases was low, suggestive of a reduced malaria burden in the area perhaps due to the population’s adherence to national malaria control and prevention strategies.


Background: Preliminary data suggest that target organ damage (TOD) and early vascular aging (EVA) may occur in children with normal blood pressure (BP).Objectives: To analyze TOD and EVA in normotensive (BP <95th percentile on ambulatory BP monitoring) type 1 diabetes children (T1D) in comparison to healthy controls (C).Subjects: 25 T1D aged 13.9 ± 2.6 years and 22 C aged 14.0 ± 3.4 years.Methods: We analyzed age- and height-related pulse wave velocity (PWV) Z-scores and expected PWV based on age, height, and mean arterial pressure (MAP). Expected vascular age based on measured PWV was calculated from pooled pediatric and adult PWV norms. Left ventricular mass index (LVMI), estimated glomerular filtration rate (eGFR), and urinary albumin/creatinine ratio (ACR) were obtained as markers of TOD.Results: T1D and C groups did not differ in anthropometry, ambulatory, LVMI, and ACR. However, median age- and height-related PWV Z-scores were higher in T1D compared to C . Mean (±SD) difference between measured and expected PWV was 0.58 ± 0.57 in T1D vs. 0.22 ± 0.59 in C, p = 0.02. The mean (±SD) difference between chronological and expected vascular age was 7.53 ± 7.74 years in T1D vs. 2.78 ± 7.01 years in C, p = 0.04.Conclusion: Increased arterial stiffness and increased intraindividual differences between expected and measured PWV as well as between chronological and expected vascular age indicate that EVA may develop in T1D children even at normal ambulatory BP levels. Elevated blood pressure (BP) represents an important cardiovascular risk factor with a direct relationship between the BP level and rates of stroke, myocardial infarction, and the risk of end-stage renal disease .In children, the BP cutoffs for increased cardiovascular risk are not clearly defined due to the lack of longitudinal studies linking childhood BP levels to long-term outcomes in adulthood . HoweverRecent studies suggest that even mild elevation of BP, below the hypertension threshold, or white coat hypertension (WCH) can lead to heart and vascular damage –8. In chPulse-wave velocity (PWV) can be used for non-invasive assessment of vascular function  and evaluation of vascular aging; it can be successfully applied to children for whom there are age- and height-specific normative data –15. IncrThe goal of our study was therefore to analyze BP and PWV in children with diabetes mellitus type 1 in comparison with healthy controls.We hypothesized that diabetic ambulatory normotensive children would show functional cardiovascular changes  and a higher vascular age compared to healthy controls even in the absence of structural changes  and that the functional changes would occur with only mild elevation of the BP level.All consecutive patients with type 1 diabetes who were referred for assessment of hypertension to Pediatric Nephrology & Hypertension Clinic, Department of Pediatrics, University Hospital Ostrava, Czechia, from January 2017 to December 2019 were enrolled in a prospective study. Out of a total of 29 patients enrolled, 25 patients with T1D were diagnosed with ambulatory normotension based on ambulatory BP monitoring (ABPM) criteria (see below), and they were included in the current study.The control group consisted of 22 healthy children (12 boys) recruited from hospital co-workers' family members.All subjects completed the office BP measurements, ABPM, echocardiography, and pulse wave velocity (PWV) measurements. No patient was treated with antihypertensive therapy at the time of the investigation. All patients had normal renal function without any significant proteinuria except for one diabetic patient with albuminuria . The demographics of both groups is shown in Inclusion criteria were as follows: written informed consent and age 10–19 years. For children with diabetes type 1, the additional inclusion criterion was diabetes duration ≥1 year. The exclusion criteria were as follows: history of other serious disorders or history of diseases affecting BP , current antihypertensive medications, or other BP-affecting issues such drug abuse or smoking.The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee. Written informed consent was obtained from all parents and patients of both study groups, as appropriate.2; BMI, weight, and height were converted into standard deviation scores (SDS), e.g., Z scores, based on reference values for healthy Czech children; see http://www.ojrech.cz/lesny/kompendium/index.htm. Weight and height were measured by a single trained nurse with precision electronic scales and fixed stadiometer.At the time of the office and ABPM measurements, the body height and weight were recorded. The body mass index (BMI) was calculated as kg/mThe office BP was measured by a single trained nurse on the same day as the ABPM (before initiating the ABPM device), according to the current European guidelines . After 1The ABPM was performed using the oscillometric device SpaceLabs 90217 . The monitor was programmed to measure the BP every 20 min during the day (6 A.M.−10 P.M.) and every 30 min during the night (10 P.M.−6 A.M.). The parents and children were instructed to keep a diary of daily activities during the ABPM measurement. However, in order to compare our results with the normative values for ABPM , we defiFor the study purposes, the following ABPM parameters were obtained and analyzed: mean arterial pressure (MAP), SBP, and DBP measured over 24 h, daytime and nighttime periods. The average absolute values for MAP, SBP, and DBP for all time periods were subsequently converted into Z-scores (SDS) using the ABPM normative data . Night-tNormotension on ABPM was defined as mean SBP and DBP and MAP <95th percentile  during 24 h, daytime and nighttime periods. Hypertension on ABPM was defined as mean SBP or DBP or MAP value ≥95th percentile  at any time period. Similarly to adult and European Society of Hypertension (ESH) guidelines, the BP load was not included in the definition of ABPM hypertension (Patients with ABPM normotension (as defined above) and office SBP or DBP SDS >1.645 were categorized as patients with WCH.via applanation tonometry using validated PulsePen device (DiaTecne s.r.l.)   s.r.l.) , 20 as d s.r.l.) , 20. AllPrior to cfPWV measurement, patients were placed in the supine position and rested for 15 min in quite temperature-comfortable room. Three electrocardiographic leads were attached. The right carotid artery was palpated and marked , and the procedure was repeated for the right femoral artery . To assess pulse wave travel distance, surface tape measurements were performed between the carotid site and the jugular notch and between the jugular notch and the femoral site. The difference between these two distances was considered the pulse travel distance , 16. PWVTo calculate the expected PWV in each child based on age, height, and MAP, we used the equation proposed by Reusz et al.  expresseThe expected PWV results were then compared with obtained PWV results (PWV difference) in each individual patient and subsequently compared between T1D and C groups.r2 = 0.95, p <0.0001; PWV in females (m/s) = 4.01 + 0.08 × age (years), r2 = 0.96, p < 0.0001. The expected age based on measured PWV (vascular age) for individual patients was then calculated as follows: patient's measured PWV – 3.99 for males /0.08. The intraindividual differences between the chronological age and the vascular age were then compared between groups.To assess vascular age of our children, we combined normative age-based PWV data for children aged 7–18 years  obtained2.7 [left ventricular mass index (LVMI)]. From the study purposes, LVMI was expressed as LVMI ratio, which was obtained by dividing the measured LVMI value by the 95th percentile of LVMI for healthy children  ultrasound systems. Measurements were performed off-line by a single experienced physician according to the guidelines of the American Society of Echocardiography . Two-dimchildren . LVH wasAll the laboratory investigations were performed on the day of ABPM. Blood draws of the patients were performed in the morning after overnight fasting. Biochemical analysis in whole blood [glycated hemoglobin (HbA1C)], serum , and the first morning urine  was measured by routine laboratory methods immediately after collection. These blood samples except HbA1C were centrifuged at 2,500 g for 6 min at 4°C. Serum concentration of total cholesterol (T-cholesterol), high-density lipoprotein cholesterol (HDL-cholesterol), low-density lipoprotein cholesterol (LDL-cholesterol), and triglycerides were measured by enzymatic methods on AU5420 analyzer . HbA1C was measured using high-performance liquid chromatography . The blood was collected in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes.Serum and urine concentrations of creatinine were determined by the enzymatic method on AU5420 analyzer . Serum concentration of cystatin C and urine albumin was measured by immunonephelometric method . The estimated glomerular filtration rate for creatinine (eGFR/creatinine) was calculated using the updated Schwartz formula . We alsoT-test  or Mann–Whitney test . In addition to the classic null hypothesis significance testing (NHST), we used estimation statistics with permutation on 5,000 resamples; results are shown as mean difference between groups, bias-corrected and accelerated confidence intervals, and permutation p-values. The magnitude of the difference between groups was estimated using Cohen's D effect size with bias-corrected accelerated 95% confidence intervals . Continuous variables were compared using Student's unpaired ntervals . Threshon = 3) as predictors of PWV (dependent variable).The relationship between PWV, diabetes, and BP was analyzed using linear regression analysis. We also performed multivariate regression analysis with only selected, deemed as most clinically important, variables   .Basic characteristics of T1D and healthy controls (C) are presented in The absolute and SDS values of office SBP and DBP were significantly higher in diabetic children compared to healthy controls . HoweverWe did not find any significant differences between diabetic children and healthy controls in LVM, LVMI, and LVMI/95th percentile .p = 0.037) and significantly higher age-related PWV SDS on standard NHST comparison    .The comparison of LVM and LVMI did not show any significant differences between the groups .r = 0.42, p = 0.04) in diabetic children    including age, presence/absence of diabetes, and 24-h SBP showed age and diabetes as significant predictors of PWV ( 0.0001) . In chil= 0.006) .Our study showed that ABPM normotensive diabetic children and adolescents had significantly increased absolute PWV as well as age- and height-related PWV SDS compared to their normotensive controls. Moreover, the intraindividual differences between measured and predicted PWV were significantly higher in T1D patients compared to healthy controls. Similarly, children with diabetes type 1 had significantly higher intraindividual differences between chronological and PWV-predicted vascular age as compared to controls, suggesting early vascular aging (EVA) in T1D patients. This is a novel finding, not previously described. There was no difference in LVM index and microalbuminuria between patients with diabetes type 1 and healthy controls.Diabetes mellitus has been proven to be a major risk factor for the development of cardiovascular disease (CVD) . Recent PWV is the most widely accepted method for assessment of arterial stiffness and vascular age , 35 in bIn adults with diabetes type 1, the increased PWV is associated with adverse cardiovascular outcomes and increased likelihood of early cardiovascular morbidity and with all-cause and cardiovascular mortality, often in connection with various comorbidities  –33. ImpoThe extent of cardiovascular morbidity in children with diabetes type 1 is not well-understood. However, many children and adolescents with diabetes type 1 fail to meet the American Diabetes Association (ADA) and International Society for Pediatric and Adolescent Diabetes (ISPAD) targets for HbA1C, SBP and DBP, LDL-cholesterol, and triglycerides , and manWhile the diagnosis of hypertension and its various forms  and proteinuria  is well-established and used by most physicians, the assessment of subclinical CV injury is still not routinely done in all children with diabetes type 1. Most centers use echocardiography to assess LVM, but the assessment of vascular stiffness using PWV is done mainly for research purposes.There are several studies describing PWV (applanation tonometry) in children and adolescents with diabetes type 1. The recent meta-analysis , which ip = 0.04). Another recent study . Our results confirmed our hypothesis that children with diabetes type 1 had higher intraindividual differences between chronological and predicted vascular age , suggestWe also measured LVM in our study population and found no difference between children with T1D and control group. This is different from some other authors who found various abnormalities in LV geometry, LVM, and LV function , 44. Thep-values and effect size estimation, which allowed us to draw conclusions from differences derived from a relatively small sample but estimated and confirmed on a large permutated sample . The use of modern statistical estimation methods can be considered a strength of our study. Secondly, in our study, we used a different applanation tonometry device (PulsePen) than most other studies in diabetics (SphygmoCor). However, the obtained PWV results from both devices are comparable, with excellent concordance between these two devices . In addition, we used estimation statistics with permutation  devices . MoreoveIn conclusion, children with diabetes type 1 and ambulatory normotension have significantly increased arterial stiffness and PWV-predicted vascular age (EVA) while having normal LVM and no significant albuminuria. The main predictors of increased vascular stiffness in children with diabetes type 1 were the HbA1C levels and systolic 24-h BP. It appears that even a small increase in ambulatory BP, even within the normal range, can contribute to the development of vascular injury and EVA in the presence of diabetes type 1.The original contributions presented in the study are included in the article/eticka.komise@fno.cz. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.The studies involving human participants were reviewed and approved by Ethics Committee, University Hospital Ostrava, 17. Listopadu 1790/5 708 52 Ostrava-Poruba e-mail: TŠu: author of the supporting grant, responsible for the data collection, controls selection, office BP measurement, ABPM, PWV measurement, calculation of Z-scores, descriptive statistics, NHST, and writing a manuscript. JS: office BP measurement, diabetic patients selection, blood, and urine samplings. JP: echocardiography. RP: multivariate analysis. TSe: author of the supporting grant and revisions of manuscript. JF: revisions, including final version of the manuscript, statistic , figures, calculation of vascular age, scientific leading, and discussion. All authors contributed to the article and approved the submitted version.This work was supported by the Ministry of Health, Czech Republic–conceptual development of research organization FNOs/2019 (MH CZ-DRO–FNOs/2019).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.