There are growing reports on the prevalence of non-falciparum species and submicroscopic infections in sub-Saharan African countries but little information is available from Cameroon.18S gene sequences were BLASTed for confirmatory diagnosis.A hospital-based cross-sectional study was carried out in four towns  from three malaria epidemiological strata  of Cameroon. Malaria parasites were detected by Giemsa light microscopy and polymerase chain reaction (PCR) assay. Non-falciparum isolates were characterized and their Plasmodium species viz. P.falciparum, P.ovalecurtisi and P.vivax, and two co-infection types  were found. The remaining infections were mono–infections with either P.falciparum or P.ovalecurtisi. All non–falciparum infections were symptomatic and microscopic. The overall proportion of submicroscopic infections was 11.8% (14/119). Most asymptomatic and submicroscopic infection cases were self-medicated with antimalarial drugs and/or medicinal plants. On analysis, P.ovalecurtisi sequences were found to be phylogenetically closer to sequences from India while P.vivax isolates appeared closer to those from Nigeria, India, and Cameroon. No G6PD-d case was found among non-falciparum infections.PCR assay detected malaria parasites in 82.4% (98/119) patients, among them 12.2% (12/98) were asymptomatic cases. Three P.vivax and P.ovalecurtisi and the absence of P.knowlesi in Cameroon. More studies are needed to address non-falciparum malaria along with submicroscopic infections for effective malaria management and control in Cameroon.This study confirms our previous work on circulation of The online version contains supplementary material available at 10.1186/s12879-022-07901-6. Anopheles mosquitoes infected with Plasmodium parasites , and a final extension (72 °C/5 min). P.knowlesi infection was confirmed by presence of 200 bp PCR product on electrophoresis gel [Primers rPLU5 and rPLU6 were used to identify esis gel . DNA sam18SSSUrRNA gene PCR results were not due to poor quality DNA, the single copy merozoite surface protein 2 (msp2) gene was amplified  was amplified and genetic polymorphism of G6PD gene was evaluated for 202A mutation by sequencing . Nucleotide and deduced amino acid sequences were aligned using CLUSTALW algorithm [PCR products of non-falciparum samples were purified using DNA GeneJET PCR purification kit , and then sequenced on an ABI3037XL genetic analyser (Applied Biosystems) with 2X coverage (sequenced from both forward and reverse directions). BLAST analysis of the n 10.0.5 . We complgorithm .The evolutionary history was inferred using Neighbour-Joining method . A bootsFever was defined as axillary temperature ≥ 37.5 °C . Parasit10-transformed before statistical analysis. The level of statistical significance was set at p-value < 0.05. Statistical analysis was performed using the statistical package for social sciences v16 for Windows  and StatView v5.0 for Windows .Data were keyed in an Excel spreadsheet , coded, and checked for consistency. Qualitative variables were presented as frequency, percentage, and confidence interval at 95% (95% CI) while quantitative variables were presented as mean ± standard deviation (SD) . Indepenvs 15.4%), 5–10 years (13.0% vs 12.7%), 10–15 years (17.4% vs 14.1%), 30–35 years (8.7% vs 8.5%), and ≥ 35 years (17.4% vs 16.8%) . The same pattern was observed between gender and age with respect to malaria epidemiological facet.The study population was mainly composed of females (60.7%) Table . The meaP.falciparum by thin smear.Based on PCR results, the overall malaria prevalence was 82.4% . Out of 86 LM-positive samples, PCR detected malaria parasites in 84 of them. Among 33 LM-negative samples, PCR-detected malaria parasites in 14 samples , although the prevalence of malaria was highest in Soudanian facet (84.6%). However, malaria infection varied significantly according to gender, with higher malaria prevalence reported in males when compared to females from Forest facet   and age . A non-significant negative correlation was found between parasitaemia and age . Parasitaemia was significantly higher in Forest facet  when compared to samples from Sahelian facet (Pette) .There was no statistically significant difference between parasitemia with respect to gender , P.ovalecurtisi , and P.vivax . P.falciparum was the only species recorded in Maroua and Mayo-Oulo, while P.ovalecurtisi and P.vivax were only found in Douala and Pette, respectively  . These infections accounted for 14.3% (14/98) of all malaria infections. The prevalence of submicroscopic infections varied between areas, with the highest values found in Mayo-Oulo . Likewise, submicroscopic infections were more prevalent in females compared to males . The proportion of asymptomatic and submicroscopic infections did not vary significantly according to patients’ age although proportions of these two types of infections were higher among those aged > 20 years (15.7%) and 5–10 years (13.3%), respectively.The proportion of asymptomatic infections was higher among females  when compared to males  but no statistically significant difference was found  by previous studies, suggesting growing evidence of its circulation in the country [P.vivax sample was found in Pette located in Sahelian facet of Cameroon. To the best of our knowledge, this is the first evidence of PCR- and sequencing-confirmed circulation of P.vivax in this area of Cameroon. Plasmodiumvivax control is tricky due to several reasons: (i) this species has dormant stages—called hypnozoites—responsible for malaria relapses many months to years after the first infection; hypnozoites are resistant to antimalarial drugs, and constitute an invisible reservoir of P.vivax parasites [P.vivax is difficult despite a few encouraging reports [Plasmodium species can modulate malaria severity [P.vivax to elicit severe malaria attacks and deaths [ country , 24, 26.arasites ; (ii) co reports  making eseverity . Also, id deaths –57.P.ovalecurtisi was found at low prevalence among study population. This study is the first to confirm clearly the presence of P.ovale subspecies in Cameroon. Current published and available data on these two subspecies in Cameroon come from a recent study on local infection [P.ovale species in Cameroon due to their role in life-threatening malaria complications viz. severe thrombocytopenia [nfection  and imponfection –60. Zhounfection . There iytopenia , severe ytopenia .P.knowlesi in Cameroon as reported in a previous study conducted in five areas of the country  [P.knowlesi in humans in sSA should not be ruled out given both high rate of human migration between individuals from sSA and SEA, and presence of available animal reservoirs in sSA. This hypothesis of human migration to explain the presence of imported Plasmodium populations in Cameroon is supported by our phylogenetic analyses. The P.vivax sample from Pette town formed a cluster with P.vivax strain of Nigerian origin. This town is located in the Far North region of Cameroon which is geographically bordered by several countries including Nigeria.This study also confirms the absence of d Limbe) . This isd Limbe) , and hasd Limbe) . A possiP.vivax and P.ovale hypnozoites. Since PQ can induce haemolytic anaemia in G6PD–d persons, testing for G6PD status especially in children, must be performed prior to PQ treatment [Keeping in mind the necessity to address non-falciparum species, this brings back on table the problem of using PQ in association with ACT or CQ in African individuals . PQ is ureatment . TestingP.ovale and P.malariae reported in an area of declining P.falciparum transmission in Tanzania [P.falciparum to non-falciparum species, if they are not addressed.Finally, the management of non-falciparum species is worth addressing in Cameroon, and it is crucial to achieve WHO’s elimination objectives . A fact Tanzania  thereby Submicroscopic infections were also reported in this study. PCR is more sensitive than LM, with a limit of detection as low as 5 parasites/µL of blood while that of LM is 50–500 parasites/µL of blood . The impP.falciparum parasites, especially to ACTs used as first line treatment [P.falciparum parasites in Rwanda and Uganda , and P.falciparum parasites with ACT-resistance associated mutations in the PfKelch13 gene in other parts of Africa  [Interestingly, self-medication practice with antimalarial and/or traditional drugs was reported in asymptomatic and/or submicroscopic infection cases. Self-medication is a commonly reported problem in Cameroonian population  which exreatment . Mostly,f Congo) –73.P.falciparum irrespective of epidemiological facet. In addition, P.ovalecurtisi and P.vivax were the two non-falciparum species found, while P.knowlesi, P.ovalecurtisi and P.malariae were not seen in this study. Malaria prevalence varied with regard to epidemiological facet with highest burden in Forest facet. All non-falciparum species cases found were symptomatic and microscopic. Asymptomatic and submicroscopic infections accounted for > 10% of all infections, and were likely due to self-medication with antimalarials and/or traditional medicines. Greater attention should be given to non-falciparum species, asymptomatic/submicroscopic infections, and self-medication in Cameroon to successfully achieve malaria control and elimination programmes.Malaria infection was highly prevalent with a predominance of Additional file 1. Primers used to discriminate human Plasmodium species, and amplify the msp2 and G6PD genes.Additional file 2. Performance of Giemsa-based microscopy incomparison with nested PCR in the identification of malaria infection.  Additional file 3. Malaria infection by age, gender and strata.  Additional file 4. Neighbor-Joining tree of P. ovale subspecies and P.vivax in comparison to NCBI sequences based on 18S rRNA gene sequences andreference strains.


Malaria is a growing problem in Africa, with prevalence varies from areas to areas due to several factors including the altitude. This study aimed to investigate the malaria distribution and its relationship with level of some blood parameters and plasma myeloperoxidase (MPO) in population of three localities with different altitudes.A total of 150 participants were recruited in each locality and facial body temperature of each was measured using a forehead digital thermometer. Blood samples were collected and used diagnose malaria parasite using the rapid test followed by Giemsa stain microscopy and have the full blood count and MPO level using a colorimetric method.falciparum malaria was 34.7%, with no difference between the three communities, but Bambili of high altitude had the highest prevalence (70.7%). A majority of the infected persons had mild malaria, with most cases being asymptomatic (temperature < 37.5 ºC). Patients had significant increase of geometric mean malaria parasite density (GMPD) in Bambili (1755 ± 216 parasites/µL) and Bamenda (1060 ± 2515 parasites/µL of blood) than patients in Santa (737 ± 799 parasites/µL). There was a significant risk to have malaria infection in Bambili  than in Santa . Bambili’ participants of 6–10 years showed a high prevalence of malaria (85.7%). GMPD was significantly different between males (p = 0.010) as well as females (p = 0.000). Participants from Santa (11.2 ± 3.2 g/dL) and Bambili (12.6 ± 2.4 g/dL) had a high haemoglobin concentration than those from Bamenda (10.6 ± 2.1 g/dL). There was a significant difference in the WBC counts and platelet counts among infected participants in the study areas. MPO level had an increasing trend among infected participants in Santa (2.378 ± 0.250), Bambili (2.582 ± 0.482) and Bamenda (2.635 ± 0.466).The overall prevalence of The results of the present study demonstrated that altitudinal variations significant impact the risk of population to have malaria with high parasitaemia and may contribute to the malaria prevalence and severity by affecting the haemoglobin concentration, WBC and platelet level and plasma MPO in population.The online version contains supplementary material available at 10.1186/s12936-022-04438-6. Plasmodium falciparum is the most virulent and deadliest parasite of the human Plasmodium species that causes falciparum malaria  x [8000WBCs].Haemoglobin concentration, total WBCs and platelet counts were obtained after performing complete blood count using a haematological analyser machine , following the protocol of the manufacturer. Blood cell indices such as haemoglobin concentration, total platelets count, WBC count were recorded. Haemoglobin concentration < 11 g/dL of blood was used to define anaemia in the patient. Anaemia was further classified as mild (Hb 10.0–10.9 g/dL), moderate (Hb 7–9.9 g/dL) and severe (Hb < 7 g/dL) . WBC couH = 5.0) were mixed with equal volumes (100 µL) of serum on a microplate. The reaction was stopped after 10 min using 0.1 M sulphuric acid and absorbance was recorded at 490 nm using an ELISA reader .Myeloperoxidase was assessed using a colometric method as adopted by Oumar et al.  with a fp ≤ 0.05.The cleaned data was uploaded into the statistical package for social sciences (SPSS) version 23  for statistical analysis ]. Most participants [27.6% . Patients from Bambili registered more positive cases of malaria [70.7% . The results (Table p = 0.003) had higher geometric mean parasite density (GMPD ± SEM) of 1755 ± 216 µL and 1060 ± 2515 µL of blood) respectively compared to patients in Santa  in the GMPD ± SEM between males and females in the entire study. The density of malaria parasite was higher in females GMPD ± SEM (1691 ± 265 µL) than males (1205 ± 1487 µL). Following the different areas, a significant  difference was observed only in Bambili, with high prevalence in females (2088 ± 305 µL) than males (1317 ± 237 µL). There was a high significant difference in the mean malaria parasitaemia in each sex group across the three study areas with male patients in Bamenda and Bambili recording higher values  than male patients in Santa. Conversely female patients in Santa and Bambili recorded significantly  higher parasitaemia than female patients in Bamenda . In the different study areas, Bambili had a significant difference in prevalence between the age groups , with patients 6–10 years old showing higher prevalence. For each age group across the three study areas, only patients aged 36–60 years old recorded significant distribution of malaria parasite  with patients in Bambili having 70.0% (14/20), Bamenda 24.4% (11/45), and Santa 5.6% (2/36) Table There was a significant distribution of p = 0.145svy) higher mean (± SD) haemoglobin concentrations of 11.9 ± 1.5 g/dL and 11.8 ± 3.5 g/dL of blood respectively, even though the post hoc test did not show any significant difference (p = 0.145svy). In patients with malaria, a significant difference was observed in Hb in the three study areas (p = 0.004). Patients that tested positive for P. falciparum in Bamenda had lower mean haemoglobin concentrations (Hb 10.6 ± 2.1 g/dL) compared to patients’ resident in Bambili (Hb 12.6 ± 2.4 g/dL). A significant difference was also observed between Hb in the malaria negative patients (p = 0.001jmp). Patients that tested negative for P. falciparum in Bambili had lower Hb (Hb 9.9 ± 4.7 g/dL) as compared to patients in Bamenda  and Santa . There was a significant difference in the haemoglobin concentration levels between patients that were positive for P. falciparum as compared to those that tested negative in Santa  and Bambili .As shown on Table p = 0.000twz) difference in the mean white blood cell count among patients resident in the different geographical study areas. Participants in Santa  and Bamenda  had higher mean WBC counts than patients in Bambili . There was a significant difference in the level of WBC in both positive  and negative  patients in all the three study areas. In the positive patients, the WBC level was higher in Santa as compared to Bambili (p = 0.000eh) and Bamenda (p = 0.006eb). It was also observed that patients from Bamenda had higher level of WBC count compared to those from Bambili (p = 0.018bh). Only patients in Santa showed a significant difference  in the mean WBC count between patients that tested positive (9.6 ± 6.9 × 103 µL) and negative  increasing trend in the mean platelet count among patients in Santa (253 ± 99 × 103µL), Bamenda (338 ± 129u × 103 µL) and Bambili (465 ± 132 × 103 µL). A similar trend was observed among patients that were positive  for P. falciparum and among those that were negative  between the study areas (Table p = 0.031), Santa  and Bambili .In addition, the results showed a significantly  variation in anaemia prevalence based on geographical location. Patients from Bamenda recorded the highest prevalence  with females in Bamenda recording the highest  higher prevalence of anaemia (21.6%) than females (17.9%) in the whole study. Males in Bamenda significantly  registered a higher prevalence 32.3%  of them were anaemic and there was a significant  more anaemic.Patients that were ≤ 5 years old and those of 61 years and above registered significantly high anaemia prevalence of  higher mean myeloperoxidase levels (2.554 ± 0.437) than patients who were negative (2.117 ± 0.862). With respect to study areas, malaria positive and negative patients showed a significant difference in MPO (p = 0.003). Participants in Santa who tested positive for P. falciparum had highly significant  MPO levels (2.378 ± 0.250) than participants that tested negative (1.721 ± 0.980).As shown on Table p = 0.001ghi) in the MPO levels. The MPO level was significantly lower in Santa (2.007 ± 0.812), as compared to Bamenda (2.604 ± 0.426 g) and Bambili (2.595 ± 0.461i). In malaria positive patients, there was no significant difference  in MPO when comparing the three different study areas. In patients who tested negative for P. falciparum, there was a significant variation  in the mean MPO levels among patients in the different geographic area, with a low MPO in participants of Santa than those of Bamenda and Bambili, but the difference was significant only in Bamenda (p = 0.05de). Independent of the geographic areas, there was a significant difference between patients with mild, moderate and severe malaria , but no difference was observed with each area with respect to the malaria categories as well as when comparing participants of the different study areas within the same malaria severity level.Comparing the levels of MPO between the study areas, there was a significant variation  results . These rThe malaria parasite density in population in the different areas was significantly different in males as well as in females. In males, the density of malaria parasite was significantly lower in Santa compared to that in Bamenda and Bambili which did not showed a difference. In contrast, in females, Bamenda, localities of low altitude had a low parasite density, followed by Santa and Bambili . In female, the results quietly indicate that the altitude may affect the density of parasite in the population. Therefore, the absence of relationship between the malaria parasite density and the altitude of the living localities may due the habit of the males as due to the crisis in the region, males are forced to change regularly the house within the three localities.P. falciparum in the entire study were asymptomatic. An experimental study associated the expression of asymptomatic falciparum malaria and related severities with a switch in the host pro-inflammatory immune response against malaria parasite toward anti-inflammatory response [P. falciparum and possible suppression of the clinical presentation of malaria defined by temperature ≥ 37.50C. This could further explain the significantly higher malaria parasitaemia observed among patients in Bambili and Bamenda though there was no significant variation in the mean malaria parasite density between these localities including the highland area of Santa. Malaria parasitaemia was significantly higher in females than in males in the entire study population. Considering the geographic settings of the study areas, male patients in Bamenda and Bambili recorded significant higher malaria parasitaemia than males in Santa. This similar trend was observed among females where those in Bambili and Santa had higher malaria parasitaemia than females in Bamenda. This suggests a possible gender based impact of altitude and climatic conditions on falciparum malaria parasitaemia.Malaria was significantly more prevalent among patients that were 6–10 years in the entire study. Reports of similar studies conducted in Tanzania  and elseresponse . This maP. falciparum in the entire study registered lower haemoglobin concentration than patients that were negative and falciparum malaria patients in Bamenda were presented with mild anaemia as a result of lower haemoglobin concentration than patients in Santa and Bambili. Similar observations were recorded among malaria patients in Myanmar [P. falciparum could be a contributing factor to the burden of anaemia especially in Bamenda and elsewhere.It was observed that altitudinal variations significantly influenced the distribution of haemoglobin concentration, white blood cells and platelet counts among the populations living in the three different localities. The results revealed that people living in the Santa and Bambili highland areas had higher haemoglobin concentrations than in the Bamenda lowland area. These results were in conformity with reports of a study in the Middle East  that rep Myanmar , the Sou Myanmar  and in B Myanmar . FemalesP. falciparum and infected patients also showed elevated levels of WBCs compared to non-infected participants. Though there was a significant increase in leucocyte levels among malaria patients in Santa, the mean WBCs level in all the populations of the different study areas were within normal and below the upper limit of normal range irrespective of their malaria status hence leukocytosis was absent in either of the populations. This suggests that altitudinal variation affects the level of WBCs among malaria patients but may not lead to serious pathologies associated with malaria infection. These suggestions coincide with results of [Participants in Santa recorded higher WBC counts than those in Bamenda and Bambili. There was a similar trend among patients that tested positive for sults of  in the Msults of  in Camersults of  that assP. falciparum across the different study areas. Malaria patients in Bamenda and Bambili recorded higher MPO levels than patients in Santa. With regards to the study areas, patients that tested positive for P. falciparum had elevated levels of MPO than non-infected patients. The implication of MPO in falciparum malaria severity is not well elucidated but [Altitude had a significant impact on the serum myeloperoxidase (MPO) levels among patients infected with ated but  reportedP. falciparum .ITNs non-users, increase levels of WBCs and platelet counts were significant predictors of falciparum malaria. Patience presented with pyrexia, anaemia and increased levels of serum MPO levels had increased likelihood of malaria infection. This suggests that these parameters could be used as significant markers of Prevalence of malaria infection was high and its distribution varied across altitudinal gradients in Mezam Division, Cameroon. The altitudinal variations had a significant impact on malaria parasitaemia. Clinical symptoms including haemoglobin concentration, WBC counts, platelet cell count and level of plasma MPO in malaria infected patients were significantly related to the altitudes. Therefore, significant attention should be given to malaria infection for both its proper diagnosis and treatment to prevent infection in Mezam Division. The role of environmental factors other than altitudes that facilitate the existence and transmission of malaria infection should also be further investigated.Additional file 1. Ethical clearance obtained for this study.Additional file 2. Questionnaire form used for collection of demographic data.Additional file 3. The raw data obtained from the field and the laboratory.


Large longitudinal studies with repeated pulse wave velocity (PWV) measurements, a direct measure of arterial stiffness, are required to realize the full potential of arterial stiffness in clinical practice. To facilitate such studies it is important to increase the power of a study by reducing within-subject variability of PWV, and to ease the use of a PWV device in clinical settings by minimizing PWV measurement difficulties.N = 35) was recorded 12 times over 3 visiting days, 7 days apart. On each day, recordings were made twice in the morning (7–10 a.m.) and afternoon (16–18 p.m.). Data were analyzed using multilevel mixed-effects models, separately for each device.We systematically investigated experimental setting and meteorological conditions, as well as physiological factors and participant characteristics, to determine whether and to what extent they affected: between- and within-subjects variability of PWV recordings, and measurement difficulties of a particular device. We conducted a 2-week longitudinal block-randomized cross-over study with two blinded observers and two commonly used devices: applanation tonometry SphygmoCor CvMS and oscillometric Arteriograph to assess carotid-femoral (cfPWV) or aortic (PWVao) PWV, respectively. Our sample had uniform and wide-spread distribution of age, blood pressures, hypertensive status and BMI. Each participant  that strongly affected both cfPWV and PWVao, other significant factors appeared to indicate a measurement approach. cfPWV as a more direct measure of arterial stiffness was additionally affected by hypertension status, outdoor temperature, interaction of MAP with outdoor temperature and the order of visit, with MAP within-subject variability contributing on average 0.27 m/s to difference in repeated measurements at 5We identified factors affecting PWV recordings and measurement-difficulties and propose how to improve PWV measuring protocols. Arterial stiffness is a phenomenon associated with vascular aging that refers to loss of arterial compliance or changes in vessel wall properties . ArteriaPulse wave velocity (PWV) measurement is considered the simplest, non-invasive, robust, and reproducible method for assessing arterial stiffness, with the carotid-femoral vascular bed regarded as the most easily accessible pathway for aortic PWV measurements , 15. BecDespite its considerable potential for cardiovascular disease prevention, measurements of PWV have limited use in clinical practice. The updated 2021 ESC Guidelines on cardiovascular disease prevention in clinical practice argues against widespread use of PWV measurement in clinics because of difficulties in measurements and precision of measurements . While rper participant = 12 recordings; Ntotal = 420), uniform distribution of participants by age, sex, BMI and hypertensive status, and multilevel mixed-effects models used in data analysis. All the recordings were performed over 2 weeks. In such a short period of time, clinically relevant changes in vascular biology are not expected, so the variability of PWV during repeated recordings can primarily be attributed to chance and confounding factors.To reach our goals we used the study design that yields strong evidence: a block-randomized cross-over longitudinal study with two observers that were blinded to each other’s readings, the largest number of repeated measurements per participant , and body mass index  to ensure even distribution across the inclusion criteria . ParticiOne person  who was initially enrolled in a study was additionally later excluded because she contracted an infection over the course of the study, leaving a total of 35 participants.The study was approved by the Ethics Committee at the University of Split School of Medicine, and all participants provided written informed consent.This is a single-blind block-randomized cross-over longitudinal study.The study took place between October 2019 and February 2020 at the University of Split School of Medicine in the Laboratory for vascular aging.Each participant was recorded 12 times in total over the course of 2 weeks, four times during each of the 3 visit days that were separated by 1 week. On each visiting day, recordings were taken in the morning (7–10 h) and afternoon (16–18 h) by the two observers.The order of devices—Sphygmocor CvMs and Arteriograph and of observers, was randomized using the block size of 4. The two observers were blinded to each other’s readings.Pulse wave measurements were taken with the two devices that use different measuring techniques—the applanation tonometer SphygmoCor CvMS  with which we collected cfPWV data and the oscillometric device Arteriograph  used to collect PWVao data.The measurements were taken in accordance with the American Heart Association’s recommendations for improving and standardizing vascular research on arterial stiffness . The obsParticipants were requested to abstain from vigorous exercise and alcohol consumption for at least 24 h prior to a recording session (morning or afternoon). They were also instructed not to eat or drink anything except water or smoke for at least 3 h before any recording session. Those taking vasoactive medicines were advised to continue taking them as usual and not to change the dosage during the study.Before the start of the study, both observers had received extensive 7-day training during which they performed approximately 40 high-quality measurements under supervision.To calibrate the pulse wave signals acquired by the SphygmoCor, we obtained brachial blood pressure measurements with the validated oscillometric sphygmomanometer .N = 1 participant).We used the subtracted distance method to calculate wave travel distance. The method was chosen over the direct method as per recommendation by the latest guideline . A largeAll participants underwent a medical history.The meteorological data: outdoor temperature, air pressure, and relative humidity, were provided by the Meteorological and Hydrological Service of Croatia’s local office, and used to estimate weather conditions on each visit day and time. The contour plot is used to show the distributions of three meteorological parameters by a visit day  for all of the participants’ visits, with relative humidity and air pressure as x and y dimensions, and temperature as a color coded z-dimension . The graThe study employs a multilevel data structure, including 35 groups  and 12 repeated measurements by a participant (level-1) totaling 420 observations. Because no random slopes are anticipated in any of the multilevel models that we developed, the sample sizes mentioned above (levels 2 and 1) are deemed adequate for estimating unbiased and accurate regression coefficients, variance components, and first level standard errors , 40, allWe used descriptive statistics to describe distribution of quantitative  and qualitative (absolute and relative frequencies) variables. To identify predictors of PWV single readings, or different types of measurement difficulties we employed several multilevel models with random intercept. Depending on the type of a dependent variable we used multilevel mixed-effects generalized linear models for continuous dependent variables like PWV readings, mixed-effects logistic regression models for dichotomous dependent variables like the occurrence of marginal-quality signals or manual signal selection, and mixed-effect Poisson regression for count data like the count of repeated attempts to record PWV values due to low-quality of a captured signal. All of the models were run with the robust estimator, which is resistant to certain types of misspecification in multilevel models such as heteroscedasticity or deviation from normality , 43, and°C), air pressure (Pa), and relative humidity (%), and participants’ characteristics—age, sex, BMI, hypertension status, MAP, SBP, DBP, HR—were all investigated for their relationship to a dependent variable by a simple multilevel regression analysis. Those independent variables that were associated with dependent variable at the p < 0.2 significance level, entered into multiple multilevel regression model. For independent variables that were non-significant in a multiple model, their contribution to the model (pseudo R2) was investigated further to decide on their inclusion in the final model.Each model was built in two steps. The experimental setting variables—order of visit, time of day, first device used in a session, and observer, meteorological variables—outdoor temperature . Thus, the school divider was effective in removing this effect of BMI.The difference in distance between the carotid and femoral sites measured with a tape vs. a school divider strongly correlated with the level of obesity as evaluated by BMI  and 5.3% (95% CI 5–6%) for cfPWV and PWVao measurements, respectively; while ICC for within-subject average cfPWV measurements was 94% (95% CI 91–97%) and 96% (95% CI 94–98%) for PWVao measurements.In both the cfPWV and PWVao models, the most prominent effects to which the largest difference in predicted PWV margins was attributed were age and MAP, in that order. The increase in PWV values with a 1-year increase in age was twice higher for PWVao than cfPWV measurements , B. HoweAs for the meteorological factors, only the outdoor temperature had a significant effect on cfPWV measurements. In cfPWV model, as mentioned earlier, outdoor temperature was involved in the interaction with MAP, suggesting that it modifies the relationship between cfPWV and MAP . PWVao mExcept for the order of visit in the cfPWV model—notably the comparison of 2nd to 1st visit values, no other experimental factor was associated with differences in cfPWV or PWVao measurements.Increased values of PWVao measurements were also associated with increased values of BMI and HR, and a female sex, whereas in the cfPWV model it was hypertensive status that was found to significantly increase cfPWV.p ≥ 0.365).During the development of multilevel mixed-effects models we also assessed if there was a significant interaction between sex and age. After including an interaction term between sex and age in the full multivariate models of both SphygmoCor and Arteriograph, and controlling for menopause status, we discovered that the interaction between age and sex is not significant  and PWVao  measurements that older age was associated with wider range of repeated PWV measurements observed in a person, whereas BMI, hypertension status or sex were not associated with this variability. However, we did find significantly larger variability of within-subject PWV measurements in menopausal than in women with menstrual cycle, for both devices . Moreover, for women who menstruate, within-subject PWV variability was comparable to men (P ≥ 0.310). In addition, we also assessed if the order of visit, the only experimental condition significantly affecting cfPWV values, is associated with decreasing variability of cfPWV measurement in a person and found this was not the case .Finally, after developing multilevel mixed-effects models that aimed to identify factors influencing values of PWV measurements, we also evaluated which, if any, participants’ characteristics are related to variability of repeated measurements in a person. We found for both cfPWV  the number of repeated measurements as a result of the SphygmoCor’s pulse wave velocity signal failing the quality control, (b) the possibility of observing the marginal quality signal by SphygmoCor with the coefficient of variation for cfPWV estimation between 6 and 10%, or (c) the Arteriograph’s oscillometric signal being manually selected for analysis .p = 0.438), or final (p = 0.276) multilevel model. The results demonstrate that it is very likely that the Arteriographs’s oscillometric signal will be manually selected for analysis in a female participant. In fact, manual adjustment was required at least once for 11 (65%) of the women, with five women requiring it on ≥8 instances out of 12 measurements. In contrast, just five males (28%) required this modification on 1–3 occasions. Measuring in the afternoons was strongly to moderately associated with a lower chance of manual adjustment, reducing the odds ratio for it by 77% (95% CI from 46 to 90%). Finally, a chance for manual adjustment differed significantly between the measurers by increasing OR for the measurer 2 by 0.91 (95% CI 0.23–1.95).As opposed to the automatic selection of the oscillometric pulse wave signal by Arteriograph device, the manual signal selection is time-consuming and subject to uncertainty. In total, we had to manually select a signal for analysis in 78 (19%) of the Arteriograph recordings. We did not find, however, that manual selection affected PWVao values when this factor was added as independent variable to the simple  of the time. Female sex increased the incidence rate ratio (IRR) of a repeated measurement by 23% (95% CI 1–50%), whereas afternoon recording time and 3rd vs. 1st visit decreased IRR by 12% (95% CI 3–21%) and 14% (95% CI 4–27%), respectively.p = 0.001).We also discovered that the same two factors—afternoon recording time and 3rd vs. 1st visit—decreased the odds of a signal with marginal quality, which we observed in 95 (23%) of measurements, by: 40% (OR 95% CI −3 to 65%) and 50% (95% CI 1–74%), respectively. When the marginal quality of a signal was added as independent variable to the simple and final multilevel model we found that it increased cfPWV values by on average 0.37 m/s  recorded PWV values, and (b) measurement difficulties associated with this recording. PWV values were repeatedly collected from the enrolled participants over the course of 2 weeks during which no clinically relevant change in PWV values is expected. The analyses were done separately for measurements acquired with two validated devices: cfPWV measurements acquired with applanation tonometry device SphygmoCor CvMS and PWVao values recorded with oscillometry Arteriograph device.We utilized the study design that provides the strong evidence: block-randomized, cross-over, longitudinal study with as many as 12 repeated measurements per participant per device, recorded with observers blinded to each other’s readings. Furthermore, our validation sample exhibited a reasonably wide range of PWV, age, BMI and brachial blood pressures values, and uniform distributions across age, sex, BMI, and hypertension status. Given that uniform distribution puts less emphasis on the center of the distribution and more on its extremes, it produces more precise validation estimates and is thus preferable as validation sample to a sample representative of an underlying population .N = 12), and that our sample had a reasonably wide range of PWV and PWV determinants, which tend to increase observed variability of PWV measurements, the agreement of repeated PWV measurements estimated in our study was comparable to what has been reported in the literature. Grillo et al. estimated within-subject CV, and ICC for cfPWV values recorded with SphygmoCor CvMS device in patients of predominantly normal weight who were hospitalized for suspected coronary artery disease. They used six repeated measurements per person and reported comparable metrics to our study with the CV of 9.5 (95% CI 7.7–11.0), and ICC of 0.85 (95% CI 0.78–0.90)  . Our ICC78–0.90) , 53. As 78–0.90) . Similar78–0.90) .Age was the factor with the largest effect on cfPWV and PWVao measurements, followed by MAP. This is consistent with the data from other studies that show a strong dependence of PWV on age and MAP , 56, 57.Other studies utilizing the SphygmoCor CvMS device reported comparable effects of age on cfPWV measurements ranging from: 0.2 m/s per 10 years , to 0.3–While we may assume that age remained constant during the 2 weeks of the study and thus did not contribute to an absolute change in a person’s repeated measurements, we also discovered that older age increased the variability of repeated measurements for both cfPWV and PWVao, thereby increasing measurement error. Grillo et al. discovered that patients with increased arterial stiffness had greater variability in repeated PWV measurements acquired with various cfPWV-estimating devices . This reThe second strongest effect on PWV recording was due to MAP, which is considered the most significant physiological variable affecting arterial stiffness , 31, 34.°C was 2.62 m/s, but at 25°C this difference was only 0.04 m/s. Individual differences in repeated measurements that are due to MAP variations are predicted to be 0.27 m/s (uncertainty 0.24–0.30) at 5°C and 0.004 m/s (0.004–0.005) m/s at 25°C. Interestingly, there was no significant main effect of outside temperature or its interaction with MAP for PWVao measurements.The expected changes in cfPWV due to MAP or outdoor temperature changes were less obvious due to the significant interaction between outdoor temperature and MAP, demonstrating that outdoor temperature moderates the relationship between cfPWV and MAP. The expected difference between predicted cfPWV assigned to lowest and highest observed MAP and adjusted for other factors at 53, CO, and N2O  per 10 bpm. The effect is similar to that reported by Tan et al., who used a hybrid applanation tonometry/oscillometric device SphygmoCor XCEL device and found an increase of cfPWV of 0.11–0.28 m/s per 10 bpm . In addiWhile current PWV estimation guidelines recommending that HR be considered as a confounding factor, there is still disagreement about the effect of HR on aortic PWV measurements, particularly considering that short-term studies investigating the relationship between heart rate and arterial stiffness reported varying results, including a positive, negative, and no association . O’RurkeOut of all the experimental conditions tested only the visit order affected the PWV measurements. Specifically, whereas the order of visits had no effect on the PWVao measurements, the cfPWV measurements acquired during the second visit were on average 0.23 m/s (95% CI 0.03–0.43) lower than those of the first visit. However, no significant difference was found when the cfPWV data of the third visit were compared with those of the first visit. One possible explanation for the lack of a trend on the third visit is a confounding of this effect by outdoor temperature. Because outdoor temperature was identified as an important factor influencing cfPWV measurements, more pronounced meteorological changes on the third visit likely masked the effect of a visit order. In particular, for some subjects, the outdoor temperature varied by 8–10°C between the third and the other two visits.The discovery of lower cfPWV values during the second visit may suggests that, despite receiving the amount of training recommended by current guidelines , both obp ≤ 0.015; 1st measurement in first visit was on average higher for 3.3 mmHg, 95% CI 0.6–6.0 than 2nd, and for 3.8 95% CI 1.0–6.5 than 1st measurement in second visit), but these differences are not large enough to be classified as white coat effect. We also found no difference in DBP between these measurements (p ≥ 0.422). A clinically significant white-coat effect is defined in terms of BPs as an office or clinic blood pressure exceeding the daytime ABPM by 20 mm Hg systolic or 10 mm Hg diastolic, either in the absence or presence of antihypertensive drug treatment  affected cfPWV or PWVao measurements although the current guideline on PWV estimation recommends that repeated/follow-up measurements should be taken at the same time of day, preferably with the same observer . SeveralThe differences in the PWV models of the two devices included participant characteristics such as sex and height which had a significant effect on PWVao, but not on cfPWV measurements; and hypertensive status, which on average increased cfPWV in hypertensive participants by 0.44 m/s but did fect PWVao measurements.Female sex on average increased PWVao by 1.56 m/s, whereas height explained on average 2.14 m/s difference in PWVao measurements between the subjects, with one cm of height increasing PWVao by 0.06 m/s (95% CI 0.02–0.09).P = 0.04) and proceeded with the definition of the reference value population by including all untreated participants, regardless of sex. Similarly, sex was not identified as significant predictor of cfPWV in the multivariable linear regression models controlling for age, MAP, HR in the study by Mitchell et al. . However, while in our final multiple model PWVao increased with the increase in height, in Jatoi’s multiple model the association did not reach significance . FurtherOverall, the findings suggest that SphygmoCor’s cfPWV values are more sensitive to the state of the arterial tree because, in addition to age and MAP, they are also dependent on hypertensive status, the interaction between MAP and outdoor temperature, outdoor temperature, and visit order; whereas Arteriograph’s PWVao values are more dependent on individual characteristics such as sex, height, and age as they are more heavily dependent on age than cfPWV.It should also be noted that our findings are device-specific and cannot be generalized to other seemingly similar techniques, such as the oscillometric Mobil-O-Graph, which calculates PWV using a formula, because the factors affecting within-subject PWV variability would obviously differ.The measurement difficulties appeared relatively frequently: from 19% of cases in which Arteriograph’s oscillometric signal had to be manually selected for analysis, to over 24% of cases in which SphygmoCor’s pulse wave signal was of marginal but acceptable quality (with a coefficient of variation for cfPWV estimates ranging from 6 to 10%), to 32% of cases in which SphygmoCor’s recordings had to be repeated. While all of these difficulties are time-consuming, we also found that one of them—a marginal quality of SphygmoCor’s signal, affected accuracy of estimated cfPWV values by raising cfPWV values on average by 0.37 m/s (95% CI 0.16–0.58 m/s). Concerning the difficulty in manually selecting the Arteriograph’s signal, which increases the uncertainty of PWVao estimation and could be expected to affect the accuracy of PWVao readings, we did not find that it was associated to a change in PWVao levels.All of aforementioned measurement difficulties were reduced when PWV measurements were performed in the afternoon (16–18 h) compared to the morning (7–10 h) sessions. The effect of time of day on need to manually select Arteriograph’s signal was strong with afternoon session reducing the OR for automatic selection by on average 75%, and reducing the probability of the event from 23% during morning sessions to 6% in the afternoon. The time of day also had moderate effect on a need to repeat a SphygmoCor measurement by reducing its IRR in the afternoon measurements on average by 12%. Finally, the OR for the marginal quality of SphygmoCor’s signal reduced by 40% in the afternoon, with a probability of such a signal decreasing from 26% during mornings to 19% in the afternoon. As time of day did not show significant effect on absolute PWV values of either cfPWV or PWVao, just by performing measurement in the afternoon measurement difficulties could be significantly reduced.Sex was another factor that affected measurement difficulties for both devices. It strongly increased the OR of manual selection of a Arteriograph’s signal by 51 times, and also increased IRR of repeated measurements by SphygmoCor by 1.23 times. The difficulties we observed in obtaining PWV estimation for female participants with both devices might be attributed to men being more likely prototype examinees due to the menstrual cycle’s effect on PWV recordings in women, which, as a result, would make signal acquisition and processing suboptimal for women. However, because the literature describes a validation sample rather than a developmental sample for prototypes, and the manufacturers do not provide a description of developmental sample, there is a lack of evidence to support or refute this assumption.We also found that the order of visits, which affected repeated cfPWV measurements, affected the occurrence of measurement difficulties with the SphygmoCor too, in such a way that both the need for repeated measurement and the occurrence of a signal with marginal quality were significantly reduced during the third visit compared with the first visit, whereas the frequency during the second visit was comparable to that during the first visit. So it seems that in terms of measurement difficulties with SphygmoCor, there is still a learning curve to be observed in the first 2 weeks.Finally, while inter-observer differences in PWV readings are not expected for oscillometric devices nor were observed in our study, we show that the need for manual selection of the Arteriograph’s signal is observer-dependent with one observer increasing the odds of it by 91%. Whether this difference between observer is due to positioning or adjusting the cuff should be explored in further studies.Considerations about the supine and sitting positions with Arteriograph.While we did not examine the effect of supine and sitting positions on Arteriograph’s PWVao measurements, Arteriograph is frequently used in clinic in a sitting position. Nurnberger et al. estimated the mean difference in PWVao between supine and sitting positions to be −0.18, with the limits of agreement ranging from −1.55 to 1.21 . Such wiPotential limitation of our study is referred to the pre-training of observers. Despite providing observers with the necessary pre-training, we discovered a learning curve with the SphygmoCor device, as measurement difficulties were significantly reduced during the third visit compared to the first. Furthermore, we also discovered that repeated cfPWV measurements recorded during second visit were on average 0.23 m/s lower than those recorded during the first visit, indicating that there may be a learning curve affecting cfPWV accuracy too, but we were unable to show this tendency for measurements collected during the third visit compared to first. Nonetheless, even if this learning curve affected repeated measurement accuracy, we do not expect it to have a significant effect on our results: the estimated difference in cfPWV between second and first visit is not large, we did not find that variability of repeated measurements taken during one visit was related to visit order, and we reported comparable agreement between repeated measurements to a study that reported this agreement using observers for which it proved they received a sufficient training period . In addiP ≤ 0.035, Mann-Whitney test). Moreover, for women who menstruate within-subject PWV variability was comparable to men (P ≥ 0.310).To capture short-term variability of PWV, we monitored within-subject PWV changes for 2 weeks only. Consequently, we were not able to control an effect of menstrual cycle on arterial stiffness by studying all women who still have menstrual periods at a similar phase in the menstrual cycle as this would imply that repeated measurements are also taken at similar phase of the cycle, which would prolong the monitoring period. However, we do not expect that effect of menstrual cycle substantially affected PWV variability in young women as we found significantly larger variability of within-subject PWV measurements in menopausal women than in women with menstrual cycle, for both devices  when differences in MAP result in smaller differences in cfPWV may reduce some of the short-term fluctuations. Also, it should be further investigated if prolonging resting time during periods with low outdoor temperature might help with reduction of short-term fluctuations.For SphygmoCor’s cfPWV measurements, we also found a significant interaction between MAP and outdoor temperature, as well as significant mean effect of temperature, which could lead to significant fluctuations in short-term repeated measurements. Recording cfPWV during a season with a higher outdoor temperature (e.g., 25Time of day or different observers who received the same amount of pre-training did not affect cfPWV or PWVao measurements. However, measurement difficulties occurred significantly less frequently in the afternoon for both instruments. To facilitate future longitudinal studies, it would be advisable that PWV is measured in the afternoon whenever possible, not necessarily with the same observer.For both devices, we found a relationship between a measurement difficulty and female sex. Whereas, the number of retaken measurements with SphygmoCor was moderately increased in women, the association with sex was very strong for manual signal selection with the Arteriograph device which was much more common in women than in men. In addition, the PWVao measurements by Arteriograph revealed that PWVao values were on average significantly higher in women than in men. This is in contrast to other devices that found higher PWV values for men, or no association with sex. Overall, the findings indicate that devices’ design may be suboptimal for women, with a possible systematic bias in Arteriograph’s measurements.The differences in factors affecting the PWV measurement in the two models most likely reflect differences in the measurement techniques of the two devices. In addition to age and MAP, cfPWV measurements which are considered a more direct measure of arterial stiffness were also influenced by hypertension status, the interaction MAP-outside temperature, temperature and the order of visits. PWVao values which were estimated from a single site were additionally influenced by patient characteristics such as sex, height, and HR, with higher PWVao values in women being specific to the Arteriograph device.The original contributions presented in this 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 the Ethical Committee of University of Split School of Medicine. The patients/participants provided their written informed consent to participate in this study.MP and AJ conceived and designed the study, analyzed the data, and wrote the draft of the manuscript. MP, BŠ, AB, PK, VĐ, and IM participated in data collection. MB, IM, and AJ supervised the data collection and the findings of the study. All the authors interpreted the data and approved the final version of the manuscript.


In the current context of tailoring interventions to maximize impact, it is important that current data of clinical epidemiology guide public health programmes and health workers in the management of severe disease. This study aimed at describing the burden of severe malaria at hospital level in two areas with distinct malaria transmission intensity.A hospital-based surveillance was established in two regional hospitals located in two areas exposed to different malaria transmission. Data on paediatric severe malaria admissions were recorded using standardized methods from August 2017 to August 2018 with an interruption during the dry season from April to June 2018.P. falciparum density was significantly higher in the LMT area compared to the HMT area: 22,861 trophozoites/µL  vs 11,291.9 trophozoites/µL . Among enrolled cases, coma was present in 70 (9.2%) participants. 196 patients (21.8%) presented with two or more convulsions episodes prior to admission. Severe anaemia was present in 448 children (49.2%). Other clinical features recorded included 184 (19.9%) cases of lethargy, 99 (10.7%) children with incoercible vomiting, 80 (8.9%) patients with haemoglobinuria, 43 (4.8%) children with severe hypoglycaemia, 37 (4.0%) cases where child was unable to drink/suck, 11 (1.2%) cases of patients with circulatory collapse/shock, and 8 cases (0.9%) of abnormal bleeding (epistaxis). The adjusted odds of presenting with coma, respiratory distress, haemoglobinuria, circulatory collapse/shock and hypoglycaemia were significantly higher (respectively 6.5 (95%CI 3.4–12.1); 1.8 (95%CI 1.0–3.2); 2.7 (95%CI 1.6–4.3); 5.9 (95%CI 1.3–27.9); 1.9 (95%CI 1.0–3.6)) in children living in the HMT area compared to those residing in the LMT area. Overall, forty-four children died during hospitalization  with the highest fatalities in children admitted with respiratory distress (26.0%) and those with hypoglycaemia (25.0%).In total, 921 children with severe malaria cases were enrolled in the study. The mean age was 33.9 (± 1.3) and 36.8 (± 1.6) months in lower malaria transmission (LMT) and higher malaria transmission (HMT) areas (p = 0.15), respectively. The geometric mean of asexual The study showed that children in the HMT area have a higher risk of presenting with coma, shock/dehydration, haemoglobinuria, hypoglycaemia, and respiratory distress. Case-fatality rate is higher among patients with respiratory distress or hypoglycaemia. Hospital surveillance provides a reliable and sustainable means to monitor the clinical presentation of severe malaria and tailor the training needs and resources allocation for case management. Despite the recent progress in malaria control in many countries across the globe, malaria remains the main public health threat in sub-Saharan Africa. Globally, there were an estimated 229 million malaria cases in 2019 in 87 malaria endemic countries, with about 94% of cases accounted for in the World Health Organization (WHO) African Region [Burkina Faso is one of those countries where progress in controlling the disease has stalled despite full-scale deployment of available interventions which include case management using artemisinin-based combination therapy (ACT), vector control through large-scale distribution of long-lasting insecticidal nets (LLINs); and seasonal malaria chemoprevention (SMC) targeting the marked seasonality of the disease transmission. The country is one of the high-burden nations as defined by the WHO and is part of the high burden to high impact initiative .The clinical epidemiology of severe, life-threatening malaria was characterised in Burkina Faso during the 1990s  at a timTo tailor interventions to maximize impact, it is important to obtain up to date information on the epidemiology of severe malaria. This study describes the current burden of severe malaria in children admitted at hospital level in two areas with distinct malaria transmission. The information on prevalence and lethality of severe malaria in hospitalized children is useful to the National Malaria Control Programme (NMCP), to health workers and may contribute to fine tuning the malaria control strategies and resources allocation.Anopheles gambiae, Anopheles arabiensis and Anopheles funestus, and Plasmodium falciparum is responsible for more than 90% of all clinical malaria cases [The study was conducted at two regional referral hospitals, the Regional Hospital of Koudougou and the Regional Hospital of Banfora, respectively. The Regional Hospital of Koudougou  is located 100 km Centre West of the capital city of Ouagadougou. Malaria transmission in this central part of the country is markedly seasonal and intense during the rainy season from June to October. The entomological inoculation rate (EIR) was estimated at 31.4 infective bites/person/year in a study conducted in the neighbouring zone of Saponé . The Bania cases , 8.The recruitment of children took place from August 2017 to August 2018 with an interruption from April to June 2018 (representing the dry season). For all consecutive children aged 0 to 15 years presenting at the paediatric emergency room at any time with a clinical presentation suggestive of severe malaria, an informed consent was obtained by study personnel from parents/legal representatives. Patients were further assessed by paediatric ward clinicians (who benefited from a refresher protocol specific training prior to the study start) and observations recorded using a standardized case report form. Data collected included the medical history, vital signs, a complete physical examination for signs of severity . Finger prick blood sample was taken for malaria smear , haemoglobin and glucose measurements using point of care devices . If the RDT was positive, anti-malarial treatment was immediately initiated based on the NMCP guidelines.Children received parenteral artesunate at 2.4 mg/kg body weight on admission (time zero), then after 12 and 24 h and then once a day, or artemether Injection at 3.2 mg/kg on admission, then 1.6 mg/kg body weight per day. In both cases, parenteral administration continued until the child improved and was able to take full course of oral ACT, using artemether-lumefantrine or artesunate-amodiaquine. Supportive therapy recommended for severe malaria includes the treatment of hypoglycaemia with dextrose when glucose < 2.2 mmol/l, blood transfusion for children with haemoglobin less than 5 g/dL. All treatments were free of charge. No adjusted dose of parenteral artesunate for children weighing less than 20 kg was adopted by the NMCP at the time of the study conduct.Plasmodium asexual forms were found, a total of two hundred thick film fields were screened for Plasmodium species other than P. falciparum. If P. falciparum was present, a count of asexual forms against leukocytes was made using a tally counter. Counting was conducted based on at least two hundred leukocytes in consistence with WHO standards. If less than 10 parasites were identified from the two hundred leukocytes screened, the count was extended to 1,000 leukocytes. On the other hand, if P. falciparum gametocytes were seen, a gametocyte count was performed against 1,000 leukocytes. All slides were read by two independent microscopists with a third reading in case of discordant results. The final result was based on the two most concordant readings. All readers are certified competent/expert by an external quality assurance system with Clinical Laboratory Services (http://www.cls.co.za), South Africa.Blood films were managed as described elsewhere . All RDTSevere malaria was defined based on presence of one or more criteria outlined in box Box 1 Features of severe malariaClinical features of severe malaria.Impaired consciousness (including unrousable coma);Prostration, i.e. generalized weakness so that the patient is unable to sit, stand or walk without assistance.Multiple convulsions: more than two episodes within 24 h;Acute pulmonary oedema and acute respiratory distress syndrome.Circulatory collapse or shock, systolic blood pressure.Clinical jaundice plus evidence of other vital organ dysfunction.Abnormal bleeding.Haemoglobinuria (dark urine or coca cola);Laboratory findings.Hypoglycaemia (< 2.2 mmol/l or < 40 mg/dl);Severe anaemia (haemoglobin < 5 g/dl).Danger signs as per NMCP guidelines.Incoercible vomiting.Lethargy.Inability to drink or suck.Data were entered into a MS ACCESS database and analysed using STATA software . Analysis included all paediatric admissions meeting the study eligibility criteria. Frequency were used for qualitative data and mean or geometric for quantitative data. The Clopper–Pearson method was used for calculating binomial confidence intervals. For computing the case fatality rates (CFR), only individuals with known in hospital outcome were included. Multivariable regression analysis was done to assess association between clinical features and transmission intensity. The significance level was set at P < 0.05.The study protocol and associated documents were reviewed and approved by CNRFP institutional bioethics committee , the Ministry of Health Ethical Committee for Biomedical Research . All study participants’ parents or legal representatives gave documented informed consent before any study procedures were performed. The study was conducted according to the principles of the declaration of Helsinki and International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines.Characteristics of enrolled children are presented in Table The mean time of illness before hospital admission was 3.0 days (95% CI 2.7–3.4) and 4.0 days (95% CI 3.5–4.5) in the LMT and the HMT areas, respectively. A high proportion of children admitted (38.4%) had also received a beta lactam antibiotic prior to hospital admission. There was a statistically significant difference between the HMT and LMT areas . Antibiotics administered include ceftriaxone (59.2%); ampicillin (29.6%) and amoxicillin (8.5%). Overall, almost half (43.1%) of children who had received an antimalarial were also given an antibiotic.The age of recruited children was 33.9 (± 1.3) and 36.8 (± 1.6) months in respectively LMT and HMT areas (p = 0.15)). For children enrolled with convulsions, the mean age was 29.5 months in LMT area compared to 38.3 in the HMT area. The difference was statistically significant.P. falciparum density was significantly higher in the LMT area compared to the HMT area . In total 58 (6.3%) of enrolled cases were harbouring P. falciparum gametocytes. The proportion of patients with gametocytaemia were 4.9% and 7.9% in LMT and HMT areas (p = 0.06), respectively. The geometric mean of gametocytes density was 44.7 gametocytes/µL (95% CI 25.4–78.7) and 81.3 gametocytes/µL (95%CI 52.2–126.5) in the LMT and HMT areas, respectively.The geometric mean of asexual Clinical presentation and overlap between the top three predominant syndromes are presented in Fig. The mean haemoglobin level was 5.7 (± 2.7) g/dL in study participants. Severe anaemia was present in 448 children (49.2%). On admission, other clinical features recorded included 8 cases (0.9%) of abnormal bleeding (epistaxis), 11 (1.2%) cases of circulatory collapse/shock, 80 (8.9%) patients with haemoglobinuria, 99 (10.7%) children with incoercible vomiting, 184 (19.9%) cases of lethargy, 37 (4.0%) children unable to drink/suck and 385 (41.8%) children with prostration. Among those with haemoglobinuria, only 7 (13.9%) have reported Quinine intake during the episode. The mean glucose level was 5.8 mmol/l (± 2.8). Severe hypoglycaemia was present in 43 (4.8%) children. The proportions of children presenting with one (01), two (02), three or more (≥ 3) severe features were 48.8%, 33.2% and 18.0%, respectively.Children living in the HMT area tend to present with three or more severe features compared to those from the LMT area . The association between clinical presentation and malaria endemicity is presented in Table Table This study investigated the burden of severe malaria in two hospitals located in two areas with different malaria transmission covered by same malaria control strategies as deployed the country NMCP.The predominant clinical presentations were anaemia (49.2%) and convulsions (21.8%); almost at the same level as reported in Burkina more than 20 years before by Modiano et al. . This isP. falciparum infection and prevalence of malaria-related anaemia [SMC consists of 4 rounds of administration of sulfadoxine/pyrimethamine + Amodiaquine given at intervals of one month to children aged between 3 and 59 months during the peak transmission season (July to October). This strategy has been shown to drastically reduce incidence of uncomplicated and severe malaria due to  anaemia –14 in ch anaemia , 16. In  anaemia . The preP. falciparum erythrocyte membrane protein 1 (PfEMP1) specific immunity which has been described to play a significant role in the susceptibility to CM [The odds of presenting with coma was significantly higher in children living in the higher malaria transmission area compared to those in lower malaria transmission area. This finding is in line with the inversely correlated geometric mean parasite. These findings corroborate previous report from Uganda , but arety to CM .Shock/dehydration was the second clinical presentation more observed in HMT area compared to LMT area (adjusted OR = 5.3 (1.1–24.6)). There are many potential causes of hypotension or shock in severe malaria. These include secondary bacterial sepsis, dehydration from vomiting or fever, haemorrhage from gastrointestinal bleeding, splenic rupture, and cardiac dysfunction. This was an unexpected finding which calls for further investigations to understand the potential causes of the difference if confirmed.P. falciparum-endemic areas who took quinine, the glucose-6-phosphate dehydrogenase (G6PD) deficiency have been described as potential causes of haemoglobinuria. Occurrence of haemoglobinuria have also been described in patients with normal erythrocyte G6PD activity who were administered quinine for severe malaria [Haemoglobinuria was one of the clinical syndromes significantly associated with the transmission intensity. The odds of presenting with haemoglobinuria was almost 3 times in HMT area although the overall incidence was similar to what was reported elsewhere in west Africa . A numbe malaria , 24. How malaria . Another malaria , 27. In The overall CFR recorded in this study is relatively high (5.0%) but remains within findings reported elsewhere –17, 28. More than half (51.4%) of cases referred by peripheral health clinics, who were admitted and managed as severe malaria cases based on HRP2 RDT results turned out to be negative by microscopy probably due to the high proportion (44.6%) among them who had received pre-referral anti-malarial treatment or alternatively to the low specificity of the HRP2 based RDT in our geographical context , 30, 31.In two areas of Burkina Faso with different ecology of malaria transmission, hospital-based surveillance of severe malaria clinical presentations has shown that children in the HMT are more significantly at risk of presenting with coma, prostration, shock/dehydration, haemoglobinuria, hypoglycaemia, and respiratory distress. Case-fatality rates are high among patients with respiratory distress and those with hypoglycaemia. Most of the deaths have occurred in children aged less than five years. Hospital surveillance provides a reliable and sustainable means to monitor the clinical presentations and lethality of severe and tailor the training needs for health workers at the frontline of cases management in different transmission patterns.


Plasmodium falciparum parasites have evolved and can no longer be detected by existing RDT. Developed by the Sysmex Corporation, the XN-31 prototype (XN-31p) is an automated haematology analyser capable of detecting Plasmodium-infected erythrocytes and providing species differentiation and stage specific parasite counts in venous blood samples without any preparation in approximately one minute. However, factors such as stable electricity supply in a temperature-controlled room, cost of the instrument and its initial set-up, and need for proprietary reagents limit the utility of the XN-31p across rural settings. To overcome some of these limitations, a hub and spoke diagnosis model was designed, in which peripheral health facilities were linked to a central hospital where detection of Plasmodium infections by the XN-31p would take place. To explore the feasibility of this concept, the applicability of capillary blood samples with the XN-31p was evaluated with respect to the effect of sample storage time and temperature on the stability of results.Simple and accurate diagnosis is a key component of malaria control programmes. Microscopy is the current gold standard, however it requires extensive training and the results largely rely on the skill of the microscopists. Malaria rapid diagnostic tests (RDT) can be performed with minimal training and offer timely diagnosis, but results are not quantitative. Moreover, some Paired capillary and venous blood samples were collected from 169 malaria-suspected outpatients in Homa Bay County Referral Hospital, Kenya. Malaria infections were diagnosed with the XN-31p, microscopy, RDT, and PCR. Capillary blood samples were remeasured on the XN-31p after 24 h of storage at either room (15–25 °C) or chilled temperatures (2–8 °C).Identical results in malaria diagnosis were observed between venous and capillary blood samples processed immediately after collection with the XN-31p. Relative to PCR, the sensitivity and specificity of the XN-31p with capillary blood samples were 0.857 and 1.000, respectively. Short-term storage of capillary blood samples at chilled temperatures had no adverse impact on parasitaemia and complete blood counts (CBC) measured by the XN-31p.These results demonstrate the potential of the XN-31p to improve routine malaria diagnosis across remote settings using a hub and spoke model.The online version contains supplementary material available at 10.1186/s12936-022-04259-7. Even though malaria is one of the oldest infectious diseases, it remains a major public health problem worldwide. In 2020, there was an estimated 241 million malaria cases and 627,000 malaria deaths globally [Plasmodium falciparum have raised questions about diagnostic accuracy [Plasmodium lactate dehydrogenase (pLDH)-detecting RDTs are also available, but they have lower sensitivity compared with HRP2-detecting RDT [In malaria diagnosis, microscopic examination of Giemsa-stained blood films relies on the visual detection of parasites, allows for estimate of parasite density, and remains the gold standard. However, training for competent microscopists takes time . Also inaccuracy –10. Plasting RDT . Polymer12. These analysers offer three measurement modes with different sample volumes and different limit of quantification. Previous studies from different malaria endemic settings showed that they had comparable performance to microscopy and RDT in malaria diagnosis and good concordance with microscopy and quantitative PCR (qPCR) in parasite counts [The automated haematology analyser XN-31 prototype (XN-31p)  is a novel malaria diagnostic support system developed for the clinical setting. The analyser is based on the principle of fluorescence flow cytometry and shares the same physical characteristics, hardware configuration, and reagent requirements with the XN-30 that is used extensively for research. Both analysers can measure a complete blood count (CBC), count malaria-infected red blood cells (iRBC), and calculate parasitaemia with a limit of quantification of 20 iRBC/μL in approximately 1 minute e counts . Howevere counts , whereasCurrently the operation of the XN-31p requires a stable electricity supply in a temperature-controlled room and specialised reagents not widely available in many remote settings. The cost of the instrument also precludes widespread deployment in developing countries. To address some of these limitations, a hub and spoke diagnosis model was designed, whereby blood samples collected in peripheral health facilities would be transported to a central hospital for malaria diagnosis by the XN-31p. The effects of transport time and temperature on sample integrity in such model were evaluated by comparing the results from capillary blood samples measured immediately after sample collection with those measured 24 h after storage at either room or chilled temperatures. Results from this study provided support for the use of the XN-31p in a hub and spoke model to cover a wider range of settings for routine malaria diagnosis.Plasmodium infections. The study was conducted in Homa Bay County Referral Hospital in Kenya between March and April 2020. The hospital is the major referral hospital in Homa Bay County, accepting patients throughout the county. As a normal clinical procedure, all outpatients were first examined by clinical officers. Once malaria was suspected, the patient was included in the study after obtaining informed consent.An observational cross-sectional study was conducted to compare the effects of blood sampling methods (venous vs. capillary) and sample storage temperature and length on the performance of the XN-31p in the detection of P. falciparum parasite prevalence in 2–10 years old in the Lake Victoria region in Kenya is estimated to be more than 30% [Homa Bay County lies along the eastern shore of Lake Victoria and is among the regions with the highest malaria prevalence in Kenya . Two raithan 30% . Long-lathan 30% . IRS hasthan 30% , and thethan 30% . A pilotthan 30% .Prior to blood sampling, a questionnaire was administered to study participants to obtain sociodemographic information and known malaria risk factors. Capillary blood (250 µL) was obtained by finger prick in a BD K2-EDTA Microtainer blood collection tube . After inversions, 70 µL of blood was spotted on Whatman ET31 Chr filter paper  and stored in a zipped plastic bag after drying at room temperature. Capillary blood was also used to prepare thick and thin blood smears for microscopic examination, and for RDT diagnosis using the SD Bioline Malaria Ag Pf/Pv RDT . A detailed protocol of capillary blood collection was shared with laboratory technicians  using the low malaria (LM) mode . In LM mBased on the results of this initial analysis, samples were divided into two groups and stored at either room or chilled temperature for 24 h. Samples in the room temperature group (8 positive and 39 negative samples) were kept in an air-conditioned room with temperature set at 22 °C, while those in the chilled temperature group (8 positive and 43 negative samples) were kept in a cooler box with ice packs. The ice packs were changed in the morning. Temperatures taken several times over a 24-h period indicated that the room was between 15 and 25 °C and the cooler box between 2 and 8 °C. After 24 h all samples were analysed again on the XN-31p using the LM mode.Plasmodium parasites, the presence of sickle cell, codocyte, dacrocyte, Howell-Jolly body , haemolysis, morphological abnormality of WBC, fibrin clots, and platelet aggregation was noted. RDT results were obtained following manufacturer’s instructions. For PCR diagnosis, DNA was extracted from a quartered blood spot (17.5 µL) using the QIAamp Blood Mini Kit  according to the manufacturer’s instructions. PCR amplification of the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene was performed as described previously [Microscopic examination was performed as described in the malaria microscopy standard operating procedure (MM-SOP-09) by the World Health Organization (WHO) . Brieflyeviously .All questionnaire data together with RDT, microscopy, and PCR results were entered into a Microsoft Excel spreadsheet and cross-checked for errors. The sensitivity, specificity, positive and negative predictive values of the XN-31p were calculated in reference to RDT, microscopy, and PCR using the R-based statistical software EZR . Concord3/μL, and the prevalence of anaemia, defined as haemoglobin (Hb) of < 11 g/dL, was 20.1%.Of 171 subjects enrolled in this study, 169 provided both venous and capillary blood samples for evaluation of the XN-31p. The demographic and clinical characteristics of the participants are described in Table Plasmodium infections, respectively. One Plasmodium malariae infection was detected by microscopy while three Plasmodium ovale and one mixed infection of P. falciparum and P. malariae were detected by PCR. On the XN-31p, Plasmodium infections were detected in the same set of 18 samples using both capillary and venous blood; all were identified as P. falciparum except in one capillary blood sample where the species could not be specified. Inconclusive results  were reported in five venous and eleven capillary blood samples by the XN-31p. Representative examples of normal and abnormal scattergrams are shown in Additional file Table Comparisons of the performance of the XN-31p with other diagnostic methods are described in Table 3 for iRBC count per μL, and between −0.036 and 0.029% for %iRBC. There were both fixed and proportional biases in the iRBC count per μL , but not in iRBC%.The concordance in parasitaemia between capillary and venous blood was evaluated by Bland–Altman analysis Fig. . The lim3 iRBC/μL and fixed and proportional biases  , and %iRBC . For iRBC count per μL, Bland–Altman analysis showed poor concordance between microscopy and XN-31p, with the limits of agreement between -90 and 210 × 10Bland–Altman analyses of the concordance in CBCs between venous and capillary blood samples are shown in Fig. Capillary blood samples were processed with the XN-31p again after 24 h at either room (15–25 °C) or chilled temperature (2–8 °C). Bland–Altman analyses indicated good concordance in both iRBC count per μL and %iRBC after 24 h, although results were more consistent for samples stored at chilled temperatures and samples with lower parasitaemia  and specific (0.986–1.000) against these conventional methods. These results are in good agreement with previous reports –14. MorePlasmodium infections by PCR is common in research but remains limited in clinical settings. Relative to PCR, the XN-31p detected fewer Plasmodium infections, resulting in a lower sensitivity (0.818–0.857) but maintaining a very high specificity (1.000). The lower diagnostic performance of the XN-31p is a disadvantage, however when compared to PCR, the XN-31p is easier to operate and provides results in a shorter period of time, making the XN-31p more suitable in clinical settings.Detection of low-density The XN-31p has three measurement modes. Whole blood (WB) and LM modes do not require any pre-treatment prior to the measurement, while PD mode needs sample dilution before the measurement. PD mode requires only 20 μL of blood and thus is useful when the amount of available sample is very limited. The applicability of capillary blood in PD mode was previously tested, however our own preliminary tests indicated low consistency in parasite counts and CBCs due to errors introduced during sample dilution. LM mode uses 130 μL of blood, which can be readily obtained through finger prick. Our comparison showed excellent concordance between venous and capillary blood samples for malaria diagnosis and species differentiation when samples were measured in LM mode. Good concordance was also observed for %iRBC, with the limits of agreement between −0.036 and 0.029%, which are well accepted in the clinical context. However, the parasite count per μL had fixed and proportional biases and the ranges for the limits of agreement were quite wide. Since there was no significant difference in RBC counts between capillary and venous blood samples ”, “Malaria?(Others)” or “Malaria?(UNC)” based on the pattern of scattergram. Unfortunately, few non-ulocytes . PCR detame area .A small number of samples in our study had inconclusive results, reported as MI-RBC Abn Scattergram by the XN-31p. In the case of capillary blood, a previous study attributed this scattergram to the presence of Howell-Jolly body or crystalized Sickle haemoglobin (HbS) . HoweverPlasmodium positive samples available for analyses were small. CBC data also showed better concordance in samples stored at chilled temperatures. Samples were kept cold in an inexpensive, food-grade insulated cooler box with ice packs that can be readily maintained in basic health facilities. Some studies showed the correlation between the distance from health facility and malaria prevalence [Operations of the XN-31p require stable electricity supply that are often unavailable in health facilities providing services in malaria endemic areas. To extend the utility of XN-31p as a malaria diagnostic tool in remote communities, capillary blood samples kept at either room or chilled temperatures after 24 h were remeasured to simulate the time and condition of blood sample transport from distant villages to Homa Bay County Referral Hospital. Generally, samples stored at 2–8 °C had better concordance in parasitaemia with their 0-h results than samples stored at room temperature, with narrower limits of agreement. The concordance would be higher if one outlier were excluded, though the numbers of evalence –30. The Plasmodium infection and measurement of CBCs, which may be useful in malaria eliminating areas. In such settings, lower malaria prevalence means fewer chances for microscopists to examine blood films and maintain competence in malaria diagnosis. Moreover, differential diagnosis of febrile diseases becomes more important in eliminating settings where malaria is no longer the main cause of fever [One of the unique features of the XN-31p is the simultaneous detection of of fever . The XN-This is the first test of the XN-31p in East Africa. The Lake Victoria basin has some of the highest malaria burden in Kenya, and previous cross-sectional surveys in Homa Bay County showed malaria prevalence of approximately 20% and 40% by microscopy and PCR, respectively , 35. MalIn summary, this study showed that in lieu of venous blood, capillary blood can be used directly without any pre-treatment or dilution for malaria diagnosis on the XN-31p automated haematology analyser. Moreover, capillary blood can be stored at chilled temperature for up to 24 h without adversely affecting malaria diagnostic and CBC results, broadening the appeal to utilise the XN-31p in a hub and spoke model as a rapid and accurate malaria diagnostic method for mass surveys and case confirmation in remote locations. The ability of the XN-31p to detect asymptomatic and submicroscopic infections and mixed-species infections needs to be investigated.Additional file 1: Fig. S1. Scattergrams illustrating (a) malaria-negative sample, (b) P. falciparum positive sample, and (c) inconclusive  by XN-31p. SFL: side fluorescence light; FSC: forward scattered light. Blue dots: non-infected RBCs, platelets, and debris; red dots: parasite-infected red blood cells; light blue dots: white blood cells. Fig. S2. Protocol for capillary blood collection. Fig. S3. Flow chart of iRBC count and %iRBC determination by microscopy. Table S1. Performance of XN-31p in comparison to conventional methods with detailed results. Table S2. Fixed and proportional biases observed in the CBCs with 24 hours stored samples