As malaria declines, innovative tools are required to further reduce transmission and achieve elimination. Mass drug administration (MDA) of artemisinin-based combination therapy (ACT) is capable of reducing malaria transmission where coverage of control interventions is already high, though the impact is short-lived. Combining ACT with ivermectin, an oral endectocide shown to reduce vector survival, may increase its impact, while also treating ivermectin-sensitive co-endemic diseases and minimising the potential impact of ACT resistance in this context.Plasmodium falciparum (Pf) parasitaemia is approximately 15%. Clusters have been randomly allocated to receive MDA with dihydroartemisinin–piperaquine and either ivermectin or placebo. The primary objective is to determine whether the addition of ivermectin MDA is more effective than dihydroartemisinin–piperaquine MDA alone in reducing the prevalence of P. falciparum parasitaemia, measured during peak transmission season after 2 years of seasonal MDA. Secondary objectives include assessing prevalence after 1 year of MDA; malaria incidence monitored through active and passive surveillance; age-adjusted prevalence of serological markers indicating exposure to P. falciparum and anopheline mosquitoes; vector parous rates, species composition, population density and sporozoite rates; prevalence of vector pyrethroid resistance; prevalence of artemisinin resistance in P. falciparum using genomic markers; ivermectin’s impact on co-endemic diseases; coverage estimates; and the safety of combined MDA.MATAMAL is a cluster-randomised placebo-controlled trial. The trial is being conducted in 24 clusters on the Bijagós Archipelago, Guinea-Bissau, where the peak prevalence of The trial has been approved by the London School of Hygiene and Tropical Medicine’s Ethics Committee (UK) (19156) and the Comite Nacional de Eticas de Saude (Guinea-Bissau) (084/CNES/INASA/2020). Results will be disseminated in peer-reviewed publications and in discussion with the Bissau-Guinean Ministry of Public Health and participating communities.NCT04844905. The use of a placebo control arm allows identification of ivermectin’s effect on malaria and neglected tropical disease transmission, above that of dihydroartemisinin–piperaquine.Blinding of participants, distributors, investigators and outcome-assessors greatly reduces the risk of bias.The geographical separation of island clusters minimises effects of contamination and spillover between clusters.Sample size of 200 over 24 clusters achieves power of 80% for the primary outcome measure.The unique setting means results may not be directly generalisable elsewhere, although malaria endemicity and transmission dynamics appear similar to those elsewhere in the region.Malaria in sub-Saharan Africa has declined dramatically since 2000, with much of the decrease due to vector control methods such as insecticide-treated nets and indoor residual spraying.Mass drug administration (MDA) with artemisinin-based combination therapy (ACT) could reduce transmission where coverage of vector-control interventions is high by impacting the human parasite reservoir,7Dihydroartemisinin–piperaquine (DP) is an efficacious and safe antimalarial.18Anopheles gambiae survival and sporozoite rates (SR).20–23 29Ivermectin (IVM) has been widely used in MDA campaigns against onchocerciasis and lymphatic filariasis (LF) in Africa for decades.Several clinical trials have demonstrated that combined IVM/ACT MDA is safe, and that it remains an effective antimalarial treatment and endectocide.The RIMDAMAL trial in Burkina Faso reported that communities receiving IVM-only MDA (150–200 µg/kg), given 5 times at 3 weekly intervals after a single dose of IVM and albendazole, saw reduced incidence of clinical malaria in young children compared with communities receiving the single dose alone.The Bijagós Archipelago lies 50 km off the Atlantic coast of Guinea-Bissau. Eighteen of the 88 tropical islands are inhabited, supporting a population of approximately 25 000 fishermen, hunter-gatherers and subsistence farmers. There is a long dry season alternating with a rainy season (June–October). The mean temperature is 27.3 °C with little monthly variation.37Plasmodium falciparum infection was 8.5% in January , 12.3% in July  and 17.5% in November  (manuscript in preparation). Infection is highest in the 5–14 years age group. Bed net coverage is estimated at 92% (95% CI 86% to 96%) with reportedly high usage (86%); however, intermittent preventative therapy in pregnancy (IPTp) coverage is lower and, in some areas, non-existent.An. gambiae sensu stricto is the predominant mosquito species during peak malaria transmission, and SR, determined by Circumsporozoite (CSP) ELISA, suggest it is the primary malaria vector.39Malaria transmission is highly seasonal, peaking at the end of the rains. Serial cross-sectional surveys, powered to estimate prevalence with ±3% precision, 80% power and 95% confidence, were conducted across the Archipelago prior to this trial and showed that, in 2018, qPCR prevalence of IVM-sensitive neglected tropical diseases (NTDs) such as LF, soil-transmitted helminths (STH) and scabies are co-endemic, the qPCR prevalence of any STH infection being 47.3%, for instance.41Malaria remains a significant public health problem on the Bijagós Archipelago despite high coverage with control measures. The discrete geographical nature of the islands and the associated limitation of population and vector movement further serve to highlight the Bijagós as an ideal study site.42MATAMAL will fill an important knowledge gap as no trial has successfully assessed the effect on malaria of IVM in addition to DP MDA.P. falciparum parasitaemia during peak malaria transmission season compared with DP MDA alone.To determine whether adjunctive IVM MDA co-administered with DP MDA significantly reduces the population-based prevalence of This is a quadruple-blind  cluster-randomised placebo-controlled trial.Twenty-four clusters have been assigned in a 1:1 ratio to one of two trial arms using restricted randomisationIntervention (DP and IVM MDA).Control (DP and IVM-placebo MDA).P. falciparum prevalence (qPCR and RDT), vector density, SMC coverage and presence of a health centre. Of 100 000 randomisations, the final randomisation was selected from the approximately 10% satisfying the criteria.Restriction variables included population, baseline This protocol was developed using SPIRIT reporting guidelines.The trial is being conducted on the Bijagós Archipelago, Guinea-Bissau . GovernmAll residents of the islands, defined as anyone sleeping on the island for the majority of a given month, will be invited to participate unless they meet any of the following exclusion criteria:Severe illness.Age under 6 months (DP).Height under 90 cm or weight under 15 kg (IVM/placebo).Pregnancy (any trimester) or breast feeding (IVM/placebo). Pregnancy (first trimester) (DP).Known hypersensitivity to either medication.Concomitant use of drugs affecting cardiac function or the corrected QT interval (DP).Loa loa (IVM/placebo).Travel to a country endemic for Residents excluded from MDA will still be eligible for participation in surveys.An extensive sensitisation campaign will take place ahead of the intervention, led by community health workers . Informed written consent will be obtained after providing written/spoken information (according to literacy) in participants’ own language. An independent witness will sign consent forms of illiterate participants. Parents/guardians will be asked to consent on behalf of all children under 18 years. Children aged 12–17 years will be invited to sign informed assent forms. Forms and participant information sheets for MDA are included as 3810.1136/bmjopen-2023-072347.supp1Supplementary data10.1136/bmjopen-2023-072347.supp2Supplementary data10.1136/bmjopen-2023-072347.supp3Supplementary data10.1136/bmjopen-2023-072347.supp4Supplementary dataThe intervention will be entire community MDA using:DP .IVM .IVM placebo .Standard National Malaria Control Programme interventions  will continue in both arms. The intervention arm will receive DP and IVM; the control arm will receive DP and placebo . A full 10.1136/bmjopen-2023-072347.supp5Supplementary dataP. falciparum infection and exhibits prophylactic activity for approximately 4 weeks.48DP is a rapidly acting artemisinin-based schizontocidal drug used to treat uncomplicated malaria. It is safe, well tolerated, efficacious in clearing Anopheles spp at concentrations present in human blood post ingestion.49IVM is an avermectin, active against a range of human parasitic infections and infestations. It is active against All treatment will be directly observed. Doses will be tablet formulations taken orally with water, with no food for 3 hours before or after. IVM and IVM–placebo will be visually identical 6 mg tablets dosed at 300 µg/kg to the nearest tablet. DP will be available at doses of 20/160 mg and 40/320 mg per tablet, administered according to bodyweight. DP can be crushed and mixed with water, and will be readministered in case of vomiting within 30 min .Before delivery to the field, an independent pharmacy team will relabel all IVM and placebo bottles with identical ‘IVM or placebo’ labels bearing pre-printed cluster codes and recoded lot numbers to prevent unblinding. Only the pharmacist and the independent statistician who conducted the randomisation and anonymised the labels will be unblinded. All laboratory samples will bear only alphanumeric codes. Statistical analysis will be conducted by a blinded statistician. Unblinding will only occur after completion of the primary analysis.P. falciparum parasitaemia in all age groups during the peak malaria transmission season after 2 years of intervention.The population-based qPCR prevalence of This was selected as the most appropriate method for quantifying malaria transmission due to its sensitivity, especially in submicroscopic infections, the reproducibility of methods and results for this well-established assay and comparability to other trials. qPCR prevalence can also be reliably obtained from dried blood spots (DBS), a cheap, simple and robust tool with good participant acceptability.P. falciparum parasitaemia in all ages, detected by qPCR, during the peak transmission season after the first year of MDA.Population-based prevalence of Plasmodium spp. lactate dehydrogenase/histidine-rich protein 2 (pLDH/HRP2) rapid diagnostic test (RDT), determined through passive surveillance of all malaria cases presenting to health facilities throughout the trial.Incidence of clinical malaria confirmed by Incidence of clinical malaria identified by RDT  during active surveillance of a cohort of children aged 5–14 years, during the intervention and peak transmission season.Incidence of malaria infection identified by qPCR and serological analysis during the same period in this cohort of children.P. falciparum.Age-adjusted prevalence of serological markers indicating recent exposure to Anopheles exposure.Prevalence of serological markers of recent An. gambiae sensu latu survival, measured in mosquitoes caught using indoor CDC light traps 7–14 days after the final MDA round in year 1 and year 2.Parity, as a measure of Mosquito species composition, population density and SR in mosquitoes caught using indoor CDC light traps.Prevalence of resistance to pyrethroids in anopheline mosquitoes using bioassay methodologies.Prevalence of resistance to artemisinin and partner drugs in humans using molecular markers of resistance.Safety of intervention through monitoring of AE.Impact on IVM-susceptible NTDs , headlice and bedbug infestation using clinical and serological parameters.Cluster-level intervention coverage estimates.MDA acceptability, feasibility and access.Cost effectiveness of adjunctive IVM in this setting.A cross-sectional survey will be conducted across all clusters beginning 4 weeks after the completion of the second year of MDA, during peak transmission. Two hundred participants will be selected by a two-step randomisation  from within a ‘yolk’ of villages in each cluster, purposively defined to capture sufficiently populated villages far from other clusters but logistically feasible to reach. Socio-demographic and GPS data will be collected alongside DBS for varATS qPCR analysis, a technique capable of detecting 0.03–0.15 parasites/μL blood, 10 times the sensitivity of standard 18S rRNA qPCR.10.1136/bmjopen-2023-072347.supp7Supplementary data10.1136/bmjopen-2023-072347.supp6Supplementary data10.1136/bmjopen-2023-072347.supp8Supplementary dataet al, mapping NTDs and Malaria on the Bijagós Archipelago of Guinea-Bissau, unpublished). These results informed a conservative estimate of the coefficient of variation of 0.46. Two hundred participants/cluster in 12 clusters/arm provides >80% power to detect a difference between arms if the primary outcome prevalence is 10% in the control arm and 5% in the intervention arm.In November 2019, mean malaria 18S qPCR prevalence across the islands was 14.8% (95% CI 14.5% to 14.9%) , baseline peak qPCR prevalence (21.2%), dominant vector species (An. gambiae), fixed time lag to onward infectiousness (12.5 days from infection to gametocyte presence) and ideal conditions such as no population movement and no contamination of participants or vectors across trial arms. A validated pharmacokinetic model was used for estimating participant IVM levels over time, and mosquito mortality was assumed to increase relative to this when feeding on a given day after receiving IVM. This model predicts control arm qPCR prevalence of 9.2% after 1 year and 6.5% at 3 years, compared with 3.9% and 0.8% in the intervention arm, an effect size of 87.8% over control , DP efficacy . A CDC light trap will be placed inside each household overnight for 3 consecutive nights, 7 days after each round of MDA in year 1 (18 clusters) and 7 days after the 3rd round of MDA in year 2 (24 clusters). There will be three further nights of trapping during the peak transmission survey. An. gambiae complex), population density (number of mosquitoes/trap/night and a proxy for human biting rate) and sporozoite detection using CSP ELISA.57These mosquitoes will also be used to estimate cluster-level and trial arm-level species composition . Cross-sectional and cohort surveys include post hoc questions on AEs. All doses will be directly observed. A population-based cross-sectional survey will be performed in all clusters during the 2023 dry season, that is, after all MDA has been delivered: 100 households will be randomly selected from household-head lists in each cluster yolk, and 1 child aged 2–10 years invited to participate from each household. Multiple children will be invited if the sample size is not met. Skin will be systematically examined to identify scabies, and impetigo in positive cases. Headlice examination and fine-tooth combing above a white cloth will occur over 10 min. Bedframes, mattresses, nets and adjacent walls will be inspected with torchlight for signs of bedbugs, or eggs, over 10 min. A stool sample (5 g) will be collected and stored in ethanol to undergo multiplex parasite PCR .67–69MDA distributors enter administration data into forms listing every householder, including absent members, and capture data on daily refusals, absences and exclusions. These forms generate a de facto census and can be used calculate monthly cluster-level coverage, absence, refusal and exclusion statistics.Qualitative and quantitative analysis of MDA acceptability, feasibility and access, including population movement, will be outlined in a separate protocol.Cost-effectiveness analysis will be detailed in a separate protocol.Data will be entered by trained MDA delivery teams under the supervision of highly trained and experienced local supervisors. Senior trial management will regularly review data entry and offer retraining as needed. Sample ID numbers will be electronically captured or double entered. A communication network will be established with remote villages to facilitate monitoring and correction of errors found on review.All analysis will be intention-to-treat. Analysis will be performed using STATA software (StataCorp) and R.Cluster-level, arm-level and overall demographic characteristics will be presented using descriptive statistics, including age, sex, bed net use, household size and village/cluster populations.Trial arms will be compared using a t-test on cluster-level malaria qPCR prevalences. A risk difference, 95% CI and p value will be presented. Analyses adjusting for age groups , bed net use and the presence of a health centre will be presented for this and all secondary outcomes.As for the primary outcome, using data from the year 1 survey.Cluster-level incidence rates will be compared using a t-test. If the distribution is markedly skewed, then the natural logarithm of each cluster-level summary will be taken. If some clusters have zero events, then 0.5 events will be added to each cluster. Rate differences, 95% CI and p value will be presented.As for outcome 2, using RDT-confirmed incidence rates from the two annual cohorts of children.As for outcome 3, using qPCR-confirmed incidence rates. Serological analysis described in outcome 5.Cluster-level serological responses to malaria antigens will be presented as both continuous median fluorescent intensity (MFI) data, a proxy for antibody titres, and binary seropositivity prevalence, with a seropositivity threshold of three standard deviations above the mean malaria-naïve MFI responses. MFI will be compared between arms using mixed effect linear regression, with a random effect for cluster. Seropositivity will be compared in the same way as the primary outcome, including analysis by age group. Cluster-level antigen-specific seroconversion rates (SCR) will be generated using serocatalytic models relying on seroprevalence, and age-seroprevalence plots will be generated by fitting data to a reversible catalytic conversion model using maximum likelihood methods. Trial arms’ SCR will be compared using a t-test.53As for outcome 5, using the gSG6 antigen and not employing SCR.Cluster-level anopheline parity will be compared between arms using a t-test. Adjustment will be made for species, MDA round, time relative to MDA date, temperature, humidity and rainfall. Parity difference, 95% CI and p value will be presented.2 and t-tests. Adjustment will be made for study month and year. Differences, 95% CIs and p values will be presented. If the distribution of densities is markedly skewed, a log transformation will be applied. SR will be compared between arms using a t-test on cluster-level SR with adjustments and covariates as described for parity analysis above. Risk difference, 95% CI and p value will be presented. EIR will be compared with a t-test on cluster level summaries.The proportion of each species identified, and mosquito densities, will be presented by cluster and arm, and compared using χSpecies composition and level of resistance will be presented using descriptive statistics. Dose–response curves will be generated for each insecticide and probit regression analysis will be performed on these data using maximum likelihood or least squares methods.The number of alleles associated with antimalarial resistance will be compared over time, before and after MDA. However, appropriate statistical tests will need to be selected depending on the allele sample size before and after MDA.Descriptive statistics will be presented on the number, nature, severity and relatedness of all reported AE, with additional details for serious AE. Cluster-level and age-group data will be presented. Rates will be compared between arms.As for the primary outcome and outcome 5.Cluster-level, arm-level and overall coverage will be presented for each month and year of intervention, and for the trial overall. The denominator for these proportions will be the total number of people recorded on MDA administration records by distributors in each cluster, whether receiving MDA or not. Refusals, exclusions and absences will be presented similarly.Qualitative and quantitative analysis of MDA acceptability, feasibility and access will be outlined in a separate protocol.Cost-effectiveness analysis will be detailed in a separate protocol.Qualitative studies report that this population almost unanimously consider malaria to be a significant problem in their homes and the region, that additional malaria control measures would be welcome, and that MDA is acceptable,All field assistants and the deputy trial manager are local residents and MDA will be delivered by ASCs within their own communities. Methods were finalised in discussion with these stakeholders, the Regional Health Directorate and MINSAP. There will be monthly feedback sessions with ASCs and staff during MDA and surveys. Trial outcomes, social science, public engagement and qualitative work will be presented to stakeholders and communities using plans developed with the contributors themselves.All necessary approvals are in place. A baseline malaria prevalence survey was completed in November 2019. Two years of MDA has successfully been delivered across all clusters, alongside two annual cohort surveys and entomological sample collection. Two annual peak transmission surveys and a dry season NTD survey have been completed. Qualitative and social science work has taken place throughout. Laboratory testing is ongoing ahead of statistical analysis.MATAMAL will be reported according to CONSORT guidance.7310.1136/bmjopen-2023-072347.supp9Supplementary dataElectronic forms are encrypted on submission to a LSHTM HERA-compliant secure server. Paper forms will be kept in limited-access locked storage at the site office. Data will be de-identified, except for consent and MDA record forms. All data will be held for a minimum of 7 years, including in LSHTM electronic repositories, whence access to de-identified data can be requested. All investigators will have access to cleaned databases.Results will be published in peer-review journals, presented at conferences and to local collaborators. Field staff, nurses and ASCs will disseminate findings to participating communities in appropriate and accessible formats. Authorship will be decided on paper-by-paper basis, to recognise significant contributions to design, conduct, analysis and reporting. Writing will not be out-sourced.


Understanding temporal and spatial dynamics of malaria transmission will help to inform effective interventions and strategies in regions approaching elimination. Parasite genomics are increasingly used to monitor epidemiologic trends, including assessing residual transmission across seasons and importation of malaria into these regions.Plasmodium falciparum samples collected from 8 neighbouring health centres between 2012 and 2018 were genotyped using molecular inversion probes (MIPs n = 1793) targeting a total of 1832 neutral and geographically informative SNPs distributed across the parasite genome. After filtering for quality and missingness, 302 samples and 1410 SNPs were retained and used for downstream population genomic analyses.In a low and seasonal transmission setting of southern Zambia, a total of 441 The analyses revealed most  infections harboured one clone (monogenomic) with some variation at local level suggesting low, but heterogenous malaria transmission. Relatedness identity-by-descent (IBD) analysis revealed variable distribution of IBD segments across the genome and 6% of pairs were highly-related (IBD ≥ 0.25). Some of the highly-related parasite populations persisted across multiple seasons, suggesting that persistence of malaria in this low-transmission region is fueled by parasites “seeding” across the dry season. For recent years, clusters of clonal parasites were identified that were dissimilar to the general parasite population, suggesting parasite populations were increasingly fragmented at small spatial scales due to intensified control efforts. Clustering analysis using PCA and t-SNE showed a lack of substantial parasite population structure.Leveraging both genomic and epidemiological data provided comprehensive picture of fluctuations in parasite populations in this pre-elimination setting of southern Zambia over 7 years.The online version contains supplementary material available at 10.1186/s12936-023-04637-9. As malaria elimination efforts continue to drive down disease burden in parts of Africa, some regions previously endemic for malaria have seen drastic reductions in overall morbidity and mortality , 2. In tParasite genomic epidemiology can be used to track transmission patterns in pre-elimination settings and help elucidate the mechanisms of persistent transmission Fig. . FollowiThese low transmission settings offer a unique opportunity to observe long-term dynamics of parasites across seasons, as the smaller number of parasites may be easier to track because of a lack of recombination with genetically distinct parasites within the mosquito. A small number of studies have attempted to observe temporal population changes in low transmission settings, and even fewer have been able to combine temporal and geospatial data to identify hotspots over space and time , 14–17. Southern Zambia has had a drastic reduction in the number of malaria cases over the past two decades . Despite2. This work was approved as part of Southern and Central Africa International Center of Excellence for Malaria Research by the Tropical Diseases Research Center, Ndola, Zambia (Ref No: TDRC/ERC/2010/14/11) and the Johns Hopkins Bloomberg School of Public Health (IRB #3467). Analyses utilizing parasite genomes from de-identified samples were deemed nonhuman subjects of research at the University of North Carolina at Chapel Hill  and Brown University .Samples were collected through passive case detection from eight health centres in and around the catchment area of Macha Hospital in Choma District, Southern Province, Zambia in an area of approximately 2000 kmPlasmodium falciparum infection by rapid diagnostic test. DNA was extracted from each DBS with a Chelex-Tween protocol [pfldh gene [P. falciparum nuclear genome [From 2012 to 2018, dried blood spots (DBS) were collected from 441 symptomatic individuals presenting to the health centres who were positive for protocol . Parasitldh gene . Samplesr genome , 25. MIPr genome . The MIPhttps://github.com/bailey-lab/MIPTools), a suite of computational tools designed to process sequencing data from MIPs. Raw reads from each MIP, identifiable using unique molecular identifiers (UMIs), were used to reconstruct sequences using MIPWrangler, and variant calling was performed on these samples using freebayes [Processing of sequencing data and variant calling was done using MIPtools  distributed across the genome . Overall COI was relatively stable over the study period, with no significant declines in recent years despite overall reduced transmission  Fig. B often tF between all 45,150 pairs of genomic regions across 1410 loci for 302 samples [kelch13 (an artemisinin resistance marker)  related parasites than those not connected by major roadways (such as Kamwanu) , multi-season networks or those spanning multiple months occurred  was conducted using all parasites , IBD analysis was able to capture different genomic signals that reflects low malaria transmission intensity in southern Zambia. Overall, most infections were monogenomic (COI = 1) and networks of identical or highly related parasites persisted across space and time. These were detected in the dry low transmission season followed by “seeding” the parasite population in the next high transmission season Figs.  and 5. TGenomic studies of local transmission and patterns of parasite connectivity in low and medium endemicity countries are becoming more common and starting to shed light on the dynamics of malaria parasite movement , 15–17. Unlike previous work in this setting , IBD wasThis work demonstrates maintenance of parasite clones through the dry season and increasing parasite clonal transmission leading to population fragmentation in recent years supporting the effectiveness of recent control efforts. Moreover, our findings did not show a significant contribution of imported malaria cases for sustainable malaria transmission in the area. However, there are multiple limitations to identifying imported cases. First, the sampling of only acute cases will limit the ability to define local transmission dynamics and identify importations. The asymptomatic reservoir likely contributes significantly to sustained low level transmission . HoweverThe success of malaria elimination in low transmission regions in Africa will depend on a deeper understanding of transmission dynamics and importation. However, there was evidence of parasite movement and connectivity at small spatial scales at the health facilities level, which helps parasites maintain a diverse genetic pool in low transmission areas, potentially creating a challenge for malaria elimination. As recommended by the WHO, defining imported vs. locally acquired cases is critical for designing and stratifying targeted interventions , 40. ThiPreliminary work suggests that these tools will still be effective in studying parasite populations in high transmission settings and patterns of parasite connectivity . In addiThis study leveraged state of the art genomic tools and analytical methods to provide a detailed snapshot of the transmission dynamics of malaria in a region on the cusp of elimination and highlights the feasibility of these methods to inform targeted interventions to achieve and sustain malaria elimination.Additional file 1: Figure S1. Study sites in Macha Region. Figure S2. Distribution of retained SNPs across Plasmodium falciparum chromosomes. Figure S3. Retained samples after filtering. Figure S4. Missingness by probe and sample. Figure S5. Complexity of infection. Figure S6. Distribution of IBD segments and selection signals across the Plasmodium falciparum genome. Figure S7. Study sites in Macha district and spatial pattern parasite relatedness. Figure S8. Network analysis of parasite relatedness across season at varying degrees of relatedness. Figure S9. Population structure of P. falciparum parasite from Macha and bordering countries. Figure S10. Spatio-temporal distribution of two outlier clonal cluster parasites described in Figure S9A.Additional file 2: Table S1. List of loci included in genome wide MIP panel and successfully genotyped. Table S2. Metadata and complexity infection for 302 successfully sequenced samples genome wide MIP panel -this data used for population genetic analysis. Table S3. List of genes with signature of selection among the 302 P. falciparum isolates.


Light microscopy and rapid diagnostic tests (RDT) have long been the recommended diagnostic methods for malaria. However, in recent years, loop-mediated isothermal amplification (LAMP) techniques have been shown to offer superior performance, in particular concerning low-grade parasitaemia, by delivering higher sensitivity and specificity with low laboratory capacity requirements in little more than an hour. In this study, the diagnostic performance of two LAMP kits were assessed head-to-head, compared to highly sensitive quantitative real time PCR (qPCR), in a non-endemic setting.® Illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of Plasmodium-DNA in 133 biobanked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their diagnostic performance compared to qPCR.In this retrospective validation study two LAMP kits; AlethiaPlasmodium DNA in 41 samples (defined as true positives), and the two LAMP methods detected 41 and 37 of those, respectively. The results from the HumaTurb Loopamp™ Malaria PDT kit were in complete congruence with the qPCR, with a sensitivity of 100% (95% CI 91.40–100%) and specificity of 100% (95% CI 96.07–100%). The Alethia® Illumigene Malaria kit had a sensitivity of 90.24% (95% CI 76.87–97.28) and a specificity of 95.65% (95% CI 89.24–98.80) as compared to qPCR.Of the 133 samples tested, qPCR detected ® illumigene Malaria kit for detection of malaria.This head-to-head comparison showed higher performance indicators of the HumaTurb Loopamp™ Malaria PDT kit compared to the AlethiaThe online version contains supplementary material available at 10.1186/s12936-023-04809-7. Plasmodium falciparum, the sensitivity is lower for other Plasmodium species as well as for low parasite density infections [Management of patients with malaria relies on rapid and accurate diagnosis to secure prompt treatment. The diagnostic methods recommended for malaria diagnosis by the World Health Organization (WHO) continue to be light microscopy and immunochromatographic rapid diagnostic tests (RDT) . While tfections –5. The dfections –8. In refections , 9–13.In LAMP, the DNA-targets are amplified without the temperature cycles of the PCR-technique. Instead, a DNA-polymerase with strand displacement activity makes multiplication of the DNA-target possible at a constant temperature. The sequences targeted by the primers lead to the formation of DNA loops during amplification, which allows a highly sensitive and specific reaction and a shorter time to detection (time from starting the DNA-amplification reaction until a threshold is reached and interpreted as a positive result) than is usual for PCR. The DNA-amplification reaction forms a white precipitation of magnesium pyrophosphate, detected as turbidity or as fluorescence when exposed to UV light . LAMP methods usually do not quantify the DNA content of the sample, which qPCR methods may through a Cq/CT-value , 14.falciparum species [Plasmodium species and parasite density. This study was designed to assess the performance of the two internationally available LAMP kits with a complete diagnostic solution for detection of Plasmodium DNA on the international market today: the Alethia® Malaria Illumigene  with reagents in the Malaria kit or Malaria PLUS kit, and the HumaTurb C + A or Humaloop M  with Loopamp™ which have several kits; Malaria Pan Detection kit and separate kits for species identification of P. falciparum or Plasmodium vivax  and specificity (99.2 – 100%) with low laboratory capacity requirements in a little more than an hour , 15–22.  species , 20, 23.00% and sis study , 25.TablThis study was performed as a retrospective laboratory validation study of the analytic performance of two LAMP assays on biobanked samples from patients with suspected malaria in Region Skåne. The main objective was to investigate which of the two LAMP kits that had the best diagnostic performance compared to highly sensitive qPCR in patients with suspected imported malaria in a non-endemic setting at the Clinical Microbiology Department of Skåne in the South of Sweden. Another aim was to assess if any of the two LAMP kits could be used to generate a semiquantitative measurement of parasitaemia level.Plasmodium-DNA in biobanked blood samples were retrospectively evaluated at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their performance compared to highly sensitive qPCR. Performance was defined as sensitivity and specificity compared to qPCR to detect DNA in the first diagnostic blood sample sent to the lab from patients with clinical suspicion of malaria.The analytical performance of two LAMP instruments and kits for detection of Plasmodium species and parasitaemia. Various ID-specialists in four hospitals had the task to perform microscopy, and for many of them, this was a task rarely performed. Both RDT and microscopy were performed for all cases of suspected malaria. After diagnosis, whole blood samples (EDTA) were routinely sent to the regional Clinical Microbiology Laboratory without further processing and stored at − 80 °C in the Region Skåne biobank 136, Klinisk mikrobiologi BD1.The Clinical Microbiology Laboratory of Region Skåne is the main laboratory for the Skåne region in the south of Sweden, serving 9 hospitals and covering a population of 1.4 million. The positivity rate of suspected malaria samples at the Clinical Microbiology Laboratory of Region Skåne during the study time was 8–10% yearly . The routine diagnostic method for malaria at the Clinical Microbiology Laboratory of Region Skåne at the time of the study was RDT and light microscopy. Blood samples were taken in EDTA-tubes from patients with suspected malaria and tested with the immunochromatographic rapid diagnostic test CareStart Malaria HRP2/pLDH (Pf/PAN) Combo Test at the point of care laboratory. In addition, microscopy of Giemsa-stained slides (irrespective of RDT result) was performed by an infectious disease specialist at the point of care laboratory, assessing The study was approved by the Swedish Ethical Review Authority (Etikprövningsmyndigheten) ID 2020-05249.Plasmodium spp. were identified through the laboratory system and selected for inclusion in the study. For every positive sample selected, the two consecutive samples that had been reported as negative for Plasmodium spp. were also included. Only one sample per patient was used. In total 51 positive samples and 102 negative samples were selected resulting in 153 included samples.Blood samples were selected among the biobanked samples previously sent to the Department of Clinical Microbiology on suspicion of malaria during the period 2018–2020. The blood samples that had once been reported as positive for Exclusion criteria were cases for which samples were not identified in the bio bank, had insufficient volume for testing in the sample tube or inconclusive information about malaria diagnosis in the clinical files.Plasmodium species from microscopy performed by clinicians. The above described patient data was collected to describe the study population from which samples were analysed in order to facilitate comparison with other settings outside of Sweden. The data is presented in Additional file Medical charts from included patients were obtained from Region Skåne medical databases. Epidemiological patient data was retrieved regarding sex, age, country of residence, country of origin, country of exposure, reason for travel, previous malaria episodes, chemoprophylaxis, time of exposure, time from symptom onset, malaria treatment initiated, presence of risk factors and comorbidities and data on parasitaemia and ® Malaria illumigene Malaria kit  and HumaTurb C + A Loopamp™ Malaria Pan Detection kit . The tests were performed according to the manufacturer’s instructions after training by the supplier. The LAMP analysis of Alethia® Illumigene Malaria kit is qualitative and is reported as positive, negative, or invalid. HumaTurb Loopamp™ Malaria PDT kit is a qualitative test that also reports time to detection. Neither of the two tested LAMP kits offer species differentiation. The LAMP analyses were performed by research personnel together with staff at the Clinical Microbiology Laboratory of Region Skåne during 2021 and 2022. Results from microscopy were blinded to the research personnel performing the LAMP assays. Invalid results were re-tested once and if the result remained invalid the sample was reported as invalid.Each of the included frozen whole blood samples obtained from the biobank were tested retrospectively with two LAMP-kits: AlethiaA total volume of 50 μL EDTA blood from each sample was used for genomic DNA extraction using the QIAamp DNA blood mini kit (Qiagen) and the protocol ‘DNA purification from Blood or Body fluids (spin protocol)’, according to manufacturer’s instructions. To reach a volume of 200 μL, starting material required for the specific extraction protocol, 150 μL of PBS 0.01 M pH 7.4 was added to 50 μL EDTA blood, according to manufacturer’s recommendation. At the final step, the genomic DNA was incubated for 5 min at room temperature prior to elution in 150 μL elution buffer and stored at −20 ℃.Plasmodium species detection and identification, a previously published multiplex, probe-based qPCR assay method was used [P. falciparum, P. vivax, Plasmodium malariae and Plasmodium ovale samples of known parasitaemia and non-template negative controls were used in each run. A cycle threshold (CT) value of 40 was used as a cut off to define positive samples. The limit of detection was 0.5–6 parasites for the different Plasmodium species. In case of inconsistent qPCR results, i.e., one out of three replicates positive, the qPCR was repeated, and the sample was analysed again in triplicate. If the result from the repeated qPCR matched the result from the first qPCR, the sample was considered as positive. Hence, one positive result out of three in the first run that was repeated in the second run was considered positive. If the repeated qPCR was negative the sample was considered negative.For was used , with th® Illumigene Malaria kit and the HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit. The non-parametric Spearman’s Rank Correlation test in IBM SPSS Statistics (version 29.0.1.0 (171)) was used to calculate all correlations; the two non-linear correlations between HumaTurb Loopamp™ Malaria PDT kit time to detection and qPCR CT value and parasitaemia as reported by microscopy as well as the linear correlation between qPCR CT value and HumaTurb Loopamp™ Malaria PDT kit time to detection.Sensitivity and specificity of LAMP techniques as compared to qPCR were calculated with the online statistical tool MedCalc . The binA total of 51 positive samples from patients diagnosed with malaria 2018–2020 were identified in the biobank list. In addition to these, 102 consecutive negative samples were selected resulting in a total of 153 samples. Of these, 5 samples could not be located in the biobank leaving a total of 148 samples that were tested with the two LAMP techniques, 47 previously registered as positive and 101 as negative. Of these samples, 14 had insufficient sample volume for qPCR, resulting in a total of 134 samples analysed by qPCR. The diagnosis of one of these samples was inconclusive with negative results in all diagnostic tests except one positive result out of six qPCR replicates (three primary replicates and three repeated replicates) with a high CT value of 38. The final clinical diagnosis of this patient was also unclear in the medical records. Therefore, this sample was excluded, resulting in a total of 133 samples analysed by both qPCR and the two LAMP kits. In total 41 samples were positive by qPCR and 92 were negative. The sample selection flowchart is detailed in Fig. The demographic characteristics of the 47 included positive cases that were tested with the two LAMP kits are presented in Additional file ® Illumigene Malaria kit and HumaTurb Loopamp™ Malaria PDT kit as compared to the reference method qPCR is presented in Tables Diagnostic performance of Alethia® Illumigene Malaria kit to qPCR there were 4 false positive and 4 false negative results leading to a sensitivity of 90.24 (95% CI 76.87–97.28) and a specificity of 95.65% (95% CI 89.24–98.80). The difference in sensitivity and specificity between the HumaTurb Loopamp™ Malaria PDT kit as compared to the Alethia® Illumigene Malaria kit is statistically significant with a p-value of 0.015 and 0.017, respectively.The results from the qPCR and HumaTurb Loopamp™ Malaria PDT kit were in complete congruence, resulting in a sensitivity of 100% (95% CI 91.40–100%) and specificity of 100% (95% CI 96.07–100%) for the HumaTurb Loopamp™ Malaria PDT kit as compared to the qPCR. When comparing the Alethia® Illumigene Malaria kit correctly identified 125 of 133 samples as either positive or negative (accuracy 94%) but failed to detect four cases of malaria and misdiagnosed four cases as positive. All four false positive results were negative in qPCR, microscopy and RDT. The clinical diagnosis for the four cases were viral infection or uncertain diagnosis. Out of the four false negative results two were positive in qPCR, microscopy and RDT with a noted parasite density of 0.2%. One of the cases had slightly inconclusive qPCR results with 2 out of 6 replicates (33.3%) positive in two independent qPCR experiments with a CT value of 40 in the first round and 38 in the second, negative microscopy and negative RDT. The clinical presentation of this case was a person with origin from an endemic area that was visiting friends and family and had had symptoms for one day. At presentation the case was diagnosed as non-malaria but three weeks later (without possible new exposure) malaria was diagnosed at another hospital. The last false negative case was positive in two of three qPCR replicates with a CT value of 40, positive in microscopy and positive in RDT. This case had used oral self-treatment before seeking care and was first interpreted as cured but was later reassessed as incompletely cured and put on malaria treatment with oral atovaquone/proguanil once more which led to clinical improvement.The AlethiaP. falciparum, P. ovale and P. vivax, along with one mixed infection with P. falciparum and P. ovale in the study samples. Both LAMP assays correctly identified one or more samples with malaria caused by all above mentioned species as positive, although one P. vivax sample and three P. falciparum samples were missed by the Alethia® Illumigene Malaria kit. The results  show that species identification by microscopy had been a challenge in the current diagnostic setting with only 33 of 41 samples correctly identified by the microscopist as compared to the qPCR  Fig. . There w® illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, was evaluated head-to-head for detection of Plasmodium DNA in 133 bio banked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden compared to qPCR. The results show that the HumaTurb Loopamp™ Malaria PDT kit had a 100% sensitivity and specificity compared to highly sensitive qPCR. The Alethia® illumigene Malaria kit correctly identified 125 of 133 samples as either positive or negative (accuracy 94%) but failed to detect four cases of malaria and misdiagnosed four samples as positive.Management of patients with malaria relies on rapid and accurate diagnosis to secure prompt treatment, also in non-endemic settings, where travelers and migrants from endemic areas may present with symptoms. In this retrospective validation study the performance of two LAMP kits; AlethiaIt is important to note that it is not possible to compare the diagnostic performance of RDT or microscopy versus the LAMP assays with this study design, since positive samples in this study were included on the basis of RDT and microscopy results which were the current laboratory methods for the malaria diagnosis in Skåne during the time of the study.This data shows a significant positive correlation between qPCR CT value and time to detection with the HumaTurb Loopamp™ Malaria PDT kit. qPCR CT value has previously been shown to correlate with level of parasitaemia . This is® Illumigene Malaria kit are problematic. It is likely that two of the cases had very low levels of circulating Plasmodium DNA and parasitaemia, indicated by qPCR CT values close to the cut off , which could have been below the limit of detection for the Alethia® Illumigene Malaria kit. In two of the samples, however, the parasite density was 0.2%  and no obvious reason for failure to detect Plasmodium DNA can be found. It is possible that the freezing of the samples for 2–4 years has damaged the parasite DNA, and thus leads to failure to detect the DNA, but this would most likely have been seen in the HumaTurb Loopamp™ Malaria PDT kit as well. Other possible reasons for false negative results could be the presence of inhibitors or the handling of the sample during the LAMP process.If the LAMP technique is to be used as a first diagnostic test to detect malaria, the 4 false negative results by the AlethiaA secondary finding is that species identification by microscopy was a challenge with only 33 of 41 positive samples previously correctly identified by the microscopist, and one case of malaria, which was positive in qPCR and one of the two LAMP techniques had been reported as negative in previous microscopy. The clinical reality in Sweden and many other non-endemic countries is that the microscopy is done at the point of care by the infectious disease doctor on call. The on-call doctor might perform malaria microscopy very rarely and might have had the microscopy training a very long time ago. This makes both sensitivity, species determination, and assessment of parasitaemia a challenge and may have affected a possible correlation between actual level of parasitaemia and time to detection by the HumaTurb Loopamp™ Malaria PDT kit. The problems associated with maintaining diagnostic skills adequately high for doctors on call outside office hours is one of the main reasons more sensitive methods are needed.® Illumigene Malaria kit has been identified when searching the literature. The high sensitivity and specificity of the LAMP tests shown in this study are in line with what have been found in other similar studies. A large systematic review and meta-analysis based on 66 studies of diagnostic accuracy of the same two LAMP methods compared to PCR, microscopy and RDT for malaria diagnosis found a sensitivity and specificity > 0.95 of both methods in most of the included studies [As previously mentioned, no other study comparing the performance of the HumaTurb Loopamp™ Malaria PDT kit and the Alethia studies . The revA strength of this study is the comparison of two LAMP methods with highly sensitive qPCR head-to head in the same samples for which “field microscopy” and RDT at the point of care were known. However, a larger sample size would have given increased power to the statistical calculations.This study is performed on samples that were frozen for 2–4 years which does not accurately represent the test material that will be used in practice, which might lead to unforeseen differences in diagnostic performance. However, all samples have been handled the same way and, according to the manufacturers, both kits may be used on frozen samples , 30. TheThe laboratory analyses were performed by trained biomedical scientists as well as non-laboratory trained hospital staff that had gone through a short training, and it is possible that handling errors by non-laboratory trained staff may have contributed to contamination. Nonetheless, since these tests are planned to be run at the point-of-care it is important that non expert laboratory trained staff can run the tests with maintained accuracy and quality.The cost-effectiveness has not been assessed in this study and is highly dependent on local factors, why it ideally should be done in each setting.This study is the first study to compare the performance of the two leading LAMP instruments for diagnosis of malaria on the same material. The results show a high performance of both instruments but a superior performance of the HumaTurb Loopamp™ Malaria PDT kit.® illumigene Malaria kit. Further studies with larger sample size to confirm these results would be beneficial. The high diagnostic sensitivity and specificity of HumaTurb Loopamp™ Malaria PDT kit along with ease of use and time to result around one hour makes this analysis suitable as a first line point of care diagnostic test for malaria in non-endemic high resource settings globally. However, the inability of these methods to perform full species differentiation and estimate the level of parasitaemia mean that they cannot yet completely replace microscopy in clinical diagnosis as this data is crucial for accurate treatment.In this head-to-head retrospective validation study of the diagnostic performance of two LAMP kits compared to qPCR, the HumaTurb Loopamp™ Malaria PDT kit had a significantly higher diagnostic performance than the AlethiaAdditional file 1. Demographic characteristics of positive cases.Additional file 2. Sequences of used primers and probes for plasmodium species detection and discrimination.


Plasmodium parasites—through visualization. Golgi staining also allowed researchers to observe its complex life cycle while documenting it. His careful observations of malaria led to the identification and characterization of its various stages, both asexual forms within human red blood cells, as well as sexual forms carried by mosquito vectors. Golgi’s research highlighted the key role mosquitoes play in malaria transmission. He demonstrated the presence of Plasmodium sporozoites within the salivary glands of infected mosquitoes, providing insight into its life cycle and the dynamics of parasite transmission. His comprehensive approach contributed significantly to our understanding of malaria as a systemic illness, leading to subsequent research efforts within this field. The Golgi Protein complex is often located within the cis-Golgi of blood parasite life cycles and mosquito stages, indicating its possible role in optimizing asexual development during blood stages. Furthermore, its expression can be conditionally repressed or its gene can be inactivated to optimize this potential role in improving its functionality for optimizing sexual development during blood stages. Camillo Golgi remains one of the leading lights of malaria research today. His innovative staining techniques, detailed observations, and insightful interpretations have laid the groundwork for subsequent discoveries and advancements in malaria studies. By deciphering intricate parasite life cycle interactions with hosts, his work has provided invaluable insights into malaria biology, pathogenesis, and epidemiology.Camillo Golgi was an esteemed Italian physician and biologist who made major advances in malaria research between the late 19th and early 20th centuries. His groundbreaking contributions in histology, especially through the development of the Golgi staining technique, revolutionized our understanding of cell structures—including  Plasmodium parasite endemic zones; many historians even debate whether or not Rome fell because of this disease [Malaria remains one of the deadliest infectious diseases ever witnessed on earth, having left lasting scars on many of our ancestors’ lives throughout history. Thanks to remarkable research conducted since the nineteenth century, malaria has begun to become less frequent throughout Europe, and was finally eliminated during the second half of the XXth century. Many ancient European regions were  disease .Over subsequent centuries, an abundance of Italian scientists, representing Italy as a whole, began researching the etiology of malaria. Camillo Golgi 1843–1926), among many notable Italian scientists who investigated this field, made significant contributions both in cell biology and malaria infection research, providing detailed accounts on Plasmodium infection processes within intraerythrocytic cycles [843–1926,Today, the parasite still thrives in subtropical and tropical regions in this vulnerable population including young children, pregnant women, and those involved in economic development activities . ReportsGolgi’s research provided much-needed insights into the complex biological cycle of malaria parasites living inside blood cells. His work shed light on their link to fever onset, providing vital data that paved the way for effective malaria therapies and prevention strategies. Simply put, Golgi provided invaluable knowledge of pathogenesis while proposing more efficient treatments and prevention methods . At firsStrongyloides stercoralis; however, he became particularly fascinated with malaria after Alphonse Laveran first identified parasites in a patient’s blood on 6 November 1880.Golgi recognized the microscope as a dynamic and effective research tool capable of producing significant findings in infectious disease research. His contributions were not limited to his own achievements alone, but included those made by exceptional disciples such as Adelchi Negri, Giovan Battista Grassi, Antonio Carini and Giuseppe Sanarelli among many others he mentored and inspired during their research careers. Golgi made many notable discoveries about Anthrax and Noteworthy is Italy’s endemicity, with devastating health and social repercussions. Laveran’s findings were validated by Rome’s scientifically established malariological school under Ettore Marchiafava and Angelo Celli; however, many questions remained, such as why fevers were occurring during various seasons and an in various locations throughout Italy, why malaria produced different outcomes, and when quinine should be administered, leaving Camillo Golgi intrigued and inspired to study malaria at the Malariological School in Rome during a brief stint at Santo Spirito Hospital during September 1885.Golgi returned to Pavia and immediately began conducting daily blood smear examinations of malaria patients at his clinical unit at the hospital. To his luck, only quartan malaria (“long fever”) existed in Pavia during the fall season, allowing for consistent observations .Plasmodium malariae’s complete dynamic cycle and clinical observations of febrile attacks before the Royal Medical Academy in Turin on 18 November 1885. He used meticulous work with daily blood smears from malaria patients to unlock the secrets behind periodic fever, something which had long baffled scientists. Golgi found that febrile attacks could be predicted by watching for signs of maturation of segmented forms of the parasite that led to febrile attacks, known as quartan fever. He concluded that when observed together, mature and segmented forms could accurately forecast when another attack would come along, something no other scientist had accomplished prior to Golgi. His incredible ability to combine clinical observations with microscopic interpretation enabled him to make significant advances in our knowledge of malaria . To address this dilemma, Golgi observed cases in Pavia during the spring of 1886 and presented his findings on 5 June 1886 to the Medical Society of Pavia where he presented what has become known today as Plasmodium vivax’s complete human schizogonic cycle as described by Golgi himself [Golgi made great strides toward understanding  himself .Plasmodium parasites in human blood, noting the coincidental relationship between chills or fever and the release of merozoites into the circulation. He also demonstrated that intermittent fevers in adults were caused by two distinct protozoan organisms—Plasmodium malariae and Plasmodium vivax—contrary to Alphonse Laveran’s belief in only “Oscillaria malariae”, thus revolutionizing the malaria diagnosis and management strategies. His discoveries greatly advanced our knowledge and provided more effective approaches [Camillo Golgi made groundbreaking contributions to malaria research from 1885 to 1892, when microbiology made strides forward. Golgi built upon the work of Ettore Marchiafava and Angelo Celli to assess the reproductive cycles of proaches ,12.Golgi combined naturalistic, biological, and clinical-pathological perspectives in 1889 to develop an innovative method for the differential diagnosis of tertian and quartan fever, independent of clinical observation, creating valuable laboratory data which could then be applied directly for clinical diagnosis. Furthermore, his accomplishments included differentiating three distinct intermittent fever types as well as establishing optimal timing for quinine administration; all this greatly advanced our knowledge of malaria while setting new standards for its diagnosis and treatment .Bacillus malariae” found in Pontin Marshes south of Rome caused malaria, thus beginning an exciting era of research that saw Italian malariologists making significant contributions despite scientific rivalries and disagreements [He played an instrumental role in disproving Klebs and Tommasi Crudeli’s assertion that the “reements .Plasmodium survival and virulence. Their results contribute to our knowledge of intracellular trafficking mechanisms within Plasmodium falciparum cells and can also potentially be used as targets for antimalarial therapy treatments targeting this protein-sorting pathway [Fluorescence microscopy, biochemical assays and genetic approaches were employed by researchers to identify PfVPS51 protein that plays an integral part in Golgi-to-endosome trafficking pathways. Researchers demonstrated that PfVPS51 localizes to the Golgi apparatus and is essential for efficient protein sorting to endosomes. Researchers discovered that disabling PfVPS51 led to defects in parasite growth and development, further supporting its importance for  pathway ,15  is yet to be investigated further, but we found evidence suggesting that it forms an association with previously uncharacterized transmembrane protein Golgi Protein 2 (GP2). Golgi Resident Protein 1’s complex with a previously uncharacterized transmembrane protein known as Golgi Protein 2 (GP2) but evidence was found regarding its involvement with Golgi Protein 2 [Malaria parasites display a less-developed Golgi apparatus compared to other eukaryotic cell types. Their Golgi apparatus is composed of dispersed, unstacked, cis- and trans-cisternae; despite its crucial role in rotein 2 ,17.The Golgi Protein complex is localized to the cis-Golgi during all stages of erythrocytic parasite life cycles as well as mosquito stages, with its expression being conditionally repressed or its gene inactivated, suggesting its significance at optimizing asexual development during blood stages. Analyses of parasite strains in which either expression was conditionally repressed and/or gene activation occurred have demonstrated this ,19. WhilMalaria parasites have been studied using electron microscopy and live-cell microscopy to reveal their ultrastructure and dynamic processes, respectively, in real time. However, this research provides key insights into dynamic processes too small to be resolved using conventional live-cell microscopy; specifically, observations were made regarding P. falciparum organelle biogenesis as well as the organization of Plasmodium cell division around microtubule-organizing centers (MTOCs).Ultrastructure expansion microscopy (U-ExM) should not be seen as a replacement for existing microscopy techniques; rather, it should be seen as an invaluable addition to our arsenal for studying malaria parasite cell biology. By filling some gaps left by electron and live-cell microscopy, respectively, U-ExM provides new insights into many parasite processes, the investigation of which would benefit greatly from U-ExM analysis; some examples have already been highlighted here, while many more await further exploration .Merozoite invasion has been extensively studied using various microscopy techniques, but U-ExM could provide invaluable insight into how organelles associate and rearrange during this process. Furthermore, the rapid disassembly of inner membrane complex IMC organelles immediately following invasion may warrant U-ExM investigation; such rapid and dramatic rearrangements exceed live-cell microscopy resolution capabilities. U-ExM offers exciting opportunities for investigating the malaria parasite biology that was once inaccessible with traditional microscopy methods. Its implementation will complement existing techniques while offering insights into key aspects of the parasite’s life cycle and cellular processes ,21.CRISPR-Cas9 screens have generated an abundance of data, revealing multiple host genes influencing Plasmodium liver-stage parasite replication and development; some genes helped, while others hindered this process. Notably, certain proteins involved in cell trafficking, the immune response, and metabolic pathways were identified as having critical functions in ensuring successful liver stage parasite development; this finding offers exciting prospects for malaria control strategies. Targeting specific host genes identified in this research could provide innovative interventions that stop malaria’s progression, and, therefore, help to avoid symptoms of malaria. The CRISPR-Cas9 screen’s power lies in its ability to reveal complex interactions between Plasmodium parasites and host cells during liver stage malaria parasite development, providing insight into malaria biology. This groundbreaking research opens the way to revolutionary therapeutic approaches against malaria, from gene editing technology and host–parasite interactions to novel antimalarial agents. All these advances hold enormous promise in our collective effort to eliminate malaria globally .T. gondii and P. falciparum share an evolutionary history and possess similar cellular processes, indicating that they should react similarly to inhibitors that affect their respective life stages. In an efficient image-based screening protocol using transgenic T. gondii expressing TgSORT-GFP and ROP1-mCherry, the ROP1-mCherry signal alone proved accurate for automated miniaturized drug screening; our aim was to identify drugs which disrupt subcellular localization, affecting parasite polarity morphology replication. Screening yielded compounds inhibiting p38-MAP kinase, such as Cpd2 (SB 203580) and Cpd3 (SB 203580 and SB 203580), respectively, known to reduce T. gondii growth [i growth .The kinase inhibitors found during our screening may target some essential parasite kinases. While p38-MAP kinase inhibitors have shown some toxicity during previous clinical trials, other drugs unrelated to kinase inhibition identified in our research appear promising for consideration. Based on our findings, we propose that combination therapies with multiple effector mechanisms might be more successful than using just one or two with identical mechanisms of action, thus making the development of resistance against multiple effector mechanisms simultaneously more challenging .Target identification and mechanism analysis are critical steps toward developing cross-species antiparasitic compounds with maximum efficacy. By connecting specific chemical scaffolds to their associated cellular phenotypes in two distinct parasites from evolutionarily related but distinct parasite families, a pathway for the comprehensive mechanistic analyses of interest can be opened. Biochemical and genetic approaches will play an integral part in targeting drug targets in the design of novel parasite inhibitors; such a comprehensive approach is key towards developing effective and targeted antiparasitic treatments .P. falciparum malaria parasite that causes the third type of malaria . Golgi’s era in the early 1890s witnessed the remarkable establishment of a taxonomy that has remained unchanged even after 130 years, specifically in relation to the genus Plasmodium and its four human species: P. falciparum, P. vivax, P. malariae, and P. ovale. Italian scientists of that time achieved an extraordinary feat in accurately describing and classifying these plasmodia, which is a rarity in the field of taxonomy, particularly when it comes to microbes. Typically, the initial observers and describers often make errors, but the Italians managed to get it right, leading to a taxonomy that has stood the test of time. Golgi’s work was completed prior to Romanowsky stain becoming available, further emphasizing his extraordinary scientific abilities. Camillo Golgi shared the Nobel Prize with Santiago Ramon y Cajal of Spain for their contributions to understanding the microstructure of the nervous system [Camillo Golgi’s diligent research allowed for an improved understanding of two prevalent plasmodia in Italy, and his findings provided invaluable assistance for Marchiafava and Celli who described, in 1892, the life cycle of the s system . Howevers system ,26.


Plasmodium falciparum, Plasmodium knowlesi, and Plasmodium malariae, but is less well-characterized with Plasmodium vivax. We describe a case of severe P. vivax malaria with hypotension, prostration, and acute kidney injury occurring 2 months after splenectomy in Papua, Indonesia. The patient was treated successfully with intravenous artesunate.Severe malaria after splenectomy has been reported with infections with  In acute malaria, the spleen has a protective role in phagocytic clearance of circulating parasites, including after antimalarial treatment.Plasmodium and Babesia parasites. Individuals who have undergone a splenectomy have an increased risk of clinical episodes of P. falciparum malaria,,P. vivax than P. falciparum.P. falciparum infections after splenectomy display higher parasitemias and a greater frequency of mature forms in circulation.P. falciparum infection after splenectomy, particularly in nonimmune patients.,,P. falciparum species, and have been reported in Plasmodium knowlesi,Plasmodium malariae,P. vivax malaria after splenectomy in malaria-endemic Papua, Indonesia.Without a functional spleen, individuals are at increased risk of severe and potentially fatal infections, especially those caused by encapsulated bacteria,Plasmodium lactate dehydrogenase (pLDH)/histidine-rich protein 2 (HRP2) and HIV rapid diagnostic tests were negative; however, routine preoperative blood smear revealed asymptomatic peripheral P. falciparum ring-stage parasitemia with a density of 443 parasites/μL. This was cleared with 3 days of oral dihydroartemisinin–piperaquine (DHP) and, after exclusion of glucose-6-phosphate dehydrogenase deficiency, a course of supervised primaquine (PQ) with a total dosage of 7 mg/kg per local postsplenectomy guidelines. The patient also received immunization with pneumococcal, meningococcal, Haemophilus, and influenza vaccinations upon recovery from splenectomy.A 48-year-old male resident of Timika, Papua, Indonesia, underwent splenectomy at Rumah Sakit Umum Daerah (RSUD) Hospital in 2021 for hypersplenism and massive splenomegaly (2.3 kg). He had a history of hepatitis C, but no clinical episodes of malaria in the preceding year. Preoperative pan-P. vivax asexual stages  at a density of 12,246 parasites/μL and low-level gametocytemia (32 parasites/μL) , total white cell (7 × 103/μL), and neutrophil counts were all normal. Kidney function testing revealed elevated creatinine (1.7 mg/dL) with normal blood urea nitrogen (12.4 mg/dL). Total bilirubin was normal (0.6 mg/dL) and cell-free hemoglobin was elevated , both measured retrospectively on frozen heparinized plasma. Liver function tests, and sodium and chloride levels were within normal limits, with mild hypokalemia. Urine output was normal, but further tests were not performed. A SARS-CoV-2 rapid antigen test was negative. During his previous splenectomy, spleen tissue had not been reimplanted, and elevated numbers of pocked red cells (30.8%) and the presence of Howell-Jolly bodies (0.4%) confirmed an absence of splenic function .During a scheduled clinical review 2 months after splenectomy, the patient was noted to have fever, headache, and myalgia. Vital signs were initially normal . Blood film revealed ites/μL) . A pan-pfunction . MicroscP. vivax malaria according to the following modified WHO criteriaThe patient was diagnosed with severe P. vivax infection after recent splenectomy, characterized by hypotension and prostration, with concomitant KDIGO stage 2 AKI. Postsplenectomy severe malaria caused by infection with P. falciparum, P. knowlesi, and P. malariae is well described, with this case showing that postsplenectomy severe malaria can also be caused by P. vivax infection.We report a case of severe malaria from P. vivax malaria after splenectomy. In both cases, the contribution of P. vivax malaria to severe anemia postsplenectomy was confounded by significant preexisting anemia at splenectomy and postulated contributory causes .,Severe anemia has also been reported with P. vivax malaria in patients with their spleen intact.Salmonella spp.Streptococcus pneumoniaeP. vivax malaria. Absence of microbiology facilities prevented testing for the presence of concomitant bacterial infection contributing to manifestations of severe malaria in this patient, and a chest radiograph was not performed to rule out concomitant bacterial pneumonia. However, because the patient recovered without any antibiotic therapy and improved with antimalarial therapy alone, we think it is likely that the severe clinical manifestations of malaria were the result of P. vivax infection alone. Nevertheless, early empirical antibiotics should be given in addition to antimalarial therapy in future patients with Plasmodium infection and severe malaria syndrome who have undergone splenectomy to cover the possibility of concomitant life-threatening bacterial infection.Severe malaria, including hypotension, prostration, and AKI, is reported with P. vivax malaria within 3 months after splenectomy,P. vivax and P. falciparum.P. vivax malaria after treatment of P. falciparum malaria is similarly described in patients with an intact spleen, and is hypothesized to be the result of relapse of preexisting P. vivax hypnozoites.P. falciparum infection, and supervised radical cure with high-dose 7-mg/kg PQ immediately after splenectomy, per local policy, suggesting that the early recurrence of P. vivax malaria was not the result of a relapse. Reinfection with P. vivax was considered more likely, given his resumption of occupational exposure as a farmer in the lowland forested area of Timika.A high frequency of P. falciparum and P. knowlesi, there is no spleen to trap and remove circulating parasites, and circulating parasitemia is greater than in patients with an intact spleen.,P. vivax malaria is associated with greater circulating and total parasite biomass, with parasite biomass-related endothelial activation and dysfunction, and with systemic inflammation.,P. vivax malaria in the same region.P. vivax biomass found in the peripheral circulation after splenectomy, endothelial activation and systemic inflammation arising from a relatively high circulating parasitemia may have contributed to disease severity in this patient. In the presence of a spleen, a large proportion of malaria-associated hemolysis occurs in the extravascular compartment, particularly the spleen. In contrast, after splenectomy, most malaria-associated hemolysis occurs in the intravascular component.P. vivax malaria.P. vivax malaria and an intact spleen,In postsplenectomy malaria from P. vivax infection after splenectomy can also cause severe malaria. The case highlights the importance of rapid diagnosis and treatment of all species causing malaria in hyposplenic populations. Postsplenectomy malaria preventative measures, including chemoprophylaxis, should be considered in areas endemic for P. vivax as well as P. falciparum.In conclusion, this case demonstrates that


Plasmodium species is necessary for making informed decisions on targeted interventions and control strategies to eliminate each of them. This study aims to describe the epidemiology of plasmodial species in Mali, where malaria is hyperendemic and seasonal. Data reports collected during high-transmission season over six consecutive years were analyzed to summarize malaria epidemiology. Malaria species and density were from blood smear microscopy. Data from 6870 symptomatic and 1740 asymptomatic participants were analyzed. The median age of participants was 12 years, and the sex ratio  was 0.81. Malaria prevalence from all Plasmodium species was 65.20% (95% CI: 60.10–69.89%) and 22.41% (CI: 16.60–28.79%) for passive and active screening, respectively. P. falciparum was the most prevalent species encountered in active and passive screening . This prevalence was followed by P. malariae  and P. ovale . Regarding frequency, P. falciparum was more frequent in symptomatic individuals . In contrast, P. malariae was more frequent in asymptomatic individuals . P. ovale remained the least frequent species (less than 1%), and no P. vivax was detected. The most frequent coinfections were P. falciparum and P. malariae (0.56%). Children aged 5–9 presented the highest frequency of P. falciparum infections (41.91%). Non-falciparum species were primarily detected in adolescents (10–14 years) with frequencies above 50%. Only P. falciparum infections had parasitemias greater than 100,000 parasites per µL of blood. P. falciparum gametocytes were found with variable prevalence across age groups. Our data highlight that P. falciparum represented the first burden, but other non-falciparum species were also important. Increasing attention to P. malariae and P. ovale is essential if malaria elimination is to be achieved.Up-to-date knowledge of key epidemiological aspects of each  P. falciparum malaria declined, P. malariae and P. ovale infections rose from two- to sixfold [The World Health Organisation (WHO) estimated the number of malaria cases to be 247 million and the number of malaria deaths to be 619,000 worldwide in 2021. In 2021, 95% of deaths due to malaria were in Africa  despite P. falciparum and P. vivax are the most significant threats among the five human-causing malaria species. In 2018, P. falciparum accounted for 99.7% of estimated malaria cases in Africa, while P. vivax predominated in America, accounting for 75% of the cases [P. malariae was the third most frequent species, with a prevalence of up to 15–40% and an overlapping distribution with P. falciparum throughout tropical Africa and malaria-endemic regions of the world [P. ovale is predominantly found in forests and humid savannah areas in West and Central Africa, with a prevalence of 4 to 10% [P. knowlesi, initially thought to infect nonhuman primates, was reported to also infect humans during the 2004 human malaria outbreak in Southeast Asia [he cases . P. malaast Asia .Plasmodium infections, contributing to 3% of malaria cases and deaths worldwide and 6% of cases in West Africa. Between 2016 and 2019, malaria cases decreased by 13%, from 384 to 334 per 1000 inhabitants [P. falciparum, P. malariae, P. ovale, and P. vivax are those causing malaria in Mali [P. falciparum, the most prevalent species, causes the most lethal episodes, and depending on the locality, this species is responsible for 82 to 95% of all malaria cases [P. malariae and P. ovale is poorly understood, hampering disease elimination.Mali is among the ten countries with the highest morbidity and mortality due to abitants . Similarabitants . The het in Mali ,15. P. fia cases . As in mfalciparum species, urging actions from the national malaria control program and decision-makers to implement specific control strategies.This study aims to fill the gap by describing the prevalence of plasmodial species encountered in a hyperendemic zone of Mali, Ntjiba, during high seasonal malaria transmission. The commune of Ntjiba, located in the Guinean Sudanese zone, is characterized by seasonal malaria transmission. Analyzing retrospective data from previous malaria field studies conducted between 2016 and 2021 in Djiba commune, this report emphasizes the burden of non-Data was gathered retrospectively using original research documents archived by the MRTC research team at the Catholic confessional center of Faladié. Faladje is the rural capital of the Ntjiba commune, located 80 km from the Malian capital Bamako. According to the local registers, the commune of Ntjiba has 23 villages with an estimated population of 23,000 inhabitants. Malaria is hyperendemic, and transmission is seasonal, mainly occurring between July and December when the rainfall is higher.Epidemiological data collected consistently from October to December for six consecutive years (2016 to 2021) were compiled. The data consisting of microscopically confirmed malaria cases were extracted from registers. Patients visiting the health center clinic with malaria symptoms were designed as passive screening cases. At the community level, asymptomatic participants invited for weekly screening at the healthcare facility were classified as active screening individuals. The passive screening group comprises all malaria-positive patients of all ages visiting our health center while the active screening group included asymptomatic volunteers aged six months and more of the selected village site population invited for malaria screening.Plasmodium species identification were assessed as previously reported on thick and thin smears, respectively [Due to logistic impediments, there was no active screening from October to December 2016 and 2017, October 2018, November 2018, and October 2021. Blood smears were read independently by two microscopists, and any discrepancies were resolved by a third microscopist. Parasitemia and For this study, the following parameters were considered: enrolment date , participants’ age and sex, type of screening (passive or active), and clinical profile (symptomatic or asymptomatic). Parasitemia was estimated as parasite stage count by microliter (parasites/µL blood). Parasitemia was categorized as low (≤2500 parasites/µL blood), moderate , and high parasitemia . Hemoglobin values less than 11.0 g/dL were classified as anemia and greater values as no anemia [Plasmodium spp. infections prevalence among screening groups, and participants’ clinical profile. The ANOVA test was used to compare variances across the different groups’ means (or averages). An alpha level of 0.05 was used for all tests to determine statistical significance.The data were recorded in Microsoft Office Access and analyzed with STATA software . Pearson’s Chi-square test was used to compare Our study used secondary data. The protocols used to populate the registers during primary data collection were all approved by the Ethics Committee of the Faculty of Medicine, Pharmacy and Odontology of the University of Bamako reference . The consent of the village communities was obtained from the local authorities before each study, and informed consent was obtained from all participants.p < 0.002). The median age of the participants was 12, ranging from 3 months to 85 years old. Children from 5 to 9 years old were the most represented, with 34.94% (CI: 28.89–39.67%) and 62.72% (CI: 61.69–89.97%) in passive and active screens, respectively (p = 0.027) and active (p < 0.001) screening.A total of 8610 participants were enrolled in this study, 6870 from passive screening (symptomatic) and 1740 from active screening (asymptomatic). The number of participants infected with at least one plasmodial species were 65.20% (CI: 60.10–69.89%) and 22.41% (CI: 16.60–28.79%) in passive and active screening, respectively . As showectively b. The dip = 0.003) and active screening  (p = 0.034)  . Malaria= 0.034) c. Preval= 0.034) .2 = 0.9309, p < 0.004). The lowest prevalence was observed in children under one year  and adults over 65 years  ), followed by 10–14  and 1–4 year olds , (R7–0.25%) .As for the active screening, malaria infections were mostly affecting people aged from 5–9  and 10–14 year olds . Only one child under one year old had parasites with no symptoms, while no asymptomatic infected individuals were detected beyond 25 years old .P. falciparum gametocytes was 1.50% (95% CI: 0.80–2.20%) (n = 97) and 0.82% (95% CI: 0.47–1.16%) (n = 16) in children (5 to 9 years) and adolescents (10 and 14 years old), respectively. For the active screening group, P. falciparum gametocytes prevalence was 0.74% (95% CI: 0.43–0.97%) (n = 61) and 0.41% (95% CI: 0.13–0.68%) (n = 34) in 5- to 9-year-old children and 10- to 14-year-old adolescents, respectively. The lowest gametocyte carriage was found in children under one year of age  and adults ) (p = 0.923).In the passive screening group, the prevalence of –0.07%)) e. The ov–0.07%)) . The meaP. falciparum represented the majority of malaria cases in symptomatic and asymptomatic individuals  than in participants with clinical symptoms  (p < 0.001)  . On the d months . No casePlasmodium species were also observed, mainly P. falciparum + P. malariae (Pf + Pm). The frequency of P. falciparum slightly decreased consistently toward the month of December. In contrast, P. malariae was more frequent in December, corresponding to the end of the high-transmission season. The differences of Plasmodium species prevalence between age groups  and over years  were both statistically significant.Coinfections of several P. ovale  and P. malariae  carriers presented anemia (hemoglobin levels < 11 g/dL), while only 18.76% (95% CI: 14.93–22.53%) with infected with P. falciparum were anaemic. One of the five mixed P. falciparum + P. malariae infections had anemia . The average parasitemia was significantly lower in P. malariae 3120 parasites/µL (SD = 3092) and P. ovale, 3002 parasites/µL (SD = 2272)   a.P. falciparum with 10,535 parasites/µL  followed by P. ovale 5400 and finally P. malariae 1429 (SD = 1032) but the difference between the mean parasite densities is not statistically significant   b.Concerning age groups, most participants between 25 and 64 years  and ≥65 years  had low parasite densities (1 to 2500 parasites/µL) . ParticiThe epidemiology of the various parasitic species of malaria found in Mali is poorly described. This study aimed to assess the epidemiological of plasmodial species in Ntjiba commune, in Mali, where malaria is hyperendemic and seasonal. All the data produced were obtained using conventional microscopy, which despite current advances in molecular diagnostic tools, remains the gold standard according to WHO . MicroscPlasmodium species over the years. However, a change could occur with climatic vagaries since we observed an increase in P. malariae cases in asymptomatic people at the end of the high-transmission season. Regrettably, the number of malaria cases in the world suffered an increment in the last two years [This study shows with clarity the high prevalence of clinical malaria in this period of each year corresponding to the peak of the transmission at the end of the rainy season, occurring between October and December in Mali. Our findings can be explained by the stability of malaria incidence at the high-transmission season and rainfall variability . It has wo years  mainly dThe high proportion of male participants infected in our data, even if there was a higher proportion of female subjects among the participants, could be explained due to sociocultural and behavioral factors that would expose them more to mosquito bites ; howeverP. falciparum being the most common (90% of the cases) regardless of the clinical profile of the participants. In our data, P. falciparum coinfections with P. malariae were more frequent than with P. ovale. Monoinfections with P. malariae were the second most frequent found in participants. Its prevalence and frequency around 2 and 5%, respectively, were seen to increase in asymptomatic people and in December, corresponding to the end of the high-transmission season. P. ovale, with a prevalence often below 1%, represented the third species of plasmodia found. Our data are consistent with a previous study carried out in this same locality [P. vivax. Although, microscopic evaluation of samples from some localities in the country suggested the presence of blood stages of P. vivax-like, later confirmed unambiguously by nested PCR [P. vivax infection, but recent studies have shown cases of infections in Duffy-negative subjects in sub-Saharan countries [Our results agree with a previous study carried out on malaria prevalence in a site with similar epidemiological characteristics in Mali , showinglocality  and withlocality . In addisted PCR . Africanountries .Plasmodium species could be found in individuals of any age; however, adolescents aged from 10 to 14 years old were most frequently infected by non-falciparum species such as P. malariae and P. ovale. Malaria appeared to be more prevalent in children 5–9 and 10–14 years old, suggesting the importance of extending SMC to those age group. In particular, we found that only P. falciparum cases displayed hyperparasitemias, regardless of the type of screening, which are linked to severe malaria cases [falciparum species, as well as their mixed infections with P. falciparum, mostly showed moderate to low parasitemia [P. malariae or P. ovale was responsible for severe malaria, as previously reported [P. falciparum infection. However, protective immunity against non-falciparum species remained to be investigated as their biology differs from that of P. falciparum [P. malariae and P. ovale infections have been shown to be responsible for anemia burden [P. falciparum infections, were found more in children aged 5 to 9 and the lowest carriage rate in those under one year and over 25 years of age, which was in agreement with previous studies [Our analysis also shows us that ia cases . Furtherreported ,39. Parareported . Studieslciparum ,42,43. Pa burden ,45, lysia burden . Gametoc studies  carried  studies .A limitation to this study is that the recorded data only covered the end of the rainy season of each year from October to December, corresponding to a part of the high-transmission season. Therefore, the results do not provide the full epidemiological information for a whole year although malaria would be detectable even at periods of very low transmission between April and June (dry season). Longitudinal parasitological and entomological surveys covering a whole year for various years would be necessary to monitor the dynamics of malaria transmission in this malaria endemic area.P. vivax infection has been detected in the population. It would therefore be necessary to combine microscopy with other tools, such qPCR, for the diagnosis of plasmodium species to closely monitor malaria infections and to assess the presence of P. vivax, because the study area has a cosmopolitan population comprising a strong community of Moors from northern Mali more likely to carry P. vivax [P. malariae and P. ovale.A second limitation is that the biological data are derived only from the results of microscopy, which may be the reason why no P. vivax , and supAs a third limitation, we found that there were few volunteers over 24 years of age who tested in the active screening compared to other age groups, representing a possible selection bias and a limitation of our study.falciparum species had low parasitemia. The frequency of P. malariae was higher at the end of high-transmission season (December) in asymptomatic carriers. P. falciparum was by far the most dominant species followed by P. malariae. P. ovale remained the third species regardless of the type of screening. Our characterization of Plasmodium species epidemiology highlights that P. falciparum represents a significant burden and that other Plasmodium species silently contribute to the malaria burden. If malaria elimination is to be achieved, awareness campaigns are needed to increase attention on P. malariae and P. ovale infections and patient care.Malaria prevalence gradually decreases over the years, and parasite density of the different species varies depending on individuals’ clinical status (symptomatic/asymptomatic) and age. Non-


Paediatric febrile illnesses pose diagnostic challenges in low-income countries. Western Uganda is endemic for both malaria and typhoid but the true prevalence of each individual disease, their co-infections and associated factors are poorly quantified.To determine the prevalence of malaria, typhoid, their co-infection, and associated factors amongst febrile children attending the paediatrics and child health department of Kampala International University Teaching Hospital (KIU-TH) in Western Uganda.Cross-sectional study used a survey questionnaire covering demographics, clinical and behavioural variables. We obtained blood for peripheral films for malaria and cultures for typhoid respectively; from 108 consecutively consented participants. Ethical approval was obtained from KIU-TH research and ethics committee . Multivariate regression analysis was performed using Stata 14.0 (StataCorp. 2015) at 95% confidence interval, regarding p < 0.05 as statistically significant.Majority of participants were males 62% (n = 67), cared for by their mothers 86.1% (n = 93). The prevalence of malaria was 25% (n = 27). The prevalence of typhoid was 3.7% (n = 4), whereas the prevalence of malaria-typhoid co-infection was 2.8% (n = 3). Using treated water from protected public taps was associated with low malaria-typhoid co-infection , whereas drinking unboiled water from open wells increased the risk for the co-infection .The prevalence of blood culture confirmed malaria-typhoid co-infection in children was lower than previously reported in serological studies. These findings emphasize the need to use gold standard diagnostic investigations in epidemiological studies. Educational campaigns should focus on the use of safe water, hygienic hand washing, and proper waste disposal; and should target mothers who mainly take care of these children. MalariaCurrent reports show increasing global trends of malaria during 2022 . The burUganda is endemic for both malaria and typhoid . TherefoP. falciparum highly endemic African region , however this association did not remain valid upon adjustment for confounding .Children who reported taking unboiled drinking water from open wells were 17 times more likely to get malaria-typhoid co-infection . Children whose source of water was public taps were 97.0% less likely to have malaria-typhoid co-infection compared to those who used open wells . This association remained statistically significant even after adjustment for confounding . There was no statistically significant association between malaria-typhoid co-infection and gender, level of education, type of accommodation and school going status .Table 7B44.1The first objective of the study was to determine the prevalence of malaria amongst febrile children attending the paediatric department of KIU-TH which was found to be 25.0%. This prevalence is lower than 36.5% reported in an Ethiopian study , though However our findings show a significant reduction in malaria prevalence from a previously reported Ugandan National average of 42.0% in 2009 . This re4.2The second objective of the study was to determine the prevalence of typhoid amongst febrile children attending the paediatric department of KIU-TH, based on blood cultures. The study established the prevalence at 3.7% and all affected participants were aged 7–12 years. This blood culture based prevalence is comparable to 2.8%  previousPreviously in Uganda, typhoid infection based on blood cultures has been largely studied only during outbreaks rather than routine in the paediatric population. Ranges between 2.6% and 22.6% were reported among adults during Kasese outbreak in Western Uganda . In a re4.3The third objective of the study was to determine the prevalence of malaria-typhoid co-infection amongst febrile children attending the paediatric department of KIU-TH. The prevalence of malaria-typhoid co-infection based on microscopy and blood culture respectively in our study population was found to be at 2.8%. Our co-infection rates are comparable to 3.5% reported in a Tanzanian study among children below 15 years  and to 2Salmonella Typhi compared to Plasmodium parasites [The co-infection rates in our study are far lower that what has been reported previously in serological studies. In their serological study in Western Uganda, Agwu et al.  reportedarasites . A consearasites ,35,52.4.4The fourth objective of the study intended to determine the factors associated with Malaria-Typhoid co-infections amongst febrile children. Upon adjusting for confounders, we found that the most crucial factor influencing this co-infection was the source of water. Using treated water from protected public taps was associated with low malaria-typhoid co-infection (p = 0.04) whereas drinking unboiled water from open public wells increased the risk for the co-infection (p = 0.037). Capturing such history could be made a routine element of screening children presenting with febrile illnesses in our settings.Other Ugandan authors have pinned contaminated water and food as main driving factors for typhoid infection , althoug4.5This study boosts of several strengths. First, the investigations used in the present study i.e., blood culture and blood side microscopy are considered gold standard in definitive diagnosis of typhoid and malaria respectively. Secondly, being a cross-sectional study, the level and quality of completeness of data could easily be controlled. Moreover, the data tool used was not only specifically designed for this study but also validated for reliability. Lastly, all positive cases of Salmonella were externally cross-examined and confirmed by an external reference laboratory.However, there were some limitations in this study. First, although blood slide and blood culture are considered gold standard for diagnosing malaria and typhoid respectively, in exceptional cases, malaria parasites may not be captured in peripheral blood smears even in presence of severe infection due to sequestration of parasitized cells in deep capillary beds. Secondly, the reported isolated case of typhoid fever in the present study was included based on presence malaria pigment in circulating neutrophils and monocytes despite having no malaria parasites in setting of suspected malarial infection. In addition, the sensitivity of blood cultures for typhoid salmonella is intrinsically moderate at only 85.0–90.0% ,53. Last5The prevalence of malaria was high in our study population compared to the national average . The preJoanitor Nakisuyi: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper. Melvis Bernis; Andrew Ndamira; Vicent Kayini: Contributed reagents, materials, analysis tools or data. Richard Mulumba: Conceived and designed the experiments. Pius Theophilus; Ezera Agwu: Performed the experiments; Contributed reagents, materials, analysis tools or data. Herman Lule: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Data will be made available on request.The study strictly followed the Uganda National Courncil for Science and Technolgy Guidelines (2014) on research involving use of human participants and in accordance with the Declaration of Helsinki . EthicalNot applicable.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.