Anopheles stephensi, an Asian malaria vector, continues to expand across Africa. The vector is now firmly established in urban settings in the Horn of Africa. Its presence in areas where malaria resurged suggested a possible role in causing malaria outbreaks. Here, using a prospective case–control design, we investigated the role of An. stephensi in transmission following a malaria outbreak in Dire Dawa, Ethiopia in April–July 2022. Screening contacts of patients with malaria and febrile controls revealed spatial clustering of Plasmodium falciparum infections around patients with malaria in strong association with the presence of An. stephensi in the household vicinity. Plasmodium sporozoites were detected in these mosquitoes. This outbreak involved clonal propagation of parasites with molecular signatures of artemisinin and diagnostic resistance. To our knowledge, this study provides the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa, highlighting the major public health threat posed by this fast-spreading mosquito. Anopheles stephensi carrying Plasmodium falciparum with diagnostic and drug resistance mutations calls for heightened vector surveillance in both urban and rural settings.Evidence that a dry-season outbreak of malaria in Dire Dawa, Ethiopia is caused by  Malaria control programs in Africa traditionally focus on rural settings, where most infections occur, but malaria is of increasing concern in urban settings2. Given the rapid urbanization in Africa3, urban malaria transmission can result in a considerable health burden4. Urban malaria is classically associated with importation from areas of intense transmission5 but can be exacerbated by the adaptation of existing malaria vectors to urban environments6 and the emergence of urban malaria vectors such as Anopheles stephensi7.The promising decline in malaria burden has slowed since 2015. This is particularly evident in Africa, the continent that carries the largest malaria prevalenceAn. stephensi is distinct from other Anopheles species that are traditional vectors in  Africa, with its preference for artificial water storage containers that are common in urban settings9. Native to the Indian subcontinent and the Persian Gulf10, An. stephensi is now rapidly expanding its geographic range westward , An. stephensi has spread across the Horn of Africa; its range now includes Ethiopia (2016)12, Sudan (2016)13, Somalia (2019)14, Eritrea (2022)15 and beyond: Yemen (2021)16, Kenya (2022)17, Ghana (2022)15 and Nigeria (2020)15. In the Horn of Africa, the vector was found firmly established18 and abundantly present in manmade aquatic habitats in the driest months of the year, when endemic vectors such as An. arabiensis are largely absent, demonstrating how well adapted the mosquito is to perennial persistence and urban ecology. This poses a risk of year-round malaria transmission. In recognition of the potentially devastating consequences of An. stephensi advancing across Africa, the World Health Organization (WHO) urgently requested more data on its distribution and released a strategy to mitigate its spread19.ard Fig. 7. First An. stephensi and widespread high prevalence of insecticide resistance, the Horn of Africa region is disproportionately affected by other emerging biological threats for malaria control, including the emergence of Plasmodium falciparum parasites with drug resistance  and histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions  that could compromise the utility of widely used rapid diagnostic tests (RDTs). Because of its abundant and species-specific expression by P. falciparum parasites, histidine-rich protein 2 (HRP2)-based RDTs are commonly used for the diagnosis of P. falciparum. Recent reports of expansion of parasites with pfhrp2/pfhrp3 gene deletions and drug resistance, together with a highly efficient invasive mosquito in the region, threaten the major gains documented in recent decades.In addition to the invasive P. falciparum and P. vivax in its native geographical range10, An. stephensi was recently confirmed as being susceptible to local parasites in Ethiopia , although direct evidence for a role of An. stephensi in this resurgence was unavailable. In this Article, following a report of a dry-season upsurge in malaria cases in Dire Dawa City, Ethiopia, where An. stephensi was recently documented8, we prospectively investigated its role in malaria transmission through responsive epidemiological and entomological surveillance  showed an overall statistically significant trend of increasing number of malaria-positive cases between 2019 and 2022 . A 12-fold increase was observed  collected from public and private health facilities ; the dormitories affected by malaria were occupied by male students only  and the additional infections detected (n = 102) in this study were found to be P. falciparum, with the exception of two P. vivax infections detected by 18S-based quantitative polymerase chain reaction (qPCR). Plasmodium infection was detected in 14 controls by 18S-based qPCR. The parasite density in these infections—which were all P. falciparum—was very low (median parasitemia was 21 parasites µl–1) and thus lies below the detection limits of conventional diagnostics. Only two of these infections had parasitemia >100 parasites µl–1 .We conducted a prospective case–control study to identify risk factors associated with this sudden rise in malaria in the city  and DDU Fig. . In the 022 Fig. . All indAnopheles in waterbodies present within a 100-m radius. Members of a case household/dormitory were more likely to be living close to An. stephensi-positive sites, defined as the presence of larvae within a 100-m radius from the household/dormitory (odds ratio (OR) 5.0, 95% confidence interval (CI) 2.8–9.4, P < 0.001), to adult mosquito resting sites  or to natural/manmade waterbodies in general . The odds of using an aerosol insecticide spray were 58% lower among members of index cases compared with controls .The results obtained from case–control analysis showed that members of index cases and controls had different levels of mosquito exposure  and RDT-detected infections . At DDU, despite all students living in close proximity , dorm-mates of malaria cases were three times more likely  compared with dorm-mates of controls . One-quarter of microscopy-positive infections (34 out of 136) were negative by HRP2-based RDT  than RDT-negative infections . Similarly, parasite densities were higher in microscopy-positive infections  than in microscopy-negative infections , strongly suggestive of pfhrp2/pfhrp3 gene deletion in these infections. Parasite density distributions were not different between university students  and city residents , malaria-infected control family members  and malaria-infected index family members .In the city, RDT Fig.  or microRDT Fig.  being deAnopheles larvae were detected in 3% (26 out of 886) of aquatic habitats, which were either artificial (n = 17) or natural (n = 9). An. stephensi was the only species detected in artificial containers (n = 414 larvae), of which the majority were metal and plastic barrels and jerrycans, and was the predominant species detected at stream edges , water storage tankers (4 out of 4), manholes (7 out of 7), inside (22 out of 508) and outside (7 out of 305) the index and control households/dormitories using Prokopack aspirators, with nearly all identified as An. stephensi  were confirmed to be this species, except four for which the ITS2-based PCR experiment failed  and An. gambiae (5 out of 16) mosquitoes  and An. gambiae s.l. , but only An. stephensi (2 out of 98) had recently fed on humans. Bloodmeal source was undetermined for one-half (n = 96) of fully engorged (n = 199) An. stephensi mosquitoes tested in this study. P. falciparum sporozoites, indicative of transmission following natural blood feeding, based on sporozoite and PCR-based detection, were confirmed only in An. stephensi .ial n = 1 or naturP. falciparum infection localities within the city demonstrated significant evidence for clustering  were detected. An. stephensi larvae and/or adult mosquitoes were more often detected near index cases (14.9%) than controls  and being above 15 years of age  were risk factors associated with P. falciparum infection positivity, while using aerosol insecticide sprays was found protective from malaria . The results further show that those individuals residing in households/dormitories with An. stephensi positivity (larvae/adult/indoor/outdoor) had a higher risk of malaria infection  compared with individuals in households/dormitories where An. stephensi was not detected.We next evaluated risk factors for being infected with um Table . Male sen = 131) of the samples. All blood samples were collected from patients before treatment was provided, and thus represent the composition of parasites in the blood. Genotyping of 131 infections at 162 microhaplotype loci by amplicon sequencing uncovered that 90% of infections were monoclonal and nearly all were closely related to other detected infections, with 98% falling into one of two distinct, nearly clonal lineages. Lineage 1 was the most common, almost completely homogeneous, observed throughout the study period, and distributed widely throughout both study sites  tested negative on HRP2-based RDT but were positive by microscopy. Lineage 1 infections did not contain pfhrp2 deletions, most were detectable by RDT , and only 2.8% (n = 3) contained the kelch 13 R622I mutation, but all had evidence of pfhrp3 deletions and the quintuple mutation in pfdhfr and pfdhps associated with antifolate resistance. Of the successfully sequenced microscopically detectable but RDT-negative infections (n = 24), some were found to be pfhrp2 and pfhrp3 double gene deleted  while the rest were only pfhrp3 gene deleted . Interestingly, most infections from lineage 2 containing the R622I mutation (11/14) exhibited incomplete antifolate resistance, lacking the pfdhfr 59 mutation. A single monoclonal infection with low relatedness within lineage 2 showed unique features: elevated pfmdr copy number, heterozygous for the pfmdr1 184 mutation, while being the only infection with a wild-type pfcrt genotype. There was no significant association between lineage 1 and lineage 2 with self-reported uptake of vector control measures , travel history, age, sex, educational level, occupation or infection detection by microscopy , a 12-fold increase in malaria incidence that was predominantly P. falciparum was observed in the city. The spatial overlap and association between malaria infection and the presence of An. stephensi, the detection of sporozoites in adult mosquitoes and the clonal propagation of parasites that we report here provide the strongest evidence so far for a role of An. stephensi in driving an urban malaria outbreak in Africa. This, to our knowledge, is the first direct evidence of the role of An. stephensi in transmitting malaria in Africa and corroborates recent reports from Djibouti of exponential increases in malaria cases in the years following detection of the species26.Our findings raise concern about urban malaria associated with the presence of 28, other African countries29 and Brazil30, and is commonly described in South East Asia31. Common explanations are increased risk due to employment and sociobehavioral factors . There may be other behavioral differences between males and females involving crepuscular activities consistent with biting times for An. stephensi, which is exophilic and exophagic32. In our setting, chewing khat outdoors is done predominately by men33 again increasing exposure to vectors. There is limited evidence for sex-associated biological differences in infection acquisition or infection consequences, with the exception of the well-established role of pregnancy in malaria risk34. The recently described longer infection duration in males compared with females35 suggests that there may be differences in infection kinetics/responses to infections between sexes that may in turn impact the epidemiology of malaria infection.The outbreak in the university campus was localized and the dormitories affected by malaria were occupied by male students only. However, in the population of Dire Dawa City, male sex and older age were predictors of malaria positivity. Higher parasite prevalence in males compared with females has been reported in EthiopiaP. falciparum infections despite the co-occurrence of P. vivax in the region. We previously demonstrated that An. stephensi is highly susceptible to Ethiopian P. vivax isolates9 and an increase in P. vivax cases coincided with a rise in An. stephensi mosquitoes in Djibouti26. Epidemiological circumstances at the start of the outbreak, notably the extent of the human infectious reservoir for Plasmodium infections, may have been more favorable for P. falciparum in our setting. In sympatric settings, it is well known that P. falciparum is more prone to epidemic expansion than P. vivax37. There is a large and increasing body of evidence (including our own work from Ethiopia)39 showing that asymptomatic P. falciparum infections can be highly infectious to mosquitoes and that the level of infectivity depends on the circulating parasite biomass . Related studies on the human infectious reservoir for P. falciparum have also demonstrated that a limited number of individuals, sometimes with asymptomatic infections, may be highly infectious to mosquitoes39. This hypothesis is supported by the limited genetic diversity of parasites detected in this study. We speculate that, at the start of the outbreak, the asymptomatic infectious reservoir for P. falciparum was larger than for P. vivax and that a small number of infected individuals may have been responsible for initiating the current outbreak. The continued increase in the proportion of P. falciparum infections between 2015 and 2022 in Dire Dawa and the timing of the outbreak supports this notion. Although sporozoite rates are difficult to compare between sites, times and species, since they depend on many factors including mosquito age and survival, the 0.5% P. falciparum sporozoite positivity that we observed is similar to that observed previously in An. stephensi in Dire Dawa and other areas in Ethiopia18 as well as sporozoite rates in An. arabiensis, a native malaria vector in Ethiopia40. We consider a comparison with other areas with markedly different parasite populations and transmission intensity less relevant although sporozoite rates of An. stephensi in Afghanistan (0.8%) and India (0.6%) are in the same range as we observed41. Higher sporozoite rates are more likely to be associated with sustained endemicity  and are typically associated with microscopy parasite prevalence between 10% and 40% (ref. 42). Continuous entomological and clinical surveillance would provide further evidence if this was the case in Dire Dawa. In contrast, asymptomatic P. vivax infections have typically too low parasite densities to infect mosquitoes43. Since P. vivax sporozoites have been detected in An. stephensi mosquitoes previously from the same setting18, it is possible that future malaria outbreaks caused by An. stephensi would also involve P. vivax.Interestingly, this outbreak only involved 44. Historical transmission levels influence the size of the submicroscopic reservoir through acquired immunity45. As Dire Dawa was previously endemic46, some low-density infections may persist and affect the interpretation of the extent of the outbreak. The relatively high-density (microscopy-detected) asymptomatic infections provided a better description of the current outbreak38.The trends in increased parasite carriage among individuals living in proximity of malaria cases were most apparent for conventional diagnostics (RDT and microscopy) but not for qPCR. This is probably to reflect the age of infections with recent infections  being more likely to be of higher parasite density while low-density infections that are detectable by qPCR to mainly reflect old infections that may have been acquired many months before the studyAn. stephensi, two other biological threats for the control of P. falciparum were identified in our study: drug resistance and diagnostic resistance. The high prevalence of parasites with the R622I mutation in the kelch13 gene is of particular concern. Although it should be noted that parasite strains were not directly tested for resistance ex vivo in the current study, a recent study identified this as a variant linked with partial drug resistance in Eritrea22. Following the first report in 2016 from northwest Ethiopia47, parasites carrying the R622I variant are reported to be expanding in the same setting48, more widely in the country49 and elsewhere in the Horn of Africa50. In addition to evidence for artemisinin-resistant parasites, mutations conferring chloroquine and antifolate resistance were common in the parasite population responsible for this outbreak. Similarly, pfhrp2 and pfhrp3 gene deletions with phenotypic evidence of RDT negativity were detected in our study. Despite its first report from Peru51, the Horn of Africa  is disproportionately affected by diagnostic challenges of infections with pfhrp2/pfhrp3 deletions. Co-occurrence of parasites with pfhrp2/pfhrp3 gene deletions and the R622I mutation was recently reported from other sites in Ethiopia49. So far, no evidence exists if the drug resistance conferring kelch13 mutation (R622I) and pfhrp2/pfhrp3 gene deletions have co-evolved in the region or if this is a matter of coincidence. Even without the evidence of co-evolution, the convergence of the three biological threats  is concerning for the region and the entire continent at large.In addition to the role for the invasive An. stephensi in an outbreak of P. falciparum infections that carry diagnostic and drug resistance markers in Ethiopia. Our data, demonstrating An. stephensi being abundant both in artificial and natural aquatic habitats in the driest months of the year, highlight how well-adapted the mosquito is to perennial persistence and urban ecology. While our outbreak investigation was performed shortly after the mosquito species was first detected in the area8, routine vector surveillance was sparse and we cannot draw firm conclusions on the timing of An. stephensi introduction in the area. Additionally, limited sensitivity of methodologies for sampling exophagic adult mosquitoes may have resulted in an underestimation of mosquito exposure and reduced precision of sporozoite prevalence estimates. Common adult mosquito collection methods have limited sensitivity for this invasive exophilic/exophagic species. Enhanced surveillance in this study revealed outdoor resting sites  that offer opportunities for targeted vector control and highlight the behavioral plasticity of this invasive mosquito which makes it less amenable to conventional control approaches. Our data on the use of protective measures  were insufficiently detailed to explore how effective these measures are against An. stephensi. Future studies should address this. Considering the very high level of resistance of An. stephensi to the major insecticides in Ethiopia54, the repellent effect of the aerosol sprays is one explanation for the protective association observed in this study55. Most sprays contain repellents such as N,N-diethyl-meta-toluamide (DEET) or permethrin. Permethrin and DEET have strong repellent effects on both Plasmodium-infected and uninfected An. stephensi mosquitoes55.In this study we concurrently examined parasite carriage and spatial clustering in humans and mosquitoes as well as genetic linkage analysis to demonstrate a highly plausible role for An. stephensi in rapidly expanding urban settings could pose a challenge to malaria control programs in Africa for four main reasons: (1) its year round persistence due to its ability to exploit manmade containers that are abundantly present in rapidly expanding urban settings; (2) its ability to evade standard vector control tools given its unique ecology and resistance to many of the currently available insecticides; (3) its ability to efficiently transmit both P. falciparum and P. vivax in the region; and (4) its confirmed role in sustaining the transmission of drug and diagnostic resistant parasites demonstrated in this study that highlights a concerning convergence of biological threats for malaria control in the Horn of Africa and beyond. There is an urgent need for intensified surveillance to identify the extent of the distribution of this vector and to develop and implement tailored control measures. While there is an increasing body of high-quality evidence of the spread of An. stephensi across the African continent, pragmatic studies on how to address this novel malaria threat are largely absent. Given increasing reports of An. stephensi in West and East Africa, the time window during which elimination of this mosquito from (parts of) Africa is possible is rapidly closing.In terms of public health consequences, the spread of 2 Dire Dawa City administrative land . The area has a warm and dry climate with low level of precipitation , and an annual temperature ranging from 19 °C to 32 °C. Malaria incidence has historically been low , with strong seasonality (August to November being the peak season), and sympatric P. falciparum and P. vivax infections.Dire Dawa, located 515 km southeast of Addis Ababa  and 311 km west of Djibouti, is a logistics hub for transportation of goods and cargo Fig. . Of its 4 states that “In some urban settings, the private sector is a major source of malaria diagnosis and treatment. However, it is poorly integrated into the surveillance system”. To give context on how much is being managed by the private sector in Dire Dawa, we have collected 4 years of data (January 2019 to May 2022) from 34 out 39 health facilities (both private and public) that are located within the city administration. This included 2 public and 5 private hospitals, 15 health centers (funded publicly) and 17 clinics (private). Some private clinics (n = 5) refused to provide data or provided incomplete data. Goro Health Center and DDU students’ clinic were selected for the current study based on the highest number of cases they reported before the start of the study (January–February 2022). In fact, together, the two health facilities reported 56% of the total cases in the city in 2022 (January–May). As in all public universities in Ethiopia, students live within campus with full and shared accommodation provided by the government. At DDU, an average of six students of the same sex and year of study share a dormitory on a three-story building that has an average of 67 dormitories. Routine healthcare service is provided in a university dedicated students’ clinic.We obtained public health data, collected through the District Health Information System 2 (DHIS2), to analyze the trend in malaria cases between 2015 and 2022. In the Ethiopian malaria case management guideline, microscopy is recommended for diagnosis at the health center level and above. RDTs are recommended to be used only at the health post level by community health extension workers, in rural settings. In all of the facilities located in Dire Dawa, microscopy was used for diagnosis. The DHIS2 data do not capture cases detected at private health facilities. The recent ‘Global framework for the response to malaria in urban areas’ by the WHOAn. stephensi on malaria, we employed a case–control study where identification of patients was done prospectively to capture co-occurrent characteristics in terms of exposure and risk factors. We recruited consecutive patients with criteria described below in a 1:2 ratio (one case:two controls) unmatched study design. Study protocol was approved by the Institutional Ethical Review Board of Armauer Hansen Research Institute (AHRI)/All Africa Leprosy Education, Research, and Training Center ethics review committee . We obtained informed written consent from all participants and guardians or parents for minors.To ascertain the effect of exposure to Anopheles mosquitoes (larvae and adult). Household/dormitory members of cases and controls who were willing to participate in the reactive case detection were included irrespective of their symptoms. Households were surveyed for mosquitoes when the head of the household and members of the dormitory gave consent to allow the study team to use mosquito collection methods in their houses/dormitories. Families of cases or controls who were not available within 72 h of recruitment of the cases or controls irrespective of their symptoms were excluded as well as individuals or family members who were unwilling/refused to give informed written consent. It is noticeable that, although the study was unmatched due to the difficulty in recruiting matched controls in geographical proximity of the cases, their general characteristics were very similar. Detailed characteristics of study participants are presented in Table Patients with (history within 48 h) fever that presented at the two health facilities and tested positive for malaria by microscopy were recruited as index cases (index) from April to July 2022. We recruited febrile patients who attended the same clinic and tested negative for malaria as controls within 72 h of when the index was identified. The index and controls were followed to their homes, and their household/dormitory members were tested for malaria and their households/dormitories were screened for 56) with prospective case identification until the stopping rule was achieved. The choice of the case:control ratio was based on a logistic regression model aimed to detect an OR of at least 2, assuming an exposure of 20% in controls at household level, where the exposure was defined as presence of An. stephensi. The power analysis was conducted in epiR package (R-cran software), and the stopping rule was set to a power of 70% for the study to be sufficiently powered to detect differences between the presence of malaria on An. stephensi exposure at household level. The controls were selected from the same population as the cases and post-stratification applied. Data from cases and controls were reviewed regularly, and final sample size was set to 290 with 101 cases and 189 controls. The recruitment of case household and control household members was done to include reactive case detection and improve the power of the study (as well as the OR minimum detection).We planned an unmatched case:control ratio of approximately 1:2 (ref. n = 34).Data on the sociodemographic, epidemiological, intervention and travel history were collected verbally using pre-tested questionnaires which were uploaded to mobile tablets using REDCap tools. The entomological survey data and intervention availability were scored by the study data collectors. Malaria case incidence data (from January 2019 to May 2022) were collected from the records of both private and public health facilities  and microscopy, and to prepare dried blood spots (DBS) on 3MM Whatman filter paper (Whatman). The remaining blood was separated into cell pellet and plasma. Slide films were confirmed by expert microscopists. Sociodemographic, epidemiological, intervention utilization, and history of travel and malaria were collected from all study participants.Finger prick blood samples (~0.5 ml), collected in BD KAnopheles mosquitoes within a 100-m radius of the index and control houses/dormitories targeting both manmade water storage containers and natural habitats including riverbeds and stream edges. We checked each aquatic habitat for 10 min from 9:00 to 11:00 and 15:00 to 17:00 for the presence of Anopheles mosquitoes’ larvae or pupae aiming for ten dips per habitat (using a standard dipper with 350 ml capacity). Characteristics of water holding containers  were recorded for each habitat  and without (n = 15) animal shelters were included  coordinates of the households and immature and adult mosquito collection sites were recorded using GARMIN handheld GPS navigator (GARMIN GPSMAP 64S).Conventional adult mosquito collection methods such as Centers for Disease Control and Prevention light traps and pyrethrum spray sheet have limited sensitivity for this invasive species mainly related with its unique resting behaviorP. falciparum and P. vivax was run using primer and probe sequences described by Hermsen57 and Wampfler58 using TaqMan Fast Advanced Master Mix (Applied Biosystems). P. falciparum parasites were quantified using standard curves generated from a serial dilution of NF54 ring stage parasites (106 to 103 parasites ml−1). For P. vivax, parasite quantification was done using plasmid constructs to infer copy numbers by running serial dilutions (107 to 103 copies µl−1) of plasmids having the amplicon. Serial dilutions of the standard curves were generated in duplicate on each plate. Multiplexed amplicon sequencing was performed on qPCR-positive samples with reagents and protocol as in Tessema et al.59. DNA was amplified for 15 or 20 cycles in multiplexed PCR, depending on parasitemia and ability to amplify, and for 15 cycles for indexing PCR. The primer pools used in this study comprised high-diversity microhaplotype targets (n = 162), polymorphisms associated with drug resistance, and targets in and adjacent to pfhrp2 and pfhrp3 to assess for gene deletion (Primer pools 1A and 5 as described in protocols.io repository)60. Amplified libraries were sequenced in a NextSeq 2000 or a MiniSeq instrument using 150PE reads with 10% PhiX.Blood samples in ethylenediaminetetraacetic acid (EDTA) tubes were used to extract genomic DNA using MagMAX magnetic bead-based technology DNA multi-sample kit on KingFisher Flex robotic extractor machine (Thermo Fisher Scientific). Fifty microliters of whole blood input was eluted in a 150 μl low-salt elution buffer. Multiplex qPCR targeting the 18S rRNA small subunit gene for Plasmodium sporozoites. Low-level expression of CSP at stages of sporogony before the parasites migrate to the salivary gland might interfere with signal detected61. Several studies have reported false positive results when targeting CSP especially in zoophilic mosquitoes63. The false positive results could lead to an overestimation of mosquito infection rates. To achieve a conservative estimate of mosquito infection rates, we implemented stringent steps as indicated below:Bisected mosquitoes: We observed previously61 that a signal detected from an earlier stage of sporogony might interfere with interpretation of sporozoite detection, probably causing false positive results. We bisected the mosquitoes anterior to the thorax–abdomen junction under a stereo microscope before processing them for infection detection64. The head and thoraces were processed and stored separately from the abdomen of the mosquitoes. We only used the head and thorax part for infection detection following homogenization in a robust semi-high-throughput mini-bead beater protocol we developed previously65. The heads and thoraces of the mosquitoes were homogenized in 150 µl molecular-grade water that contains 0.2 g zirconium bead (1 mm diameter) using a Mini-Bead Beater 96. Part of the homogenate (50 µl) was used for nucleic acid extraction using cetyl trimethyl ammonium bromide62; 100 µl grinding buffer  was added to the remaining that was used to screen samples for circumsporozoite in bead-based assay.Circumsporozoite bead-based assay: We adopted the most advanced (highly sensitive) bead-based assay for infection detection in mosquitoes66 by targeting CSP. Antibody-coupled magnetic beads and biotinylated secondary antibodies were obtained from the Centers for Disease Control and Prevention, Division of Parasitic Diseases and Malaria, Entomology Branch, Atlanta, GA, USA, and implemented as described before7 and were run using MagPix immunoanalyzer .Quality control to reduce cross-reactivity: The bead-based assay we adopted may eliminate false negatives due to lower limit of detection than previous ELISA-based assays66 but also brings a challenge of enhanced detection of cross-reacting proteins. To reduce this chance, mosquito homogenate was boiled at 100 °C before processing to eliminate false positives that may be caused by heat-unstable cross-reactive proteins to strengthen the validity of the results. To ascertain this specificity issue, we have included colony-maintained An. arabiensis and An. stephensi mosquitoes fed on sugar solution and patients’ blood in direct membrane feeding assays  that were used as negative and positive controls, respectively. Plasmodium-infected mosquitoes were used as positive controls along with sugar-fed mosquitoes as negative controls in every extraction round -based protocol that targets circumsporzoite protein (CSP) that is expressed on the surface of 67. A multiplex PCR assay that amplifies the cytochrome b gene based on Kent and Norris68 was used for bloodmeal source analysis. We have introduced slight modifications to improve product size separation on gel electrophoresis. The multiplex of cow and dog was separately done from the multiplex of goat and human. The optimized PCR thermal cycler conditions were: 5 min at 95 °C as an initial denaturation followed by 40 cycles of denaturation at 95 °C for 60 s, annealing at 56 °C for 60 s for cow and dog multiplex, and 62 °C for goat and human multiplex, followed by an extension at 72 °C for 60 s, and 1 cycle of the final extension at 72 °C for 7 min.Nucleic acid was extracted from the abdomen of fully engorged mosquitoes for bloodmeal source identification following the same procedure used for the head and thoraces using a cetyltrimethylammonium bromide (CTAB)-based method as described beforeAnopheles morphological identification was done following a recently published protocol that targets the ITS2 gene69. An. stephensi diagnostic amplicon of 438 bp size was expected while a universal amplicon of varying sizes (>600 bp), depending on the length of ITS2 in a particular species, was expected in this multiplex protocol .Study data collection tools (mobile application version 5.20.11) were prepared and managed using REDCap electronic data capture tools hosted at AHRI. CSV files exported from REDCap were analyzed using STATA 17 (StataCorp), RStudio v.2022.12.0.353 , QGIS v.3.22.16 , GraphPad Prism 5.03 (GraphPad Software) and RStudio using packages lme4  and dciferSociodemographic: sex, age, educational level and occupationHousehold characteristics: main materials used for building the household, fuel source, water source and presence of water bodies near the household/dormitory, and presence of livestockIntervention: presence, number, and condition of bed nets, use of bed nets, use of smoke repellents or aerosol mosquito spray, and history of insecticide residual sprayDiagnosis and treatment: malaria test result by RDTs and microscopy, temperature, presence of symptoms and treatment history, and pregnancy statusHuman behavior: travel history, health seeking behavior, sleeping and waking time, and sleeping placeEntomological survey: mosquito collection method and time of collection, mosquito species detected and density, Anopheles species detected and density, abdominal status of mosquitoes detected, type of aquatic habitat near the household/dormitory, and type and characteristics of water sources detected within 100-m radius around the household/dormitoryWe collected the following variables in this study:P. falciparum were subjected to filtering, demultiplexing and allele inference using a Nextflow-based pipeline71. We used cut adapt to demultiplex reads for each locus based on the locus primer sequences , filter reads by length (100 base pairs) and quality . We used dada2 to infer variants and remove chimeras. Reads with a Phred quality score of less than 5 were truncated. The leftmost base was trimmed and trimmed reads of less than 75 base pairs were filtered out. Default values were used for all other parameters. We then aligned alleles to their reference sequence and filtered out reads with low alignment. We masked homopolymers and tandem repeats to avoid false positives.FASTQ files from multiplexed amplicon sequencing of P. falciparum genotypes in diverse loci using Dcifer with default settings70. Pairwise relatedness was only considered between samples where the lower 95% CI of estimated relatedness was greater than 0.1. Point estimates of pairwise relatedness that satisfied this threshold were then binned into low, medium and high relatedness at greater than 0.2, 0.5 and 0.9 respectively. Samples were then clustered based on pairwise relatedness. Drug resistance marker genotypes were extracted from loci of interest. Evidence of pfhrp2 and pfhrp3 deletions were identified from a drop in normalized coverage in amplicons within and surrounding pfhrp2 and pfhrp3. Complexity of infection was estimated by taking the 0.97 quantile (fifth highest number) of observed alleles across loci to minimize the impact of false positives on estimates.Pairwise relatedness analysis was performed on We used standard case–control analyses to examine the association between risk factors and malaria infection. It calculates point estimates and CIs for the OR along with the significance level based on the chi-squared test. Continuous variables were presented as median and IQR. Tests of association between two categorical variables were performed using chi-squared test on contingency tables. Mann–Kendall statistical test was used to test for monotonic (increasing or decreasing) trends of malaria cases using the secondary data obtained from the private and public health facilities at the city and DDU.2 area), clustering of prevalence data was assessed in the city only. The prevalence of malaria by RDT and/or microscopy was calculated for each household. Global and local Moran’s I calculations were used to estimate the level of spatial autocorrelation within household prevalence data. The statistical strength of association for global Moran’s I was calculated using Monte-Carlo methods based on 9,999 times permutations of the prevalence data. The Euclidean distance from the river to every site where adult or larval An. stephensi were located were calculated in meters.As the dormitories within the university study site were located within a small area  to account for intra-class correlation72. The covariates included for the multi-level logistic regression analysis with random effect are listed in detail in Supplementary Table P value lower than 0.3 within these models  and the Bayes information criteria (BIC) values, with the top model being the one with the lowest AIC value73. Variable selection was repeated for three different response variables: model 1 with response RDT/microscopy, model 2 with response RDT/microscopy/qPCR, and, finally, model 3 with response qPCR. As a result, only five of the 12 factors assessed for individual and household characteristics  were included for the final model  enabled us to control for unknown variations by including them as random effects in the model. In fact, individuals living in the same household may share exposures that can determine similarities in malaria transmission as well as in the larger setting (city versus university).After model selection with several model outcomes and distribution  is vector of p explanatory variables or covariates measured on the i individual and on the j household (cluster), β is vector of fixed regression coefficients or parameters and uj is a random effect varying over household and case control.Let Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-023-02641-9.Supplementary InformationSupplementary information.Reporting SummarySource Data Tables 1–3This file contains the source dataset for the three tables that are part of the main text of the paper. It contains three tabs that are labeled: Source_Data_Table_1, Source_Data_Table_2 and Source_Data_Table_3.Source Data Fig. 1An. stephensi distribution (native and invasive range) and detection of Plasmodium infection for Fig. 1a and geographic location of the study area  for Fig. 1b source data.Global Source Data Fig. 2Source data obtained from the national DHIS2 repository for Dire Dawa between January 2013 and May 2022 and data collected during the study period from the study participants infection status  in separate Excel sheet for Fig. 2a–e.Source Data Fig. 3Anopheles mosquito presence  and overlap of Plasmodium infection detected in study participants and An. stephensi in the respective households  and pairwise genetic relatedness data  and their clustering .Source data on the geographic location of infections detected in the study households , Source Data Extended Data Table 1Source data collected from 34 private and public health facilities located in Dire Dawa for the period between January 2019 and May 2022.Source Data Extended Data Table 2Source data on symptom status (axillary temperature ≥37.5 °C) by study site and participant category.Source Data Extended Data Table 3Sociodemographic, intervention utilization and malaria predisposing factors related to malaria infection source data.Source Data Extended Data Table 4Infection status  source data.Source Data Extended Data Table 5Source data on infection status .Source Data Extended Data Table 6Source data related to adult and larvae mosquito surveys. Individual adult mosquito data including date, method and place of collection, and abdominal status included.Source Data Extended Data Table 7Source data related to adult and larvae mosquito surveys. Individual adult mosquito data including date, method and place of collection, and abdominal status included.Source Data Extended Data Table 8Source data related to adult and larvae mosquito surveys. Individual adult mosquito data including date, method and place of collection, and abdominal status included.Source Data Extended Data Table 9Source data collected during the study period from the study participants on infection status (as measured by RDTs and microscopy), sociodemographic status, intervention utilization and malaria predisposing factors related to parasite lineages.


In Cameroon, malaria contributes significantly to the morbidity and mortality of children under 5 years old. In order to encourage adequate treatment-seeking in health facilities, user fee exemptions for malaria treatment have been instituted. However, many children are still brought to health facilities in the late stage of severe malaria. This study sought to determine the factors affecting the hospital treatment-seeking time of guardians of children under 5 years within the context of this user fee exemption.This was a cross-sectional study conducted at three randomly selected health facilities of the Buea Health District. A pre-tested questionnaire was used to collect data on the treatment-seeking behaviour and time of guardians, as well as potential predictors of this time. Hospital treatment sought after 24 h of noticing symptoms was denoted as delayed. Continuous variables were described using medians while categorical variables were described using percentages. A multivariate regression analysis was used to determine the factors affecting malaria treatment-seeking time of guardians. All statistical tests were done at a 95% confidence interval.Most of the guardians made use of pre-hospital treatments, with self-medication being practiced by 39.7% (95% CI 35.1–44.3%) of them. A total of 193 (49.5%) guardians delayed seeking treatment at health facilities. Reasons for delay included financial constraints and watchful waiting at home, during which guardians waited and hoped their child could get better without requiring medicines. Guardians with estimated monthly household incomes denoted as low/middle were significantly more likely  to delay seeking hospital treatment. The occupation of guardians was another significant determinant of treatment-seeking time . Also, guardians with tertiary education were less likely  to delay seeking hospital treatment.This study suggests that despite user fee exemption, other factors such as educational and income levels of guardians affect malaria treatment-seeking time for children aged under five. Therefore, these factors should be considered when enacting policies aimed at increasing access of children to health facilities. Despite recent reductions in its burden, malaria in sub-Saharan Africa (SSA) continues to be a major public health problem in children . In 2018The clinical presentation of malaria in children usually ranges from the asymptomatic carriage of parasites, to a febrile disease that may evolve into a severe life-threatening illness . InfecteDelay in seeking treatment for malaria is described as when treatment is sought more than 24 h after recognition of symptoms . To avoiTreatment-seeking time for children is determined by the treatment-seeking behaviour of their guardians, which is in turn determined by several social factors inherent to guardians , 11, 13.In an effort to reduce the child mortality rate, the government of Cameroon has adopted several policies to reduce the burden of malaria—a major cause of child mortality in Cameroon . One of Since the implementation of the malaria user fee exemption policy in 2011, there has been an increase in malaria-related consultations for children aged 5 years and below , 17. DesThe aim of this study was to determine the factors affecting the seeking of hospital-based treatment by guardians of children under 5 years with malaria, in the context of a user fee exemption, in the Buea Health District.The Buea Health District (BHD) is located in the southwest region of Cameroon. This region has a total of 18 health districts, with that of Buea situated on the eastern slopes of Mount Cameroon, with a total surface area of 870 sq km. Buea has an equatorial climate with two seasons: the dry and wet seasons. Malaria transmission is most prominent during the wet season. In 2017, the BHD had an estimated population of 169,745 inhabitants; children under 5 years made up 13.6% of the population. The BHD is made up of seven health areas: Buea road, Muea, Molyko, Bova, Buea Town, Bokwaongo and Tole . Figure This study was carried out in three health facilities of BHD, selected through a stratified probabilistic sampling technique. The district has public, private and confessional health facilities, each having the capacity to diagnose and treat malaria in children. This study ran from January 2020 to July 2020, making a total of seven months.This was a hospital-based, cross-sectional-study. The target population was the guardians of children under 5 years old with malaria, presenting at health facilities in BHD during the time of the study. Included were consenting adult guardians of children aged 5 years and under, with a diagnosis of malaria. Excluded were guardians of children who had malaria and another chronic or acute illness, guardians having more than one sick child at the hospital, and guardians with children referred from other health facilities.With the prevalence of malaria in children estimated at 27.4% in the southwest region of Cameroon , the tarCochran formula:Substituting the values, A multistage sampling technique was used to select three health facilities in which the study was conducted. All the health facilities of the district were first divided into seven clusters, each representing a health area. Three health areas  were then selected by balloting. Subsequently, one health facility was selected by a second stage of balloting, from each of the selected clusters. The study was then conducted in these three selected health facilities, that is, the Muea Sub-divisional Hospital from the Muea health area, Solidarity Hospital from the Molyko health area, and the Buea Regional Hospital from the Buea road health area.Table A consecutive sampling technique was employed to recruit participants from the three selected health facilities.Data were collected with the help of a pre-tested structured questionnaire. Pre-testing was carried out on 27 participants from Molyko Health Centre, located in the study health district. During pre-testing, participants were asked to remark on their comprehension of the questions. After the process, some questions were rephrased in order to ease comprehension. This questionnaire  carried out daily onsite supervision of data collection, and checked all answered questionnaires for consistency. Whenever inconsistencies were noted, the PI made sure the right information was obtained by interviewing the participant again. Data collectors received a financial motivation during training and as data collectors.The pre-tested structured questionnaire was administered to consenting guardians of children under 5 years with malaria, who fulfilled the inclusion criteria. In a situation where more than one guardian existed, priority was given to the guardian present at the beginning of the child’s illness. The questionnaires were administered in English or Pidgin English, depending on the language the respondent best understood.Questionnaires were checked daily for completeness and consistency by the PI. Code numbers were used to identify participants, and data were entered into Epi Info version 7.0 before being exported to Statistical Package for Social Sciences (SPSS) version 20.0 for analysis.p-values < 0.2 were considered candidates for the multivariate logistic model. All statistical tests were done at a 95% confidence interval, and p-values < 0.05 were considered statistically significant.Continuous variables were first checked for normality using the Kolmogorov–Smirnov test, and normality transformations attempted, before being described using medians and interquartile ranges. Correlation techniques were used to evaluate relationships between continuous variables. Categorical variables were expressed using percentages, contingency tables, pie charts, and bar charts. The Pearson Chi-squared test was used to assess statistical significance of the categorical variables after making sure the smallest expected value exceeded five. In cases where this condition was not fulfilled, the likelihood ratio was considered. A multivariate logistic regression analysis was then carried out using treatment-seeking time (classified as delayed or early) as the outcome variable. The procedure for developing the multivariate model was as follows: a bivariate logistic analysis was first conducted for each of the independent variables, and then all those variables with A total of 402 guardians were recruited, of which 12 were excluded as their children were found to have other infections and chronic co-morbidities. A total of 390 guardians were finally included. Out of the 390 participants, 270 (69.2%) were female. The median age of the participants was 32 years, with an interquartile range of 7.3. As shown on Table Table p = 0.000) of participants employed some other form of treatment before seeking hospital-based care. As shown on Table With respect to treatment-seeking behaviour, 242 guardians representing 62.1% . Table A total of 193 [ (49.5%); 95% CI 44.4–54.6%; Statistical relationships between the socio-demographic characteristics of guardians and the malaria treatment-seeking time for their children are depicted on Table p = 0.000) was noted when these two variables were compared.Additionally, treatment-seeking time of guardians for their sick children was found to increase with their household size. A positive and moderate strength correlation  was found between the number of children a guardian has under his/her care and their treatment-seeking time for malaria.Furthermore, a positive, moderate strength correlation . Guardians with a household size of ≤ 5 persons were found to be less likely to delay seeking care for their children  . Guardians with at most five children under their care were less likely to seek treatment late   less likely to delay in seeking hospital treatment for their children with malaria. Additionally, guardians who had attained tertiary education were found to be 0.315 times  less likely to delay in seeking hospital treatment for their children with malaria. Guardians with an estimated household income level of low/middle were 3.794 times more likely to delay in seeking treatment compared to those with income estimated as high.Table This study sought to determine the factors favouring delay by guardians seeking hospital-based treatment for their children with malaria, despite the existence of a user fee exemption. It provides baseline information that could be used to strengthen policies geared towards access to health facilities.A majority (62.1%) of guardians in the study administered some form of treatment to their children before seeking hospital-based care. This is similar to findings from other studies carried out in Nigeria, Laos, Ethiopia, and Cameroon, where guardians were noted to use homemade remedies, drugs from roadside vendors and/or herbs before seeking hospital-based treatment when their child did not get better –27. EvenSimilar to a study in the general population of Cameroon depicting high levels of self-medication in adults , this stAbout half (50.5%) of guardians in the study brought their children to hospital-based care for malaria within 24 h of noticing symptoms of illness. This is in contrast to other studies in Southern Ghana and Eastern Nigeria that reported 11% and 22%, respectively, of guardians seeking hospital treatment within 24 h , 30. ThiSeeking hospital treatment after 24 h was noted in 49.5% of the participants. The main reasons mentioned for this delay were financial constraints, followed by the need to observe the sick child for a while, hoping he/she got better. This latter reason was mostly given by those who administered some non-hospital-based treatment to their children. A similar study in Nigeria also mentioned watchful waiting as a major reason why guardians delayed seeking hospital treatment for their children with malaria . In the p-values less than 0.2 were used to create a multivariate logistic regression model, adjusting for potential confounders. It was noted that the educational level of guardians was a significant predictor of the time taken to seek care at a health facility. Guardians with a tertiary level of education were less likely to delay in seeking treatment for their children. This conforms to similar studies in Nigeria [Factors affecting malaria treatment-seeking time were classified as early (≤ 24 h) or delayed (≥ 25 h), in the context of a user fee exemption. After a bivariate analysis was run, predictors with  Nigeria , 32. ThiThe monthly household income proved to be a predictor of malaria treatment-seeking time by guardians. Guardians with an estimated monthly income above 100,000 F CFA were less like to delay seeking treatment for their children. Chukwuocha et al. . This coThis study showed that most (73.6%) guardians who sought early treatment for their children were female. Though gender was not a statistically significant determinant of early treatment-seeking, this trend could be that most caregivers are female and are, therefore, more predisposed to accompany their children to the hospital.The time taken by guardians to seek hospital treatment was an estimate and could have been affected by a recall bias. The study did not take into consideration the ability of guardians to recognize malaria and its severity in their children. This could have played a role in affecting treatment-seeking behaviour and time to seek hospital treatment. However, to the best of our knowledge, this study is the first in Cameroon to determine the factors affecting the time taken by guardians to seek malaria treatment in health facilities for children under 5 years, in the context of a user fee exemption.Despite the current policy on free malaria treatments for children under 5 years old in health facilities, most guardians of children with malaria remain reluctant to seek adequate treatment in health facilities. This delay is found to be determined by social and economic factors inherent in guardians. The current user fee exemption policy is therefore not sufficient to stimulate considerable malaria treatment-seeking in health facilities. There is a need to ameliorate the social and economic welfare of the population in order to decrease delay in seeking malaria treatment from health facilities.


Anopheles mosquito-borne disease caused by five different eukaryotic protozoa parasites. Amoebiasis is a parasitic infection caused by Entamoeba histolytica. Both diseases are widespread in Liberia. A returning traveler was diagnosed and treated for malaria, and 20 days later, an amoebic liver abscess was discovered, meaning that the malaria infection masked the amoebic infection, which emphasizes the importance of a complete examination of returning travelers, especially for those returning from Sub-Saharan Africa, where coinfections are more common. Herein, we propose that the relationship between Malaria and amoebic liver abscesses should be explored by researching the effects of malaria on ferritin levels and the immune components in the liver and whether it helps the emergence of hepatic amoebic abscesses or not.Malaria is an infectious  Anopheles mosquito-borne disease caused by five different eukaryotic protozoa parasites of the genus Plasmodia, one of which is Plasmodium falciparum, and it is responsible for 90% of global deaths from malaria [Malaria is an infectious  malaria .The infection is usually associated with a travel history to infected countries, of which is Liberia, where the entire population is at high risk of catching the disease . MalariaEntamoeba histolytica and is transmitted via the fecal-oral route. The infection varies in severity from asymptomatic cases to complicated ones that may include liver abscesses [On the other hand, amoebiasis is a parasitic infection caused by bscesses .Amoebiasis remains a global problem that is especially present in developing countries such as Syria and Liberia, where it mainly spreads through contaminated water supplies  Around 5Like malaria, amoebiasis is commonly associated with a travel history to infected regions including Liberia. Most patients who develop liver abscesses also develop fever, right hypochondriac pain, and digestive symptoms. The symptoms usually manifest 2–4 weeks after infection .Travelers to infected regions are advised to follow precautionary measures. For preventing malaria, protection from the mosquito bite is usually achieved by applying insect repellents on naked skin such as diethyltoluamide (DEET) and using insecticide-treated nets or clothing . InfectiFor preventing amoebiasis, we wash vegetables before consumption, maintain good hygiene, drink clean water, and avoid unhygienically prepared beverages , 4.Here, we present a case combining these two concurrent diseases in 20 days.A 65-year-old, Middle-Eastern male was admitted with a high fever, chills, and vomiting. The symptoms had begun while he was in Liberia, and he came back to Syria as soon as his fever worsened. The patient had no other clinical symptoms and no medical history of other illnesses or drug consumption.Plasmodium falciparum, and the test was run twice in two different labs. The patient was treated accordingly with artemisinin, 4 tablets per dose twice a day for three days (each tablet contains 20 mg artemether and 120 mg lumefantrine).A blood test was performed, and a blood smear was examined under the light microscope . He was Twenty days after malaria treatment, the patient continued to suffer from fever, shivers, and general fatigue. Clinical presentation included pale skin, mucosa, and right hypochondriac pain without jaundice. A blood test with further lab examinations was performed .Plasmodium falciparum organisms, while a computed tomography (CT) scan and abdominal echography were also scheduled.Blood smears were run for three consecutive days to detect The smears returned negative, and the CT scan revealed liver abscesses Figures  and 2; tEchinococcus granulosus antibodies and Entamoeba histolytica antibodies, and the test rendered positive results for Entamoeba histolytica.A blood test was performed searching for The abscess was drained as a therapeutic and diagnostic measure, followed by a course of 500 mg intravenous metronidazole every 8 hours for 14 days (Flagyl), to complete the treatment. After improvement, the patient was discharged and instructed to contact his physician if any of his symptoms relapsed, which did not occur.Malaria is an endemic infection in Liberia, making it one of the most important differential diagnoses for our patient's symptoms on his first visit, meaning it should be ruled out first, especially with the presence of thrombocytopenia , 8. OtheE. histolytica is most frequently symptomatic, Entamoeba dispar is largely considered nonpathogenic, and Entamoeba moshkovskii has unclear pathogenicity. E. histolytica's invasiveness arises from its proton and tissue lysing properties [Three species of intestinal amoeba exist. operties . Risk faoperties , 4.P. falciparum and E. histolytica was previously recorded, predominantly in Sub-Saharan Africa [Entamoeba histolytica, which uses iron in ferritin to grow and multiply [Albeit coinfection of n Africa . It remamultiply , 12.Plasmodium falciparum's role in causing the failure of dendritic cells' function [Plasmodium falciparum parasites, which prevent harmful immunopathology and suppress inflammatory responses. One study even suggests that the low levels of plasmodium can downregulate signals needed for antigen-presenting cell (APC) activation [, Entamoeba histolytica has a surface molecule named lipopeptidophosphoglycan (LPPG) which binds to TLR2 and TLR4 on the surface of dendritic cells. This shows that LPPG activates APC and therefore is essential in regulating the inflammatory response in amoebiasis [Another connection might be through function . Liver dtivation . On the oebiasis . This miE. histolytica being the third most detected intestinal pathogen in travelers returning from low-income countries presenting with infectious gastrointestinal disease [No further tests were run after the malaria infection was confirmed. However, had the admitting doctor screened for other infectious diseases endemic to Liberia, the amoebic infection could have been noticed earlier, leading to a better treatment plan and prognosis, especially with  disease .Here, we stress the importance of a complete examination, and we suggest a cost-benefit study to decide whether symptomatic returning travelers should be screened for the most prevalent endemic infections even if a diagnosis was reached.Physical examination should always be thorough, and all signs and symptoms should be accounted for even if a lab diagnosis was made. While treating a returning traveler suffering from an infectious disease, it is imperative to check for other infectious diseases endemic to the area, especially for travelers returning from Sub-Saharan Africa, where coinfections are more common.Furthermore, we recommend a wait-and-see approach besides precise examinations as the second infection might manifest itself after treatment of the first.Finally, we propose that the relationship between malaria and amoebic liver abscesses be explored by researching the effects of malaria on ferritin levels and the immune components in the liver and whether it makes the host susceptible to amoebic liver abscesses.


There is conflicting literature regarding the long-term effect of anthracycline treatment on arterial stiffness. This study assessed local arterial stiffness using ultrafast ultrasound imaging (UUI) in anthracycline treated childhood cancer survivors, at rest and during exercise.2) and 21 healthy controls (mean age 26.00 ± 8.91 years) were included. Participants completed a demographic survey, fasting bloodwork for cardiovascular biomarkers, and performed a submaximal exercise test on a semi-supine bicycle. Pulse wave velocity (PWV) was measured in the left common carotid artery by direct pulse wave imaging using UUI at rest and submaximal exercise. Both PWV at the systolic foot (PWV-SF) and dicrotic notch (PWV-DN) were measured. Central  PWV was obtained by applanation tonometry. Carotid measurements were taken by conventional ultrasound. Measures were compared using two-tailed Students t-test or Chi-squared test, as appropriate.20 childhood cancer survivors (mean age 21.02 ± 9.45 years) treated with anthracyclines (mean cumulative dose 200.7 ± 126.80 mg/mp > 0.05) between childhood cancer survivors and healthy controls in demographic parameters , blood biomarkers , cardiovascular parameters  or PWV measured by UUI at rest or at exercise. There was also no difference in the cardiovascular adaptation between rest and exercise in the two groups (p > 0.05). Multivariate analysis revealed age (p = 0.024) and LDL-c (p = 0.019) to be significant correlates of PWV-SF in childhood cancer survivors, in line with previously published data.There was no statistically significant difference (We did not identify a significant impact of anthracycline treatment in young survivors of childhood cancer on local arterial stiffness in the left common carotid artery as measured by UUI. Every year, approximately 1,000 Canadian children are diagnosed with cancer , 3. WhilArterial stiffness is a biomarker of vascular health and a predictor of cardiovascular events in adults . There aThe aim of the current study was to use UUI to measure local PWV in the left carotid artery at rest and during an exercise in childhood cancer survivors treated with anthracyclines and compare the findings to healthy controls. The hypothesis was that local arterial stiffness in CCS would be increased at rest and demonstrate an abnormal response to exercise. This is the first study evaluating arterial stiffness in childhood cancer survivors using UUI.This study included 20 childhood cancer survivors (CCS) and 21 healthy volunteers (HV). Participants were recruited by the department of echocardiography at the Hospital for Sick Children, Toronto. The study was approved by the research ethics committee (REB #1000060834) and all participants gave informed consent. The CCS group inclusion criteria were: age <18 years at time of cancer diagnosis, currently in remission from cancer, and treated with anthracyclines with the final dose completed at least 3 years before inclusion. No participants had significant congenital heart disease or cardiomyopathy. Healthy volunteers were age-matched and had no history of cardiovascular disease.Demographic parameters including age, sex, weight, and height were obtained. Blood pressure  measurements, conventional ultrasound acquisitions of the left common carotid artery (CCA), UUI acquisitions of the left CCA, and carotid-femoral PWV measurements by applanation tonometry on the right were taken at baseline  and at submaximal exertion during an exercise test, at 75 W see . OfflineParticipants who consented in advance to blood work fasted overnight. Measurements included total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-c), fasting glucose, HbA1c, insulin, and high sensitivity C-Reactive protein (hs-CRP).A Dinamap® ProCare oscillometric device  was used to measure systolic, diastolic, and mean arterial pressure and simultaneous heart rate in triplicate. Established guidelines for BP measurement were followed with measurements in the right arm using an appropriate cuff size with the arm at heart level after 5 min of rest with the participant seated . OscilloA modified Bruce Protocol was used on a semi-supine bicycle ergometer (Ergoline USA), such that patients were sitting and leaning back at about a 120° angle with their neck stable and exposed  equipped with a 11l linear transducer. Carotid IMT and carotid artery distensibility were assessed see . The acqCentral (aortic) PWV was obtained by applanation tonometry . It was measured as the transit time of the pulse wave between the right carotid and right femoral artery . The traThe left CCA was assessed by UUI using a programmable ultrafast ultrasound system  equipped with a GE 11l linear transducer . The probe was placed in a longitudinal orientation with its distal end about 2 cm proximal to the carotid bifurcation of the CCA. The length of the arterial segment was about 40 mm. After the probe was set up and the sequence was initiated, the UUI acquisition was triggered automatically by the peak of the R wave on ECG. Each acquisition lasted 709 ms and covered at least one cardiac cycle. Each measurement was taken at least twice to ensure reproducibility. Raw ultrasound data were beamformed into In-phase/Quadrature datasets (IQ), and processed using MATLAB R2019a . Offline analysis of PWV on MATLAB used well verified post-processing methods, as follows. The arterial walls were first localized on the IQ images. The anterior and posterior CCA were manually delineated and approximated by a second order polynomial line. The radial velocity of the wall was then derived by a frame-to-frame IQ auto-correlation (Loupas estimator) , 21. Thet-test, or Chi-squared test, as appropriate. Non-parametric measures were compared with the Mann–Whitney U test. Correlations were analyzed using Pearson correlation analysis. Multiple regression was performed on independent variables showing a linear correlation to PWV to identify significant determinants of PWV. All statistical analyses were performed using MedCalc . A p-value <0.05 was considered to indicate statistical significance. Bonferroni correction was applied where suitable.Results are presented as mean ± standard deviation (SD). Normality was defined by the Shapiro-Wilk test. Parametric measures were compared using unpaired two-tailed or paired Students p = 0.10). 2. Only six CCS participants (30%) had an anthracycline dose exceeding 250 mg/m2. None of the participants had significant known cardiac abnormalities or familial cardiomyopathies, pre-existing cardiovascular conditions, kidney disease, or type 1 diabetes. They were all non-smokers. Five participants (25%) received radiation therapy. No participants were receiving or had previously received cardiac medications, other than one participant who received Dexrazoxane. This study included 20 CCS treated with anthracyclines, aged 20.9 ± 9.2 years, and 21 HV, aged 26 ± 8.91 years (p < 0.001) and HV group (p < 0.01). However, there was no difference in PWV-SF between CCS and HV groups at rest (p = 0.56) or during exercise (p = 0.46).p = 0.02), but not in the HV group (p = 0.07). There was no difference in PWV-DN between CCS and HV groups at rest (p = 0.50) or during exercise (p = 0.76).p = 0.007), BMI (p = 0.017), BP , IMT (p = 0.039), and systolic and diastolic carotid diameter (p < 0.001 for both).p = 0.020) and B-index (p = 0.020).PWV-DN in the CCS group had no significant correlations. In the HV group, PWV-DN correlated to minimum (diastolic) carotid diameter (p = 0.024) and LDL-c (p = 0.019) remained significantly correlated to PWV-SF. In the HV group, age (p = 0.048), SBP and DBP carotid diameter  remained significantly correlated to PWV-SF.Multiple regression was conducted for the correlated factors described above at rest. In the CCS group, age (p = 0.016), height (p = 0.025), IMT (p = 0.001), and min (diastolic) carotid diameter (p = 0.015). PWV-SF in the CCS group additionally correlated with B-index (p = 0.031), and LDLc (p = 0.031).p = 0.010), height (p = 0.010) and diastolic carotid diameter (p = 0.015).During exercise, PWV-DN correlated with max (systolic) carotid diameter in both groups. PWV-DN in the HV group additionally correlated with weight (p < 0.01), but not in other parameters.Regarding the intra-observer variability, 15 pulse waves  obtained by UUI were re-analyzed and are depicted in Regarding the inter-observer variability, 34 pulse waves were re-analyzed , and are depicted in In this study, UUI measurement of local left carotid PWV was used to assess arterial stiffness in CCS treated with anthracyclines both at rest and at the end of submaximal exercise study. Overall, we observed no significant differences in PWV measurements between CCS and healthy controls either at rest or after exercise. However, the correlation between PWV and certain factors did differ between the two groups. Importantly, PWV-SF in CCS was not correlated with BMI, IMT, BP, and carotid diameter, all of which have been shown to be correlated to the PWV of healthy individuals in previous studies –27. ThisWe estimated both global (by tonometry) and local (by UUI) PWV. We observed no significant difference between local PWV-SF measurements of the left common carotid artery and global carotid-femoral tonometry measurements in either group. Based on the hypothesized mechanism of vascular injury by anthracyclines leading to endothelial dysfunction, there is no evidence that any particular arteries will be targeted, but it is reasonable to hypothesize that arteries with high dependence on endothelial control may show more dysfunction . In factThis study analyzed the vascular effects of anthracyclines exclusively in the chronic setting, with an average of 13.71 ± 7.63 years since therapy completion. Cardiotoxicity due to anthracyclines is typically classified as acute/early (effects during treatment), subacute (within a year of treatment), or chronic (over a year after exposure) . Signifi2 is related to a significantly increased risk of cardiac disease  having a cumulative dose exceeding 250 mg/m2. As such, it is possible that the threshold for high risk of cardiotoxicity is translated to vascular toxicity.The cardiac toxicity of anthracyclines is much better studied and understood compared to vascular toxicity. While the exact mechanism continues to be studied, it is thought that the mechanism of cardiac toxicity involves the activation of cell death pathways and inhibition of mitochondrial biogenesis . Specifi disease . The ave2. Cifra et al. demonstrated dynamic myocardial response to exercise in a similar CCS population, and demonstrated a similar response in HR and BP  as in this current study  were taken during stage 3 of the Bruce protocol (75 Watts) for all patients. This stage was at different levels of personal exertion for each participant. Finally, this was a cross-sectional study which evaluated participants at a single point in time. As mentioned in the discussion, longer-term effects of anthracyclines and long-term follow up are not known for vascular toxicity. Ideally, participants could have been tracked multiple times over several years to determine the trajectory of vascular toxicity.2. The use of UUI for studying the impact of anthracyclines in arterial stiffness is an interesting and emerging field of research. As such, additional studies with large cohorts are needed to confirm our observations.Overall, there were no significant differences found between left common carotid stiffness measured by UUI in HV and CCS populations in our study. This continued to be true under cardiovascular stress, induced by an exercise trial. While there is a documented risk of cardiotoxicity with anthracycline treatment, this study does not provide evidence that anthracyclines alter vascular properties in a CCS population treated with an average dose <250 mg/m


Plasmodium vivax malaria that resulted in severe disease, a magnitude rarely seen in previous literature. Our patient was a 35-year-old healthy woman who presented to the emergency department with abdominal pain, nausea, vomiting, and fever. Further workup revealed severe thrombocytopenia with prolonged prothrombin (PT) and partial thromboplastin time (PTT). An initial thick smear failed to detect any Plasmodium species, but a thin smear revealed P. vivax. The patient's hospital stay was complicated by septic shock requiring intensive care unit (ICU) admission. This unique case represents P. vivax as the causative agent of severe malaria even in healthy, immunocompetent patients. Malaria has various causative agents that can have a spectrum of disease manifestations, some potentially fatal. Various species have been established as etiologies of malaria, though our understanding of the severity of various species is changing. We present a unique case of P. falciparum, P. vivax, P. ovale, and P. malaria [Malaria is a potentially fatal disease transmitted from the bite of a female Anopheles spp mosquito. The Anopheles mosquito’s saliva contains Plasmodium, which is a single-celled eukaryotic organism. Despite advances in preventive and diagnostic measures, the World Health Organization (WHO) reported an estimated 247 million cases and 619 thousand deaths in 2022 alone [ malaria .The clinical course of malaria can be varied. Following the initial infective bite, 7-30 days often pass before the onset of symptoms, which is known as the incubation period. Initial symptoms of uncomplicated disease are typically nonspecific, often including fevers, chills, malaise/fatigue, tachycardia, abdominal pain, and/or diarrhea. Fevers may initially occur at irregular intervals, but later in the course of the infection, classic febrile paroxysms may ensue (depending on the species) . On physThe gold standard of diagnosis is a Giemsa-stained blood smear . Thick sP. falciparum and is a medical emergency, with mortality approaching 100% without appropriate treatment. Studies have shown that although P. vivax has a higher incidence, P. falciparum is associated with significantly higher rates of hospitalization and death [Severe malaria is classically associated with nd death . Young and death . Charactnd death . The WHO recommends combined artemisinin therapy for treating severe malaria, though the regimen varies based on regional sensitivity patterns. For patients who meet the criteria for severe malaria, the first step is to initiate intravenous (IV) or intramuscular (IM) artesunate . After tP. vivax rarely presents with severe malaria of this magnitude. The patient presented herein was found to have septic shock and disseminated intravascular coagulopathy (DIC), which are uncommon complications for this etiology of malaria. Additionally, an initial thin smear was sent as part of the routine hematologic analysis, but given its poor sensitivity for malaria, it failed to detect any organisms. This case displays the importance of awareness of severe P. vivax malaria and how timely diagnosis can lead to potentially life-saving treatment.This case is unique as A 35-year-old Hispanic female with no past medical history presented to the emergency department in New York City with a chief complaint of aching upper abdominal pain for four days. The abdominal pain was initially abrupt but progressively worsened, and it was associated with fatigue, fever, chills, nausea, vomiting, and “black” stools. The patient believed that her symptoms stemmed from the consumption of “wet rice” earlier in the week. In a further interview, it was revealed that about 1 month prior to the presentation, the patient traveled through the jungle as she migrated from Ecuador to the United States.P. vivax as the causative organism of infection.On physical examination, the patient was in acute distress and had upper abdominal tenderness to palpation, without any guarding or rebound. The patient had a fever of 101.3°F and a blood pressure of 89/49 mmHg. The blood pressure remained hypotensive despite a 4L fluid bolus, prompting critical care admission for septic shock of unknown etiology. Blood cultures, stool pathogens, viral panels, and peripheral blood smear were all ordered and the patient was initiated on empiric broad-spectrum antibiotics. CT abdomen and pelvis did not reveal any acute abdominopelvic pathology, and routine blood work revealed severe thrombocytopenia with prolonged PT and PTT, suggestive of disseminated intravascular coagulopathy (DIC)  admission for septic shock and DIC which is atypical given her age and medical history. Our case report serves to raise awareness among healthcare providers of the possible severity of P. vivax and the importance of timely diagnosis and treatment.This case of a 35-year-old female with