Vincarostine A, a novel anti-malarial trimeric monoterpenoid indole alkaloid from Catharanthus roseus.

A novel trimeric monoterpenoid indole alkaloid, vincarostine A (1) consisting of an aspidosperma-iboga-aspidosperma type skeleton, was isolated from the whole plant of Catharanthus roseus. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and CD spectrum. Vincarostine A (1) showed anti-malarial activity.

Persistence of Anti-SE36 Antibodies Induced by the Malaria Vaccine Candidate BK-SE36/CpG in 5-10-Year-Old Burkinabe Children Naturally Exposed to Malaria.

Information on the dynamics and decline/persistence of antibody titres is important in vaccine development. A recent vaccine trial in malaria-exposed, healthy African adults and children living in a malaria hyperendemic and seasonal area (Ouagadougou, Burkina Faso) was the first study in which BK-SE36/CpG was administered to different age groups. In 5- to 10-year-old children, the risk of malaria infection was markedly lower in the BK-SE36/CpG arm compared to the control arm. We report here data on antibody titres measured in this age-group after the high malaria transmission season of 2021 (three years after the first vaccine dose was administered). At Year 3, 83% of children had detectable anti-SE36 total IgG antibodies. Geometric mean antibody titres and the proportion of children with detectable anti-SE36 antibodies were markedly higher in the BK-SE36/CpG arm than the control (rabies) arm. The information obtained in this study will guide investigators on future vaccine/booster schedules for this promising blood-stage malaria vaccine candidate.

Computed tomography-guided percutaneous needle biopsy for middle mediastinal tumors with retroaortic paravertebral approach: A case report.

A 74-year-old man was referred to our hospital for a close examination of a mediastinal mass. Contrast-enhanced CT showed a middle mediastinal tumor. We planned to perform a CT-guided percutaneous needle biopsy of the tumor using a retroaortic paravertebral approach to avoid transpulmonary puncture. A coaxial blunt-tip needle with a side hole was used to create space in the mediastinum and avoid azygos vein injury. After injecting normal saline, a blunt-tip needle was advanced through the space between the aorta and the vertebral body to the anterior surface of the tumor, and tissue was obtained. The patient was discharged the following day with no complications. For percutaneous middle mediastinal tumor biopsy, the retroaortic paravertebral approach may be a safe, effective route.

Ceramicines U-Z from Chisocheton ceramicus and structure-antimalarial activity relationship study.

Ceramicines are a series of limonoids which were isolated from the barks of Malaysian Chisocheton ceramicus (Meliaceae), and were known to show various biological activity. Six new limonoids, ceramicines U-Z (1-6), with a cyclopentanone[α]phenanthrene ring system with a ß-furyl ring at C-17 were isolated from the barks of C. ceramicus. Their structures were determined on the basis of the 1D and 2D NMR analyses, and their absolute configurations were investigated by CD spectroscopy. Ceramicine W (3) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC50 value of 1.2 µM. In addition, the structure-antimalarial activity relationship (SAR) of the ceramicines was investigated to identify substituent patterns that may enhance activity. It appears that ring B and the functional groups in the vicinity of rings B and C are critical for the antimalarial activity of the ceramicines. In particular, bulky ester substituents with equatorial orientation at C-7 and C-12 greatly increase the antimalarial activity.

Immune tolerance caused by repeated P. falciparum infection against SE36 malaria vaccine candidate antigen and the resulting limited polymorphism.

The call for second generation malaria vaccines needs not only the identification of novel candidate antigens or adjuvants but also a better understanding of immune responses and the underlying protective processes. Plasmodium parasites have evolved a range of strategies to manipulate the host immune system to guarantee survival and establish parasitism. These immune evasion strategies hamper efforts to develop effective malaria vaccines. In the case of a malaria vaccine targeting the N-terminal domain of P. falciparum serine repeat antigen 5 (SE36), now in clinical trials, we observed reduced responsiveness (lowered immunogenicity) which may be attributed to immune tolerance/immune suppression. Here, immunogenicity data and insights into the immune responses to SE36 antigen from epidemiological studies and clinical trials are summarized. Documenting these observations is important to help identify gaps for SE36 continued development and engender hope that highly effective blood-stage/multi-stage vaccines can be achieved.

Safety and immunogenicity of BK-SE36/CpG malaria vaccine in healthy Burkinabe adults and children: a phase 1b randomised, controlled, double-blinded, age de-escalation trial.

Background: BK-SE36/CpG is a recombinant blood-stage malaria vaccine candidate based on the N-terminal Plasmodium falciparum serine repeat antigen5 (SE36), adsorbed to aluminium hydroxide gel and reconstituted, prior to administration, with synthetic oligodeoxynucleotides bearing CpG motifs. In healthy Japanese adult males, BK-SE36/CpG was well tolerated. This study assessed its safety and immunogenicity in healthy malaria-exposed African adults and children. Methods: A double-blind, randomised, controlled, age de-escalating clinical trial was conducted in an urban area of Ouagadougou, Burkina Faso. Healthy participants (n=135) aged 21-45 years (Cohort 1), 5-10 years (Cohort 2) and 12-24 months (Cohort 3) were randomised to receive three vaccine doses (Day 0, 28 and 112) of BK-SE36/CpG or rabies vaccine by intramuscular injection. Results: One hundred thirty-four of 135 (99.2%) subjects received all three scheduled vaccine doses. Vaccinations were well tolerated with no related Grade 3 (severe) adverse events (AEs). Pain/limitation of limb movement, headache in adults and fever in younger children (all mild to moderate in intensity) were the most frequently observed local and systemic AEs. Eighty-three of BK-SE36/CpG (91%) recipients and 37 of control subjects (84%) had Grade 1/2 events within 28 days post vaccination. Events considered by the investigator to be vaccine related were experienced by 38% and 14% of subjects in BK-SE36/CpG and control arms, respectively. Throughout the trial, six Grade 3 events (in 4 subjects), not related to vaccination, were recorded in the BK-SE36/CpG arm: 5 events (in 3 subjects) within 28 days of vaccination. All serious adverse events (SAEs) (n=5) were due to severe malaria (52-226 days post vaccination) and not related to vaccination. In all cohorts, BK-SE36/CpG arm had higher antibody titres after Dose 3 than after Dose 2. Younger cohorts had stronger immune responses (12-24-month-old > 5-10 years-old > 21-45 years-old). Sera predominantly reacted to peptides that lie in intrinsically unstructured regions of SE36. In the control arm, there were no marked fold changes in antibody titres and participants' sera reacted poorly to all peptides spanning SE36. Conclusion: BK-SE36/CpG was well-tolerated and immunogenic. These results pave the way for further proof-of-concept studies to demonstrate vaccine efficacy. Clinical trial registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1921, PACTR201701001921166.

Antimalarial ceramicines Q-T from Chisocheton ceramicus.

Ceramicines are a series of limonoids that were isolated from the bark of Malaysian Chisocheton ceramicus (Meliaceae) and were known to show various biological activity. Four new limonoids, ceramicines Q-T (1-4) were isolated from the barks of C. ceramicus, and their structures were determined on the basis of the 1D and 2D NMR analyses in combination with calculated 13C chemical shift data. Ceramicines Q-T (1-4) were established to be new limonoids with a cyclopentanone[α]phenanthren ring system with a ß-furyl ring at C-17, and without a tetrahydrofuran ring like ceramicine B, which is characteristic of known ceramicines. Ceramicine R (2) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC50 value of 2.8 µM.

Plasmodium falciparum infection coinciding with the malaria vaccine candidate BK-SE36 administration interferes with the immune responses in Burkinabe children.

Background: A vaccine targeting the erythrocyte stages of Plasmodium falciparum could play a role in preventing clinical disease. BK-SE36 is a promising malaria vaccine candidate that has shown a good safety profile and immunological responses during field evaluations. It was observed that repeated natural infections could result in immune tolerance against SE36 molecule. Methods: The primary trial was conducted to assess the safety and immunogenicity of the BK-SE36 in two cohorts of children aged 25-60 months (Cohort 1) and 12-24 months (Cohort 2). Immunization was at full dose (1.0 mL) administered at 0, 1, and 6 months. Blood samples were collected before each vaccination for immunological assessments and detection of Plasmodium falciparum infection by microscopy. Blood samples were further collected one month post each vaccination to evaluate immunogenicity. Results: Of seventy-two (72) subjects that have received BK-SE36 vaccination, 71 had available blood smears during vaccination days. One month post Dose 2, the geometric mean of SE36 antibodies was 263.2 (95% CI: 178.9-387.1) in uninfected individuals compared to 77.1 (95% CI: 47.3-125.7) in infected participants. The same trend was observed one-month post booster dose. Participants uninfected at the time of booster vaccination had significantly higher GMTs compared to those who were infected (424.1 (95% CI: 301.9-595.8) vs. 92.8 (95% CI: 34.9-246.6), p = 0.002. There was a 14.3 (95% CI: 9.7-21.1) and 2.4 (95% CI: 1.3-4.4) fold-change, respectively, in uninfected and infected participants between one-month post Dose 2 and booster. The difference was statistically significant (p < 0.001). Conclusion: Concomitant infection by P. falciparum during BK-SE36 vaccine candidate administration is associated with reduced humoral responses. However, it is to be noted that the BK-SE36 primary trial was not designed to investigate the influence of concomitant infection on vaccine-induced immune response and should be interpreted cautiously. Trial registration: WHO ICTRP, PACTR201411000934120.

The Impact of Sequestration on Artemisinin-Induced Parasite Clearance in Plasmodium falciparum Malaria in Africa.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum is spreading in Southeast Asia and Africa. In vivo susceptibility to artemisinin is studied by looking at the rate of decline of peripheral parasitemia (parasite clearance half-life). However, parasites that are adhered/sequestered to the endothelium and undetectable in the peripheral blood are not considered in the estimation of parasite clearance. Here, we evaluated the influence of sequestration on in vivo artemisinin efficacy in Uganda, where artemisinin resistance is spreading. METHODS: We analyzed 133 patients with P. falciparum malaria included in an in vivo study on artemisinin efficacy in northern Uganda in 2018 and 2019. The parasite clearance half-life was estimated from peripheral parasitemia after artemisinin monotherapy. P. falciparum histidine-rich protein 2 (PfHRP2) was measured in pretreatment plasma. The number of sequestered parasites was estimated from PfHRP2 concentration and peripheral parasitemia. RESULTS: The estimated number of sequestered parasites per plasma volume ranged from 0 to 2 564 000/µL. Inflammation, thrombocytopenia, and dyslipidemia were significantly associated with sequestration independent of peripheral parasitemia. The median parasite clearance half-lives were 1.65 hours in patients infected with Pfkelch13 wild-type parasites (n = 104) and 3.95 hours in those with A675V artemisinin-resistant mutant (n = 18). In the multivariable model for the wild-type population, 1 000 000/µL of sequestered parasites were estimated to delay parasite clearance by 16.8% (95% confidence interval, 5.1%-28.5%), although it was not clear in the A675V population. CONCLUSIONS: In patients with P. falciparum malaria without artemisinin-resistant mutations, intensive sequestration delays parasite clearance after treatment, which may contribute to reduced artemisinin efficacy.

Safety and immunogenicity of BK-SE36 in a blinded, randomized, controlled, age de-escalating phase Ib clinical trial in Burkinabe children.

Background: A blood-stage vaccine targeting the erythrocytic-stages of the malaria parasite Plasmodium falciparum could play a role to protect against clinical disease. Antibodies against the P. falciparum serine repeat antigen 5 (SE47 and SE36 domains) correlate well with the absence of clinical symptoms in sero-epidemiological studies. A previous phase Ib trial of the recombinant SE36 antigen formulated with aluminum hydroxyl gel (BK-SE36) was promising. This is the first time the vaccine candidate was evaluated in young children below 5 years using two vaccination routes. Methods: Safety and immunogenicity of BK-SE36 was assessed in a double-blind, randomized, controlled, age de-escalating phase Ib trial. Fifty-four Burkinabe children in each age cohort, 25-60 or 12-24 months, were randomized in a 1:1:1 ratio to receive three doses of BK-SE36 either by intramuscular (BK IM) or subcutaneous (BK SC) route on Day 0, Week 4, and 26; or the control vaccine, Synflorix® via IM route on Day 0, Week 26 (and physiological saline on Week 4). Safety data and samples for immunogenicity analyses were collected at various time-points. Results: Of 108 subjects, 104 subjects (96.3%) (Cohort 1: 94.4%; Cohort 2: 98.1%) received all three scheduled vaccine doses. Local reactions, mostly mild or of moderate severity, occurred in 99 subjects (91.7%). The proportion of subjects that received three doses without experiencing Grade 3 adverse events was similar across BK-SE36 vaccines and control arms (Cohort 1: 100%, 89%, and 89%; and Cohort 2: 83%, 82%, and 83% for BK IM, BK SC, and control, respectively). BK-SE36 vaccine was immunogenic, inducing more than 2-fold change in antibody titers from pre-vaccination, with no difference between the two vaccination routes. Titers waned before the third dose but in both cohorts titers were boosted 6 months after the first vaccination. The younger cohort had 2-fold and 4-fold higher geometric mean titers compared to the 25- to 60-month-old cohort after 2 and 3 doses of BK-SE36, respectively. Conclusion: BK-SE36 was well tolerated and immunogenic using either intramuscular or subcutaneous routes, with higher immune response in the younger cohort. Clinical Trial Registration: https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=934, identifier PACTR201411000934120.

Detection of histidine-rich protein 2- and/or 3-deleted Plasmodium falciparum using the automated hematology analyzer XN-31: A proof-of-concept study.

Rapid diagnostic tests (RDTs) based on immunochromatographic detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) have been frequently used for malaria diagnosis. The HRP2-based RDTs are highly sensitive and easy to use; however, their sensitivity may be low in detecting P. falciparum strains carrying deletion of the pfhrp2 and pfhrp3 genes encoding HRP2 and HRP3, respectively. The automated hematology analyzer XN-31, developed by Sysmex (Kobe, Japan) to aid in malaria diagnosis, has higher sensitivity than RDTs owing to a unique automated nucleic acid staining technology that has shown great potential in clinical settings. In this study, we compared the performance of the XN-31 analyzer and two RDTs to detect pfhrp2- and/or pfhrp3-deleted parasites cultured in vitro. The analyses showed that the analyzer was not only as sensitive to pfhrp2- and/or pfhrp3-deleted strains as it was to the wild-type strain but also had higher sensitivity than the RDTs. These results suggested that the XN-31 analyzer is useful for rapid and reliable detection of pfhrp2- and/or pfhrp3-deleted parasites in clinical settings.

Meta-Analysis of Human Antibodies Against Plasmodium falciparum Variable Surface and Merozoite Stage Antigens.

Concerted efforts to fight malaria have caused significant reductions in global malaria cases and mortality. Sustaining this will be critical to avoid rebound and outbreaks of seasonal malaria. Identifying predictive attributes that define clinical malaria will be key to guide development of second-generation tools to fight malaria. Broadly reactive antibodies against variable surface antigens that are expressed on the surface of infected erythrocytes and merozoites stage antigens are targets of naturally acquired immunity and prime candidates for anti-malaria therapeutics and vaccines. However, predicting the relationship between the antigen-specific antibodies and protection from clinical malaria remains unresolved. Here, we used new datasets and multiple approaches combined with re-analysis of our previous data to assess the multi-dimensional and complex relationship between antibody responses and clinical malaria outcomes. We observed 22 antigens (17 PfEMP1 domains, 3 RIFIN family members, merozoite surface protein 3 (PF3D7_1035400), and merozoites-associated armadillo repeats protein (PF3D7_1035900) that were selected across three different clinical malaria definitions (1,000/2,500/5,000 parasites/µl plus fever). In addition, Principal Components Analysis (PCA) indicated that the first three components (Dim1, Dim2 and Dim3 with eigenvalues of 306, 48, and 29, respectively) accounted for 66.1% of the total variations seen. Specifically, the Dim1, Dim2 and Dim3 explained 52.8%, 8.2% and 5% of variability, respectively. We further observed a significant relationship between the first component scores and age with antibodies to PfEMP1 domains being the key contributing variables. This is consistent with a recent proposal suggesting that there is an ordered acquisition of antibodies targeting PfEMP1 proteins. Thus, although limited, and further work on the significance of the selected antigens will be required, these approaches may provide insights for identification of drivers of naturally acquired protective immunity as well as guide development of additional tools for malaria elimination and eradication.

Chukranoids A-I, isopimarane diterpenoids from Chukrasia velutina.

Bioactivity guided separation of Chukrasia velutina root methanolic extract led to the isolation of nine new isopimarane diterpenoids, chukranoids A-I (1-9). The absolute configuration was then assigned by comparing the experimental CD spectra and the calculated CD spectra. Chukranoids A-I (1-9) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain. It seems that conjugate system in the isopimarane skeleton may influence their antimalarial activity.

Caloforines A-G, coumarins from the bark of Calophyllum scriblitifolium.

Bioactivity-guided separation of the methanol extract of Calophyllum scriblitifolium bark led to the isolation of five new pyranocoumarins, caloforines A-E (1-5) and two new coumarins, caloforines F and G (6 and 7). Their structures were elucidated by 1D and 2D NMR spectroscopy, and their absolute configurations were investigated by a combination of CD spectroscopy and DFT calculation. Caloforines A-F (1-6) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain.

African-specific polymorphisms in Plasmodium falciparum serine repeat antigen 5 in Uganda and Burkina Faso clinical samples do not interfere with antibody response to BK-SE36 vaccination.

BK-SE36, based on Plasmodium falciparum serine repeat antigen 5 (SERA5), is a blood-stage malaria vaccine candidate currently being evaluated in clinical trials. Phase 1 trials in Uganda and Burkina Faso have demonstrated promising safety and immunogenicity profiles. However, the genetic diversity of sera5 in Africa and the role of allele/variant-specific immunity remain a major concern. Here, sequence analyses were done on 226 strains collected from the two clinical trial/follow-up studies and 88 strains from two cross-sectional studies in Africa. Compared to other highly polymorphic vaccine candidate antigens, polymorphisms in sera5 were largely confined to the repeat regions of the gene. Results also confirmed a SERA5 consensus sequence with African-specific polymorphisms. Mismatches with the vaccine-type SE36 (BK-SE36) in the octamer repeat, serine repeat, and flanking regions, and single-nucleotide polymorphisms in non-repeat regions could compromise vaccine response and efficacy. However, the haplotype diversity of SERA5 was similar between vaccinated and control participants. There was no marked bias or difference in the patterns of distribution of the SE36 haplotype and no statistically significant genetic differentiation among parasites infecting BK-SE36 vaccinees and controls. Results indicate that BK-SE36 does not elicit an allele-specific immune response.

Walsogynes H-O from Walsura chrysogyne.

Eight new limonoids, walsogynes H-O (1-8) were isolated from the barks of Walsura chrysogyne, and their structures were determined on the basis of the 1D and 2D NMR data. Walsogynes H-M (1-6) and O (8) were concluded to be 11,12-seco limonoids with a dodecahydro-1H-naphtho[1,8-bc:3,4-c']difuran skeleton, and walsogyne N (7) to be 11,12-seco limonoid sharing a unique dodecahydronaphtho[1,8-bc:5,4-b'c']difuran skeleton. Walsogynes H-O (1-8) exhibited potent antimalarial activity against Plasmodium falciparum 3D7 strain with IC50 value of 2.5, 2.6, 1.6, 2.5, 1.5, 2.6, 2.1, and 1.1 µM, respectively.

Can Deep Learning-Based Volumetric Analysis Predict Oxygen Demand Increase in Patients with COVID-19 Pneumonia?

Background and Objectives: This study aimed to investigate whether predictive indicators for the deterioration of respiratory status can be derived from the deep learning data analysis of initial chest computed tomography (CT) scans of patients with coronavirus disease 2019 (COVID-19). Materials and Methods: Out of 117 CT scans of 75 patients with COVID-19 admitted to our hospital between April and June 2020, we retrospectively analyzed 79 CT scans that had a definite time of onset and were performed prior to any medication intervention. Patients were grouped according to the presence or absence of increased oxygen demand after CT scan. Quantitative volume data of lung opacity were measured automatically using a deep learning-based image analysis system. The sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) of the opacity volume data were calculated to evaluate the accuracy of the system in predicting the deterioration of respiratory status. Results: All 79 CT scans were included (median age, 62 years (interquartile range, 46-77 years); 56 (70.9%) were male. The volume of opacity was significantly higher for the increased oxygen demand group than for the nonincreased oxygen demand group (585.3 vs. 132.8 mL, p < 0.001). The sensitivity, specificity, and AUC were 76.5%, 68.2%, and 0.737, respectively, in the prediction of increased oxygen demand. Conclusion: Deep learning-based quantitative analysis of the affected lung volume in the initial CT scans of patients with COVID-19 can predict the deterioration of respiratory status to improve treatment and resource management.

Evidence of Artemisinin-Resistant Malaria in Africa.

BACKGROUND: In the six Southeast Asian countries that make up the Greater Mekong Subregion, Plasmodium falciparum has developed resistance to derivatives of artemisinin, the main component of first-line treatments for malaria. Clinical resistance to artemisinin monotherapy in other global regions, including Africa, would be problematic. METHODS: In this longitudinal study conducted in Northern Uganda, we treated patients who had P. falciparum infection with intravenous artesunate (a water-soluble artemisinin derivative) and estimated the parasite clearance half-life. We evaluated ex vivo susceptibility of the parasite using a ring-stage survival assay and genotyped resistance-related genes. RESULTS: From 2017 through 2019, a total of 14 of 240 patients who received intravenous artesunate had evidence of in vivo artemisinin resistance (parasite clearance half-life, >5 hours). Of these 14 patients, 13 were infected with P. falciparum parasites with mutations in the A675V or C469Y allele in the kelch13 gene. Such mutations were associated with prolonged parasite clearance half-lives (geometric mean, 3.95 hours for A675V and 3.30 hours for C469Y, vs. 1.78 hours for wild-type allele; P<0.001 and P = 0.05, respectively). The ring-stage survival assay showed a higher frequency of parasite survival among organisms with the A675V allele than among those with the wild-type allele. The prevalence of parasites with kelch13 mutations increased significantly, from 3.9% in 2015 to 19.8% in 2019, due primarily to the increased frequency of the A675V and C469Y alleles (P<0.001 and P = 0.004, respectively). Single-nucleotide polymorphisms flanking the A675V mutation in Uganda were substantially different from those in Southeast Asia. CONCLUSIONS: The independent emergence and local spread of clinically artemisinin-resistant P. falciparum has been identified in Africa. The two kelch13 mutations may be markers for detection of these resistant parasites. (Funded by the Japan Society for the Promotion of Science and others.).

Assessment of Mixed Plasmodium falciparum sera5 Infection in Endemic Burkitt Lymphoma: A Case-Control Study in Malawi.

BACKGROUND: Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in Africa and is linked to Plasmodium falciparum (Pf) malaria infection, one of the most common and deadly childhood infections in Africa; however, the role of Pf genetic diversity is unclear. A potential role of Pf genetic diversity in eBL has been suggested by a correlation of age-specific patterns of eBL with the complexity of Pf infection in Ghana, Uganda, and Tanzania, as well as a finding of significantly higher Pf genetic diversity, based on a sensitive molecular barcode assay, in eBL cases than matched controls in Malawi. We examined this hypothesis by measuring diversity in Pf-serine repeat antigen-5 (Pfsera5), an antigenic target of blood-stage immunity to malaria, among 200 eBL cases and 140 controls, all Pf polymerase chain reaction (PCR)-positive, in Malawi. METHODS: We performed Pfsera5 PCR and sequencing (~3.3 kb over exons II-IV) to determine single or mixed PfSERA5 infection status. The patterns of Pfsera5 PCR positivity, mixed infection, sequence variants, and haplotypes among eBL cases, controls, and combined/pooled were analyzed using frequency tables. The association of mixed Pfsera5 infection with eBL was evaluated using logistic regression, controlling for age, sex, and previously measured Pf genetic diversity. RESULTS: Pfsera5 PCR was positive in 108 eBL cases and 70 controls. Mixed PfSERA5 infection was detected in 41.7% of eBL cases versus 24.3% of controls; the odds ratio (OR) was 2.18, and the 95% confidence interval (CI) was 1.12-4.26, which remained significant in adjusted results (adjusted odds ratio [aOR] of 2.40, 95% CI of 1.11-5.17). A total of 29 nucleotide variations and 96 haplotypes were identified, but these were unrelated to eBL. CONCLUSIONS: Our results increase the evidence supporting the hypothesis that infection with mixed Pf infection is increased with eBL and suggest that measuring Pf genetic diversity may provide new insights into the role of Pf infection in eBL.