Risk of Malaria Following Untreated Subpatent Plasmodium falciparum Infections: Results Over 4 Years From a Cohort in a High-Transmission Area in Western Kenya.

BACKGROUND: People with suspected malaria may harbor Plasmodium falciparum undetected by rapid diagnostic test (RDT). The impact of these subpatent infections on the risk of developing clinical malaria is not fully understood. METHODS: We analyzed subpatent P. falciparum infections using a longitudinal cohort in a high-transmission site in Kenya. Weighted Kaplan-Meier models estimated the risk difference (RD) for clinical malaria during the 60 days following a symptomatic subpatent infection. Stratum-specific estimates by age and transmission season assessed modification. RESULTS: Over 54 months, we observed 1128 symptomatic RDT-negative suspected malaria episodes, of which 400 (35.5%) harbored subpatent P. falciparum. Overall, the 60-day risk of developing clinical malaria was low following all episodes (8.6% [95% confidence interval, 6.7%-10.4%]). In the low-transmission season, the risk of clinical malaria was slightly higher in those with subpatent infection, whereas the opposite was true in the high-transmission season (low-transmission season RD, 2.3% [95% confidence interval, .4%-4.2%]; high-transmission season RD, -4.8% [-9.5% to -.05%]). CONCLUSIONS: The risk of developing clinical malaria among people with undetected subpatent infections is low. A slightly elevated risk in the low-transmission season may merit alternate management, but RDTs identify clinically relevant infections in the high-transmission season.

Plasmodium vivax Prevalence in Semiarid Region of Northern Kenya, 2019.

In urban and rural areas of Turkana County, Kenya, we found that 2% of household members of patients with Plasmodium falciparum infections were infected with P. vivax. Enhanced surveillance of P. vivax and increased clinical resources are needed to inform control measures and identify and manage P. vivax infections.

Analytic optimization of Plasmodium falciparum marker gene haplotype recovery from amplicon deep sequencing of complex mixtures.

Molecular epidemiologic studies of malaria parasites commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (ama1, csp, msp7, sera2, and trap) from DBS containing mixtures of DNA from 1 to 10 known P. falciparum reference strains across 3 parasite densities in triplicate (n=270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148/179) of false positives while removing only 8% (67/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with <1.5 genomes/µL (OR: 3.88, CI: 1.82-8.27, vs. high-density samples with ≥75 genomes/µL) and in samples with lower read depth (OR per 10,000 reads: 0.61, CI: 0.54-0.69). Furthermore, minority haplotypes within a sample were more likely to be missed than dominant haplotypes (OR per 0.01 increase in proportion: 0.96, CI: 0.96-0.97). Finally, in clinical samples the percent concordance across markers for multiplicity of infection ranged from 40%-80%. Taken together, our observations indicate that, with sufficient read depth, haplotypes can be successfully recovered from DBS while limiting the false positive rate.

Symptomatic malaria enhances protection from reinfection with homologous Plasmodium falciparum parasites.

A signature remains elusive of naturally-acquired immunity against Plasmodium falciparum. We identified P. falciparum in a 14-month cohort of 239 people in Kenya, genotyped at immunogenic parasite targets expressed in the pre-erythrocytic (circumsporozoite protein, CSP) and blood (apical membrane antigen 1, AMA-1) stages, and classified into epitope type based on variants in the DV10, Th2R, and Th3R epitopes in CSP and the c1L region of AMA-1. Compared to asymptomatic index infections, symptomatic malaria was associated with reduced reinfection by parasites bearing homologous CSP-Th2R (adjusted hazard ratio [aHR]:0.63; 95% CI:0.45-0.89; p = 0.008) CSP-Th3R (aHR:0.71; 95% CI:0.52-0.97; p = 0.033), and AMA-1 c1L (aHR:0.63; 95% CI:0.43-0.94; p = 0.022) epitope types. The association of symptomatic malaria with reduced hazard of homologous reinfection was strongest for rare epitope types. Symptomatic malaria provides more durable protection against reinfection with parasites bearing homologous epitope types. The phenotype represents a legible molecular epidemiologic signature of naturally-acquired immunity by which to identify new antigen targets.

Symptomatic malaria enhances protection from reinfection with homologous Plasmodium falciparum parasites.

A signature remains elusive of naturally-acquired immunity against Plasmodium falciparum . We identified P. falciparum in a 14-month cohort of 239 people in Kenya, genotyped at immunogenic parasite targets expressed in the pre-erythrocytic (circumsporozoite protein, CSP) and blood (apical membrane antigen 1, AMA-1) stages, and classified into epitope type based on variants in the DV10, Th2R, and Th3R epitopes in CSP and the c1L region of AMA-1. Compared to asymptomatic index infections, symptomatic malaria was associated with a reduced reinfection by parasites bearing homologous CSP-Th2R (adjusted hazard ratio [aHR]:0.63; 95% CI:0.45-0.89; p=0.008) CSP-Th3R (aHR:0.71; 95% CI:0.52-0.97; p=0.033), and AMA-1 c1L (aHR:0.63; 95% CI:0.43-0.94; p=0.022) epitope types. The association of symptomatic malaria with reduced risk of homologous reinfection was strongest for rare epitope types. Symptomatic malaria more effectively promotes functional immune responses. The phenotype represents a legible molecular epidemiologic signature of naturally-acquired immunity by which to identify new antigen targets.

Plasmodium falciparum Genetic Diversity in Coincident Human and Mosquito Hosts.

Population genetic diversity of Plasmodium falciparum antigenic loci is high despite large bottlenecks in population size during the parasite life cycle. The prevalence of genetically distinct haplotypes at these loci, while well characterized in humans, has not been thoroughly compared between human and mosquito hosts. We assessed parasite haplotype prevalence, diversity, and evenness using human and mosquito P. falciparum infections collected from the same households during a 14-month longitudinal cohort study using amplicon deep sequencing of two antigenic gene fragments (ama1 and csp). To a prior set of infected humans (n = 1,175/2,813; 86.2% sequencing success) and mosquito abdomens (n = 199/1,448; 95.5% sequencing success), we added sequences from infected mosquito heads (n = 134/1,448; 98.5% sequencing success). The overall and sample-level parasite populations were more diverse in mosquitoes than in humans. Additionally, haplotype prevalences were more even in the P. falciparum human population than in the mosquito population, consistent with balancing selection occurring at these loci in humans. In contrast, we observed that infections in humans were more likely to harbor a dominant haplotype than infections in mosquitoes, potentially due to removal of unfit strains by the human immune system. Finally, within a given mosquito, there was little overlap in genetic composition of abdomen and head infections, suggesting that infections may be cleared from the abdomen during a mosquito's lifespan. Taken together, our observations provide evidence for the mosquito vector acting as a reservoir of sequence diversity in malaria parasite populations. IMPORTANCE Plasmodium falciparum is the deadliest human malaria parasite, and infections consisting of concurrent, multiple strains are common in regions of high endemicity. During transitions within and between the parasite's mosquito and human hosts, these strains are subject to population bottlenecks, and distinct parasite strains may have differential fitness in the various environments encountered. These bottlenecks and fitness differences may lead to differences in strain prevalence and diversity between hosts. We investigated differences in genetic diversity and evenness between P. falciparum parasites in human and mosquito hosts collected from the same households during a 14-month longitudinal study in Kenya. Compared to human parasite populations and infections, P. falciparum parasites observed in mosquito populations and infections were more diverse by multiple population genetic metrics. This suggests that the mosquito vector acts as a reservoir of sequence diversity in malaria parasite populations.

The risks and benefits of early venous thromboembolism prophylaxis after elective spinal surgery: A single-centre experience.

BACKGROUND: There is a wide ranging incidence of venous thromboembolism after surgery and it continues to be a major cause of morbidity after spinal procedures.This study's aim was to investigate the relationship between timing and administration of venous thromboembolism (VTE) pharmacologic chemoprophylaxis after spinal surgery and the resulting VTE and bleeding complications by reviewing current practices and outcomes at a high-volume single institution to better define opportunities for perioperative intervention to prevent VTE without increasing bleeding complications. METHODS: All patients who underwent elective one or two-stage lumbar spinal fusion procedures were identified. A logistic regression was used to evaluate (1) risk of symptomatic VTE within 30 days of surgery and (2) bleeding-related complications. The odds of developing a VTE as well as bleeding-related complications were compared among the three treatment groups: no chemoprophylaxis, chemoprophylaxis < 24h of surgery and chemoprophylaxis given > 24h post-surgery. RESULTS: When adjusted for doses administered, the odds of developing a postoperative VTE within 30 days were 0.189 (95% confidence interval (0.044, 0.808)) in patients who received anticoagulation < 24h postoperatively, compared to those who received no anticoagulation (p = 0.025). There was no difference in bleeding rates. CONCLUSION: Patients undergoing elective spinal surgery who received anticoagulation within 24h of the conclusion of their procedure had an 81% reduction in the odds of developing a deep vein thrombosis within 30 days with no significant difference in bleeding complications.

Plasmodium falciparum importation does not sustain malaria transmission in a semi-arid region of Kenya.

Human movement impacts the spread and transmission of infectious diseases. Recently, a large reservoir of Plasmodium falciparum malaria was identified in a semi-arid region of northwestern Kenya historically considered unsuitable for malaria transmission. Understanding the sources and patterns of transmission attributable to human movement would aid in designing and targeting interventions to decrease the unexpectedly high malaria burden in the region. Toward this goal, polymorphic parasite genes (ama1, csp) in residents and passengers traveling to Central Turkana were genotyped by amplicon deep sequencing. Genotyping and epidemiological data were combined to assess parasite importation. The contribution of travel to malaria transmission was estimated by modelling case reproductive numbers inclusive and exclusive of travelers. P. falciparum was detected in 6.7% (127/1891) of inbound passengers, including new haplotypes which were later detected in locally-transmitted infections. Case reproductive numbers approximated 1 and did not change when travelers were removed from transmission networks, suggesting that transmission is not fueled by travel to the region but locally endemic. Thus, malaria is not only prevalent in Central Turkana but also sustained by local transmission. As such, interrupting importation is unlikely to be an effective malaria control strategy on its own, but targeting interventions locally has the potential to drive down transmission.

Impact of Sickle Cell Trait Hemoglobin on the Intraerythrocytic Transcriptional Program of Plasmodium falciparum.

Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening falciparum malaria in African children. Despite this clear protection, the molecular mechanisms by which HbAS confers these protective phenotypes remain incompletely understood. As a forward genetic screen for aberrant parasite transcriptional responses associated with parasite neutralization in HbAS red blood cells (RBCs), we performed comparative transcriptomic analyses of Plasmodium falciparum in normal (HbAA) and HbAS erythrocytes during both in vitro cultivation of reference parasite strains and naturally occurring P. falciparum infections in Malian children with HbAA or HbAS. During in vitro cultivation, parasites matured normally in HbAS RBCs, and the temporal expression was largely unperturbed of the highly ordered transcriptional program that underlies the parasite's maturation throughout the intraerythrocytic development cycle (IDC). However, differential expression analysis identified hundreds of transcripts aberrantly expressed in HbAS, largely occurring late in the IDC. Surprisingly, transcripts encoding members of the Maurer's clefts were overexpressed in HbAS despite impaired parasite protein export in these RBCs, while parasites in HbAS RBCs underexpressed transcripts associated with the endoplasmic reticulum and those encoding serine repeat antigen proteases that promote parasite egress. Analyses of P. falciparum transcriptomes from 32 children with uncomplicated malaria identified stage-specific differential expression: among infections composed of ring-stage parasites, only cyclophilin 19B was underexpressed in children with HbAS, while trophozoite-stage infections identified a range of differentially expressed transcripts, including downregulation in HbAS of several transcripts associated with severe malaria in collateral studies. Collectively, our comparative transcriptomic screen in vitro and in vivo indicates that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Because HbAS consistently protects from severe P. falciparum, modulation of these responses may offer avenues by which to neutralize P. falciparum parasites. IMPORTANCE Sickle-trait hemoglobin (HbAS) confers nearly complete protection from severe, life-threatening malaria, yet the molecular mechanisms that underlie HbAS protection from severe malaria remain incompletely understood. Here, we used transcriptome sequencing (RNA-seq) to measure the impact of HbAS on the blood-stage transcriptome of Plasmodium falciparum in in vitro time series experiments and in vivo samples from natural infections. Our in vitro time series data reveal that, during its blood stage, P. falciparum's gene expression in HbAS is impacted primarily through alterations in the abundance of gene products as opposed to variations in the timing of gene expression. Collectively, our in vitro and in vivo data indicate that P. falciparum adapts to HbAS by altering its protein chaperone and folding machinery, oxidative stress response, and protein export machinery. Due to the persistent association of HbAS and protection from severe disease, these processes that are modified in HbAS may offer strategies to neutralize P. falciparum.

Impact of asymptomatic Plasmodium falciparum infection on the risk of subsequent symptomatic malaria in a longitudinal cohort in Kenya.

Background: Asymptomatic Plasmodium falciparum infections are common in sub-Saharan Africa, but their effect on subsequent symptomaticity is incompletely understood. Methods: In a 29-month cohort of 268 people in Western Kenya, we investigated the association between asymptomatic P. falciparum and subsequent symptomatic malaria with frailty Cox models. Results: Compared to being uninfected, asymptomatic infections were associated with an increased 1 month likelihood of symptomatic malaria (adjusted hazard ratio [aHR]: 2.61, 95% CI: 2.05 to 3.33), and this association was modified by sex, with females (aHR: 3.71, 95% CI: 2.62 to 5.24) at higher risk for symptomaticity than males (aHR: 1.76, 95% CI: 1.24 to 2.50). This increased symptomatic malaria risk was observed for asymptomatic infections of all densities and in people of all ages. Long-term risk was attenuated but still present in children under age 5 (29-month aHR: 1.38, 95% CI: 1.05 to 1.81). Conclusions: In this high-transmission setting, asymptomatic P. falciparum can be quickly followed by symptoms and may be targeted to reduce the incidence of symptomatic illness. Funding: This work was supported by the National Institute of Allergy and Infectious Diseases (R21AI126024 to WPO, R01AI146849 to WPO and SMT).

Sickle-trait hemoglobin reduces adhesion to both CD36 and EPCR by Plasmodium falciparum-infected erythrocytes.

Sickle-trait hemoglobin protects against severe Plasmodium falciparum malaria. Severe malaria is governed in part by the expression of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that are encoded by var genes, specifically those variants that bind Endothelial Protein C Receptor (EPCR). In this study, we investigate the effect of sickle-trait on parasite var gene expression and function in vitro and in field-collected parasites. We mapped var gene reads generated from RNA sequencing in parasite cultures in normal and sickle-cell trait blood throughout the asexual lifecycle. We investigated sickle-trait effect on PfEMP1 interactions with host receptors CD36 and EPCR using static adhesion assays and flow cytometry. Var expression in vivo was compared by assembling var domains sequenced from total RNA in parasites infecting Malian children with HbAA and HbAS. Sickle-trait did not alter the abundance or type of var gene transcripts in vitro, nor the abundance of overall transcripts or of var functional domains in vivo. In adhesion assays using recombinant host receptors, sickle-trait reduced adhesion by 73-86% to CD36 and 83% to EPCR. Similarly, sickle-trait reduced the surface expression of EPCR-binding PfEMP1. In conclusion, Sickle-cell trait does not directly affect var gene transcription but does reduce the surface expression and function of PfEMP1. This provides a direct mechanism for protection against severe malaria conferred by sickle-trait hemoglobin. Trial Registration: ClinicalTrials.gov Identifier: NCT02645604.

Exposure to Diverse Plasmodium falciparum Genotypes Shapes the Risk of Symptomatic Malaria in Incident and Persistent Infections: A Longitudinal Molecular Epidemiologic Study in Kenya.

BACKGROUND: Repeated exposure to malaria infections could protect against symptomatic progression as people develop adaptive immunity to infections acquired over time. METHODS: We investigated how new, recurrent, and persistent Plasmodium falciparum infections were associated with the odds of developing symptomatic compared with asymptomatic malaria. Using a 14-month longitudinal cohort in Western Kenya, we used amplicon deep sequencing of 2 polymorphic genes (pfama1 and pfcsp) to assess overlap of parasite genotypes (represented by haplotypes) acquired within an individual's successive infections. We hypothesized infections with novel haplotypes would increase the odds of symptomatic malaria. RESULTS: After excluding initial infections, we observed 534 asymptomatic and 88 symptomatic infections across 186 people. We detected 109 pfcsp haplotypes, and each infection was classified as harboring novel, recurrent, or persistent haplotypes. Incident infections with only new haplotypes had higher odds of symptomatic malaria when compared with infections with only recurrent haplotypes [odds ratio (OR): 3.24; 95% confidence interval (CI), 1.20-8.78], but infections with both new and recurrent haplotypes (OR: 0.64; 95% CI: 0.15-2.65) did not. Assessing persistent infections, those with mixed (persistent with new or recurrent) haplotypes (OR: 0.77; 95% CI: 0.21-2.75) had no association with symptomatic malaria compared with infections with only persistent haplotypes. Results were similar for pfama1. CONCLUSIONS: These results confirm that incident infections with only novel haplotypes were associated with increased odds of symptomatic malaria compared with infections with only recurrent haplotypes but this relationship was not seen when haplotypes persisted over time in consecutive infections.

Genotyping cognate Plasmodium falciparum in humans and mosquitoes to estimate onward transmission of asymptomatic infections.

Malaria control may be enhanced by targeting reservoirs of Plasmodium falciparum transmission. One putative reservoir is asymptomatic malaria infections and the scale of their contribution to transmission in natural settings is not known. We assess the contribution of asymptomatic malaria to onward transmission using a 14-month longitudinal cohort of 239 participants in a high transmission site in Western Kenya. We identify P. falciparum in asymptomatically- and symptomatically-infected participants and naturally-fed mosquitoes from their households, genotype all parasites using deep sequencing of the parasite genes pfama1 and pfcsp, and use haplotypes to infer participant-to-mosquito transmission through a probabilistic model. In 1,242 infections (1,039 in people and 203 in mosquitoes), we observe 229 (pfcsp) and 348 (pfama1) unique parasite haplotypes. Using these to link human and mosquito infections, compared with symptomatic infections, asymptomatic infections more than double the odds of transmission to a mosquito among people with both infection types (Odds Ratio: 2.56; 95% Confidence Interval (CI): 1.36-4.81) and among all participants (OR 2.66; 95% CI: 2.05-3.47). Overall, 94.6% (95% CI: 93.1-95.8%) of mosquito infections likely resulted from asymptomatic infections. In high transmission areas, asymptomatic infections are the major contributor to mosquito infections and may be targeted as a component of transmission reduction.

Computer-based simulation to reduce EHR-related chemotherapy ordering errors.

BACKGROUND: The electronic health record (EHR) is a contributor to serious patient harm occurring within a sociotechnical system. Chemotherapy ordering is a high-risk task due to the complex nature of ordering workflows and potential detrimental effects if wrong chemotherapeutic doses are administered. Many chemotherapy ordering errors cannot be mitigated through systems-based changes due to the limited extent to which individual institutions are able to customize proprietary EHR software. We hypothesized that simulation-based training could improve providers' ability to identify and mitigate common chemotherapy ordering errors. METHODS: Pediatric hematology/oncology providers voluntarily participated in simulations using an EHR testing ("Playground") environment. The number of safety risks identified and mitigated by each provider at baseline was recorded. Risks were reviewed one-on-one after initial simulations and at a group "lunch-and-learn" session. At three-month follow-up, repeat simulations assessed for improvements in error identification and mitigation, and providers were surveyed about prevention of real-life safety events. RESULTS: The 8 participating providers identified and mitigated an average of 5.5 out of 10 safety risks during the initial simulation, compared 7.4 safety risks at the follow up simulation (p=0.030). Two of the providers (25%) reported preventing at least one real-world patient safety event in the clinical setting as a result of the initial training session. CONCLUSIONS: Simulation-based training may reduce providers' susceptibility to chemotherapy ordering safety vulnerabilities within the EHR. This approach may be used when systems-based EHR improvements are not feasible due to limited ability to customize local instances of proprietary EHR software.

High-resolution micro-epidemiology of parasite spatial and temporal dynamics in a high malaria transmission setting in Kenya.

Novel interventions that leverage the heterogeneity of parasite transmission are needed to achieve malaria elimination. To better understand spatial and temporal dynamics of transmission, we applied amplicon next-generation sequencing of two polymorphic gene regions (csp and ama1) to a cohort identified via reactive case detection in a high-transmission setting in western Kenya. From April 2013 to July 2014, we enrolled 442 symptomatic children with malaria, 442 matched controls, and all household members of both groups. Here, we evaluate genetic similarity between infected individuals using three indices: sharing of parasite haplotypes on binary and proportional scales and the L1 norm. Symptomatic children more commonly share haplotypes with their own household members. Furthermore, we observe robust temporal structuring of parasite genetic similarity and identify the unique molecular signature of an outbreak. These findings of both micro- and macro-scale organization of parasite populations might be harnessed to inform next-generation malaria control measures.

Direct Detection of Unamplified Pathogen RNA in Blood Lysate using an Integrated Lab-in-a-Stick Device and Ultrabright SERS Nanorattles.

Direct detection of genetic biomarkers in body fluid lysate without target amplification will revolutionize nucleic acid-based diagnostics. However, the low concentration of target sequences makes this goal challenging. We report a method for direct detection of pathogen RNA in blood lysate using a bioassay using surface-enhanced Raman spectroscopy (SERS)-based detection integrated in a "lab-in-a-stick" portable device. Two levels of signal enhancement were employed to achieve the sensitivity required for direct detection. Each target sequence was tagged with an ultrabright SERS-encoded nanorattle with ultrahigh SERS signals, and these tagged target sequences were concentrated into a focused spot for detection using hybridization sandwiches with magnetic microbeads. Furthermore, the washing process was automated by integration into a "lab-in-a-stick" portable device. We could directly detect synthetic target with a limit of detection of 200 fM. More importantly, we detected plasmodium falciparum malaria parasite RNA directly in infected red blood cells lysate. To our knowledge, this is the first report of SERS-based direct detection of pathogen nucleic acid in blood lysate without nucleic acid extraction or target amplification. The results show the potential of our integrated bioassay for field use and point-of-care diagnostics.

Importance of hereditary and selected environmental risk factors in the etiology of inflammatory breast cancer: a case-comparison study.

BACKGROUND: To assess the importance of heredity in the etiology of inflammatory breast cancer (IBC), we compared IBC patients to several carefully chosen comparison groups with respect to the prevalence of first-degree family history of breast cancer. METHODS: IBC cases (n = 141) were compared to non-inflammatory breast cancer cases (n = 178) ascertained through George Washington University (GWU) with respect to the prevalence of first-degree family history of breast cancer and selected environmental/lifestyle risk factors for breast cancer. Similar comparisons were conducted with subjects from three case-control studies: breast cancer cases (n = 1145) and unaffected controls (n = 1142) from the Cancer Genetic Markers of Susceptibility (CGEMS) study, breast cancer cases (n = 465) and controls (n = 9317) from the Women's Health Initiative (WHI) study, and ovarian cancer cases (n = 260) and controls (n = 331) from a study by University of Toronto (UT). RESULTS: The frequency of first-degree breast cancer family history among IBC cases was 17.0 % compared to 24.4 % for GWU breast cancer cases, 23.9 % and 17.9 % for CGEMS breast cancer cases and controls, respectively, 16.9 % and 12.6 % for WHI breast cancer cases and controls, respectively, and 24.2 % and 11.2 % for UT ovarian cancer cases and controls, respectively. IBC cases had a significantly lower prevalence of parous women than WHI breast cancer cases (OR = 0.46, 95 % CI:0.27-0.81) and controls (OR = 0.31, 95 % CI:0.20-0.49). Oral contraceptive use was significantly higher among IBC cases compared to WHI breast cancer cases (OR = 7.77, 95 % CI:4.82-12.59) and controls (OR = 8.14, 95 % CI:5.28-12.61). IBC cases had a significantly higher frequency of regular alcohol consumption (≥1 drink per day) compared to WHI controls (OR = 1.84, 95 % CI:1.20-2.82) and UT controls (OR = 1.86, 95 % CI:1.07-3.22) and higher (statistically non-significant) prevalence (21.3 %) compared to breast cancer cases from GWU (18.2 %) and WHI (15.2 %). CONCLUSIONS: The prevalence of first-degree breast cancer family history among IBC cases was lower compared to breast and ovarian cancer cases but higher than unaffected individuals. Our multiple-case inflammatory and non-inflammatory breast cancer families may reflect aggregation of common genetic and/or environmental factors predisposing to both types of breast cancer. Our findings that oral contraceptive use and regular alcohol consumption may be associated with IBC warrant further investigations.

The predatory ecology of Deinonychus and the origin of flapping in birds.

Most non-avian theropod dinosaurs are characterized by fearsome serrated teeth and sharp recurved claws. Interpretation of theropod predatory ecology is typically based on functional morphological analysis of these and other physical features. The notorious hypertrophied 'killing claw' on pedal digit (D) II of the maniraptoran theropod Deinonychus (Paraves: Dromaeosauridae) is hypothesized to have been a predatory adaptation for slashing or climbing, leading to the suggestion that Deinonychus and other dromaeosaurids were cursorial predators specialized for actively attacking and killing prey several times larger than themselves. However, this hypothesis is problematic as extant animals that possess similarly hypertrophied claws do not use them to slash or climb up prey. Here we offer an alternative interpretation: that the hypertrophied D-II claw of dromaeosaurids was functionally analogous to the enlarged talon also found on D-II of extant Accipitridae (hawks and eagles; one family of the birds commonly known as "raptors"). Here, the talon is used to maintain grip on prey of subequal body size to the predator, while the victim is pinned down by the body weight of the raptor and dismembered by the beak. The foot of Deinonychus exhibits morphology consistent with a grasping function, supportive of the prey immobilisation behavior model. Opposite morphological trends within Deinonychosauria (Dromaeosauridae + Troodontidae) are indicative of ecological separation. Placed in context of avian evolution, the grasping foot of Deinonychus and other terrestrial predatory paravians is hypothesized to have been an exaptation for the grasping foot of arboreal perching birds. Here we also describe "stability flapping", a novel behaviour executed for positioning and stability during the initial stages of prey immobilisation, which may have been pivotal to the evolution of the flapping stroke. These findings overhaul our perception of predatory dinosaurs and highlight the role of exaptation in the evolution of novel structures and behaviours.

Reanalysis of "Raptorex kriegsteini": a juvenile tyrannosaurid dinosaur from Mongolia.

The carnivorous Tyrannosauridae are among the most iconic dinosaurs: typified by large body size, tiny forelimbs, and massive robust skulls with laterally thickened teeth. The recently described small-bodied tyrannosaurid Raptorex kreigsteini is exceptional as its discovery proposes that many of the distinctive anatomical traits of derived tyrannosaurids were acquired in the Early Cretaceous, before the evolution of large body size. This inference depends on two core interpretations: that the holotype (LH PV18) derives from the Lower Cretaceous of China, and that despite its small size, it is a subadult or young adult. Here we show that the published data is equivocal regarding stratigraphic position and that ontogenetic reanalysis shows there is no reason to conclude that LH PV18 has reached this level of maturity. The probable juvenile status of LH PV18 makes its use as a holotype unreliable, since diagnostic features of Raptorex may be symptomatic of its immature status, rather than its actual phylogenetic position. These findings are consistent with the original sale description of LH PV18 as a juvenile Tarbosaurus from the Upper Cretaceous of Mongolia. Consequently, we suggest that there is currently no evidence to support the conclusion that tyrannosaurid skeletal design first evolved in the Early Cretaceous at small body size.

Predatory functional morphology in raptors: interdigital variation in talon size is related to prey restraint and immobilisation technique.

Despite the ubiquity of raptors in terrestrial ecosystems, many aspects of their predatory behaviour remain poorly understood. Surprisingly little is known about the morphology of raptor talons and how they are employed during feeding behaviour. Talon size variation among digits can be used to distinguish families of raptors and is related to different techniques of prey restraint and immobilisation. The hypertrophied talons on digits (D) I and II in Accipitridae have evolved primarily to restrain large struggling prey while they are immobilised by dismemberment. Falconidae have only modest talons on each digit and only slightly enlarged D-I and II. For immobilisation, Falconini rely more strongly on strike impact and breaking the necks of their prey, having evolved a 'tooth' on the beak to aid in doing so. Pandionidae have enlarged, highly recurved talons on each digit, an adaptation for piscivory, convergently seen to a lesser extent in fishing eagles. Strigiformes bear enlarged talons with comparatively low curvature on each digit, part of a suite of adaptations to increase constriction efficiency by maximising grip strength, indicative of specialisation on small prey. Restraint and immobilisation strategy change as prey increase in size. Small prey are restrained by containment within the foot and immobilised by constriction and beak attacks. Large prey are restrained by pinning under the bodyweight of the raptor, maintaining grip with the talons, and immobilised by dismemberment (Accipitridae), or severing the spinal cord (Falconini). Within all raptors, physical attributes of the feet trade off against each other to attain great strength, but it is the variable means by which this is achieved that distinguishes them ecologically. Our findings show that interdigital talon morphology varies consistently among raptor families, and that this is directly correlative with variation in their typical prey capture and restraint strategy.