Hypertensive Disorders of Pregnancy and Risk of Early Brain Abnormalities on Magnetic Resonance Imaging at Term among Infants Born at ≤32 Weeks' Gestational Age.

OBJECTIVE: To evaluate the proximal effects of hypertensive disorders of pregnancy (HDP) on a validated measure of brain abnormalities in infants born at ≤32 weeks' gestational age (GA) using magnetic resonance imaging at term-equivalent age. STUDY DESIGN: In a multisite prospective cohort study, 395 infants born at ≤32 weeks' GA, underwent 3T magnetic resonance imaging scan between 39 and 44 weeks' postmenstrual age. A single neuroradiologist, blinded to clinical history, evaluated the standardized Kidokoro global brain abnormality score as the primary outcome. We classified infants as HDP-exposed by maternal diagnosis of chronic hypertension, gestational hypertension, pre-eclampsia, or eclampsia. Linear regression analysis identified the independent effects of HDP on infant brain abnormalities, adjusting for histologic chorioamnionitis, maternal smoking, antenatal steroids, magnesium sulfate, and infant sex. Mediation analyses quantified the indirect effect of HDP mediated via impaired intrauterine growth and prematurity and remaining direct effects on brain abnormalities. RESULTS: A total of 170/395 infants (43%) were HDP-exposed. Adjusted multivariable analyses revealed HDP-exposed infants had 27% (95% CI 5%-53%) higher brain abnormality scores than those without HDP exposure (P = .02), primarily driven by increased white matter injury/abnormality scores (P = .01). Mediation analyses showed HDP-induced impaired intrauterine growth significantly (P = .02) contributed to brain abnormality scores (22% of the total effect). CONCLUSIONS: Maternal hypertension independently increased the risk for early brain injury and/or maturational delays in infants born at ≤32 weeks' GA with an indirect effect of 22% resulting from impaired intrauterine growth. Enhanced prevention/treatment of maternal hypertension may mitigate the risk of infant brain abnormalities and potential neurodevelopmental impairments.

Effects of Early Enteral to Parenteral Protein Ratios on Brain Volume and Somatic Growth in Very Low Birth Weight Infants.

OBJECTIVE: To evaluate whether a higher proportion of enteral vs parenteral protein ratio (E:P ratio) in the first 28 days after birth is associated with increased brain volume and somatic growth in very low birth weight (VLBW; birth weight <1500 g) infants. STUDY DESIGN: This was a retrospective analysis of a subcohort of VLBW infants (n = 256, gestational age mean 28.07 [SD 2.17] weeks, birth weight 1038.80 [SD 262.95] grams) from the Cincinnati Infant Neurodevelopment Early Prediction Study, a regional prospective study of infants born at ≤32 weeks' gestation. Brain magnetic resonance imaging was obtained at term-equivalent age. Macronutrient intake and growth metrics for the first 28 days were collected retrospectively. The primary outcome was total brain tissue volume. The relationships between E:P ratio, total and regional brain tissue volumes, and somatic growth were analyzed by multivariable linear regression models; composite variables were used to adjust for potential confounders including pregnancy risk factors and initial severity of illness. RESULTS: Higher E:P ratio was associated with increased total brain tissue volume but was not associated with change in head circumference z score. In secondary analyses, higher E:P ratio was associated with increased weight velocity. There were no significant associations between E:P ratio and change in weight or length z scores or regional brain volumes. CONCLUSIONS: Higher E:P ratio in the first 28 days was positively associated with total brain volume and weight gain. Promoting the provision of enteral over parenteral protein may improve brain and somatic growth in VLBW infants.

Diagnostic performance and comparison of ultrasensitive and conventional rapid diagnostic test, thick blood smear and quantitative PCR for detection of low-density Plasmodium falciparum infections during a controlled human malaria infection study in Equatorial Guinea.

BACKGROUND: Progress towards malaria elimination has stagnated, partly because infections persisting at low parasite densities comprise a large reservoir contributing to ongoing malaria transmission and are difficult to detect. This study compared the performance of an ultrasensitive rapid diagnostic test (uRDT) designed to detect low density infections to a conventional RDT (cRDT), expert microscopy using Giemsa-stained thick blood smears (TBS), and quantitative polymerase chain reaction (qPCR) during a controlled human malaria infection (CHMI) study conducted in malaria exposed adults (NCT03590340). METHODS: Blood samples were collected from healthy Equatoguineans aged 18-35 years beginning on day 8 after CHMI with 3.2 × 103 cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ Challenge, strain NF54) administered by direct venous inoculation. qPCR (18s ribosomal DNA), uRDT (Alere™ Malaria Ag P.f.), cRDT [Carestart Malaria Pf/PAN (PfHRP2/pLDH)], and TBS were performed daily until the volunteer became TBS positive and treatment was administered. qPCR was the reference for the presence of Plasmodium falciparum parasites. RESULTS: 279 samples were collected from 24 participants; 123 were positive by qPCR. TBS detected 24/123 (19.5% sensitivity [95% CI 13.1-27.8%]), uRDT 21/123 (17.1% sensitivity [95% CI 11.1-25.1%]), cRDT 10/123 (8.1% sensitivity [95% CI 4.2-14.8%]); all were 100% specific and did not detect any positive samples not detected by qPCR. TBS and uRDT were more sensitive than cRDT (TBS vs. cRDT p = 0.015; uRDT vs. cRDT p = 0.053), detecting parasitaemias as low as 3.7 parasites/µL (p/µL) (TBS and uRDT) compared to 5.6 p/µL (cRDT) based on TBS density measurements. TBS, uRDT and cRDT did not detect any of the 70/123 samples positive by qPCR below 5.86 p/µL, the qPCR density corresponding to 3.7 p/µL by TBS. The median prepatent periods in days (ranges) were 14.5 (10-20), 18.0 (15-28), 18.0 (15-20) and 18.0 (16-24) for qPCR, TBS, uRDT and cRDT, respectively; qPCR detected parasitaemia significantly earlier (3.5 days) than the other tests. CONCLUSIONS: TBS and uRDT had similar sensitivities, both were more sensitive than cRDT, and neither matched qPCR for detecting low density parasitaemia. uRDT could be considered an alternative to TBS in selected applications, such as CHMI or field diagnosis, where qualitative, dichotomous results for malaria infection might be sufficient.

Spatial and Frequency Specific Artifact Reduction in Optically Pumped Magnetometer Recordings.

BACKGROUND: Magnetoencephalography (MEG) based on optically pumped magnetometers (OPMs) opens up new opportunities for brain research. However, OPM recordings are associated with artifacts. We describe a new artifact reduction method, frequency specific signal space classification (FSSSC), to improve the signal-to-noise ratio of OPM recordings. METHODS: FSSSC was based on time-frequency analysis and signal space classification (SSC). SSC was accomplished by computing the orthogonality of the brain signal and artifact. A dipole phantom was used to determine if the method could remove artifacts and improve accuracy of source localization. Auditory evoked magnetic fields (AEFs) from human subjects were used to assess the usefulness of artifact reduction in the study of brain function because bilateral AEFs have proven a good benchmark for testing new methods. OPM data from empty room recordings were used to estimate magnetic artifacts. The effectiveness of FSSSC was assessed in waveforms, spectrograms, and covariance domains. RESULTS: MEG recordings from phantom tests show that FSSSC can remove artifacts, normalize waveforms, and significantly improve source localization accuracy. MEG signals from human subjects show that FSSC can reveal auditory evoked magnetic responses overshadowed and distorted by artifacts. The present study demonstrates FSSSC is effective at removing artifacts in OPM recordings. This can facilitate the analyses of waveforms, spectrograms, and covariance. The accuracy of source localization of OPM recordings can be significantly improved by FSSSC. CONCLUSIONS: Brain responses distorted by artifacts can be restored. The results of the present study strongly support that artifact reduction is very important in order for OPMs to become a viable alternative to conventional MEG.

Efficacy, Safety, and Durability of Voretigene Neparvovec-rzyl in RPE65 Mutation-Associated Inherited Retinal Dystrophy: Results of Phase 1 and 3 Trials.

PURPOSE: To report the durability of voretigene neparvovec-rzyl (VN) adeno-associated viral vector-based gene therapy for RPE65 mutation-associated inherited retinal dystrophy (IRD), including results of a phase 1 follow-on study at year 4 and phase 3 study at year 2. DESIGN: Open-label phase 1 follow-on clinical trial and open-label, randomized, controlled phase 3 clinical trial. PARTICIPANTS: Forty subjects who received 1.5×1011 vector genomes (vg) of VN per eye in at least 1 eye during the trials, including 11 phase 1 follow-on subjects and 29 phase 3 subjects (20 original intervention [OI] and 9 control/intervention [CI]). METHODS: Subretinal injection of VN in the second eye of phase 1 follow-on subjects and in both eyes of phase 3 subjects. MAIN OUTCOME MEASURES: End points common to the phase 1 and phase 3 studies included change in performance on the Multi-Luminance Mobility Test (MLMT) within the illuminance range evaluated, full-field light sensitivity threshold (FST) testing, and best-corrected visual acuity (BCVA). Safety end points included adverse event reporting, ophthalmic examination, physical examination, and laboratory testing. RESULTS: Mean (standard deviation) MLMT lux score change was 2.4 (1.3) at 4 years compared with 2.6 (1.6) at 1 year after administration in phase 1 follow-on subjects (n = 8), 1.9 (1.1) at 2 years, and 1.9 (1.0) at 1 year post-administration in OI subjects (n = 20), and 2.1 (1.6) at 1 year post-administration in CI subjects (n = 9). All 3 groups maintained an average improvement in FST, reflecting more than a 2 log10(cd.s/m2) improvement in light sensitivity at 1 year and subsequent available follow-up visits. The safety profile was consistent with vitrectomy and the subretinal injection procedure, and no deleterious immune responses occurred. CONCLUSIONS: After VN gene augmentation therapy, there was a favorable benefit-to-risk profile with similar improvement demonstrated in navigational ability and light sensitivity among 3 groups of subjects with RPE65 mutation-associated IRD, a degenerative disease that progresses to complete blindness. The safety profile is consistent with the administration procedure. These data suggest that this effect, which is nearly maximal by 30 days after VN administration, is durable for 4 years, with observation ongoing.

Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial.

BACKGROUND: Phase 1 studies have shown potential benefit of gene replacement in RPE65-mediated inherited retinal dystrophy. This phase 3 study assessed the efficacy and safety of voretigene neparvovec in participants whose inherited retinal dystrophy would otherwise progress to complete blindness. METHODS: In this open-label, randomised, controlled phase 3 trial done at two sites in the USA, individuals aged 3 years or older with, in each eye, best corrected visual acuity of 20/60 or worse, or visual field less than 20 degrees in any meridian, or both, with confirmed genetic diagnosis of biallelic RPE65 mutations, sufficient viable retina, and ability to perform standardised multi-luminance mobility testing (MLMT) within the luminance range evaluated, were eligible. Participants were randomly assigned (2:1) to intervention or control using a permuted block design, stratified by age (<10 years and ≥10 years) and baseline mobility testing passing level (pass at ≥125 lux vs <125 lux). Graders assessing primary outcome were masked to treatment group. Intervention was bilateral, subretinal injection of 1·5 × 1011 vector genomes of voretigene neparvovec in 0·3 mL total volume. The primary efficacy endpoint was 1-year change in MLMT performance, measuring functional vision at specified light levels. The intention-to-treat (ITT) and modified ITT populations were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, number NCT00999609, and enrolment is complete. FINDINGS: Between Nov 15, 2012, and Nov 21, 2013, 31 individuals were enrolled and randomly assigned to intervention (n=21) or control (n=10). One participant from each group withdrew after consent, before intervention, leaving an mITT population of 20 intervention and nine control participants. At 1 year, mean bilateral MLMT change score was 1·8 (SD 1·1) light levels in the intervention group versus 0·2 (1·0) in the control group (difference of 1·6, 95% CI 0·72-2·41, p=0·0013). 13 (65%) of 20 intervention participants, but no control participants, passed MLMT at the lowest luminance level tested (1 lux), demonstrating maximum possible improvement. No product-related serious adverse events or deleterious immune responses occurred. Two intervention participants, one with a pre-existing complex seizure disorder and another who experienced oral surgery complications, had serious adverse events unrelated to study participation. Most ocular events were mild in severity. INTERPRETATION: Voretigene neparvovec gene replacement improved functional vision in RPE65-mediated inherited retinal dystrophy previously medically untreatable. FUNDING: Spark Therapeutics.

Extremely preterm children exhibit increased interhemispheric connectivity for language: findings from fMRI-constrained MEG analysis.

Children born extremely preterm are at significant risk for cognitive impairment, including language deficits. The relationship between preterm birth and neurological changes that underlie cognitive deficits is poorly understood. We use a stories-listening task in fMRI and MEG to characterize language network representation and connectivity in children born extremely preterm (n = 15, <28 weeks gestation, ages 4-6 years), and in a group of typically developing control participants (n = 15, term birth, 4-6 years). Participants completed a brief neuropsychological assessment. Conventional fMRI analyses revealed no significant differences in language network representation across groups (p > .05, corrected). The whole-group fMRI activation map was parcellated to define the language network as a set of discrete nodes, and the timecourse of neuronal activity at each position was estimated using linearly constrained minimum variance beamformer in MEG. Virtual timecourses were subjected to connectivity and network-based analyses. We observed significantly increased beta-band functional connectivity in extremely preterm compared to controls (p < .05). Specifically, we observed an increase in connectivity between left and right perisylvian cortex. Subsequent effective connectivity analyses revealed that hyperconnectivity in preterms was due to significantly increased information flux originating from the right hemisphere (p < 0.05). The total strength and density of the language network were not related to language or nonverbal performance, suggesting that the observed hyperconnectivity is a "pure" effect of prematurity. Although our extremely preterm children exhibited typical language network architecture, we observed significantly altered network dynamics, indicating reliance on an alternative neural strategy for the language task.

The essential activities of the bacterial sigma factor.

Transcription is the first and most heavily regulated step in gene expression. Sigma (σ) factors are general transcription factors that reversibly bind RNA polymerase (RNAP) and mediate transcription of all genes in bacteria. σ Factors play 3 major roles in the RNA synthesis initiation process: they (i) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. Recent structural studies have demonstrated that when σ factors bind promoter DNA, they capture 1 or more nucleotides that are flipped out of the helical DNA stack and this stabilizes the promoter open-complex intermediate that is required for the initiation of RNA synthesis. This review describes the structure and function of the σ70 family of σ proteins and the essential roles they play in the transcription process.

The atypical two-subunit σ factor from Bacillus subtilis is regulated by an integral membrane protein and acid stress.

Extracytoplasmic function (ECF) σ factors constitute a major component of the physicochemical sensory apparatus in bacteria. Most ECF σ factors are co-expressed with a negative regulator called an anti-σ factor that binds to its cognate σ factor and sequesters it from productive association with core RNA polymerase (RNAP). Anti-σ factors constitute an important element of signal transduction pathways that mediate an appropriate transcriptional response to changing environmental conditions. The Bacillus subtilis genome encodes seven canonical ECF σ factors and six of these are co-expressed with experimentally verified anti-σ factors. B. subtilis also expresses an ECF-like atypical two-subunit σ factor composed of subunits SigO and RsoA that becomes active after exposure to certain cell-wall-acting antibiotics and to growth under acidic conditions. This work describes the identification and preliminary characterization of a protein (RsiO, formerly YvrL) that constitutes the anti-σ factor cognate to SigO-RsoA. Synthesis of RsiO represses SigO-RsoA-dependent transcription initiation by binding the N-terminus of SigO under neutral (pH 7) conditions. Reconstitution of the SigO-RsoA-RsiO regulatory system into a heterologous host reveals that the imposition of acid stress (pH 5.4) abolishes the ability of RsiO to repress SigO-RsoA-dependent transcription and this correlates with loss of RsiO binding affinity for SigO. A current model for RsiO function indicates that RsiO responds, either directly or indirectly, to increased extracytoplasmic hydrogen ion concentration and becomes inactivated. This results in the release of SigO into the cytoplasm, where it productively associates with RsoA and core RNAP to initiate transcription from target promoters in the cell.

Characterization of a protein-protein interaction within the SigO-RsoA two-subunit σ factor: the σ70 region 2.3-like segment of RsoA mediates interaction with SigO.

σ factors are single subunit general transcription factors that reversibly bind core RNA polymerase and mediate gene-specific transcription in bacteria. Previously, an atypical two-subunit σ factor was identified that activates transcription from a group of related promoters in Bacillus subtilis. Both of the subunits, named SigO and RsoA, share primary sequence similarity with the canonical σ70 family of σ factors and interact with each other and with RNA polymerase subunits. Here we show that the σ70 region 2.3-like segment of RsoA is unexpectedly sufficient for interaction with the amino-terminus of SigO and the ß' subunit. A mutational analysis of RsoA identified aromatic residues conserved amongst all RsoA homologues, and often amongst canonical σ factors, that are particularly important for the SigO-RsoA interaction. In a canonical σ factor, region 2.3 amino acids bind non-template strand DNA, trapping the promoter in a single-stranded state required for initiation of transcription. Accordingly, we speculate that RsoA region 2.3 protein-binding activity likely arose from a motif that, at least in its ancestral protein, participated in DNA-binding interactions.

Functional reconstitution of an unusual Firmicutes σ factor into a Gram-negative heterologous host.

Sigma (σ) factors are single-subunit proteins that reversibly bind RNA polymerase and play an important role in the transcription initiation process. An unusual 2-subunit σ factor, consisting of proteins SigO and RsoA, activates transcription from a group of related promoters in Bacillus subtilis. These 2 proteins specifically interact with each other and with RNA polymerase subunits. This system is widespread among species in several Bacillus-related genera, but otherwise appears restricted to the Firmicutes. Here, we reconstituted SigO-RsoA, and a cognate promoter, into the distantly related heterologous host Escherichia coli to examine whether this system can function in bacteria outside of the Firmicutes. We show that these proteins can productively associate with E. coli RNA polymerase and activate transcription, demonstrating that there are no structural barriers to function. In parallel, we tested a wide array of protein-protein interaction mutations and promoter mutations that impact SigO-RsoA function in both B. subtilis and E. coli and conclude that the SigO-RsoA system behaves, in most instances, similarly in both genetic backgrounds. These data raise the possibility of genetically isolating the system in this heterologous host and away from unknown B. subtilis factors that may also be playing a role in SigO-RsoA regulatory pathways, thus facilitating further study of the system. As a result of this work, we also provide a comprehensive mutational analysis of a SigO-RsoA promoter and report the preliminary identification of amino acids in SigO that play a role in mediating the SigO-RsoA protein-protein interaction.

Entering the Next Phase: Predicting Biological Effects of Biomolecular Condensates.

Biomolecular condensates are increasingly recognized as important drivers of cellular function; their dysregulation leads to pathology and disease. We discuss three questions in terms of the impending utility of data-driven techniques to predict condensate-driven biological outcomes, i.e., the impact of cellular state changes on condensates, the effect of condensates on biochemical processes within, and condensate properties that result in cellular dysregulation and disease.

Structural connectivity at term equivalent age and language in preterm children at 2 years corrected.

We previously reported interhemispheric structural hyperconnectivity bypassing the corpus callosum in children born extremely preterm (<28 weeks) versus term children. This increased connectivity was positively associated with language performance at 4-6 years of age in our prior work. In the present study, we aim to investigate whether this extracallosal connectivity develops in extremely preterm infants at term equivalent age by leveraging a prospective cohort study of 350 very and extremely preterm infants followed longitudinally in the Cincinnati Infant Neurodevelopment Early Prediction Study. For this secondary analysis, we included only children born extremely preterm and without significant brain injury (n = 95). We use higher-order diffusion modelling to assess the degree to which extracallosal pathways are present in extremely preterm infants and predictive of later language scores at 22-26 months corrected age. We compare results obtained from two higher-order diffusion models: generalized q-sampling imaging and constrained spherical deconvolution. Advanced MRI was obtained at term equivalent age (39-44 weeks post-menstrual age). For structural connectometry analysis, we assessed the level of correlation between white matter connectivity at the whole-brain level at term equivalent age and language scores at 2 years corrected age, controlling for post-menstrual age, sex, brain abnormality score and social risk. For our constrained spherical deconvolution analyses, we performed connectivity-based fixel enhancement, using probabilistic tractography to inform statistical testing of the hypothesis that fibre metrics at term equivalent age relate to language scores at 2 years corrected age after adjusting for covariates. Ninety-five infants were extremely preterm with no significant brain injury. Of these, 53 had complete neurodevelopmental and imaging data sets that passed quality control. In the connectometry analyses adjusted for covariates and multiple comparisons (P < 0.05), the following tracks were inversely correlated with language: bilateral cerebellar white matter and middle cerebellar peduncles, bilateral corticospinal tracks, posterior commissure and the posterior inferior fronto-occipital fasciculus. No tracks from the constrained spherical deconvolution/connectivity-based fixel enhancement analyses remained significant after correction for multiple comparisons. Our findings provide critical information about the ontogeny of structural brain networks supporting language in extremely preterm children. Greater connectivity in more posterior tracks that include the cerebellum and connections to the regions of the temporal lobes at term equivalent age appears to be disadvantageous for language development.

Early and consistent safe sleep practices in the neonatal intensive care unit: a sustained regional quality improvement initiative.

OBJECTIVE: To increase compliance with standardized safe sleep recommendations for patients in a cohort of regional level III/IV neonatal intensive care units (NICUs) in accordance with recently revised guidelines issued by the American Academy of Pediatrics (AAP). STUDY DESIGN: A regional quality improvement (QI) initiative led by a multidisciplinary task force standardized safe sleep criteria across participating NICU sites. Universal and unit-specific interventions were implemented via Plan-Do-Study-Act (PDSA) cycles with evaluation of compliance through routine crib audits, run chart completion, and Pareto chart analysis. RESULTS: Following QI implementation, compliance with safe sleep guidelines for eligible NICU infants improved from 34% to 90% from October 2019 through September 2022. CONCLUSION: Compliance with early, consistent modeling of safe sleep practices nearly tripled in this cohort of regional NICUs. A standardized, timely approach to safe sleep transition demonstrated dramatic and sustained improvement in the practice and modeling of safe sleep behaviors in the NICU.

Metabolic acclimation to excess light intensity in Chlamydomonas reinhardtii.

There are several well-described acclimation responses to excess light in green algae but the effect on metabolism has not been thoroughly investigated. This study examines the metabolic changes during photoacclimation to high-light (HL) stress in Chlamydomonas reinhardtii using nuclear magnetic resonance and mass spectrometry. Using principal component analysis, a clear metabolic response to HL intensity was observed on global metabolite pools, with major changes in the levels of amino acids and related nitrogen metabolites. Amino acid pools increased during short-term photoacclimation, but were especially prominent in HL-acclimated cultures. Unexpectedly, we observed an increase in mitochondrial metabolism through downstream photorespiratory pathways. The expression of two genes encoding key enzymes in the photorespiratory pathway, glycolate dehydrogenase and malate synthase, were highly responsive to the HL stress. We propose that this pathway contributes to metabolite pools involved in nitrogen assimilation and may play a direct role in photoacclimation. Our results suggest that primary and secondary metabolism is highly pliable and plays a critical role in coping with the energetic imbalance during HL exposure and a necessary adjustment to support an increased growth rate that is an effective energy sink for the excess reducing power generated during HL stress.

Loop-mediated isothermal amplification identifies nematode Leidynema in the hindgut of non-pest cockroach.

Cockroach microbiome studies generally focus on pest cockroach species belonging to the Blattidae and Ectobiidae families. There are no reports characterizing the gut microbiome of non-pest cockroach species Blaberus discoidalis (family Blaberidae), which is commonly used as a food source for insectivorous animals. We discovered the parasitic nematode Leidynema appendiculata in the B. discoidalis hindgut during initial work characterizing the gut microbiome of this organism. To determine the proportion of the B. discoidalis colony that was colonized by L. appendiculata, 28 S rDNA was amplified using two Methods: endpoint polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). B. discoidalis colonies were raised on three diet types (control, high fibre, and high fat and salt) for 21 days before dissection. Each individual was sexed during dissection to identify potential sex-based effects of colonization. Data collected were analysed to determine if diet and sex impacted parasite colonization patterns. LAMP detected a higher proportion of parasite positive samples when compared to endpoint PCR. No sex- or diet-based differences in L. appendiculata colonization were found. This study adds to the limited existing knowledge of the B. discoidalis gut microbiome.