Sanger sequencing and deconvolution of polyclonal infections: a quantitative approach to monitor drug-resistant Plasmodium falciparum.

BACKGROUND: Anti-malarial drug resistance in Plasmodium falciparum is a major public health problem in malaria-endemic regions. Although various technical improvements in sequencing methods have been introduced to identify SNPs, the conventional approach with current tools does not discriminate mixed infections, and thus can be improved for more sensitive surveillance of anti-malarial resistance to better inform control strategies. METHODS: We developed a computational approach for deconvolution of chromatograms generated by standard Sanger sequencing of PCR amplicons in order to quantify molecular marker variants of anti-malarial drug resistance genes [Plasmodium falciparum dihydropteorate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr)]. We validated this computational approach using mixtures of V1/S and FCR3 at varying proportions between 0 and 100%, then applied it to field samples collected in Doneguebougou, Mali in 2018. We determined the mean fraction of resistance alleles in individual samples, as well as the prevalence of infections carrying resistant parasites. FINDINGS: We observed a highly significant correlation between the predicted and measured proportions of V1/S and FCR3 alleles in mixed laboratory samples (all p < 0.001). Among field samples, the mean fraction of resistant Pfdhps alleles was 4.7% 431V, 95.9% 436F/A, 49.9% 437G, 0.0% 540E, 1.2% 581G and 1.5% 613S/T; corresponding prevalences were 50.0%, 100%, 72.5%, 0.0%, 25.0%, and 12.5%, respectively. The mean fraction of resistant Pfdhfr alleles was 0.6% 16V, 11.1% 50R, 89.0% 51I, 98.3% 59R, 74.7% 108T/N, 8.6% 140L and 8.7% 164L; corresponding prevalences were 12.5%, 75.0%, 100%, 100%, 100%, 50.0%, and 28.6%, respectively. We identified two new point mutations on the Pfdhps gene at codons D484T and D545N. INTERPRETATION: Computational deconvolution of sequencing chromatograms can discriminate varying proportions of antimalarial drug-sensitive versus -resistant alleles. This cost-effective and quantitative variant-sequencing approach will be useful for population-based surveys that characterize mixed infections at the individual level to survey known and unknown mutations in P. falciparum drug-resistance genes. FUNDING: This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). HM was supported by the African Postdoctoral Training Initiative (APTI) Fellowship program jointly managed by the US NIH, The African Academy of Sciences (AAS) and Bill & Melinda Gates Foundation (BMGF); Grant Reference Number: APTI-18-01.

Structure-guided design of VAR2CSA-based immunogens and a cocktail strategy for a placental malaria vaccine.

Placental accumulation of Plasmodium falciparum infected erythrocytes results in maternal anemia, low birth weight, and pregnancy loss. The parasite protein VAR2CSA facilitates the accumulation of infected erythrocytes in the placenta through interaction with the host receptor chondroitin sulfate A (CSA). Antibodies that prevent the VAR2CSA-CSA interaction correlate with protection from placental malaria, and VAR2CSA is a high-priority placental malaria vaccine antigen. Here, structure-guided design leveraging the full-length structures of VAR2CSA produced a stable immunogen that retains the critical conserved functional elements of VAR2CSA. The design expressed with a six-fold greater yield than the full-length protein and elicited antibodies that prevent adhesion of infected erythrocytes to CSA. The reduced size and adaptability of the designed immunogen enable efficient production of multiple variants of VAR2CSA for use in a cocktail vaccination strategy to increase the breadth of protection. These designs form strong foundations for the development of potent broadly protective placental malaria vaccines.

Relationships Between Vocal Fold Adduction Patterns, Vocal Acoustic Quality, and Vocal Effort in Individuals With and Without Hyperfunctional Voice Disorders.

OBJECTIVES/HYPOTHESIS: Increased vocal effort and aberrant vocal quality are often attributed to vocal fold hyperadduction in hyperfunctional voice disorders. However, there are currently no established methods to quantify vocal fold adduction beyond subjective descriptors in this clinical population. Furthermore, relationships between vocal fold adduction patterns, vocal effort severity, and vocal quality are not well characterized. Therefore, the objectives of this study were to (1) quantify vocal fold adduction, applying a previously validated method developed for patients with vocal fold paralysis, and (2) correlate these measures with acoustic vocal quality and self-perceived measures of vocal effort severity. METHODS: A deep learning program, Automated Glottic Action Tracking using artificial Intelligence, was used to track glottic angle configurations and vocal fold adduction velocities on laryngoscopic videos across 60 laryngoscopies (20 primary muscle tension dysphonia [pMTD], 20 phonotraumatic lesions, and 20 healthy controls). Voice samples were also acquired, and cepstral peak prominence (CPP) and H1-H2 acoustic measures were used to quantify vocal quality. Participants were also asked to rate their vocal effort on a 100 mm visual analog scale. RESULTS: There were no significant group differences in glottic angle configurations or vocal fold adduction velocities, although there were trends toward increased peak vocal fold adduction velocities in patients with hyperfunctional voice disorders compared to controls. Vocal effort was significantly higher in the two hyperfunctional groups compared to controls. CPP was significantly lower in the pMTD group, but there were no group differences in acoustic parameters between any of the other groups or for H1-H2 values. CONCLUSION: Despite significantly more vocal effort reported in patients with hyperfunctional voice disorders, there were no significant group differences in vocal fold adduction patterns. These findings suggest other physiologic mechanisms may also be responsible for the symptoms and genesis of pMTD and benign vocal fold lesions.

A deployable curriculum with 3D printed skills trainers for altered airway management.

BACKGROUND: Altered Airway Anatomy (AAA), including tracheostomies and laryngectomies, may represent an area of unease for non-Otolaryngology trainees, due to a lack of exposure, structured education, or dedicated training in altered airway management. Inability to effectively stabilize an altered airway is associated with significant risk of patient morbidity and mortality. This study aims to assess the efficacy of a concise curriculum using three-dimensional (3D) printed airway models for skill training in improving Anesthesiology trainees' competency in AAA management. METHODS: A prospective cohort of 42 anesthesiology residents at a tertiary care institution were guided through a 75-min curriculum on AAA, including case discussion, surgical video, and hands-on practice with tracheostomy and laryngectomy skills trainers. Pre- and post- course surveys assessing provider confidence (Likert scale) and knowledge (multiple choice questions) were administered. Additionally, an observed skills competency assessment was performed. RESULTS: Self-perceived confidence improved from a summative score across all domains of 23.65/40 pre-course to 36.39/40 post-course (n = 31, p < 0.001). Technical knowledge on multiple choice questions improved from 71 to 95% (n = 29, p < 0.001). In the completed skills competency assessment, 42/42 residents completed 5/5 assessed tasks successfully, demonstrating objective skills-based competency. CONCLUSIONS: This study demonstrates an improvement in anesthesiology resident self-assessed confidence, objective knowledge, and skills based competency surrounding management of patients with AAA following a 75-min simulation-based curriculum.

New Medical Device and Therapeutic Approvals in Otolaryngology: State of the Art Review of 2022.

Objective: To review new drugs and devices relevant to otolaryngology approved by the Food and Drug Administration (FDA) in 2022. Data Sources: Publicly available FDA data on drugs and devices approved in 2022. Review Methods: A preliminary screen was conducted to identify drugs and devices relevant to otolaryngology. A secondary screen by members of the American Academy of Otolaryngology-Head and Neck Surgery's (AAO-HNS) Medical Devices and Drugs Committee differentiated between minor updates and new approvals. The final list of drugs and devices was sent to members of each subspecialty for review and analysis. Conclusion: A total of 1251 devices and 37 drugs were identified on preliminary screening. Of these, 329 devices and 5 drugs were sent to subspecialists for further review, from which 37 devices and 2 novel drugs were selected for further analysis. The newly approved devices spanned all subspecialties within otolaryngology. Many of the newly approved devices aimed to enhance patient experience, including over-the-counter hearing aids, sleep monitoring devices, and refined CPAP devices. Other advances aimed to improve surgical access, convenience, or comfort in the operating room and clinic. Implications for Practice: Many new devices and drugs are approved each year to improve patient care and care delivery. By staying up to date with these advances, otolaryngologists can leverage new innovations to improve the safety and quality of care. Given the recent approval of these devices, further studies are needed to assess long-term impact within the field of otolaryngology.

Optical Flow Analysis of Paralaryngeal Muscle Movement.

OBJECTIVES: The paralaryngeal muscles are thought to be hyperfunctional with phonation in patients with primary muscle tension dysphonia (pMTD). However, objective, quantitative tools to assess paralaryngeal movement patterns lack. The objectives of this study were to (1) validate the use of optical flow to characterize paralaryngeal movement patterns with phonation, (2) characterize phonatory optical flow velocities and variability of the paralaryngeal muscles before and after a vocal load challenge, and (3) compare phonatory optical flow measures to standard laryngoscopic, acoustic, and self-perceptual assessments. METHODS: Phonatory movement velocities and variability of the paralaryngeal muscles at vocal onsets and offsets were quantified from ultrasound videos and optical flow methods across 42 subjects with and without a diagnosis of pMTD, before and after a vocal load challenge. Severity of laryngoscopic mediolateral supraglottic compression, acoustic perturbation, and ratings of vocal effort and discomfort were also obtained at both time points. RESULTS: There were no significant differences in optical flow measures of the paralaryngeal muscles with phonation between patients with pMTD and controls. Patients with pMTD had significantly more supraglottic compression, higher acoustic perturbations, and higher vocal effort and vocal tract discomfort ratings. Vocal load had a significant effect on vocal effort and discomfort but not on supraglottic compression, acoustics, or optical flow measures of the paralaryngeal muscles. CONCLUSION: Optical flow methods can be used to study paralaryngeal muscle movement velocity and variability patterns during vocal productions, although the role of the paralaryngeal in pMTD diagnostics (e.g., vocal hyperfunction) remains suspect. LEVEL OF EVIDENCE: 2 Laryngoscope, 134:1792-1801, 2024.

Dissecting Clinical Features of COVID-19 in a Cohort of 21,312 Acute Care Patients.

COVID-19 has resulted in over 645 million hospitalization and 7 million deaths globally. However, many questions still remain about clinical complications in COVID-19 and if these complications changed with different circulating SARS-CoV-2 strains. We analyzed a 2.5-year retrospective cohort of 47,063 encounters for 21,312 acute care patients at five Central Texas hospitals and define distinct trajectory groups (TGs) with latent class mixed modeling, based on the World Health Organization COVID-19 Ordinal Scale. Using this TG framework, we evaluated the association of demographics, diagnoses, vitals, labs, imaging, consultations, and medications with COVID-19 severity and broad clinical outcomes. Patients within 6 distinct TGs differed in manifestations of multi-organ disease and multiple clinical factors. The proportion of mild patients increased over time, particularly during Omicron waves. Age separated mild and fatal patients, though did not distinguish patients with severe versus critical disease. Male and Hispanic/Latino demographics were associated with more severe/critical TGs. More severe patients had a higher rate of neuropsychiatric diagnoses, consultations, and brain imaging, which did not change significantly in severe patients across SARS-CoV-2 variant waves. More severely affected patients also demonstrated an immunological signature of high neutrophils and immature granulocytes, and low lymphocytes and monocytes. Interestingly, low albumin was one of the best lab predictors of COVID-19 severity in association with higher malnutrition in severe/critical patients, raising concern of nutritional insufficiency influencing COVID-19 outcomes. Despite this, only a small fraction of severe/critical patients had nutritional labs checked (pre-albumin, thiamine, Vitamin D, B vitamins) or received targeted interventions to address nutritional deficiencies such as vitamin replacement. Our findings underscore the significant link between COVID-19 severity, neuropsychiatric complications, and nutritional insufficiency as key risk factors of COVID-19 outcomes and raise the question of the need for more widespread early assessment of patients' neurological, psychiatric, and nutritional status in acute care settings to help identify those at risk of severe disease outcomes.

Relationships Between Laryngoscopic Analysis Metrics of Supraglottic Compression and Vocal Effort in Primary Muscle Tension Dysphonia.

PURPOSE: Supraglottic compression is thought to underlie vocal effort in patients with primary muscle tension dysphonia (pMTD). However, the relationship between supraglottic compression and vocal effort in this clinical population remains unclear. Gold standard laryngoscopic assessment metrics for supraglottic compression are also lacking. The goals of this study were to identify metrics proposed in the literature that could distinguish patients diagnosed with pMTD from typical voice users and determine their relationships to the vocal effort. METHODS: Flexible laryngeal endoscopy was performed on 50 participants (25 pMTD, 25 controls). The presence of supraglottic compression was characterized using a categorical (nominal) scale and severity was quantified on ordinal and continuous scales. The three laryngoscopic metrics were correlated with self-perceived ratings of vocal effort on a 100 mm visual analog scale. RESULTS: Inter-rater reliability was strongest for the continuous scale (P's < 0.0001) compared to categorical (P's < 0.001) and ordinal (P's < 0.001) scales. The presence of different supraglottic compression patterns varied in both groups, and there were no significant group differences on categorical (P's > 0.05) scales. Mediolateral (M-L) supraglottic compression was significantly greater in the pMTD group (P < 0.0001), and anteroposterior (A-P) compression was significantly greater in the control group (P = 0.001) using continuous scales. There were no significant relationships between any of the three laryngoscopic metric types and vocal effort ratings (P's > 0.05), except for a significantly positive relationship between anterior-posterior compression on the ordinal scale and vocal effort in the control group (P = 0.047). CONCLUSIONS: Continuous scales are reliable and valid for distinguishing individuals with pMTD from those without voice disorders, especially occupational voice users. M-L supraglottic compression may be a better indicator of pMTD than A-P compression. However, the poor correlation between supraglottic compression and vocal effort suggests that one may not influence the other. Future studies should focus on other mechanisms underlying vocal effort in patients with pMTD.

MtrA modulates Mycobacterium tuberculosis cell division in host microenvironments to mediate intrinsic resistance and drug tolerance.

The success of Mycobacterium tuberculosis (Mtb) is largely attributed to its ability to physiologically adapt and withstand diverse localized stresses within host microenvironments. Here, we present a data-driven model (EGRIN 2.0) that captures the dynamic interplay of environmental cues and genome-encoded regulatory programs in Mtb. Analysis of EGRIN 2.0 shows how modulation of the MtrAB two-component signaling system tunes Mtb growth in response to related host microenvironmental cues. Disruption of MtrAB by tunable CRISPR interference confirms that the signaling system regulates multiple peptidoglycan hydrolases, among other targets, that are important for cell division. Further, MtrA decreases the effectiveness of antibiotics by mechanisms of both intrinsic resistance and drug tolerance. Together, the model-enabled dissection of complex MtrA regulation highlights its importance as a drug target and illustrates how EGRIN 2.0 facilitates discovery and mechanistic characterization of Mtb adaptation to specific host microenvironments within the host.

Preclinical evaluations of Pfs25-EPA and Pfs230D1-EPA in AS01 for a vaccine to reduce malaria transmission.

Malaria transmission-blocking vaccine candidates Pfs25-EPA and Pfs230D1-EPA target sexual stage development of Plasmodium falciparum parasites in the mosquito host, thereby reducing mosquito infectivity. When formulated on Alhydrogel, Pfs25-EPA has demonstrated safety and immunogenicity in a phase 1 field trial, while Pfs230D1-EPA has shown superior activity to Pfs25-EPA in a phase 1 US trial and has entered phase 2 field trials. Development continues to enhance immunogenicity of these candidates toward producing a vaccine to reduce malaria transmission (VRMT) with both pre-erythrocytic (i.e., anti-infection) and transmission-blocking components. GSK Adjuvant Systems have demonstrated successful potency in pre-erythrocytic vaccine trials and might offer a common platform for VRMT development. Here, we describe preclinical evaluations of Pfs25-EPA and Pfs230D1-EPA nanoparticles with GSK platforms. Formulations were stable after a series of assessments and induced superior antibody titers and functional activity in CD-1 mice, compared to Alhydrogel formulations of the same antigens.

Malaria transmission-blocking vaccines Pfs230D1-EPA and Pfs25-EPA in Alhydrogel in healthy Malian adults; a phase 1, randomised, controlled trial.

BACKGROUND: Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians. METHODS: We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4·5, and 16·5 of either 47 µg Pfs25, 40 µg Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 µg Pfs25 plus 40 µg Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462. FINDINGS: Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 µg), Pfs230D1-EPA/Alhydrogel (15 µg) or comparator, followed by Pfs25-EPA/Alhydrogel (16 µg) plus Pfs230D1-EPA/Alhydrogel (15 µg) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 µg Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 µg Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 µg Pfs25-EPA/Alhydrogel plus 40 µg Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77·8 [95% CI 56·9-106·3], 146·4 [108·3-198·0], and 410·2 [301·6-558·0]; Pfs25 geometric mean 177·7 [130·3-242·4] and 315·7 [209·9-474·6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74·5% [66·6-82·5]) and Pfs25 plus Pfs230D1 (68·6% [57·3-79·8]), after the third dose and after the fourth dose (88·9% [81·7-96·2] for Pfs230D1 and 85·0% [78·4-91·5] Pfs25 plus Pfs230D1) but not for Pfs25 (58·2% [49·1-67·3] after the third dose and 58·2% [48·5-67·9] after the fourth dose). Pfs230D1 transmission-reducing activity (73·7% [64·1-83·3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0·013 to 0·014], Pfs230D1 22 [2%] of 954 [-0·005 to 0·027], and combination 11 [1%] of 940 [-0·024 to 0·002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0·024 to 0·001). INTERPRETATION: Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness. FUNDING: Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.

Acute Cricoarytenoid Joint Fixation Following Routine Intubation: A Case Report.

Prolonged intubation is a common cause of injury to the posterior larynx often resulting in cricoarytenoid joint (CAJ) fixation and posterior glottic stenosis (PGS). We present a case of respiratory failure due to acute bilateral CAJ fixation and PGS following only 2 days of intubation for routine cardiac surgery. A tracheostomy was placed due to critical airway obstruction. Clinicians should remain vigilant for laryngeal injury presenting as CAJ fixation and PGS. Prompt surgical consultation is advised as early intervention is associated with reduced morbidity.

Quantification of False Vocal Fold Hyperfunction During Quiet Breathing in Muscle Tension Dysphonia.

BACKGROUND/OBJECTIVES: False vocal fold (FVF) hyperfunction during phonation is thought to be a diagnostic sign of primary muscle tension dysphonia (pMTD). However, hyperfunctional patterns with phonation are also observed in typical speakers. This study tested the hypothesis that FVF posturing during quiet breathing, as measured by the curvature of FVF, could differentiate patients with pMTD from typical speakers. METHODS: Laryngoscopic images were collected prospectively in 30 subjects with pMTD and 33 typical speakers. Images were acquired at the end of expiration and maximal inspiration during quiet breathing, during sustained /i/, and during loud phonation before and after a 30-min vocal loading task. The FVF curvature (degree of concavity/convexity) was quantified using a novel curvature index (CI, >0 for hyperfunctional/convex, <0 for "relaxed"/concave) and compared between the two groups. RESULTS: At end-expiration, the pMTD group adopted a convex FVF contour, whereas the control group adopted a concave FVF contour (mean CI 0.123 [SEM 0.046] vs. -0.093 [SEM 0.030], p = 0.0002) before vocal loading. At maximal inspiration, the pMTD group had a neutral/straight FVF contour, whereas the control group had a concave FVF contour (mean CI 0.012 [SEM 0.038] vs. -0.155 [SEM 0.018], p = 0.0002). There were no statistically significant differences in FVF curvature between groups in either the sustained voiced or loud conditions. Vocal loading did not change any of these relationships. CONCLUSIONS: A hyperfunctional posture of the FVFs during quiet breathing especially at end-expiration may be more indicative of a hyperfunctional voice disorder than supraglottic constriction during voicing. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:3449-3454, 2023.

High-velocity power training has similar effects to traditional resistance training for functional performance in older adults: a systematic review.

QUESTIONS: What is the effect of high-velocity power training (HVPT) compared with traditional resistance training (TRT) on functional performance in older adults? What is the quality of intervention reporting for the relevant literature? DESIGN: Systematic review and meta-analysis of randomised controlled trials. PARTICIPANTS: Older adults (aged > 60 years), regardless of health status, baseline functional capacity or residential status. INTERVENTIONS: High-velocity power training with the intent to perform the concentric phase as quickly as possible compared with traditional moderate-velocity resistance training performed with a concentric phase of ≥ 2 seconds. OUTCOME MEASURES: Short Physical Performance Battery (SPPB), Timed Up and Go test (TUG), five times sit-to-stand test (5-STS), 30-second sit-to-stand test (30-STS), gait speed tests, static or dynamic balance tests, stair climb tests and walking tests for distance. The quality of intervention reporting was assessed with the Consensus on Exercise Reporting Template (CERT) score. RESULTS: Nineteen trials with 1,055 participants were included in the meta-analysis. Compared with TRT, HVPT had a weak-to-moderate effect on change from baseline scores for the SPPB (SMD 0.27, 95% CI 0.02 to 0.53; low-quality evidence) and TUG (SMD 0.35, 95% CI 0.06 to 0.63; low-quality evidence). The effect of HVPT relative to TRT for other outcomes remained very uncertain. The average CERT score across all trials was 53%, with two trials rated high quality and four rated moderate quality. CONCLUSION: HVPT had similar effects to TRT for functional performance in older adults, but there is considerable uncertainty in most estimates. HVPT had better effects on the SPPB and TUG, but it is unclear whether the benefit is large enough to be clinically worthwhile.

Extrinsic Laryngeal Muscle Tension in Primary Muscle Tension Dysphonia with Shear Wave Elastography.

OBJECTIVES: It has been assumed that patients with primary muscle tension dysphonia (pMTD) have more extrinsic laryngeal muscle (ELM) tension, but tools to study this phenomenon lack. Shear wave elastography (SWE) is a potential method to address these shortcomings. The objectives of this study were to apply SWE to the ELMs, compare SWE measures to standard clinical metrics, and determine group differences in pMTD and typical voice users before and after vocal load. METHODS: SWE measurements of the ELMs from ultrasound examinations of the anterior neck, supraglottic compression severities from laryngoscopic images, cepstral peak prominences (CPP) from voice recordings, and self-perceptual ratings of vocal effort and discomfort were obtained in voice users with (N = 30) and without (N = 35) pMTD, before and after a vocal load challenge. RESULTS: ELM tension significantly increased from rest-to-voiced conditions in both groups. However, the groups were similar in their ELM stiffness levels at SWE at baseline, during vocalization, and post-vocal load. Levels of vocal effort and discomfort and supraglottic compression were significantly higher and CPP was significantly lower in the pMTD group. Vocal load had a significant effect on vocal effort and discomfort but not on laryngeal or acoustic patterns. CONCLUSION: SWE can be used to quantify ELM tension with voicing. Although the pMTD group reported significantly higher levels of vocal effort and vocal tract discomfort and, on average, exhibited significantly more severe supraglottic compression and lower CPP values, there were no group differences in levels of ELM tension using SWE. LEVEL OF EVIDENCE: 2 Laryngoscope, 133:3482-3491, 2023.

Use of Motion Capture Technology to Study Extrinsic Laryngeal Muscle Tension and Hyperfunction.

OBJECTIVES: Patients with primary muscle tension dysphonia (pMTD) commonly report paralaryngeal pain and discomfort, and extrinsic laryngeal muscle (ELM) tension and hyperfunction are commonly implicated. However, quantitative physiological metrics to study ELM movement patterns for the characterization of pMTD diagnosis and monitoring of treatment progress are lacking. The objectives of this study were to validate motion capture (MoCap) technology to study ELM kinematics, determine whether MoCap could distinguish ELM tension and hyperfunction between individuals with and without pMTD, and investigate relationships between common clinical voice metrics and ELM kinematics. METHODS: Thirty subjects (15 with pMTD and 15 controls) were recruited for the study. Sixteen markers were placed on different anatomical landmarks on the chin and anterior neck. Movements across these regions were tracked during four voice and speech tasks using two three-dimensional cameras. Movement displacement and variability were determined based on 16 key-points and 53 edges. RESULTS: Intraclass correlation coefficients demonstrated high intra- and inter-rater reliability (p's < 0.001). Other than greater movement displacements around the thyrohyoid space during longer phrasing (reading passage, 30-s diadochokinetics) and more movement variability in patients with pMTD, kinematic patterns between groups were similar across the 53 edges for the four voice and speech tasks. There were also no significant correlations between ELM kinematics and standard voice metrics. CONCLUSION: Results demonstrate the feasibility and reliability of MoCap for the study of ELM kinematics. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:3472-3481, 2023.

Mycobacterium tuberculosis Gene Expression Associated With Fluoroquinolone Resistance and Efflux Pump Inhibition.

BACKGROUND: We evaluated the relationship between response to efflux pump inhibition in fluoroquinolone-resistant Mycobacterium tuberculosis (Mtb) isolates and differences in gene expression and expression quantitative trait loci (eQTL). METHODS: We determined ofloxacin minimum inhibitory concentration (MIC) for ofloxacin-resistant and -susceptible Mtb isolates without and with the efflux pump inhibitor verapamil. We performed RNA sequencing (RNA-seq), whole genome sequencing (WGS), and eQTL analysis, focusing on efflux pump, transport, and secretion-associated genes. RESULTS: Of 42 ofloxacin-resistant Mtb isolates, 27 had adequate WGS coverage and acceptable RNA-seq quality. Of these 27, 7 had >2-fold reduction in ofloxacin MIC with verapamil; 6 had 2-fold reduction, and 14 had <2-fold reduction. Five genes (including Rv0191) had significantly increased expression in the MIC fold change >2 compared to <2 groups. Among regulated genes, 31 eQTLs (without ofloxacin) and 35 eQTLs (with ofloxacin) had significant allele frequency differences between MIC fold change >2 and <2 groups. Of these, Rv1410c, Rv2459, and Rv3756c (without ofloxacin) and Rv0191 and Rv3756c (with ofloxacin) have previously been associated with antituberculosis drug resistance. CONCLUSIONS: In this first reported eQTL analysis in Mtb, Rv0191 had increased gene expression and significance in eQTL analysis, making it a candidate for functional evaluation of efflux-mediated fluoroquinolone resistance in Mtb.

Examining Use and Effectiveness of Teletherapy for Patients with Dysphonia.

OBJECTIVE(S)/HYPOTHESIS: Virtual therapy (teletherapy) for patients with dysphonia has become ubiquitous in the COVID-19 era. However, barriers to widespread implementation are evident, including unpredictable insurance coverage attributed to limited evidence supporting this approach. In our single-institution cohort, our objective was to show strong evidence for use and effectiveness of teletherapy for patients with dysphonia. STUDY DESIGN: Single institution, retrospective cohort study. MATERIAL AND METHODS: This was an analysis of all patients referred for speech therapy with dysphonia as primary diagnosis from 4/1/2020 to 7/1/2021 and in whom all therapy sessions were delivered in a teletherapy format. We collated and analyzed demographics and clinical characteristics and adherence to the teletherapy program. We assessed changes in perceptual assessments and vocal capabilities (GRBAS, MPT), patient-reported outcomes (V-RQOL), and metrics of session outcomes (complexity of vocal tasks, carry-over of target voice) pre- and post-teletherapy using student's t test and chi-square test. RESULTS: Our cohort included 234 patients (mean [SD] age 52 [20] years) residing a mean (SD) distance of 51.3 (67.1) miles from our institution. The most common referral diagnosis was muscle tension dysphonia (n = 145, 62.0% patients). Patients attended a mean (SD) of 4.2 (3.0) sessions; 68.0% (n = 159) of patients completed four or more sessions and/or were deemed appropriate for discharge from teletherapy program. Statistically significant improvements were seen in complexity and consistency of vocal tasks with consistent gains in carry-over of target voice for isolated tasks and connected speech. CONCLUSIONS: Teletherapy is a versatile and effective approach for treatment of patients with dysphonia of varying age, geography, and diagnoses.