Uncialamycin-based antibody-drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect.

Antibody-drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin [Formula: see text], have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.

RNA Controls PolyQ Protein Phase Transitions.

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophysical properties of phase-separated compartments.

Impact of cystic fibrosis multidisciplinary virtual clinics on patient experience, time commitments and costs.

BACKGROUND AND AIMS: The experience of outpatient care may differ for select patient groups. This prospective study evaluates the adult patient experience of multidisciplinary outpatient cystic fibrosis (CF) care with videoconferencing through telehealth compared with face-to-face care the year prior. METHODS: People with CF without a lung transplant were recruited. Patient-reported outcomes were obtained at commencement and 12 months into the study, reflecting both their face-to-face and telehealth through videoconferencing experience, respectively. Three patient cohorts were analysed: (i) participants with a regional residence, (ii) participants with a nonregional including metropolitan residence and (iii) participants with colonised multiresistant microbiota. RESULTS: Seventy-four patients were enrolled in the study (mean age, 37 ± 11 years; 50% male; mean forced expiratory volume in the first second of expiration, 60% [standard deviation, 23]) between February 2020 and May 2021. No differences between models were observed in the participants' rating of the health care team, general and mental health rating, and their confidence in handling treatment plans at home. No between-group differences in the Cystic Fibrosis Questionnaire - Revised (CFQ-R) were observed. Travel duration and the cost of attending a clinic was significantly reduced, particularly for the regional group (4 h, AU$108 per clinic; P < 0.05). A total of 93% respondents preferred to continue with a hybrid approach. CONCLUSION: In this pilot study, participants' experience of care and quality of life were no different with face-to-face and virtual care between the groups. Time and cost-savings, particularly for patients living in regional areas, were observed. Most participants preferred to continue with a hybrid model for outpatient care.

Does physical activity mediate the associations between blue space and mental health? A cross-sectional study in Australia.

BACKGROUND: Research has begun to examine whether blue space is beneficial to mental health. While results are promising, it is difficult to know which aspects of mental health or mental ill-health may benefit most. Physical activity has been proposed as one potential mechanism via which blue space may be associated with better mental health. However, very few studies have examined mechanisms. We examined associations between blue space proximity and a range of mental health outcomes and examined which of these associations were mediated by physical activity. METHODS: 350 participants (M = 38.74, SD = 14.92, 70% female) self-reported their weekly physical activity and completed measures of depression, anxiety, and psychological wellbeing. We then used GIS software to calculate blue space proximity (i.e., coastal and inland), and structural equation modelling with mediation paths to determine the role of physical activity in the associations between bluespace and mental health. RESULTS: Physical activity partially mediated the associations between coastal proximity and depression (ß = 0.02, 95% CI = 0.001, 0.05), anxiety (ß = 0.03, 95% CI = 0.01, 0.06), and wellbeing (ß = - 0.03, 95% CI = - 0.08, - 0.01), and fully mediated the associations between inland water proximity and depression (ß = 0.02, 95% CI = 0.003, 0.05), anxiety (ß = 0.03, 95% CI = 0.01, 0.07), and wellbeing (ß = - 0.03, 95% CI = - 0.07, - 0.01). CONCLUSION: While physical activity appears to explain associations between inland blue space and mental health outcomes, it only partially explains the association between coastal blue space and mental health, suggesting other mechanisms may play a role and even inactive exposure may be beneficial.

Development of a psychosocial oncology core curriculum for multidisciplinary education and training: Initial content validation using the modified Delphi Method.

OBJECTIVE: Despite its four decade history, the multidisciplinary specialty of psychosocial oncology (PSO) has no official consensus on core content. In 2014, the American Psychosocial Oncology Society (APOS) Board charged the APOS Professional Education Committee with outlining curricular content needed for core competence. METHODS: Content validation was completed using a four-phase modified Delphi Method. During Phase I, a Professional Education Committee subgroup proposed domains and items, which were rated by the APOS Fellows and Board via online survey. During Phase II, Fellows completed a second, revised survey. Phase III incorporated early career members. Developmental and diversity items were integrated into each domain. In Phase IV, a larger group of subject matter experts were surveyed, with feedback incorporated. Validation across phases involved average rating thresholds, intraclass correlations, and final percent agreement. RESULTS: The Delphi Method supported 12 content domains: Cancer Basics, Psychosocial Oncology, Professional Development, Ethics, Emotional/Psychological Concerns, Sexuality and Relationship Concerns, Spiritual/Religious Concerns, Healthcare Communication and Decision Making, Social/Practical Problems, Caregiver Concerns, Cognitive Concerns, Physical Symptoms and Psychosocial Assessment/Treatment. High levels of agreement were achieved across domains (86%-100%) and items, with no significant rating differences by discipline. CONCLUSIONS: This proposed core content can enhance and standardize education and training in PSO including APOS' Virtual Psychosocial Oncology Core Curriculum, focused on broadly expanding the PSO workforce, particularly in underserved areas. Next steps include development of core competencies and establishment of online training modules based on this content validation.

Depressive Symptoms and Drinking to Cope in Relation to Alcohol Use Outcomes among White and Black/African American College Students.

OBJECTIVE: Prior research shows that Black/African American adults experience more negative alcohol use consequences than White adults, despite lower alcohol consumption. Research also shows that Black/African Americans experience higher rates of depression, which can increase risk for alcohol consumption and alcohol use disorder (AUD) through drinking to cope. We examined associations between depressive symptoms and drinking to cope with alcohol consumption and AUD symptoms among White and Black/African American college students. METHODS: Participants completed an online survey during the fall and spring semester of their first year of college (N = 2,168, 62.8% female, 75.8% White). Path analyses were conducted to examine whether depressive symptoms and drinking to cope mediated the association between race/ethnicity and alcohol use outcomes, and whether race/ethnicity moderated the associations between depressive symptoms, drinking to cope, and alcohol use outcomes. RESULTS: Results indicated that Black/African Americans had lower levels of depressive symptoms, which were associated with lower drinking to cope, and in turn associated with lower alcohol consumption and AUD symptoms. Multigroup analysis indicated that the pattern of associations between depressive symptoms, drinking to cope, and alcohol use outcomes were largely similar between White and Black/African American college students and between males and females, except that the association between depressive symptoms and drinking to cope appeared to be stronger for Whites than for Black/African American students. CONCLUSION: Depressive symptoms and drinking to cope are risk factors in relation to alcohol use outcomes among White and Black/African American college students and partially account for the link between race/ethnicity and alcohol use outcomes.Supplemental data for this article is available online at https://doi.org/10.1080/10826084.2022.2034871 .

Design, Synthesis, and Biological Evaluation of Tubulysin Analogues, Linker-Drugs, and Antibody-Drug Conjugates, Insights into Structure-Activity Relationships, and Tubulysin-Tubulin Binding Derived from X-ray Crystallographic Analysis.

Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/ß interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations.

Liquid buccal mucosa graft endoscopic urethroplasty: a validation animal study.

PURPOSE: To validate a novel method of urethral stricture treatment using liquid buccal mucosal grafts (LBMG) to augment direct vision internal urethrotomy (DVIU) in an animal model. MATERIALS AND METHODS: A rabbit stricture model was used to test this method. Strictures were induced in 26 rabbits using electroresection of urethral epithelium. The animals were randomized into two groups: Group-1, treated with DVIU and LBMG in fibrin glue, and Group-2, DVIU with fibrin glue only. LBMG was prepared by suspension of mechanically minced buccal mucosa micrografts in fibrin glue. This LBMG-fibrin glue mixture was later injected into the urethrotomies of Group-1 animals. All animals were killed at 24 weeks after repeat retrograde urethrogram (RUG) and urethroscopy by surgeon blinded to the treatment arm. Radiographic images and histological specimens were reviewed by a radiologist and a pathologist, respectively, blinded to the treatment arm. Stricture treatment was considered a success if a diameter measured on RUG increased by ≥ 50% compared to pre-treatment RUG diameter. Histological specimens were assessed for the presence of BMG engraftment. RESULTS: In Group-1, 8/12(67%) animals demonstrated engraftment of LBMG, compared to none in Group-2 (p = 0.0005). 7/12(58%) in Group-1 showed radiographic resolution/improvement of strictures compared to 5/13 Group-2 rabbits (38%, p = 0.145). The median percent change for the Group-1 was 59%, compared to 41.6% for Group-2 (p = 0.29). CONCLUSION: This proof-of-concept study demonstrates feasibility of LBMG for endoscopic urethral stricture repairs. Further studies are needed to establish the role of this novel concept in treatment of urethral strictures.

Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching.

Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.

You Can Only Go Full Speed for So Long: The Career Development of Psychologists Working in Psychosocial Oncology Settings.

Cancer centers have adopted a holistic approach to cancer treatment to better meet the psychosocial needs of cancer survivors. However, the current number of psychosocial providers in oncology is inadequate to meet the growing demand and psychosocial providers may face barriers in accessing oncology-specific training. The current study aims to explore the career development of psychologists working in oncology to inform training and workplace supports, as well as to inform training for health psychologists interested in other sub-specialties. Interviews were conducted with 20 psychologists with oncology work experience. Data were analyzed using the consensual qualitative research method. Results indicated three primary domains: (a) factors influencing entry into the field, (b) factors influencing ongoing career decision-making, and (c) factors influencing success in psychosocial oncology. The complexities of these domains are discussed; suggestions for supporting psychologists interested in psychosocial oncology at individual as well as systemic levels are provided.

Age-related differences in neuropsychological assessment of fetal alcohol spectrum disorder: a cross-sectional study.

This cross-sectional study examined 6 key areas of neuropsychological functioning (cognitive, academic, attention, executive function, adaptive skills) comparing adolescents and school-age children with prenatal alcohol exposure (PAE). The aims were: (i) to examine which neuropsychological measures were predictive of an FASD diagnosis in adolescents and school-age children with PAE, and (ii) to compare the neuropsychological performance of adolescents and children diagnosed with FASD. Hierarchical logistic regressions determined that the Full-Scale IQ, Verbal Comprehension and Perceptual Reasoning indices, basic reading and math skills, adaptive functioning at school, and components of executive functioning (dependent on age) improved the probability of an accurate FASD diagnosis in both groups: 9.1% to 19.2% for adolescents and 10.9% to 19.4% for school-age children (61.5%-80.9% correct classifications overall). For the age comparison analyses (ANOVAs/MANOVAs), a significant difference was observed in the cognitive domain, as well as with basic math skills (trend) in the sample diagnosed with FASD, with lower scores observed for adolescents across these measures. These findings provide further evidence for age differences in neuropsychological assessment as well as increased neuropsychological difficulties in adolescence by comparison with childhood with FASD. Longitudinal studies will be needed to make further inferences about developmental changes in neuropsychological functioning in FASD.

Biophysical characterization of organelle-based RNA/protein liquid phases using microfluidics.

Living cells contain numerous membrane-less RNA/protein (RNP) bodies that assemble by intracellular liquid-liquid phase separation. The properties of these condensed phase droplets are increasingly recognized as important in their physiological function within living cells, and also through the link to protein aggregation pathologies. However, techniques such as droplet coalescence analysis or standard microrheology do not always enable robust property measurements of model RNA/protein droplets in vitro. Here, we introduce a microfluidic platform that drives protein droplets into a single large phase, which facilitates viscosity measurements using passive microrheology and/or active two-phase flow analysis. We use this technique to study various phase separating proteins from structures including P granules, nucleoli, and Whi3 droplets. In each case, droplets exhibit simple liquid behavior, with shear rate-independent viscosities, over observed timescales. Interestingly, we find that a reported order of magnitude difference between the timescale of Whi3 and LAF-1 droplet coalescence is driven by large differences in surface tension rather than viscosity, with implications for droplet assembly and function. The ability to simultaneously perform active and passive microrheological measurements enables studying the impact of ATP-dependent biological activity on RNP droplets, which is a key area for future research.

Clinical impact of the interstitial lung disease multidisciplinary service.

BACKGROUND AND OBJECTIVE: Multidisciplinary discussions (MDDs) have been shown to improve diagnostic accuracy in interstitial lung disease (ILD) diagnosis. However, their clinical impact on patient care has never been clearly demonstrated. We describe the effect that an ILD multidisciplinary service has upon the diagnosis and management of patients with suspected ILD. METHODS: Patients at two specialized centres with suspected ILD underwent ILD multidisciplinary team review (ILD-MDT) (standard ILD clinic visit and diagnostic review at ILD-MDD). We compared changes in ILD diagnosis and management at referral to those following the ILD-MDT. RESULTS: Ninety patients, 60% males (54/90), aged 67.3 years (SD = 11.4) were reviewed for suspected ILD. Overall, the ILD-MDT resulted in a change in specific ILD diagnosis in 48/90 (53%) patients. Of the 27 patients referred with a diagnosis of idiopathic pulmonary fibrosis (IPF), the diagnosis was changed at MDD in 10 patients. In contrast, seven patients had their diagnosis changed to IPF. There was also a significant reduction in 'unclassifiable' diseases and disease behaviour classifications provided additional information beyond ILD diagnosis. CONCLUSION: Dedicated tertiary ILD-MDT service has an important clinical impact on the care of the ILD patient, with frequent changes in ILD diagnosis and subsequent management. Further research to investigate long-term clinical outcomes of ILD-MDT is required.

The merozoite surface protein 1 complex is a platform for binding to human erythrocytes by Plasmodium falciparum.

Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2-4 × 10(-7) m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.

Proinflammatory milieu in combat-related PTSD is independent of depression and early life stress.

BACKGROUND: Chronic inflammation may be involved in combat-related post-traumatic stress disorder (PTSD) and may help explain comorbid physical diseases. However, the extent to which combat exposure per se, depression, or early life trauma, all of which are associated with combat PTSD, may confound the relationship between PTSD and inflammation is unclear. METHODS: We quantified interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and C-reactive protein (CRP) in 51 combat-exposed males with PTSD and 51 combat-exposed males without PTSD, and assessed PTSD and depression severity as well as history of early life trauma. To decrease the possibility of Type I errors, we summed standardized scores of IL-1ß, IL-6, TNFα, IFNγ and CRP into a total "pro-inflammatory score". PTSD symptom severity was assessed with the Clinician Administered PTSD Scale (CAPS) rating scale. RESULTS: Subjects with PTSD had significantly higher pro-inflammatory scores compared to combat-exposed subjects without PTSD (p=0.006), and even after controlling for early life trauma, depression diagnosis and severity, body mass index, ethnicity, education, asthma/allergies, time since combat and the use of possibly confounding medications (p=0.002). Within the PTSD group, the pro-inflammatory score was not significantly correlated with depressive symptom severity, CAPS total score, or with the number of early life traumas. CONCLUSIONS: Combat-related PTSD in males is associated with higher levels of pro-inflammatory cytokines, even after accounting for depression and early life trauma. These results, from one of the largest studies of inflammatory cytokines in PTSD to date, suggest that immune activation may be a core element of PTSD pathophysiology more so than a signature of combat exposure alone.

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1.

Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actin's polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly, having a substantially reduced filament-binding loop and C-terminal α4 helix. Despite this divergence in structure, we demonstrate that PfADF1 is capable of efficient actin filament severing. Furthermore, this severing occurs despite PfADF1's low binding affinity for filaments. Comparative structural analysis along with biochemical and microscopy evidence establishes that severing is reliant on the availability of an exposed basic residue in the filament-binding loop, a conserved minimal requirement that defines AC-mediated filament disassembly across eukaryotic cells.

The operational development of diagnostic seasickness criteria through a human cyber-physical system.

Thresholds that guide diagnoses of probable and acceptable seasickness levels on board ships are scarcely reported in literature. Motion sickness incidence and motion sickness dose value thresholds exist, but are defined for specific environments, such as naval, or offered merely as optional criteria for ship performance metrics. The presented work communicates a novel means of developing seasickness diagnostic criteria during ship operation, based on observations from shipboard measurement systems and seafarers using an innovative platform. The innovative platform provides personalised seasickness criteria that are accessible during ship operation to estimate the probable level of seasickness on board. Results are compared to that from a traditional method of data acquisition and analyses, post operation, revealing a similar trend in diagnostic threshold magnitudes (13-85 m/s1.5) that can be applicable to voyages with different durations (0.5-6 hr) considering desired levels of seasickness (10-50 %). The seasickness criteria are envisioned to be pertinent for the prediction of probable seasickness levels based on sea state forecasts and ship motion estimation.

Risk Factors for Early Treatment Discontinuation Due to Toxicity Among Patients With Metastatic Castration-resistant Prostate Cancer Receiving Androgen Receptor-targeted Therapy.

OBJECTIVES: Androgen receptor-targeted therapies (ARTs) improve survival outcomes in patients with metastatic castration-resistant prostate cancer (mCRPC); however, a significant portion of patients discontinue treatment for various reasons including treatment-related toxicity. We aim to describe reasons for ART treatment discontinuation and identify predictors associated with increased risk of treatment discontinuation due to toxicity. METHODS: We performed a single-institution retrospective review of patients with mCRPC receiving ART between 2010 and 2021. Our primary aim was to identify risk factors for treatment discontinuation due to toxicity. Our secondary aim was to describe ART discontinuation patterns among patients with mCRPC. RESULTS: One hundred thirty-three patients with mCRPC started and discontinued ARTs. Fourteen patients (10.5%) discontinued treatment due to toxicity. Common reasons for treatment discontinuation include Prostate Specific Antigen test progression, radiographic progression, toxicity, and death. Significant predictors of treatment discontinuation due to toxicity on bivariate analysis and multivariate analysis included patient-reported falls (odds ratio [OR]: 7.67, CI: [1.31-40.42]; P =0.016), rash (OR: 13.4, CI: [1.35-134.81]; P =0.026), and weakness (OR: 4.16, CI: [1.15-15.0]; P =0.019). CONCLUSIONS: Our work presents the first description of ART treatment discontinuation and its causes in the real-world setting, as well as patient-reported side effects. Most patients with mCRPC discontinued treatment due to the progression of disease and a minority of patients discontinued secondary to treatment toxicity. Initial multivariable analysis suggests that patient-reported weakness, falls, and rash were associated with a higher likelihood of treatment discontinuation due to toxicity. Early monitoring of this population can prolong the duration of treatment and prevent unnecessary treatment burden.

Mycoplasma genitalium incidence, organism load, and treatment failure in a cohort of young Australian women.

BACKGROUND: Mycoplasma genitalium (MG) is an emerging sexually transmitted infection (STI) that is potentially associated with reproductive tract sequelae in women. This study aimed to estimate MG incidence and treatment failure and provide estimates of organism load in infection. METHODS: 1110 women aged 16-25 years were recruited from primary care clinics in Australia. Women were tested for MG at baseline, 6 months, and 12 months, and MG organism load was measured by quantitative polymerase chain reaction (PCR). MG-positive cases were screened for MG 23S ribosomal RNA (rRNA) gene point mutations shown to confer azithromycin resistance using high-resolution melt following PCR. RESULTS: MG incidence rate was 1.3 per 100 person-years (n=14; 95% confidence interval [CI], .8-2.3); women reporting 3 or more sex partners in the last 12 months had an increased rate of incident infection (rate ratio [RR], 5.1; 95% CI, 1.3-19.6]). There were 3 cases of MG reinfection (0.8 per 100 person-years [95% CI, .1-.9]. Organism load was higher for prevalent than incident infection (P=.04). There were 3 cases of treatment failure (9.4% [95% CI, 2.0-25.0]); organism load was higher in cases with treatment failure than in successfully treated cases (P<.01). An MG 23S rRNA mutation was detected in 5 cases (3 cases of treatment failure and 2 successfully treated). CONCLUSIONS: Although MG incidence was relatively low, testing should be recommended for women considered to be at increased risk based on sexual history. Our results also suggest that organism load might be important in azithromycin treatment failure.