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1.
J Gen Intern Med ; 37(4): 949-953, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35060003

RESUMO

A majority of Americans favor universal health insurance, but there is uncertainty over how best to achieve this goal. Whatever the insurance design that is implemented, additional details that must be considered include breadth of services covered, restrictions and limits on volumes of services, cost-sharing for individuals, and pricing. In the hopes that research can inform this ongoing debate, we review evidence supporting different models for achieving universal coverage in the US and identify areas where additional research and stakeholder input is needed. Key areas in need of further research include how care should be organized, how costs can be reduced, and what healthcare services universal insurance should cover.


Assuntos
Serviços de Saúde , Cobertura Universal do Seguro de Saúde , Acessibilidade aos Serviços de Saúde , Humanos , Seguro Saúde , Pesquisa
2.
J Gen Intern Med ; 37(13): 3396-3403, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35112278

RESUMO

BACKGROUND: Transgender individuals are less likely to have had a primary care visit in the last year than cisgender individuals. While the importance of multidisciplinary clinics for transgender care has been established, little is known about the healthcare experiences of transgender patients with these clinics. OBJECTIVE: To describe how patients experience transgender clinics and how these experiences compare to those experiences in other settings. PARTICIPANTS: Twenty-one adult patients of a gender health program. DESIGN AND APPROACH: Semi-structured interviews of transgender patients. The interviews focused on two domains: healthcare experiences and relationships with healthcare providers. KEY RESULTS: Overall, transgender patients expressed a need for healthcare services, particularly for primary care, that are partially met by the comprehensive care clinic model. Limitations in access included the lack of willing providers, where the patients live, and long wait times for appointments. Participants recounted a range of experiences, both positive and negative, with providers outside of the transgender clinic, but only positive experiences to share about providers from the transgender clinic. CONCLUSION: Outside specialty transgender settings, many patients had negative experiences with providers who were unwilling or unable to provide care. This study speaks to the need for primary care providers who can and will treat transgender patients, as well as the need for healthcare spaces that feel safe to transgender patents.


Assuntos
Pessoas Transgênero , Adulto , Atenção à Saúde , Identidade de Gênero , Pessoal de Saúde , Humanos , Pesquisa Qualitativa
3.
Age Ageing ; 51(1)2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34850811

RESUMO

INTRODUCTION: hospital transfers and admissions are critical events in the care of nursing home residents. We sought to determine hospital transfer rates at different ages. METHODS: a cohort of 1,187 long-stay nursing home residents who had participated in a Centers for Medicare and Medicaid demonstration project. We analysed the number of hospital transfers of the study participants recorded by the Minimum Data Set. Using a modern regression technique, we depicted the annual rate of hospital transfers as a smooth function of age. RESULTS: transfer rates declined with age in a nonlinear fashion. Rates were the highest among residents younger than 60 years of age (1.30-2.15 transfers per year), relatively stable between 60 and 80 (1.17-1.30 transfers per year) and lower in those older than 80 (0.77-1.17 transfers per year). Factors associated with increased risk of transfers included prior diagnoses of hip fracture (annual incidence rate ratio or IRR: 2.057, 95% confidence interval (CI): [1.240, 3.412]), dialysis (IRR: 1.717, 95% CI: [1.313, 2.246]), urinary tract infection (IRR: 1.755, 95% CI: [1.361, 2.264]), pneumonia (IRR: 1.501, 95% CI: [1.072, 2.104]), daily pain (IRR: 1.297, 95% CI: [1.055,1.594]), anaemia (IRR: 1.229, 95% CI [1.068, 1.414]) and chronic obstructive pulmonary disease (IRR: 1.168, 95% CI: [1.010,1.352]). Transfer rates were lower in residents who had orders reflecting preferences for comfort care (IRR: 0.79, 95% CI: [0.665, 0.936]). DISCUSSION: younger nursing home residents may require specialised interventions to reduce hospital transfers; declining transfer rates with the oldest age groups may reflect preferences for comfort-focused care.


Assuntos
Casas de Saúde , Transferência de Pacientes , Fatores Etários , Idoso , Hospitalização , Hospitais , Humanos , Medicare , Estados Unidos/epidemiologia
4.
J Gen Intern Med ; 36(5): 1189-1196, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33140276

RESUMO

BACKGROUND: Patients who undergo the complex series of transitions from the hospital to a skilled nursing facility (SNF) back to home represent a unique patient population with multiple comorbidities and impaired functional abilities. The needs and outcomes of patients who are discharged from the hospital to SNF before returning home are understudied in care transitions scholarship. OBJECTIVE: To study the patient and caregiver challenges and perspectives on transitions from the hospital to the SNF and back to home. DESIGN: Between 48 h and 1 week after discharge from the SNF, semi-structured interviews were performed with a convenience sample of patients and caregivers in their homes. Within 1 to 2 weeks after the baseline interview, follow-up interviews were performed over the phone. PARTICIPANTS: A total of 39 interviewees comprised older adults undergoing the series of transitions from hospital to skilled nursing facility to home and their informal caregivers. MAIN MEASURES: A constructionist, grounded-theory approach was used to code the interviews, identify major themes and subthemes, and develop a theoretical model explaining the outcomes of the SNF to home transition. KEY RESULTS: The mean age of the patients was 76.6 years and 64.8 years for the caregivers. Four major themes were identified: comforts of home, information needs, post-SNF care, and independence. Patients noted an extended time away from home and were motivated to return to and remain in the home. Information needs were variably met and affected post-SNF care, including medication management, appointments, and therapy gains and setbacks. Interviewees identified independent function at home as the most important outcome of the transition home. CONCLUSIONS: Post-SNF in home support is needed rapidly after discharge from the SNF to prevent adverse outcomes. In-home support needs to be highly individualized based on a patient's and caregiver's unique situation and needs.


Assuntos
Cuidadores , Instituições de Cuidados Especializados de Enfermagem , Idoso , Hospitais , Humanos , Alta do Paciente
5.
BMC Health Serv Res ; 21(1): 492, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34030672

RESUMO

BACKGROUND: Centers for Medicare and Medicaid Services (CMS) funded demonstration project to evaluate financial incentives for nursing facilities providing care for 6 clinical conditions to reduce potentially avoidable hospitalizations (PAHs). The Optimizing Patient Transfers, Impacting Medical Quality, and Improving Symptoms: Transforming Institutional Care (OPTIMISTIC) site tested payment incentives alone and in combination with the successful nurse-led OPTIMISTIC clinical model. Our objective was to identify facility and resident characteristics associated with transfers, including financial incentives with or without the clinical model. METHODS: This was a longitudinal analysis from April 2017 to June 2018 of transfers among nursing home residents in 40 nursing facilities, 17 had the full clinical + payment model (1726 residents) and 23 had payment only model (2142 residents). Using CMS claims data, the Minimum Data Set, and Nursing Home Compare, multilevel logit models estimated the likelihood of all-cause transfers and PAHs (based on CMS claims data and ICD-codes) associated with facility and resident characteristics. RESULTS: The clinical + payment model was associated with 4.1 percentage points (pps) lower risk of all-cause transfers (95% confidence interval [CI] - 6.2 to - 2.1). Characteristics associated with lower PAH risk included residents aged 95+ years (- 2.4 pps; 95% CI - 3.8 to - 1.1), Medicare-Medicaid dual-eligibility (- 2.5 pps; 95% CI - 3.3 to - 1.7), advanced and moderate cognitive impairment (- 3.3 pps; 95% CI - 4.4 to - 2.1; - 1.2 pps; 95% CI - 2.2 to - 0.2). Changes in Health, End-stage disease and Symptoms and Signs (CHESS) score above most stable (CHESS score 4) increased the risk of PAH by 7.3 pps (95% CI 1.5 to 13.1). CONCLUSIONS: Multiple resident and facility characteristics are associated with transfers. Facilities with the clinical + payment model demonstrated lower risk of all-cause transfers compared to those with payment only, but not for PAHs.


Assuntos
Medicare , Casas de Saúde , Idoso , Idoso de 80 Anos ou mais , Hospitalização , Humanos , Transferência de Pacientes , Instituições de Cuidados Especializados de Enfermagem , Estados Unidos
6.
Brain Behav Immun ; 86: 22-29, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31059804

RESUMO

There is now reliable evidence that early psychosocial stress exposures are associated with behavioral health in children; the degree to which these same kinds of stress exposures predict physical health outcomes is not yet clear. We investigated the links between economic adversity, family and caregiving stress in early childhood and several markers of immune function in early adolescence. The sample is derived from the Family Life Project, a prospective longitudinal study of at-risk families. Socio-demographic and psychosocial risks have been assessed at regular intervals since the children were first assessed at 2 months of age. When the children were early adolescents, we conducted an in-depth health assessment of a subsample of families; blood samples were collected from venipuncture for interleukin(IL)-6, Tumor Necrosis Factor (TNF)-alpha, and C-reactive protein (CRP), as well as glucocorticoid resistance. Results indicated limited but reliable evidence of an association between early risk exposure and inflammation in adolescence. Specifically, caregiver depressive symptoms in early childhood predicted elevated CRP almost a decade later, and the prediction was significant after accounting for multiple covariates such as socio-economic adversity, health behaviors and body mass index. Our findings provide strong but limited evidence that early stress exposures may be associated with inflammation, suggesting one mechanism linking early stress exposure to compromised behavioral and somatic health.


Assuntos
Experiências Adversas da Infância , Cuidadores/psicologia , Depressão , Violência Doméstica , Saúde da Família , Inflamação/etiologia , Estresse Psicológico , Adolescente , Proteína C-Reativa/análise , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interleucina-6/sangue , Estudos Longitudinais , Masculino , Estudos Prospectivos , Fatores de Risco , Fator de Necrose Tumoral alfa/sangue
9.
J Gen Intern Med ; 32(2): 199-203, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27704367

RESUMO

We assembled a cross-cutting team of experts representing primary care physicians (PCPs), home care physicians, physicians who see patients in skilled nursing facilities (SNF physicians), skilled nursing facility medical directors, human factors engineers, transitional care researchers, geriatricians, internists, family practitioners, and three major organizations: AMDA, SGIM, and AGS. This work was sponsored through a grant from the Association of Subspecialty Physicians (ASP). Members of the team mapped the process of discharging patients from a skilled nursing facility into the community and subsequent care of their outpatient PCP. Four areas of process improvement were identified, building on the prior work of the AMDA Transitions of Care Committee and the experiences of the team members. The team identified issues and developed best practices perceived as feasible for SNF physician and PCP practices to accomplish. The goal of these consensus-based recommended best practices is to provide a safe and high-quality transition for patients moving between the care of their SNF physician and PCP.


Assuntos
Consenso , Continuidade da Assistência ao Paciente/normas , Qualidade da Assistência à Saúde/normas , Instituições de Cuidados Especializados de Enfermagem/organização & administração , Cuidado Transicional/organização & administração , Benchmarking , Centers for Medicare and Medicaid Services, U.S. , Hospitalização , Humanos , Alta do Paciente/estatística & dados numéricos , Readmissão do Paciente/economia , Atenção Primária à Saúde/organização & administração , Instituições de Cuidados Especializados de Enfermagem/economia , Cuidado Transicional/economia , Estados Unidos
10.
Dev Psychopathol ; 29(5): 1589-1600, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29162168

RESUMO

There is now a clear focus on incorporating, and integrating, multiple levels of analysis in developmental science. The current study adds to research in this area by including markers of the immune and neuroendocrine systems in a longitudinal study of temperament in infants. Observational and parent-reported ratings of infant temperament, serum markers of the innate immune system, and cortisol reactivity from repeated salivary collections were examined in a sample of 123 infants who were assessed at 6 months and again when they were, on average, 17 months old. Blood from venipuncture was collected for analyses of nine select innate immune cytokines; salivary cortisol collected prior to and 15 min and 30 min following a physical exam including blood draw was used as an index of neuroendocrine functioning. Analyses indicated fairly minimal significant associations between biological markers and temperament at 6 months. However, by 17 months of age, we found reliable and nonoverlapping associations between observed fearful temperament and biological markers of the immune and neuroendocrine systems. The findings provide some of the earliest evidence of robust biological correlates of fear behavior with the immune system, and identify possible immune and neuroendocrine mechanisms for understanding the origins of behavioral development.


Assuntos
Citocinas/sangue , Hidrocortisona/análise , Temperamento/fisiologia , Feminino , Humanos , Lactente , Estudos Longitudinais , Masculino , Saliva/química
12.
Brain Behav Immun ; 48: 265-73, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25862953

RESUMO

BACKGROUND: Quality of the parent-child relationship is a robust predictor of behavioral and emotional health for children and adolescents; the application to physical health is less clear. METHODS: We investigated the links between observed parent-child relationship quality in an interaction task and antibody response to meningococcal conjugate vaccine in a longitudinal study of 164 ambulatory 10-11 year-old children; additional analyses examine associations with cortisol reactivity, BMI, and somatic illness. RESULTS: Observed Negative/Conflict behavior in the interaction task predicted a less robust antibody response to meningococcal serotype C vaccine in the child over a 6 month-period, after controlling for socio-economic and other covariates. Observer rated interaction conflict also predicted increased cortisol reactivity following the interaction task and higher BMI, but these factors did not account for the link between relationship quality and antibody response. CONCLUSIONS: The results begin to document the degree to which a major source of child stress exposure, parent-child relationship conflict, is associated with altered immune system development in children, and may constitute an important public health consideration.


Assuntos
Formação de Anticorpos , Emoções , Vacinas Meningocócicas/imunologia , Relações Pais-Filho , Vacinação/psicologia , Criança , Feminino , Humanos , Masculino , Saúde Mental
13.
WMJ ; 114(5): 185-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26726338

RESUMO

BACKGROUND: Safe hospital discharges have become a major focus in the national discussion on transitions of care and care coordination. Education on the hospital discharge process is evolving as the needs of trainees are better understood. PURPOSE: This study is a cross-sectional survey of residents in a Midwestern residency program about their confidence in safely discharging patients from the hospital, including how they have or have not learned to do so. METHODS: An anonymous paper questionnaire was distributed to a convenience sample of interns and residents at a weekly meeting of the residency program. RESULTS: Most residents reported a general confidence in their abilities to safely discharge patients from the hospital; however, further probing revealed that their confidence breaks down when required to competently perform specific tasks of the discharge process such as activity restrictions or facilitation of home care. More than 50% of house staff surveyed responded that their education in many specific aspects of the discharge process are lacking. CONCLUSION: Interdisciplinary care education, and the discharge summary in particular, warrant further scrutiny as a care transition tool and means of teaching safe hospital discharge to trainees. We present a questionnaire that may serve useful as an anonymous tool to gauge residents' educational needs.


Assuntos
Competência Clínica , Educação de Pós-Graduação em Medicina , Medicina Interna/educação , Internato e Residência , Sumários de Alta do Paciente Hospitalar , Adulto , Continuidade da Assistência ao Paciente , Estudos Transversais , Feminino , Humanos , Masculino , Inquéritos e Questionários , Wisconsin
14.
Dev Psychopathol ; 26(4 Pt 2): 1567-76, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25422979

RESUMO

Research findings in psychoneuroimmunology document reliable, bidirectional linkages among psychological processes, the nervous system, and the immune system. However, available data are based almost entirely on animal and adult human studies; the application to children and adolescents is uncertain. We capitalized on the experimental leverage provided by a routine vaccination to examine the link between mood symptoms and the immune response to a vaccine challenge in early adolescence. One hundred twenty-six 11-year-olds for whom vaccine response data were available were assessed at prevaccination and 4 weeks, 3 months, and 6 months following vaccination; self-report ratings of depression and anxiety as well as measures of psychosocial and somatic risk were assessed prior to vaccine response. Analyses indicated that children's internalizing mood symptoms were associated with elevated and persistently higher antibody responses, with evidence extending to two of the four serogroups. The associations remained after controlling for multiple possible confounders (social class, body mass index, sleep, psychosocial risk, and pubertal status). The observed enhanced vaccine response associated with depressive and anxious symptoms in early adolescence may reflect an important developmental difference in immune system-brain interplay between adults and children, and it underscores the need for further developmental studies of psychoneuroimmunology.


Assuntos
Ansiedade/imunologia , Depressão/imunologia , Vacinas Meningocócicas/imunologia , Criança , Feminino , Humanos , Masculino
15.
Clin Geriatr Med ; 40(2): 321-331, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38521602

RESUMO

LGBTQ + older adults have a high likelihood of accessing nursing home care. This is due to several factors: limitations performing activities of daily living and instrumental activities of daily living, restricted support networks, social isolation, delay seeking assistance, limited economic resources, and dementia. Nursing home residents fear going in the closet, which can have adverse health effects. Cultivating an inclusive nursing home culture, including administration, staff, and residents, can help older LGBTQ + adults adjust and thrive in long-term care.


Assuntos
Assistência de Longa Duração , Minorias Sexuais e de Gênero , Humanos , Idoso , Atividades Cotidianas , Cuidados Semi-Intensivos , Casas de Saúde
17.
Brain Behav Immun ; 32: 21-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23439080

RESUMO

Prenatal anxiety has been linked with altered immune function in offspring in animal studies, but the relevance for human health is unknown. We examined prenatal maternal anxiety as a predictor of adaptive immunity in infants at 2 and 6 months of age as part of a prospective longitudinal study. The humoral immune response to hepatitis B vaccine was assessed at 2 months (n=80) and 6 months (n=76) of age. Prenatal anxiety predicted lower hepatitis B antibody titers at 6 months of age independent of obstetric and socio-demographic covariates; the effects were limited to those infants who had not completed the 3-dose vaccine series (for transformed titer values, r=-.36, p<.05). Cell-mediated immune responses at 2 (n=56) and 6 (n=54) months of age were examined by ELISpot assays for interferon(IFN)-γ, interleukin(IL)-2, and IL-4 responder cell frequencies to three antigens: hepatitis B surface antigen, tetanus toxoid, and phytohaemagglutinin (PHA). Prenatal maternal anxiety was associated with reduced IFN-γ and increased IL-4 responder cell frequencies at 6 months of age, independent of obstetric and socio-demographic covariates. No effect of prenatal anxiety was found on adaptive immunity at 2 months of age. The findings provide the first demonstration in humans that prenatal anxiety alters adaptive immunity in the infant.


Assuntos
Imunidade Adaptativa/imunologia , Ansiedade/imunologia , Ansiedade/psicologia , Complicações na Gravidez/imunologia , Complicações na Gravidez/psicologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Adulto , Ritmo Circadiano/fisiologia , Citocinas/metabolismo , Feminino , Glucocorticoides/metabolismo , Hepatite , Antígenos da Hepatite B/análise , Vacinas contra Hepatite B , Humanos , Hidrocortisona/sangue , Imunização , Recém-Nascido , Masculino , Testes Neuropsicológicos , Gravidez , Estudos Prospectivos , Escalas de Graduação Psiquiátrica , Fatores Socioeconômicos , Linfócitos T/imunologia , Adulto Jovem
18.
J Appl Gerontol ; 42(2): 194-204, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36205006

RESUMO

BACKGROUND: Incomplete communication between staff and providers may cause adverse outcomes for nursing home residents. The Situation-Background-Assessment-Recommendation (SBAR) tool is designed to improve communication around changes in condition (CIC). An adapted SBAR was developed for the Centers for Medicare and Medicaid Services demonstration project, OPTIMISTIC, to increase its use during a resident CIC and to improve documentation. METHODS: Four Plan-Do-Study-Act (PDSA) cycles to develop and refine successive protocol implementation of the OPTIMISTIC SBAR were deployed in four Indiana nursing homes. Use of SBAR, documentation quality, and participant surveys were assessed pre- and post-intervention implementation. RESULTS: OPTIMISTIC SBAR use and documentation quality improved in three of the four buildings. Participants reported improved collaboration between nurses and providers after SBAR intervention. CONCLUSION: Successive PDSA cycles implementing changes in an OPTIMISTIC SBAR protocol for resident CIC led to an increase in SBAR use, improved documentation, and better collaboration between nursing staff and providers.


Assuntos
Medicare , Recursos Humanos de Enfermagem , Idoso , Estados Unidos , Humanos , Casas de Saúde , Comunicação , Avaliação de Programas e Projetos de Saúde
19.
J Am Med Dir Assoc ; 23(1): 105-110, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34181908

RESUMO

OBJECTIVES: Potentially avoidable hospitalizations are harmful to nursing home residents. Despite extensive care transitions research, no studies have described transfers originating outside the nursing home (eg, visiting family members or at a dialysis center). This article describes 82 out-of-facility (community) transfers and compares them to transfers originating within the nursing home (direct transfers). DESIGN: Secondary data analysis with multivariable model for community transfer risk factors. SETTING AND PARTICIPANTS: Eighty-two community transfers and 1362 transfers originating in the nursing home, involving 870 residents enrolled in the OPTIMISTIC demonstration project between January 1, 2015, and June 30, 2016. METHODS: Transfers were compared using data from the Minimum Data Set and root cause analyses performed at time of transfer. Multivariable associations were assessed at the transfer level to define risk factors for community transfers. Project nurses collected data on community transfers to inform a root cause analysis. RESULTS: Residents with community transfers were younger (74.4 years vs 78.2 years), with lower prevalence of cognitive impairment (44.8% vs 70.3%) and higher rates of heart failure (38.7% vs 23.3%) than residents with direct transfers. Community transfers were more likely due to cardiovascular illness (31.2% vs 8.7%), whereas less likely to be for cognitive, behavioral, and psychiatric concerns (11.7% vs 22.7%). Nearly half (46%) of community transfers originated at dialysis centers. Residents transferred outside the nursing home were less likely to have documented limitations to care such as a do not resuscitate code status. Communication during community transfers was identified on root cause analyses as a potential area for improvement. CONCLUSIONS AND IMPLICATIONS: Community transfers were more likely to occur in younger residents with higher rates of cardiovascular disease and lower rates of cognitive impairment. Improved communication between nursing home staff and outside providers as well as more extensive advance care planning for residents with cardiovascular disease may reduce community transfers.


Assuntos
Planejamento Antecipado de Cuidados , Transferência de Pacientes , Hospitalização , Humanos , Casas de Saúde , Instituições de Cuidados Especializados de Enfermagem
20.
Microbiol Spectr ; 10(3): e0263021, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35612314

RESUMO

As increasing evidence emerges that interstrain genetic diversity among Candida albicans clinical isolates underpins phenotypic variation compared to the reference isolate SC5314, new genetic tools are required to interrogate gene function across strain backgrounds. Here, the SAT1-flipper plasmid was reengineered to contain a C. albicans codon optimized hygromycin B resistance gene (CaHygB). Cassettes were PCR-amplified from both SAT1-flipper and CaHygB-flipper plasmids using primers with homologous sequences flanking target genes of interest to serve as repair templates. Ribonucleoprotein (RNP) complexes containing proprietary CRISPR RNAs (crRNAs), universal transactivating CRISPR RNA (tracrRNA), and Cas9 protein were assembled in vitro and transformed, along with both repair templates, by electroporation into C. albicans. Homozygous deletion of the ADE2 gene results in red-pigmented colonies and this gene was used to validate our approach. Both in SC5314 and a variety of clinical isolates (529L, JS15, SJCA1, TW1), homozygous gene targeting was nearly 100% when plating on media containing nourseothricin and hygromycin B with transformation efficiencies exceeding 104 homozygous deletion mutants per µg of DNA. A gene reversion system was also employed with plasmids pDUP3 and pDIS3 engineered to contain the ADH1 terminator and an overlap extension PCR-mediated approach combined with CRISPR-Cas9 targeting at the NEUT5 neutral locus. A variety of single or compound mutants (Δ/Δals3, Δ/Δcph1 Δ/Δefg1, Δ/Δece1) and their revertant strains were constructed and phenotypically validated by a variety of assays, including biofilm formation, hyphal growth, and macrophage IL-1ß response. Thus, we have established a cloning-free, modular system for highly efficient homozygous gene deletion and reversion in diverse isolates. IMPORTANCE Recently, phenotypic heterogeneity in Candida albicans isolates has been recognized as an underappreciated factor contributing to gene diversification and broadly impacts strain-to-strain antifungal resistance, fitness, and pathogenicity. We have designed a cloning-free genetic system for rapid gene deletion and reversion in C. albicans clinical isolates that interlaces established recyclable genetic systems with CRISPR-Cas9 technology. The SAT1-flipper was reengineered to contain CaHygB encoding resistance to hygromycin B. Using a modular PCR-mediated approach coupled with in vitro ribonucleoprotein assembly with commercial reagents, both SAT1- and CaHygB-flipper cassettes were simultaneously integrated at loci with high efficiency (104 transformants per µg DNA) and upward of 99% homozygous gene targeting across a collection of diverse isolates of various anatomical origin. Revertant strains were constructed by overlap extension PCR with CRISPR-Cas9 targeted integration at the NEUT5 locus. Thus, this facile system will aid in unraveling the genetic factors contributing to the complexity of intraspecies diversity.


Assuntos
Sistemas CRISPR-Cas , Candida albicans , Candida albicans/genética , Homozigoto , Higromicina B , Ribonucleoproteínas/genética , Deleção de Sequência
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