Impact of dementia on 30-, 180-, and 365-day mortality during the first pandemic wave in older adults seen in spanish emergency departments diagnosed with or without COVID-19.

OBJECTIVES: To assess whether dementia is an independent predictor of death after a hospital emergency department (ED) visit by older adults with or without a COVID-19 diagnosis during the first pandemic wave. METHOD: We used data from the EDEN-Covid (Emergency Department and Elderly Needs during Covid) cohort formed by all patients ≥65 years seen in 52 Spanish EDs from March 30 to April 5, 2020. The association of prior history of dementia with mortality at 30, 180 and 365 d was evaluated in the overall sample and according to a COVID-19 or non COVID diagnosis. RESULTS: We included 9,770 patients aged 78.7 ± 8.3 years, 51.1% men, 1513 (15.5%) subjects with prior history of dementia and 3055 (31.3%) with COVID-19 diagnosis. 1399 patients (14.3%) died at 30 d, 2008 (20.6%) at 180 days and 2456 (25.1%) at 365 d. The adjusted Hazard Ratio (aHR) for age, sex, comorbidity, disability and diagnosis for death associated with dementia were 1.16 (95% CI 1.01-1.34) at 30 d; 1.15 at 180 d (95% CI 1.03-1.30) and 1.19 at 365 d (95% CI 1.07-1.32), p < .001. In patients with COVID-19, the aHR were 1.26 (95% CI: 1.04-1.52) at 30 days; 1.29 at 180 d (95% CI: 1.09-1.53) and 1.35 at 365 d (95% CI: 1.15-1.58). CONCLUSION: Dementia in older adults attending Spanish EDs during the first pandemic wave was independently associated with 30-, 180- and 365-day mortality. This impact was lower when adjusted for age, sex, comorbidity and disability, and was greater in patients diagnosed with COVID-19.

A multidomain intervention against cognitive decline in an at-risk-population in Germany: Results from the cluster-randomized AgeWell.de trial.

INTRODUCTION: We investigated the effectiveness of a multidomain intervention to preserve cognitive function in older adults at risk for dementia in Germany in a cluster-randomized trial. METHODS: Individuals with a Cardiovascular Risk Factors, Aging, and Dementia (CAIDE) risk score ≥ 9 aged 60 to 77 years were recruited. After randomization of their general practitioner (GP), patients received a multidomain intervention (including optimization of nutrition and medication, and physical, social, and cognitive activity) or general health advice and GP treatment as usual over 24 months. Primary outcome was global cognitive performance (composite z score, based on domain-specific neuropsychological tests). RESULTS: Of 1030 participants at baseline, n = 819 completed the 24-month follow-up assessment. No differences regarding global cognitive performance (average marginal effect = 0.010, 95% confidence interval: -0.113, 0.133) were found between groups at follow-up. Perceived restrictions in intervention conduct by the COVID-19 pandemic did not impact intervention effectiveness. DISCUSSION: The intervention did not improve global cognitive performance. HIGHLIGHTS: Overall, no intervention effects on global cognitive performance were detected. The multidomain intervention improved health-related quality of life in the total sample. In women, the multidomain intervention reduced depressive symptoms. The intervention was completed during the COVID-19 pandemic.

Exploring FeLV-Gag-Based VLPs as a New Vaccine Platform-Analysis of Production and Immunogenicity.

Feline leukemia virus (FeLV) is one of the most prevalent infectious diseases in domestic cats. Although different commercial vaccines are available, none of them provides full protection. Thus, efforts to design a more efficient vaccine are needed. Our group has successfully engineered HIV-1 Gag-based VLPs that induce a potent and functional immune response against the HIV-1 transmembrane protein gp41. Here, we propose to use this concept to generate FeLV-Gag-based VLPs as a novel vaccine strategy against this retrovirus. By analogy to our HIV-1 platform, a fragment of the FeLV transmembrane p15E protein was exposed on FeLV-Gag-based VLPs. After optimization of Gag sequences, the immunogenicity of the selected candidates was evaluated in C57BL/6 and BALB/c mice, showing strong cellular and humoral responses to Gag but failing to generate anti-p15E antibodies. Altogether, this study not only tests the versatility of the enveloped VLP-based vaccine platform but also sheds light on FeLV vaccine research.

Progressive Local Accumulation of Self-Assembled Nanoreactors in a Hydrogel Matrix through Repetitive Injections of ATP.

Cellular functions are regulated with high spatial control through the local activation of chemical processes in a complex inhomogeneous matrix. The development of synthetic macroscopic systems with a similar capacity allows fundamental studies aimed at understanding the relationship between local molecular events and the emergence of functional properties at the macroscopic level. Here, we show that a kinetically stable inhomogeneous hydrogel matrix is spontaneously formed upon the local injection of ATP. Locally, ATP templates the self-assembly of amphiphiles into large nanoreactors with a much lower diffusion rate compared to unassembled amphiphiles. The local depletion of unassembled amphiphiles near the injection point installs a concentration gradient along which unassembled amphiphiles diffuse from the surroundings to the center. This allows for a progressive local accumulation of self-assembled nanoreactors in the matrix upon repetitive cycles of ATP injection separated by time intervals during which diffusion of unassembled amphiphiles takes place. Contrary to the homogeneous matrix containing the same components, in the inhomogeneous matrix the local upregulation of a chemical reaction occurs. Depending on the way the same amount of injected ATP is administered to the hydrogel matrix different macroscopic distributions of nanoreactors are obtained, which affect the location in the matrix where the chemical reaction is upregulated.

Specific properties of shRNA-mediated CCR5 downregulation that enhance the inhibition of HIV-1 infection in combination with shRNA targeting HIV-1 rev.

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

[Impact of the HEARTS Initiative in a second-level health institution in ColombiaImpacto da iniciativa HEARTS em um estabelecimento de atenção secundária à saúde na Colômbia]. / Impacto de la Iniciativa HEARTS en una institución de salud de segundo nivel en Colombia.

Objective: To identify the impact of the HEARTS initiative on patients with high blood pressure treated in a Colombian hospital. Methods: Quasi-experimental, retrospective study between 2017 and 2019 with hypertensive patients over 18 years of age included in the HEARTS strategy and treated at the Santa Mónica Hospital in the municipality of Dosquebradas, department of Risaralda. The unit of analysis was medical history. Blood pressure targets (defined in the HEARTS initiative as a patient with systolic blood pressure <140 mmHg and diastolic pressure <90 mmHg) were assessed at the time of admission to the HEARTS program and one year later. Sociodemographic, pharmacological, clinical, and blood pressure targets were included, as was compliance with the recommendations of the initiative. Descriptive analysis was performed, and binary logistic regression was applied (p <0.05). Results: A total of 372 patients were studied, of whom 262 were women (70.4%). The mean age was 66.3 ± 12.2 years. In the first consultation, 285 patients (76.6%) presented blood pressure figures within the target range; in the second consultation this was achieved by 84.1% of patients (n=313, mean difference: 7.5%, 95% CI: 1.8 to 13.1; p=0.005). After implementation of the HEARTS initiative, 77.4% of patients (n=288) continued with the initial treatment. Following HEARTS recommendations increases the likelihood of being in the target range (p=0.033; OR= 2,688; CI= 1.081 - 6.684). Conclusions: Implementation of the HEARTS initiative favorably impacted blood pressure figures in patients with hypertension and decreased cardiovascular risk.

Objetivo: Identificar o impacto da iniciativa HEARTS nos pacientes com hipertensão arterial atendidos em um hospital colombiano. Métodos: Estudo quase-experimental, retrospectivo entre 2017 e 2019, com pacientes hipertensos maiores de 18 anos incluídos na estratégia HEARTS, atendidos no Hospital Santa Mónica do município de Dosquebradas, departamento de Risaralda. A unidade de análise foi a história clínica. As metas de pressão arterial (definidas na iniciativa HEARTS como pressão arterial sistólica <140 mmHg e pressão diastólica <90 mmHg) foram avaliadas no momento de ingresso no programa HEARTS e um ano depois. Foram incluídas variáveis sociodemográficas, farmacológicas, clínicas, metas de pressão arterial e adesão à iniciativa. Foram realizadas análises descritivas e foi aplicada a regressão logística binária (p<0,05). Resultados: Foram analisados 372 pacientes, dos quais 262 eram mulheres (70,4%). A média de idade foi de 66,3 ± 12,2 anos. Na primeira consulta de controle, 285 pacientes (76,6%) apresentaram valores de pressão arterial dentro das metas; na segunda consulta, as metas haviam sido alcançadas por 84,1% dos pacientes (n=313 diferença média: 7,5%, IC95%: 1,8 a 13,1, p=0,005). Após a implementação da iniciativa HEARTS, 77,4% dos pacientes (n=288) continuaram com o manejo inicial. O cumprimento das recomendações da HEARTS aumenta a probabilidade de estar nas metas (p=0,033; OR= 2,688; IC= 1,081 - 6,684). Conclusões: A implementação da iniciativa HEARTS teve um impacto favorável nos valores da pressão arterial em pacientes com hipertensão e diminuiu o risco cardiovascular.

Types of leisure time physical activities (LTPA) of community-dwelling persons who have been screened positive for dementia.

BACKGROUND: To (a) describe the pattern of leisure time physical activities (LTPA) in community-dwelling persons who have been screened positive for dementia and (b) determine the health-related and sociodemographic factors associated with LTPA. METHODS: Data of the general practitioner-based, randomized, controlled intervention trial, DelpHi-MV (Dementia: life- and person-centered help in Mecklenburg-Western Pomerania) were used. Patients aged 70 years or older, who lived at home and had a DemTect< 9 were informed about the study by their General practitioners and invited to participate. Data from 436 participants with complete baseline data were used. Standardized, computer-assisted assessments were made during face-to-face interviews at the participants' homes. RESULTS: Two hundred thirty-eight patients (54.6%) carried out LTPA (men 58.4%, women 51.8%). Physically active patients mentioned one to two different activities; diversity of LTPA was higher for men than for women. The most-frequently mentioned types of activity were gardening (35.3%), cycling (24.1%) and mobility training (12.4%); there was only a statistically significant difference between men and women in cycling, χ2(1) = 21.47, p < .001. The odds of LTPA increased with increasing quality of life (OR = 2.41), lower impairments in activities of daily living (OR = 0.85), and living in a rural environment (OR = 2.02). CONCLUSIONS: Our findings suggest that people who have been screened positive for dementia living in a rural area are more likely to be active than people living in an urban area. Following studies should investigate whether this difference has an effect on the progression of dementia. TRIAL REGISTRATION: ClinicalTrial.gov Identifier NCT01401582 .

Curcumin Inhibits In Vitro SARS-CoV-2 Infection In Vero E6 Cells through Multiple Antiviral Mechanisms.

Due to the scarcity of therapeutic approaches for COVID-19, we investigated the antiviral and anti-inflammatory properties of curcumin against SARS-CoV-2 using in vitro models. The cytotoxicity of curcumin was evaluated using MTT assay in Vero E6 cells. The antiviral activity of this compound against SARS-CoV-2 was evaluated using four treatment strategies (i. pre-post infection treatment, ii. co-treatment, iii. pre-infection, and iv. post-infection). The D614G strain and Delta variant of SARS-CoV-2 were used, and the viral titer was quantified by plaque assay. The anti-inflammatory effect was evaluated in peripheral blood mononuclear cells (PBMCs) using qPCR and ELISA. By pre-post infection treatment, Curcumin (10 µg/mL) exhibited antiviral effect of 99% and 99.8% against DG614 strain and Delta variant, respectively. Curcumin also inhibited D614G strain by pre-infection and post-infection treatment. In addition, curcumin showed a virucidal effect against D614G strain and Delta variant. Finally, the pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) released by PBMCs triggered by SARS-CoV-2 were decreased after treatment with curcumin. Our results suggest that curcumin affects the SARS-CoV-2 replicative cycle and exhibits virucidal effect with a variant/strain independent antiviral effect and immune-modulatory properties. This is the first study that showed a combined (antiviral/anti-inflammatory) effect of curcumin during SARS-CoV-2 infection. However, additional studies are required to define its use as a treatment for the COVID-19.

Candida isolates causing candidemia show different degrees of virulence in Galleria mellonella.

We aim to assess intra- and interspecies differences in the virulence of Candida spp. strains causing candidemia using the invertebrate Galleria mellonella model. We studied 739 Candida spp. isolates (C. albicans [n = 373], C. parapsilosis [n = 203], C. glabrata [n = 92], C. tropicalis [n = 53], and C. krusei [n = 18]) collected from patients with candidemia admitted to Gregorio Marañon Hospital (Madrid, Spain). Species-specific infecting inocula (yeast cells/larva) were adjusted (5 × 105 [C. albicans, and C. tropicalis], 2 × 106-5 × 106 [C. parapsilosis, C. glabrata, and C. krusei]) and used to infect 10 larvae per isolate; percentage of survival and median survival per isolate were calculated. According to the interquartile range of the median survival, isolates with a median survival under P25 were classified as of high-virulence and isolates with a median survival over P75 as of low virulence. The median survival of larvae infected with different species was variable: C. albicans (n = 2 days, IQR <1-3 days), C. tropicalis (n = 2 days, IQR 1.5-4 days), C. parapsilosis (n = 2 days, IQR 2-3.5 days), C. glabrata (n = 3 days, IQR 2-3 days), and C. krusei (n = 7 days, 6.5->8 days) (P < .001). Differences in virulence among species were validated by histological examination (day +1 post-infection) in the larvae infected by the isolates of each virulence category and species. Virulence-related gene expression in C. albicans isolates did not reach statistical significance. We report species-specific virulence patterns of Candida spp. and show that isolates within a given species have different degrees of virulence in the animal model.

High frequency of HPV genotypes 59, 66, 52, 51, 39 and 56 in women from Western Mexico.

BACKGROUND: Human papillomavirus infection is an important factor associated with cervical cancer (CC) development. The prevalence and genotype distribution vary greatly worldwide. Examining local epidemiological data constitutes an important step towards the development of vaccines to prevent CC. In this work, we studied the prevalence of HPV genotypes in women from Western Mexico with the COBAS 4800 and/or Linear Array Genotyping Test (LA). METHODS: The samples analysed in this study represent a population from Western Mexico, which includes six different states. Our approach was first to test for HPV in cervical samples from women who attended their health clinic for routine gynaecological studies (open-population, n = 3000) by utilizing COBAS 4800. Afterwards, 300 of the HPV-positive samples were randomly selected to be genotyped with LA; finally, we genotyped samples from women with cervical intraepithelial neoplasia grade 1 (CIN 1, n = 71) and CC (n = 96) with LA. Sociodemographic data of the diverse groups were also compared. RESULTS: The overall HPV prevalence among the open-population of women as determined by COBAS 4800 was 12.1% (n = 364/3000). Among the HPV-positive samples, single infections (SI) with HPV16 were detected in 12.4% (n = 45/364), SI with HPV18 were detected in 1.4%, and infection with at least one of the genotypes included in the high-risk HPV pool was detected in 74.5% of the cases. LA analysis of the samples showed that in addition to HPV genotypes 16 and 18, there was a high prevalence of HPV genotypes 59, 66, 52, 51, 39 and 56 in women from Western Mexico. With respect to the sociodemographic data, we found statistically significant differences in the number of pregnancies, the use of hormonal contraceptives and tobacco intake. CONCLUSIONS: Our data indicate that there is a high prevalence of HPV genotypes which are not covered by the vaccines currently available in Mexico; therefore, it is necessary to include HPVs 59, 66, 51, 39 and 56 in the design of future vaccines to reduce the risk of CC development. It is also essential to emphasize that the use of hormonal contraceptives and tobacco smoking are risk factors for CC development in addition to the presence of HPV.

Components of the transitional care model (TCM) to reduce readmission in geriatric patients: a systematic review.

BACKGROUND: Demographic changes are taking place in most industrialized countries. Geriatric patients are defined by the European Union of Medical Specialists as aged over 65 years and suffering from frailty and multi-morbidity, whose complexity puts a major burden on these patients, their family caregivers and the public health care system. To counteract negative outcomes and to maintain consistency in care between hospital and community dwelling, the transitional of care has emerged over the last several decades. Our objectives were to identify and summarize the components of the Transitional Care Model implemented with geriatric patients (aged over 65 years, with multi-morbidity) for the reduction of all-cause readmission. Another objective was to recognize the Transitional Care Model components' role and impact on readmission rate reduction on the transition of care from hospital to community dwelling (not nursing homes). METHODS: Randomized controlled trials (sample size ≥50 participants per group; intervention period ≥30 days), with geriatric patients were included. Electronic databases (MEDLINE, CINAHL, PsycINFO and The Cochrane Central Register of Controlled Trials) were searched from January 1994 to December 2019 published in English or German. A qualitative synthesis of the findings as well as a systematic assessment of the interventions intensities was performed. RESULTS: Three articles met the inclusion criteria. One of the included trials applied all of the nine Transitional Care Model components described by Hirschman and colleagues and obtained a high-intensity level of intervention in the intensities assessment. This and another trial reported reductions in the readmission rate (p < 0.05), but the third trial did not report significant differences between the groups in the longer follow-up period (up to 12 months). CONCLUSIONS: Our findings suggest that high intensity multicomponent and multidisciplinary interventions are likely to be effective reducing readmission rates in geriatric patients, without increasing cost. Components such as type of staffing, assessing and managing symptoms, educating and promoting self-management, maintaining relationships and fostering coordination seem to have an important role in reducing the readmission rate. Research is needed to perform further investigations addressing geriatric patients well above 65 years old, to further understand the importance of individual components of the TCM in this population.

Oligosaccharides: Defense Inducers, Their Recognition in Plants, Commercial Uses and Perspectives.

Plants have innate immune systems or defense mechanisms that respond to the attack of pathogenic microorganisms. Unlike mammals, they lack mobile defense cells, so defense processes depend on autonomous cellular events with a broad repertoire of recognition to detect pathogens, which compensates for the lack of an adaptive immune system. These defense mechanisms remain inactive or latent until they are activated after exposure or contact with inducing agents, or after the application of the inductor; they remain inactive only until they are affected by a pathogen or challenged by an elicitor from the same. Resistance induction represents a focus of interest, as it promotes the activation of plant defense mechanisms, reducing the use of chemical synthesis pesticides, an alternative that has even led to the generation of new commercial products with high efficiency, stability and lower environmental impact, which increase productivity by reducing not only losses but also increasing plant growth. Considering the above, the objective of this review is to address the issue of resistance induction with a focus on the potential of the use of oligosaccharides in agriculture, how they are recognized by plants, how they can be used for commercial products and perspectives.

[Healthcare of the future-Insights and strategy for (dementia) health services research]. / Gesundheitsversorgung der Zukunft ­ Erkenntnisse und Strategie der (Demenz­)Versorgungsforschung.

BACKGROUND: Due to the demographic change healthcare for older people is becoming more important. A key strategic document for the near future is the national dementia strategy (NDS), which defines four fields of action including promoting excellent research on dementia. The NDS will guide and influence the further development of dementia healthcare research in the coming years. OBJECTIVE: The current research on specific NDS topics is presented and an outlook on expected developments is given. MATERIAL AND METHODS: This article provides a narrative review in which concepts and examples for selected sections of the NDS are presented: funding and promotion of healthcare research, development of evidence-based prevention and healthcare concepts and transfer into routine care, support for people with dementia (PwD) and their caregiver, cross-sectoral networking, participation in dementia research and networks in healthcare research. These were analyzed with respect to future developments and concretized based on current healthcare and promotion models. RESULTS: Insights are given into the healthcare concept of dementia care management, rethinking regional healthcare models such as medicine and e­health. The innovation fund and research practice networks are described as examples of current structural methods of evidence-based design of future healthcare. CONCLUSION: The NDS represents an ambitious agenda with very comprehensive goals and topics for the improvement of healthcare for PwD and will probably significantly influence healthcare research and thus healthcare in the future. Overarching, mutually influencing and strengthening components on the way to improvement of the situation for PwD and the healthcare system are translation, participation and networking in research.

Detection of Echinocandin-Resistant Candida glabrata in Blood Cultures Spiked with Different Percentages of FKS2 Mutants.

Infections caused by the coexistence of Candida glabrata echinocandin-resistant and echinocandin-susceptible cells may be possible, and the detection of FKS mutants when the proportions of FKS mutants are underrepresented poses a problem. We assessed the role of EUCAST and methods directly performed on positive blood cultures-Etest (ETDIR) and anidulafungin-containing agar plate assays-for detecting resistance in C. glabrata isolates containing different amounts of echinocandin-susceptible and -resistant Candida glabrata isolates. We studied 10 pairs of C. glabrata isolates involving parental echinocandin-susceptible isolates and isogenic echinocandin-resistant FKS mutant isolates. Three inocula per pair (1 × 103 to 5 × 103, 1 × 102 to 5 × 102, and 10 to 50 CFU/ml) spanning suspensions with different amounts of susceptible/resistant isolates (9/1, 5/5, and 1/9 proportions for each the three inocula) were prepared. The suspensions were spiked in Bactec bottles and incubated until they were positive, and the three methods were compared. The EUCAST method showed echinocandin resistance when the bottles were spiked with susceptible/resistant isolates at 5/5 and 1/9 proportions; the results for the suspensions with a 9/1 proportion of susceptible/resistant isolates were susceptible for three pairs. We observed with the ETDIR resistance to both echinocandins in all pairs (resistance to micafungin and anidulafungin; MICs, ≥0.064 mg/liter and ≥0.125 mg/liter, respectively) and a double ring of growth inhibition in two pairs. The anidulafungin-containing plates showed fungal growth in the 90 spiked blood cultures at 48 h. Testing of echinocandin susceptibility with the ETDIR directly on the positive blood culture bottles is a reliable and rapid method to detect echinocandin resistance in C. glabrata On the other hand, resistance can be missed with the EUCAST method when resistant isolates are underrepresented.

MSH2 Gene Point Mutations Are Not Antifungal Resistance Markers in Candida glabrata.

The high rates of antifungal resistance in Candida glabrata may be facilitated by the presence of alterations in the MSH2 gene. We aimed to study the sequence of the MSH2 gene in 124 invasive C. glabrata isolates causing incident episodes of candidemia (n = 81), subsequent candidemia episodes (n = 9), endocarditis (n = 2), and in vitro-generated echinocandin-resistant isolates (n = 32) and assessed its relationship with genotypes, acquisition of antifungal resistance in vivo and in vitro, and patient prognosis. The MSH2 gene was sequenced, and isolates were genotyped using six microsatellite markers and multilocus sequence typing (MLST) based on six housekeeping genes. According to EUCAST, isolates causing candidemia (n = 90) were echinocandin susceptible, and four of them were fluconazole resistant (MIC ≥64 mg/liter). One isolate obtained from a heart valve was resistant to micafungin and anidulafungin (MICs, 2 mg/liter and 1 mg/liter, respectively). MSH2 gene mutations were present in 44.4% of the incident isolates, the most common being V239L. The presence of MSH2 mutations was not correlated with in vitro or in vivo antifungal resistance. Microsatellite and MLST revealed 27 genotypes and 17 sequence types, respectively. Fluconazole-resistant isolates were unrelated. Most MSH2 mutations were found in cluster isolates; conversely, some mutations were found in more than one genotype. No clinical differences, including previous antifungal use, were found between patients infected by wild-type MSH2 gene isolates and isolates with any point mutation. The presence of MSH2 gene mutations in C. glabrata isolates causing candidemia is not correlated with specific genotypes, the promotion of antifungal resistance, or the clinical outcome.

Growth kinetics in Candida spp.: Differences between species and potential impact on antifungal susceptibility testing as described by the EUCAST.

We studied the growth kinetic parameters of clinically relevant Candida species to verify the differences between species following the incubation and medium conditions recommended by the EUCAST. We analyzed 705 susceptible Candida spp. from patients with candidemia and Candida glabrata isolates resistant to echinocandins or fluconazole (n = 38) and calculated the average growth rate, maximum peak, time to maximum rate, and lag phase. We also examined inter- and intra-species differences, as well as the percentage of isolates reaching an optical density of 0.2 over time. Interspecies differences in growth phases and kinetic parameters were found. C. glabrata was the fastest growing species and the lag phase of C. parapsilosis was longer than that of the other species considered in this study. Strain-to-strain variations were found between species. A positive correlation between the average growth rate and maximum peak was determined. Echinocandin-resistant C. glabrata isolates had significantly lower average growth rate but higher time to maximum rate in comparison to wild-type C. glabrata isolates. Incubation periods of 12-15 hours allowed reaching the 0.2 optical density threshold in 100% of C. glabrata, C. tropicalis, and C. krusei isolates. We show differences in kinetic parameters between Candida spp. C. glabrata was the fastest growing species and C. parapsilosis showed the longest lag phase. Resistance to echinocandins may affect the growth kinetic curve. Speeding up antifungal susceptibility results could be possible for some isolates, particularly C. glabrata, C. tropicalis, and C. krusei.

Resistance to Echinocandins in Candida Can Be Detected by Performing the Etest Directly on Blood Culture Samples.

We examined the rapid evaluation of susceptibility to echinocandins in Candida spp. using the Etest performed directly on positive blood cultures and anidulafungin-containing agar plates. We prospectively collected 80 positive blood cultures (Bactec-FX system, Becton-Dickinson, Cockeysville, MD, USA) with echinocandin-susceptible Candida spp. (n = 60) and echinocandin-intermediate Candida parapsilosis (n = 20) from patients with candidemia. Additionally, blood culture bottles of nonfungemic/bacteremic patients were spiked with 35 echinocandin-resistant Candida species isolates. A total of 2 to 4 drops of medium from each bottle were stroked directly onto both RPMI 1640 agar plates with micafungin and anidulafungin Etest strips (ETDIR) and Sabouraud agar plates containing 2 mg/liter of anidulafungin. The isolates were tested according to the EUCAST method and Etest standard (ETSD). Essential and categorical agreement between the methods was calculated. The essential agreement and categorical agreement between the EUCAST method and ETDIR and ETSD were both >97.4%. The essential agreement between ETDIR and the EUCAST method for both echinocandins was >97%. The categorical agreement between the FKS sequence and ETDIR was 97.4%. The ETDIR MICs of anidulafungin and micafungin (≥0.19 mg/liter and ≥0.064 mg/liter, respectively) effectively separated all susceptible FKS wild-type isolates from the resistant FKS mutant isolates. The categorical agreement (62.6%) between the EUCAST method and growth on anidulafungin-containing plates was poor, with the best agreement observed for Candida glabrata (94.2%). When performed directly on positive blood cultures from patients with candidemia, the Etest with micafungin and anidulafungin is a reliable procedure for the rapid testing of susceptibility to echinocandins in Candida species isolates.

Mutant Prevention Concentration and Mutant Selection Window of Micafungin and Anidulafungin in Clinical Candida glabrata Isolates.

We report the mutant prevention concentration (MPC) and mutant selection window (MSW) for micafungin and anidulafungin administered to treat Candida glabrata We also determine the mutation frequency. We studied 20 echinocandin-susceptible, fluconazole-intermediate, and FKS wild-type C. glabrata isolates. Adjusted inocula were stroked directly onto Sabouraud agar plates containing different concentrations of micafungin or anidulafungin and visually inspected daily for up to 5 days of incubation. Individual colonies growing on the plates containing echinocandins at 1 mg/liter were selected for antifungal susceptibility testing. The FKS genes of the resulting individual phenotypically resistant colonies were sequenced, and the MPC, MSW, and mutation frequency were determined. Biofilm was quantified, and the growth kinetics and virulence (Galleria mellonella model) of the resulting individual FKS mutant colonies were studied. For micafungin and anidulafungin, we found similar results for the MPC (0.06 to 2 mg/liter and 0.25 to 2 mg/liter, respectively), MSW (0.015 to 2 mg/liter for both echinocandins), and mutation frequency (3.7 × 10-8 and 2.8 × 10-8, respectively). A total of 12 isolates were able to grow at 1 mg/liter on echinocandin-containing plates, yielding a total of 32 phenotypically resistant colonies; however, FKS2 mutations (ΔF658, S663P, W715L, and E655A) were observed only in 21 colonies. We did not find differences in biofilm formation, the kinetic parameters studied, or the median survival of larvae infected by wild-type isolates and the resulting individual FKS2 mutant colonies. Echinocandin concentrations lower than 2 mg/liter can lead to selection of resistance mutations in C. glabrata isolates in vitro.

Low and constant micafungin concentrations may be sufficient to lead to resistance mutations in FKS2 gene of Candida glabrata.

We studied the ability of five echinocandin-susceptible C. glabrata isolates to acquire in vitro resistance to anidulafungin and micafungin. All isolates became phenotypically resistant after 2-4 days of exposure to low and constant micafungin concentrations (P < .05). Mutations in the HS1 region of the FKS2 gene were found in all isolates. The acquisition of resistance was not related to the previous use of antifungal treatment in the patients or the presence of mutations at MSH2 gene. We found differences (P < .0001) in the median survival of Galleria mellonella larvae infected with FKS2 mutant isolates (5 days) and wild-type isolates (3 days).

In Vitro Exposure to Increasing Micafungin Concentrations Easily Promotes Echinocandin Resistance in Candida glabrata Isolates.

We assessed the in vitro susceptibility of five echinocandin-susceptible Candida glabrata isolates after exposure to micafungin. The direct exposure to plates at different micafungin concentrations resulted in the inhibition of growth at 0.062 µg/ml. The progressive exposure was performed on plates using 0.031 µg/ml of micafungin and sequential propagation on plates containing the next 2-fold concentration; the MICs of micafungin and anidulafungin increased sequentially, and all the isolates became echinocandin resistant, showing fks2 mutations.