Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

IMPORTANCE: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C . glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C . glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C . glabrata blood isolates ( n =1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. Importance: Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Whole-genome sequencing confirms a persistent candidaemia clonal outbreak due to multidrug-resistant Candida parapsilosis.

OBJECTIVES: Although perceived as a rare clinical entity, recent studies have noted the emergence of MDR C. parapsilosis (MDR-Cp) isolates from single patients (resistant to both azole and echinocandins). We previously reported a case series of MDR-Cp isolates carrying a novel FKS1R658G mutation. Herein, we identified an echinocandin-naive patient infected with MDR-Cp a few months after the previously described isolates. WGS and CRISPR-Cas9 editing were used to explore the origin of the new MDR-Cp isolates, and to determine if the novel mutation confers echinocandin resistance. METHODS: WGS was applied to assess the clonality of these isolates and CRISPR-Cas9 editing and a Galleria mellonella model were used to examine whether FKS1R658G confers echinocandin resistance. RESULTS: Fluconazole treatment failed, and the patient was successfully treated with liposomal amphotericin B (LAMB). WGS proved that all historical and novel MDR-Cp strains were clonal and distant from the fluconazole-resistant outbreak cluster in the same hospital. CRISPR-Cas9 editing and G. mellonella virulence assays confirmed that FKS1R658G confers echinocandin resistance in vitro and in vivo. Interestingly, the FKS1R658G mutant showed a very modest fitness cost compared with the parental WT strain, consistent with the persistence of the MDR-Cp cluster in our hospital. CONCLUSIONS: Our study showcases the emergence of MDR-Cp isolates as a novel threat in clinical settings, which undermines the efficacy of the two most widely used antifungal drugs against candidiasis, leaving only LAMB as a last resort. Additionally, surveillance studies and WGS are warranted to effectively establish infection control and antifungal stewardship strategies.

Karyon: a computational framework for the diagnosis of hybrids, aneuploids, and other nonstandard architectures in genome assemblies.

BACKGROUND: Recent technological developments have made genome sequencing and assembly highly accessible and widely used. However, the presence in sequenced organisms of certain genomic features such as high heterozygosity, polyploidy, aneuploidy, heterokaryosis, or extreme compositional biases can challenge current standard assembly procedures and result in highly fragmented assemblies. Hence, we hypothesized that genome databases must contain a nonnegligible fraction of low-quality assemblies that result from such type of intrinsic genomic factors. FINDINGS: Here we present Karyon, a Python-based toolkit that uses raw sequencing data and de novo genome assembly to assess several parameters and generate informative plots to assist in the identification of nonchanonical genomic traits. Karyon includes automated de novo genome assembly and variant calling pipelines. We tested Karyon by diagnosing 35 highly fragmented publicly available assemblies from 19 different Mucorales (Fungi) species. CONCLUSIONS: Our results show that 10 (28.57%) of the assemblies presented signs of unusual genomic configurations, suggesting that these are common, at least for some lineages within the Fungi.

Candidemia Among Coronavirus Disease 2019 Patients in Turkey Admitted to Intensive Care Units: A Retrospective Multicenter Study.

Background: We evaluated the epidemiology of candidemia among coronavirus disease 2019 (COVID-19) patients admitted to intensive care units (ICUs). Methods: We conducted a retrospective multicenter study in Turkey between April and December 2020. Results: Twenty-eight of 148 enrolled patients developed candidemia, yielding an incidence of 19% and incidence rate of 14/1000 patient-days. The probability of acquiring candidemia at 10, 20, and 30 days of ICU admission was 6%, 26%, and 50%, respectively. More than 80% of patients received antibiotics, corticosteroid, and mechanical ventilation. Receipt of a carbapenem (odds ratio [OR] = 6.0, 95% confidence interval [CI] = 1.6-22.3, P = .008), central venous catheter (OR = 4.3, 95% CI = 1.3-14.2, P = .02), and bacteremia preceding candidemia (OR = 6.6, 95% CI = 2.1-20.1, P = .001) were independent risk factors for candidemia. The mortality rate did not differ between patients with and without candidemia. Age (OR = 1.05, 95% CI = 1.01-1.09, P = .02) and mechanical ventilation (OR = 61, 95% CI = 15.8-234.9, P < .0001) were independent risk factors for death. Candida albicans was the most prevalent species overall. In Izmir, Candida parapsilosis accounted for 50% (2 of 4) of candidemia. Both C parapsilosis isolates were fluconazole nonsusceptible, harbored Erg11-Y132F mutation, and were clonal based on whole-genome sequencing. The 2 infected patients resided in ICUs with ongoing outbreaks due to fluconazole-resistant C parapsilosis. Conclusions: Physicians should be aware of the elevated risk for candidemia among patients with COVID-19 who require ICU care. Prolonged ICU exposure and ICU practices rendered to COVID-19 patients are important contributing factors to candidemia. Emphasis should be placed on (1) heightened infection control in the ICU and (2) developing antibiotic stewardship strategies to reduce irrational antimicrobial therapy.

PhylomeDB V5: an expanding repository for genome-wide catalogues of annotated gene phylogenies.

PhylomeDB is a unique knowledge base providing public access to minable and browsable catalogues of pre-computed genome-wide collections of annotated sequences, alignments and phylogenies (i.e. phylomes) of homologous genes, as well as to their corresponding phylogeny-based orthology and paralogy relationships. In addition, PhylomeDB trees and alignments can be downloaded for further processing to detect and date gene duplication events, infer past events of inter-species hybridization and horizontal gene transfer, as well as to uncover footprints of selection, introgression, gene conversion, or other relevant evolutionary processes in the genes and organisms of interest. Here, we describe the latest evolution of PhylomeDB (version 5). This new version includes a newly implemented web interface and several new functionalities such as optimized searching procedures, the possibility to create user-defined phylome collections, and a fully redesigned data structure. This release also represents a significant core data expansion, with the database providing access to 534 phylomes, comprising over 8 million trees, and homology relationships for genes in over 6000 species. This makes PhylomeDB the largest and most comprehensive public repository of gene phylogenies. PhylomeDB is available at http://www.phylomedb.org.

[Atypical presentation of diabetic foot in post-COVID-19 patients]. / Presentación atípica de pie diabético en pacientes pos-COVID-19.

Medical attention of patients with a diabetic foot has been disrupted since de COVID-19 pandemic began, because health systems had to provide care to those patients affected by this disease to the detriment of the control of chronic diseases. Several reports show an increase in amputations during the pandemic, primarily due to the lack of health controls in patients suffering from diabetes or diabetic foot. This could have resulted in later consultation and more severe presentations. We describe three medical cases that had recently been affected by COVID-19 and developed a rare and rapidly evolving diabetic foot that required a major amputation. One possible explanation for this atypical presentation could be that COVID-19 predisposes patients to vein and arterial thrombosis due to systemic inflammation, platelet activation, endothelial dysfunction and stasis from prolonged immobility. This could have exacerbated chronic ischemia secondary to diabetes in which metabolic disturbances often seen in these patients predispose to atherosclerosis.

La atención de pacientes con pie diabético se vio afectada luego de la aparición de COVID-19, ya que los sistemas de salud debieron dar prioridad a la atención de dicha enfermedad y se restringió el control de las enfermedades crónicas. Existen algunas comunicaciones sobre el aumento de las amputaciones durante la pandemia, el cual fue atribuido principalmente a la falta de controles de los pacientes con diabetes y pie diabético. Esto pudo haber influido en la consulta tardía y en la aparición de presentaciones más graves. Presentamos los casos de tres pacientes con reciente enfermedad por COVID-19 que desarrollaron una forma atípica de pie diabético grave y rápidamente evolutiva cuyo desenlace fue la amputación mayor. Una posible explicación estaría relacionada a que los pacientes con COVID-19 tienen mayor riesgo de enfermedad trombótica, tanto venosa como arterial, debido a la intensa respuesta inflamatoria, la activación plaquetaria, la disfunción endotelial y la estasis sanguínea por inmovilización, que se asocia a esta enfermedad. Esto podría agravar la isquemia crónica que desarrollan las personas con diabetes, causada por el estado metabólico anormal que favorece la ateroesclerosis de todo el árbol vascular.

Presentación atípica de pie diabético en pacientes pos-COVID-19 / Atypical presentation of diabetic foot in post-COVID-19 patients

Resumen La atención de pacientes con pie diabético se vio afectada luego de la aparición de COVID-19, ya que los sistemas de salud debieron dar prioridad a la atención de dicha enfermedad y se restringió el control de las enfermedades crónicas. Existen algunas comunicaciones sobre el aumento de las amputaciones durante la pandemia, el cual fue atribuido principalmente a la falta de controles de los pacientes con diabetes y pie diabético. Esto pudo haber influido en la consulta tardía y en la aparición de presentaciones más graves. Presentamos los casos de tres pacientes con reciente enfermedad por COVID-19 que desarrollaron una forma atípica de pie diabético grave y rápidamente evolutiva cuyo desenlace fue la amputación mayor. Una posible explicación estaría relacionada a que los pacientes con COVID-19 tienen mayor riesgo de enfermedad trombótica, tanto venosa como arterial, debido a la intensa respuesta inflamatoria, la activación plaquetaria, la disfunción endotelial y la estasis sanguínea por inmovilización, que se asocia a esta enfermedad. Esto podría agravar la isquemia crónica que desarrollan las personas con diabetes, causada por el estado metabólico anormal que favorece la ateroesclerosis de todo el árbol vascular.

Abstract Medical attention of patients with a diabetic foot has been disrupted since de COVID-19 pandemic began, because health systems had to provide care to those patients affected by this disease to the detriment of the control of chronic diseases. Several reports show an increase in amputations during the pandemic, primarily due to the lack of health controls in patients suffering from diabetes or diabetic foot. This could have resulted in later consultation and more severe presentations. We describe three medical cases that had recently been affected by COVID-19 and developed a rare and rapidly evolving diabetic foot that required a major amputation. One possible explanation for this atypi cal presentation could be that COVID-19 predisposes patients to vein and arterial thrombosis due to systemic inflammation, platelet activation, endothelial dysfunction and stasis from prolonged immobility. This could have exacerbated chronic ischemia secondary to diabetes in which metabolic disturbances often seen in these patients predispose to atherosclerosis.

Pareto optimality between growth-rate and lag-time couples metabolic noise to phenotypic heterogeneity in Escherichia coli.

Despite mounting evidence that in clonal bacterial populations, phenotypic variability originates from stochasticity in gene expression, little is known about noise-shaping evolutionary forces and how expression noise translates to phenotypic differences. Here we developed a high-throughput assay that uses a redox-sensitive dye to couple growth of thousands of bacterial colonies to their respiratory activity and show that in Escherichia coli, noisy regulation of lower glycolysis and citric acid cycle is responsible for large variations in respiratory metabolism. We found that these variations are Pareto optimal to maximization of growth rate and minimization of lag time, two objectives competing between fermentative and respiratory metabolism. Metabolome-based analysis revealed the role of respiratory metabolism in preventing the accumulation of toxic intermediates of branched chain amino acid biosynthesis, thereby supporting early onset of cell growth after carbon starvation. We propose that optimal metabolic tradeoffs play a key role in shaping and preserving phenotypic heterogeneity and adaptation to fluctuating environments.

Neurogranin Expression Is Regulated by Synaptic Activity and Promotes Synaptogenesis in Cultured Hippocampal Neurons.

Neurogranin (Ng) is a calmodulin (CaM)-binding protein that is phosphorylated by protein kinase C (PKC) and is highly enriched in the dendrites and spines of telencephalic neurons. It is proposed to be involved in regulating CaM availability in the post-synaptic environment to modulate the efficiency of excitatory synaptic transmission. There is a close relationship between Ng and cognitive performance; its expression peaks in the forebrain coinciding with maximum synaptogenic activity, and it is reduced in several conditions of impaired cognition. We studied the expression of Ng in cultured hippocampal neurons and found that both protein and mRNA levels were about 10% of that found in the adult hippocampus. Long-term blockade of NMDA receptors substantially decreased Ng expression. On the other hand, treatments that enhanced synaptic activity such as long-term bicuculline treatment or co-culture with glial cells or cholesterol increased Ng expression. Chemical long-term potentiation (cLTP) induced an initial drop of Ng, with a minimum after 15 min followed by a slow recovery during the next 2-4 h. This effect was most evident in the synaptosome-enriched fraction, thus suggesting local synthesis in dendrites. Lentiviral expression of Ng led to increased density of both excitatory and inhibitory synapses in the second and third weeks of culture. These results indicate that Ng expression is regulated by synaptic activity and that Ng promotes the synaptogenesis process. Given its relationship with cognitive function, we propose targeting of Ng expression as a promising strategy to prevent or alleviate the cognitive deficits associated with aging and neuropathological conditions.

Restricción del crecimiento intrauterino: Complicaciones inmediatas unidad neonatal, Hospital de San José, Bogotá DC / Immediate complications of intrauterine growth restriction neonatal unit - Hospital de San José Bogotá DC

Identificar las complicaciones de los recién nacidos (RN) con restricción del crecimiento intrauterino (RCIU) en un período de dos años y describir la mortalidad, características maternas y tiempo de inicio del aporte enteral. Métodos: estudio observacional descriptivo en todos los neonatos hospitalizados en la unidad de RN con RCIU en el Hospital de San José, Bogotá, DC, Colombia, entre abril 2009 y marzo 2011. Resultados: de 7.263 nacimientos, se identificaron 140 hospitalizados con RCIU, de estos 81 simétrico (57.8%) y 59 asimétrico (42.2%). Las complicaciones más frecuentes fueron dificultad respiratoria (50.7%), ictericia (48.5%) e hipoglicemia (17.8%). Se registraron cinco muertes (3.5%) y seis casos de enterocolitis necrosante (4.2%). El estrato socioeconómico bajo (56.4%) y la desnutrición (23.5%) fueron las características maternas más frecuentes. El inicio del aporte enteral fue más común en las primeras 24 horas de vida (77.1%), sin relación directa con el desarrollo de enterocolitis. Conclusión: es muy importante conocer los factores de riesgo que conducen a RCIU y sus complicaciones. La desnutrición materna y estrato socioeconómico pueden jugar un papel importante en su desarrollo y en el futuro el control puede ser la base para la prevención.

To describe the immediate (neonatal) complications of intrauterine growth restriction (IUGR), alongsidewith mortality rates, maternal factors and the initiation time of enteric feeding, in a two-year period. Methods: anobservational descriptive trial was conducted including the newborn babies with IUGR hospitalized in the neonatalunit at Hospital de San José, Bogotá, DC, Colombia, between April 2009 and March 2011. Results: out of 7,263 births140 neonates were diagnosed with IUGR and were admitted to the neonatal unit, 81 were classified as symmetricIURG (57.8%) and 59 as asymmetric IUGR (42.2%). The most common complications were respiratory distresssyndrome (50.7%), jaundice (48.5%) and hypoglycemia (17.8%). Five deaths were registered (3.5%) and six cases ofnecrotizing enterocolitis (4.2%) were identified. The most significant maternal factors associated with IUGR were alow socioeconomic status (56.4%) and poor nutrition (23.5%). Enteric support was most often initiated during the first24 hours after birth (77.1%), with no direct relation with the development of enterocolitis. Conclusions: it is extremelyimportant to know the factors that can contribute to IUGR and subsequent complications. Maternal malnutrition andlow socioeconomic status may play an important role in the development of IUGR. Adequate prenatal care may bethe base for prevention in the future.

Variabilidad del flujo respiratorio pico en asmáticos persistentes moderados y severos tratados con intal / Variabilty of The Respiratory Flux In Persistent Moderate and Severe Asthmatic Patients Treated With Intal

Se realizó un estudio de intervención con el objetivo de evaluar la eficacia del tratamiento con Intal en niños con asma bronquial persistente, moderada o severa perteneciente al área urbana del Policlínico Docente “Luis Aldana Palomino”, ubicado en el municipio Amancio, en el período comprendido entre agosto del 2003 y agosto 2004. El universo lo constituyeron 51 niños asmáticos, remitidos a una consulta previamente establecida con este fin, previa coordinación con el médico de la familia; la muestra quedó conformada por 34 pacientes según criterios de inclusión. Hubo predominio del asma persistente moderada sobre la severa y de las edades entre los 10 y 18 años. La capacidad para los juegos mejoró considerablemente al año de tratamiento, así como la variabilidad del flujo espiratorio pico(AU)

In the urban area belonging to “Luis Aldana Palomino” Policlinic, from the municipality of Amancio , an intervention study was carried out, in order to evaluate Intal treatment effectiveness in children with persistent, moderate or severe bronchial asthma, from August 2003 to August 2004. The universal set consisted of 51 asthmatic children, referred to a clinic, previously established for that goal; 34 patients were taken as sample according to inclusion criteria. Persistent and moderate asthma and 10-to-18- year age group predominated. The capability to play was improved after one year of treatment, as well as the maximum expiratory flow rate(AU)

Tratamiento con Intal del asma moderada y severa en niños: ausencias a la escuela / Treatment of Moderate and Severe Asthma with Intal in Children: school absenteeism

Se realizó un estudio de intervención con el objetivo de evaluar la eficacia del tratamiento con Intal en niños con asma bronquial persistente moderada o severa perteneciente al área urbana del Policlínico Docente “Luis Aldana Palomino”, ubicado en el municipio Amancio, en el período comprendido entre agosto del 2003 y agosto 2004. El universo lo constituyeron 51 niños asmáticos, remitidos a una consulta previamente establecida con este fin, previa coordinación con el médico de la familia; la muestra quedó conformada por 34 pacientes según criterios de inclusión. El número de ataques por año disminuyó considerablemente al año del tratamiento. Casi todos los pacientes dejaron de tener disnea matinal luego del tratamiento. Igualmente al final del estudio habían mejorado considerablemente la tos nocturna y las ausencias a la escuela(AU)

In the urban area belonging to “Luis Aldana Palomino” Policlinic, from the municipality of Amancio , an intervention study was carried out, in order to evaluate Intal treatment effectiveness in asthmatic children, from August 2003 to August 2004. The universal set consisted of 51 asthmatic children, referred to a clinic previously established for that goal, and 34 patients were taken as sample according to inclusion criteria. The number of asthma attacks decreased after one year of treatment. At the end, almost the majority of the patients did not present troubles to breath in the morning; the absences to school and nocturnal cough turned better too(AU)

Fijación interna rigida craneocervical: analisis experimental en el componente occipital / Craneocervical rigid internal fixation: experimental analysis in the occipital component

En este estudio experimental se buscó identificar las zonas de seguridad de la fosa posterior para fijación interna rígida desde el punto de vista del riesgo de compromiso de los senos venosos asi como tambien aquellas zonas con mayor estabilidad ósea. Se analizó la anatomía occipital, las mediciones del calibre óseo en las zonas estratégicas de fijación en cráneos desecados, asi como tambien en pacientes mediante tomografias. Se encontraron variaciones izquierdas y derechas en la ubicación del seno occipital asi como en la arquitectura osea aun en un mismo cráneo. Las variaciones anatómicas y la simetría de la fosa posterior hacen imposible predecir con seguridad el grosor del cráneo en las zonas estratégicas. Se recomienda efectuar la fijación a nivel de la línea curva occipital inferior evitando la linea media por la presencia variable del seno occipital.