Evaluación del sistema MALDI-TOF VITEK MSTM para la identificación rápida de microorganismos de interés clínico

RESUMEN: La identificación de los microorganismos ha sido una de las principales tareas del microbiólogo a través de los tiempos. La espectrometría de masas (EM) ha irrumpido con fuerza en los laboratorios, en principio, para la identificación de microorganismos. La motivación de la tesis fue demostrar que la espectrometría de masas MALDI-TOF es una técnica fiable, rápida y precisa que permite identificar a nivel de especie la mayor parte de los microorganismos presentes en muestras de origen humano clínicamente relevantes, de manera que puede utilizarse en la rutina diaria de un laboratorio de microbiología clínica como alternativa o complemento a las técnicas tradicionales. Para validar esta hipótesis se planteó el objetivo inicial de evaluar el sistema MALDI-TOF VITEK MSTM (bioMérieux, Marcy l'Etoile, Francia) en una colección de cepas que incluía una variedad de especies representativa de las aisladas con más frecuencia en nuestro laboratorio. A continuación, se evaluó la aplicación de la técnica para resolver la identificación a nivel de especie de 3 grupos de microorganismos: el complejo Acinetobacter baumannii (ACB), Rhodococcus equi / Dietzia spp. y Candida spp.. Finalmente concluir que la tecnología MALDI-TOF es una buena metodología para la actividad rutinaria en laboratorios de microbiología clínica, ya que identifica una amplia gama de especies bacterianas y levaduras de importancia clínica, con rapidez y precisión.ABSTRACT: The identification of microorganisms has been one of the main tasks of the microbiologist over time. Mass spectrometry (MS) has entered laboratories strongly, in principle, for the identification of microorganisms. The aim of the thesis was to demonstrate that mass spectrometry MALDI-TOF is a reliable, fast and accurate technique that allows the identification at species level of most microorganisms present in clinically relevant human samples. so it can be used in the daily routine of a clinical microbiology laboratory as an alternative or complement to traditional techniques. To validate this hypothesis, the initial objective of evaluating the MALDI-TOF VITEK MSTM system (bioMérieux, Marcy l'Etoile, France) was set out in a collection of strains that included a variety of species representative of those most frequently isolated in our laboratory. The application of the technique to resolve the species-level identification of 3 groups of microorganisms was then evaluated: Acinetobacter baumannii complex (ACB), Rhodococcus equi/Dietzia spp. and Candida spp.. Finally, conclude that MALDI-TOF technology is a good methodology for routine activity in clinical microbiology laboratories, identifying a wide range of clinically important bacterial and yeast species, quickly and accurately

Post-traumatic Endophthalmitis Caused by Nocardia nova

Introduction: Nocardia nova complex has been associated with infections in both immunocompetent and immunocompromised patients. Infection can be localized or disseminated, affecting skin and soft tissues, the respiratory system, bones and joints, the circulatory system and especially the central nervous system. Ocular infections such as keratitis, scleritis, conjunctivitis, dacryocystitis, orbital cellulitis and endophthalmitis due to Nocardia spp. are infrequently reported, and usually described after penetrating corneal trauma or ocular contact with plants and soils. Case presentation: An immunocompetent male presented with a history of penetrating ocular trauma that had evolved to infectious endophthalmitis, which was refractory to different antibiotic treatments. No micro-organisms were isolated from repeated conjunctival smear and corneal scraping cultures between the ocular trauma (August 2014) and the endophthalmitis diagnosis (November 2015). After this period, N. nova sensu stricto was isolated in aqueous humour aspirate. Treatment was adjusted and clinical improvement was obtained after an adequate microbiological procedure, including an optimal sampling and an antimicrobial-susceptibility testing report. Conclusion: Nocardia identification to the species level and performance of antimicrobial-susceptibility tests are both essential tools for treatment adjustment and clinical improvement

Sternal wound infection caused by Gordonia bronchialis: identification by MALDI-TOF MS

Introduction: Gordonia spp. infections are uncommon. However, a few clinical cases have been reported in the literature, particularly those involving immunocompromised hosts. Advanced microbiology diagnosis techniques, such as matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS), have been recently introduced in clinical microbiology laboratories in order to improve microbial identification, resulting in better patient management. Case presentation: Here, we present a new clinical case of persistent wound infection caused by Gordonia bronchialis in a 64-year-old woman after a mitral valve replacement, using two MALDI-TOF-based systems for identifying this micro-organism. Conclusion: Both MALDI-TOF systems were able to identify Gordonia spp.; thus, providing a useful tool that overcomes the current limitations of phenotypic identification associated with this micro-organism. Although the technique validation deserves additional verification, our study provides guidance about MALDI-TOF as a fast and easy method for Gordonia spp. identification

A multicentre study investigating parameters which influence direct bacterial identification from urine

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification

Epidemiologic and clinical impact of acinetobacter baumannii colonization and infection: A reappraisal

Abstract: Acinetobacter baumannii is one of the most important antibiotic-resistant nosocomial bacteria. We investigated changes in the clinical and molecular epidemiology of A. baumannii over a 10-year period. We compared the data from 2 prospective multicenter cohort studies in Spain, one performed in 2000 (183 patients) and one in 2010 (246 patients), which included consecutive patients infected or colonized by A. baumannii. Molecular typing was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The incidence density of A. baumannii colonization or infection increased significantly from 0.14 in 2000 to 0.52 in 2010 in medical services (p<0.001). The number of non-nosocomial health careassociated cases increased from 1.2% to 14.2%, respectively (p<0.001). Previous exposure to carbapenems increased in 2010(16.9% in 2000 vs 27.3% in 2010, p¼0.03). The drugs most frequently used for definitive treatment of patients with infections were carbapenems in 2000 (45%) and colistin in 2010 (50.3%). There was molecular-typing evidence of an increase in the frequency of A. baumannii acquisition in non-intensive care unit wards in 2010 (7.6% in 2000 vs 19.2% in 2010, p¼0.01). By MSLT, the ST2 clonal group predominated and increased in 2010. This epidemic clonal group was more frequently resistant to imipenem and was associated with an increased risk of sepsis, although not with severe sepsis or mortality. Some significant changes were noted in the epidemiology of A. baumannii, which is increasingly affecting patients admitted to conventional wards and is also the cause of non-nosocomial health care-associated infections. Epidemic clones seem to combine antimicrobial resistance and the ability to spread, while maintaining their clinical virulence.ACKNOWLEDGMENTS: The authors thank the Platform for Genotyping of Pathogens and Public Health (Institut Pasteur, Paris, France) for coding MLST alleles and profiles as available at www. pasteur.fr/mlst. The authors are grateful for the support of the Spanish Group for Nosocomial Infections (GEIH) and the Spanish Group for Antimicrobial Mechanisms of Action and Resistance (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) for their support in the development of the project

Automatic classification of Candida species using Raman spectroscopy and machine learning

One of the problems that most affect hospitals is infections by pathogenic microorganisms. Rapid identification and adequate, timely treatment can avoid fatal consequences and the development of antibiotic resistance, so it is crucial to use fast, reliable, and not too laborious techniques to obtain quick results. Raman spectroscopy has proven to be a powerful tool for molecular analysis, meeting these requirements better than traditional techniques. In this work, we have used Raman spectroscopy combined with machine learning algorithms to explore the automatic identification of eleven species of the genus Candida, the most common cause of fungal infections worldwide. The Raman spectra were obtained from more than 220 different measurements of dried drops from pure cultures of each Candida species using a Raman Confocal Microscope with a 532 nm laser excitation source. After developing a spectral preprocessing methodology, a study of the quality and variability of the measured spectra at the isolate and species level, and the spectral features contributing to inter-class variations, showed the potential to discriminate between those pathogenic yeasts. Several machine learning and deep learning algorithms were trained using hyperparameter optimization techniques to find the best possible classifier for this spectral data, in terms of accuracy and lowest possible overfitting. We found that a one-dimensional Convolutional Neural Network (1-D CNN) could achieve above 80 % overall accuracy for the eleven classes spectral dataset, with good generalization capabilities.This work was supported by the R + D projects INNVAL19/17 (funded by Instituto de Investigación Valdecilla-IDIVAL), PID2019-107270RB-C21 (funded by MCIN/ AEI /10.13039/501100011033) and by Plan Nacional de I + D + and Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0007), CIBERINFEC (CB21/13/00068), CIBER-BBN (BBNGC1601), cofinanced by European Development Regional Fund “A way to achieve Europe”. A. A. O.-S was financially supported by the Miguel Servet II program (ISCIII-CPII17-00011)

Is Routine Prophylaxis Against Pneumocystis jirovecii Needed in Liver Transplantation? A Retrospective Single-Centre Experience and Current Prophylaxis Strategies in Spain

In liver transplant (LT) recipients, Pneumocystis jirovecii pneumonia (PJP) is most frequently reported before 1992 when immunosuppressive regimens were more intense. It is uncertain whether universal PJP prophylaxis is still applicable in the contemporary LT setting. We aimed to examine the incidence of PJP in LT recipients followed at our institution where routine prophylaxis has never been practiced and to define the prophylaxis strategies currently employed among LT units in Spain. All LT performed from 1990 to October 2019 were retrospectively reviewed and Spanish LT units were queried via email to specify their current prophylaxis strategy. During the study period, 662 LT procedures were carried out on 610 patients. Five cases of PJP were identified, with only one occurring within the first 6 months. The cumulative incidence and incidence rate were 0.82% and 0.99 cases per 1000 person transplant years. All LT units responded, the majority of which provide prophylaxis (80%). Duration of prophylaxis, however, varied significantly. The low incidence of PJP in our unprophylaxed cohort, with most cases occurring beyond the usual recommended period of prophylaxis, questions a one-size-fits-all approach to PJP prophylaxis. A significant heterogeneity in prophylaxis strategies exists among Spanish LT centres.Funding: This study was supported by the Health Research Institute Marqués de Valdecilla. IDIVAL. Santander. NEXT VAL17/07 grant to José Ignacio Fortea Ormaechea

Efficient differentiation of Corynebacterium striatum, Corynebacterium amycolatum and Corynebacterium xerosis clinical isolates by multiplex PCR using novel species-specific primers

A multiplex-PCR (mPCR) assay was designed with species-specific primers which generate amplicons of 226 bp, 434 bp and 106 bp for differentiating the species C. striatum, C. amycolatum, and C. xerosis, respectively. mPCR results were 100% in agreement with identifications achieved by 16S rRNA and rpoB gene sequencing and by VITEK-MS.This work was supported by grants from FAPESB (JCB0031/2013) and CAPES (PROCAD 071/2013)

Epigenetic profiling linked to multisystem inflammatory syndrome in children (MIS-C): A multicenter, retrospective study

Background Most children and adolescents infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain asymptomatic or develop a mild coronavirus disease 2019 (COVID-19) that usually does not require medical intervention. However, a small proportion of pediatric patients develop a severe clinical condition, multisystem inflammatory syndrome in children (MIS-C). The involvement of epigenetics in the control of the immune response and viral activity prompted us to carry out an epigenomic study to uncover target loci regulated by DNA methylation that could be altered upon the appearance of MIS-C. Methods Peripheral blood samples were recruited from 43 confirmed MIS-C patients. 69 non-COVID-19 pediatric samples and 15 COVID-19 pediatric samples without MIS-C were used as controls. The cases in the two groups were mixed and divided into discovery (MIS-C= 29 and non-MIS-C= 56) and validation (MIS-C= 14 and non-MIS C = 28) cohorts, and balanced for age, gender and ethnic background. We interrogated 850,000 CpG sites of the human genome for DNA methylation variants. Findings The DNA methylation content of 33 CpG loci was linked with the presence of MIS-C. Of these sites, 18 (54.5%) were located in described genes. The top candidate gene was the immune T-cell mediator ZEB2; and others highly ranked candidates included the regulator of natural killer cell functional competence SH2D1B; VWA8, which contains a domain of the Von Willebrand factor A involved in the pediatric hemostasis disease; and human leukocyte antigen complex member HLA-DRB1; in addition to pro-inflammatory genes such as CUL2 and AIM2. The identified loci were used to construct a DNA methylation profile (EPIMISC) that was associated with MIS-C in both cohorts. The EPIMISC signature was also overrepresented in Kawasaki disease patients, a childhood pathology with a possible viral trigger, that shares many of the clinical features of MIS-C. Interpretation We have characterized DNA methylation loci that are associated with MIS-C diagnosis. The identified genes are likely contributors to the characteristic exaggerated host inflammatory response observed in these patients. The described epigenetic signature could also provide new targets for more specific therapies for the disorder.Copyright (c) 2022 The Author(s). Published by Elsevier Ltd

Epidemiological and clinical characteristics of Streptococcus tigurinus endocarditis

Background: Streptococcus tigurinus was recently described as a new streptococcal species within the viridans group streptococci (VGS). The objectives of the present work were to analyse the clinical and microbiological characteristics of S. tigurinus isolated from patients with bacteraemias, to determine the prevalence of S. tigurinus among VGS endocarditis in Spain, and to compare the clinical characteristics and outcomes of endocarditis caused by S. tigurinus and other VGS. Methods: Retrospective nationwide study, performed between 2008 and 2016 in 9 Spanish hospitals from 7 different provinces comprising 237 cases of infective endocarditis. Streptococcal isolates were identified by sequencing fragments of their 16S rRNA, sodA and groEL genes. Clinical data of patients with streptococcal endocarditis were prospectively collected according to a pre-established protocol. Results: Patients with endocarditis represented 7/9 (77.8%) and 26/86 (30.2%) of the bacteraemias caused by S. tigurinus and other VGS, respectively (p < 0.001), in two of the hospital participants. Among patients with streptococcal endocarditis, 12 different Streptococcus species were recognized being S. oralis, S. tigurinus and S. mitis the three more common. No relevant statistical differences were observed in the clinical characteristics and outcomes of endocarditis caused by the different VGS species. Conclusions: In this multicenter study performed in Spain, S. tigurinus showed a higher predilection for the endocardial endothelium as compared to other VGS. However, clinical characteristics and outcomes of endocarditis caused by S. tigurinus did not significantly differ from endocarditis caused by other oral streptococci.JMM received a personal 80:20 research grant from the Institut d’InvestigacionsBiomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain during 2017–19. Nofunding entity played any role in the design of the study and data collection,analysis, and interpretation of data and in writing the manuscript