Commercial real time PCR implementation for rapid diagnosis of onychomycosis: A new workflow in a clinical laboratory.

INTRODUCTION: Onychomycosis is a frequent and underdiagnosed condition. Approximately 90% of toenail onychomycosis infections are caused by dermatophytes, but classical diagnosis based on culture and microscopy observation is slow and has low sensitivity. Both limitations can be solved incorporating molecular techniques to routine diagnosis of onychomycosis. OBJECTIVE: Prospective evaluation of the utility of incorporating in the clinical laboratory workflow a commercial real time PCR (qPCR) for dermatophytes detection in nails after potassium hydroxide direct observation screening. MATERIALS AND METHODS: 152 nail samples were included (34 KOH negative and 118 KOH positive) and processed by culture and qPCR. RESULTS: In the negative KOH group, only one dermatophyte grew in culture and three were detected by qPCR. In the group of positive KOH, 57 dermatophytes grew in culture and 81 were detected by qPCR. In this group, 25% of diagnosed dermatophytes were detected only by qPCR. The sensitivity of qPCR compared to culture is 92.8% and time of response decreases from days to hours. CONCLUSION: Based in our results, we propose a workflow algorithm for a clinical laboratory that eliminates culture for qPCR positive samples.

PCR a tiempo real comercial para diagnóstico rápido de las onicomicosis: un nuevo algoritmo de trabajo en el laboratorio clínico / Commercial real time PCR implementation for rapid diagnosis of onychomycosis: A new workflow in a clinical laboratory

Introduction: Onychomycosis is a frequent and underdiagnosed condition. Approximately 90% of toenail onychomycosis infections are caused by dermatophytes, but classical diagnosis based on culture and microscopy observation is slow and has low sensitivity. Both limitations can be solved incorporating molecular techniques to routine diagnosis of onychomycosis. Objective: Prospective evaluation of the utility of incorporating in the clinical laboratory workflow a commercial real time PCR (qPCR) for dermatophytes detection in nails after potassium hydroxide direct observation screening. Materials and methods: 152 nail samples were included (34 KOH negative and 118 KOH positive) and processed by culture and qPCR. Results: In the negative KOH group, only one dermatophyte grew in culture and three were detected by qPCR. In the group of positive KOH, 57 dermatophytes grew in culture and 81 were detected by qPCR. In this group, 25% of diagnosed dermatophytes were detected only by qPCR. The sensitivity of qPCR compared to culture is 92.8% and time of response decreases from days to hours. Conclusion: Based in our results, we propose a workflow algorithm for a clinical laboratory that eliminates culture for qPCR positive samples.(AU)

Introducción: La onicomicosis es una patología frecuentemente infradiagnosticada. Aproximadamente el 90% de las infecciones en las uñas del pie están causadas por dermatofitos, pero el diagnóstico microbiológico clásico basado en cultivo y microscopia es lento y tiene una baja sensibilidad. Ambas limitaciones pueden resolverse incorporando técnicas moleculares al diagnóstico de la onicomicosis. Objetivo: Evaluación prospectiva de la utilidad de incorporar en un laboratorio clínico una PCR a tiempo real (qPCR) comercial para detección de dermatofitos en uñas tras cribado por examen directo con hidróxido de potasio (KOH). Materiales y métodos: Se incluyeron 152 muestras de uñas (34 KOH negativas y 118 KOH positivas) y se procesaron mediante cultivo y qPCR. Resultados: En el grupo KOH negativo, solo un dermatofito creció en cultivo y 3 se detectaron mediante qPCR. En el grupo KOH positivo, 57 dermatofitos crecieron en cultivo y 81 se detectaron por qPCR. En este grupo, el 25% de los dermatofitos diagnosticados se detectaron únicamente mediante qPCR. La sensibilidad de la qPCR comparada con el cultivo es del 92,8% y el tiempo de respuesta disminuye de días a horas. Conclusión: En base a nuestros resultados, proponemos un algoritmo de flujo de trabajo para los laboratorios de microbiología clínica, que elimina el cultivo para aquellas muestras con qPCR positiva.(AU)

Retrospective search of SARS-CoV-2 in respiratory samples in Vallès Occidental (Barcelona, Spain) before the first case was reported / Búsqueda retrospectiva del SARS-CoV-2 en muestras respiratorias en el Vallès Occidental (Barcelona, España) antes de que se notificara el primer caso

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Retrospective search of SARS-CoV-2 in respiratory samples in Vallès Occidental (Barcelona, Spain) before the first case was reported / Búsqueda retrospectiva del SARS-CoV-2 en muestras respiratorias en el Vallès Occidental (Barcelona, España) antes de que se notificara el primer caso

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Direct bacterial identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry: A systematic review and meta-analysis / Identificación bacteriana directa a partir de hemocultivos positivos usando espectrometría de masas MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight): una revisión sistemática y metaanálisis

INTRODUCTION: The rapid identification of bacteraemia-causing pathogens could assist clinicians in the timely prescription of targeted therapy, thereby reducing the morbidity and mortality of this infection. In recent years, numerous techniques that rapidly and directly identify positive blood cultures have been marketed, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) being one of the most commonly used. METHODS: The aim of this systematic review and meta-analysis was to evaluate the accuracy of MALDI-TOF (Bruker(R)) for the direct identification of positive blood culture bottles. RESULTS: A meta-analysis was performed to summarize the results of the 32 studies evaluated. The overall quality of the studies was moderate. For Gram-positive bacteria, overall rates of correct identification of the species ranged from 0.17 to 0.98, with a cumulative rate (random-effects model) of 0.72 (95% CI: 0.64-0.80). For Gram-negative bacteria, correct identification rates ranged from 0.66 to 1.00, with a cumulative effect of 0.92 (95% CI: 0.88-0.95). For Enterobacteriaceae, the rate was 0.96 (95% CI: 0.94-0.97). CONCLUSION: MALDI-TOF mass spectrometry shows high accuracy for the correct identification of Gram-negative bacteria, particularly Enterobacteriaceae, directly from positive blood culture bottles, and moderate accuracy for the identification of Gram-positive bacteria (low for some species)

INTRODUCCIÓN: La identificación rápida de los patógenos causantes de bacteriemia orienta a los clínicos a prescribir con mayor celeridad un tratamiento dirigido y reducir así la morbimortalidad de dicha infección. Durante los últimos años, han aparecido en el mercado numerosas técnicas con la intención de cubrir esta necesidad, que logran una identificación rápida y directa a partir de los frascos de hemocultivos positivos. La espectrometría de masas matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) es una de las tecnologías más utilizadas en este campo. MÉTODOS: El objetivo de ese estudio es realizar una revisión sistemática y metaanálisis que evalúe la precisión de MALDI-TOF MS (Bruker) para la identificación directa a partir de frascos de hemocultivos positivos. RESULTADOS: El metaanálisis fue realizado para sintetizar los resultados de los 32 estudios evaluados. La calidad total de los estudios fue moderada. Para las bacterias grampositivas, el ratio total de identificaciones correctas a nivel de especie fue del 0,17 al 0,98 con un ratio acumulativo (modelo de efectos aleatorios) de 0,72 (IC 95%: 0,64-0,80). Para las bacterias gramnegativas, el rango de identificaciones correctas fue del 0,66 al 1,00 con un efecto acumulativo de 0,92 (IC 95%: 0,88-0,95), llegando a un 0,96 (IC 95%: 0,94-0,97) en Enterobacteriaceae. CONCLUSIONES: La espectrometría de masas MALDI-TOF muestra una alta precisión para la correcta identificación de bacterias gramnegativas realizada directamente a partir de los frascos de hemocultivos positivos, siendo mayor en el grupo de las enterobacterias. Para grampositivas, la precisión es moderada, llegando a ser baja para alguna especie

Direct bacterial identification from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry: A systematic review and meta-analysis.

INTRODUCTION: The rapid identification of bacteraemia-causing pathogens could assist clinicians in the timely prescription of targeted therapy, thereby reducing the morbidity and mortality of this infection. In recent years, numerous techniques that rapidly and directly identify positive blood cultures have been marketed, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) being one of the most commonly used. METHODS: The aim of this systematic review and meta-analysis was to evaluate the accuracy of MALDI-TOF (Bruker®) for the direct identification of positive blood culture bottles. RESULTS: A meta-analysis was performed to summarize the results of the 32 studies evaluated. The overall quality of the studies was moderate. For Gram-positive bacteria, overall rates of correct identification of the species ranged from 0.17 to 0.98, with a cumulative rate (random-effects model) of 0.72 (95% CI: 0.64-0.80). For Gram-negative bacteria, correct identification rates ranged from 0.66 to 1.00, with a cumulative effect of 0.92 (95% CI: 0.88-0.95). For Enterobacteriaceae, the rate was 0.96 (95% CI: 0.94-0.97). CONCLUSION: MALDI-TOF mass spectrometry shows high accuracy for the correct identification of Gram-negative bacteria, particularly Enterobacteriaceae, directly from positive blood culture bottles, and moderate accuracy for the identification of Gram-positive bacteria (low for some species).

Identificación y determinación de sensibilidad a antibióticos de aislados de hemocultivos a partir de subcultivos de corta incubación / Identification and antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures

Introducción: La espectrometría de masas Matrix-Assisted Laser Desorption-Ionization Time-of-Flight(MALDI-TOF) permite la identificación rápida de los microorganismos causantes de bacteriemia. Se requieren métodos fiables y rápidos que permitan acortar el tiempo necesario hasta disponer de los resultados de sensibilidad a antibióticos de los aislados de hemocultivos. Métodos: Se evalúa la fiabilidad de un método que combina la identificación con MALDI-TOF y el estudio de sensibilidad en paneles de microdilución inoculados a partir de un subcultivo incubado durante solo 4h. Resultados: La concordancia de los resultados de sensibilidad a antibióticos de la técnica evaluada frente a la técnica de referencia fue del 99,3%, sin que se observaran errores máximos. Conclusión: La inoculación de paneles de microdilución a partir de un subcultivo de solo 4h de incubación es un método fiable y fácil de realizar que permite acortar el tiempo de informe de hemocultivos positivos (AU)

Introduction: Mass spectrometry Matrix-Assisted Laser Desorption-Ionization Time-of-Flight (MALDI-TOF) helps in the rapid identification of microorganisms causing blood stream infection. Rapid and reliable methods are required to decrease the turnaround time for reporting antimicrobial susceptibility results from blood culture isolates. Methods: An evaluation was performed on the reliability of a method for antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures. Results: The agreement between the evaluated and standard methods was 99.3%. The major and minor error rates were 0.4% and 0.3%, respectively, and no very major errors were observed. Conclusion: The inoculation of briefly incubated solid medium cultures into antimicrobial susceptibility testing panels is an easy and reliable technique, and helps to decrease the turnaround time for reporting antimicrobial susceptibility results of positive blood cultures (AU)

Identification and antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures. / Identificación y determinación de sensibilidad a antibióticos de aislados de hemocultivos a partir de subcultivos de corta incubación.

INTRODUCTION: Mass spectrometry Matrix-Assisted Laser Desorption-Ionization Time-of-Flight (MALDI-TOF) helps in the rapid identification of microorganisms causing blood stream infection. Rapid and reliable methods are required to decrease the turnaround time for reporting antimicrobial susceptibility results from blood culture isolates. METHODS: An evaluation was performed on the reliability of a method for antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures. RESULTS: The agreement between the evaluated and standard methods was 99.3%. The major and minor error rates were 0.4% and 0.3%, respectively, and no very major errors were observed. CONCLUSION: The inoculation of briefly incubated solid medium cultures into antimicrobial susceptibility testing panels is an easy and reliable technique, and helps to decrease the turnaround time for reporting antimicrobial susceptibility results of positive blood cultures.

[Application of mass spectrometry to bacterial identification]. / Aplicación de la espectrometría de masas en la identificación de bacterias.

Correct and rapid identification of bacteria is essential for the correct diagnosis and treatment of infected patients. Until a few years ago, biochemical, colorimetric or even antibiotic sensitivity tests were used to identify genera and species. The main limitations of these methods were the time needed for their performance and the difficulty of distinguishing between microorganisms that were little reactive, highly similar, or difficult to culture. Many of these problems have been solved by the introduction of mass spectrometry (MS) in the laboratory with the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight). Knowledge of the strengths and weaknesses of this technology is essential to be able to take maximum advantage of this technique. Not all microorganisms can be identified with the same ease and reliability by MALDI-TOF and microbiologists need to know how to interpret the results obtained with this technique and the available alternatives in order to identify the microorganisms causing the most problems. This article aims to summarise the available information on the correct identification of the main human pathogenic bacteria through the use of MALDI-TOF MS, focusing on Gram-negative, Grampositive and anaerobic microorganisms. The main factors that must be taken into account for the reliable identification of any bacterium are the conditions for culture, sample preparation with the ideal extraction method and especially the use of a correct and updated database.

Aplicación de la espectrometría de masas en la identificación de bacterias / Application of mass spectrometry to bacterial identification

Una identificación correcta y rápida de las bacterias es esencial para un diagnóstico y un tratamiento adecuado de los pacientes con infecciones. Hasta hace pocos años se utilizaban pruebas bioquímicas, colorimétricas o incluso de sensibilidad antibiótica para la identificación a niveles de género y especie. Las principales limitaciones de estos métodos son el tiempo necesario para su realización y la dificultad para diferenciar microorganismos poco reactivos, muy parecidos entre ellos o de difícil crecimiento. Desde la introducción en el laboratorio de la espectrometría de masas (EM) con el uso de MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight), muchos de estos problemas se han solventado. Para poder sacar el máximo rendimiento a esta tecnología se han de conocer sus puntos fuertes y sus limitaciones. No todos los microorganismos se identifican con la misma facilidad o fiabilidad mediante MALDI-TOF y es obligación del microbiólogo saber cómo interpretar los resultados que de este se obtienen y las alternativas disponibles para lograr identificar los microorganismos que generan más problemas. Este trabajo pretende hacer una recopilación de la información disponible sobre la correcta identificación de las principales bacterias patógenas humanas mediante el uso de la EM MALDI-TOF, centrándose en gramnegativos, grampositivos y microorganismos anaerobios. Las condiciones del cultivo, la preparación de la extensión con el método de extracción idóneo y, sobre todo, el uso de una correcta y actualizada base de datos son los principales factores que hay que tener en cuenta para la identificación fiable de cualquier bacteria

Correct and rapid identification of bacteria is essential for the correct diagnosis and treatment of infected patients. Until a few years ago, biochemical, colorimetric or even antibiotic sensitivity tests were used to identify genera and species. The main limitations of these methods were the time needed for their performance and the difficulty of distinguishing between microorganisms that were little reactive, highly similar, or difficult to culture. Many of these problems have been solved by the introduction of mass spectrometry (MS) in the laboratory with the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight). Knowledge of the strengths and weaknesses of this technology is essential to be able to take maximum advantage of this technique. Not all microorganisms can be identified with the same ease and reliability by MALDI-TOF and microbiologists need to know how to interpret the results obtained with this technique and the available alternatives in order to identify the microorganisms causing the most problems. This article aims to summarise the available information on the correct identification of the main human pathogenic bacteria through the use of MALDI-TOF MS, focusing on Gram-negative, Grampositive and anaerobic microorganisms. The main factors that must be taken into account for the reliable identification of any bacterium are the conditions for culture, sample preparation with the ideal extraction method and especially the use of a correct and updated database

First reported case of a corneal abscess caused by Massilia timonae / Primer caso descrito de absceso corneal causado por Massilia timonae

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