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)

Métodos microbiológicos para la monitorización de la limpieza, desinfección y esterilización de dispositivos médicos / Microbiological monitoring of medical devices after cleaning, disinfection and sterilisation

El uso de los dispositivos semicríticos reutilizables se ha extendido en la práctica médica actual tanto con fines diagnósticos como terapéuticos. Sin embargo, la reutilización de estos instrumentos conlleva el riesgo de una transmisión cruzada de microorganismos de un paciente a otro. El proceso de limpieza y desinfección de estos dispositivos es complejo, largo, caro y muy sensible a que se produzcan fallos. En el presente documento se analizan los aspectos epidemiológicos de las infecciones asociadas a la reutilización de los dispositivos semicríticos, y el papel del laboratorio de Microbiología en la monitorización del proceso de limpieza y desinfección de los mismos a través de los controles microbiológicos. Se revisan las recomendaciones de diferentes sociedades científicas sobre la pertinencia de dichos controles y se establecen recomendaciones específicas para la toma y el procesamiento de las muestras, la interpretación de los resultados y las medidas a tomar en función de los resultados obtenidos

The use of reusable semi-critical devices has been extended in current medical practice for both diagnostic and therapeutic purposes. However, reuse of these instruments carries the risk of cross-transmission of microorganisms from one patient to another. The process of cleaning and disinfecting these devices is complex, long, expensive and very error-prone. This paper analyses the epidemiological aspects of infections associated with the reuse of semi-critical devices and the role of the Microbiology laboratory in monitoring the cleaning and disinfecting process through microbiological controls. The recommendations of different scientific societies on the relevance of such controls are reviewed and specific recommendations are proposed for the taking and processing of the samples, interpretation of the results and measures to be taken depending on the results obtained

Microbiological monitoring of medical devices after cleaning, disinfection and sterilisation. / Métodos microbiológicos para la monitorización de la limpieza, desinfección y esterilización de dispositivos médicos.

The use of reusable semi-critical devices has been extended in current medical practice for both diagnostic and therapeutic purposes. However, reuse of these instruments carries the risk of cross-transmission of microorganisms from one patient to another. The process of cleaning and disinfecting these devices is complex, long, expensive and very error-prone. This paper analyses the epidemiological aspects of infections associated with the reuse of semi-critical devices and the role of the Microbiology laboratory in monitoring the cleaning and disinfecting process through microbiological controls. The recommendations of different scientific societies on the relevance of such controls are reviewed and specific recommendations are proposed for the taking and processing of the samples, interpretation of the results and measures to be taken depending on the results obtained.

Estudio comparativo de concordancia y costes entre la prueba de la tuberculina y QuantiFERON ®-TB Gold In-Tube en el diagnóstico de la infección latente tuberculosa en contactos de pacientes con tuberculosis pulmonar / Comparative study of concordance and costs between tuberculin skin test and QuantiFERON ®-TB Gold In-Tube in the diagnosis of latent tuberculosis infection among contacts of patients with pulmonary tuberculosis

Introducción: El diagnóstico de la infección latente tuberculosa (ILT) es posible realizarlo mediante la prueba de la tuberculina (PT) o bien a través de las denominadas técnicas de interferon-γ release assays (IGRAS, «análisis de liberación del interferón-γ»), siendo QuantiFERON®-TB Gold In-Tube (QF-G-IT) la más usada. Los IGRAS permiten evitar algunos inconvenientes de la PT, especialmente la reacción cruzada con la vacuna con bacilo de Calmette-Guérin (BCG). No obstante, también presentan algunos problemas, como son los derivados del coste de la técnica, así como el ser un método de laboratorio que precisa una infraestructura y experiencia adecuadas. No existe un claro consenso sobre cuál de las técnicas debería utilizarse de forma prioritaria para el diagnóstico de la ILT. Método: Se trata de un estudio comparativo entre la PT y la QF-G-IT en nuestra cohorte de contactos de pacientes con tuberculosis pulmonar durante el período de estudio (n = 101). Se realizó un análisis de la concordancia global y por grupos según los contactos estuvieran vacunados con BCG o no. Se realizó, además, un estudio de costes de ambas técnicas y de las estrategias diagnósticas basadas en ellas. Resultados: La concordancia entre la PT y la QF-G-IT fue aceptable en el global de la muestra, pero muy buena en el grupo de no vacunados. Se registraron muy pocos casos de valores indeterminados. El estudio de costes mostró que la PT era más económica que la QF-G-IT; sin embargo, al analizar el coste de las estrategias según cada técnica, la PT mostró un mayor coste-beneficio. Conclusión: Aconsejamos considerar QF-G-IT como la única y preferente técnica para el diagnóstico de la ILT en contactos convivientes, basados en una buena concordancia general entre ambas técnicas (más aún si eliminamos el efecto de la vacuna) y un estudio de costes favorable a QF-G-IT (AU)

Introduction: Recently diagnosis of latent tuberculosis infection (LTBI) can be made using the tuberculin skin test (TST) or by techniques known as interferon-γ release assays (IGRAS), being QuantiFERON®-TB Gold In-Tube (QF-G-IT) the most used. The IGRAS avoid some drawbacks of the TST, especially cross-reaction with bacillus Calmette-Guérin (BCG) vaccine, but also present some problems such as those arising from cost and the need of having an adequate infrastructure and experience. There is no clear consensus on which technique should be preferentially used for the diagnosis of LTBI. Methods: This is a comparative study between the TST and QT-G-IT in a cohort of contacts of patients with pulmonary tuberculosis during the study period. An analysis of global agreement and groups was performed according to whether the contacts were vaccinated with BCG or not. A study of costs of both techniques and diagnostic strategies based on these techniques was performed. Results: The agreement between TST and QF-G-IT was acceptable in the whole sample yet it was very good in the unvaccinated group. Few cases of indeterminate values were recorded. The cost study showed that TST was cheaper than QF-G-IT; however when we analyzed the cost of the strategies according to each technique, the QF-G-IT showed a better cost-benefit. Conclusion: We suggest considering QF-G-IT as the only preferred technique for the diagnosis of LTBI in household contacts, based on good overall agreement between the 2 techniques (even if we eliminate the effect of the vaccine) and a cost analysis favorable to QF-G-IT (AU)

[Comparative study of concordance and costs between tuberculin skin test and QuantiFERON(®)-TB Gold In-Tube in the diagnosis of latent tuberculosis infection among contacts of patients with pulmonary tuberculosis]. / Estudio comparativo de concordancia y costes entre la prueba de la tuberculina y QuantiFERON(®)-TB Gold In-Tube en el diagnóstico de la infección latente tuberculosa en contactos de pacientes con tuberculosis pulmonar.

INTRODUCTION: Recently diagnosis of latent tuberculosis infection (LTBI) can be made using the tuberculin skin test (TST) or by techniques known as interferon-γ release assays (IGRAS), being QuantiFERON(®)-TB Gold In-Tube (QF-G-IT) the most used. The IGRAS avoid some drawbacks of the TST, especially cross-reaction with bacillus Calmette-Guérin (BCG) vaccine, but also present some problems such as those arising from cost and the need of having an adequate infrastructure and experience. There is no clear consensus on which technique should be preferentially used for the diagnosis of LTBI. METHODS: This is a comparative study between the TST and QT-G-IT in a cohort of contacts of patients with pulmonary tuberculosis during the study period. An analysis of global agreement and groups was performed according to whether the contacts were vaccinated with BCG or not. A study of costs of both techniques and diagnostic strategies based on these techniques was performed. RESULTS: The agreement between TST and QF-G-IT was acceptable in the whole sample yet it was very good in the unvaccinated group. Few cases of indeterminate values were recorded. The cost study showed that TST was cheaper than QF-G-IT; however when we analyzed the cost of the strategies according to each technique, the QF-G-IT showed a better cost-benefit. CONCLUSION: We suggest considering QF-G-IT as the only preferred technique for the diagnosis of LTBI in household contacts, based on good overall agreement between the 2 techniques (even if we eliminate the effect of the vaccine) and a cost analysis favorable to QF-G-IT.

[Diabetic foot osteomyelitis: is conservative treatment possible?]. / Osteomielitis de pie diabético: ¿es posible un manejo conservador?

INTRODUCTION: The aim of the present study is to determine the proportion of foot ulcers, complicated by osteomyelitis in diabetic patients, that heal without amputation. Furthermore, an attempt is made to analyze the main clinical and microbiological characteristics of episodes, and to identify potential predictive factors leading to the failure of conservative treatment. METHODS: A prospective observational study was carried out between 2007 and 2009 on diabetic patients with a foot lesion and attending a diabetic foot clinic. A percutaneous bone biopsy was required to be included in the study. RESULTS: A total of 81 episodes of diabetic foot osteomyelitis in 64 patients were evaluated. Staphylococcus aureus (28/81) and coagulase negative Staphylococcus (22/81) were the most frequent organisms isolated. Among the gramnegative group (34/81), non-fermenting gram negative bacteria were the most prevalent organisms isolated (14/81). Conservative treatment was successful in 73% of episodes. After a logistic regression analysis using the most significant prognostic variables, only lesion size greater than 2cm independently predicted failure of conservative treatment. Culture guided antibiotic treatment was associated with a better prognosis. CONCLUSION: Conservative treatment, including culture-guided antibiotics, is successful without amputation in a large proportion of diabetic patients with diabetic foot osteomyelitis. Considering empiric therapy directed at non-fermenting gramnegative bacteria could be advisable in some cases, because they are frequently isolated in our setting.

[Evolution of the sensitivity of 235 strains of Helicobacter pylori from 1995 to 1998 and impact of antibiotic treatment]. / Evolución de la sensibilidad de 235 cepas de Helicobacter pylori entre 1995 y 1998 e impacto del tratamiento antibiótico.

BACKGROUND: The aim of this study was to investigate the sensitivity of Helicobacter pylori to the antibiotics used in its eradication over a period of four years and to determine the influence of previous treatment on sensitivity. MATERIAL AND METHODS: During the period from 1995 to 1998 we determined the sensitivity of 235 consecutive Helicobacter pylori isolates to amoxicillin, metronidazole, clarythromycin and tetracycline by means of E-test methodology. The MIC values found were related with the prior use of eradicating treatment. RESULTS: The percentage of resistant strains were as follows: 23.5% to metronidazole, 12.9% to clarythromycin and 0.7% to tetracycline; none of the strains was resistant to amoxicillin. There were no significant changes in percentage of resistance to the drugs studied over the 4-year period. Resistance to metronidazole and clarythromycin was significantly higher (p 5 0.03 and p < 0.001 respectively) in strains isolated from patients who had received previous treatment. CONCLUSIONS: Monitorization of H. pylori sensitivity to the drugs used in its eradication is particularly important in patients who have undergone prior treatment.

Evolución de la sensibilidad de 235 cepas de Helicobacter pylori entre 1995 y 1998 e impacto del tratamiento antibiótico / Sensitivity of 235 strains of Helicobacter pylori from 1995 to 1998 and impact of antibiotic treatment

FUNDAMENTO. Conocer la sensibilidad de Helicobacter pylori a los antibióticos utilizados en el tratamiento erradicador, su evolución temporal y la influencia de la utilización previa de dicho tratamiento. MATERIAL Y MÉTODOS. Se estudió mediante E-test la sensibilidad a amoxicilina, metronidazol, claritromicina y tetraciclina de 235 aislamientos consecutivos de Helicobacter pylori durante el período comprendido entre 1995 y 1998, y se relacionaron los valores de concentración mínima inhibitoria (CMI) hallados con la utilización previa de tratamiento erradicador. RESULTADOS. Se observó un porcentaje de resistencia a metronidazol de 23,5 por ciento, de 12,9 por ciento a claritromicina y de 0,7 por ciento a tetraciclina; ninguna de las cepas fue resistente a amoxicilina. No se observaron cambios significativos en los porcentajes de resistencia a lo largo del período estudiado. Los porcentajes de resistencia a metronidazol y claritromicina fueron significativamente más elevados (p 0,03 y p < 0,001, respectivamente) en las cepas aisladas de los pacientes que habían recibido tratamiento. CONCLUSIONES. Es necesaria la monitorización de la sensibilidad de H. pylori a los antibióticos utilizados en el tratamiento erradicador, especialmente en los pacientes previamente tratados Helicobacter pylori isolates to amoxicillin, metronidazole, clarythromycin and tetracycline by means of E-test methodology. The MIC values found were related with the prior use of eradicating treatment. RESULTS. The percentage of resistant strains were as follows: 23.5 por ciento to metronidazole, 12.9 por ciento to clarythromycin and 0.7 por ciento to tetracycline; none of the strains was resistant to amoxicillin. There were no significant changes in percentage of resistance to the drugs studied over the 4-year period. Resistance to metronidazole and clarythromycin was significantly higher (p 0.03 and p < 0.001 respectively) in strains isolated from patients who had received previous treatment. CONCLUSIONS. Monitorization of H. pylori sensitivity to the drugs used in its eradication is particularly important in patients who have undergone prior treatment (AU)