Do specific antimicrobial stewardship interventions have an impact on carbapenem resistance in Gram-negative bacilli? A multicentre quasi-experimental ecological study: time-trend analysis and characterization of carbapenemases.

BACKGROUND: Carbapenem-resistant Gram-negative bacilli (CR-GNB) are among the most threatening microorganisms worldwide and carbapenem use facilitates their spread. Antimicrobial stewardship programmes (ASPs) can help to optimize the use of antibiotics. This study evaluates the impact of a multifaceted educational ASP on carbapenem use and on the epidemiology of CR-GNB. METHODS: We conducted a quasi-experimental, time-series study in seven hospitals, from January 2014 to September 2018. The key intervention was composed of educational interviews promoting the appropriate use of carbapenems. The primary endpoints were carbapenem consumption and incidence density (ID) of CR-GNB. All non-duplicated CR-GNB clinical isolates were tested using phenotypic assays and PCR for the presence of carbapenemases. Joinpoint regression and interrupted time-series analyses were used to determine trends. RESULTS: A decrease in carbapenem consumption throughout the study period [average quarterly percentage change (AQPC) -1.5%, P < 0.001] and a -8.170 (-16.064 to -0.277) level change following the intervention were observed. The ID of CR-Acinetobacter baumannii decreased (AQPC -3.5%, P = 0.02) and the overall ID of CR-GNB remained stable (AQPC -0.4%, P = 0.52). CR-GNB, CR-Pseudomonas aeruginosa and CR-A. baumannii IDs per hospital correlated with the local consumption of carbapenems. The most prevalent carbapenem resistance mechanisms were OXA-23 for CR-A. baumannii (76.1%), OXA-48 for CR-Klebsiella pneumoniae (66%) and no carbapenemases for CR-P. aeruginosa (91.7%). The epidemiology of carbapenemases was heterogeneous throughout the study, especially for carbapenemase-producing Enterobacteriaceae. CONCLUSIONS: In conclusion, a multifaceted, educational interview-based ASP targeting carbapenem prescribing reduced carbapenem use and the ID of CR-A. baumannii.

[Application of mass spectrometry in mycobacteria]. / Aplicación de la espectrometría de masas en micobacterias.

To date, more than 170 species of mycobacteria have been described, of which more than one third may be pathogenic to humans, representing a significant workload for microbiology laboratories. These species must be identified in clinical practice, which has long been a major problem due to the shortcomings of conventional (phenotypic) methods and the limitations and complexity of modern methods largely based on molecular biology techniques. The aim of this review was to briefly describe different aspects related to the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) for the identification of mycobacteria. Several difficulties are encountered with the use of this methodology in these microorganisms mainly due to the high pathogenicity of some mycobacteria and the peculiar structure of their cell wall, requiring inactivation and special protein extraction protocols. We also analysed other relevant aspects such as culture media, the reference methods employed (gold standard) in the final identification of the different species, the cut-off used to accept data as valid, and the databases of the different mass spectrometry systems available. MS has revolutionized diagnosis in modern microbiology; however, specific improvements are needed to consolidate the use of this technology in mycobacteriology.

[Microbiological diagnosis of urinary tract infections]. / Diagnóstico microbiológico de las infecciones urinarias.

Urinary tract infections (UTI) are the most common infectious diseases observed in primary care; up to one-third of women will have at least one symptomatic UTI by age 24, and more than one-half of women will be affected by the end of life. In addition, UTIs represent 40% of nosocomial infections, and being usually associated with urinary catheters. Although urine cultures would not be indicated in all cases, these samples are the most abundant in the laboratories of clinical microbiology. Thus, the working protocols applied to these samples have an important impact in the performance of the laboratory. The samples are collected by mid stream urine, and 60-70% of them are negative culture. At present, several commercial systems have been introduced in order to simplify and automate this process. A urine culture with ≥ 10(5) CFU/ml has classically been considered as positive, although lower counts are valued in certain clinical settings. Factors related to this count e.g. methods to obtain urine, conservation of the sample or use of chemical preservatives as well as low counts are critical points to be discussed in detail. The development of antimicrobial resistance logically affects uropathogens, mainly Escherichia coli, which remains the most frequently isolated in urine cultures. The aim of this paper is to review the most innovating aspects influencing the microbiological diagnosis of UTI.

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

Use of MALDI-TOF MS for Identification of Nontuberculous Mycobacterium Species Isolated from Clinical Specimens.

The aim of this study was to compare the results obtained for identification by MALDI-TOF of nontuberculous mycobacteria (NTM) isolated in clinical samples with those obtained by GenoType Mycobacterium CM/AS (common mycobacteria/additional species). A total of 66 Mycobacterium isolates from various clinical specimens (mainly respiratory) were tested in this study. They were identified using MALDI-TOF Bruker from strains isolated in Lowenstein, following the recommended protocol of heat inactivation and extraction, and were simultaneously analyzed through hybridization by GenoType Mycobacterium from liquid culture MGIT. Our results showed that identification by MALDI-TOF was correct in 98.4% (65/66) of NTM isolated in our clinical practice (M. avium, M. intracellulare, M. abscessus, M. chelonae, M. fortuitum, M. mucogenicum, M. kansasii, and M. scrofulaceum). MALDI-TOF was found to be an accurate, rapid, and cost-effective system for identification of mycobacteria species.

Evaluación de una prueba rápida para la detección de PBP2a en Staphylococcus aureus / Evaluation of a rapid assay for detection of PBP2a Staphylococcus aureus

INTRODUCCIÓN: En los últimos años se ha producido un incremento de las infecciones producidas por Staphylococcus aureus resistente a meticilina (SARM). En comparación con las producidas por S. aureus sensible a meticilina (SASM), las infecciones por SARM requieren estancias hospitalarias más prolongadas y presentan mayor mortalidad. La detección rápida de la resistencia a la meticilina por la adquisición del gen mecA que codifica la proteína fijadora de penicilina (PBP2a) es crucial para evitar la diseminación nosocomial e instaurar una correcta terapia antimicrobiana. Nos proponemos evaluar el test inmunocromatográfico rápido para la detección de PBP2a directamente de colonias de S. aureus, PBP2a SA Culture Colony Test(R) (ICPBP2a). MATERIAL Y MÉTODOS: En 107 cepas de S. aureus se estudió la resistencia a meticilina mediante las siguientes pruebas: el sistema automatizado Vitek2(R) (bioMérieux), CHROMagar MRSA II(R) (BD Becton Dickinson ), difusión con disco de cefoxitina, la ICPBP2a (AlereTM) y como método de referencia, la detección molecular del gen mecA. RESULTADOS: La sensibilidad y especificidad para las pruebas de detección fueron para la difusión en agar con disco de cefoxitina 100% y 100% respectivamente, Vitek2(R) 100% y 100%, CHROMagarTM MRSA II 100% y 96%, y la ICPBP2a 98,25% y 100%. CONCLUSIÓN: La inmunocromatografía para la detección de PBP2a es una técnica rápida, fácil y económica. Resulta muy útil para el manejo de brotes hospitalarios

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen causing both healthcare-associated and community-acquired infection. Rapid and accurate detection of this pathogen is crucial for the use of appropriate antimicrobial therapy and the control of nosocomial spread. METHODS: A total of 107 S. aureus strains were assayed for methicillin resistance: Vitek2(R) (bioMérieux), CHROMagarTM MRSA II (BD Becton Dickinson), disk diffusion in agar for cefoxitin 30 μg and immunochromatography PBP2a SA Culture Colony Test (AlereTM). The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. RESULTS: Sensitivity and specificity were: disk diffusion for cefoxitin 100% and 100% respectively, Vitek2(R) 100 and 100%, CHROMagarTM MRSA II 100 and 96%, and ICPBP2a detection 98,25% and 100%. CONCLUSION: ICPBP2a Culture Colony Test (AlereTM) is fast, efficient and economical technique for detection of penicillin binding protein 2a (PBP2a) from isolates. This assay is a useful tool for the management of hospital outbreaks

Evaluación de un ensayo inmunocromatográfico para la detección de carbapenemasa OXA-48 / Evaluation of an immunochromatographic test for the detection of OXA-48 carbapenemase

Introducción. La detección y diferenciación de los distintos tipos de carbapenemasas es crucial para el control y diseminación de las mismas. OXA-48 es la carbapenemasa más frecuente en España y en nuestro medio. El objetivo del estudio fue evaluar el nuevo test inmunocromatográfico OXA-48 Card letitest (Coris, BioConcept Belgium) para detectar esta carbapenemasa a partir de medios sólidos. Material y Métodos. Durante el último año se han aislado 151 cepas productoras de carbapenemasas, de las cuales 136 presentaban OXA-48 (126 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 5 Escherichia coli, 4 Enterobacter cloacae) y 15 productoras de otras carbapenemasas. Estas 15 cepas junto con otras 73 con distintos mecanismos de resistencia: 54 productoras de β-lactamasas de espectro extendido y 19 con otros mecanismos, fueron utilizadas como controles negativos. Resultados. Las 136 cepas portadoras de OXA-48 resultaron positivas en la prueba OXA-48 Card letitest y las 88 especies utilizadas como controles fueron negativos, por lo que la sensibilidad y especificidad de la prueba OXA-48 Card letitest fue del 100%. Discusión. La OXA-48 Card letitest resulta ser una prueba fácil, rápida, segura y barata (aproximadamente unos 6 Euros por test) que puede utilizarse en los laboratorios de Microbiología para confirmar la producción de carbapenemasa OXA-48 a partir de los aislamientos clínicos (AU)

Introduction. Detection and differentiation of various types of carbapenemases is crucial to their control and dissemination. OXA -48 is the most common carbapenemase in Spain and in our environment. The aim of this study is the evaluation of a new immunochromatographic test OXA-48 Card letitest (Coris, BioConcept Belgium) to detect this carbapenemase from solid media. Material and Methods. During the last year 151 strains of carbapenemase producing bacteria have been isolated, of which 136 were OXA-48 (126 Klebsiella pneumoniae, 1 Klebsiella oxytoca, 5 Escherichia coli, 4 Enterobacter cloacae), and 15 producing other carbapenemases . These 15 strains with other 73 carrying other resistance mechanisms (54 extended-spectrum β-lactamases producers and 19 with other mechanisms) were used as negative controls. Results. One hundred and thirty six strains carrying OXA- 48 were positive with the test OXA-48 Card letitest and the 88 species used as controls were negative, resulting in a sensitivity and specificity of 100%. Discussion. The OXA-48 Card letitest is simple, quick, safe and cheap (approx. 6Euros/test) and can be used in microbiology laboratories to confirm the production of OXA-48 carbapenemase in clinical isolates (AU)

Aplicación de la espectrometría de masas en micobacterias / Application of mass spectrometry in mycobacteria

Hasta la actualidad se han descrito más de 170 especies de micobacterias. Más de un tercio de ellas pueden ser patógenas para el ser humano, lo que supone una carga importante de trabajo en los laboratorios de microbiología. Su identificación se hace necesaria en la práctica clínica y ha supuesto durante años un importante problema debido a las carencias de los métodos fenotípicos o tradicionales y a la limitación y complejidad de las herramientas más modernas, basadas en su mayoría en técnicas de biología molecular. El objetivo de esta revisión es realizar una breve descripción de los diferentes aspectos relacionados con la utilización de la espectrometría de masas (EM) MALDI-TOF (matrix-assisted laser desorption ionization time-off light) en la identificación de las micobacterias. Son varias las dificultades encontradas en la aplicación del método en estos microorganismos, derivadas fundamentalmente del elevado carácter patógeno de algunos miembros, que hace necesaria su inactivación, y la peculiar estructura de su pared celular, que requiere protocolos especiales de extracción de las proteínas. Otros temas de relevancia -como son los medios de cultivo de los que se parte, los métodos de referencia utilizados (gold standard) en la identificación final de las distintas especies y los puntos de corte que deben asumirse para aceptar un resultado como válido- son también motivo de análisis, así como las bases de datos de los diferentes sistemas disponibles. La EM es una técnica que ha revolucionado el diagnóstico de la microbiología moderna; sin embargo, futuras y puntuales mejoras son necesarias para consolidar su uso en la identificación micobacteriológica

To date, more than 170 species of mycobacteria have been described, of which more than one third may be pathogenic to humans, representing a significant workload for microbiology laboratories. These species must be identified in clinical practice, which has long been a major problem due to the shortcomings of conventional (phenotypic) methods and the limitations and complexity of modern methods largely based on molecular biology techniques. The aim of this review was to briefly describe different aspects related to the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) for the identification of mycobacteria. Several difficulties are encountered with the use of this methodology in these microorganisms mainly due to the high pathogenicity of some mycobacteria and the peculiar structure of their cell wall, requiring inactivation and special protein extraction protocols. We also analysed other relevant aspects such as culture media, the reference methods employed (gold standard) in the final identification of the different species, the cut-off used to accept data as valid, and the databases of the different mass spectrometry systems available. MS has revolutionized diagnosis in modern microbiology; however, specific improvements are needed to consolidate the use of this technology in mycobacteriology

Serosurvey study of Toscana virus in domestic animals, Granada, Spain.

Toscana virus (TOSV) is transmitted by infected sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis in humans. It remains unclear if there are animal reservoirs able to maintain the virus through the cold months of the year, when the vector is not circulating. From May to October of 2006 and 2007, we conducted a serosurvey study on domestic animals from Granada province (southern Spain). TOSV was investigated in 1186 serum samples from horses, goats, pigs, cats, dogs, sheep, and cows by serology (indirect fluorescence assay), viral culture, and RT-polymerase chain reaction. Specific anti-TOSV antibodies were detected in 429 (36.2%) serum samples. The highest seropositivity rates were observed in cats (59.6%) and dogs (48.3%). These results suggest that an important percentage of the domestic animals have been infected by TOSV. Significantly different seroprevalence rates were detected in goats among distinct geographical areas. All viral cultures were negative. TOSV was detected by RT-polymerase chain reaction in only one serum sample from a goat. Thus, the studied animals do not seem to act as reservoirs for TOSV; otherwise, they could be amplifying hosts for the virus.

Diagnóstico microbiológico de las infecciones urinarias / Microbiological diagnosis of urinary tract infections

La infección del tracto urinario es la infección bacteriana más frecuente observada en el ámbito ambulatorio: 1 de cada 3 mujeres desarrollará una infección urinaria que requerirá tratamiento con antibióticos antes de los 24 años y, al menos, el 50% una infección del tracto urinario durante su vida. Además, las infecciones del tracto urinario representan el 40% de las infecciones nosocomiales, usualmente asociadas a sondajes urinarios. Aunque el cultivo de orina no estaría indicado en todos los casos, estas muestras son las más abundantes en los laboratorios de microbiología clínica, y la carga de trabajo que conllevan tiene una repercusión importante en el funcionamiento del laboratorio. Se trata fundamentalmente de orinas ambulatorias de “micción media”, de las cuales el 60-70% son cultivo negativo. En la actualidad existen sistemas comerciales disponibles con objeto de automatizar y simplificar su procesamiento. Clásicamente se han considerado positivas orinas con recuento ≥ 105 UFC/ml, aunque se valoran recuentos más bajos en determinadas situaciones clínicas. Factores relacionados con este recuento, como el modo de obtención de la orina, su conservación y el uso de conservantes químicos, así como el significado de estos bajos recuentos son puntos críticos que se analizarán detalladamente. El desarrollo de resistencias antimicrobianas afecta, lógicamente, a los uropatógenos, fundamentalmente Escherichia coli, que sigue siendo el más frecuentemente aislado. El objetivo de este texto es hacer una revisión de los aspectos más relevantes que influyen en el diagnóstico microbiológico de las infecciones del tracto urinario

Urinary tract infections (UTI) are the most common infectious diseases observed in primary care; up to one-third of women will have at least one symptomatic UTI by age 24, and more than one-half of women will be affected by the end of life. In addition, UTIs represent 40% of nosocomial infections, and being usually associated with urinary catheters. Although urine cultures would not be indicated in all cases, these samples are the most abundant in the laboratories of clinical microbiology. Thus, the working protocols applied to these samples have an important impact in the performance of the laboratory. The samples are collected by mid stream urine, and 60-70% of them are negative culture. At present, several commercial systems have been introduced in order to simplify and automate this process. A urine culture with ≥ 105 CFU/ml has classically been considered as positive, although lower counts are valued in certain clinical settings. Factors related to this count e.g. methods to obtain urine, conservation of the sample or use of chemical preservatives as well as low counts are critical points to be discussed in detail. The development of antimicrobial resistance logically affects uropathogens, mainly Escherichia coli, which remains the most frequently isolated in urine cultures. The aim of this paper is to review the most innovating aspects influencing the microbiological diagnosis of UTI