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Resumen Introduccion El objetivo fue realizar la validacion clinica del sistema molecular AMR Direct Flow Chip® para la deteccion de genes de resistencia a antimicrobianos partiendo de aislados bacterianos en cultivo, asi como de hisopos de muestras nasales o rectales. Metodos El ensayo AMR es una PCR multiplex seguida de hibridacion reversa tipo dot blot en arrays de ADN completamente automatizada mediante la plataforma HS24, con un tiempo de realizacion de 3 h. Se realizo la validacion preclinica con 104 cepas bacterianas caracterizadas y posteriormente se analizaron 210 muestras de hisopos nasales o rectales. Resultados La sensibilidad y la especificidad del ensayo preclinico fueron del 100%, identificando correctamente las 104 cepas. En la validacion clinica, la sensibilidad fue del 100% y la especificidad fue del 100% en muestras rectales y del 97% en hisopos nasales. Conclusiones El sistema AMR Direct Flow Chip® es un sistema rapido y eficaz para la deteccion de microorganismos multirresistentes a partir de muestras rectales y nasales.

In the last decades, microbiology laboratories have undergone unprecedented technological changes which have revolutionized the diagnosis of infectious diseases. They have last generation technology which allows precise, rapid and effective diagnosis of a wide range of infectious diseases, while also providing valuable information on the antibiotic sensitivity/resistance of the causal microorganism, thereby notably reducing the morbidity and mortality of the patients and at the same time improving in-hospital patient management. Several studies have shown that with the increase in the use of rapid diagnostic techniques this technological advancement has a greater impact when accessibility to the microbiology laboratory is possible beyond the usual 7 h workday. This document discusses the need for clinical microbiology services to work 24 h a day, 7 days a week (24/7).

Resumen Introduccion La diarrea por Clostridioides difficile es un importante problema de salud publica, cuyo tratamiento es complejo. La transferencia de microbiota fecal (TMF) se postula como una terapia util para prevenir recidivas. Material y metodos Se analizaron seis muestras fecales, una procedente del donante y cinco del paciente antes y despues de la TMF. Se amplifico y secuencio el gen 16Sr mediante secuenciacion masiva y se estudio la diversidad y composicion taxonomica. Resultados La diversidad aumento en las muestras post-TMF, y se identificaron dos clusteres, uno formado por las muestras no patologicas (donante y paciente post-TMF), y otro por la muestra patologica. Los resultados obtenidos a traves Qiime2 y Bioconductor fueron similares. Conclusion El analisis realizado demostro un incremento en la diversidad taxonomica del paciente tras la TMF, sugiriendo su utilidad. Ademas, los resultados obtenidos con Qiime2 y Bioconductor reflejaron la importancia de unificar los analisis bioinformaticos.

Background Pseudomonas aeruginosa neurosurgical meningitis is a rare entity, usually related with intraventricular catheters and associated with high mortality rates. We describe the clinical characteristics, treatment and outcomes of a series of neurosurgical meningitis caused by P. aeruginosa along 1990–2016. Methods Descriptive, retrospective study of all postsurgical meningitis due to P. aeruginosa related to intraventricular catheters in Hospital Universitario Central de Asturias, between 1990 and 2016. Clinical features, therapeutic approaches and prognostic factors were analyzed statistically. A binary logistic regression analysis was performed to determine the factors influencing the infection mortality. Result 51 episodes from 51 different patients with CSF culture positive for P. aeruginosa were reviewed. Seventeen patients (33.3%) died as a direct consequence of the infection. Univariate analysis showed that mortality was higher in the group of patients treated with ceftazidime (12 vs. 15, p = 0.068, OR 3.040 [0.877–10.544]) and lower in patients whom had received intrathecal therapy (2 vs. 13, p = 0.050, OR 4.64 [0.80–34.93]), without differences observed between those patients treated with aminoglycosides or with colistin. Any patient treated with colistin died (0 vs. 6, p = 0.067, OR: not defined). In the multivariate analysis mortality was only significant higher for patients without catheter withdrawal (p = 0.014) and lower for those patients who received intrathecal therapy (p = 0.05) or adequate empirical treatment (p = 0.006). Conclusions The mortality of P. aeruginosa meningitis is high especially in infections without catheter withdrawal and in patients for whom the intrathecal route of administration was not used. Catheter withdrawal was an independent factor of good outcome in our series.

Resumen Introduccion La aplicacion de los puntos de corte establecidos por el European Committee on Antimicrobial Susceptibility Testing (EUCAST) en comparacion con los del Clinical and Laboratory Standards Institute (CLSI) modifica los criterios de interpretacion de la sensibilidad de algunos antimicrobianos y esto conduce a cambios en los informes de sensibilidad antibiotica acumulada. Metodos Analisis de la influencia de la aplicacion del EUCAST en 10.359 aislados clinicos de Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus y Enterococcus spp. Resultados Al aplicar los puntos de corte del EUCAST, la mayoria de los porcentajes de sensibilidad a antimicrobianos no se altero o lo hizo de forma muy leve; sin embargo, se observo una disminucion de la sensibilidad a los aminoglucosidos en bacilos gramnegativos, especialmente a la amicacina en Pseudomonas aeruginosa (23,2%), aunque solo el 5,7% fueron totalmente resistentes; ademas, disminuyo notablemente el porcentaje de aislados sensibles a aztreonam. Es de destacar el aumento de cepas de Staphylococcus aureus resistentes a clindamicina (51,5%) y a aminoglucosidos (gentamicina 43,1%). Conclusiones El cambio de los criterios del CLSI a los de EUCAST en algunos patogenos supone una alteracion en los porcentajes de resistencia a algunos antimicrobianos y, por tanto, en la epidemiologia local de la resistencia. Estos cambios deben realizarse por un grupo multidisciplinar, que analice la influencia de los nuevos datos en los protocolos de tratamiento empirico de cada centro.

Cumulative reports on antimicrobial susceptibility tests data are important for selecting empirical treatments, as an educational tool in programs on antimicrobial use, and for establishing breakpoints defining clinical categories. These reports should be based on data validated by clinical microbiologists using diagnostic samples (not surveillance samples). In order to avoid a bias derived from including several isolates obtained from the same patient, it is recommended that, for a defined period, only the first isolate is counted. A minimal number of isolates per species should be presented: a figure of >=30 isolates is statistically acceptable. The report is usually presented in a table format where, for each cell, information on clinically relevant microorganisms-antimicrobial agents is presented. Depending on particular needs, multiple tables showing data related to patients, samples, services or special pathogens can be prepared.

The introduction of mass spectrometry through MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) in the diagnosis of bacteraemia and fungaemia has represented a revolution due to the rapidity and reliability of the results that it can offer to microbiology services and laboratories through analysis of the mass spectrum of the bacterial protein directly from positive blood culture bottles. These data are more useful if they are used in conjunction with other techniques able to identify the antibiotic resistance pattern of the microorganism. There is a need for a process of standardising sample processing protocols and for perfecting the identification of the agents causing bacteraemia, especially in some species of Gram-positive cocci and in polymicrobial processes. The introduction of this methodology provides rapid information that is highly important for the clinical management of bacteraemia. The availability of a multidisciplinary working group that applies all this information quickly and correctly in hospitals will improve the quality of care, reduce antibiotic expenditure and hospital stay and help to control the serious problem of antibiotic resistance.

INTRODUCTION An increase in whooping cough in most of the developed countries has been detected in the last decade. OBJECTIVE To determine whether the administration of dTpa vaccine instead of DTPa fifth dose is contributing to the appearance of these cases. METHODS A descriptive study based on cases of whooping cough reported during an epidemic period in the city of Alicante in the first 5 months of 2014. Only pertussis cases confirmed by PCR were included in the study, and only those vaccinated with 5 doses were included in the analysis of the period of protection. RESULTS A total of 104 cases of pertussis confirmed by PCR were reported, with 85 cases (82%) having had 5 doses of vaccine. The mean time and standard deviation (SD) of protection was 2.1±1.1 years with dTpa, and 5.1±1.5 years with DTPa (p

Current diagnosis of malaria is based on the combined and sequential use of rapid antigen detection tests (RDT) of Plasmodium and subsequent visualization of the parasite stained with Giemsa solution in a thin and thick blood smears. If an expert microscopist is not available, should always be a sensitive RDT to rule out infection by Plasmodium falciparum, output the result immediately and prepare thick smears (air dried) and thin extensions (fixed with methanol) for subsequent staining and review by an expert microscopist. The RDT should be used as an initial screening test, but should not replace microscopy techniques, which should be done in parallel. The diagnosis of malaria should be performed immediately after clinical suspicion. The delay in laboratory diagnosis (greater than 3 hours) should not prevent the initiation of empirical antimalarial treatment if the probability of malaria is high. If the first microscopic examination and RDT are negative, they must be repeated daily in patients with high suspicion. If suspicion remains after three negative results must be sought the opinion of an tropical diseases expert. Genomic amplification methods (PCR) are useful as confirmation of microscopic diagnosis, to characterize mixed infections undetectable by other methods, and to diagnose asymptomatic infections with submicroscopic parasitaemia.

The introduction of mass spectrometry through MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) in the diagnosis of bacteraemia and fungaemia has represented a revolution due to the rapidity and reliability of the results that it can offer to microbiology services and laboratories through analysis of the mass spectrum of the bacterial protein directly from positive blood culture bottles. These data are more useful if they are used in conjunction with other techniques able to identify the antibiotic resistance pattern of the microorganism. There is a need for a process of standardising sample processing protocols and for perfecting the identification of the agents causing bacteraemia, especially in some species of Gram-positive cocci and in polymicrobial processes. The introduction of this methodology provides rapid information that is highly important for the clinical management of bacteraemia. The availability of a multidisciplinary working group that applies all this information quickly and correctly in hospitals will improve the quality of care, reduce antibiotic expenditure and hospital stay and help to control the serious problem of antibiotic resistance.

Determination of HIV-1 tropism is mandatory before using CCR5 antagonists in clinical practice. One drug of this class, maraviroc, has been approved for the treatment of HIV infection. The phenotypic assay, TrofileTM, was clinically validated in the clinical development program of maraviroc and has been widely used to select candidates for maraviroc therapy. Phenotypic tests, however, have the disadvantage of being complex, are costly and time-consuming, and their accessibility is limited, which hampers their routine use in clinical diagnosis. Genotypic assays, based on sequencing the third hypervariable (V3 loop) of the viral gene env, interpreted according to various genotypic bioinformatic tools, such as geno2pheno and PSSM, are faster and cheaper than phenotypic assays, and are also more accessible. In retrospective analyses of the maraviroc pivotal trials, genotypic methods using either conventional ("bulk") or deep-sequencing technology predicted virologic response to maraviroc similarly to phenotypic assays and are now included within several European recommendations to guide the clinical use of CCR5 antagonists.

Cumulative reports on antimicrobial susceptibility tests data are important for selecting empirical treatments, as an educational tool in programs on antimicrobial use, and for establishing breakpoints defining clinical categories. These reports should be based on data validated by clinical microbiologists using diagnostic samples (not surveillance samples). In order to avoid a bias derived from including several isolates obtained from the same patient, it is recommended that, for a defined period, only the first isolate is counted. A minimal number of isolates per species should be presented: a figure of=30 isolates is statistically acceptable. The report is usually presented in a table format where, for each cell, information on clinically relevant microorganisms-antimicrobial agents is presented. Depending on particular needs, multiple tables showing data related to patients, samples, services or special pathogens can be prepared.

Introduccion La finalidad de este estudio es comparar la actividad de ciprofloxacino, levofloxacino, moxifloxacino, gatifloxacino y ofloxacino frente a Pseudomonas aeruginosa mediante la determinacion de la concentracion preventiva de mutantes. Metodos Se estudiaron 34 aislados clinicos de P. aeruginosa productores de infecciones nosocomiales. La determinacion de la concentracion preventiva de mutantes (CPM) se realizo mediante el inoculo de 10 10 UFC/ml en placas de Mueller-Hinton con concentraciones seriadas de antibioticos. Los microorganismos se agruparon en funcion de si el paciente habia recibido tratamientos previos con fluoroquinolonas. Resultados La fluoroquinolona que presenta menores valores de CPM es ciprofloxacino, seguida de levofloxacino. Al analizar las cepas de pacientes con tratamientos previos con fluoroquinolonas, se observa que aunque las cepas siguen siendo sensibles segun los criterios del Clinical and Laboratory Standards Institute (CLSI), todos los farmacos muestran menos actividad. Conclusiones Aunque el microorganismo sea sensible a las fluoroquinolonas por los metodos clasicos, debe valorarse la idoneidad de administrar tratamientos con estos compuestos en infecciones graves si el enfermo ha sido previamente tratado con ellos.

Introduccion En los ultimos anos observamos la aparicion, cada vez mas frecuente, de bacterias, y singularmente de Escherichia coli, productoras de betalactamasas de espectro extendido (BLEE), que confieren resistencia a las cefalosporinas (excepto cefamicinas) y aztreonam. Estas bacterias son frecuentemente resistentes tambien a antibioticos no betalactamicos, lo que plantea un importante problema clinico. Metodos Estudio descriptivo de las cepas de E. coli productoras de BLEE aisladas en todo tipo de muestras en dos hospitales del sur de la provincia de Alicante en un periodo de 57 meses (de enero de 1999 a septiembre de 2003), con especial atencion a su origen (intrahospitalario o extrahospitalario), a las corresistencias a antibioticos no betalactamicos y a la evolucion cronologica de su incidencia. Resultados Respectivamente, 3 y 2,25% de las cepas de E. coli aisladas en cada hospital son productoras de BLEE (3,83 y 2,85% de las cepas procedentes de ingresados y 2,74 y 2,1% de las de pacientes ambulatorios). El 30,73 y 24,58% de las cepas productoras de BLEE se aislaron en pacientes hospitalizados. En ambos hospitales se encontraron porcentajes de corresistencia a ciprofloxacino, gentamicina y cotrimoxazol muy superiores en cepas productoras de BLEE. Conclusion El porcentaje de cepas de E. coli productoras de BLEE es elevado en nuestro medio, pero es mas notable su clara tendencia al incremento. Es resenable el elevado porcentaje de cepas productoras procedentes del ambito extrahospitalario. Por ultimo, resaltamos los elevados indices de corresistencia a antibioticos no betalactamicos.

BACKGROUND Tuberculosis in immigrants is an emerging disease in industrialized countries. METHOD We retrospectively analyzed the clinical and epidemiological characteristics of culture-positive tuberculosis cases in the immigrant and native populations. RESULTS One hundred and five cases of tuberculosis were observed; 22 cases (21%) were in immigrants. The 2002 incidence was 64.3 cases per 100,000 immigrants. Mean age of the affected immigrants was 28.5 years lower than that of the affected native population (p < 0.001). The rate of HIV coinfection was 9.1%. In 81.8% (18) of immigrants, presentation was exclusively pulmonary, a higher percentage than in the native population (59.2%) (p = 0.05). Loss to follow-up was more common among immigrants (35%) than among the native population (9.2%) (p < 0.001). CONCLUSIONS Tuberculosis in the immigrant population generally involves the lungs, appears in younger subjects and is a public heath problem because of the high rate of losses to follow-up.

Fundamento Salmonella spp., como productor de gastroenteritis y otros cuadros clinicos, sigue constituyendo un importante problema para la salud publica mundial. Por ello, el conocimiento de su epidemiologia a nivel general y local mediante la serotipificacion se considera uno de los principales pilares para su control sanitario. Material y metodo Se ha estudiado un total de 15.181 muestras fecales y el resto de las recibidas en nuestro servicio procedentes de otras localizaciones, pertenecientes tanto a pacientes ingresados como del area sanitaria que al mismo corresponden. El aislamiento de Salmonella se ha realizado a partir de medios convencionales agar MacConkey, Salmonella-Shigella, selenito F. Su identificacion se ha llevado a cabo mediante paneles en metodos automatizados Microscan y Wider I. La serotipificacion se ha efectuado con sueros polivalentes y monoespecificos (Difco) y confirmacion por el Laboratorio Nacional de Referencia de Salmonella y Shigella de Espana (LNRSSE). Resultados En los coprocultivos y hemocultivos se detecta el 96,4% de Salmonella spp. y se destaca la presencia en liquido cefalorraquideo, esputo, biopsia rectal, exudado vaginal y otras localizaciones. En un total de 1.290 pacientes se aislaron 37 serotipos diferentes, siendo Enteritidis y Typhimurium los mas frecuentes. Se destaca la presencia del serotipo Virchow en los anos 1994-1999, aislado tanto en heces como en el liquido encefalorraquideo, asi como la presencia de serotipos poco habituales, como Blockley, London, Give y Mikawasina, entre otros. Conclusiones La cada vez mas frecuente aparicion de nuevos serotipos, asi como la necesidad de una mas amplia tipificacion, como el fagotipo, para obtener una completa informacion epidemiologica, hace necesaria una mayor capacidad de tipificacion en nuestros laboratorios, asi como la posibilidad de contar con centros de referencia como el LNRSSE.

An increase in whooping cough in most of the developed countries has been detected in the last decade.To determine whether the administration of dTpa vaccine instead of DTPa fifth dose is contributing to the appearance of these cases.A descriptive study based on cases of whooping cough reported during an epidemic period in the city of Alicante in the first 5 months of 2014. Only pertussis cases confirmed by PCR were included in the study, and only those vaccinated with 5 doses were included in the analysis of the period of protection.A total of 104 cases of pertussis confirmed by PCR were reported, with 85 cases (82%) having had 5 doses of vaccine. The mean time and standard deviation (SD) of protection was 2.1±1.1 years with dTpa, and 5.1±1.5 years with DTPa (p.001). In the protection, adjusted for age, it was observed that, after 3 years, only 47.6% of people vaccinated with dTpa were still protected, while people vaccinated with DTPa were 100% protected (P.001).This study found that people who were properly vaccinated against pertussis and received their last re-vaccination dose with dTpa had a shorter period of protection than those who were vaccinated with DTPa.

Determination of HIV-1 tropism is mandatory before using CCR5 antagonists in clinical practice. One drug of this class, maraviroc, has been approved for the treatment of HIV infection. The phenotypic assay, TrofileTM, was clinically validated in the clinical development program of maraviroc and has been widely used to select candidates for maraviroc therapy. Phenotypic tests, however, have the disadvantage of being complex, are costly and time-consuming, and their accessibility is limited, which hampers their routine use in clinical diagnosis. Genotypic assays, based on sequencing the third hypervariable (V3 loop) of the viral gene env, interpreted according to various genotypic bioinformatic tools, such as geno2pheno and PSSM, are faster and cheaper than phenotypic assays, and are also more accessible. In retrospective analyses of the maraviroc pivotal trials, genotypic methods using either conventional ("bulk") or deep-sequencing technology predicted virologic response to maraviroc similarly to phenotypic assays and are now included within several European recommendations to guide the clinical use of CCR5 antagonists.

Despite their pivotal role in the Spanish healthcare system, clinical microbiology laboratories are experiencing difficult times and tough challenges. The following changes are required to adapt to the new situation: a) the use of molecular diagnostics to provide rapid diagnosis; b) the development of diagnostic capabilities to identify emerging or imported infectious diseases; c) the ability to advise on the interpretation of microbiological results; d) encouragement of the implantation of point-of-care testing and assessment of its performance and development; e) the implantation of quality control systems in the laboratory; f) the implementation of laboratory information systems to support real-time communication between hospital and community clinicians, public health laboratories and managers; g) the design of networking systems with professionals from other disciplines, and h) the promotion of training and teaching programs. Only if they are well prepared will clinical microbiology laboratories be able to implant the new technologies, be recognized as a cornerstone of the healthcare system, and achieve better recognition by society at large, hospital administrators and healthcare authorities.

In the last years, we have verified the increasing emergence of bacteria, specially Escherichia coli, that produce expanded spectrum beta-lactamases (ESBL), enzymes which confer resistance to all cephalosporins (except cephamycins) and aztreonam. These bacteria are frequently resistant also to non-beta-lactam antibiotics, a fact which poses an important clinical problem.Descriptive study of ESBL-producing strains of E. coli isolated in all kind of specimens in two hospitals of Southern Alicante (Spain), throughout a period of 57 months (January 1999 to September 2003), paying a close attention to their origin (outpatients or admitted patients), co-resistance to non beta-lactam antibiotics and evolution of their incidence.Respectively, 3% and 2.25% of E. coli strains isolated in each hospital produce ESBL (3.83% and 2.85% of strains from admitted and 2.74% and 2.1% from outpatients). 30.73% and 24.58% of strains ESBL were isolated in admitted patients. We found in both hospitals much higher percentages of co-resistance to ciprofloxacin, gentamicin and trimetoprim-sulfamethoxazole in ESBL-producing strains.The percentage of ESBL-producing E. coli is high in our environment, but it is even more noteworthy its clear trend to increase. It is very remarkable the high percentage of ESBL-producing strains isolated from outpatients. Finally, we emphasize the high percentages of co-resistance to non-beta-lactam antibiotics.

Tuberculosis in immigrants is an emerging disease in industrialized countries.We retrospectively analyzed the clinical and epidemiological characteristics of culture-positive tuberculosis cases in the immigrant and native populations.One hundred and five cases of tuberculosis were observed; 22 cases (21%) were in immigrants. The 2002 incidence was 64.3 cases per 100,000 immigrants. Mean age of the affected immigrants was 28.5 years lower than that of the affected native population (p0.001). The rate of HIV coinfection was 9.1%. In 81.8% (18) of immigrants, presentation was exclusively pulmonary, a higher percentage than in the native population (59.2%) (p = 0.05). Loss to follow-up was more common among immigrants (35%) than among the native population (9.2%) (p0.001).Tuberculosis in the immigrant population generally involves the lungs, appears in younger subjects and is a public heath problem because of the high rate of losses to follow-up.

As a producer of gastro-enteritis and other symptoms, Salmonella spp. Remains an important problem for world public health. Epidemiological knowledge at both general and local level by means of serotypification is considered one of the fundamental aspects for its control.We studied 15.181 stool samples, and the others specimens. Isolation using the usual routine media, agar MacConkey, Salmonella-Shigella, selenito F. Identification using the automated Microscan and wider I method, serotypification with multi-purpose and monospecific serums (Difco), and confirmation of Salmonella and Shigella by the National Reference Laboratory, from the LNRSSE.Although 96.6% of Salmonella spp. Is detected in cultures of faeces and blood, it is also noted in LCR, sputum, rectal biopsy and vaginal secretions among other sites. In total of 1290 patients, 37 different serotypes were isolated, the most frequent of which were Enteritidis and Typhimurium. The presence of Virchow, isolated in both faeces and LCR, was notable in the years 94-99, as was as the presence of less typical serotypes, such as Blockley, London, Give and Mikawasima, among others.