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Resumen Introducción: Las enfermedades producidas por micobacterias son de gran importancia clínica y epidemiológica presentando el complejo Mycobacterium tuberculosis (MTBc) una morbi-mortalidad mayor que la producida por micobacterias no tuberculosas (MNTB). La identificación tradicional está basada en sus características fenotípicas mediante procesos laboriosos e incapaces en algunos casos de distinguir entre especies. Actualmente, la mayoría de las técnicas utilizadas se basan en métodos moleculares que tienen alta veracidad, pero son complejas y de alto costo. La espectrometría de masas con desorción/ionización láser asistida por una matriz asociada a tiempo de vuelo (MALDI-TOF MS) se basa en la comparación del espectro proteico producido con respecto al de una base de datos de referencia. Objetivo: Evaluar el rendimiento de MALDI-TOF MS en la identificación de micobacterias comparado con métodos moleculares: Material y Métodos: Se analizaron 28 aislados de nueve especies distintas mediante MALDI-TOF MS. Resultados: Se identificó correctamente 78,5% de las aislados (22/28), concordante en 100% (9/9) de MNTB de crecimiento rápido, 60% (9/15) en las MNTB de crecimiento lento y 100% (4/4) de MTBc. Todas las especies no identificadas (6/6) pertenecen al complejo M. avium/intracellulare. Conclusión: MALDI-TOD MS es una metodología rápida, fácil y de bajo costo, con adecuada veracidad respecto a los métodos moleculares.

Resumen La tuberculosis (TBC) extra-pulmonar alcanza al 26,2% de los casos totales de TBC en Chile. El cultivo es el método estándar de oro, pero es lento. La técnica Xpert® MTB/RIF permite detectar Mycobacterium tuberculosis complex (MTBc) por RPC en tiempo real en menos de 3 h, sin embargo, ha sido validada sólo para muestras respiratorias. El objetivo de este estudio fue determinar la utilidad de la prueba Xpert® MTB/RIF en la detección de MTBc en muestras extra-pulmonares en comparación con un estándar de oro combinado consistente en un cultivo de micobacterias positivo (medio sólido y líquido) y/o un método molecular validado positivo (q-RPC, Cobas® TaqMan-MTB). Se analizaron 50 muestras extra-pulmonares, de las cuales 25 fueron definidas positivas y 25 negativas para MTBc en base a estándar de oro combinado. Las 25 muestras definidas positivas tuvieron un resultado positivo por Xpert® MTB/RIF; de las 25 muestras definidas negativas, 24 tuvieron un resultado negativo y una de ellas un resultado positivo. Se obtuvo una concordancia global entre Xpert® MTB/RIF y el estándar de oro combinado de 98%. La prueba Xpert® MTB/RIF fue capaz de detectar 12 casos de TBC extra-pulmonar con baciloscopia negativa y 3 casos con cultivo negativo. El método Xpert® MTB/RIF ha demostrado tener una sensibilidad similar al q-RPC para detectar MTBc en muestras extra-pulmonares y permite reducir sustancialmente el tiempo de diagnóstico.

Introducción: La identificación de pacientes con Staphylococcus aureus resistente a meticilina (SARM) y Enterococcus resistente a vancomicina (ERV), permite limitar su diseminación usando aislamiento en cohorte y precauciones de contacto. Los resultados de los cultivos microbiológicos demoran 3 a 5 días, lo que retrasa el retiro de las precauciones y agrega costos económicos. Objetivos: Implementar técnica de reacción de polimerasa en cadena en tiempo real (RPC), GeneXpert R, para SARM y ERV y comparar tiempos de respuesta y costos en relación al uso de microbiología convencional. Material y Métodos: Se compararon dos períodos, uno en que se usó solo RPC (grupo RPC) y otro histórico, en el que se usó microbiología tradicional (grupo estándar) Resultados: Se confirmó SARM y/o ERV en 29,9% de los pacientes del grupo RPC, y en 9,6% del grupo estándar. Los tiempos de respuesta fueron 15 ± 9 h (grupo RCP) y 53 ± 23 h (grupo estándar). Los costos directos por paciente fueron de USD 245 en el grupo RPC y de USD 530 en el grupo estándar. Discusión: La RPC en tiempo real, GeneXpert, para SAMR y ERV tuvo un alto impacto alto clínico que justifica su incorporación.

Introducción: Los cultivos clínicos detectan sólo un tercio de los pacientes colonizados con enterobacterias resistentes a carbapenémicos, su identificación temprana permitiría implementar precauciones de contacto y así interrumpir su transmisión. En nuestro hospital no hay descritas infecciones causadas por enterobacterias productoras de carbapenemesas Objetivo: Realizar vigilancia de colonización intestinal por enterobacterias productoras de carbapenemasas en pacientes ingresados a nuestro hospital. MaterialyMétodos: Se realizaron cultivos de vigilancia rectal en pacientes con cinco o más días de hospitalización en una unidad de paciente crítico y cuidados especiales de nuestro hospital, en forma mensual, desde julio a diciembre del año 2011. Posteriormente se realizaron tests fenotípicos (test de Hodge modificado y test de ácido fenilborónico) para detectar enterobacterias productoras de carbapenemasas y búsqueda confirmatoria de seis carbapenemasas del grupo A y B de la clasificación de Ambler mediante reacción de polimerasa en cadena (RPC). Resultados: Durante este periodo, se llevaron a cabo 241 vigilancias rectales; de éstas, 38 enterobacterias resultaron resistentes o con susceptibilidad intermedia a carbapenémicos por método de dilución en agar, las que correspondieron a 30 pacientes estudiados. Todas las RPC fueron negativas. Conclusión: A pesar del significativo número de cepas resistentes a carbapenémicos, en los pacientes analizados no se encontraron enterobacterias productoras de carbapenemasas. Destacamos la importancia de la realización de cultivos de vigilancia antes de tener casos clínicos sintomáticos detectados.

Extra-pulmonary tuberculosis (TB) represents the 26.2% of total TB cases in Chile. Culture is the gold standard method, but the process is extremely slow. Xpert®MTB/RIF technique detects Mycobacterium tuberculosis complex (MTBc) through real time PCR in less than 3 h. However, it has been validated only for respiratory specimens. We aimed to determine the performance of Xpert®MTB/RIF test in detecting MTBc in extra-respiratory specimens compared with a combined gold standard consisting in a positive (liquid and solid) mycobacterial culture and/or a positive validated molecular method (q-RPC, Cobas®TaqMan®-MTB). Fifty extra-respiratory specimens were analyzed, from which 25 were positive and 25 negative for MTBc based on the combined gold standard. The 25 positive specimens had a positive result by Xpert®MTB/RIF; from the 25 negative specimens, 24 had a negative result and one had a positive result. We obtained an overall concordance of 98% between Xpert®MTB/RIF and the combined gold standard. Xpert®MTB/RIF test was able to detect 12 smear-negative specimens and 3 culture-negative specimens, all of them corresponding to extra-pulmonary TB cases. Xpert®MTB/RIF showed similar sensitivity to q-RPC in detecting MTBc in extra-respiratory specimens. This procedure allowed a substantial reduction in the time of diagnosis.

Antecedentes: El diagnóstico de meningitis tuberculosa (MTBC) se ve limitado por la ausencia de técnicas diagnósticas rápidas y precisas en líquido cefalorraquídeo (LCR). En este estudio evaluamos la respuesta inmunoló-gica de anticuerpos anti-antígeno A60 de Mycobacterium tuberculosis en LCR en comparación a la determinación de adenosina deaminasa (ADA). Métodos: Un total de 63 muestras de LCR fueron estudiadas mediante ELISA indirecto para detección de IgG, IgM e IgA anti-A60. Estas muestras incluyeron 17 casos de MTBC confirmada y 46 controles con otras infecciones. Resultados: Los títulos de IgG, IgM e IgA anti A-60 resultaron significativamente superiores en casos de MTBC versus controles (p > 0,01). El anticuerpo con mej or poder discriminatorio resultó IgM, con un área bajo la curva ROC de 0,928 (95%IC 0,8340,978), comparado a 0,863 (95% IC: 0,752-0,936) para ADA (p = NS). La sensibilidad de IgM anti-A60 (nivel de corte > 0,06 U/ml) fue de 94,1% versus 88,2% para ADA (nivel de corte > 6,2 U/ml), p = NS. Ambos IgM anti-A60 y ADA presentaron la misma especificidad baja-moderada (80,4%). Dos casos de MMTBC fueron correctamente identificados por IgM anti-A60 pero no por ALDA. Conclusión: La detección de anticuerpos anti-A60 (IgM) puede ser de ayuda en el diagnostico de MTBC en forma complementaria a la determinación de ALDA. La baja especificidad de ambos tests constituye su principal limitante.

SUMMARYStreptococcus pyogenescauses a variety of infections because of virulence factors such as capsular hyaluronic acid and M protein. The aim of this study was to determineemmtypes and capsule phenotype in 110 isolates ofS. pyogenesfrom patients with invasive (sterile sites) and non-invasive (mainly pharyngitis) infections in Chile, and the relationship between both virulence factors. The most abundant types found wereemm12,emm1,emm4andemm28and their distribution was similar to that seen in Latin America and developed countries, but very different from that in Asia and Pacific Island countries. Ten of 16emmtypes identified in pharyngeal isolates were found in sterile-site isolates, and three of nineemmtypes of sterile-site isolates occurred in pharyngeal isolates; threeemmsubtypes were novel. The amount of hyaluronic acid was significantly higher in sterile-site isolates but did not differ substantially amongemmtypes. Only three isolates were markedly capsulate and two of them had mutations in thecsrRgene that codes for a repressor of capsule synthesis genes. We found a non-random association betweenemmtypes andcsrRgene alleles suggesting that horizontal gene transfer is not freely occurring in the population.

Introducción: Los sistemas automatizados han facilitado el flujo de trabajo, mejorado la estandarización, la trazabilidad, disminuido el error humano y la carga de trabajo en los laboratorios. A pesar de que la microbiología ha permanecido poco automatizada, en los últimos años han aparecido nuevas herramientas para la automatización de la etapa pre analítica. Objetivos: Evaluar el desempeño de un sistema automatizado de siembra de urocultivos y la concordancia con la siembra manual convencional en el recuento semicuantitativo de colonias. Materiales y Métodos: 495 muestras de orinas fueron sembradas según nuestro protocolo habitual y comparadas con las placas de CPS® obtenidas con PREVI™ Isola en cuanto a positividad/negatividad, muestras polimicrobianas, especies de bacterias aisladas, recuentos y necesidad de resembrar. Resultados: Hubo concordancia en 98,97% de los positivos y negativos, en 99,39% de las muestras polimicrobianas, en 99,76% de las especies aisladas y en 98,56% de los recuentos. La necesidad de resiembra disminuyo de 12,1% a un 1,1% usando este sistema automatizado. Discusión: El desempeño de PREVI™ Isola fue el esperado, mejorando el aislamiento bacteriano y el tiempo requerido y representa una buena herramienta para la automatización de laboratorios.

Automated systems have simplified laboratory workflow, improved standardization, traceability and diminished human errors and workload. Although microbiology laboratories have little automation, in recent years new tools for automating pre analytical steps have appeared.To assess the performance of an automated streaking machine for urine cultures and its agreement with the conventional manual plating method for semi quantitative colony counts.495 urine samples for urinary culture were inoculated in CPS® agar using our standard protocol and the PREVI™ Isola. Rates of positivity, negativity, polymicrobial growth, bacterial species, colony counts and re-isolation requirements were compared.Agreement was achieved in 98.97% of the positive/negative results, in 99.39% of the polymicrobial growth, 99.76% of bacterial species isolated and in 98.56 % of colony counts. The need for re-isolation of colonies decreased from 12.1% to 1.1% using the automated system.PREVI™ Isola's performance was as expected, time saving and improving bacterial isolation. It represents a helpful tool for laboratory automation.

La incidencia de coqueluche en Chile varia entre 4,1 y 7,5 por 100.000 habitantes. La deteccion de Bordetella pertussis se realiza por RPC-tiempo real (Q-RPC) dirigida a la secuencia de insercion IS481. Sin embargo, esta secuencia se encuentra tambien en el genoma de B. bronchiseptica y B. holmesii. Este ultimo es tambien un patogeno respiratorio que produce un cuadro similar a B. pertussis. Sin embargo, es importante diferenciar entre estas especies porque en pacientes inmunosuprimidos B. holmesii tiene mayor tendencia a causar bacteriemia y ademas es menos susceptible a eritromicina. El objetivo de este trabajo es determinar, prospectiva y retrospectivamente, la presencia de B. holmesii en muestras informadas positivas para B. pertussis en el periodo 2010-2011. Durante ese periodo ingresaron al laboratorio 1. 994 muestras de hisopado nasofaringeo para RPC de Bordetella sp., de las cuales 224 fueron positivas. El analisis por Q-RPC dirigido al gen recA de B. holmesii de las 224 muestras positivas determino una prevalencia de B. holmesii de 0,6% (12/1994). Debido al comportamiento mas agresivo en inmunosuprimidos y al patron de resistencia de B. holmesi, se decide incorporar la deteccion de rutina de B. pertussis y B. holmesii en todas las muestras en que se detecta inicialmente la presencia de Bordetella sp.

Identification of patients with methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE) is essential to limit the spread of these agents, through the use of isolation and contact precautions. Traditional microbiology has a long turn around time (3-5 days) extending the time of isolation, increasing complexity and cost of these patients.To implement a new real time polymerase chain reaction (PCR) GeneXpert R for SAMR and VRE detection. To compare costs and turn around time of PCR versus traditional cultures.Two periods were compared, in the first, traditional microbiology (standard group) was used, and in the second, only PCR was used (PCR group).MRSA or VRE were identified in 29.9% of patients in the PCR group and in 9.6% in the standard group. Turn around time was 15 ± 9 hours in PCR group and 53 ± 23 hours in standard group. PCR group had a net cost of USD 245 per patient and standard group USD 530 per patient.PCR technique GeneXpert R for MRSA and VRE had a positive impact in the management of these patients and justifies its inclusion.

Clinical cultures detect only one-third of colonized patients with carbapenem-resistant Enterobacteriaceae. Early identification and contact precautions implementation would help to interrupt transmission. In our hospital no carbapenemase-producing enterobacteria infections have been described.To perform stool surveillance cultures in patients hospitalized in critical care unit with the purpose to detect carbapenemase-producing Enterobacteriacea.Rectal swabs were obtained of patients after five or more days of hospital stay, on a monthly basis from July to December 2011. Phenotypic assays (modified test Hodge and phenylboronic acid test) and polymerase chain reaction (PCR) searching for six carbapenemases of group A and B of Ambler's classification were performed.During this period, 241 surveillance rectal cultures were performed. Thirty eight enterobacteria isolated from 30 patients presented a decreased susceptibility to carbapenems by agar dilution method. All PCR were negative.We found that despite the significant number of resistant isolates, patients hospitalized in our institution are not colonized with carbapenemase-producing Enterobacteriaceae. We highlight the importance of screening before having the problem in place.

The main goal of this work was to identify the mechanisms responsible for carbapenem resistance in 61 Chilean clinical isolates of Enterobacteriaceae (Enterobacter spp., Serratia marcescens, Morganella morganii, Escherichia coli and Klebsiella pneumoniae) with reduced susceptibility to at least one carbapenem (ertapenem, imipenem or meropenem). All of the isolates were analysed for the presence of carbapenemases, extended spectrum β-lactamases (ESBLs), AmpC enzymes and outer-membrane proteins. None of the isolates exhibited carbapenemase activity nor did they have any of the carbapenemase genes that were screened for. Most of the 61 strains produced at least one ESBL and/or one AmpC enzyme and either lost their porins or had altered porins according to sequence analysis. The distribution of ESBLs and AmpC enzymes was different among the species studied. Resistance in K. pneumoniae and E. coli isolates was associated with ESBLs; in M. morganii isolates, resistance was attributed to overexpression of an AmpC enzyme; and in Enterobacter spp. isolates, resistance was associated with both types of enzymes. In K. pneumoniae isolates, porin integrity was more a determinant of carbapenem resistance than the presence of ESBLs, whereas in isolates of Enterobacter spp., M. morganii and S. marcescens, the presence of an overexpressed AmpC enzyme was associated with higher imipenem and meropenem MIC values. Therefore, carbapenem resistance in Chilean isolates is not due to true carbapenemases but rather to a combination of porin loss/alteration and β-lactamase activity. The fact that carbapenemases were not detected in this study is unique, given that many countries in the region have already reported the presence of these enzymes.

Extended-spectrum-β-lactamases (ESBL) are plasmid-encoded enzymes that confer resistance to multiple antimicrobials. ESBL-producing enterobacteria that cause bacteremia limit therapeutic options and increase mortality.To perform a clinical and molecular description of bacteremia caused by ESBL-producing enterobacteria.We retrospectively studied the cases of bacteremia due to ESBL-producing Escherichia coli, Klebsiella pneumoniae and Proteus spp in adults admitted to a university hospital during the years 2004-2007. We reviewed the clinical records and antimicrobial susceptibility patterns. Molecular typing was performed by polymerase chain reaction and study of clonality by pulsed-field electrophoresis.We found a prevalence of 9.8% ESBL in enterobacteria causing bacteremia. Decreased susceptibility to quinolones and aminoglycosides was observed, without resistance to carbapenems. The predominant ESBL types were CTX-M (96%), TEM (62%) and GES (28%). 79% of the strains presented more than one type of ESBL. Clinical analysis revealed high prevalence of risk factors, previous use of antimicrobials and of invasive devices. There was no significant clonality.The presence of ESBLs in bloodstream infections is a clinical problem that must be considered when choosing empiric therapy.

Macrolide and lincosamide resistance in Streptococcus pyogenes is due to the acquisition of mef, ermB and ermA genes, which confer different resistance phenotypes, namely M, MLSBconstitutive and MLSBinducible respectively. The last report of resistance in Chile was done in the period 1990-1998, in which resistance to macrolides was 5.4%, with M phenotype as the predominant one.To characterize the evolution of erythromycin and clindamycin resistance and their associated genes in S. pyogenes strains isolated from patients with invasive and non-invasive infections in the period 1996 to 2005.Resistance to erythromycin and clindamycin was determined in 1,282 clinical isolates using the disk diffusion test. Resistant isolates were analyzed by polymerase chain reaction (PCR) for the presence of the above mentioned resistance genes.Global resistance to erythromycin and clindamycin was 3.5 and 0.7% respectively. Eighty percent of the resistant strains possessed the M. phenotype.Resistance levels of S. pyogenes have decreased in Chile in the last years. Most resistant strains have M phenotype in contrast to many countries in which the MLSB constitutive phenotype is the predominant one.

Background : Macrolide and lincosamide resistance in Streptococcus pyogenes is due to the acquisition of mef, ermB and ermA genes, which confer different resis- tance phenotypes, namely M, MLSBconstitut ive and MLSBinducible respectively. The last report of resistance in Chile was done in the period 1990-1998, in which resistance to macrolides was 5.4%, with M phenotype as the predominant one. Aim : To characterize the evolution of erythromycin and clindamycin resistance and their associated genes in S. pyogenes strains iso lated from patients with invasive and non- invasive infections in the period 1996 to 2005. Material and Methods: Resistance to erythromycin and clindamycin was determined in 1,282 clinical isolates using the disk diffusion test. Resistant isolates were analyzed by polymerase chain reaction (PCR) for the presence of the above mentioned resistance genes. Results : Global resistance to erythromycin and clindamycin was 3.5 and 0.7% respectively. Eighty percent of the resistant strains possessed the M. phenotype. Conclusions : Resistance levels of S. pyogenes have decreased in Chile in the last years. Most resistant strains have M phenotype in contrast to many countries in which the MLSB constitutive phenotype is the predominant one

Diagnosis of tuberculous meningitis (TBM) is hampered by the lack of rapid and accurate diagnostic tools. We evaluated the immunological response to Mycobacterium tuberculosis anti-A60 antibodies in cerebrospinal fluid (CSF) in comparison to adenosine deaminase (ADA) determination, for the diagnosis of TBM.A total of 63 CSF samples were analyzed by indirect ELISA for the detection of anti- A60 IgG, IgM and IgA. These include samples from 17 patients with confirmed TBM and 46 control patients with other infections.The mean individual anti-A60 IgM, IgG and IgA CSF antibody titers were significantly higher in TBM in comparison with control groups (p0.01). The best discriminatory CSF antibody for confirming TBM diagnosis was IgM, with an area under the receiver operating characteristic curve of 0.928 (95%CI 0.834-0.978), compared to 0.863 (95% CI: 0.752-0.936) for ADA testing (p = NS). The sensitivity of anti- A60 IgM CSF antibody titers (cutoff0.06 U/ml) was 94.1% compared to 88.2% for ADA (cutoff6.2 U/ml), p = NS. Both anti A60 IgM and ADA showed the same moderate specificity (80.4%). Two cases of TBM were correctly identified by anti-A60 IgM but missed by ADA.The ELISA test for anti-antigen A60 antibodies (IgM) is a rapid and sensitive tool for the rapid diagnosis of TBM that can be a complement to ALDA determination. The specificity of both tests is still a limitation in TBM diagnosis.

Commercial polymerase chain reaction (PCR) kits are widely accepted for analysis of smear positive respiratory specimens, but the sensitivity is variable for smear negative ones.To assess the PCR method usefulness in smear negative respiratory and non respiratory specimens.We compared the PCR results (AMPLICOR MTB test, Roche) of 235 specimens subjected to culture in Loewenstein-Jensen agar (as the gold standard).181 (76%) were respiratory and 54 (24%) extra-respiratory specimens. The sensitivity was 88%) and 50%, respectively, specificity and PPV was 100%in both cases. NPV was 99.4%in respiratory specimens and 96.1% in non-respiratory specimens.The good performance of this PCR in smear negative respiratory specimens allows the clinician to take decisions based on the result of this exam. In extra-respiratory specimens the contribution is important only when the PCR result is positive.

Introduccion: Los metodos comerciales de reaccion de polimerasa en cadena (RPC) estan validados para el diagnostico de Mycobacterium tuberculosis solo en muestras respiratorias con baciloscopia positiva. En muestras con baciloscopia negativa, la sensibilidad es variable. Objetivo: Evaluar el comportamiento de la RPC en uso en muestras con baciloscopia negativa. Metodo: Se compararon los resultados de 235 muestras analizadas por cultivo en agar Loewenstein-Jensen ("estandar de oro") y RPC (AMPLICOR MTB test®, Roche). Resultados: Se analizaron 181 muestras respiratorias y 54 muestras extra-respiratorias. La sensibilidad de la RPC fue de 88 y 50%), respectivamente, la especificidad y el VPP fueron, en ambos casos, de 100%o, y el VPN fue de 99,4%o en muestras respiratorias y de 96,1%) en extra-respiratorias. Conclusiones: El buen desempeno de esta RPC en muestras respiratorias con baciloscopia negativa permite al medico clinico tomar decisiones en base al resultado de este examen, fundamentadamente. En muestras extra-respiratorias, solo pueden sacarse conclusiones frente a resultados de RPC positivos.

Background: Metallo-s-lactamases (MBL) confer high resistance to carbapenems in Pseudomonas aeruginosa (Psae). They are encoded in mobile elements of different genes (VIM, IMP, SMP, GIM), along with other resistance genes. Aim: To detect the presence of MBL in imipenem resistant Psae strains. Material and methods: Fifty-nine imipenem resistant Psae strains isolated from January 2004 to August 2005 in a University Clinical Hospital, were included. The presence of MBL was studied by Etest (phenotypic) and genotypic polymerase chain reaction (PCR) methods. To rule out a nosocomial outbreak, MBL positive strains, were studied by pulse field gel electrophoresis. Results: The presente of MBL was detected in eleven strains. AH were type VIM and were not clonally related. There was no concordance between phenotypic and genotypic MBL detecting methods. AH the strains were also multiresistant. Conclusions: The presence of MBL was detected in 19% of imipenem resistant Psae strains

Several studies have concluded that it is inappropriate to perform stool cultures (SC) to inpatients that have stayed hospitalized three days or more because nosocomial diarrhea is not due to enteric pathogenic bacteria that are searched through this exam. The aim of this paper was to analyze the SC yield performed to patients hospitalized in the UC Health Net since January to December 2002 and the rate of positive results obtained depending on the length of hospitalization in order to define if international guidelines are useful to the national reality. During twelve months 3474 SC were evaluated, 458 (13.2%) belonged to inpatients. Of them 16 (3.5%) were positive, 13 were obtained on the first day, 2 at the second day and 1 at the fifth day of hospitalization. Only 1/190 (0.5) SC obtained after two days of hospitalization yielded a positive result. These results reinforce the international recommendation of not performing SC to inpatients that have stayed more than three days at the hospital; besides, considering previous figures, we suggest to make a cutoff at day two of hospitalization, after two days there is no a significant clinical impact. This strategy means to reduce 41% of these analyses and to save the corresponding money.