RESUMEN
Abstract The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.
Resumen El objetivo de este estudio fue comparar el desempeño de dos sistemas MALDI-TOF MS en la identificación de bacterias anaerobias estrictas de interés clínico. La secuenciación del gen 16S ARNr fue el método de referencia utilizado cuando se observaron discrepancias o inconsistencias entre plataformas. Se recuperaron 333 aislados de muestras clínicas de diferentes centros de la Ciudad Autónoma de Buenos Aires entre 2016 y 2021. Los aislados se identificaron por duplicado mediante dos sistemas MALDI-TOF MS: el BD Bruker Biotyper (Bruker Daltonics, Bremen, Alemania) y el Vitek MS (bioMèrieux, Marcy-l'Etoile, Francia). A través del sistema Vitek MS, los mismos fueron identificados a nivel de especie o complejo de especies en un 65,5% (n: 218) y de género en un 71,2% (n: 237), mientras que no se identificaron en un 29,4% (n: 98) y fue incorrecta en el 5,1% (n: 17). Mediante el sistema Bruker Biotyper, dichos valores fueron del 85,3% (n: 284), del 89,7% (n: 299), del 14,1% (n: 47) y del 0,6% (n: 2), respectivamente. La diferencia entre ambos métodos fue estadísticamente significativa (p<0,0001). En conclusión, los sistemas MALDI-TOF MS aceleran la identificación microbiana. Son especialmente útiles para los microorganismos de crecimiento lento, como las bacterias anaerobias, que son difíciles de identificar con los métodos tradicionales. El sistema Bruker demostró ser más preciso que el Vitek MS. Para que estos métodos sean realmente efectivos es fundamental actualizar las bases de datos de ambos sistemas e incrementar el número de espectros de referencia dentro de las plataformas.
RESUMEN
The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.
Asunto(s)
Bacterias Anaerobias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bacterias Anaerobias/genética , ARN Ribosómico 16S/genética , ArgentinaRESUMEN
Two metallo-ß-lactamase-producing Klebsiella pneumoniae (HA30 and HA31) were isolated in a hospital in Argentina during 2018. K. pneumoniae HA30 was isolated from a rectal swab during the epidemiological surveillance for carbapenemase-producing strains, while K. pneumoniae HA31 was collected from the same patient 4 days after hospitalization. The aim of the present study was to identify the clonal relationships and resistome of these two NDM-producing K. pneumoniae strains isolated from a patient with a fatal outcome. Whole-genome sequencing (WGS) was performed using Illumina MiSeq-I, and subsequent analysis involved genome assembly, annotation, antibiotic resistance gene identification, multilocus sequence typing (MLST), and plasmid characterization using bioinformatics tools. Conjugation assays to E. coli J53 was conducted as previously described. K. pneumoniae HA30 exhibited extensively drug-resistant phenotype, while HA31 was multidrug-resistant as defined by Magiorakos et al., including both resistance to carbapenems, aminoglycosides and ciprofloxacin with blaNDM-5, blaCTX-M-15 and rmtB genes found in both strains. MLST analysis showed that both strains belonged to ST11, differing by only 4 cgSNPs, indicating that K. pneumoniae HA30 and HA31 were the same strain. Conjugation assays revealed that K. pneumoniae HA31 strain possessed a transferable plasmid to E. coli J53. Bioinformatics studies identified that the same strain colonizing an inpatient during hospital admission subsequently caused the infection leading to a fatal outcome, being the first report of blaNDM-5, rmtB and blaCTX-M-15 genes in a K. pneumoniae ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.
Asunto(s)
Escherichia coli , Klebsiella pneumoniae , Humanos , Klebsiella pneumoniae/genética , Tipificación de Secuencias Multilocus , Genómica , Antibacterianos/farmacología , beta-Lactamasas/genéticaRESUMEN
Abstract This study aimed to assess the impact of the implementation of a rapid multiplex molecular FilmArray Respiratory Panel (FRP) on the medical management of immunocompromised patients from a community general hospital. We conducted a single-center, retrospective, and before-after study. Two periods were evaluated: before the implementation of the FRP (pre-FRP) from April 2017 to May 2018 and after the implementation of the FRP (post-FRP) from January to July 2019. The inclusion criteria were immunocompromised patients over 18 years of age with suspected acute respiratory illness tested by conventional diagnostic meth-ods (pre-FRP) or the FilmArray™ Respiratory Panel v1.7 (post-FRP). A total of 142 patients were included, 64 patients in the pre-FRP and 78 patients in the post-FRP. The positive detec-tion rate was significantly higher in the post-FRP (63% vs. 10%, p <0.01). There were more patients receiving antimicrobial treatment in the pre-FRP compared with the post-FRP period (94% vs. 68%, p <0.01). A decrease in beta-lactam (89% vs. 61%, p <0.01) and macrolide (44% vs. 13%, p < 0.01) prescriptions were observed in the post-FRP. No differences were observed in oseltamivir use (22% vs. 13%, p = 0.14), changes in antimicrobial treatment, hospital admission rate, days-reduction in droplet isolation precautions, hospital length of stay (LOS), admission to intensive care unit (ICU), LOS in ICU, treatment failure and 30-day mortality. The implementa-tion of the FRP impacted patient care by improving diagnostic yield and optimizing antimicrobial treatment in immunocompromised adult patients.
Resumen El objetivo de este estudio fue evaluar el impacto de la implementación del panel respiratorio FilmArray® (FRP), un sistema automatizado de PCR multiplex, en el estándar de cuidado de pacientes adultos inmunocomprometidos en un hospital general. Es un estudio retrospectivo de un único centro con diseno antes/después. Los periodos evaluados fueron abril 2017-mayo 2018, previo a la implementación del FRP (pre-FRP), y enero 2019-julio 2019, luego de la implementación (post-FRP). Los criterios de inclusión fueron pacientes mayores de 18 años inmunocomprometidos con sospecha de infección respiratoria aguda a los que se les realizó, en pre-FRP, diagnóstico por métodos convencionales, y en post-FRP, el panel respiratorio FRP versión 1.7. Se incluyeron un total de 142 pacientes, 64 en pre-FRP y 78 en post-FRP. La tasa de positividad fue significativamente mayor en post-FRP frente a pre-FRP (63 vs. 10%, p<0,01). Hubo más pacientes con tratamiento antimicrobiano en pre-FRP que en post-FRP (94 vs. 68%, p <0,01). En pre-FRP hubo más pacientes tratados con betalactámicos (89 vs. 61%, p <0,01) y macrólidos (44 vs. 13%, p < 0,01). No se observaron diferencias significativas en el uso de oseltamivir (22 vs. 13%, p = 0,14), cambios en los tratamientos, número de hospitalizaciones, uso de aislamientos, duración de la estadía hospitalaria, ingreso a la unidad de cuidados intensivos, estadía en dicha unidad, falla de tratamiento y mortalidad a 30 días. El uso de FRP contribuyó a la atención del paciente mejorando el rendimiento diagnóstico y optimizando la terapia antimicrobiana en pacientes adultos inmunocomprometidos.
RESUMEN
Toxin-producing Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. However, it is now recognized as a cause of diarrhea in the community. This single-center study aimed to determine the epidemiological origin of CDI cases between January 2014 and December 2019 and to compare demographic characteristics, comorbidities, risk factors, severity, and mortality of community CDI with healthcare facility-associated CDI. There were 52 CDI cases from the community (34.4%). Community patients were significantly younger (53 yo vs. 65 yo), less comorbid (Charlson Index 1.65 vs. 3.98), and less severe (only one case). The main risk factor was the use of antibiotics in the previous 90 days (65%). However, we did not find any known risk factor in 7 patients.
Asunto(s)
Clostridioides difficile , Infecciones por Clostridium , Infecciones Comunitarias Adquiridas , Infección Hospitalaria , Humanos , Infecciones Comunitarias Adquiridas/epidemiología , Hospitales Generales , Argentina/epidemiología , Infecciones por Clostridium/epidemiología , Diarrea/epidemiología , Infección Hospitalaria/epidemiologíaRESUMEN
This study aimed to assess the impact of the implementation of a rapid multiplex molecular FilmArray Respiratory Panel (FRP) on the medical management of immunocompromised patients from a community general hospital. We conducted a single-center, retrospective, and before-after study. Two periods were evaluated: before the implementation of the FRP (pre-FRP) from April 2017 to May 2018 and after the implementation of the FRP (post-FRP) from January to July 2019. The inclusion criteria were immunocompromised patients over 18 years of age with suspected acute respiratory illness tested by conventional diagnostic methods (pre-FRP) or the FilmArray™ Respiratory Panel v1.7 (post-FRP). A total of 142 patients were included, 64 patients in the pre-FRP and 78 patients in the post-FRP. The positive detection rate was significantly higher in the post-FRP (63% vs. 10%, p<0.01). There were more patients receiving antimicrobial treatment in the pre-FRP compared with the post-FRP period (94% vs. 68%, p<0.01). A decrease in beta-lactam (89% vs. 61%, p<0.01) and macrolide (44% vs. 13%, p<0.01) prescriptions were observed in the post-FRP. No differences were observed in oseltamivir use (22% vs. 13%, p=0.14), changes in antimicrobial treatment, hospital admission rate, days-reduction in droplet isolation precautions, hospital length of stay (LOS), admission to intensive care unit (ICU), LOS in ICU, treatment failure and 30-day mortality. The implementation of the FRP impacted patient care by improving diagnostic yield and optimizing antimicrobial treatment in immunocompromised adult patients.
Asunto(s)
Antiinfecciosos , Infecciones del Sistema Respiratorio , Adulto , Humanos , Adolescente , Antibacterianos/uso terapéutico , Estudios Retrospectivos , Estudios Controlados Antes y Después , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Prescripciones , Huésped InmunocomprometidoRESUMEN
OBJECTIVES: The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution. METHODS: Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed. RESULTS: Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions. CONCLUSION: Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to ß-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Escherichia coli , Escherichia coli/genética , Farmacorresistencia Bacteriana Múltiple/genética , beta-Lactamasas/genética , Antibacterianos/farmacología , Klebsiella pneumoniae , Genómica , HospitalesRESUMEN
OBJECTIVES: The emergence of blaKPC-2 within nosocomial settings has become a major public health crisis worldwide. Our aim was to perform whole-genome sequencing (WGS) of three KPC-producing Gram-negative bacilli (KPC-GNB) strains isolated from a hospitalized patient to identify acquired antimicrobial resistance genes (ARGs). METHODS: WGS was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Bioinformatics analysis was done using Resfinder, AMRFinder, ISFinder, plasmidSPAdes, PlasmidFinder, MOB-suite, PLSDB database, and IntegronFinder. Conjugation assays were performed to assess the ability of blaKPC-2 to transfer via a plasmid-related mobilization mechanism. RESULTS: High-risk clone KPC-producing Klebsiella pneumoniae sequence type (ST) 258 (HA3) was colonizing an inpatient who later was infected by KPC-producing Escherichia coli ST730 (HA4) and subsequently by KPC-producing K. pneumoniae ST11 (HA15) during hospitalization. Although belonging to different species, both strains causing infections harbored the same gene configuration for dissemination of blaKPC-2 in related IncM1 plasmids recently found in other KPC-GNB isolated from Hospital Alemán at Ciudad Autónoma de Buenos Aires. Conjugation assays revealed that only pDCVEA4-KPC from E. coli HA4 was successfully transferred with a conjugation frequency of 3.66 × 101. CONCLUSIONS: Interchange of multidrug-resistant K. pneumoniae lineages ST258 replaced by ST11 in the framework of colonization and infection by KPC-GNB of an inpatient from our institution was found. In addition, the transfer of the gene configuration of blaKPC-2 between infecting strains may have occurred in the nosocomial environment, but we cannot rule out that the event took place in vivo, within the patient, during hospitalization.
Asunto(s)
Infección Hospitalaria , Infecciones por Klebsiella , Humanos , Antibacterianos/farmacología , beta-Lactamasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Pandemias , Pacientes Internos , Infecciones por Klebsiella/epidemiología , Farmacorresistencia Bacteriana , Plásmidos/genética , Klebsiella pneumoniae , Hospitalización , Infección Hospitalaria/epidemiologíaRESUMEN
OBJECTIVES: Enterobacter cloacae complex (ECC) has awakened interest recently because of its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence types (STs) which represent high-risk clones, there is substantial clonal diversity in the ECC. This work aimed to perform whole-genome sequencing (WGS), genomic analysis, and phylogenetic studies of a Klebsiella pneumoniae carbapenemase (KPC) -producing multidrug-resistant (MDR) ECC isolate from Argentina. METHODS: We analysed the genome of an MDR KPC-producing ECC strain isolated from a urine sample from a patient in a hospital in Argentina. The WGS was done by Illumina MiSeq-I (Illumina, San Diego, CA). The genome was assembled with SPAdes 3.9.0, and annotated with PROKKA, RAST, and Blast. Plasmids were identified with PlasmidFinder. Antibiotic resistance genes were detected using RESfinder, CARD, and Blastn. STs were identified with pubMLST. RESULTS: The strain was identified as Enterobacter hormaechei, an important emerging human pathogen. No ST could be assigned; six of seven alleles of multilocus sequence typing (MLST) were the same as for E. hormaechei ST66, which is a high-risk clone. We found multiple acquired antibiotic resistance genes, including blaKPC-2 in an IncM1 plasmid, and a secretion system VI, which can favour the prevalence of ECC strains while competing with other bacteria. CONCLUSION: Because of its MLST profile being so close to that of E. hormaechei ST66, the acquisition of multiple resistance genes, and the presence of the secretion systems, the potential of this strain for becoming a new high-risk clone cannot be discarded.
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Enterobacter cloacae , Infecciones por Enterobacteriaceae , Humanos , Enterobacter cloacae/genética , Tipificación de Secuencias Multilocus , Infecciones por Enterobacteriaceae/microbiología , Filogenia , Klebsiella pneumoniae/genética , Antibacterianos/farmacología , Células ClonalesRESUMEN
Resumen Trichophyton benhamiae es un dermatofito zoofílico. Puede causar tinea corporis, tinea faciei y tinea capitis. Se caracteriza por producir lesiones inflamatorias, sobre todo en niños. El objetivo de esta publicación es describir 7 casos clínicos de pacientes pediátricos atendidos entre julio del 2019 y enero del 2020 en nuestra institución. A los pacientes se les solicitó estudio micológico convencional, con posterior confirmación con MALDI-TOF MS y secuencia-ción del ADN ribosomal. Se aisló e identificó T. benhamiae como agente etiológico; el nexo epidemiológico fue el contacto con cobayos. Estas son las primeras descripciones de infecciones causadas por T. benhamiae en Argentina. Al realizar estudios micológicos convencionales, este agente puede confundirse con otros dermatofitos, por lo tanto, se requieren herramientas como MALDI-TOF MS o la secuenciación para llegar a un diagnóstico definitivo. Es importante contar con datos epidemiológicos, como el contacto con mascotas no tradicionales, para una presunción diagnóstica adecuada.
Trichophyton benhamiae is a zoonotic dermatophyte that can cause tinea corporis, tinea faciei and tinea capitis, producing inflammatory lesions, especially in children. In this publication, we describe 7clinical cases of pediatric patients that occurred in our institution between July 2019 and January 2020. All patients underwent a conventional mycological study. The identification of fungi isolates was confirmed by MALDI-TOF MS and sequencing of the ribosomal DNA. T. benhamiae was identified as the etiological agent, whose epidemiological link in all cases was the contact with Guinea pigs. This is the first description of infections caused by T. benhamiae in Argentina. This dermatophyte can be misidentified as other more frequent dermatophytes when performing conventional studies. Molecular technology should be used to reach a definitive diagnosis. It is important to have epidemiological data from patients such as contact with non-traditional pets, especially Guinea pigs, for an adequate presumptive diagnosis of this dermatophytosis.
RESUMEN
Abstract Human parechovirus (HPeV) is one of the members of the family Picornaviridae that has been associated with fever of unknown origin, gastroenteritis, clinical sepsis, meningitis, orencephalitis in very young infants. HPeV detection is not routinely performed in most clinical microbiology laboratories in Argentina and, therefore, its real prevalence is unknown. We here report three cases of HPeV CNS infection that presented to our hospital with different clinical features after the implementation of a multiplex PCR meningitis/encephalitis panel. Molecular diagnostic techniques could help improve patient care and understand the real prevalence of this infection in Argentina.
Resumen Los parechovirus humanos (HPeV) son virus de la familia Picornaviridae, que se han asociado a diferentes cuadros clínicos, como fiebre de origen desconocido, gastroenteritis, sepsis, meningitis o encefalitis en ninos pequeños. Su detección no está disponible de rutina en la mayoría de los laboratorios de nuestro país, por lo que su prevalencia es desconocida. Reportamos 3 casos de infección del sistema nervioso central por HPeV con diferentes características clínicas, que se presentaron luego de la implementación de un panel molecular para el diagnóstico sindrómico de meningitis/encefalitis. Las técnicas de diagnóstico molecular podrían ayudar a mejorar el abordaje y el cuidado de estos pacientes, así como también a conocer la prevalencia de esta infección en Argentina.
RESUMEN
Trichophyton benhamiae is a zoonotic dermatophyte that can cause tinea corporis, tinea faciei and tinea capitis, producing inflammatory lesions, especially in children. In this publication, we describe 7clinical cases of pediatric patients that occurred in our institution between July 2019 and January 2020. All patients underwent a conventional mycological study. The identification of fungi isolates was confirmed by MALDI-TOF MS and sequencing of the ribosomal DNA. T. benhamiae was identified as the etiological agent, whose epidemiological link in all cases was the contact with Guinea pigs. This is the first description of infections caused by T. benhamiae in Argentina. This dermatophyte can be misidentified as other more frequent dermatophytes when performing conventional studies. Molecular technology should be used to reach a definitive diagnosis. It is important to have epidemiological data from patients such as contact with non-traditional pets, especially Guinea pigs, for an adequate presumptive diagnosis of this dermatophytosis.
Asunto(s)
Arthrodermataceae , Tiña , Animales , Argentina/epidemiología , Arthrodermataceae/genética , ADN Ribosómico , Cobayas , Tiña/diagnóstico , Tiña/epidemiología , Tiña/veterinaria , Trichophyton/genéticaRESUMEN
Human parechovirus (HPeV) is one of the members of the family Picornaviridae that has been associated with fever of unknown origin, gastroenteritis, clinical sepsis, meningitis, or encephalitis in very young infants. HPeV detection is not routinely performed in most clinical microbiology laboratories in Argentina and, therefore, its real prevalence is unknown. We here report three cases of HPeV CNS infection that presented to our hospital with different clinical features after the implementation of a multiplex PCR meningitis/encephalitis panel. Molecular diagnostic techniques could help improve patient care and understand the real prevalence of this infection in Argentina.
Asunto(s)
Parechovirus , Infecciones por Picornaviridae , Sepsis , Argentina , Niño , Humanos , Lactante , Técnicas de Diagnóstico Molecular , Parechovirus/genética , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/epidemiología , Sepsis/diagnóstico , Sepsis/epidemiologíaRESUMEN
Resumen El panel BCID2 de BioFire® (BioFire, Salt Lake City, EE.UU.) utiliza un análisis de PCR múltiple a partir de hemocultivos positivos con resultados en una hora. El objetivo de este estudio fue determinar el desempeño del método a partir de hemocultivos positivos de pacientes sépticos en 5 hospitales de la Argentina. Se incluyeron 121 pacientes y 124 episodios. Con respecto a la identificación microbiana, la sensibilidad global y la correspondiente a los microorganismos incluidos en la base de datos fue del 94% y 97% respectivamente. La sensibilidad del BCID2 para detectar CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B fue del 100% y la especificidad fue del 99% para NDM y VIM y del 100% para el resto. Esto llevó a cambios en el tratamiento antimicrobiano en 57/98 episodios (58%). El panel BCID2 es una herramienta importante para la adecuación del tratamiento antimicrobiano de pacientes con sepsis.
Abstract The BioFire® BCID2 panel (BioFire, Salt Lake City, UT) uses multiplex PCR analysis from positive blood cultures with results within one hour. The objective of this study was to determine the performance of the method from positive blood cultures of septic patients in 5 hospitals in Argentina. A total of 121 patients and 124 episodes were included. With regard to microbial identification, the global sensitivity and that corresponding to the microorganisms included in the database was 94% and 97%, respectively. The sensitivity of BCID2 to detect CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B was 100% and the specificity was 99% for NDM and VIM and 100% for the rest. This led to changes in antimicrobial treatment in 57/98 episodes (58%). The BCID2 panel is an important tool for the adequacy of antimicrobial treatment of patients with sepsis.
Resumo Estudo multicêntrico argentino sobre a utilidade do painel BCID2 do Sistema FilmArray™ na detecção de bacteremia O painel BCID2 de BioFire® B (BioFire, Salt Lake City, EUA) utiliza uma análise de PCR múltipla de hemoculturas positivas com resultados em uma hora. O objetivo deste estudo foi determinar o desempenho do método a partir de hemoculturas positivas de pacientes sépticos em 5 hospitais da Argentina. Cento e vinte e um pacientes e 124 episódios foram incluídos. No que se refere à identificação microbiana, a sensibilidade global e correspondente aos microrganismos incluídos na base de dados foi de 94% e 97%, respectivamente. A sensibilidade do BCID2 para detectar CTX-M, KPC, NDM, VIM, IMP, mecA/C, vanA/B foi de 100% e a especificidade foi de 99% para NDM e VIM e 100% para o resto. Isso levou a mudanças no tratamento antimicrobiano em 57/98 episódios (58%). O painel BCID2 é uma ferramenta importante para a adequação do tratamento antimicrobiano de pacientes com sepse.
Asunto(s)
Estudio Multicéntrico , Bacteriemia , Caribdotoxina , Descanso , Diagnóstico , Cultivo de Sangre , MétodosRESUMEN
After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma ß-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.
Asunto(s)
Actinas/metabolismo , Antígenos de Protozoos/metabolismo , Movimiento Celular/genética , Citoesqueleto/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Tubulina (Proteína)/metabolismo , Animales , Antígenos de Protozoos/genética , Transporte Biológico , Microscopía Inmunoelectrónica , Microtúbulos/metabolismo , Simulación del Acoplamiento Molecular , Proteómica , Proteínas Protozoarias/genética , Vesículas Secretoras/metabolismo , Toxoplasma/genética , Toxoplasmosis/parasitología , Toxoplasmosis/patología , Vacuolas/parasitologíaRESUMEN
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microorganisms in clinical laboratories because it is rapid, relatively simple to use, accurate, and can be used for a wide number of microorganisms. Several studies have demonstrated the utility of this technique in the identification of yeasts; however, its performance is usually improved by the extension of the database. Here we developed an in-house database of 143 strains belonging to 42 yeast species in the MALDI Biotyper platform, and we validated the extended database with 388 regional strains and 15 reference strains belonging to 55 yeast species. We also performed an intra- and interlaboratory study to assess reproducibility and analyzed the use of the cutoff values of 1.700 and 2.000 to correctly identify at species level. The creation of an in-house database that extended the manufacturer's database was successful in view of no incorrect identification was introduced. The best performance was observed by using the extended database and a cutoff value of 1.700 with a sensitivity of .94 and specificity of .96. A reproducibility study showed utility to detect deviations and could be used for external quality control. The extended database was able to differentiate closely related species and it has potential in distinguishing the molecular genotypes of Cryptococcus neoformans and Cryptococcus gattii.
Asunto(s)
Bases de Datos Factuales , Hongos/química , Hongos/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Argentina , Bases de Datos como Asunto , Proteínas Fúngicas/análisis , Hongos/clasificación , Micosis/diagnóstico , Micosis/microbiología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60µM for l-dihydroorotate, and a K(m)=29µM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91µM, 96µM, and 60µM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs.
Asunto(s)
Mitocondrias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Proteínas Protozoarias/química , Pirimidinas/biosíntesis , Toxoplasma/enzimología , Secuencia de Aminoácidos , Vías Biosintéticas , Clonación Molecular , Secuencia Conservada , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Técnicas de Inactivación de Genes , Cinética , Datos de Secuencia Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Proteolisis , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Papulosquamous diseases are a heterogeneous group of disorders whose etiology primarily is unknown. The nosology of these disorders is based on a descriptive morphology of clinical lesions characterized by scaly papules and plaques. The major entities in this group include psoriasis, parapsoriasis (including pityriasis lichenoides et varioliformis acuta), lichen planus, lichen nitidus, lichen striatus, pityriasis rosea, pityriasis rubra pilaris, seborrheic dermatitis, and the Gianotti-Crosti syndrome. Many other conditions may become papulosquamous and should be considered in the differential diagnosis.