Detection of toxigenic Clostridioides --#1;Clostridium--#3; difficile: Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates / Detección de Clostridioides --#1;Clostridium--#3; difficile toxigénico: utilidad de dos métodos de enzimoinmunoensayo comerciales y una PCR en las muestras de materia fecal y en los respectivos aislamientos

The best laboratory diagnostic approach to detect Clostridioides --#1;Clostridium--#3; difficile infection (CDI) is a subject of ongoing debate. With the aim of evaluating four laboratory diagnostic methods, 250 unformed stools from patients with suspected CDI submitted to nine medical center laboratories from November 2010 to December 2011, were studied using: (1) an immunochromatographic rapid assay test that combines the qualitative determination of glutamate dehydrogenase (GDH) plus toxins A and B (QAB), the CDIFF QUIK CHEK COMPLETE assay; (2) an enzyme immunoassay for qualitative determination of toxins A and B, the RIDASCREENTC. difficile Toxin A/B assay (RAB); (3) a PCR for the toxin B gene assay (PCR); and (4) the toxigenic culture (TC).C. difficile isolates from direct toxin negative stools by QAB, RAB and PCR were evaluated for toxigenicity by the same direct tests, in order to assess the contribution of the TC (QAB-TC, RAB-TC, PCR-TC). A combination of the cell culture cytotoxicity neutralization assay (CCCNA) in stools, and the same assay on isolates from direct negative samples (CCCNA-TC) was considered the reference method (CCCNA/CCCNA-TC). Of the 250 stools tested, 107 (42.8%) were positive by CCCNA/CCCNA-TC. The GDH and PCR/PCR-TC assays were the most sensitive, 91.59% and 87.62%, respectively. The QAB, RAB, QAB/QAB-TC and RAB/RAB-TC had the highest specificities, ca. 95%. A negative GDH result would rule out CDI, however, its low positive likelihood ratio (PLR) of 3.97 indicates that a positive result should always be complemented with the detection of toxins. If the RAB, QAB, and PCR assays do not detect toxins from direct feces, the toxigenic culture should be performed. In view of our results, the most accurate and reliable methods to be applied in a clinical microbiology laboratory were the QAB/QAB-TC, and RAB/RAB-TC, with PLRs >10 and negative likelihood ratios <0.30.

El mejor procedimiento para realizar el diagnóstico de laboratorio de la infección causada por Clostridioides --#1;Clostridium--#3; difficile (ICD) es aún objeto de debate. Con el fin de evaluar cuatro métodos diagnósticos de laboratorio, se estudiaron 250 muestras de heces diarreicas provenientes de pacientes con sospecha de ICD remitidas a los laboratorios de nueve centros médicos entre noviembre de 2010 y diciembre de 2011. Dichas muestras se analizaron mediante los siguientes métodos:1) un ensayo rápido inmunocromatográfico que combina la detección cualitativa de la glutamato deshidrogenasa (GDH) y de las toxinas Ay B (QAB), CDIFF QUIK CHEK COMPLETE;2) un enzimoinmunoanálisis para la determinación cualitativa de las toxinas A/B, RIDASCREENTC. difficile Toxin A/B (RAB);3) un método molecular basado en PCR para la detección del gen que codifica la toxina B (PCR) y 4) el cultivo toxigénico (TC). Como método de referencia se utilizó la combinación del ensayo de citotoxicidad sobre cultivo de células con la neutralización de toxina mediante anticuerpo específico en los filtrados de las heces (CCCNA) y el mismo método en sobrenadantes de aislamientos de C. difficile (CCCNA-TC). La toxigenicidad de las cepas aisladas de muestras directas negativas con QAB, RAB y PCR se evaluó con los mismos métodos, con el propósito de detectar la contribución del TC (QAB-TC, RAB-TC, PCR-TC). De las 250 muestras estudiadas, 107 (42,8%) fueron positivas por CCCNA/CCCNA-TC. Los métodos GDH y PCR/PCR-TC fueron los más sensibles: 91,59 y 87,62%, respectivamente. Los métodos QAB, RAB, QAB/QAB-TC y RAB/RAB-TC mostraron las mayores especificidades, del 95%, aproximadamente. Un resultado negativo para GDH excluiría la ICD, pero su baja razón de verosimilitud positiva (PLR), que fue 3,97, indica que un resultado positivo debe complementarse con la detección de toxinas. Cuando no se detectan toxinas directas por RAB, QAB ni PCR, debería realizarse el TC. De acuerdo con nuestros resultados, los métodos más precisos y confiables para ser aplicados en un laboratorio de microbiología clínica son QAB/QAB-TC y RAB/RAB-TC, con una PLR> 10 y una razón de verosimilitud negativa < 0,30.

Detection of toxigenic Clostridioides [Clostridium] difficile: Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates.

The best laboratory diagnostic approach to detect Clostridioides [Clostridium] difficile infection (CDI) is a subject of ongoing debate. With the aim of evaluating four laboratory diagnostic methods, 250 unformed stools from patients with suspected CDI submitted to nine medical center laboratories from November 2010 to December 2011, were studied using: (1) an immunochromatographic rapid assay test that combines the qualitative determination of glutamate dehydrogenase (GDH) plus toxins A and B (QAB), the CDIFF QUIK CHEK COMPLETE assay; (2) an enzyme immunoassay for qualitative determination of toxins A and B, the RIDASCREEN™ C. difficile Toxin A/B assay (RAB); (3) a PCR for the toxin B gene assay (PCR); and (4) the toxigenic culture (TC). C. difficile isolates from direct toxin negative stools by QAB, RAB and PCR were evaluated for toxigenicity by the same direct tests, in order to assess the contribution of the TC (QAB-TC, RAB-TC, PCR-TC). A combination of the cell culture cytotoxicity neutralization assay (CCCNA) in stools, and the same assay on isolates from direct negative samples (CCCNA-TC) was considered the reference method (CCCNA/CCCNA-TC). Of the 250 stools tested, 107 (42.8%) were positive by CCCNA/CCCNA-TC. The GDH and PCR/PCR-TC assays were the most sensitive, 91.59% and 87.62%, respectively. The QAB, RAB, QAB/QAB-TC and RAB/RAB-TC had the highest specificities, ca. 95%. A negative GDH result would rule out CDI, however, its low positive likelihood ratio (PLR) of 3.97 indicates that a positive result should always be complemented with the detection of toxins. If the RAB, QAB, and PCR assays do not detect toxins from direct feces, the toxigenic culture should be performed. In view of our results, the most accurate and reliable methods to be applied in a clinical microbiology laboratory were the QAB/QAB-TC, and RAB/RAB-TC, with PLRs >10 and negative likelihood ratios <0.30.

Evaluation of phenotypic and genotypic markers for clinical strains of Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic pathogen that is rapidly evolving toward multidrug resistance and is involved in various nosocomial infections that are often severe. It strongly prompts the epidemiological study of A. baumannii infections. However, there is no a generally accepted typing scheme. Different genotypic and phenotypic procedures were evaluated for the characterization of clinical isolates of A. baumannii isolated from a community hospital of Rosario, Argentina (Hospital de Emergencias Clemente Alvarez, HECA), during a period of four years. These included PCR with degenerate oligonucleotide primers (DO-PCR), repetitive extragenic palindromic-PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and antibiotyping. Amongst individual methods, DO-PCR and PFGE were found the most suitable methods to discriminate A. baumannii clinical isolates [discriminatory indexes (D) of 0.98 and 0.96, respectively]. On the other hand, both antibiotyping and REP-PCR were much less discriminatory (D: 0.86 and 0.77, respectively). The combination of antibiotyping with any of the above genotypic procedures was found to largely increase D. In particular, the combination of DO-PCR and antibiotyping provided the best discriminatory method for epidemiological studies of A. baumannii. Combination of the different genotypic and phenotypic procedures allowed the inference of genetic relationships and dissemination of multidrug-resistant A. baumannii clones in HECA in the period 1994-1999. One particular strain, which showed sensibility to carbapenems, was found widely distributed in this hospital during 1994-1996. A different strain, showing additional resistance to carbapenems, rapidly disseminated in HECA in coincidence with the introduction of imipenem therapy in 1997.

Evaluation of phenotypic and genotypic markers for clinical strains of acinetobacter baumannii

Acinetobacter baumannii es un importante patógeno oportunista. Este microorganismo adquiere con facilidad resistencia a antimicrobianos, involucrándose en infecciones nosocomiales generalmente graves. Estas características promueven el análisis epidemiológico de las infecciones provocadas por el mismo. Sin embargo, no hay aún un esquema de tipificación generalmente aceptado para este patógeno. Hemos evaluado en este trabajo diferentes procedimientos fenotípicos y genotípicos para la caracterización de aislamientos clínicos de A. baumannil aislados en un Hospital Público de Rosario (Hospital de Emergencias Clemente Alvarez, HECA), durante un período de cuatro años. Estos incluyeron PCR con oligonucleótidos degenerados (OD-PCR), PCR empleando cebadores homólogos a secuenciais palindrómicas extragénicas repetitivas (REP-PCR), electroforesis en geles de agarosa con campo pulsado (PFGE) y ensayo de susceptibilidad a antimicrobianos. OD-OCR y PFGE, entre los métodos individuales, fueron los métodos de mayor poder discriminatorio (índice discriminatorio, D, de 0.98 y 0.96; respectivamente). Por otra parte, el antibiotipo y REP-PCR presentaron menor discriminacíon (D: 0.86 y 0.77; respectivamente). La combinación de antibiotipo con cada uno de los procedimientos genotípicos mencionados originó un aumento importante en los índices discriminatorios de cada método. En particular, la combinación de OD-PCR y antibiotipo constituvó la mejor metodología para el estudio epidemiológico de A.baumannii. Así, la combinación de los procedimientos feno- y genotípicos mencionados permitó inferir las relaciones genéticas y la diseminación de clones de A.baumannii multirresistentes en el HECA en el período 1994-99. Una cepa particular, sensible a imipenem, estuvo ampliamente diseminada en el hospital durante 1994-1996. Por otra parte, un clon diferente, con resistencia adicional a carbapenemes, se diseminó rapidamente en el hospital en 1997, en coincidencia con la introducción de imipenem como terapia antibiótica.

Evaluation of phenotypic and genotypic markers for clinical strains of acinetobacter baumannii

Acinetobacter baumannii es un importante patógeno oportunista. Este microorganismo adquiere con facilidad resistencia a antimicrobianos, involucrándose en infecciones nosocomiales generalmente graves. Estas características promueven el análisis epidemiológico de las infecciones provocadas por el mismo. Sin embargo, no hay aún un esquema de tipificación generalmente aceptado para este patógeno. Hemos evaluado en este trabajo diferentes procedimientos fenotípicos y genotípicos para la caracterización de aislamientos clínicos de A. baumannil aislados en un Hospital Público de Rosario (Hospital de Emergencias Clemente Alvarez, HECA), durante un período de cuatro años. Estos incluyeron PCR con oligonucleótidos degenerados (OD-PCR), PCR empleando cebadores homólogos a secuenciais palindrómicas extragénicas repetitivas (REP-PCR), electroforesis en geles de agarosa con campo pulsado (PFGE) y ensayo de susceptibilidad a antimicrobianos. OD-OCR y PFGE, entre los métodos individuales, fueron los métodos de mayor poder discriminatorio (índice discriminatorio, D, de 0.98 y 0.96; respectivamente). Por otra parte, el antibiotipo y REP-PCR presentaron menor discriminacíon (D: 0.86 y 0.77; respectivamente). La combinación de antibiotipo con cada uno de los procedimientos genotípicos mencionados originó un aumento importante en los índices discriminatorios de cada método. En particular, la combinación de OD-PCR y antibiotipo constituvó la mejor metodología para el estudio epidemiológico de A.baumannii. Así, la combinación de los procedimientos feno- y genotípicos mencionados permitó inferir las relaciones genéticas y la diseminación de clones de A.baumannii multirresistentes en el HECA en el período 1994-99. Una cepa particular, sensible a imipenem, estuvo ampliamente diseminada en el hospital durante 1994-1996. Por otra parte, un clon diferente, con resistencia adicional a carbapenemes, se diseminó rapidamente en el hospital en 1997, en coincidencia con la introducción de imipenem como terapia antibiótica. (AU)

Evaluation of phenotypic and genotypic markers for clinical strains of Acinetobacter baumannii.

Acinetobacter baumannii is an important opportunistic pathogen that is rapidly evolving toward multidrug resistance and is involved in various nosocomial infections that are often severe. It strongly prompts the epidemiological study of A. baumannii infections. However, there is no a generally accepted typing scheme. Different genotypic and phenotypic procedures were evaluated for the characterization of clinical isolates of A. baumannii isolated from a community hospital of Rosario, Argentina (Hospital de Emergencias Clemente Alvarez, HECA), during a period of four years. These included PCR with degenerate oligonucleotide primers (DO-PCR), repetitive extragenic palindromic-PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and antibiotyping. Amongst individual methods, DO-PCR and PFGE were found the most suitable methods to discriminate A. baumannii clinical isolates [discriminatory indexes (D) of 0.98 and 0.96, respectively]. On the other hand, both antibiotyping and REP-PCR were much less discriminatory (D: 0.86 and 0.77, respectively). The combination of antibiotyping with any of the above genotypic procedures was found to largely increase D. In particular, the combination of DO-PCR and antibiotyping provided the best discriminatory method for epidemiological studies of A. baumannii. Combination of the different genotypic and phenotypic procedures allowed the inference of genetic relationships and dissemination of multidrug-resistant A. baumannii clones in HECA in the period 1994-1999. One particular strain, which showed sensibility to carbapenems, was found widely distributed in this hospital during 1994-1996. A different strain, showing additional resistance to carbapenems, rapidly disseminated in HECA in coincidence with the introduction of imipenem therapy in 1997.

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides

Epidemioiogicai studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemioiogically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemioiogically-unrelated isolates (ciassified into 5 different serotypes) resulted in ll distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.(AU)

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides

Epidemioiogicai studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemioiogically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemioiogically-unrelated isolates (ciassified into 5 different serotypes) resulted in ll distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.