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.

First national survey of antibiotic susceptibility of the Bacteroides fragilis group: emerging resistance to carbapenems in Argentina.

The antibiotic susceptibility rates of 363 clinical Bacteroides fragilis group isolates collected from 17 centers in Argentina during the period from 2006 to 2009 were as follows: piperacillin-tazobactam, 99%; ampicillin-sulbactam, 92%; cefoxitin, 72%; tigecycline, 100%; moxifloxacin, 91%; and clindamycin, 52%. No metronidazole resistance was detected in these isolates during this time period. Resistance to imipenem, doripenem, and ertapenem was observed in 1.1%, 1.6%, and 2.3% of B. fragilis group strains, respectively. B. fragilis species showed a resistance profile of 1.5% to imipenem, 1.9% to doripenem, and 2.4% to ertapenem. This is the first report of carbapenem resistance in Argentina. The cfiA gene was present in 8 out of 23 isolates, all of them belonging to the B. fragilis species and displaying reduced susceptibility or resistance to carbapenems (MICs ≥ 4 µg/ml). Three out of eight cfiA-positive isolates were fully resistant to carbapenems, while 5 out of 8 isolates showed low-level resistance (MICs, 4 to 8 µg/ml). The inhibition by EDTA was a good predictor of the presence of metallo-ß-lactamases in the fully resistant B. fragilis strains, but discrepant results were observed for low-level resistant isolates. B. fragilis was more susceptible to antimicrobial agents than other Bacteroides species. Bacteroides vulgatus species was the most resistant to ampicillin-sulbactam and piperacillin-tazobactam, and B. thetaiotaomicron/ovatus strains showed the highest level of resistance to carbapenems, with an unknown resistance mechanism. B. vulgatus and the uncommon non-Bacteroides fragilis species were the most resistant to moxifloxacin, showing an overall resistance rate of 15.1%.

[First Argentine consensus guidelines for in vitro antimicrobial susceptibility testing of clinically relevant anaerobic bacteria in humans/ Anaerobic Subcommittee of the Asociación Argentina de Microbiología]. / Primer consenso argentino para el estudio de la sensibilidad in vitro a los antimicrobianos de las bacterias anaerobias de importancia clínica en humanos.

Through time, anaerobic bacteria have shown good susceptibility to clinically useful antianaerobic agents. Nevertheless, the antimicrobial resistance profile of most of the anaerobic species related to severe infections in humans has been modified in the last years and different kinds of resistance to the most active agents have emerged, making their effectiveness less predictable. With the aim of finding an answer and for the purpose of facilitating the detection of anaerobic antimicrobial resistance, the Anaerobic Subcommittee of the Asociación Argentina de Microbiología developed the First Argentine consensus guidelines for in vitro antimicrobial susceptibility testing of clinically relevant anaerobic bacteria in humans. This document resulted from the compatibilization of the Clinical and Laboratory Standards Institute recommendations, the international literature and the work and experience of the Subcommittee. The Consensus document provides a brief taxonomy review, and exposes why and when anaerobic antimicrobial susceptibility tests should be conducted, and which antimicrobial agents can be used according to the species involved. The recommendations on how to perform, read and interpret in vitro anaerobic antimicrobial susceptibility tests with each method are exposed. Finally, the antibiotic susceptibility profile, the classification of antibiotics according to their in vitro activities, the natural and acquired mechanisms of resistance, the emerging resistance and the regional antibiotic resistance profile of clinically relevant anaerobic species are shown.

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.