Re: Enrichment broth and detection of extended-spectrum-beta-lactamase-producing bacteria in throat and rectal surveillance cultures of intensive care unit patients.

We read with interest the article by Murk et al. (4). The authors conclude that a simple overnight preenrichment step improves the detection of extended-spectrum-beta-lactamase (ESBL)-positive strains and permits earlier recognition and isolation of patients who carry these strains. We would like to address some concerns we have about the validity of these data. First, it is of major importance to use controlled and standardized conditions in the evaluation of culture-based screening protocols. During a 2-month period, 500 surveillance specimens were collected from 88 mechanically ventilated intensive care unit (ICU) patients. The patients’ swabs were first streaked on a beta-lactamase screening agar (BLSE) (AES Chemunex, Bruz cedex, France) and then inserted into 5 ml of antibiotic-free Trypticase soy broth (TSB) for overnight incubation at 37°C. The following day, 100 l of the enriched samples was subcultured onto BLSE. Although the inoculum size is a very important parameter in this type of evaluation, the authors did not use the same inoculum for both methods. Therefore, a potentially larger inoculum in the TSB could have influenced the sensitivities of the methods evaluated, e.g., an increased sensitivity for the enrichment protocol. Second, the authors did not use a defined, genotypic gold standard for the detection of ESBL. For the confirmation of ESBL-producing isolates, the double-disc synergy test and Etest were used. One patient harbored an ESBL-positive strain of Achromobacter xylosoxidans, but to our knowledge, there are no guidelines for phenotypic ESBL detection for this species. Resistance mechanisms other than ESBL, such as hyperproduction of SHV-1, may give false-positive confirmation tests. Therefore, confirmation of phenotypic results by genotypic characterization of resistance mechanisms should have been confirmed by PCR assays targeting bla genes with amplicon sequencing. Third, the authors showed in their analyses of the 500 surveillance specimens collected from 88 ICU patients that with enrichment, the number of cultures that yielded ESBL-positive bacteria (n 20) was twice that of cultures without enrichment (n 10), which is a statistically significant difference (McNemar’s test; P 0.006). However, when paired binary responses (cultured with enrichment versus cultured without enrichment) are compared using McNemar’s test, it is assumed that the paired responses are independent of each other (3). Subsequently, the test accounts for the correlation within the paired responses in the variance of the test statistic. The 500 paired responses of the surveillance specimens do not seem to fulfill this independence assumption, as individual patients have contributed more than one paired response (patient 1 contributed 4 cultured swabs; patient 2, 14 swabs; patient 3, 19 swabs; patient 4, 4 swabs; patient 5, 2 swabs; patient 6, 23 swabs; patient 7, 6 swabs; patient 8, 9 swabs; patient 9, 8 swabs). When a patient contributes more than one paired response to the study sample, the variability of the observations for the patient is less than the variability among different patients. Analyzing these 500 paired responses as independent, without accounting for the clustering of paired responses (where the cluster is the patient), will result in an underestimated standard error and P value (1, 2). It would be interesting to find out if the highly significant difference in ESBL-positive cultures with and without the enrichment step would remain after accounting for the apparent clustering that is present in this sample of paired responses. There are several statistical methods that could be used in the analysis of clustered matched-pair data (3). Determining the optimal ESBL screening protocol is clearly of the utmost importance. Studies like those conducted by Murk et al. will hopefully set the basis for carrying out prospective research specially designed to avoid recognizable biases.