Citations

An evaluation was undertaken to determine the optimal method for testing the susceptibilities of 100 clinical isolates and two reference strains of Enterococcus spp. to vancomycin in vitro. Six testing methods were studied by using the following media and incubation times: agar screen with the Synergy Quad Plate (Remel, Lenexa, Kans.), an in-house-prepared brain heart infusion (BHI) agar plate, and an in-house-prepared Mueller-Hinton (MH) agar plate, all incubated for 24 or 48 h; broth microdilution (Sensititre Just One Strip; AccuMed International, Inc., West Lake, Ohio) with BHI or cation-adjusted MH broth incubated for 24 or 48 h; agar dilution with BHI or MH agar incubated for 24 or 48 h; epsilometer test (E test; AB BioDisk, Solna, Sweden) with BHI or MH agar incubated for 24 or 48 h; disk diffusion with BHI or MH agar incubated for 24 or 48 h; and the automated Vitek method with the gram-positive susceptibility Staphylococcus aureus card and R02.03 software (bioMerieux, Inc., Hazelwood, Mo.). Growth failures occurred with MH media (n = 6) but not with BHI media. One growth failure occurred with the Vitek method. Results for each testing method for each Enterococcus strain were interpreted as susceptible, intermediate, or resistant according to current National Committee for Clinical Laboratory Standards (NCCLS) criteria and compared to the vancomycin resistance genotype (i.e., vanA, vanB, vanC-1, or vanC-2/3). For all methods, extension of the incubation time from 24 h to 48 h either produced no difference in the results or gave poorer results. The following methods produced no very major or major interpretive errors: broth microdilution with BHI media incubated for 24 h, agar dilution with BHI media incubated for 24 or 48 h, and E test with BHI media incubated for 24 or 48 h. Unacceptable frequencies of very major errors (> 1%) occurred with all methods for which MH media were used. Minor interpretive errors were frequent with all methods. These minor interpretive errors also occurred most frequently with Enterococcus strains with vanC genes, which encoded low-level vancomycin resistance (MIC < or = 8 microg/ml), as opposed to Enterococcus strains which possessed vanA or vanB genes, which encoded higher-level vancomycin resistance (MIC > or = 64 microg/ml). Modification of NCCLS breakpoints, especially for motile Enterococcus spp. (E. casseliflavus, E. flavescens, and E. gallinarum), may resolve this problem; however, in the current study, one E. faecalis strain and one E. faecium strain carried only the vanC gene. The agar screen method may also require reformulation. The current agar screen plate contains 6 microg of vancomycin per ml, which may not detect all low-level resistance associated with vanC genotypes. Nevertheless, the clinical significance of this low-level vancomycin resistance remains unknown.

Reliable nucleic acid extraction techniques for blood specimens are required for the sensitive detection of viral DNA. Standardized procedures for processing blood specimens for the molecular detection of herpesviruses (cytomegalovirus [CMV], herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus [EBV]) have not been established. Three methods were used to extract DNA from blood specimens from healthy donors and asymptomatic immunocompromised patients: (i) IsoQuick treatment of whole blood, (ii) extraction of the peripheral blood leukocytes by lysis (lysis buffer and proteinase K), and (iii) extraction of peripheral blood leukocytes with phenol-chloroform (sodium docecyl sulfate solution and proteinase K). All blood specimens from 25 healthy blood donors were negative for CMV, herpes simplex virus and varicella-zoster virus nucleic acid sequences, regardless of the extraction method, while three samples (12%) extracted by the lysis technique were positive for EBV DNA. Of 25 blood samples from asymptomatic immunocompromised patients, CMV and EBV each were detected in nine specimens by lysis extraction, four each by IsoQuick and four (CMV) and six (EBV) by the phenol-chloroform method. Our results indicate that the lysis method is optimal for the detection of CMV and EBV DNA sequences by PCR from the leukocytic fraction of blood specimens. DNA of these viruses is frequently present in blood specimens from asymptomatic immunocompromised patients and occasionally from healthy donors.

Cytomegalovirus (CMV) viremia is a widely used laboratory marker of CMV disease following transplantation and is additionally used to trigger preemptive antiviral therapy. Despite this, the optimal method for diagnosing CMV viremia in transplantation recipients remains unknown. To determine the sampling frequency and blood volume required for the optimal diagnosis of viremia by shell vial assay, a prospective study of 46 viremic transplantation recipients was conducted. Blood specimens (2.5 and 5 ml) were collected twice, 3 h apart, at a median of 1.4 days (range, 1 to 3 days) after the triggering shell vial-positive blood had been collected. Considering a single 2.5-ml specimen, an average of only 40% of previously viremic patients had documented CMV in their blood: this increased to 50% when a second 2.5-ml sample of blood was collected 3 h later. The yields of two 2.5-ml versus two 5-ml samples were 50 versus 61%, respectively. Viremia as detected by shell vial assay is intermittent, and increasing the frequency and volume of blood sampling increases its diagnosis. These results have implications in diagnosis of CMV infection and its preemptive therapy.

Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.

We prospectively studied the utility of the amplification of cytomegalovirus (CMV) DNA in the sera of liver transplant recipients in order to predict symptomatic CMV infection, thus enabling preemptive therapy with antiviral agents. Serum samples obtained at biweekly intervals from 20 sequential liver transplant recipients for at least 8 weeks following transplantation were tested by the PCR amplification procedure. Results were correlated with blood and urine cultures, histopathological findings from infected organs, and clinical manifestations. Six patients (30%) developed symptomatic CMV infection; in five (83%) of these patients, CMV DNA was detected prior to symptomatic CMV infection, and in one (17%) of these patients, CMV DNA was detected at the time of symptomatic CMV infection. CMV DNA was detected a mean of 13 days (range, 0 to 23 days) prior to the onset of symptomatic CMV infection. In addition, CMV DNA was detected in the sera of four of five patients with asymptomatic viremia and two patients with asymptomatic viruria. Lastly, the PCR was negative for sera from seven patients with no evidence of CMV infection. We found that PCR was able to detect the presence of CMV DNA in the sera of liver transplant recipients at a sensitivity of 92% and a specificity of 100% for CMV infection, while the sensitivity and specificity for symptomatic infection were 100 and 57%, respectively.

ABSTRACT Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of Staphylococcus lugdunensis among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as S. lugdunensis .

ABSTRACT The 16S rRNA sequences of enterococcal species E. faecium , E. faecalis , E. gallinarum , E. casseliflavus/flavescens , E. dispar , E. pseudoavium , E. sulfureus , E. malodoratus , E. raffinosus , E. cecorum , E. hirae , E. saccharolyticus , E. seriolicida , E. mundtii , E. avium , E. durans , E. columbae , and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium . The implications of this finding are discussed.

Metagenomic shotgun sequencing for the identification of pathogens is being increasingly utilized as a diagnostic method. Interpretation of large and complicated data sets is a significant challenge, for which multiple commercial tools have been developed. Three commercial metagenomic shotgun sequencing tools, CosmosID, One Codex, and IDbyDNA, were compared to determine whether they result in similar interpretations of the same sequencing data.

Objectives/HypothesisCreate a competency‐based assessment tool for pediatric tracheotomy.Study DesignBlinded, modified, Delphi consensus process.MethodsUsing the REDCap database, a list of 31 potential items was circulated to 65 expert surgeons who perform pediatric tracheotomy. In the first round, items were rated as “keep” or “remove,” and comments were incorporated. In the second round, experts were asked to rate the importance of each item on a seven‐point Likert scale. Consensus criteria were determined a priori with a goal of 7 to 25 final items.ResultsThe first round achieved a response rate of 39/65 (60.0%), and returned questionnaires were 99.5% complete. All items were rated as “keep,” and 137 comments were incorporated. In the second round, 30 task‐specific and seven previously validated global rating items were distributed, and the response rate was 44/65 (67.7%), with returned questionnaires being 99.3% complete. Of the Task‐Specific Items, 13 reached consensus, 10 were near consensus, and 7 did not achieve consensus. For the 7 previously validated global rating items, 5 reached consensus and two were near consensus.ConclusionsIt is feasible to reach consensus on the important steps involved in pediatric tracheotomy using a modified Delphi consensus process. These items can now be considered to create a competency‐based assessment tool for pediatric tracheotomy. Such a tool will hopefully allow trainees to focus on the important aspects of this procedure and help teaching programs standardize how they evaluate trainees during this procedure.Level of Evidence5 Laryngoscope, 130:2700–2707, 2020

AbstractIntroductionConventional Z‐scores are generated by subtracting the mean and dividing by the standard deviation. More recent methods linearly correct for age, sex, and education, so that these “adjusted” Z‐scores better represent whether an individual's cognitive performance is abnormal. Extreme negative Z‐scores for individuals relative to this normative distribution are considered indicative of cognitive deficiency.MethodsIn this article, we consider nonlinear shape constrained additive models accounting for age, sex, and education (correcting for nonlinearity). Additional shape constrained additive models account for varying standard deviation of the cognitive scores with age (correcting for heterogeneity of variance).ResultsCorrected Z‐scores based on nonlinear shape constrained additive models provide improved adjustment for age, sex, and education, as indicated by higher adjusted‐R2.DiscussionNonlinearly corrected Z‐scores with respect to age, sex, and education with age‐varying residual standard deviation allow for improved detection of non‐normative extreme cognitive scores.