Clinical evaluation of repetitive sequence-based polymerase chain reaction using the Diversi-Lab System for strain typing of vancomycin-resistant enterococci.

The reliability of the Diversi-Lab System, an automated method of microbial strain typing using repetitive sequence-based polymerase chain reaction (rep-PCR), was evaluated by comparing results with those obtained by pulsed-field gel electrophoresis (PFGE). Ninety-five clinical isolates of vancomycin-resistant enterococci (VRE; 13 groups, 2-17 isolates per group) sent to Associated Regional and University Pathologists (ARUP) Laboratories for typing were tested by both methods. Rep-PCR and PFGE results were concordant for 83 isolates: all 32 isolates in 6 of the groups and 51 of the 63 isolates in the other 7 groups. Clustering of the remaining 12 isolates differed. With the Diversi-Lab System, analysis is objective, and results are available in 4 h, compared with a more subjective analysis and a 2- to 3-day turnaround time for PFGE. The Diversi-Lab System may be a viable alternative to PFGE for typing VRE in clinical reference laboratories.

Temporal trends of invasive disease due to Streptococcus pneumoniae among children in the intermountain west: emergence of nonvaccine serogroups.

BACKGROUND: Use of the heptavalent pneumococcal conjugate vaccine (PCV-7 [Prevnar]) has been associated with decreased a incidence of invasive pneumococcal disease (IPD) among children in the United States. METHODS: Cases of IPD in children < 18 years of age insured by or receiving health care from Intermountain Health Care during 1996-2003 were identified. Isolates of S. pneumoniae from children with IPD treated at Primary Children's Medical Center (PCMC; Salt Lake City, UT) during 1997-2003 were serogrouped. Temporal trends of IPD, serogroup distribution of pneumococci, and antibiotic resistance among pneumococci were analyzed. RESULTS: A total of 1535 cases of IPD were identified. The rate of IPD decreased 27% after the introduction of PCV7. Among children with IPD who were cared for at PCMC, disease in 73% was caused by PCV7 serogroups in 1997-2000, compared with 50% in 2001-2003 (P < .001), and the percentage of isolates resistant to penicillin decreased from 34% in 1997-2000 to 22% in 2001-2003 (P = .04). The percentage of IPD cases that were empyema increased from 16% to 30% (P = .015), and the percentage of severe cases of IPD increased from 57% to 71% (P = .026). Children with IPD due to non-PCV7 serogroups were older, were more likely to have parapneumonic empyema, and had longer hospital stays. CONCLUSIONS: The incidence of IPD in the IMW decreased by 27% after the introduction of the PCV7 vaccine. During the postvaccine period (2001-2003), there were significant decreases in the proportion of cases of IPD caused by PCV7 and antibiotic-resistant serogroups. These benefits were accompanied by a significant increase in the proportion of IPD cases due to non-PCV7 serogroups, with increases in the incidence of empyema and severe IPD.

Clinical evaluation of the DiversiLab microbial typing system using repetitive-sequence-based PCR for characterization of Staphylococcus aureus strains.

The DiversiLab System, which includes microfluidics-based detection, reagent kits, and software for data processing and analysis, is an automated method using repetitive sequence-based PCR (rep-PCR) for microbial strain typing. To assess the reliability of the DiversiLab System for strain characterization of Staphylococcus aureus, we tested clinical isolates sent to ARUP Laboratories for typing and compared results to those of pulsed field electrophoresis (PFGE) aided by the cluster analysis provided by BioNumerics software. spa typing was performed when the results of these two methods for an outbreak were not concordant. The study included 89 S. aureus isolates (65 mecA positive, 24 mecA negative) from 19 outbreaks (2 to 11 isolates/outbreak). The DiversiLab and PFGE-BioNumerics results were concordant for 15 of the 19 outbreaks. For the remaining four outbreaks, there was partial concordance between the two methods. spa typing results were the same as or more similar to rep-PCR results for three of those outbreaks and were more similar to PFGE results for one. With regard to performance, the DiversiLab system was considerably less labor intensive than PFGE and provided results in less than 24 h, compared with 2 to 3 days for PFGE. Additionally, the Web-based DiversiLab software provides standardized comparisons among isolates almost instantaneously and generates user-friendly, customized reports.

Evaluation of a modified gen-probe amplified direct test for detection of Mycobacterium tuberculosis complex organisms in cerebrospinal fluid.

Laboratory evidence for tuberculous meningitis is difficult to acquire due to the low numbers of organisms present in cerebrospinal fluid (CSF) and the presence of nucleic acid amplification inhibitors. The Amplified Mycobacterium tuberculosis Direct Test (MTD) is sensitive and specific for the direct detection of M. tuberculosis complex in respiratory samples but has not been approved for CSF. We evaluated a modified version of the current MTD, optimized for use with CSF samples. Samples were prepared by spiking CSF with various numbers of M. tuberculosis complex organisms. The modified MTD performance was compared with results obtained using a purified RNA sample extracted using the Qiagen RNeasy Protect Bacteria Mini Kit. By use of CSF artificially spiked with M. tuberculosis complex, the sensitivity of the modified MTD was 100% (six of six) for CSF samples containing approximately 600 CFU/ml, 78% (seven of nine) for approximately 60 CFU/ml, 50% (three of six) for 6 CFU/ml, and 17% (one of six) for samples with <1 CFU/ml. The specificity of the modified MTD method was 100% (22 of 22). The sensitivity of the Qiagen MTD method was 100% for CSF samples containing approximately 600 CFU/ml (six of six) and approximately 60 CFU/ml (nine of nine), 50% for samples with approximately 6 CFU/ml (three of six), and 50% for samples with <1 CFU/ml (three of six). The specificity of the Qiagen MTD method was 86% (19 of 22). With the Qiagen MTD method, however, initial results were equivocal for 14 of the 27 (52%) positive samples, requiring repeat analysis, whereas with the modified MTD, only 1 of 27 (4%) was equivocal. The modified MTD for CSF samples was less time-consuming and less expensive and resulted in considerably fewer equivocal results than the Qiagen MTD method did.

Comparison of the Denka Seiken slide agglutination method to the quellung test for serogrouping of Streptococcus pneumoniae isolates.

This study compared a slide agglutination test (Denka Seiken, Tokyo, Japan) to the "gold standard" quellung reaction (Pneumotest; Statens Serum Institut, Copenhagen, Denmark) for the serogrouping of pneumococci. Two hundred clinical isolates of Streptococcus pneumoniae were used for the comparison. Each assay was performed according to the manufacturer's instructions. There was an overall agreement of 95.7% between the two methods. Only 4 of 10 isolates of serogroup 22 were detected with the slide agglutination assay. Two isolates that were untypeable by the Pneumotest method were typed as serogroups 6 and 31 by the slide agglutination method. The Pneumotest method was unable to type 22 isolates, and the slide agglutination method was unable to type 16 isolates. The slide agglutination method compares favorably with the Pneumotest method and is easier to perform and to interpret.