A rapid and high-throughput screening approach for methicillin-resistant Staphylococcus aureus based on the combination of two different real-time PCR assays.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that has been responsible for major nosocomial epidemics worldwide. For infection control programs, rapid and adequate detection of MRSA is of great importance. We developed a rapid and high-throughput molecular screening approach that consists of an overnight selective broth enrichment, followed by mecA, mecC, and S. aureus-specific (SA442 gene) real-time PCR assays, with subsequent confirmation using a staphylococcal cassette chromosome mec element (SCCmec)-orfX-based real-time PCR assay (GeneOhm MRSA assay) and culture. Here, the results of the screening approach over a 2-year period are presented. During this period, a total of 13,387 samples were analyzed for the presence of MRSA, 2.6% of which were reported as MRSA positive. No MRSA isolates carrying the mecC gene were detected during this study. Based on the results of the real-time PCR assays only, 95.2% of the samples could be reported as negative within 24 h. Furthermore, the performance of these real-time PCR assays was evaluated using a set of 104 assorted MRSA isolates, which demonstrated high sensitivity for both the combination of mecA and mecC with SA442 and the BD GeneOhm MRSA assay (98.1% and 97.1%, respectively). This molecular screening approach proved to be an accurate method for obtaining reliable negative results within 24 h after arrival at the laboratory and contributes to improvement of infection control programs, especially in areas with a low MRSA prevalence.

Rapid enterovirus molecular testing in cerebrospinal fluid reduces length of hospitalization and duration of antibiotic therapy in children with aseptic meningitis.

We studied the potential benefits of introducing a rapid enterovirus molecular test in children with enterovirus meningitis. The 2 groups of pediatric patients were comparable with respect to clinical and laboratory data, but differed in availability of enterovirus test results. In the control group, the results were available within 3 to 7 days, whereas in the study group, rapid enterovirus molecular test results were available within 3 to 24 hours. The median duration of hospitalization and the duration of antibiotics were significantly reduced to, respectively, 2 days and 1 day in the study group when compared with the control group (P < 0.001). Mean costs per patient calculation showed an average reduction of more than US $1450 (P < 0.001).

Evaluation of 5'-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools.

5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with reference strains of E. coli (n=40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx(1c), stx(1d), and stx(2f)), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log(10) and 3.42 to 4.63 log(10) CFU/g of stool for 5'-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation of

Comparison of COBAS AMPLICOR Neisseria gonorrhoeae PCR, including confirmation with N. gonorrhoeae-specific 16S rRNA PCR, with traditional culture.

A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased the positive predictive value from 54.8 to 96.6%.

Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori.

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

Does the declining prevalence of Helicobacter pylori unmask patients with idiopathic peptic ulcer disease? Trends over an 8 year period.

OBJECTIVES: Recent studies have suggested that the prevalence of Helicobacter pylori infection in patients with ulcer disease who were not using non-steroidal anti-inflammatory drugs (NSAIDs) has been overestimated. The decreasing prevalence of H. pylori could lead to a relative increase in the number of patients with this idiopathic peptic ulcer disease (IPUD). This study aimed to investigate the prevalence of IPUD and any possible trends. DESIGN AND METHODS: The reports of all upper gastro-intestinal endoscopies performed in a Dutch regional hospital over the period 1991 to 1998 were reviewed. If a gastric and/or duodenal ulcer had been diagnosed, data concerning possible H. pylori infection (culture, histology, rapid in-house urease test) were retrieved. If H. pylori tests were negative, hospital files were examined for possible use of NSAIDs or other rare causes of ulcer disease. When these were not found, stored biopsy specimens were tested for H. heilmanii by using the polymerase chain reaction technique. RESULTS: Ulcer disease was diagnosed in 405 patients who had undergone endoscopy (159 with gastric ulcer, 235 with duodenal ulcer, and 11 with both gastric and duodenal ulcer). H. pylori infection was found in 349 of these patients (86.2%). Thirty-three of the 56 H. pylori negative patients used NSAIDs and three patients had Crohn's disease, leaving 20 patients with IPUD (4.9%, 12 gastric ulcer and eight duodenal ulcer). Time trends over the study period showed a decrease of H. pylori associated peptic ulcer disease (P <0.002) and an increase of NSAID associated peptic ulcer disease (P <0.0005). The prevalence of IPUD remained stable (P=0.978). CONCLUSIONS: The prevalence of patients with H. pylori negative ulcer disease significantly decreased in our study population due to an increase in the number of patients with NSAID associated peptic ulcer disease. IPUD was rare and its prevalence did not increase over a period of 8 years.

Prospective study of use of PCR amplification and sequencing of 16S ribosomal DNA from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting.

We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 x 10(2) to 2 x 10(2) CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and gram-positive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.

Approach to treatment of dyspepsia in primary care: a randomized trial comparing "test-and-treat" with prompt endoscopy.

BACKGROUND: The value of the "test-and-treat" strategy in the approach to dyspepsia has been evaluated only in a few secondary care studies. Most patients with dyspepsia, however, are treated by their primary care physician. This study evaluated the test-and-treat strategy in primary care. METHODS: Patients consulting their general practitioners for dyspepsia were randomized to either direct open-access endoscopy with Helicobacter pylori testing or a test-and-treat strategy by H pylori serology. In the 12-month follow-up period, any additional treatment or referral for investigations was left at the discretion of the general practitioner. At the end of the study, data were collected concerning the number of endoscopies, changes in symptom severity and quality of life, patient satisfaction, and the use of medical resources. RESULTS: Two hundred seventy patients were enrolled (129 who received endoscopy and 141 in the test-and-treat group). The prevalence of H pylori infection was 38.3% and 37.2% in the test-and-treat and endoscopy groups, respectively. In the test-and-treat group, 46 patients (33%) were referred for endoscopy during follow-up. Improvement in symptom severity, quality of life, and patient satisfaction was comparable in both groups. Patients in the test-and-treat group paid more dyspepsia-related visits to their general practitioner (P =.005). Patients in the endoscopy group were more often prescribed proton pump inhibitors (P =.007), whereas patients in the test-and-treat group were more often prescribed prokinetic drugs (P =.005). CONCLUSIONS: The test-and-treat strategy proved to be as effective and safe as prompt endoscopy. Only a minority of patients were referred for endoscopy after the test-and-treat approach.

Molecular patchwork: Chromosomal recombination between two Helicobacter pylori strains during natural colonization.

Genetic analysis of two Helicobacter pylori strains isolated from a single gastric biopsy showed evidence of extensive horizontal gene transfer. Several large recombinations were identified in the rdxA gene, which is involved in metronidazole resistance.