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In JoVE (1)
Other Publications (11)
- American Journal of Clinical Pathology
- Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America
- Biodegradation
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
- Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
- Journal of Clinical Microbiology
Articles by Elizabeth Marlowe in JoVE
JoVE Immunology and Infection
A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures
1Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Cente, 2Pasadena, CA, Southern California Permanente Medical Group, 3Detroit, Detroit Medical Center, 4Woburn, MA, AdvanDx
A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).
Other articles by Elizabeth Marlowe on PubMed
Practical Therapeutic Application of the Oxoid PBP2' Latex Agglutination Test for the Rapid Identification of Methicillin-resistant Staphylococcus Aureus in Blood Cultures
The Oxoid PBP2' latex agglutination test (OLA; Oxoid, Basingstoke, England) was evaluated in a controlled prospective study examining Staphylococcus aureus from 25 positive blood cultures. Subcultures of positive blood cultures with coagulase-positive, gram-positive cocci in clusters were batched, and the OLA was performed at the end of the working day, once growth was seen on the plate. Results were sent to the infectious disease pharmacist for therapy evaluation, and the 24-hour minimum inhibitory concentration (MIC) was confirmed the next day. Blood culture OLA results correlated 100% with oxacillin MIC results for the patient, and results were available in as little as 3 hours after the blood culture was positive. The mean time difference between the OLA and MIC reports was 19.4 hours. This test allowed same-day resistance marker reporting and was easily incorporated into the work flow of the clinical laboratory.
Gastrointestinal Microflora Studies in Late-onset Autism
Some cases of late-onset (regressive) autism may involve abnormal flora because oral vancomycin, which is poorly absorbed, may lead to significant improvement in these children. Fecal flora of children with regressive autism was compared with that of control children, and clostridial counts were higher. The number of clostridial species found in the stools of children with autism was greater than in the stools of control children. Children with autism had 9 species of Clostridium not found in controls, whereas controls yielded only 3 species not found in children with autism. In all, there were 25 different clostridial species found. In gastric and duodenal specimens, the most striking finding was total absence of non-spore-forming anaerobes and microaerophilic bacteria from control children and significant numbers of such bacteria from children with autism. These studies demonstrate significant alterations in the upper and lower intestinal flora of children with late-onset autism and may provide insights into the nature of this disorder.
Application of a Reverse Transcription-PCR Assay to Monitor Regulation of the Catabolic NahAc Gene During Phenanthrene Degradation
Biodegradation of polycyclic aromatic hydrocarbons (PAH), such as phenanthrene, in environmental samples is often limited by low bioavailability which results from a combination of low aqueous solubility and/or high sorption. The purpose of this study was to investigate the influence of agents that increase PAH bioavailability on expression of the PAH catabolic gene nahAc. Phenanthrene was used as a model PAH and Pseudomonasputida PpG7, which contains the NAH7 plasmid that encodes the genes responsible for naphthalene and phenanthrene degradation, was used as a model degrader. PAH bioavailability was altered by the addition of two biosurfactants, rhamnolipid and hydroxypropyl-beta-cyclodextrin (HPCD). Gene expression was determined by extraction of bacterial mRNA followed by RT-PCR amplification of two transcripts; nahAc, a naphthalene dioxygenase gene, and rpoD, a housekeeping gene. Results indicate that the lag period preceding nahAc gene induction decreased from 312 to 48 h in the presence of biosurfactants. Expression of the nahAc gene, as measured by RT-PCR, in the presence of surfactants was bimodal on a temporal basis, indicating that induction stopped briefly during biodegradation. Cessation of induction could have resulted from the up-regulation of alternate pathways or the accumulation of toxic intermediates. In contrast, expression of the rpoD gene was maintained throughout the duration of each experiment. This research demonstrates that the use of a gene expression assay to monitor the impact of substrate bioavailability on substrate utilization provides unique information concerning the biodegradation process that cannot be obtained from more traditional biodegradation assays such as cell growth or substrate disappearance. Gene expression assays also have the potential for use in assessing the impact of other environmental factors on biodegradation.
Conventional and Molecular Methods for Verification of Results Obtained with BacT/Alert Nonvent Blood Culture Bottles
A strategy comparing molecular and conventional methods for verification of the BacT/Alert nonvent blood culture bottles (Organon Teknika, Durham, N.C.) was performed with seeded isolates. The bottles were evaluated with 12 common organisms from bloodstream infections. Overall, the bottles were equivalent as determined by conventional and molecular methods.
Application of an RRNA Probe Matrix for Rapid Identification of Bacteria and Fungi from Routine Blood Cultures
One of the most important functions of the clinical microbiology laboratory is the identification of the etiology of sepsis. For this study, aliquots from 405 positive blood cultures were tested against a unique array of DNA probes directed against rRNA subsequences of bacteria and fungi for identification. Another 280 samples that were negative after 5 days of incubation were also tested. Blood culture bottles were incubated in a BacT/Alert3D instrument. For the rRNA assay, a 0.4-ml aliquot was removed, and the cells were pelleted by centrifugation. The pellet was washed and frozen at -70 degrees C. Analysis of the pellet involved a lysis step and then the addition of samples to the reaction wells containing the probes in a microtiter plate format. Analysis was performed by using a hybridization protection assay. Results were taken through a series of deductive steps to obtain species, or in some cases genus, identification. Batch sample preparation required approximately 15 min, and sample analysis required another 60 min. Probe results were compared to conventional biochemical identifications. The probe test was negative for the 280 samples that were negative by the BacT/Alert 3D system and for another 21 samples that were false positive (the instrument signaled, but there was no growth). Microorganisms from the remaining 384 signal-positive samples included 60 Enterobacteriaceae, 10 Pseudomonas aeruginosa, 10 other gram-negative bacteria, 40 Staphylococcus aureus, 152 coagulase-negative staphylococci, 28 streptococci, 22 enterococci, 21 other gram-positive bacteria, 8 anaerobes, and 16 yeast organisms. Seventeen cultures were polymicrobial, and one was gram positive and culture negative. Discordance between probe and conventional identification results was noted for only 12 (1.75%) samples. This novel rapid molecular approach to the identification of bacteria and yeast in blood cultures was highly sensitive (100%) and specific (96%).
Sensitivity and Specificity of a Rapid RRNA Gene Probe Assay for Simultaneous Identification of Staphylococcus Aureus and Detection of MecA
rRNA gene sequences were used for identification and target adequacy controls in a DNA probe assay to identify isolates as Staphylococcus and, more specifically, as S. aureus within 1 hour. mecA status was simultaneously determined using a specific DNA probe. The target adequacy control guarded against false-negative mecA results.
Performance of the GeneXpert Enterovirus Assay for Detection of Enteroviral RNA in Cerebrospinal Fluid
The GeneXpert Dx System allows for automated extraction, processing, amplification and real-time detection of target nucleic acids.
Clostridium Difficile Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms
The incidence of Clostridium difficile infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert C. difficile assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (tcdB). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert C. difficile assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert C. difficile assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert C. difficile assay was statistically superior to the EIA (P, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (P, 0.0363). Combining the GDH and Xpert C. difficile assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert C. difficile assay was completed, on average, in less than 1 h. Xpert C. difficile testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual- and multiple-test algorithms evaluated in this study.
Multicenter Evaluation of a New Shortened Peptide Nucleic Acid Fluorescence in Situ Hybridization Procedure for Species Identification of Select Gram-negative Bacilli from Blood Cultures
A shortened protocol for two peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assays for the detection of Gram-negative bacilli from positive blood cultures was evaluated in a multicenter trial. There was 100% concordance between the two protocols for each assay (368 of 368 and 370 of 370 results) and 99.7% (367 of 368 and 369 of 370 results) agreement with routine laboratory techniques.
Impact of Strain Type on Detection of Toxigenic Clostridium Difficile: Comparison of Molecular Diagnostic and Enzyme Immunoassay Approaches
A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites' standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two- and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P = 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P = 0.004, respectively, Fisher's exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.
Evaluation of the Cepheid Xpert MTB/RIF Assay for Direct Detection of Mycobacterium Tuberculosis Complex in Respiratory Specimens
A total of 217 specimens submitted for routine smear and culture from three different sites within the western United States were used to evaluate the GeneXpert MTB/RIF assay (for research use only) (Cepheid, Sunnyvale, CA). Overall agreement compared to culture was 89% (98% for smear positives and 72% for smear negatives) for detection of Mycobacterium tuberculosis.
