Clinical Diagnostic Microbiology Research Articles

Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service.

The aim of the present study was to develop a broad-range PCR based on bacterial 16S rDNA for use in the routine diagnostic clinical microbiology service. The optimization and validation of the assay for use on clinical specimens from normally sterile sites is described, and preliminary results are reported on the use of the assay in the clinical diagnosis of bacterial infection in 382 paediatric specimens over a 2-year period. These results are compared to those obtained by standard culture techniques and show increased diagnosis of bacterial infection when both culture and PCR are used together; 16S rDNA PCR provided the sole evidence of pathogenic infection in 71 cases. Key stages in the assay development and potential pitfalls of the technique are highlighted and the improvement the assay offers in the diagnosis of infection in the paediatric setting is discussed.

Stains in microbiology and molecular biology

H. J. Conn of the New York Agricultural Experiment Station was the founder and leader of the Commission on the Standardization of Biological Stains. This Commission ultimately evolved into the present Biological Stain Commission. Conn did much to determine the chemistry of stains and to promote their applications in biology. Interestingly, Conn was a microbiologist and co-authored an early textbook entitled `` Bacteriology ’’ (Williams and Wilkins, 1st ed., 1923). It was ® tting for the Biological Stain Commission, at its 79th annual meeting, to convene a scienti® c session devoted to microbiological stains and stains employed in contemporary molecular biology. The most important stain in microbiology is the Gram stain. The mechanism of this empirical stain remained unresolved until Terry Beveridge and his colleagues (University of Guelph) developed seminal techniques to show that the Gram-positive character was due to physical entrapment of the dye. During the 1980s, Beveridge et al. published the de® nitive work on the Gram stain mechanism. In the scienti® c session, Dr Beveridge reviewed the history of the Gram stain and provided a critical description of its mechanism. During the past several decades important new stains have been discovered that proved to have signi® cant use in the clinical diagnostic microbiology laboratory. Dyes are available that speci® cally stain capsules, ¯ agella, spores, nucleic acids, polyphosphates, polysaccharides, lipids and proteins. Dr Berneice Madison (Centers for Disease Control, Atlanta) described the most commonly employed stains in diagnostic microbiology. She not only provided a lucid discussion of the general properties of the stains, but gave speci® c applications and de® ned their limits and reliabilities as well. During the past three decades, molecular biology has emerged as an essential component of all aspects of biology and medicine. Molecular biology assumed its elevated position partially because techniques were developed for electrophoresis and blotting. These techniques required the use of new and sensitive stains to detect minute amounts of protein and/or nucleic acids. Dr Lisa Williams (University of Louisville) gave an elegant overview of most of the stains utilized to detect molecules on gels and blots. Her presentation not only provided a background on the mechanisms of the individual dyes and stains, but also included examples of their use. Science is incremental. The genius of Conn and others early in the 20th century is now paying large dividends for scientists in the 21st century.

Raman spectroscopic method for identification of clinically relevant microorganisms growing on solid culture medium.

Routine clinical microbiological identification of pathogenic microorganisms is largely based on nutritional and biochemical tests. In the case of severely ill patients, the unavoidable time delay associated with such identification procedures can be fatal. We present a novel identification method based on confocal Raman microspectroscopy. With this approach it is possible to obtain Raman spectra directly from microbial microcolonies on the solid culture medium, which have developed after only 6 h of culturing for the most commonly encountered organisms. Due to the limited thickness of microcolonies, some of the underlying culture medium is sampled together with the bacteria. Spectra measured at different depths in a microcolony contain different amounts of the medium signal. A mathematical routine, involving vector algebra, is described for the nonsubjective correction of spectra for variable signal contributions of the medium. To illustrate the possibilities of our approach for the identification of microorganisms, Raman spectra were collected from 6-h microcolonies of five bacterial strains on solid culture medium. The classification results show that confocal Raman microspectroscopy has great potential as a powerful new tool in clinical diagnostic microbiology.

Infectious disease physicians rate microbiology services and practices.

Recent years have seen increasing emphasis on cost containment and quality improvement in clinical laboratory activities. Modifying those activities to enhance clinical relevance is one strategy that should be satisfying to both laboratory scientists and administrators. This guest commentary describes one approach to quality improvement--the use of user surveys to identify areas for improvement. As an initial attempt to define such areas in clinical diagnostic microbiology, infectious disease specialists, targeted for their particular interest and expertise in microbiology laboratory results, were polled and their responses were analyzed. Some of these data have been presented previously (E. J. Baron, D. P. Francis, and K. M. Peddecord, abstr. C-170, p. 520, in Abstracts of the 94th General Meeting of the American Society for Microbiology, 1994; K. M. Peddecord, E. J. Baron, D. P. Francis, and A. S. Benenson, abstr. C-172, p. 520, in Abstracts of the 94th General Meeting of the American Society for Microbiology, 1994; K. M. Peddecord, E. J. Baron, D. P. Francis, and J. A. Drew, Am. J. Clin. Pathol. 105:58-64, 1996). The discussion includes our recommendations for the use of these survey responses, and their limitations, as stimuli to initiate reexamination of certain microbiology laboratory practices in the interest of developing more cost-effective and clinically relevant protocols.

Detection of Chlamydia trachomatis in clinical specimens Comparison of culture, direct antigen detection, DNA probe hybridization and PCR

Isolation of Chlamydia trachomatis by cell culture is labor intensive and depends on the presence of live organisms which limits time and conditions of transport. Rapid tests that detect either C. trachomatis antigen or nucleic acids do not need viable cells but lack sufficient sensitivity. PCR is the most promising technology as yet but is subject to contamination due to amplicon carry over and it is not practical for use in diagnostic laboratories. The first commercially available PCR assay, Amplicor, was developed by Roche Molecular Systems. The assay includes UNG sterilization to prevent false positive results due to amplicon carry over and was designed for use in diagnostic laboratories. We have compared this PCR test with culture, antigen detection (Chlamydiazyme including confirmatory assay, Abbott) and DNA-RNA hybridization (GenProbe PACE 2, Diagnostic Products Corporation) to detect C. trachomatis in urogenital specimens. It was concluded that Amplicor was the most sensitive and specific assay. The assay appeared rapid, easy to perform and less expensive than culture. We found Amplicor very suitable for use in a clinical diagnostic microbiology laboratory.

Laboratory Diagnostic Methods for Central Nervous System Infections

Infections of the CNS have a high mortality, and rapid laboratory diagnosis and adequate antimicrobial therapy are critically important for their management. New techniques assist the clinical microbiology laboratory to isolate and identify micro-organisms more rapidly and accurately than with the use of classic procedures. Microbial identification using immunologic and DNA hybridization techniques has importantly reduced the time needed for the diagnosis of infectious diseases. Culture, however, is still the standard method to confirm the identity of an organism isolated from CSF or CNS sites. With the increase in the number of antimicrobial agents and the recognition of resistance in many isolates, antimicrobial susceptibility testing has become extremely important in the selection of optimal antimicrobial therapy. Communication between the physician and the clinical microbiology laboratory is essential for optimum patient care.

Sandra Fessia, Priscilla Fawcett, Carol MaCvaugh and Sharon Ryan, Diagnostic Clinical Microbiology – A Benchtop Perspective. XIII + 221 S., 36 Abb., 30 Tab. Philadelphia – London – Toronto – Montreal – Sydney – Tokyo 1988. W. B. Saunders Company (Harcourt Brace Jovanovich Inc.). $ 12.50. ISBN: 0-7216-2326-3

Journal of Basic MicrobiologyVolume 30, Issue 4 p. 232-232 Book Review Sandra Fessia, Priscilla Fawcett, Carol MaCvaugh and Sharon Ryan, Diagnostic Clinical Microbiology – A Benchtop Perspective. XIII + 221 S., 36 Abb., 30 Tab. Philadelphia – London – Toronto – Montreal – Sydney – Tokyo 1988. W. B. Saunders Company (Harcourt Brace Jovanovich Inc.). $ 12.50. ISBN: 0-7216-2326-3 A. Stelzner, A. Stelzner JenaSearch for more papers by this author A. Stelzner, A. Stelzner JenaSearch for more papers by this author First published: 1990 https://doi.org/10.1002/jobm.3620300403AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume30, Issue41990Pages 232-232 RelatedInformation

Tube dilution antimicrobial susceptibility testing: efficacy of a microtechnique applicable to diagnostic laboratories.

A microtechnique for determining antibiotic susceptibilities by the serial dilution method was evaluated in a clinical diagnostic microbiology laboratory. As compared with the standard tube method, an agreement of 94% was achieved for determining minimal inhibitory concentration with +/- one tube dilution as the criterion of comparison. The experience with this system suggests that it could easily be incorporated into diagnostic laboratories as a routine procedure.

Tube Dilution Antimicrobial Susceptibility Testing: Efficacy of a Microtechnique Applicable to Diagnostic Laboratories

A microtechnique for determining antibiotic susceptibilities by the serial dilution method was evaluated in a clinical diagnostic microbiology laboratory. As compared with the standard tube method, an agreement of 94% was achieved for determining minimal inhibitory concentration with +/- one tube dilution as the criterion of comparison. The experience with this system suggests that it could easily be incorporated into diagnostic laboratories as a routine procedure.