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Bacterial DNA Testing


Here are some insights from an interesting article published in the Journal of Clinical Microbiology. Although the reported 80 % sterile samples refers to blood (which is an ideal media for most microbial pathogens) they might have some relevance to other clinical samples such as EPS (with the percentage being higher).

Journal of Clinical Microbiology, August 1998, p. 2169-2172, Vol. 36, No. 8

Preventing Antibiotic Resistance through Rapid Genotypic Identification of Bacteria and of Their Antibiotic Resistance Genes in the Clinical Microbiology Laboratory
Michel G. Bergeron* and Marc Ouellette
Centre de Recherche en Infectiologie de l'Universite Laval and Division of Microbiology, Faculty of Medicine, Universite Laval, Quebec, Canada G1V 4G2
Speed is the essence when one deals with bacterial infections. Although the Gram stain can sometimes be helpful, presently, diagnosis in the clinical microbiology laboratory is only confirmatory because a clinical decision has been made long before (usually 48 h) the identity of the organism responsible for the infection and its susceptibility to antibiotics become available.
With the actual state-of-the-art technology, which dates back to the last century, we cannot even tell accurately before 18 to 24 h whether a clinical sample has bacteria or not. This is of importance because no bacteria can be grown out of more than 80% of all normally sterile clinical samples sent to clinical microbiology laboratories (4).
The lack of a timely response by the laboratory has consequences on antibiotic usage and prescription. Patients must be treated empirically. When severe or nosocomial infections are suspected, they are often treated with broad-spectrum antibiotics. The increased use of broad-spectrum antibiotics is not restricted to hospitalized patients in intensive care units or patients seen in emergency rooms, however. Indeed, a recent American survey has indicated that toxic and expensive broad-spectrum antibiotics are prescribed more frequently for the treatment of common infections by office-based physicians (14).
Clearly, with 80% of normally sterile specimens received in the microbiology laboratory not growing any microorganism, several patients are receiving antibiotics even if they do not have a bacterial infection because there are no accurate ways of determining before the next day whether the clinical sample harbors bacteria. In line with this latter argument, a recent study in Spain has indicated that on any particular day, the number of antibiotic prescriptions exceeded by three times the number of bacterial infections diagnosed (3).
Moreover, microbiologic results are available so slowly that physicians rarely consult them unless the patient is not responding to the given antibiotic. If physicians could have in hand the identity of the microorganism and its resistance profile from the microbiology laboratory at the same time that they have the biochemistry and hematology results, antibiotic prescription rates could go down dramatically, and when antibiotics are needed, more targeted and inexpensive antibiotics could be used.
On the other hand, whether you are using phenotypic or genotypic identification systems, the presence of bacteria or even the absence of bacteria in the clinical specimens does not necessarily mean the presence or the absence of infection because clinical judgment should always prevail.
Rapid identification of microorganisms as a means of decreasing the emergence of antimicrobial resistance.
The advances in sample preparation, DNA-based amplification techniques, and product detection have evolved to the extent that it is now possible to identify microorganisms directly from clinical specimens in 1 h (13). Moreover, as these DNA-based tests evolve, their sensitivity will allow the detection of a single copy of the genome of a microorganism. If the precise identification of the microbial agents responsible for infections were available within 1 h when the results of other laboratory tests are available to the physician, it would have a major impact on the management and treatment of patients.
The use of universal probes based either on the rRNA gene (11) or on some other conserved region of microorganism genomes should indicate whether or not the patient is infected with a bacterium. Because more than 80% of normally sterile clinical specimens (blood, cerebrospinal fluid, joint fluid, etc.) sent to the microbiology laboratory are not "infected" or do not harbor bacteria (4), the use of universal primers should permit determination in 1 h of whether or not the patient suffers from a bacterial infection.
Obviously, universal probes would not be useful for sputum or surgical wound specimens or specimens from other nonsterile clinical sites. Provided that appropriate controls are included and relevant sensitivity is reached, the absence of amplification products would suggest the absence of bacterial infections and the use of antibiotics could be avoided. In contrast, the detection of an amplification product with universal primers would indicate that a bacterium is present. However, it would not provide information on the nature of the bacterium and hence on the antibiotic to be used. Therefore, universal primers are useful for screening negative samples but are of limited value for orienting the choice of antibiotics in the case of a positive reaction.
There are now specific DNA probes or amplification primers for almost every relevant pathogenic organisms (8, 26), and these primers can be used to identify the bacteria present in clinical specimens. Because multiple bacteria can be isolated from different sites, it would be advisable to carry out reactions under multiplex conditions, i.e., with more than one pair of primers per reaction. It would be possible to discriminate the amplicon either by size on agarose gel electrophoresis or with a different fluorochrome if fluorescence was to be used as the detection method. It should also be possible to decrease substantially the number of primers by generating genus-specific or even group-specific PCR primers.
This approach has the benefit on the one hand of decreasing the complexity of the amplification reactions and on the other of increasing the proportion of bacteria detected. With group-, genus-, and species-specific amplification primers it should be possible to detect most microorganisms responsible for any type of infection. Nevertheless, there will always be the rare uncommon pathogen that is responsible for an infection but that may not be detected with the available primers. Because the universal primers would have detected the presence of an infection but none of the genus- or species-specific primers would have produced an amplification product, it would indicate that the infection is due to an uncommon pathogen. In those rare instances, culture may be requested if species determination was thought to be useful, but with time, most microorganisms could be identified by DNA-based tests. Rapid bacterial identification would be of major benefit to the clinician, but because the antibiotic susceptibility profile is an important parameter in the management of infections, we believe that a rapid identification system will fully blossom only when both bacterial identification and the resistance profile are provided simultaneously.


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