|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,
Preventing Antibiotic Resistance through Rapid Genotypic Identification
of Bacteria and of Their Antibiotic Resistance Genes in the Clinical
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
|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).
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).
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
|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
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