DƯỢC LÍ Goodman & Gilman's The Pharmacological Basis of Therapeutics 12th, 2010

1370
SECTION VII
CHEMOTHERAPY OF MICROBIAL DISEASES
A
Response
B
Response
100
80
60
40
20
0
100
80
60
40
20
E max
0
0 100
[Antimicrobial]
Figure 48–3. Changes in sigmoid E max
model with increases in
drug resistance. Increase in resistance may show changes in IC 50
(panel A: the IC 50
increases from 70 (orange line) to 100 (green
line), to 140 (blue line)) or decrease in E max
(panel B: efficacy
decreases from full response (orange line) to 70% (green line))
broth medium that contains a culture of the test microorganism.
The lowest concentration of the agent that prevents
visible growth after 18-24 hours of incubation is
known as the minimum inhibitory concentration (MIC).
Automated systems also use a broth-dilution method. The
optical density of a broth culture of the clinical isolate incubated in
the presence of drug is determined. If the density of the culture
exceeds a threshold optical density, then growth has occurred at that
concentration of drug. The MIC is the concentration at which the
optical density remains below the threshold.
The disk-diffusion technique provides only qualitative or
semi-quantitative information on antimicrobial susceptibility. The
test is performed by applying commercially available filter-paper
disks impregnated with a specific amount of the drug onto an agar
surface, over which a culture of the microorganism has been
streaked. After 18-24 hours of incubation, the size of the clear zone
of inhibition around the disk is measured. The diameter of the zone
depends on the activity of the drug against the test strain.
Standardized values for zone sizes for each bacterial species and
antibiotic permit classification of the clinical isolate as either resistant
or susceptible. A variant of the disk diffusion tests is the Epsilometer
test, or E-test. A rectangular test strip impregnated with changing
concentrations of antimicrobial agent, usually across 15 dilutions,
is placed on an agar plate that has a heavy inoculum of test
organism. The drug concentrations are printed along this long test
strip. The cultures are then incubated under favorable conditions for
24 hours, 48 hours, or 5 days, depending on the test organism. There
is no growth with higher concentrations and heavy microbial growth
where there is lower drug concentration, so that a clear elliptical zone
is formed that bisects the test strip at the MIC. This test has the virtue
of determining an actual MIC value, rather than the dichotomous
categorization of “susceptible” or “resistant.” There are test strips
for hundreds of antibacterial agents as well as for some antifungal
agents active against Candida species.
Sometimes, a biochemical or immunological reaction that
leads to a color change is used to detect susceptibility. As an example,
methicillin resistance in Staphylococcus aureus (methicillinresistant
S. aureus, or MRSA) occurs because of acquisition of a
low-affinity penicillin-binding protein 2’ (2a), or PBP2’ (PBP2a).
The PBP2’ is extracted from isolates of the MRSA, and supernatant
mixed with a latex reagent with monoclonal antibody against PBP2’.
Visible clumping within 3 minutes indicates presence of PBP2’, indicating
MRSA.
Fungi. For fungi that are yeasts (i.e., Candida), susceptibility
testing methods are similar to those used for
bacteria. However, the definitions of MIC differ based
on drug and the type of yeast, so there are cutoff points
of 50% decrease in turbidity compared to controls at
24 hours, or 80% at 48 hours, or total clearance of the
turbidity. Susceptibility tests and MICs for triazoles
have been extensively shown to correlate with clinical
outcomes.
Standardized tests for echinocandin antifungals and amphotericin
B-based compounds are available. However, the correlation
of these tests with clinical outcomes is still weak. Susceptibility
tests for molds have also been developed, but the clinical correlations
are still being examined. Different terminology from MICs
has been adopted when evaluating echinocandins against molds
because the fungal burden can not be measured by counting discrete
cells in molds, given that hyphae will break up into unpredictable
numbers of discrete fungi when under antifungal pressure.
Furthermore, echinocandins often do not completely inhibit mold
growth, but instead cause damage reflected by morphological
changes in hyphae. Thus, the minimum effective concentration
(MEC) for echinocandins is the lowest drug concentration at which
short, stubby, and highly branched hyphae are observed on microscopic
examination.
Viruses. In HIV phenotypic assays, the patient’s HIV-
RNA is extracted from plasma, and genes for targets of
antiretroviral drugs such as reverse transcriptase and
protease are amplified. The genes are then inserted into
a standard HIV vector that lacks analogous gene
sequences to produce a recombinant virus, which is
co-incubated with drug of interest in a mammalian cell